WO2014166994A1

WO2014166994A1 - Nano-microdelivery systems for oral delivery of an active ingredient

Info

Publication number: WO2014166994A1
Application number: PCT/EP2014/057150
Authority: WO
Inventors: Ioannis S. CHRONAKIS; Lars JØRGENSEN
Original assignee: Danmarks Tekniske Universitet
Priority date: 2013-04-09
Filing date: 2014-04-09
Publication date: 2014-10-16

Abstract

A composition for oral delivery of one or more active ingredients in the form of a lipid nano-micro-delivery system comprising a lipid nano-micro-structure comprising at least one lipid and at least one active ingredient, said at least one active ingredient being immobilized in said lipid nano-micro-structure; and at least one enzyme.

Description

Nano-microdelivery systems for oral delivery of an active ingredient Field of the invention

The present invention relates to a composition for oral delivery of one or more active ingredients. More particularly, the present invention relates to a lipid nano-micro-delivery system comprising one or more active ingredients and at least one enzyme, wherein the active ingredient(s) may be delivered through the oral cavity by a controlled delivery.

Background of the invention

Controlled delivery of an active ingredient via the oral route is of immense importance for a range of delivery applications. Incorporation of active ingredients into nano-to-microscale delivery systems may alter the rate and efficiency of delivery. Moreover, controlled release of these active ingredients immobilised at nano-micro-delivery systems, wherein their release can be triggered by external stimuli such as pH, light and temperature is highly preferred for achieving a desired specific delivery.

Enzymatically-sensitive drug-delivery platforms are of great interest in medical areas. For instance, enzymatically-sensitive controlled release platforms have been previously investigated for drug delivery, cf. Thornton PD, Heise A: Highly specific dual enzyme- mediated payload release from peptide-coated silica particles. J Am Chem Soc 2010, 132: 2024-2028; Venkatesh S, Wower J, Byrne ME: Nucleic acid therapeutic carriers with on- demand triggered release. Bioconjug Chem 2009, 20: 1773-1782; and Schlossbauer A, Kecht J, Bein T: Biotin-avidin as a protease-responsive cap system for controlled guest release from colloidal mesoporous silica. Angew Chem Int Ed Engl 2009, 48: 3092-3095.

WO 2007/113665 A2 discloses a polymerized solid lipid nanoparticle system comprising lipids and long chain fatty acids, a therapeutic protein or peptide, an adjuvant, a lectin, at least one polymer, and a pharmaceutically acceptable carrier. Administration of said system may be oral, sublingual or buccal.

WO 2010/114901 Al discloses methods and compositions for the controlled release of a drug from a liposome carrier.

WO 2012/088059 A2 relates to a facile method for crosslinking and incorporating bioactive molecules into electrospun fiber scaffolds, wherein said scaffolds are crosslinked with an acrylate. US 2009/0291133 Al discloses methods and compositions for enhancing transdermal delivery of a bioactive agent.

US 4,880,634 relates to lipid nano-pellets as excipient system for perorally administered drugs, wherein the excipient system is in the form of an ultrafine aqueous, colloidal suspension of lipid nano-pellets comprised of lipids and a surfactant of which the particle diameters of the nano-pellets range from 50-1,000 nm.

US 5,885,486 relates to solid lipid particles, particles of bioactive agents and methods for the manufacture and use thereof. More particularly the document relates to the preparation of suspensions of colloidal solid lipid particles of predominantly anisometrical shape with the lipid matrix being in a stable polymorphic modification and of suspensions of micron and submicron particles of bioactive agents.

However, there is a need in the art for enzymatically-sensitive oral delivery platforms that facilitate controlled release of active ingredients from lipid nano-micro-structures in the presence of an enzyme. There is a need in the art for enzymatically-sensitive oral delivery platforms that facilitate controlled release of active ingredients from lipid nano-micro- structures, wherein said lipid nano-micro-structures may be utilised both in the solid state, semi-solid state and/or in the dispersed state. More particularly, there is a need in the art for enzymatically-sensitive lipid nano-micro-structures which can be used as oral delivery platforms of active ingredient(s). There is also a need in the art for enzymatically-sensitive lipid nano-micro-structures that do not have a single diffusion behaviour of an active ingredient(s) but also has an enzyme- controlled release kinetics of the active ingredient(s) over time.

Object of the invention

It is an object of embodiments of the invention to provide a composition for controlled oral delivery of one or more active ingredients.

It is a further object of embodiments of the invention to provide a composition that incorporates enzyme(s) to facilitate the controlled oral delivery of one or more active ingredients.

It is a further object of embodiments of the invention to provide a composition of lipid nano- micro-structures using enzyme-triggered controlled delivery of said one or more active ingredients. Summary of the invention

It has been found by the present inventor(s) that by providing a lipid nano-micro-structure with incorporating enzyme(s) it has surprisingly turned out that a controlled oral delivery of one or more active ingredients may be obtained.

So, in a first aspect the present invention relates to a composition for controlled oral delivery of at least one active ingredient comprising: i) a lipid nano-micro-structure comprising a core region and a shell region or a multilayer shell region, said lipid nano-micro-structure comprising at least one lipid and at least one active ingredient, said at least one active ingredient being immobilised in said lipid nano-micro-structure; and

ii) at least one enzyme, wherein said lipid nano-micro-structure is selected from the group comprising lipid nano- micro-particles, lipid nano-micro-laminated particles, lipid nano-micro-fibres, nano-micro- particles encapsulated/immobilised in lipid nano-micro-fibres, lipid nano-micro-particles encapsulated/immobilised in polymer nano-microfibers, lipid nano-micro-particles encapsulated/immobilised in polymer nano-micro-particles, and nano-micro-structured lipid carriers (N-MLC's), lipid drug conjugate (LDC) nano-micro-particles, polymer - lipid hybrid nano-microparticles (PLNM), and any combinations thereof, preferably wherein said lipid nano-micro-structure is selected from the group comprising solid lipid nano- micro-particles and lipid nano-micro-fibres,

In a second aspect the present invention relates to a method for the preparation of a composition in the form of lipid nano-micro-particles according to the invention, comprising the steps of: i) Providing said at least one lipid in a melted state, optionally by heating to above the phase transition temperature thereof or providing said at least one lipid dispersed in a solvent;

ii) Dispersing or dissolving said at least one active ingredient in the melted lipid, or in the lipid dispersed in a solvent;

iii) Optionally adding at least one surfactant, optionally at least one excipient and optionally at least one mucoadhesive compound and optionally at least one porogen compound neat or in a solution thereof, preferably in an aqueous solution thereof; iv) mixing and homogenising to obtain a nano-micro-structure comprising said at least one active ingredient; wherein at least one enzyme is added either before, during and/or after the step of mixing and homogenising to obtain an enzyme-containing nano-micro-structure.

In a third aspect the present invention relates to a method for the preparation of a composition in the form of lipid nano-micro-fibres, comprising the steps of: i) Providing said at least one lipid in a solvent, preferably an organic solvent or an organic solvent/aqueous system or in a supercritical fluid;

ii) Optionally adding at least one surfactant, at least one excipient and optionally at least one mucoadhesive compound and optionally at least one porogen compound;

iii) Optionally heating to above the main phase transition temperature;

iv) Dispersing or dissolving said at least one active ingredient in the mixture obtained in step ii);

v) Applying an electrical field to obtain a nano-micro-structure; wherein at least one enzyme is added either before, during and/or after the step of applying an electrical field to obtain an enzyme-containing nano-micro-structure.

In a fourth aspect the present invention relates to a use of the composition according to the invention for oral delivery of said at least one active ingredient.

Legends to the figure

Fig. 1 shows the release of nicotine from solid lipid nanoparticles (SLN) with and without lipase enzyme, and at different enzyme levels;

Fig. 2 shows a Cryo-TEM (Tunnelling Electron Microscope) image of SLN particles prepared according to Example 1;

Fig. 3 shows aspirin release from SLN with and without lipase;

Fig. 4 shows release behavior of the flavor compound (4-Hydroxy-2,5-dimethyl-3(2 - )- furanone) from electrospun lipid fibers with or without enzyme (phospholipase, A_x or phospholipase D); Fig . 5 shows electrospun lipid fibers containing the flavor compound 4-Hydroxy-2,5-dimethyl- 3(2H)-furanone of example 3 visualized using scanning electron microscope at a

magnification of 250X; and

Fig . 6 shows the same electrospun lipid fibers containing the flavor compound 4-Hydroxy- 2,5-dimethyl-3(2H)-furanone of example 3 visualized using scanning electron microscope at a magnification of lOOOx.

Detailed disclosure of the invention

Definitions

In the present context the term "oral delivery" refers to delivery to or via the oral cavity. Exemplary oral delivery routes include i .a . buccal, sublingual, gingival and gastrointestinal delivery.

In the present context the term "solid lipid" is a lipid that is solid at room temperature and also at physiological body temperature.

In the present context the term "nano-micro-structure" refers to a structure the size of which is in the nanometer and micrometer range, such as in the range 1 nm - 1000 μιη, such as 10 nm - 1000 μιη, such as 10 nm - 100 μιη, such as 10 nm - 10 μιη, such as 10 nm - 1 μιη, preferably in the range 1 nm - 1 μιη . The term also refers to a structure of any form, such as a sphere/beads, a fiber structure or any other shape wherein their average diameter is in the nanometer and micrometer range. The term also refers to a combination of fiber(s) and particles/beads; in particular where particle(s) structures are encapsulated / immobilised within fiber structures. This could e.g . be obtained using electrostatic processing methods (such as electrospray, electrocoextrusion and/or electrospinning, etc.) .

In the present context the term "fiber", "fibre", and "fiber structure" refers to a structure having an oblong shape, i.e. wherein the length is at least 3 times the cross-section.

The term "solid lipid nano-micro-particle" or "SLN" refers to solid lipid nano-micro-particles having a solid lipid core matrix.

The term "nano-micro-structured lipid carriers (N-MLC's)" is well-known in the art and disclosed in e.g . (R.H. Muller,M . Radtke, S.A. Wissing, 'Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) in cosmetic and dermatological preparations', Advanced Drug Delivery Reviews , Volume 54, Supplement, 1 November 2002, Pages S131-S155 as well in Nanoparticulates As Drug Carriers By V. P. Torchilin (editor) Imperial College Press, 2006 , chapters 9 & 10). Nano-microstructured lipid carriers refer to structures that comprise a less organized solid lipid matrix, i.e. by blending a fluid lipid with the solid lipid. Generally, N-MLC's shows a higher loading capability for the immobilizing components than SLN-M.

The term "Lipid Drug Conjugate (LDC) Nano-micro-particles" is well-known in the art and refers to nano-micro-particles created through formation of insoluble lipid-drug conjugates either by salt formation or covalent linking like ester linkage. The formed LDC could be mixed into aqueous surfactant solution for preparation of SLN's by homogenization or other methods, cf. eg. Shaji J., Jain V. Solid Lipid Nanoparticles: A novel carrier for Chemoterapy, International Journal of Pharmacy and Pharmaceutical Sciences, Vol 2, Suppl 3, (1-10) 2010 and Olbrich C, Gebner A, Kayser O, Muller RH. Lipid-drug conjugate (LDC) nanoparticles as novel carrier system for the hydrophilic antitrypanosomal drug diminazene diaceturate. J Drug Target 2002; 10: 387-96. Although the term "LDC" implies drug-containing conjugates it is to be understood that the term "LDC" as used in the present context includes any active ingredient incorporated into lipid-active ingredient conjugates.

The term '"Polymer - Lipid hybrid Nano-micro-particles" (PLNM) refers to nano-micro- particles involving formation of complexation of compounds and an ionic polymer, cf. Shaji, supra; Wong HL, Bendayan R, Rauth AM, Wu XY. Development of solid lipid nanoparticles containing ionically-complexed chemotherapeutic drugs and chemosensitizers. J Pharm

Sci.,2004; 93: 1993-2004; Wong HL, Rauth AM, Bendayan R, Manias JL, Ramaswamy M, Liu Z et al. A new polymer-lipid hybrid nanoparticle system increases cytotoxicity of doxorubicin against multidrug resistant human breast cancer cells. Pharm Res 2006; 23 : 1574-85; Wong HL, Bendayan R, Rauth AM, Xue HY, Babakhanian K, Wu XY. A mechanistic study of enhanced doxorubicin uptake and retention in multidrug resistant breast cancer cells using a polymer-lipid hybrid nanoparticle (PLN) system. J Pharmacol Exp Ther 2006; 317: 1372-81; and Li Y, Taulier N, Rauth AM, Wu XY. Screening of lipid carriers and characterization of drug- polymer complex for the rational design of polymer-lipid hybrid nanoparticles. Pharm Res 2006; 23: 1877-87. Thus, charges on compounds are neutralized with polymer counter-ion and the formed complex is encapsulated into solid lipid nano-micro-particles. Using this approach the encapsulation efficiency could be increased from typical 20-35 % to over 80 % and it is contemplated that the inclusion of ionic polymer in PLNM may accelerate the release rate of active ingredient.

In the present context the term "diameter" of the nano-micro-structure refers to the average diameter of the structure in question. Thus in connection with spheres the diameter is the average diameter of the spheres in question, and in connection with fibres the diameter is the average width of the fibres in question.

In the present context the term "enzyme" refers to a protein compound which is present in the composition according to the invention and which imparts desired properties thereto. The enzyme is used to facilitate the modification of the nano-micro-structure and thereby control the release (rate and amount) of the active ingredient from the nano-micro-structure.

Enzymes (or biocatalysts) may be obtained from animal tissue, plants and micro-organisms (e.g microbes such as yeast, bacteria or fungi). The term enzyme also includes

conjugated proteins - that contain a protein (apoenzyme) and a non-protein (coenzyme) part as well as zymogens, i.e. inactive precursors of enzymes.

In the present context the term "controlled oral delivery" means that the at least one active ingredient is released at a rate which is different from the rate at which said at least one active ingredient would be released if no enzyme were present. In one embodiment a controlled release will result in a slower release rate, such as a release rate which is at least 10% slower than a non-controlled release, such as at least 20 % slower, 25% slower, 30% slower, 40% slower, such as 50% slower, such as 60% slower, such as 70% slower, such as 80% slower, such as 90% slower, preferably at least 10% slower. In another embodiment a controlled release will result in a faster release rate, such as a release rate which is at least 10% faster than a non-controlled release, such as at least 20 % faster, 25% faster, 30% faster, 40% faster, such as 50% faster, such as 60% faster, such as 70% faster, such as 80% faster, such as 90% faster, preferably at least 10 % faster.

In the present context the term "core region" of a lipid nano-micro-structure refers to the interior or the space inside the lipid nano-micro-structure.

In the present context the term "shell region" of a lipid nano-micro-structure refers to the exterior or the outer surface of said lipid nano-micro-structure.

In the present context the term "nano-micro-laminated lipid structure" refers to a lipid nano- micro-structure consisting of a lipid core and a multilayer shell. The multilayer shell consists of at least two layers and can be designed to further control the release of the at least one active ingredient. In the present context the terms "mucoadhesive" and "mucoadhesion" refers to the concept of a composition adhering to a mucous membrane. Mucoadhesive compounds facilitate mucoadhesion by their specific properties. In the present context the term "excipient" refers to a compound which may be present in the composition according to the invention and which imparts desired properties thereto. An excipient may be used to regulate hydrophilicity and/or amphiphilicity and thereby control the release of the active ingredient from the nano-micro-structure and may also be used as "carrier polymer", e.g. as known in the electrostatic processing field.

In the present context the term "porogen" refers to a compound with pore-generating properties. The size of the porogen particles will affect the size of the pores in a polymer in question, while the polymer to porogen ratio is directly correlated to the amount of porosity of the final structure. Non-limiting examples of porogens are inorganic salts such as sodium chloride, and carbohydrate crystals, such as crystals of saccharose.

Specific embodiments of the invention

The at least one active ingredient to be delivered may be selected from a broad range of ingredients or their mixture.

In an embodiment of the invention the at least one active ingredient is selected from nicotine and nicotine salts and caffeine.

In another embodiment of the invention the at least one active ingredient comprises a flavour component. Although the range of flavours is almost limitless, flavours commonly fall into several broad categories, such as fruit flavours, spice flavours, and mint flavours. Flavours may be synthetic and natural or any combinations thereof. Non-limiting examples thereof include the following. Fruit flavours include lemon, orange, lime, grapefruit, tangerine, strawberry, apple, cherry, raspberry, blackberry, blueberry, banana, pineapple, cantaloupe, muskmelon, watermelon, grape, currant, mango, kiwi and mixtures thereof. Spice flavours include cinnamon, vanilla, clove, chocolate, nutmeg, coffee, liqorice, eucalyptus, ginger, lemongrass, cardamon, thyme, rosemary, anise and mixtures thereof. Mint flavours include spearmint, peppermint, wintergreen, basil, corn mint, menthol and mixtures thereof.

In an embodiment of the invention said flavour component is selected from the group consisting of one or more components of vanilla, such as vanillin, one or more components of spearmint such as R-carvone, one or more components of orange, such as limonene, menthol, one or more components of nutmeg, such as eugenol, of one or more components of eucalyptus, such as eucalyptol, one or more components of cinnamon , such as

cinnamaldehyde, one or more components of thyme, such as thymol, one or more components of anise, such as anisole, one or more components of lemon or lime, such as citral. In another embodiment of the invention the at least one active ingredient comprises aromatic substances, flavoring agents (flavor oils and flavor extracts) and essential oils, which are known to persons skilled in the art. In a particularly preferred embodiment, the flavoring agents include all types of mints, including peppermint, spearmint, wintergreen, menthol, in the form of flavor oils and flavor extracts, as well as citrus based flavors, cinnamon, essential oils, such as menthol, eucalyptol, thymol, camphor, menthyl, salicylate, also known as wintergreen, and phenol, etc

In another embodiment of the invention the at least one active ingredient may be selected from any of the below: Oral pain relievers/anesthetics such as benzocaine, lidocaine, tetracaine, butacaine sulfate, benzyl alcohol, hexylreorcinol, menthol, phenol, phenolate sodium, salicylic alcohol, dyclonine HCI, hexylresorcinols, aspirin, acetaminophen, and the like;

Agents for the prevention of caries, which include, for example, fluoride, stannous fluoride, sodium fluoride, monofluorophosphate (MFP), and the like; Antigingivitis/antiplaque agents, such as triclosan, quaternary ammonium compounds (e. g. , cetyl pyridinium chloride and domiphen bromide), essential oils (e. g. , eucalyptol, menthol, menthyl salicylate and thymol), phenol, stannous fluoride, and zinc salts (e. g. zinc citrate), polydimethylsiloxine;

Agents for relieving dry mouth, including pilocarpine, cevimeline, flavor oils (e. g.mint, citrus, etc.);

Polyols (e. g. xylitol, mannitol, sorbitol, maltitol, etc), gums, such as xanthan gum, cellulosics (e. g. sodium carboxymehylcellulose, hydroxyethylcellulose, etc), enzymes (e. g. glucose oxidase, lactoperoxidase, and lysozyme, etc. ) mucopolysaccharides, glycomannin, and fruit acids (citric acid, apple/malic acid, tartaric acid, etc.); Desensitizers such as potassium nitrate, strontium nitrate, calcium oxalate 2-hydroxyethyl methacrylate, and the like;

Whitening agents such as carbamide peroxide, and perhydrol urea; Antiviral agents such as acyclovir, famciclovir, penciclovir, valacyclovir and docosanol; Antibiotics/antifungals, such as polymyxin B and neomycin, clindamycin, penicillin, ketoconazole, clotrimazole, miconazole, chlorhexidine, and the like;

Cough suppressants and anti-tussives, including camphor, menthol and eucalyptus oil;

Expectorants such as guaifenesin (glyceryl guaiacolate);

Demulscents such as pectin, gelatine, glycerine, linseed, tragacanth and marshmallow; Anti-inflammatory agents such as hydrocortisone and prednisone;

Antioxidants such as EGCG (epigallocatechin gallate), ursolic acid, rosemary extract, grape seed extract, pine bark extract, co-enzyme Q-10, superoxide dismutase, lutein, lycopene, astaxathin, alpha lipoic acid, tocopherol, resveratrobioperene, carotenes, flavonoids and the like;

Bronchodilators such as albuterol, terbutaline, theophylline, ephedrine, epinephrine, and the like; .

Antihistamines such as diphenhydramine HCI/citrate, chlorpheniramine maleate,

brompheniramine maleate, clemastin fumarate, doxylamine succinate, phenindamine tartrate, tripolidine HCI, thonzylamine HCI, pyrilamine maleate, and dexchlorpheniramine maleate;

Decongestants such as pseudoephedrine HCI and phenylpropanolamine HCI;

Herbal compounds such as ephedra, feverfew, parthenolide, chamomile, licorice and derivatives, slippery elm, grape seed extract, garlic, acidophilus, bee propolis, chlorophyll, alfalfa, cardamom Echinacea, myrrh, peppermint, rosemary, and sage;

Odor neutralizers such as zinc salts, chlorophyll, and the like;

Enzymes such as but not limited primary dried yeast, lysozyme, lactoferrin, glucose oxidase, lactoperoxidases, dextranases, oxidases, etc. ;

Vitamins such as tocopherol-vitamin E, retinal, ascorbic acid-vitamin C, vitamin D, including vitamin D₂ and D₃, vitamin B_x, vitamin B₂, vitamin B₃, pro-B₅, B₅, B_i2, vitamin B₉, vitamin K and salts/esters thereof; Ionic forms of minerals such as calcium, phosphorus, potassium, sulfur, sodium,

chlorine, magnesium, and the like. Ionic forms of important trace or minor minerals and metals, such as iron, cobalt, copper, zinc, molybdenum, magnesium; manganese, iodine, selenium and the like; Amino acids such as, Arginine, Asparagine Aspartic acid, Cysteine, Glutamic acid, Glutamine, Glycine, Histidine, Isoleucine, Methionine, Leucine, Lysine, Ornithine, Phenylalanine,

Threonine, Tryptophan, Tyrosine, Valine, Proline, Serine;

Further non-limiting examples of drug(s) that may preferably be included in the oral composition of the present invention, for example, anti-hypertensive agents, anti-cancer agents, etoposide, therapeutic biomolecules, and the like;

Genes; such as liver genes and the like;

Vaccines for viral diseases, for bacteria diseases, cancer vaccines, carriers for vaccines, and the like;

Biologically active molecules selected from but not limited to DNA, mRNA, iRNA, microRNA, oligonucleotides and antisense sequence;

Growth factors such as cytokines and hormones (such as progesterone, insulin, etc) and the like;

Active cultures such as probiotic (lactic acid bacteria, bifidobacteria, etc.,) and prebiotic cultures and the like; Bioactive proteins such as BSA, gelatine/collagen, casein and milk proteins, soy, wheat, fish proteins and bioactive peptides such as fish peptides, C-peptide and the like;

Omega-3 fatty acids (PUFA) such as hexadecatrienoic acid (HTA), alpha-linolenic acid (ALA), stearidonic acid (SDA), dietary fats and the like;

Supplements, like fat soluble such as phytosterols, etc and non-fat soluble such as choline bitartrate, etc. Other as echinacea, flax seed oil, ginkgo biloba, golden seal, inositol, spirulina extract, polyphenols, flavanols (such as proanthocyanidin, etc.), hormones supplements (steroids such as DHEA, pregnenolone, the hormone melatonin, etc), chondroitin sulfate and the like. In an embodiment of the invention the at least one active ingredient comprises any combination/mixture of any of the above compounds.

The amount of active ingredient may vary widely depending on the actual active ingredient used . However, typically an amount in the range 0.1 - 70 % by weight of the lipid nano- micro-structure is used, such as about 1-50% by weight, such as 1- 20 % by weight.

The composition according to the invention comprises at least one enzyme component. Although the range of enzymes is almost limitless, enzymes commonly fall into several broad categories, wherein the substrates of such enzymes are either of lipidic, carbohydrate or protein origin . Enzymes may be synthetic or natural or any combinations thereof. Non- limiting examples thereof include lipases, carbohydrases and peptidases. Non-limiting examples thereof include the following : Amylases, Amyloglucosidase, Glucoamylase, Aminopeptidase, Catalase, Cellulase, dextranases, Ficin (peptide hydrolase), Hemicellulase, a-galactosidase, Gelatinase, β-glucanases, glucose oxidase, Invertase, Glucose isomerase,, Esterase-lipase; Phopholipases (phospholipase, phospholipase A_lr phospholipase A₂, phospholipase C, phospholipase D) ; triacylglycerol lipase; galactolipase; lipid-phosphate phosphatase; Lysophospholipase; diacylglycerollipase; and acyltransferase; lipoprotein lipase; lipase with bile salts, Animal lipase (triacylglycerol hydrolase), lactase,

lactoperoxidases, Lysozyme, Maltase, Oligosaccharidases,oxidases, Pancreatin (peptide hydrolase), Papain, Pectinases, pectolytic enzymes (e.g . polygalacturonase and pectin methylesterase), Proteases, (I, II, III, IV, V), Pepsin, Phytase, Trypsin (chemotrypsin) (peptide hydrolase), xylanases, and any combination/mixture of the above enzymes.

In an embodiment of the invention the enzyme is a lipase. A lipase may be useful to control the release of an active ingredient by modifying the structure of the nano-micro-structure. A suitable lipase for use in the invention is a Thermomyces lanuginosus lipase. In an embodiment of the invention at least one enzyme is present in the 'shell region' or the exterior of the solid lipid nano-micro-structure. The enzymes may thus be present in the form of or as part of a coating of the solid lipid nano-micro-structure. Thus the enzyme may be incorporated as a solution or dispersion after application of any further excipients or may be added together with any other excipients. Under appropriate conditions (such as wetting, swelling, corrosion and diffusion of water after oral use, etc), the kinetics of the enzymatic degradation and/or modification of the nano-micro-structure shell may facilitate the release rate and released amount of the active ingredient loaded in the core region of the solid lipid nano-micro-structure. In another embodiment of the invention at least one enzyme is present in the 'core region' or the interior of the solid lipid nano-micro-structure. Under appropriate conditions after the oral use, the release rate and amount of the active ingredient will be triggered by the kinetics of the solid lipid nano-micro-structure degradation and/or modification facilitated by the at least one enzyme.

In another embodiment of the invention the at least one enzyme is exclusively or additionally immobilized at the lipid nano-micro-structure by post-addition or post-treatment.

In another embodiment of the invention the at least one enzyme is exclusively or additionally immobilized at the lipid nano-micro-structure by post-surface attachment. Post-surface attachment includes physical adsorption or covalent attachment of enzyme(s) on prepared lipid nano-micro-structures.

In another embodiment of the invention at least one enzyme is present in the 'core region' or the interior of the solid lipid nano-micro-structure and the same or another enzyme is present in the 'shell region' or the exterior of the solid lipid nano-micro-structure. By having an enzyme both in the core region and in the shell region it is possible to tailor the release of the at least one active ingredient which may itself be present either at the interior and/or the exterior of the lipid nano-micro-structure. Thus by way of a non-limiting example a glucanase may be incorporated in the shell region or in a multilayer shell region or the exterior of a nano-micro-laminated lipid structure containing e.g. a carbohydrate such as chitosan as excipient together with e.g. nicotine as active ingredient and incorporating a flavour as another active ingredient in the 'core region' or the interior of a lipid nano-micro-tructure together with an enzyme, such as a lipase. The lipase at an appropriate enzyme 'level' will gradually degrade the lipid structure and release the flavor ingredient accordingly; (an example is shown at the Figure 1, where the presence of different enzyme levels initiate different release kinetics of the active compound, as desired according to the usage).

Alternatively, the lipid 'core-region' can be produced for example with an appropriate lipid thickness/diameter (of the nano-micro-particle or nano-micro-fibers, etc.), which then will control the enzymatic degradation and release kinetics of the active compound, as preferred. Thereby a relatively fast modification of the shell region of the nano-micro-structure may take place to release the nicotine rapidly while a delayed release of the flavour ingredient may take place.

In another embodiment of the invention the at least one active ingredient is present in separate lipid nano-micro-structures from the lipid nano-micro-structure incorporating the at least one enzyme. Hereby it is possible to control the release, the activity and thus the efficiency of the enzyme, consequently allowing a controlled modification of the lipid matrix where the active ingredient is incorporated.

In another embodiment of the invention at least one enzyme is present in the form of a separately added enzyme in a dispersed and/or solid state after the preparation of the solid lipid nano-micro-structure. The ability of the loaded enzyme to access by

wetting/swelling/corrosion/diffusion, etc., the lipid nano-micro-structure's shell and/or core, and to control the kinetics of modification of the nano-micro-structure will facilitate the control of the release rate and the released amount of the active ingredient.

In another embodiment of the invention at least one enzyme is present in separate nano- micro-structures from the lipid nano-micro-structure incorporating the at least one active ingredient. Thus said separate nano-micro-structures may be either lipid nano-micro- structures or nano-micro-structures of non-lipid origin, such as carbohydrate origin, such as starch nano-micro-structures.

The amount or level of enzyme in the lipid nano-micro-structures according to the invention varies widely depending on a variety of factors, such as the enzyme(s) in question, the active ingredient(s), the desired release, the temperature, the pH, the light, the chemical structure of the enzyme, the chemical structure of the lipid nano-micro-structure, the secondary structure of the lipid nano-micro-structure and the morphology of the lipid-nano-micro- structure. Lipid nano-micro-structures may take the form of either particles, fibres or nano-micro- structured lipid carriers (N-MLC's) or a combination thereof, such as lipid nano-micro- particles encapsulated/immobilised in nano-micro-fibres or lipid nano-micro-particles encapsulated/immobilised in polymer nano-micro-particles, or nano-micro-particles encapsulated/immobilised in lipid nano-micro-fibres, lipid drug conjugate (LDC) nano-micro- particles, polymer - lipid hybrid nano-micro-particles (PLNM), or a combination of lipid nano- micro-structures wherein one of the active components is immobilized in separate lipid nano- micro-structures from the lipid nano-micro-structures immobilizing the other component.

The lipid nano-micro-structure is preferably either solid lipid nano-micro-particles or lipid nano-micro-fibres. The solid lipid nano-micro-particles may be prepared by methods known in the art, e.g. as disclosed in Parhi et al, "Production of Solid Lipid Nanoparticles-Drug Loading and Release Mechanism", J. Chem. Pharm. Res., 2010, 2(l) : 211-227. Lipid nano-micro-fibres may be prepared by electrospinning, e.g. as disclosed in McKee et al, "Phospholipid Nonwoven Electrospun Membranes" Science, 2006, 311 : 353-355. Solid lipid nano-micro-beads may be prepared by electrospraying or by using electrocoextrusion, e.g. as disclosed in N. J. Zuidam and E. Shimoni Chapter 2, Overview of Microencapsulates for Use in Food Products or Processes and Methods to Make Them, and N.J. Zuidam and V.A. Nedovic (eds.),

Encapsulation Technologies for Active Food Ingredients and Food Processing, DOI 10.1007/ 978-l-4419-1008-0_2, Springer Science& Business Media, LLC 2010.

Lipid nano-micro-particles encapsulated/immobilised in nano-micro-fibres may be prepared using electrostatic processing methods such as electrospray, electrocoextrusion and/or electrospinning, etc. In one embodiment lipid nano-micro-particles are added to a polymer solution and electrospun, electrosprayed or coelectroextruded together with the polymer solution into fibres or into other shapes, such as beads.

In an embodiment of the invention the lipid nano-micro-structures are in the form of Lipid Drug Conjugate (LDC) Nano-micro-particles' produced through formation of insoluble lipid- (active ingredient) conjugates either by salt formation or covalent linking like ester linkage. In an embodiment of the invention the formed LDC could be mixed into aqueous surfactant solution for preparation of SLN's by homogenization or other methods.

In another embodiment of the invention the lipid nano-micro-structures are in the form of Polymer - Lipid hybrid Nano-micro-particles' (PLNM) which involves formation of

complexation of active ingredient and an ionic polymer. Charges on active ingredients are neutralized with polymer counter-ion and the formed complex is encapsulated into solid lipid nano-micro-particles.

In an embodiment of the invention the at least one active ingredient is present in the form of encapsulated material in the lipid nano-micro-structures.

In another embodiment of the invention the at least one active ingredient is present in the form of a coating on lipid nano-micro-structures such as lipid nano-micro-fibres. This may be obtained e.g. by using coaxial electrospinning, or coaxial electrospray of colloidal

suspensions, or electrocoextrusion of different liquids from coaxial capillaries.

In an embodiment of the invention said one active ingredient is a nicotine component selected from the group consisting of nicotine base and a salt of nicotine, such as nicotine bitartrate. The use of a salt of nicotine, such as nicotine bitartrate, may be beneficial in order to change the mechanism of absorption through the cell layers. In an embodiment of the invention said lipid nano-microstructure comprises at least one lipid selected from the group consisting of fatty acids, esters and fatty mono-, di-, and

triglycerides thereof, partial glycerides, fatty alcohols and their esters and ethers, natural and synthetic waxes such as bees wax and carnauba wax, wax alcohols and their esters, hydrogenated vegetable oils, hard paraffins, phospholipids, sterols and sterol derivatives, and mixtures of any of the above lipids.

In an embodiment of the invention said at least one lipid is selected from the group consisting of C₈-24 fatty acids, C₈-24 fatty mono-, di-, or triglycerides, such as C₁₀.₂2 fatty acids, Cio-22 fatty mono-, di-, or triglycerides, such as saturated Cio-22 fatty acids and Cio-22 fatty mono-, di-, or triglycerides, and mixtures of any of the above lipids.

In an embodiment of the invention said at least one lipid is selected from the group consisting of capric, lauric, myristic, palmitic, stearic, and arachidic acids and mono-, di- and triglycerides thereof, preferably selected from trimyristin, tripalmitin, tristearin, tricaprin, myristic acid, palmitic acid, stearic acid, and behenic acid, and mixtures of any of the above lipids.

In an embodiment of the invention the lipid nano-micro-structures are lipid nano-micro- particles, said lipid nano-micro-particles comprising at least one lipid selected from the group consisting of C₈-24 fatty acids, C₈-24 fatty mono-, di-, or triglycerides, such as Cio-22 fatty acids, Cio-22 fatty mono-, di-, or triglycerides, such as saturated Cio-22 fatty acids and Cio-22 fatty mono-, di-, or triglycerides, and mixtures of any of the above lipids, preferably selected from the group consisting of capric, lauric, myristic, palmitic, stearic, and arachidic acids and mono-, di- and triglycerides thereof, preferably selected from trimyristin, tripalmitin, tristearin, tricaprin, myristic acid, palmitic acid, stearic acid, and behenic acid, and mixtures of any of the above lipids. In an embodiment of the invention the lipid is a phospholipid preferably selected from the group consisting of phosphatidylcholine (PC), phosphatidylethanolamine (PE),

phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidic acid (PA), DPG (bisphosphatidyl glycerol), PEOH (phosphatidyl alcohol), cholesterol, ergosterol and lanosterol, and mixtures of any of the above lipids, preferably

phosphatidylcholine (PC)-containing mixtures.

In an embodiment of the invention the lipid nano-micro-structures are lipid nano-micro- fibres, said lipid nano-micro-fibres comprising at least one lipid selected from the group consisting of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidic acid (PA), DPG (bisphosphatidyl glycerol), PEOH (phosphatidyl alcohol), cholesterol, ergosterol and lanosterol, and mixtures of any of the above lipids, preferably phosphatidylcholine (PC)- containing mixtures.

In an embodiment of the invention the composition comprises at least one surfactant, wherein said at least one surfactant is selected from the group consisting of ionic, non-ionic, and amphoteric surfactants, preferably selected from the group consisting of non-ionic surfactants. Non-limiting examples thereof include polyvinyl alcohol (PVA), polyoxyethylene esters and ethers, such as Tween ®80, SPAN®80 and Triton X-100, lecithin, sodium docecyl sulfate (SDS), copolymers of polyoxyethylene oxide and polyoxypropylene oxide, such as Poloxamer® 188, etc.

In an embodiment of the invention the at least one surfactant is present in a ratio of from about 1 : 0.005 to about 1 : 10 lipid nano-micro-structure:surfactant, preferably in the ratio of about 1 : 0.01 to about 1 : 0.1.

Thus the use of a surfactant(s) may stabilize the nano-micro-structure and prevent agglomeration of the individual nano-micro-particles. The use of a surfactant(s) may also control the morphology of the nano-micro-structures that produced using electrostatic processing methods.

In an embodiment of the invention at least one porogen is present. Porogen leaching from the lipid nano-micro-structures can be used to control the nano-micro-pore size and porosity by selecting suitable amount of the added porogens, thus further controlling the release over time of the active ingredient. Modification of porogen surface to volume ratio can be used to optimize the permeability of the lipid nano-micro-structure and thus control the release of the active ingredient.

In an embodiment of the invention the composition comprises nicotine and a flavor component, wherein the nicotine component is immobilized in separate lipid nano-micro- structures from the lipid nano-micro-structures immobilizing the flavor component. By incorporating the nicotine in separate lipid nano-micro-structures from the flavor component the release of both the nicotine component and the flavor component may be controlled to obtain e.g. a fast release of nicotine to satisfy the nicotine craving and a longer lasting flavor release to obtain a pleasant taste in the mouth.

In an embodiment of the invention the nicotine component is immobilized in solid lipid nano- micro-particles and the flavor component is immobilized in lipid nano-micro-fibres. Thereby it is possible to design the delivery of the nicotine and flavor to obtain the desired release profile.

In another embodiment of the invention the composition comprises nicotine and a flavor component, wherein both components are present in the same lipid nano-micro-structures. Thereby it is possible to mask any undesired taste of the nicotine.

In another embodiment of the invention the composition comprises nicotine and a flavor component, wherein a flavor component is encapsulated in the lipid nano-micro-structure and the nicotine is present as a coating on the surface thereof. Thereby a fast release of nicotine and a longer-lasting release of flavor can be obtained. In an embodiment of the invention the composition further comprises at least one excipient, preferably wherein said excipient is a hydrophilic polymer and/or amphiphilic polymer, preferably selected from the group consisting of pectin, chitosan, dextran, pullulan, carrageenan, starch, amylose, amylopectin, maltodextrin, cellulose acetate, sodium alginate, gellan, logust bean gum, guar gum, xanthan, konjac gum, tara gum, lipopolysaccharides, polyvinyl alcohol (PVA), polyacrylic acid (PAA), polyethylene oxide (PEO), methyl cellulose

(MC), sodium carboxy methylcellulose (SCMC), hydroxy propyl cellulose (HPC), cyclodextrins; hyaluronic acid; proteins such as BSA, gelatin, casein, milk proteins, soy, wheat, fish proteins; preferably selected from the group consisting of pectin, PEO, PVA and PAA. Such an excipient may further impart a desired hydrophilicity to the composition and may further control the dissolution rate and may thereby control the release of an active ingredient and to result in swelling and/or erosion of the lipid nano-micro-structure.

In an embodiment of the invention the composition further comprises a compound having mucoadhesive properties in order to further control the delivery of the nicotine and/or flavor component to the desired place of delivery. A mucoadhesive compound may e.g. the included in the composition in the form of a coating of the lipid nano-micro-structure by polymer absorption or chemical crosslinking.

Non-limiting examples of a mucoadhesive compound include a compound selected from the group consisting of pectin, chitosan, sodium alginate, polyvinyl alcohol (PVA), polyacrylic acid (PAA), methyl cellulose (MC), sodium carboxy methylcellulose (SCMC), hydroxy propyl cellulose (HPC), N-acetylglucosamine, glucan (e.g. oat beta glucan) preferably selected from the group consisting of pectin, PVA and PAA.

In an embodiment of the invention a composition in the form of lipid nano-micro-particles or nano-microlipid carriers (N-MLC's) is prepared by one of the following methods. High pressure homoaenization

Hot Homoaenization (enzymeis) in the core region^')

1) One or more lipids in a melted state are provided, optionally by heating to above the phase transition temperature thereof. Thus heating may not be necessary when the lipid(s) in question is/are in a liquid state at room temperature. However, in other embodiments heating will be applied in order to provide the lipid(s) in a melted state. Alternatively the lipids are provided dispersed in a solvent.

2) The at least one active ingredient is dissolved or dispersed in the melted lipid, or in the lipid dispersed in a solvent.

3) any optional ingredients, such as surfactant(s), excipient(s) and/or mucoadhesive compound(s) and/or porogen(s) are added to the lipid dispersion either neat or as a solution, preferably an aqueous solution thereof, and mixed.

4) optionally adjusting the temperature;

5) dispersing said at least one enzyme;

6) thereafter the mixture obtained is subjected to high pressure homogenization in a manner known per se, at an elevated temperature,

7) thereafter the mixture obtained is cooled, to obtain the lipid nano-micro-particles or N-MLC's comprising an enzyme at the core region; and

8) Optionally adding at least one enzyme at the above nano-micro-structure by post- addition or post-treatment and/or by post-surface attachment at the above nano- micro-structure, such as by physical adsorption or covalent attachment of enzyme(s).

Hot Homoaenization (enzymeis) in the shell region)

2) The at least one active ingredient is dissolved or dispersed therein or in the lipid

dispersed in a solvent.

4) optionally adjusting the temperature;

5) thereafter the mixture obtained is subjected to high pressure homogenization in a manner known per se, at an elevated temperature; 6) thereafter the mixture obtained is cooled or the temperature is adjusted;

7) at least one enzyme is dispersed in the above dispersion to obtain the lipid nano- micro-particles or N-MLC's comprising an enzyme at the shell region; and

Cold Homoqenization

1) One or more lipids in a melted state are provided, optionally by heating to above the phase transition temperature thereof. Thus heating may not be necessary when the lipid in question is/are in a liquid state at room temperature. However, in other embodiments heating will be applied in order to provide the lipid(s) in a melted state

2) the at least one active ingredient is dissolved or dispersed therein and rapidly cooled afterwards;

3) said at least one enzyme is dispersed in the solution/dispersion obtained;

4) the above solid lipid mixture is milled to nano-micron size;

5) optionally, the above mixture is mixed with any optional ingredients, such as

surfactant(s), excipient(s) and/or mucoadhesive compound(s) and/or porogen(s) that are added to the mixture either neat or as a solution, preferably as an aqueous solution thereof,

6) thereafter the mixture obtained is subjected to high pressure homogenisation below the melting temperature of the lipid(s), to obtain the lipid nano-micro-particles or N- MLC's;

7) Optionally adding at least one enzyme at the above nano-micro-structure by post- addition or post-treatment and/or by post-surface attachment at the above nano- micro-structure, such as by physical adsorption or covalent attachment of enzyme(s).

Microemulsion technique

1) One or more lipids in a melted state are provided, optionally by heating to above the phase transition temperature thereof. Thus heating may not be necessary when the lipid in question is in a liquid state at room temperature. However, in other embodiments heating will be applied in order to provide the lipid(s) in a melted state

2) the at least one active ingredient is dissolved or dispersed therein

3) dispersing said at least one enzyme;

4) separately, any optional ingredients, such as surfactant(s), excipient(s) and/or

mucoadhesive compound(s) and/or porogen(s) in aqueous solution or dispersion are also heated to this temperature 5) the above two solutions or dispersions are then mixed together under mild stirring

6) the above hot mixture (pre-emulsion) is added to a cold water solution under mild stirring to obtain the lipid nano-micro-particles or N-MLC's;

Solvent emulsification-evaporation technique

1) One or more lipids and the at least one active ingredient are dissolved or dispersed in an water immiscible organic solvent;

2) dispersing said at least one enzyme;

3) any optional ingredients, such as surfactant(s), excipient(s) and/or mucoadhesive compound(s) and/or porogen(s) in aqueous solution or dispersion are added to the above mixture;

4) the resulting solution or dispersion is emulsified using high speed homogenization;

5) thereafter the organic solvent is removed (by e.g. rotary evaporator, etc.) to obtain the lipid nano-micro-particles or N-MLC's;

6) Optionally adding at least one enzyme at the above nano-micro-structure by post- addition or post-treatment and/or by post-surface attachment at the above nano- micro-structure, such as by physical adsorption or covalent attachment of enzyme(s).

Solvent emulsification-diffusion technique

1) Any optional ingredients, such as surfactant(s), excipient(s) and/or mucoadhesive compound(s) and/or porogen(s) in aqueous-solvent solution or dispersion of an aqueous-solvent is used, where the solvent is partially miscible with water,

2) one or more lipids and the at least one active ingredient are dissolved or dispersed in the above mixture, optionally with the use of heating;

3) optionally adjusting the temperature;

4) dispersing said at least one enzyme;

5) The above solution or dispersion is stirred to obtain an emulsion

6) The above emulsion is diluted with water to allow diffusion of the solvent to the

aqueous phase lipid nano-micro-particles or N-MLC's are created by dilution of the above emulsion; and

7) removal of the solvent (by e.g. rotary evaporator, etc.);

8) Optionally adding at least one enzyme at the above nano-micro-structure by post- addition or post-treatment and/or by post-surface attachment at the above nano- micro-structure, such as by physical adsorption or covalent attachment of enzyme(s). Melting dispersion method (Hot melt encapsulation method)

1) One or more lipid(s) in a melted state are provided, optionally by heating to above the phase transition temperature thereof. Thus heating may not be necessary when the lipid in question is in a liquid state at room temperature. However, in other embodiments heating will be applied in order to provide the lipid(s) in a melted state;

2) the at least one active ingredient is dissolved or dispersed therein; the above mixture then regarded as oil phase;

3) simultaneously an aqueous phase containing any optional ingredients, such as

surfactant(s), excipient(s) and/or mucoadhesive compound(s) and/or porogen(s) was also heated to same temperature as the oil phase;

4) Dispersing said at least one enzyme to the aqueous phase and /or to the oil phase;

5) the above oil phase added in to a small volume of the aqueous phase and the

resulting emulsion stirred at high speed stirring;

6) the resulting emulsion is cooled to below the phase transition temperature thereof to obtain the lipid nano-micro-particles or N-MLC's;

High shear homoqenization and/or ultrasonication technique

1) One or more lipids and the at least one enzyme and the at least one active ingredient are mixed in the melted state, optionally by heating to above the phase transition temperature of the lipid(s). Thus heating may not be necessary when the lipid(s) in question is/are in a liquid state at room temperature. However, in other embodiments heating will be applied in order to provide the lipid(s) in a melted state;

2) any optional ingredients, such as surfactant(s), excipient(s) and/or mucoadhesive compound(s) and/or porogen(s) in aqueous solution or dispersion are heated and added to the above lipid-active ingredient mixture

3) the above solution or dispersion is emulsified in the melted state by probe sonication or using high speed stirring;

4) the above solution or dispersion is cooled to below the phase transition temperature thereof to obtain the lipid nano-micro-particles or N-MLC's;

5) Optionally adding at least one enzyme at the above nano-micro-structure by post- addition or post-treatment and/or by post-surface attachment at the above nano- micro-structure, such as by physical adsorption or covalent attachment of enzyme(s). Double Emulsion technique

1) The at least one active ingredient is dissolved or dispersed with any optional

ingredients, such as surfactant(s), excipient(s) and/or mucoadhesive compound(s) and/or porogen(s) in an aqueous solution or dispersion;

2) optionally adjusting the temperature;

3) dispersing said at least one enzyme;

4) the above solution or dispersion is emulsified with one or more lipid in the melted state, optionally by heating to above the phase transition temperature thereof. Thus heating may not be necessary when the lipid(s) in question is/are in a liquid state at room temperature. However, in other embodiments heating will be applied in order to provide the lipid(s) in a melted state.

5) the above solution or dispersion mixture is then stirred and filtered to obtain the lipid nano-micro-particles or N-MLC's;

Membrane contactor technique

1) One or more lipids and the at least one active ingredient are mixed in the melted state, optionally by heating to above the phase transition temperature of the lipid(s). Thus heating may not be necessary when the lipid(s) in question is/are in a liquid state at room temperature. However, in other embodiments heating will be applied in order to provide the lipid(s) in a melted state.

2) optionally adjusting the temperature;

3) dispersing said at least one enzyme;

4) the above mixture is pressed through membrane pores into a continuous flow of an aqueous solution or dispersion containing any optional ingredients, such as surfactant(s), excipient(s) and/or mucoadhesive compound(s) and/or porogen(s);

5) the above setup is kept above the lipid(s) melting temperature

6) afterwards, the outlet mixture is cooled below the lipid(s) melting temperature to obtain the lipid nano-micro-particles or N-MLC's;

7) Optionally adding at least one enzyme at the above nano-micro-structure by post- addition or post-treatment and/or by post-surface attachment at the above nano- micro-structure, such as by physical adsorption or covalent attachment of enzyme(s). Solvent injection technique

1) One or more lipid and the at least one active ingredient and the at least one enzyme are dissolved or dispersed in an aqueous-miscible solvent or mixture thereof;

2) optionally adjusting the temperature;

3) the above solution or dispersion is pressed through a needle into a stirred aqueous phase, containing any optional ingredients, such as surfactant(s), excipient(s) and/or mucoadhesive compound(s) and/or porogen(s);

4) the above solution or dispersion is then filtered to remove excess of one or more lipids to obtain the lipid nano-micro-particles or N-MLC's;

5) Optionally adding at least one enzyme at the above nano-micro-structure by post- addition or post-treatment and/or by post-surface attachment at the above nano- micro-structure, such as by physical adsorption or covalent attachment of enzyme(s).

Supercritical fluid technology

Gas/supercritical/antisolvent (GAS/SAS)

1) One or more lipids and the at least one enzyme and the at least one active ingredient are dissolved or dispersed in an organic solvent with any optional ingredients, such as surfactant(s), excipient(s) and/or mucoadhesive compound(s) and/or porogen(s);

2) a supercritical fluid is dissolved in the above organic solvent mixture, causing liquid expansion;

3) a precipitate is formed, due to the reduction of the ability of the solvent to dissolve the above mixture;

4) lipid nano-micro-particles or N-MLC's are formed thereby;

Gas saturated solution iPGSS)

1) One or more lipids and optionally the at least one enzyme and the at least one active ingredient are dissolved or dispersed in an solution with any optional ingredients, such as surfactant(s), excipient(s) and/or mucoadhesive compound(s) and/or porogen(s);

2) a supercritical fluid is dissolved at the above solution or dispersion mixture;

3) the above solution or dispersion is subjected to a rapid depressurization, creating lipid nano-micro-particles or N-MLC's; 4) Optionally adding at least one enzyme at the above nano-micro-structure by post- addition or post-treatment and/or by post-surface attachment at the above nano- micro-structure, such as by physical adsorption or covalent attachment of enzyme(s).

In any of the above methods the order of addition of the at least one active ingredient, the at least one enzyme, and any optional ingredients, such as surfactant(s), excipient(s), mucoadhesive compound(s) and/or porogen(s) is not decisive and may be reversed as known to a person skilled in the art.

In an embodiment of the invention a composition in the form of lipid nano-micro-fibres are prepared by providing at least one lipid in a solvent, preferably an organic solvent, an organic solvent/aqueous system or in a supercritical fluid. As organic solvents may be mentioned acetone, acetic acid, l,l,l,3,3,3-hexafluoro-2-propanol (HFIP), trifluoroacetic acid (TFA), trifluoroethanol, dichloromethane (DCM), chloroform, ethanol, formic acid, N,N-dimethyl formamide (DMF), hexafluoroacetone (HFA), among other, and as supercritical fluids may be mentioned carbon dioxide and water. The choice of solvent depends on the electrostatic processing conditions specific for the composition, as known in the art. Any optional ingredients, such as surfactant(s), excipient(s), mucoadhesive compound(s) and/or porogen(s) are added and the at least one active ingredient is dissolved or dispersed in the mixture obtained. Furthermore at least one enzyme is added neat or dissolved or dispersed in a solvent, such as a solvent as above. Thereafter an electrical field is applied in a manner known per se to obtain a nano-micro-structure in the form of nano-micro-fibres (or other shapes as known in the art).

The order of addition of the at least one active ingredient, the at least one enzyme, and any optional ingredients, such as surfactant(s), excipient(s), mucoadhesive compound(s) and/or porogen(s) is not decisive and may be reversed as known to a person skilled in the art. In an embodiment of the invention a composition in the form of lipid nano-micro-fibres are prepared by providing at least one lipid in an organic solvent or organic/aqueous solvent or in a supercritical fluid. Any optional ingredients, such as surfactant(s), excipient(s), and mucoadhesive compound(s) and porogen(s) are added. Optionally heating to above the main phase transition temperature is performed, and the at least one active ingredient is dissolved or dispersed in the mixture obtained. Furthermore at least one enzyme is added neat or dissolved or dispersed in a solvent, such as a solvent as above. Thereafter an electrical field is applied in a manner known per se to obtain a nano-micro-structure in the form of nano- micro-fibres (or other shapes as known in the art). In an embodiment of the invention the application of an electrical field is obtained by coaxial electrohydrodynamic processing. Electrohydrodynamic processing methods include for example i) electrospinning and emulsion electrospinning, ii) electrospray (of oppositely charged droplets, or colloidal suspensions), and iii) electrocoextrusion (of different liquids from coaxial capillaries).

The order of addition of the at least one active ingredient, the at least one enzyme, and any optional ingredients, such as surfactant(s), excipient(s), mucoadhesive compound(s) and/or porogen(s) is not decisive and may be reversed as known to a person skilled in the art.

In another embodiment of the invention a composition in the form of lipid nano-micro-fibres are prepared as above by adding the enzyme via co-axial electrostatic processing device to obtain the enzyme attached at the shell or outer surface of the lipid nano-micro-structure.

In an embodiment of the invention the composition is for use in chewing gums, lozenges, strips, orally dispersible powders, oral liquid dispersions, mouth sprays, mouth wash, pouches for oral use, lotions, liniments, tablets, capsules, pastes, gels, films and pills.

The composition according to the invention may be incorporated in any desired delivery form contemplated for oral delivery.

The invention is disclosed in more detail below in the form of specific, non-limiting examples thereof.

Example 1

Composition

Lipids:

Stearic acid 400 mg

Hydrogenated sunflower oil : 200 mg

Surfactants:

Polyvinylalcohol : 25 mg Tween®80 : 25 mg Active ingredient: Nicotine base: 20 mg

The SLN's of example 1 were prepared by melting the lipids to 80°C and dissolving nicotine base therein. Tween® 80 was dissolved in 1 ml of water and the solution was mixed with the melted lipids by vortexing at 2000 rpm to obtain a crude pre-emulsion.

The mixed solution was transferred by syringe into a flask containing a solution of PVA in 50 ml of hot water at 85 °C. The solution was subjected to homogenizing at 8000 rpm for 25 minutes to obtain solid lipid nanoparticles (SLN) containing nicotine. The above dispersion of solid lipid nanoparticles were split and retained at a temperature of 0-5 °C. Then, different amounts of lipase from Thermomyces lanuginosus were incorporated to these dispersions (at this temperature the enzyme is inactivated). At 0-5 °C, the lipase was allowed to adhere to the surface of the SLN for 5 min. After that the release of the active ingredient (nicotine) was measured as typical at 37 ± 0.5 °C. As it appears from figure 1, the release rate of nicotine was increased in a dose dependent manner by the incorporation of lipase. Figure 2 shows a cryo-TEM image of the SLN particles in question.

Example 2

Composition

Lipids: Stearic acid 400 mg DY114: 200 mg Surfactants: Polyvinylalcohol: 50 mg

Tween®85: 5 mg Active ingredient:

Aspirin: 33 mg Enzyme:

Lipase from Candida sp. The SLN's of example 1 were prepared by melting the lipids to 80°C and dissolving aspirin base therein. Tween® 85 was dissolved in 1 ml of water and the solution was mixed with the melted lipids by vortexing at 2000 rpm to obtain a crude pre-emulsion.

The mixed solution was transferred by syringe into a flask containing a solution of PVA in 50 ml of hot water at 85 °C. The solution was subjected to homogenizing at 8000 rpm for 30 minutes to obtain solid lipid nanoparticles (SLN) containing aspirin.

The above dispersion of solid lipid nanoparticles were split and retained at a temperature of 0-5 °C. Then, lipase was incorporated into these dispersions (at this temperature the enzyme is inactivated). At 0-5 °C, the lipase was allowed to adhere to the surface of the SLN for 5 min. After that the release of the active ingredient (aspirin) was measured at 37 ± 0.5 °C.

Fig. 3 shows a comparison of aspirin release from SLN with and without lipase measured at dialysis membranes with 3 ml solution of example 2 at 37 ± 0.5 °C with magnetic stirring.

Example 3

Inner needle composition : Lipids:

Asolectin available from Sigma-Aldrich, a mixture of phospholipids from soybean, 2 g Solvent:

Isooctane 1.92 mL

Active ingredient (flavour) : 20 mg 4-Hydroxy-2,5-dimethyl-3(2 - )-furanone

Outer needle composition :

Isooctane

Enzyme: Phospholipase, A_x from Thermomyces lanuginosus or Phospholipase D from Arachis hypogaea (peanut)

Co-axial electrospinning was applied in this example. The inner needle composition was mixed with slow magnetic stirring for 24 hours and then allowed to rest for 1 hour before being electrospun. Parameters for electrospinning were 0.05 ml/min, distance 10 cm and voltage 35 kV.

The outer needle composition is isooctane; all solvents evaporate during electrospinning leaving only lipid fibers left made of asolectin and 4-Hydroxy-2,5-dimethyl-3(2H)-furanone. Image of fibers can be found in figures 5 and 6. Figures 5 and 6 show electrospun lipid fibers containing the flavor compound 4-Hydroxy-2,5-dimethyl-3(2H)-furanone visualized using scanning electron microscope sputter coated with gold for 10 seconds at 40 mA at a distance of 10 cm at magnifications of 250X and lOOOx, respectively.

Example 3a:

The electrospun lipid fibers are then subjected to a post-treatment with Phospholipase A_x from Thermomyces lanuginosus (commercial name Lecitase™ Ultra, product of Novozymes Corp). A solution of enzyme is allowed to adhere to the surface at 0-5° C for 5 min. After that the release of the active ingredient (furanone) was measured at 37 ± 0.5 °C.

Example 3b:

The electrospun lipid fibers are then subjected to a post-treatment with Phospholipase D from Arachis hypogaea (peanut). A solution of enzyme is allowed to adhere to the surface at 0-5° C for 5 min. After that the release of the active ingredient (furanone) was measured at 37 ± 0.5 °C. The release behavior of the flavor compound (4-Hydroxy-2,5-dimethyl-3(2 - )-furanone) from electrospun lipid fibers with or without enzyme (phospholipase, A_x with 18 LU/mg lipid or phospholipase D with 0.1 LU/mg lipid) appears from fig. 4 measured at 6-8kDa dialysis membranes with 4 mL water inside in a 200 mL water volume. The temperature was controlled at 37±0.5 °C with magnetic stirring. Phospholipase A_x from Thermomyces lanuginosus and phospholipase D from Arachis hypogaea (peanut).

List of references

Thornton PD, Heise A: Highly specific dual enzyme-mediated payload release from peptide- coated silica particles. J Am Chem Soc 2010, 132: 2024-2028. Venkatesh S, Wower J, Byrne ME: Nucleic acid therapeutic carriers with on-demand triggered release. Bioconjug Chem 2009, 20: 1773-1782.

Schlossbauer A, Kecht J, Bein T: Biotin-avidin as a protease-responsive cap system for controlled guest release from colloidal mesoporous silica. Angew Chem Int Ed Engl 2009, 48: 3092-3095. WO 2007/113665 A2

WO 2010/114901 Al

WO 2012/088059 A2

US 2009/0291133 Al

US 4,880,634

US 5,885,486

Claims

1. A composition for controlled oral delivery of at least one active ingredient comprising : i) a lipid nano-micro-structure comprising a core region and a shell region or a

multilayer shell region, said lipid nano-micro-structure comprising at least one lipid and at least one active ingredient, said at least one active ingredient being immobilized in said lipid nano-micro-structure; and

ii) at least one enzyme , wherein said lipid nano-micro-structure is selected from the group comprising lipid nano- micro-particles, lipid nano-micro-laminated particles, lipid nano-micro-fibres, nano- micro-particles encapsulated/immobilized in lipid nano-micro-fibres, lipid nano-micro- particles encapsulated/immobilized in polymer nano-micro-fibers, lipid nano-micro- particles encapsulated/immobilized in polymer nano-micro-particles, nano-micro- structured lipid carriers (N-MLC's), lipid drug conjugate (LDC) nano-micro-particles, polymer - lipid hybrid nano-micro-particles (PLNM), and any combinations thereof, preferably wherein said lipid nano-micro-structure is selected from the group comprising solid lipid nano-micro-particles and lipid nano-micro-fibres.

2. The composition according to claim 1, wherein said at least one active ingredient is selected from the group consisting of nicotine and nicotine salts, caffeine, a flavor component, oral pain relievers/anesthetics, agents for the prevention of caries,

antigingivitis/antiplaque agents, agents for relieving dry mouth, essential oils, flavor oils, polyols, gums, enzymes, fruit acids, desensitizers, whitening agents, antiviral agents, antibiotics/antifungals, cough suppressants and anti-tussives, expectorants, demulscents, anti-inflammatory agents, antioxidants, bronchodilators, antihistamines, decongestants, herbal compounds, odor neutralizers, vitamins, metals, minerals, growth factors and hormones, amino acids, anti-hypertensive agents, anti-cancer agents, etoposide, therapeutic biomolecules, biologically active molecules, genes, vaccines, active cultures, bioactive proteins, omega-3 and omega-6 fatty acids, dietary fats, supplements, hormones, supplements, and any mixtures thereof.

3. The composition according to any one of the preceding claims, wherein said enzyme is selected from the group consisting of Amylases, Amyloglucosidase, Glucoamylase,

Aminopeptidase, Catalase, Cellulase, dextranases, Ficin (peptide hydrolase), Hemicellulase, a-galactosidase, Gelatinase, β-glucanases, glucose oxidase, Invertase, Glucose isomerase, Esterase-lipase; Phopholipases (phospholipase, phospholipase A_lr phospholipase A₂, phospholipase C, phospholipase D) ; triacylglycerol lipase; galactolipase; lipid-phosphate phosphatase; Lysophospholipase; diacylglycerollipase; acyltransferase; lipoprotein lipase; lipase with bile salts, Animal lipase (triacylglycerol hydrolase), Lactase, lactoperoxidases, Lysozyme, Maltase, Oligosaccharidases, oxidases, Pancreatin (peptide hydrolase), Papain, Pectinases, pectolytic enzymes (e.g . polygalacturonase and pectin methylesterase), Proteases, (I, II, III, IV, V), Pepsin, Phytase, Trypsin (chemotrypsin) (peptide hydrolase), xylanases, and any combination/mixture of the above enzymes, preferably selecred from the group consisting of a lipase, a phospholipase, a carbohydrase, an amylase, a xylanase, a pectinase, a peptidase and a protease.

4. The composition according to any one of the preceding claims, wherein said at least one enzyme is present at the shell region of the lipid nano-microstructure.

5. The composition according to any one of the preceding claims, wherein said at least one enzyme is present at the core region of the lipid nano-microstructure.

6. The composition according to any one of the preceding claims, wherein said at least one enzyme is present separately from the lipid nano-microstructure.

7. The composition according to anyone of the preceding cla ims, wherein said lipid nano- microstructure comprises at least one lipid selected from the group consisting of fatty acids, fatty esters and fatty mono-, di-, and triglycerides thereof, partial glycerides, fatty alcohols and their esters and ethers, natural and synthetic waxes such as bees wax and carnauba wax, wax alcohols and their esters, hydrogenated vegetable oils, hard paraffins,

phospholipids, sterols and sterol derivatives, and mixtures of any of the above lipids, preferably wherein said at least one lipid is selected from the group consisting of C fatty acids and C₈-₂₄ fatty mono-, di-, or triglycerides, such as C fatty acids and C fatty mono-, di-, or triglycerides, such as saturated C fatty acids and C fatty mono-, di-, or triglycerides, and mixtures of any of the above lipids, preferably wherein said at least one lipid is selected from the group consisting of capric, lauric, myristic, palmitic, stearic, and arachidic acids and mono-, di- and triglycerides thereof, preferably trimyristin, tripalmitin, tristearin, tricaprin, myristic acid, palmitic acid, stearic acid, and behenic acid, and mixtures of any of the above lipids.

8. The composition according to any one of the preceding claims further comprising at least one surfactant, wherein said at least one surfactant is selected from the group consisting of ionic, non-ionic, and amphoteric surfactants, preferably selected from the group consisting of non-ionic surfactants such as polyvinyl alcohol (PVA), polyoxyethylene esters and ethers, lecithin, sodium docecyl sulfate (SDS), copolymers of polyoxyethylene oxide and

polyoxypropylene oxide, preferably PVA, polyoxyethylene esters and ethers, lecithin wherein the at least one surfactant is present in a ratio of from about 1 : 0.005 to about 1 : 10 lipid nano-micro-structure: surfactant, preferably in the ratio of about 1 : 0.01 to about 1 : 0.1.

9. The composition according to anyone of the preceding claims further comprising at least one excipient, preferably wherein said excipient is a hydrophilic polymer and/or amphiphilic polymer, preferably selected from the group consisting of polysaccharides/carbohydrates such as pectin, chitosan, dextran, pullulan, carrageenan, starch, amylose, amylopectin, cellulose acetate, sodium alginate, logust bean gum, guar gum, gellan, xanthan, tara gum, konjac gum, methyl cellulose (MC), sodium carboxy methylcellulose (SCMC), hydroxy propyl cellulose (HPC); cyclodextrins; hyaluronic acid; proteins such as BSA, gelatine, casein, milk proteins, soy, wheat, fish proteins; polyvinyl alcohol (PVA), polyacrylic acid (PAA), polyethylene oxide (PEO), preferably selected from the group consisting of chitosan, starch, amylose, amylopectin, pectin, MC, SCMC, HPC, PEO, PVA and PAA and/or further comprising at least one mucoadhesive compound selected from the group consisting of pectin, chitosan, sodium alginate, polyvinyl alcohol (PVA), polyacrylic acid (PAA), methyl cellulose (MC), sodium carboxy methylcellulose (SCMC), hydroxy propyl cellulose (HPC), N- acetylglucosamine, glucan (e. g. , oat beta-glucan), preferably selected from the group consisting of pectin, PVA and PAA.

10. The composition according to any one of the preceding claims for use in chewing gums, lozenges, strips, orally dispersible powders, oral liquid dispersions, mouth sprays, mouth wash, pouches for oral use, lotions, liniments, tablets, capsules, creams, pastes, gels, films and pills.

11. A method for the preparation of a composition in the form of lipid nano-micro-particles according to any one of the preceding claims, comprising the steps of: i) Providing said at least one lipid in a melted state, optionally by heating to above the phase transition temperature thereof or providing said at least one lipid dispersed in a solvent;

ii) Dispersing or dissolving said at least one active ingredient in the melted lipid, or in the lipid dispersed in a solvent ;

iii) Optionally adding at least one surfactant, optionally at least one excipient and optionally at least one mucoadhesive compound and optionally at least one porogen compound neat or in a solution thereof, preferably in an aqueous solution thereof;

iv) mixing and homogenizing to obtain a nano-micro-structure comprising said at least one active ingredient; wherein at least one enzyme is added either before, during and/or after the step of mixing and homogenizing to obtain an enzyme-containing nano-micro-structure.

12. A method for the preparation of a composition in the form of lipid nano-micro-fibres according to any one of claims 1-10, comprising the steps of: i) Providing said at least one lipid in a solvent, preferably an organic solvent or an organic solvent/aqueous system or in a supercritical fluid;

Optionally adding at least one surfactant, at least one excipient, and optionally at least one mucoadhesive compound and/or optionally at least one porogen compound;

iii) Optionally heating to above the main phase transition temperature;

iv) Dispersing or dissolving at least one active ingredient in the mixture obtained in step ii); and

13. The method according to claim 12, wherein the application of an electrical field is obtained by coaxial electrohydrodynamic processing.

14. The method according to any one of claims 11-13, comprising the further step of adding at least one enzyme at the above nano-micro-structure by further post-addition or post- treatment.

15. The method according to any one of claims 11-14, wherein said further step of adding at least one enzyme is by further post-surface attachment, such as by physical adsorption or covalent attachment of enzyme(s).

16. A use of the composition according to any one of claims 1-10 for oral delivery of said at least one active ingredient.