WO2015118549A1

WO2015118549A1 - Encapsulated biologically active agents

Info

Publication number: WO2015118549A1
Application number: PCT/IL2015/050149
Authority: WO
Inventors: Saul Koder
Original assignee: F&C Licorice Ltd.
Priority date: 2014-02-10
Filing date: 2015-02-10
Publication date: 2015-08-13

Abstract

The present invention relates to a platform for enhanced bioavailability and delivery of biologically active compounds. Specifically, the invention provides an orally administrable composition comprising an encapsulated biologically active agent and encapsulated inhibitors of gut enzymatic and/or transport activity. The invention further relates to pharmaceutical and nutraceutical compositions, as well as to consumable products comprising the orally administrable composition.

Description

ENCAPSULATED BIOLOGICALLY ACTIVE AGENTS

FIELD OF THE INVENTION

This invention relates to orally administrable formulations for the controlled release of a biologically active agent (e.g. a drug), and to consumable products and food supplements comprising them.

BACKGROUND OF THE INVENTION

Turmeric (curcuma longa L.) a member of the ginger family is extensively used in Ayurveda, Unani and Siddha medicine in India as home remedy for various diseases. Curcumin is the principal curcuminoid in turmeric. Curcuminoids are polyphenolic compounds that give turmeric its yellow color.

Clinically, curcumin has already been used to reduce post-operative inflammation. Safety evaluation studies indicate that both turmeric and curcumin have the potential for the development of modern medicine for the treatment of various diseases.

Orally administered Curcumin exhibits low bioavailability due to a poor absorption and a rapid metabolism in vivo (Anand et al., 2007). Bioavailability studies in humans reported variable but always low plasma levels of curcumin ranging from 0.016-11 μπιοι/L after acute or chronic supplementation of the pure compound at dosage ranging from 2 to 12g. All the pharmacokinetic studies concord that once absorbed, curcumin undergoes extensive reduction, most likely through alcohol dehydrogenase, followed by conjugation at various tissue sites mainly in the liver, kidney and intestinal mucosa. Thus curcumin glucuronides, sulphated and hexahydrocurcumin, are the major curcumin metabolites; glucuronides representing in many cases up to 99% of total conjugates in the plasma. Anyway less than 1% curcumin ingested is retrieved in the blood, trace amounts are generally found principally metabolized in urines and from 40 up to 75% of curcumin ingested is excreted in unchanged form in faeces. Kurien and Scofield, 2009 disclose encapsulation of curcumin in PEG. Curcumin is heated in order to increase its solubility and facilitate encapsulation. Shaikh et al, 2009 describe nanoencapsulation of curcumin. In both cases curcumin is encapsulated as a single agent.

A clinical study by Cheng and colleagues (2001) shows that after oral intake of 8 grams of curcumin, serum concentrations of the drug peaked 1-2 hours after intake to a level of about 1.75μΜ and declined gradually within 12 hours.

Shoba and colleagues administered 2 grams of pure curcumin powder to fasting volunteers resulting in low curcumin concentrations detected in plasma (less than lOng/ml) one hour post dosing. Co-ingestion of curcumin with 20 mg of the pepper constituent 1-piperoylpiperidine appeared to increase curcumin's bioavailability.

SUMMARY OF THE INVENTION

The present invention provides a platform for enhanced bioavailability and delivery of biologically active compounds.

Accordingly, by a first of its aspects, the present invention provides an orally administrable composition, said composition comprising:

(a) microcapsules each consisting of an encapsulation agent and at least one biologically active agent; and

(b) microcapsules each consisting of an encapsulation agent and at least one inhibitor of gut enzymatic and/or transport activity.

In one embodiment, the at least one biologically active agent and the at least one inhibitor of gut enzymatic and/or transport activity are released in the gut in a sustained manner.

In another embodiment, the at least one biologically active agent is released in the gut in a sustained manner and the at least one inhibitor of gut enzymatic and/or transport activity is released in the gut in a partially sustained manner.

In certain embodiments the microcapsules are being formulated so as to allow differential release rates of said at least one biologically active agent and said at least one inhibitor of gut enzymatic and/or transport activity.

In one embodiment the at least one inhibitor of gut enzymatic and/or transport activity is released in the gut prior to the biologically active agent. The at least one inhibitor of gut enzymatic and/or transport activity and the biologically active agent may be released gradually or in a pulse mode.

In certain embodiments the coating material comprises a multilayered coating.

In certain embodiments said encapsulation agent is selected from the group consisting of resistant starch, fat coatings, mono and diglycerides, wax, shellac, cellulose based coatings, and any combination thereof.

Specifically said cellulose based coatings are selected from the group consisting of Methyl cellulose (MC), Hydroxypropyl cellulose (HPC), Ethyl Cellulose (EC), Hydroxy ethyl cellulose (HEC), hydroxy propyl methyl cellulose (HPMC), carboxy methyl cellulose (CMC), Hydroxypropyl methyl cellulose acetate, cellulose acetate phthalate, hydroxymethyl cellulose phthalate, cellulose trimellitate, hydroxymethyl cellulose acetate succinate and any combination thereof.

In certain embodiments said encapsulation agent further comprises a plasticizer

In certain embodiments said encapsulation agent further comprises Hydrogenated Vegetable oils.

The microcapsules may comprise between about 5% and about 20% w/w encapsulation agent.

In certain embodiments said biologically active agent is selected from the group consisting of a curcuminoid, digoxin, antibiotics (e.g. cyclosporine A), anti neoplastic agent, and viral inhibitors (e.g. HIV protease inhibitors).

In certain embodiments said inhibitor inhibits the activity of an enzyme selected from the group consisting of UGT (UDP-glucuronosyltransferase) enzyme family, sulfotransferase enzymes, alcohol dehydrogenase, and p450 enzymes.

In specific embodiments said inhibitor is selected from the group consisting of Piperine, Quercetin, Genistein, Glabridin, and any combination thereof.

In a specific embodiment the orally administrable composition of the invention comprises the inhibitors Piperine, Quercetin, Genistein, and a curcuminoid.

In another aspect, the present invention provides an orally administrable composition for the controlled release of a curcuminoid wherein said composition comprises microcapsules consisting of at least one encapsulation agent and a curcuminoid.

In certain embodiments said microcapsules comprise between about 60% and about 90% curcumin and between about 10% and 30% encapsulation agent. In certain embodiments said composition is a solid composition.

In another aspect, the present invention provides a pharmaceutical or nutraceutical composition comprising the orally administrable compositions of the invention.

In certain embodiments said pharmaceutical composition is for the treatment of inflammatory diseases, infectious diseases, or cancer.

In certain embodiments the composition of the invention further comprises a carrier, exipient or diluents.

In another aspect, the present invention provides a consumable product comprising the orally administrable composition of the invention.

In certain embodiments said product is selected from the group consisting of dry pasta, bread and bread crisps.

In another one of its aspects, the present invention provides a method for increasing the bioavailability of a biologically active agent, said method comprising:

(a) preparing microcapsules each consisting of an encapsulation agent and said biologically active agent;

(b) preparing microcapsules each consisting of an encapsulation agent and at least one inhibitor of gut enzymatic and/or transport activity; and

(c) mixing said microcapsules obtained in step (a) and in step (b) to obtain a composition suitable for oral administration;

wherein when orally administered the biologically active agent and the at least one inhibitor of gut enzymatic and/or transport activity are released in the gut in a sustained manner.

In certain embodiments the composition obtained in step (c) is the composition of the invention as described above.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to understand the invention and to see how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

Fig. 1 is a graph demonstrating the content (as a percent of total weight) of bisdesmethoxycurcumin, desmethoxycurcumin, curcumin and total curcuminoids in dry pasta (A) and cooked pasta (B). Fig. 2 is a graph demonstrating the content (as a percent of total weight) of bisdesmethoxycurcumin (A), desmethoxycurcumin (B), curcumin (C) and total curcuminoids (D) levels in bread crisps baked at 180 ° C for 15 and 30 minutes.

Fig. 3 is a graph demonstrating the mean concentration of curcumin and metabolized curcumin (total curcumins) in subjects' sera collected over the experiment day. The subject groups consumed bread supplemented with free curcumine (Free); bread supplemented with ecapsulated-curcumin (Enc Cure); and bread supplemented with encapsulated curcumin + enhancers, namely gut enzyme inhibitors (Enc Cure + Enhanc). (n = 10).

Fig. 4 is a graph demonstrating the mean concentration of curcumin and metabolized curcumin (total curcumin metabolites) in subjects' urine collected 24 hours after the commencement of the experiment. The subject groups consumed bread supplemented with free curcumin (FC); bread supplemented with encapsulated- curcumin (EC); and bread supplemented with encapsulated curcumin + enhancers, namely gut enzyme inhibitors (EC + EN), (n = 10).

Fig. 5 is a graph demonstrating the mean concentration of total curcumin phenolic acids in subjects' urine collected 24 hours after the commencement of the experiment. The subject groups consumed bread supplemented with free curcumin (FC); bread supplemented with ecapsulated-curcumin (EC); and bread supplemented with encapsulated curcumin + enhancers, namely gut enzyme inhibitors (EC + EN), (n = 10).

DETAILED DESCRIPTION OF EMBODIMENTS

The cells lining the intestine are equipped with metabolizing enzymes that convert biologically active compounds into non active metabolites, and molecular transporters that pump these compounds or their metabolite byproducts out of the intestinal lining back into the intestine. These enzymatic mechanisms result in a reduction in drug availability and thus compromise their therapeutic effect.

The present invention provides a method for increasing the bioavailability of biologically active compounds (e.g. drugs) which are orally ingested, using microencapsulation as a vector for compound delivery.

The microencapsulation of a drug in a coating allows an attenuated time release mode of absorption through the gut. Without wishing to be bound by theory, the encapsulation attenuates the release of the drug within the digestive system. Specifically it allows "safe passage" through areas of the digestive system which are rich in digestive enzymes (e.g. the stomach). The capsule is degraded with time and the drug is finally released into the intestine in an area which contains a less degrading enzymatic activity.

In one aspect, the invention thus provides an orally administrable composition comprising a mixture of microcapsules, wherein said composition comprises at least two types of microcapsules, a first being microcapsules comprising the biologically active agent (i.e. drug) and the second being microcapsules comprising an inhibitor of gut enzymatic and/or transport activity. In one embodiment of the invention, the microcapsules in the composition are constructed so as to allow differential release rates of the active agent and the inhibitor of the gut enzymatic and/or transport activity. Furthermore, the microcapsules can be constructed to allow differential release rate of the inhibitor of the gut enzymatic and/or transport activity, so as to spread the release of these compounds throughout the gastrointestinal tract.

As used herein the term "orally administrable composition" or "orally administrable formulation" refers to compositions that may be administered orally in any form suitable for oral delivery such as a tablet, powder, capsule or caplet. The compositions may be administered as such or incorporated into a consumable product.

Preferably, the orally administrable composition of the invention is for the controlled release of a drug.

As used herein the term "controlled-release" or "sustained-release" or "released in a sustained manner" refers to the gradual release of the drug and/or the gut enzyme inhibitors in the gastrointestinal (GI) system as opposed to their immediate exposure to the GI environment.

As used herein the term "partially sustained manner" relates to a composition of the invention in which some of the gut enzyme inhibitors in the composition are not encapsulated thereby capable of exerting their inhibitory activity immediately upon entry into the GI system.

The terms "gastrointestinal (GI) system ", "gastrointestinal tract" and "gut" are used herein interchangeably. As used herein, the term "microcapsules " refers to active agents (e.g. a drug such as curcumin, or an inhibitor of gut enzyme activity) coated with an encapsulating agent.

As used herein the term "encapsulation" and in particular "microencapsulation" refers to a process wherein a core composed of the compound of interest in solid, liquid or gaseous form is covered by a thin film of a coating agent. The encapsulation generates a physical barrier having different morphological and resistance characteristics depending on the coating material (also termed herein "encapsulation agent"). Most microcapsules have diameters between a few micrometers and a few millimeters.

Techniques for microencapsulation of compounds intended for human or animal consumption are well known in the art (see for example in Handbook of Food Preservation, Second Edition, Chapter 22 "Encapsulation, Stabilization, and Controlled Release of Food Ingredients and Bioactives" by Pegg and Shahidi, pages 510-568) and depend on the physical and chemical properties of the material to be encapsulated.

Such techniques include, but are not limited to spray drying, spray cooling and spray chilling, fluidized bed coating, extrusion, centrifugal extrusion, lyophilization, coacervation, centrifugal suspension separation, cocrystalization, liposome entrapment, interfacial polymerization, inclusion complexation (molecular inclusion).

In a specific embodiment, as shown in the Examples below, the encapsulation is performed in a fluid bed coater. The fluid bed coater may have various volume capacities (e.g. 4 inch, 6 inch, 9 inch, 12 inch, 18 inch etc) - depending on the required/produced quantity. Determination of the inlet and outlet temperatures of the device, as well as the drying period is well within the knowledge of a person skilled in the art.

Encapsulation further encompasses nanoparticulate delivery systems (nanoencapsulation), which relate to techniques for generating nano scale capsules, i.e. in the range of 1-100 nm.

Encapsulation is largely used in the pharmaceutical industry as well as in the food industry. Among others, encapsulation serves to allow a controlled release of bioactive ingredients in various parts of the gastro intestinal tract.

In accordance with the invention the encapsulation is performed using an encapsulation agent (also termed herein "coating material"). Non-limiting examples of an encapsulation agent are resistant starch, Hydrogenated Vegetable oils (HVO), fat coatings, mono and di glycerides, wax, shellac, cellulose-based compounds, e.g. Methyl cellulose (MC), Hydroxypropyl cellulose (HPC), Ethyl Cellulose (EC), Hydroxy ethyl cellulose (HEC), hydroxypropyl methyl cellulose (HPMC), carboxy methyl cellulose (CMC), Hydroxypropyl methyl cellulose acetate, and combinations thereof. These coatings are gradually dissolved in the gut in a time-dependent manner.

In one specific embodiment, the encapsulation agent is a cellulose-based compound (also termed herein cellulose-based coating).

Additional examples of cellulose-based coatings are cellulose acetate phthalate, hydroxymethyl cellulose phthalate, cellulose trimellitate, and hydroxymethyl cellulose acetate succinate. These coatings are pH sensitive and may dictate differential dissolution in the gut based on the local pH.

The coating of the active agent may further comprise at least one plasticizer. The at least one plasticizer may be any compound that acts as a plasticizer and is compatible with the encapsulation agent. Non limiting examples of piasticizers include acetvlated monoglycerides, medium chain triglycerides, dibutyl sebacate etc. in one specific embodiment castor oil serves as a plasticizer for ethylcellulose based coating.

In one embodiment, wherein Ethyl Cellulose is used as a coating agent the plasticizer may be in an amount of between about 0.5% and about 25% (w/w) of the Ethyl Cellulose.

Certain coating agents may be applied without addition of any plasticizer.

In the coating process the encapsulation agent may be dissolved in a suitable solvent which is compatible with the encapsulation agents' characteristics. The solvent may be an aqueous solvent or an organic solvent. In one specific example, wherein the coating material is a cellulose-based compound organic solvents may be used for dissolving the coating material, including, but are not limited to, ethanol, toluene, acetone, methanol or any mixture thereof. In one specific embodiment the organic solvents used for the encapsulation are acetone and methanol which are compatible for use with ethyl cellulose.

The encapsulation agent(s) may be dissolved in the suitable organic solvent to achieve a solution having between about 3% and 15% solids, e.g. 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% solids. In one specific embodiment the encapsulation agent is dissoved in acetone and methanol to obtain a solution having 4% solids.

Certain coating agents may be applied without dissolving in an organic solution.

The core may be of 50 - 3000 microns, the coating may be between 5% - 80%, and may contain additional components e.g. oil or a combination of the above substances, for example a cellulose derivative and wax or hydorgenated vegetable oil (HVO) for example hydrogenated palm oil, or a combination of a cellulose derivative, HVO and a plasticizer.

In one embodiment the composition comprises an encapsulated drug as a single agent.

In another embodiment, the composition comprises an encapsulated drug in combination with at least one encapsulated gut enzyme inhibitor.

As used herein the term "biologically active compound" or "biologically active agent" refers to a compound with physiological activity in a subject's body, for example a drug. In accordance with the present invention, the drug may be any pharmaceutically active ingredient which is taken orally and may be compromised by the activity of gut enzymes. Non-limiting examples of such drugs include: curcumin, digoxin, antibiotics (e.g. cyclosporine A), anti neoplastic agents and anti viral agents (e.g. HIV protease inhibitors).

In one specific embodiment the drug is a curcuminoid i.e. curcumin, and curcumin derivatives such as, but not limited to, demethoxycurcumin (also termed desmethoxycurcumin) and bisdemethoxycurcumin (also termed bisdesmethoxycurcumin), or a mixture of the three compounds.

As used herein the term "Curcumin " relates to diferuloylmethane [(IE, 6E)-1 , 7-bis (4-hydroxy- 3-methoxyphenyl) -1 , 6- heptadiene-3, 5- dione], and has the chemical structure de icted below (C2₁H2₀O₆):

It is a polyphenolic compound having a molecular weight of 368.37 and a melting point of 183°C. Curcumin is relatively insoluble in water, but dissolves in acetone, dimethylsulphoxide and ethanol. It is unstable at basic pH, and degrades within 30 minutes to trans-6-(40-hydroxy-30-methoxyphenyl)-2, 4-dioxo-5-hexanal, ferulic acid, feruloylmethane and vanillin.

Curcumin has been largely investigated in the last few years showing a broad array of biological activities, such as those described in Aggarwal et al (Phytopharmaceuticals in Cancer Chemoprevention (2005) 349-387). A growing body of literature has demonstrated the antioxidant, anti inflammatory, anti-carcinogenic, and anti-infectious activity of curcumin based on the ability of this compound to regulate a number of cellular signal transduction pathways (Calabrese et al., 2008).

Curcumin is readily conjugated in the intestine and liver to form curcumin glucuronides and curcumin sulfates or reduced to hexahydrocurcumin (Ireson et al. (2002) Cancer Epidemiol. Biomarkers Prev. 11 (1), pp: 105-111).

Accordingly, in certain embodiments the microcapsules comprise between about 70% and about 90% Curcumin (also referred to herein as Curcumin Particles (CP)) and between about 5% and about 20% Coating material (CM). In certain embodiments the microcapsules further comprise between about 10% and 20% Hydrogenated vegetable oil (HVO).

In specific embodiments the microcapsules comprise 90% CP and 10% CM; 85% CP and 15% CM; 76.5% CP, 8.5% CM and 15%HVO; or 72.25% CP, 12.75% CM and 15% HVO.

Examples of converting enzymes which may act in the gut on an orally taken drug include, but are not limited to: The UGT (UDP-glucuronosyltransferase) enzyme family; sulfotransferase enzymes; alcohol dehydrogenase; and p450 enzymes.

Non limiting examples of gut enzyme inhibitors include but are not limited to Piperine (which inhibits UGT (UDP-glucuronosyltransferase), p450 enzymes, P- glycoprotein and CYP3A4 (Rajinder et al The J. of Pharmacology and Experimental Therapeutics Vol. 302, No.2 pages 645-650)), Quercetin (which inhibits sulfotransferase enzymes), Genistein (which inhibits alcohol dehydrogenase), and Glabridin (which inhibits p450 enzymes). All these gut enzyme inhibitors are commercially available.

In certain embodiments wherein the composition comprises an encapsulated drug and an encapsulated enzyme inhibitor the release mode would be two fold, namely, the enzyme inhibitor is released first while the drug is released at a later stage. Moreover, the various enzyme inhibitors may also be released sequentially thereby allowing sequential inhibition of the relevant gut enzymes. The sequential release of the compounds may be gradual and continuous or in a pulse mode. In the pulse mode the enzyme inhibitor is release in a first pulse and the drug is released in a second belated pulse. As elaborated below, the sequential release is achieved by differential coating of the various components.

In one embodiment, the encapsulation of the drug and the enzyme inhibitor is performed using a multilayered coating. The multilayered coating will allow release of the active compounds at different times, different areas of the intestine and at specific release rates. In one specific embodiment said drug is encapsulated in a multilayered coating comprising for example one layer of ethyl cellulose (optionally including a plasticizer) and a second layer being HVO (Hydrogenated Vegetable Oil); while the enzyme inhibitor is coated with a single layer coating allowing an enhanced disintegration of the coating in the gut and an enhanced release of the enzyme inhibitor prior to the release of the drug. Furthermore, the drug and/or the enzyme inhibitor may each be coated with different coating material allowing gradual release of the drug and/or the enzyme inhibitor within the gut.

The present invention thus provides microcapsules encapsulating bioactive compounds, which in the absence of the coating are normally absorbed at the upper portion of the gut. The encapsulation allows these compounds to reach the lower gut and to exert their beneficial action, without being inactivated prematurely.

The examples provided herein refer to curcumin but are applicable to any orally administered drug. Examples of drugs known to be metabolized by gut enzymes, e.g. P450 are well known in the art and may be found for example in "Text book of personalized Medicine" - Kewal K. Jain; Springer Science business madia; 2009; pp 73- 74.

Accordingly, in one specific embodiment the present invention provides encapsulated curcumin. Encapsulation may be performed by any method known in the art, for example, a fluidized bed coater - using a Wurster process. In accordance with the invention curcumin may be obtained from various sources including, but not limited to, Oleoresin Turmeric, or curcuma root. In one embodiment, curcumin content in the microcapsule is in the range of about 60% to about 90%. In one embodiment, curcumin content in the microcapsule is at least 80%. In another embodiment, curcumin content in the microcapsule is at least 85%. The remaining constituents of the microcapsule include the coating material and optionally additional active ingredients e.g. gut enzyme inhibitors which enhance curcumin activity.

In one embodiment, the content of the encapsulated gut enzyme inhibitor in the composition is between about 5% and 10%. In one specific embodiment the content of the encapsulated gut enzyme inhibitor in the composition is between 8.5%. In another specific embodiment said gut enzyme inhibitors include at least one of Piperine, Quercitin or Genistein or any combination thereof. In one specific embodiment said gut enzyme inhibitors are provided in equal proportions. In another embodiment said gut enzyme inhibitors are provided in unequal proportions.

The gut enzyme inhibitors (at least one or any combination of the above listed inhibitors) may be divided into aliquots and coated with different concentrations of the coating material, e.g. ethyl cellulose (optionally including a plasticizer). For example, with 0% Ethyl cellulose (no coating), 5% Ethyl cellulose, 10% Ethyl cellulose, 15% Ethyl cellulose, 20% Ethyl cellulose, 25% Ethyl cellulose, or any intermediate percentage of the coating material. The composition may therefore include at least one, at least two, at least three, at least four, at least five or at least 6 types of aliquots of the differentially coated gut enzyme inhibitors, in any combination thereof. For example, the composition may include equal proportions of gut enzyme inhibitors coated with 0%, 5%, 10% and 15% ethyl cellulose (optionally including a plasticizer). In such case the gut enzyme inhibitors are released in a partially sustained manner. In another example, the composition may include equal proportions of gut enzyme inhibitors coated with 5%, 10%, 15% and 20% ethyl cellulose (optionally including a plasticizer). In certain embodiments, the composition includes unequal proportions of the differentially coated gut enzyme inhibitors.

In one non-limiting example of the invention Curcumin is encapsulated with a double coating, the first being ethyl cellulose (optionally including a plasticizer) and the second being HVO while the gut enzyme inhibitors are divided into aliquots and coated with 5%, 10%, 15%, and 20% ethyl cellulose (optionally including a plasticizer). Ethyl cellulose may be obtained from commercial sources, for example, Ethocel 100 (purchased from Ashland).

In certain embodiments the composition of the invention is provided as a solid composition in a form such as a tablet, powder, granules, a capsule, or a caplet. The compositions may conveniently be presented in unit dosage form and prepared by standard methods well known in the art. In other embodiments, the composition may be provided in soft gel, syrup, suspension, emulsion or solution in water or non-aqueous media, lozenges (including liquid-filled) or chews. It should be noted that liquid may compromise the long term stability of the coating and hence dry formulations of the composition of the invention are preferred. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be included in the composition as well.

In certain embodiments the composition further comprises a suitable carrier or excipient. As used herein the term "acceptable carrier, excipient or diluent" includes any and all solvents, dispersion media, antibacterial and antifungal agents and the like, as known in the art. The carrier can be solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Each carrier should be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the subject. Except as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated.

In one aspect the microcapsules of the invention are incorporated into a consumable product, i.e. food or beverage for human or animal consumption.

In one embodiment, the microcapsules of the invention are incorporated into starch-based material.

In one embodiment, the microcapsules of the invention are incorporated into dry pasta.

In another embodiment, the microcapsules of the invention are incorporated into bread or bread crisps.

The present invention therefore pertains to consumable products containing the microcapsules of the invention. The consumable product (e.g. pasta, bread or bread crisps) is prepared by a regular procedure known in the art while partially replacing flour with the composition of the invention.

In one embodiment, said consumable products have a curcumin content of between about 0.5% to 5% w/w. In one specific embodiment, said consumable products have a curcumin content of about 1% or 1.2% w/w.

There are potential nutraceutical uses for compositions according to embodiments of the invention, for example the compositions may be formulated into supplements sold in health food stores and pharmacies. In one specific embodiment curcumin content in said composition for use as food supplement is between about 85% and 95%. As used herein the term "nutraceutical composition " refers to compositions of the invention which are provided as food supplements.

In another aspect, the present invention provides a pharmaceutical composition comprising encapsulated gut enzyme inhibitors and an encapsulated drug.

In particular, the present invention provides a pharmaceutical composition comprising encapsulated gut enzyme inhibitors and an encapsulated curcumionoid. Said pharmaceutical composition may be used for the treatment of any disease known to be affected by curcumin e.g. inflammatory diseases, infectious diseases, or cancer.

In one specific embodiment the present invention provides a pharmaceutical composition comprising encapsulated gut enzyme inhibitors and encapsulated curcumin for the treatment of cancer.

In said pharmaceutical composition the gut enzyme inhibitor content in the microcapsule is between about 5% and 10%. In one specific embodiment said gut enzyme inhibitors include at least one of Piperine, Quercitin or Genistein or any combination thereof. In one specific embodiment said gut enzyme inhibitors are provided in equal proportions. In another embodiment said gut enzyme inhibitors are provided in unequal proportions.

In a non-limiting example Curcumin is encapsulated with a double coating, the first being ethyl cellulose (optionally including a plasticizer) and the second being HVO while the gut enzyme inhibitors are divided into aliquots and coated with 5% ethyl cellulose, 10% ethyl cellulose, 15% ethyl cellulose and 20% ethyl cellulose. In certain embodiments the ethyl cellulose is Ethocel 100. Examples

Example 1: Production of encapsulated curcumin

Different types of curcumin-containing microcapsules were produced, having varying proportions of their components i.e. the active material Oleoresin Turmeric (as Curcumin > 95%), the cellulose derivatives (Ethocel 10 or Ethocel 100), the castor oil and the Hydrogenated Vegetable Oil, as follows:

Active material: Oleoresin Turmeric (as Curcumin > 95%) - 85% ± 2% (of the total mixture); and

Coating material: Cellulose derivatives (total of 15% ± 2% of the total mixture) including: Ethocel 10 (13.2%) + Castor oil (1.8%).

Active material: Oleoresin Turmeric (as Curcumin > 95%) - 85% ± 2%; and Coating material: Cellulose derivatives (total of 15% ± 2%) including: Ethocel 100 (13.2%) + Castor oil (1.8%).

Active material: Oleoresin Turmeric (as Curcumin > 95%) - 90% ± 2%; and Coating material: Cellulose derivatives (total of 10% ± 2%) including: Ethocel 100 (8.8%) + Castor oil (1.2%).

Active material: Oleoresin Turmeric (as Curcumin > 95%) - 80% ± 2%; and Coating material: Cellulose derivatives (total of 20% ± 2%) including: Ethocel 10 (17.6%) + Castor oil (2.4%).

Active material: Oleoresin Turmeric (as Curcumin > 95%) - 76.5% ± 2%; and Coating material: Cellulose derivatives (total of 8.5% ± 2%) including: Ethocel 100 (7.48%) + Castor oil (1.02%); + Hydrogenated Vegetable Oil 15% ± 2%.

Active material: Oleoresin Turmeric (as Curcumin > 95%) - 72.25% ± 2%; and Coating material: Cellulose derivatives (total of 12.75% ± 2%) including: Ethocel 100 (11.22%) + Castor oil (1.53%); + Hydrogenated Vegetable Oil 15% ± 2%. Active material: Oleoresin Turmeric (as Curcumin > 95%) - 72.25% ± 2%; and Coating material: Cellulose derivatives (total of 12.75% ± 2%) including: Ethocel 10 (11.22%) + Castor oil (1.53%); + Hydrogenated Vegetable Oil (15% ± 2%).

Active material: Oleoresin Turmeric (as Curcumin > 95%) - 68.0% ± 2%; and Coating material: Cellulose derivatives (total of 17% ± 2%) including: Ethocel 10 (14.96%) + Castor oil (2.04%); + Hydrogenated Vegetable Oil (15% ± 2%).

The encapsulation was performed by spraying curcumin with a solution of Ethocel 100 and castor oil in acetone and methanol in a fluid bed coater as described in Example 2. Some of the compositions were further encapsulate with a second layer of HVO as described in Example 2 below.

Example 2: Production of compositions comprising encapsulated curcumin + encapsulated gut enzyme inhibitors

Next, a composition comprising both curcumin and gut enzyme inhibitors was produced. The combination of Ethocel 100, castor oil and HVO was selected for the coating.

Step 1 - Encapsulation of curcumin with 10% (w/w) cellulose derivatives as a first layer:

Preparation of a solution with 4% solids: Ethocel 100 (purchased from Ashland) and castor oil (purchased from Henry Lamotte Oils GMBH) as a plasticizer were dissolved in acetone (purchased from Sasol) and methanol (purchased from Sasol), according the following formulation:

• Ethylcellulose (Ethocel 100): 3.52% (88% of solids)

• Castor oil: 0.48% (12% of solids)

• Acetone BP (British Pharmacopeia): 76.8% (80% of solvent mix)

• Methanol BP: 19.2% (20% of solvent mix) The coating process was performed in a 6 inch (6") fluid bed coater with a bottom spraying mechanism (produced by Coating Place Inc.) as follows:

• for each kg of Oleoresin Turmeric (as Curcumin having a content of bioactive compounds higher than 95% (purchased from Synthite)), 2.78kg of solution was taken.

• inlet temperature was 45°-65°C.

• outlet temperature was 31 °-40°C (preferably at 36°C±2).

• At the end of the spraying process, the composition was dried for 5-10 minutes.

Step 2 - Encapsulation of curcumin with 15% of HVO as a second layer: o The Hydrogenated Vegetable Oil (palm oil, purchased from Wilmar) was melted and held at a temperature of 100°-110°C.

o The encapsulated curcumin obtained in step 1 was further coated as follows:

• Encapsulated curcumin from step 1 : 85%

• Hydrogenated Vegetable Oil: 15% o The coating process was also performed in a 6" fluid bed coater with a bottom spraying mechanism with the following parameters:

• inlet temperature of 40°-50°C.

• outlet temperature of 39°-46° (preferably at 43°C±2).

Step 3 - Encapsulation of gut enzyme inhibitors with 5%, 10%, 15%, and 20% (w/w) of cellulose derivatives: o Preparation of a solution with 4% solids: Ethocel 100 with 2% of castor oil as a piasticizer is dissolved in a system of acetone and methanol, according to the formulation described in step I.

o The coating process of the gut enzyme inhibitors is performed in a similar manner to that of curcumin in step 1. o The gut enzyme inhibitors (Piperine (purchased from Sabinsa), Quercetin (as Quercitin dihydrate purchased from Alalim) and Genistein (as Soy isoflavones purchased from Solbar) are coated with the 4% solid solution (described in step 1) to coating levels of 5%, 10%, 15%, and 20%. The coating could be performed on the mixture of the 3 gut enzyme inhibitors or on each inhibitor individually.

The mixture of gut enzyme inhibitors was divided to four equal portions: one coated with 5% Ethocel 100, the second coated with 10% Ethocel 100, the third coated with 15% Ethocel 100 and the fourth coated with 20% Ethocel 100.

The encapsulated curcumin and the encapsulated gut enzyme inhibitors were mixed to obtain a composition.

In one exemplary embodiment, the composition included:

Oleoresin Turmeric (as Curcumin > 95%) - 70.0% ± 2%;

Coating material, including:

Cellulose derivatives (total of 7.77% ± 2%) including: Ethocel 100 (6.84%) and Castor oil (0.93%); and

Hydrogenated Vegetable Oil (HVO) (13.73% ± 2%)

Gut enzyme inhibitors (Piperine, Quercitin and Genistein in equal proportions coated with 5%, 10%, 15% and 20% Ethocel 100) - 8.5% ; and

The composition may be incorporated into consumable products as described below.

Example 3: Analytical procedure to quantify curcuminoids in starch based material

Curcuminoids content in dry pasta and bread crisps was determined after extraction with ethanol and following analysis by HPLC equipped with UV-VIS detector. A novel extraction procedure was developed in order to obtain a recovery of near 100%. Each sample was extracted after freeze drying and grinding. Briefly, 50 mg of each sample were weighted and a pre-digestion step with 30 μΐ of Viscozyme™ mix (purchased from Fluka) and 1 ml of acidified water (pH = 3.0) was performed in a shaking bath for 16 hours at 40 °C. This pre-digestion step was necessary for 2 main reasons 1) during the pasta and bread crisps preparation process curcumin forms non covalent complexes with starch; 2) in food products enriched with encapsulated curcumin, coating material can be either intact or partially destroyed by food processing. Both factors may affect extraction efficiency, making curcuminoids partially inaccessible during solvent extraction. After enzymatic digestion, extraction was carried out for three times with 10 ml of ethanol and one more time with 5 ml ethanol. After solvent addition, samples were vortexed for 2 minutes and then centrifuged at 4000 rpm for 10 minutes at 4 °C. All upper phases were collected together and total volume was adjusted to 40 ml. After ultracentrifugation at 12000 rpm for 5 minutes at 4 °C, 20 μΐ of extract was injected. Chromatographic separation was performed with Shimadzu 10A VP HPLC system, using a Prodigy 5u ODS3 100 A column, size 250 x 4.60 mm purchased by Phenomenex. Elution was carried out with isocratic method with 50% A phase (0.1 M formic acid in water) and 50% B phase (acetonitrile) and carrier flow was 0.800 ml/min. Curcuminoids were detected by UV- vis detector at a wavelength of 262 nm.

Example 4: Analytical procedure to quantify curcuminoids in human plasma, urine and feces

Extraction of curcumin and its metabolites curcumin glucuronide, curcumin sulfate and hexahydrocurcumin from serum and urine was performed by method described by Shaikh et al. (2009) with some modifications. For serum, 500 μΐ of sample were extracted twice with 2 ml of ethyl acetate, vortexed 30 sec and centrifuged at 4000 rpm for 10 minutes at 4 °C. Upper phases were collected together and total extract was dried under nitrogen flow. Dried extract was re-suspended with 50 μΐ of methanol. For urine samples the same procedure was employed but with 3 ml of sample and 4 extraction steps. Curcumin and curcumin sulfate extraction from feces was carried out by a method reported by Sharma et al. (2001) with some modifications. 2 ml of acetonitrile: water 70:30 mix were added to 1 ml of fecal suspension (10% w/v in PBS containing 2mM BHT). After shaking by vortex 30 sec and centrifugation at 4000 rpm for 10 minutes at 4 °C, curcuminoids in the upper phase were separated from other fecal constituents by CI 8 solid phase extraction and eluted from the cartridge with acetonitrile (2 ml). Finally, 30 μΐ of curcuminoids extract from feces, and re-suspended extracts from serum and urine, were injected into LC/MS/MS system, that consisted in PerkinElmer Series 200 LC system, coupled with API3000 LC/MS/MS system. Curcumin and metabolites separation was accomplished using the same column, liquid phases (with A:B phases ratio of 30:70), and carrier flow used for curcuminoids analysis in food products.

RESULTS

Example 5: Production of dry pasta with curcumin in free and encapsulated form

Dough was prepared with the addition of the functional bioactive ingredient (i.e. curcumin in a free or encapsulated form, with or without inhibitors of gut enzymatic activity). A longer pre hydration step of semolina and the functional ingredient was necessary. Laminated pasta was produced as well as an extruded product (Rigatoni).

Sensory acceptance in curcumin-enriched pasta

Two kinds of curcumin-enriched pasta shaped like Italian "tagliatelle" were made: one with curcumin in encapsulated form (curcumin content about 85%), and the other with a free curcumin powder (curcumin content 95%).

These two products were made by partially replacing flour with the curcumin- based ingredient, to obtain a curcumin content corresponding to 1 % in the final product. The drying process, to reach a moisture content of 12 % was made at low temperature (25-32°C) and for a long time (24 hours) to reduce, as much as possible, the degradation of bioactive ingredient.

Data indicate that colour intensity and pungent smell were more pronounced in pasta containing free curcumin powder. No significant differences were found for sweet taste whereas the encapsulation process masked the unpleasant curcumin bitter taste perception.

This bitter taste, which is a typical feature of curcumin based products, was one of the main difficulties for the consumer acceptance of this kind of products. In fact, the pasta enriched with encapsulated curcumin was preferred by the large majority of the subjects.

Curcumin stability to the preparation process

To evaluate curcuminoids stability, chemical analysis of dry pasta and cooked pasta was performed. The pasta was subjected to extraction as described above and its content was analyzed using HPLC.

Data reported in Figure 1 indicated that there was no significant difference in curcuminoids stability between pasta enriched with encapsulated curcumin and free curcumin, both in dry or cooked pasta.

Example 6: Production of bread crisps with curcumin in free and encapsulated form

To gain further information on thermal stability bread crisps containing curcumin were also produced. In this product much higher temperature (up to 180°C) is reached during the preparation process and the differences in the degree of thermal degradation between free and encapsulated curcumin can be better appreciated. Bread crisps toasted at 180° C for 15 and 30 minutes, with free curcumin and four kinds of encapsulated curcumin were made (1.2% final content).

The types of encapsulated curcumin are as follows:

• 76.5% ± 2% curcumin + Coating material: Cellulose derivatives (8.5% ± 2%) including Ethocel 100 (7.48%) + Castor oil (1.02%); + Hydrogenated Vegetable Oil 15% ± 2%.

• 90% ± 2% curcumin + Coating material: Cellulose derivatives (10% ± 2%) including Ethocel 100 (8.8%) + Castor oil (1. 2%).

• 85% ± 2% curcumin + Coating material: Cellulose derivatives (15% ± 2%) including Ethocel 100 (13.2%) + Castor oil (1.8%).

• 72.25% ± 2% curcumin + Coating material: Cellulose derivatives (12.75% ± 2%) including Ethocel 100 (11.22%) + Castor oil (1.53%); + Hydrogenated Vegetable Oil 15% ± 2%.

Table 1 shows the ingredients for preparing bread crisps with free and encapsulated curcumin. Table 1

Sample Ingredients amount for lOOg of dough

CP - curcumin particles; CM - coating material; HVO - Hydrogenated Vegetable Oil

As shown in Figure 2, encapsulation with a higher percentage coating material (12.75% and 15%) is associated with higher curcuminoids stability in the enriched product, on dry basis, compared to the product enriched with free curcumin. Particularly, this protective effect is more appreciable when toasting is carried out for 30 minutes. With regards to HVO addition, for microcapsules with high percentage of coating material, it seems to be an additional protective agent for both toasting times. This additional protection is not kept during 30 minutes toasting in microcapsules containing a lower percentage of coating material.

Bio availability studies in human subjects

The bioavailability of the microencapsulated compounds compared to free curcumin was examined in human subjects.

Three types of bread containing curcuminoids in free form, in microencapsulated form and in microencapsulated form + microencapsulated enzyme inhibitors (2g per lOOg serving) were produced.

Ten healthy subjects with no type of gastrointestinal disorders, no recent body weight changes, or any drug therapy, were selected and enrolled to participate in the study.

In the study, volunteers were randomized to receive bread with free curcumin, with encapsulated curcumin or with encapsulated curcumin and encapsulated gut enzyme inhibitors. Briefly, the volunteers had a polyphenol-free diet 2 days before the experiment and throughout. In particular the volunteers had to avoid certain foods such as fruits and fruit juices, vegetables, chocolate, coffee, tea, whole grains and legumes while bread, pasta, rice, fish, milk and dairy products, meat and meat products were allowed.

Blood samples were collected from the volunteers before and at 0.5, 1, 2, 4, 6 and 24 hours after the consumption of 100 g of the curcumin-enriched bread.

Urine and feces samples were also collected. Two supplements, lOOg each, of the curcumin-enriched bread were consumed by the subjects at the experiment day. Thus in the experiment days subjects ingested totally 6 g curcumin.

All biological samples were analyzed to evaluate the concentration of curcuminoids and metabolites using the methods previously described.

Analysis of the level of curcumin and its various metabolites (curcumin glucuronide, esahydroxy curcumin glucuronide and desmethoxy curcumin) in the subjects' sera revealed that consumption of bread containing encapsulate curcumin + the gut enzyme inhibitors (Piperine, Quercitin and Genistein) resulted in an increased and prolonged level of curcumin and its metabolites in the sera as compared with encapsulated curcumin alone and free curcumin (see Fig. 3).

As shown in Figure 4, analysis of total curcumin metabolite content in urine samples 24 hours after curcumin consumption revealed that the highest level was found in subjects who consumed encapsulated curcumin + the gut enzyme inhibitors (Piperine, Quercitin and Genistein, also termed the "enhancer").

Interestingly, consumption of encapsulated curcumin and to an even higher extent, consumption of encapsulated curcumin + enzyme inhibitors significantly increased the concentration of phenolic acids (chlorogenic acid, Ferulic acid, Vanillic acid, diHPA, HPA and HPP) in urine samples obtained 24 hours after curcumin consumption (Figure 5).

Urine and blood data indicated that curcumin encapsulation determined a slowed but continuous absorption of the compound over a supplementation day.

A lower 24h fecal excretion was found after consumption of encapsulated curcumin + enzyme inhibitors (0.52 ng) as compared with free curcumin (31.32 ng). Interestingly, the level of total phenolic acids in faeces samples obtained 24 hours after consumption of encapsulated curcumin + enzyme inhibitors were significantly lower as compared with the phenolic acid content in faeces samples obtained 24 hours after consumption of free curcumin (51.46ng compared with 0.13ng, respectively)

The overall results obtained in the bioavailability tests suggest that encapsulated curcumin + enzyme inhibitors elicits its bioactivity over a longer time-period compared to the free-compound, or the encapsulate curcumin without an enzyme inhibitor.

References

Aggarwal et al: Phytopharmaceuticals in Cancer Chemoprevention (2005) 349-387

Anand et al: Bioavailability of curcumin: problems and promises. Mol Pharm. 2007 Nov-Dec; 4(6):807-18.

Calabrese et al: Curcumin and the cellular stress response in free radical-related diseases. Mol. Nutr. Food Res. 2008; 52:1062-73

Cheng et al: Phase I clinical trial of curcumin, a chemopreventive agent, in patients with high-risk or pre-malignant lesions. Anticancer Res. (2001) 21, pp: 2895-2900

Kurien BT and Scofield RH: Increasing aqueous solubility of curcumin for improving bioavailability. Trends Pharmacol Sci. 2009 Volume 30, Issue 7, pages 334-335.

Shaikh J et al: Nanoparticle encapsulation improves oral bioavailability of curcumin by at least 9-fold when compared to curcumin administered with piperine as absorption enhancher. Eu J Pharm Sci 2009, Vol 37, 223-30.

Sharma R.A. et al: Pharmacodynamic and pharmacokinetic study of oral Curcuma extract in patients with colorectal cancer. Clin Cancer Res 2001, Vol 7, 1894-1900.

Shoba et al: Influence of piperine on the pharmacokinetics of curcumin in animals and human volunteers. (1998) Planta Med 64, pp: 353-356.

Claims

CLAIMS:

1. An orally administrable composition, said composition comprising:

2. An orally administrable composition in accordance with claim 1, wherein said at least one biologically active agent and said at least one inhibitor of gut enzymatic and/or transport activity are released in the gut in a sustained manner.

3. An orally administrable composition in accordance with claim 1 wherein said at least one biologically active agent is released in the gut in a sustained manner and said at least one inhibitor of gut enzymatic and/or transport activity is released in the gut in a partially sustained manner.

4. An orally administrable composition in accordance with any one of the preceding claims wherein said microcapsules are being formulated so as to allow differential release rates of said at least one biologically active agent and said at least one inhibitor of gut enzymatic and/or transport activity.

5. An orally administrable composition in accordance with any one of the preceding claims wherein said at least one inhibitor of gut enzymatic and/or transport activity is released in the gut prior to the biologically active agent.

6. An orally administrable composition in accordance with any one of the preceding claims wherein the at least one inhibitor of gut enzymatic and/or transport activity and the biologically active agent are released gradually or in a pulse mode.

7. An orally administrable composition in accordance with any one of the preceding claims wherein said microcapsules comprise a multilayered coating.

8. An orally administrable composition in accordance with any one of the preceding claims wherein said encapsulation agent is selected from the group consisting of resistant starch, fat coatings, mono and diglycerides, wax, shellac, cellulose based coatings, and any combination thereof.

9. An orally administrable composition in accordance with claim 8 wherein said cellulose based coatings are selected from the group consisting of Methyl cellulose (MC), Hydroxypropyl cellulose (HPC), Ethyl Cellulose (EC), Hydroxy ethyl cellulose (HEC), hydroxy propyl methyl cellulose (HPMC), carboxy methyl cellulose (CMC), Hydroxypropyl methyl cellulose acetate, cellulose acetate phthalate, hydroxymethyl cellulose phthalate, cellulose trimellitate, hydroxymethyl cellulose acetate succinate and any combination thereof.

10. An orally administrable composition in accordance with any one of the preceding claims wherein said encapsulation agent further comprises a plasticizer.

11. An orally administrable composition in accordance with any one of the preceding claims wherein said encapsulation agent further comprises Hydrogenated Vegetable oils.

12. An orally administrable composition in accordance with any one of the preceding claims wherein said microcapsules comprise between about 5% and about 20% w/w encapsulation agent.

13. An orally administrable composition in accordance with any one of the preceding claims wherein said biologically active agent is selected from the group consisting of a curcuminoid, digoxin, antibiotics, anti neoplastic agents, and viral inhibitors.

14. An orally administrable composition in accordance with any one of the preceding claims wherein said inhibitor inhibits the activity of an enzyme selected from the group consisting of UGT (UDP-glucuronosyltransferase) enzyme family, sulfotransferase enzymes, alcohol dehydrogenase, and p450 enzymes.

15. An orally administrable composition in accordance with any one of the preceding claims wherein said at least one inhibitor is selected from the group consisting of Piperine, Quercetin, Genistein, Glabridin, and any combination thereof.

16. An orally administrable composition in accordance with any one of the preceding claims wherein said inhibitor is a combination of Piperine, Quercetin, Genistein, and wherein said biologically active agent is a curcuminoid.

17. An orally administrable composition for the controlled release of a curcuminoid wherein said composition comprises microcapsules consisting of at least one encapsulation agent and a curcuminoid.

18. An orally administrable composition in accordance with claim 17 wherein said microcapsules comprise between about 60% and about 90% curcumin and between about 10% and 30% said at least one encapsulation agent.

19. An orally administrable composition in accordance with any one of the preceding claims, wherein said composition is a solid composition.

20. A pharmaceutical or nutraceutical composition comprising the orally administrable composition in accordance with any one of claims 1-19.

21. A pharmaceutical composition according to claim 20 for the treatment of inflammatory diseases, infectious diseases, or cancer.

22. An orally administrable composition in accordance with any one of claims 1-21, wherein said composition further comprises a carrier, exipient or diluents therefore.

23. A consumable product comprising the orally administrable composition in accordance with any one of claims 1-18.

24. A consumable product in accordance with claim 23 wherein said product is selected from the group consisting of dry pasta, bread and bread crisps.

25. A method for increasing the bioavailability of a biologically active agent, said method comprising:

26. The method of claim 25 wherein the composition obtained in step (c) is the orally administrable composition of any one of claims 1-22.