Arterial plaque control with enzymatic cyclodextrin inclusion complex formulations

Abstract

The invention provides oral cyclodextrin inclusion complex formulations in which inclusion complexes of borneol and trans-cinnamaldehyde are provided together with nattokinase and with an enzyme having a cyclodextrin-degrading activity capable of digesting cyclodextrins, so that upon delivery of the formulation to a target tissue the CD- degrading enzyme is activated and releases the borneol and trans-cinnamaldehyde from the cyclodextrins. These cyclodextrin inclusion complex formulations may be used for treating atheromatous plaque and related diseases.

Description

ARTERIAL PLAQUE CONTROL WITH ENZYMATIC CYCLODEXTRIN INCLUSION COMPLEX FORMULATIONS FIELD OF THE INVENTION

[0001] The invention is in the field of biochemical constructs for delivery of therapeutic organic compounds as inclusions within cyclodextrins, in formulations that include enzymes having therapeutic and cyclodextrin-degrading activities. BACKGROUND OF THE INVENTION

[0002] Arterial plaque (atheroma) buildup, also called atheromatous plaque, involves the deposition within the lumen of arteries of recalcitrant deposits made up of cholesterol, calcium and other cellular material, including macrophages and connective tissue. The atheroma may progressively occlude the artery, stenosis or restenosis, and may rupture. In this way, atheromatous plaque has an etiological role in a variety of increasingly prevalent diseases, such as atherosclerosis, coronary artery disease, carotid artery disease, peripheral artery disease, myocardial infarction and stroke.

[0003] Cyclodextrins are non-reducing cyclic glucose oligosaccharides, frequently the product of cyclomaltodextrin glucanotransferase (E.C. 2.4.1.19; CGTase) catalyzed degradation of starch. Cyclodextrins may have a variety of structures (see Saenger et al., Chem. Rev.98 (1998) 1787-1802), including three common cyclodextrins with 6, 7 or 8 D-glucopyranonsyl residues (α-, β-, and γ-cyclodextrin respectively) linked in a ring by α- 1,4 glycosidic bonds. The frustoconical shape of cyclodextrins forms a cavity or lumen, with the cavities having different diameters depending on the number of glucose units. The scale of selected cyclodextrin (CD) structures is set out in Table 1. Larger cyclodextrins such as cyclomaltononaose (δ-CD) and cyclomaltodecaose (ε-CD) are also possible, as well as a variety of cyclodextrin-based supra-molecular structures (see Zhang and Ma, Adv Drug Deliv Rev.2013 Aug;65(9):1215-33). Table 1: cyclodextrin structures Cyclodextrin Lumen diameter (nm)Inner rim Outer rim α, (glucose)6 0.45 0.53 β, (glucose)7 0.60 0.65 γ, (glucose)8 0.75 0.85

[0004] Cyclodextrins are generally amphipathic, with the wider rim of the lumen displaying the 2- and 3-OH groups and the narrower rim displaying 6-OH. These hydrophilic hydroxyl groups are accordingly on the outside of the lumen, whereas the inner surface is generally hydrophobic and lined with the anomeric oxygen atoms and the C3-H and C5-H hydrogen atoms. In aqueous solution, this hydrophobic lumen may contain water molecules, for example about 3 (α-CD), 7 (β-CD) or 9 (γ-CD) poorly held but low entropy, and hence relatively easily displaceable water molecules. Thus, otherwise hydrophilic cyclodextrins may bind retain one or more suitably-sized molecules within, or partially within, the lumen of the CD, forming a cyclodextrin inclusion body or complex. For example, non-polar aliphatic and aromatic compounds, including drugs, such as lipophilic drugs, may be bound so as to increase the water solubility of normally hydrophobic compounds or minimize undesirable properties such as odor or taste in certain food additives. For this reason, cyclodextrin inclusions are widely used in the pharmaceutical, food and cosmetic fields (see Hedges, Chem. Rev. 98 (1998) 2035- 2044). Cyclodextrins have for example been used in a variety of sustained release drug preparations, such as for inclusion complexes of a medical compound with a hydrophobic cyclodextrin derivative (U.S. Patent No.4,869,904).

[0005] Cyclodextrins may be chemically modified in a wide variety of ways. For example, to modify the inclusion specificity, physical and chemical properties of the cyclodextrin. Hydroxyl groups of a CD may for example be derivatized. For example, two modified CDs have been used in a number of pharmaceutical products: SBE-β-CD, or Captisol, a polyanionic variably substituted sulfobutyl ether of β-CD, and HP-β-CD, a modified CD commercially developed by Janssen. Additional CD derivatives include sugammadex or Org-25969, in which the 6-hydroxy groups on γ-CD have been replacedby carboxythio acetate ether linkages, and hydroxybutenyl-β-CD. Alternative forms of cyclodextrin include: 2,6-Di-O-methyl-β-CD (DIMEB), 2-hydroxylpropyl-β-cyclodextrin (HP-β-CD), randomly methylated-β-cyclodextrin (RAMEB), sulfobutyl ether β-cyclodextrin (SBE-β-CD), and sulfobutylether-γ-cyclodextrin (SBEγCD), sulfobutylated beta- cyclodextrin sodium salt, sulfobutylated beta-cyclodextrin sodium salt, (2-Hydroxypropyl)- alpha-cyclodextrin, (2-Hydroxypropyl)-beta-cyclodextrin, (2-Hydroxypropyl)-gamma- cyclodextrin, DIMEB-50 Heptakis(2,6-di-O-methyl)-beta-cyclodextrin, TRIMEB Heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin, methyl-beta-cyclodextrin, octakis(6- deoxy-6-iodo)-gamma-cyclodexrin, and, octakis(6-deoxy-6-bromo)-gamma-cyclodexrin. Although CDs such as these have been developed with favorable pharmacological and toxicological profiles, there is the potential that, following administration, residual CDs may perturb the pharmacokinetic properties of drugs, including coadministered drugs, particularly after parenteral administration (see Stella and He, Toxicol Pathol January 2008 vol.36 no.130-42).

[0006] Cyclodextrins are variably susceptible to enzymatic digestion. For example, γ- CD is relatively easily hydrolyzed by α-amylases whereas α-cyclodextrin is more poorly hydrolyzed. CD based therapeutics generally depend on the activity of endogenous amylases to digest the CD. There is however significant variability in amylase activity between patients. For example, patients with pancreatic insufficiency, cystic fibrosis, celiac disease or Crohn's disease, may lack normal amounts of amylase. Similarly, patients, particularly geriatric patients, may be deficient in gastric acid production and thereby fail to create conditions of appropriately low pH in the duodenum to properly trigger release of pancreatic amylase. A similar effect may result from the increasing common use of antacids, histamine-2 blockers, proton pump inhibitors or alternative acid blockers.

[0007] A variety of microbial cyclodextrin digesting enzymes have been identified. CD- degrading enzymes include cyclomaltodextrinase (or cyclodextrinase, or CDase, EC 3.2.1.54), maltogenic amylase (EC 3.2.1.133), neopullulanase (EC 3.2.1.135), which have been reported to be capable of hydrolyzing CDs and in some cases additional substrates such as pullulan, and starch. Cyclodextrinase (CDase) catalyzes the hydrolysis of CDs to form linear oligosaccharides of α-1,4-linkages, and it can accordinglyrelease substances from CD inclusion complexes. A CDase from Bacillus macerans was reported in 1968, and many CDases from bacteria have since been characterized, such as enzymes from Bacillus sp., Thermoanaerobacter ethanolicus strain 39E, Flavobacterium sp. , and Klebsiella oxytoca strain M5a1. Archaea CDases have been characterized from Archaeoglobus fulgidus, Thermococcus sp. B1001, Thermococcus sp. CL1, Thermofilum pendens, and Pyrococcus furiosus. The structure of the CDase from Flavobacterium sp. has been characterized in detail (see Sun et al., Archaea, Volume 2015 (2015), Article ID 397924, reporting the identification of a gene encoding a cyclodextrinase from Thermococcus kodakarensis KOD1 (CDase-Tk)).

[0008] Nattokinase is a naturally occurring serine protease (UniProt P35835; EC number 3.4.21.62) produced by Bacillus subtilis var natto in the course of fermentation, traditionally in the course of producing the fermented soybean food product commonly known as nattō.

[0009] Borneol is a naturally occurring bicyclic terpene derivative (rel-(1R,2S,4R)- 1,7,7-trimethylbicyclo[2.2.1]heptan-2-ol), occurring naturally as d-borneol and l-borneol enantiomers.

[0010] Trans-cinnamaldehyde, the trans isomer of cinnamaldehyde ((2E)-3- phenylprop-2-enal), is a naturally occurring phenylpropanoid that gives cinnamon its characteristic flavour and odor. SUMMARY

[0011] Therapeutic formulations are provided which include nattokinase, and in which borneol and trans-cinnamaldehyde are provided as guest molecules in cyclodextrin inclusion complexes. These formulations are provided for use in controlling arterial plaque (atheroma), and accordingly for treating conditions in which arterial plaque formation has an etiological role, such as atherosclerosis, coronary artery disease, carotid artery disease and peripheral artery disease. Formulations may for example be used so as to decrease atheromatous plaque in a subject.

[0012] A biologically acceptable carrier may be provided for the cyclodextrin inclusion complex, so that the guest molecule is stably retained by the cyclodextrin within the biologically acceptable carrier. An enzyme may also be provided in the vehicle, having acyclodextrin-degrading activity capable of digesting the cyclodextrin retaining the guest molecule. The CD-degrading enzyme may be formulated so that the cyclodextrin- degrading activity is activated on delivery of the vehicle to a target so as to release the guest molecule from the cyclodextrin cavity.

[0013] In alternative aspects of the delivery vehicle, the CD-degrading enzyme may be co-formulated with the cyclodextrin inclusion complex or the CD-degrading enzyme may be co-packaged in the delivery vehicle with the cyclodextrin inclusion complex. When the CD-degrading enzyme is co-packaged, the delivery vehicle may further include a biochemically acceptable carrier for the CD-degrading enzyme.

[0014] The CD-degrading enzyme may for example be an amylase, a cyclodextrinase, maltogenic amylase or neopullulanase. An amylase may for example be a mammalian salivary amylase or a pancreatic amylase, or an amylase of fungal, or bacterial origin. A cyclodextrinase may for example be a microbial cyclodextrinase.

[0015] The cyclodextrin may for example be a CD derivative, such as a hydrophobic alkylated cyclodextrin or a mixed methylated / ethylated cyclodextrin.

[0016] The ratio of the cyclodextrin to the guest molecule may for example be 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4 or 1:5, although a wide range of alternative values for this parameter are also possible, including non-integer ratios.

[0017] The cyclodextrin may for example be an alpha, beta or gamma cyclodextrin, although again a very wide range of alternative CD structures may be used. The biologically acceptable carrier may be a pharmaceutically acceptable carrier. The delivery vehicle may be formulated for sustained release of the guest molecules, and / or other active agents. In this way, the invention provides alternative embodiments in which CD delivery vehicles may be formulated and used as a medicaments.

[0018] Methods are provided for treating patients to reduce atheromatous plaque, or for treating a disease which is characterized by atheromatous plaque, for example in a mammalian subject, such as a human patient, including obese subjects. DETAILED DESCRIPTION

[0019] The present formulations may be used so as to treat atheromatous plaque, for example for treating or preventing a disease which can be treated or prevented byameliorating atheromatous plaque in a mammal. Atheromatous plaque contributes to the pathogenesis of a wide range of conditions, including atherosclerosis, coronary artery disease, carotid artery disease and peripheral artery disease.

[0020] Specific formulations may for example include nattokinase and a cyclodextrin inclusion complex formulation of borneol and trans-cinnamaldehyde (TCA), and these may be co-formulated with a CD degrading enzyme. In select embodiments, borneol and TCA are provided in a beta CD inclusion, formulated together with an amylase.

[0021] In select embodiments, the CD-degrading enzyme provided in the vehicle may be formulated so that the cyclodextrin-degrading activity is activated on delivery of the vehicle to a target so as to release the guest molecule from the cyclodextrin cavity. CD- degrading enzyme activation may for example be accomplished in a medicament, for example for oral delivery, in a dry dosage form, such as a capsule or tablet, in which the CD-degrading enzyme is admixed, so that the CD-degrading enzyme will not be active until activated by moisture in the gastrointestinal tract of a host. Similarly, a wide variety of time release matrices and formulations are known, which may be adapted for use in CD delivery vehicles so as to orchestrate the appropriate activation of the CD-degrading CD-degrading enzyme upon delivery to the target.

[0022] In various aspects, CD delivery vehicles may have the CD-degrading enzyme co-formulated with the cyclodextrin inclusion complex, as for example discussed above, or the CD-degrading enzyme may be co-packaged in the delivery vehicle with the cyclodextrin inclusion complex. In the case of co-packaging, the delivery vehicle may for example include a biochemically acceptable carrier for the CD-degrading enzyme – distinct from the carrier for the CD inclusion complex. For example, delivery vehicles may be provided with separated compartments containing the CD inclusion complex and the CD-degrading enzyme, so that the delivery vehicle will be made up of a CD inclusion complex compartment connected to a CD-degrading enzyme compartment. Mechanisms may be provided for the combined release of the CD inclusion complex and the CD- degrading enzyme from the respective compartments in the delivery vehicle. For example, syringes may be provided having distinct compartments of this kind that are discharged by a common discharge mechanism, such as a mechanism that cooperatively displaces pistons in each compartment so as to discharge aliquots of CD inclusioncomplex and CD-degrading enzyme, so that the enzyme and the complex may then be comingled to activate the enzymatic release of the guest molecule from the CD. Vehicles of this kind may for example be used to dispense a topical cream or other surface-active formulations. A wide variety of delivery vehicles of this kind may be adapted from devices that are known for dispensing two-part compositions such as epoxy resins, two-part medicaments or dental formulations, as for example disclosed in U.S. Patent Nos. 4538920, 8100295, 8308340, 8875947, 8499976 and International Patent Publications WO2007041266 and WO2000021842.

[0023] There are a wide variety of techniques available to prepare CD inclusion complexes, as for example described in: Chaudhary & Patel, IJPSR, 2013, Vol.4(1): 68- 76; Carneiro et al., 2019, Int. J. Mol. Sci. 2019, 20, 642; US Patent Publications US20090029020; US20090214446; US Patent Nos 5,070,081; 5,552,378; 5,674,854, and 8,658,692. A common approach is known as the kneading method, which involves mixing CDs with water or an aqueous alcohol to provide a paste. The bioactive molecule may then be added to the paste and kneaded for a specified time. The kneaded mixture may then be dried and passed through sieve if desired. Alternatively, a slurry method involves steps of: mixing the bioactive molecule and the cyclodextrin, adding a suitable amount of water to the mixture, typically with vigorous mixing, until a paste or a slurry is formed; continuing the mixing with further addition of water if necessary to maintain the paste or the slurry consistency, for a suitable period of time, such as 15 minutes, to form the inclusion complex; and, drying the product of this final step. Other ingredients, such as emulsifiers, may facilitate the formation of inclusion complexes, for example processes that involve steps of: dry blending a cyclodextrin and an emulsifier (e.g., pectin); combining the dry blend of cyclodextrin and the emulsifier with a solvent such as water in a reactor, and agitating; adding the guest molecule and stirring (e.g., for approximately 5 to 8 hours); optionally cooling the reaction mixture with stirring; and emulsifying the mixture prior to drying the cyclodextrin inclusion complex to form a powder. Other known approaches to preparing CD inclusions involve lyophilization, microwave irradiation, and a supercritical fluid antisolvent technique.

[0024] The CD delivery vehicles of the invention can be provided alone or in combination with other compounds (for example, nucleic acid molecules, smallmolecules, peptides, or peptide analogues), in the presence of a carrier, such as a liposome, an adjuvant, or any pharmaceutically or biologically acceptable carrier. Select embodiments include medicaments in a form suitable for administration to animal hosts, such as mammals, for example, humans. As used herein “pharmaceutically acceptable carrier” or “excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The carrier can be suitable for any appropriate form of administration, including topical, subcutaneous, intradermal, intravenous, parenteral, intraperitoneal, intramuscular, sublingual, inhalational, intratumoral or oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the biologically active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0025] Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer the delivery vehicles to subjects. Any appropriate route of administration may be employed, for example, parenteral, intravenous, intradermal, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, intracisternal, intraperitoneal, intranasal, inhalational, aerosol, topical, intratumoral, sublingual or oral administration. Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; for intranasal formulations, in the form of powders, nasal drops, or aerosols; and for sublingual formulations, in the form of drops, aerosols or tablets.

[0026] Cyclodextrin-degrading or digesting enzymes may for example be formulated for oral delivery. Enteric enzyme formulations may for example be provided, such as submicron particle formulations prepared by emulsion solvent evaporation (Sharma et al., Pharm Dev Technol. 2013 May-Jun;18(3):560-9). Similarly, delivery vehicles may beformulated as hydrogels (see US Patent Publication 20140094433), or medicated gums (see US Patent Publication 20130022652).

[0027] Methods well known in the art for making formulations are found in, for example, “Remington’s Pharmaceutical Sciences” (20th edition), ed. A. Gennaro, 2000, Mack Publishing Company, Easton, PA. Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide / glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for include ethylene- vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.

[0028] Pharmaceutical compositions of the present invention may be in any form which allows for the composition to be administered to a patient. For example, the composition may be in the form of a solid, liquid or gas (aerosol). Typical routes of administration include, without limitation, oral, topical, parenteral, sublingual, rectal, vaginal, and intranasal. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, epidural, intrasternal injection or infusion techniques. Pharmaceutical composition of the invention are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient. Compositions that will be administered to a patient take the form of one or more dosage units, where for example, a tablet, capsule or cachet may be a single dosage unit, and a container of the compound in aerosol form may hold a plurality of dosage units.

[0029] Materials used in preparing the pharmaceutical compositions should be pharmaceutically pure and non-toxic in the amounts used. The inventive compositions may include one or more compounds (active ingredients) known for a particularly desirable effect. It will be evident to those of ordinary skill in the art that the optimaldosage of the active ingredient(s) in the pharmaceutical composition will depend on a variety of factors. Relevant factors include, without limitation, the type of subject (e.g., human), the particular form of the active ingredient, the manner of administration and the composition employed.

[0030] In general, the pharmaceutical composition includes a delivery vehicle of the present invention as described herein, in admixture with one or more carriers. The carrier(s) may be particulate, so that the compositions are, for example, in tablet or powder form. The carrier(s) may be liquid, with the compositions being, for example, an oral syrup or injectable liquid. In addition, the carrier(s) may be gaseous, so as to provide an aerosol composition useful in, e.g., inhalatory administration.

[0031] When intended for oral administration, the composition is preferably in either solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.

[0032] As a solid composition for oral administration, the composition may be formulated into a powder, granule, compressed tablet, pill, capsule, cachet, chewing gum, wafer, lozenges, or the like form. Such a solid composition will typically contain one or more inert diluents or edible carriers. In addition, one or more of the following adjuvants may be present: binders such as syrups, acacia, sorbitol, polyvinylpyrrolidone, carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin, and mixtures thereof; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; fillers such as lactose, mannitols, starch, calcium phosphate, sorbitol, methylcellulose, and mixtures thereof; lubricants such as magnesium stearate, high molecular weight polymers such as polyethylene glycol, high molecular weight fatty acids such as stearic acid, silica, wetting agents such as sodium lauryl sulfate, glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin, a flavoring agent such as peppermint, methyl salicylate or orange flavoring, and a coloring agent.

[0033] When the composition is in the form of a capsule, e.g., a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or a fatty oil.

[0034] The composition may be in the form of a liquid, e.g., an elixir, syrup, solution, aqueous or oily emulsion or suspension, or even dry powders which may be reconstituted with water and / or other liquid media prior to use. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, preferred compositions contain, in addition to the present compounds, one or more of a sweetening agent, thickening agent, preservative (e.g., alkyl p-hydoxybenzoate), dye / colorant and flavor enhancer (flavorant). In a composition intended to be administered by injection, one or more of a surfactant, preservative (e.g., alkyl p- hydroxybenzoate), wetting agent, dispersing agent, suspending agent (e.g., sorbitol, glucose, or other sugar syrups), buffer, stabilizer and isotonic agent may be included. The emulsifying agent may be selected from lecithin or sorbitol monooleate.

[0035] The liquid pharmaceutical compositions of the invention, whether they be solutions, suspensions or other like form, may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer’s solution, isotonic sodium chloride, fixed oils such as synthetic mono or digylcerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. An injectable pharmaceutical composition is preferably sterile.

[0036] The pharmaceutical composition may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment, cream or gel base. The base, for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickening agents may be present in a pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or iontophoresis device. Topicalformulations may contain a concentration of the biologically active compound of from about 0.1 to about 25% w / v (weight per unit volume).

[0037] The composition may be intended for rectal administration, in the form, e.g., of a suppository which will melt in the rectum and release the drug. The composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient. Such bases include, without limitation, lanolin, cocoa butter and polyethylene glycol. Low- melting waxes are preferred for the preparation of a suppository, where mixtures of fatty acid glycerides and / or cocoa butter are suitable waxes. The waxes may be melted, and the aminocyclohexyl ether compound is dispersed homogeneously therein by stirring. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool and thereby solidify.

[0038] The composition may include various materials which modify the physical form of a solid or liquid dosage unit. For example, the composition may include materials that form a coating shell around the active ingredients. The materials which form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredients may be encased in a gelatin capsule or cachet.

[0039] The pharmaceutical composition of the present invention may consist of gaseous dosage units, e.g., it may be in the form of an aerosol. The term aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system which dispenses the active ingredients. Aerosols of compounds of the invention may be delivered in single phase, bi-phasic, or tri-phasic systems in order to deliver the active ingredient(s). Delivery of the aerosol includes the necessary container, activators, valves, subcontainers, and the like, which together may form a kit.

[0040] The biologically active compounds may be in the form of the free base or in the form of a pharmaceutically acceptable salt such as the hydrochloride, sulfate, phosphate, citrate, fumarate, methanesulfonate, acetate, tartrate, maleate, lactate, mandelate, salicylate, succinate and other salts known in the art. The appropriate salt would be chosen to enhance bioavailability or stability of the compound for the appropriate mode of employment (e.g., oral or parenteral routes of administration).

[0041] A composition intended to be administered by injection can be prepared by combining the delivery vehicle of the present invention with water, and preferably buffering agents, so as to form a solution. The water is preferably sterile pyrogen-free water. A surfactant may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with the aminocyclohexyl ether compound so as to facilitate dissolution or homogeneous suspension of the aminocyclohexyl ether compound in the aqueous delivery system. Surfactants are desirably present in aqueous compositions of the invention because the aminocyclohexyl ether compounds according to the present invention may be hydrophobic. Other carriers for injection include, without limitation, sterile peroxide-free ethyl oleate, dehydrated alcohols, propylene glycol, as well as mixtures thereof.

[0042] Suitable pharmaceutical adjuvants for the injecting solutions include stabilizing agents, solubilizing agents, buffers, and viscosity regulators. Examples of these adjuvants include ethanol, ethylenediaminetetraacetic acid (EDTA), tartrate buffers, citrate buffers, and high molecular weight polyethylene oxide viscosity regulators. These pharmaceutical formulations may be injected intramuscularly, epidurally, intraperitoneally, or intravenously.

[0043] The present invention also provides kits that contain a pharmaceutical composition which includes one or more delivery vehicles. The kit also includes instructions for the use of the pharmaceutical. Preferably, a commercial package will contain one or more unit doses of the pharmaceutical composition. For example, such a unit dose may be an amount sufficient for the preparation of an intravenous injection. It will be evident to those of ordinary skill in the art that compounds which are light and / or air sensitive may require special packaging and / or formulation. For example, packaging may be used which is opaque to light, and / or sealed from contact with ambient air, and / or formulated with suitable coatings or excipients.

[0044] An “effective amount” of a CD inclusion complex delivery vehicle according to the invention includes a therapeutically effective amount or a prophylactically effective amount. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of a delivery vehicle may vary according to factors suchas the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount may also be one in which any toxic or detrimental effects of the delivery vehicle or active compound are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, a prophylactic dose is used in subjects prior to or at an earlier stage of disease, so that a prophylactically effective amount may be less than a therapeutically effective amount. For any particular subject, the timing and dose of treatments may be adjusted over time (e.g., timing may be daily, every other day, weekly, monthly) according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.

[0045] In select embodiments, the present invention provides a composition or medicament that includes one or more biologically active molecules, selected from biologically active compounds or a solvate, pharmaceutically acceptable salt, ester, amide, complex, chelate, stereoisomer, stereoisomeric mixture, geometric isomer, crystalline or amorphous form, metabolite, metabolic precursor or prodrug thereof, including isolated enantiomeric, diastereomeric and geometric isomers thereof, and mixtures thereof, in combination with a pharmaceutically acceptable carrier, diluent or excipient, and further provides a method for the manufacture of such a composition or medicament.

[0046] Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. The word "comprising" is used herein as an open-ended term, substantially equivalent to the phrase "including, but not limited to", and the word "comprises" has a corresponding meaning. As used herein, the singular forms"a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a thing" includes more than one such thing.

[0047] Citation of references herein is not an admission that such references are prior art to the present invention. Any priority document(s) and all publications, including but not limited to patents and patent applications, cited in this specification are incorporated herein by reference. All documents cited or referenced in herein cited documents, together with any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual publication were specifically and individually indicated to be incorporated by reference herein and as though fully set forth herein. The invention includes all embodiments and variations substantially as hereinbefore described and with reference to the examples and drawings.

[0048] In some embodiments, the invention excludes steps that involve medical or surgical treatment. EXAMPLE

[0049] An exemplary CD inclusion complex formulation is described below, with a clinical example evidencing amelioration of arterial plaque in a human subject.

[0050] Borneol and trans cinnamaldehyde (TCA) are formulated together in a borneol / TCA formulation as follows. Borneol, mw 154 g / mol, in screened powder form is melted into TCA liquid, mw 132 g / mol, at low heat in a 1:1 wt ratio. Dry beta CD, mw 1,134 g / mol, is suspended in an aqueous slurry in an amount that is approximately 10x the amount of each of borneol and TCA by weight. The borneol / TCA mixture is gradually added the beta CD slurry with mixing. The resulting borneol / TCA CD inclusion slurry is dried to provide a stock borneol / TCA CD powder that is approximately 10% by weight borneol and TCA.

[0051] Capsules were prepared from a nattokinase powder and the stock borneol / TCA CD powder (“BOR / TCA-POW”) as follows:440 mg 0 capsules Nattokinase Powder ..........................................105 mg BOR / TCA-POW .................................................250 mg E4M 20% / K100 M / 80% blend* .........................70 mg Amylase .............................................................5 mg Ca Laurate (flow agent) ....................................10 mg * E4M cellulose (hydroxypropylmethylcellulose) and K100 M (hypromellose, HPMC), to provide for sustained release, putatively a sustained release of 10-12 hours. Example 1

[0052] A male subject was treated with the 440 mg capsules prepared as described above, twice daily, for approximately 10 months. The subject had an over 5 year history of annual carotid artery ultrasound screening, with a consistent result of “mild” carotid artery plaque build up. Following the course of treatment, a color flow ultrasound showed no evidence of remaining plaque.

Claims

CLAIMS 1. A method of treating or preventing atheromatous plaque in a mammal, or treating or preventing a disease which can be treated or prevented by ameliorating atheromatous plaque in the mammal, or of decreasing atheromatous plaque in the mammal, comprising orally administering to the mammal an effective amount of a cyclodextrin (CD) inclusion complex formulation, comprising: a borneol CD inclusion complex; a trans- cinnamaldehyde (TCA) CD inclusion complex; a nattokinase; and, a cyclodextrin degrading enzyme.

2. The method of claim 1, wherein the borneol CD inclusion complex is a beta CD inclusion complex and the trans-cinnamaldehyde CD inclusion complex is a beta CD inclusion complex.

3. The method of claim 1 or 2, wherein the CD inclusion complex formulation is a single formulation comprising a combined borneol / TCA CD inclusion complex and the nattokinase, co-formulated with the cyclodextrin degrading enzyme.

4. The method of any one of claims 1-3, wherein the mammal is a human patient, and wherein the disease is atherosclerosis, coronary artery disease, carotid artery disease or peripheral artery disease.

5. The method of any one of claims 1-4, further comprising monitoring the mammal for arterial atheroma.

6. The method of any one of claims 1-5, wherein the CD inclusion complex formulation further comprises a time release agent.

7. The method of claim 6, wherein the time release agent comprises a hydroxypropylmethylcellulose and / or a hypromellose.

8. The method of claim 7, wherein the hydroxypropylmethylcellulose is E4M hydroxypropylmethylcellulose and / or the hypromellose is K100 M hypromellose.

9. The method of any one of claims 1-8, wherein the cyclodextrin degrading enzyme is an amylase, a cyclodextrinase, a microbial cyclodextrinase, a maltogenic amylase or neopullulanase.

10. The method according to claim 9, wherein the amylase is a mammalian salivary amylase, a mammalian pancreatic amylase or a microbial amylase.

11. The method of any one of claims 1-10, wherein CD inclusion complex formulation further comprises a pharmaceutically acceptable carrier.

12. The method of claim 11, wherein the pharmaceutically acceptable carrier is calcium laurate.

13. The method of any one of claims 1-12, wherein the CD-degrading enzyme is formulated so that a cyclodextrin-degrading activity of the CD-degrading enzyme is activated on delivery of the CD inclusion complex formulation to a target tissue so as to release borneol and TCA from the borneol / TCA CD inclusion complex; and wherein the target tissue is a gastrointestinal (GI) tract of the mammal.

14. The method of any one of claim 1-13, wherein the mammal is a human.

15. The method of any one of claims 1-14, wherein the CD inclusion complex formulation is formulated for sustained release of the nattokinase and of the borneol and TCA from the borneol / TCA CD inclusion complex.

16. A cyclodextrin (CD) inclusion complex formulation, comprising: nattokinase, a borneol CD inclusion complex, and a trans-cinnamaldehyde CD inclusion complex; co- formulated with a cyclodextrin degrading enzyme.

17. The CD inclusion complex formulation of claim 16, wherein the borneol CDinclusion complex is a beta CD inclusion complex, and the trans-cinnamaldehyde CD inclusion complex is a beta CD inclusion complex; optionally wherein the borneol CD inclusion complex and the trans-cinnamaldehyde CD inclusion complex are provided in the form of a combined borneol / TCA CD inclusion complex.

18. The CD inclusion complex formulation of claims 16 or 17, for use for treating a disease that is: atherosclerosis, coronary artery disease, carotid artery disease or peripheral artery disease.

19. The CD inclusion complex formulation of any one of claims 16-18, wherein the CD inclusion complex formulation further comprises a time release agent.

20. The CD inclusion complex formulation of claim 19, wherein the time release agent comprises a hydroxypropylmethylcellulose and / or a hypromellose.

21. The CD inclusion complex formulation of claim 20, wherein the hydroxypropylmethylcellulose is E4M hydroxypropylmethylcellulose and / or the hypromellose is K100 M hypromellose.

22. The CD inclusion complex formulation of any one of claims 16-21, wherein the cyclodextrin degrading enzyme is an amylase, a cyclodextrinase, a microbial cyclodextrinase, a maltogenic amylase or neopullulanase.

23. The CD inclusion complex formulation of claim 22, wherein the amylase is a mammalian salivary amylase, a mammalian pancreatic amylase or a microbial amylase.

24. The CD inclusion complex formulation of any one of claims 16-23, wherein CD inclusion complex formulation further comprises a pharmaceutically acceptable carrier.

25. The CD inclusion complex formulation of claim 24, wherein the pharmaceutically acceptable carrier is calcium laurate.

26. The CD inclusion complex formulation of any one of claims 16-25, wherein the CD- degrading enzyme is formulated so that a cyclodextrin-degrading activity of the CD- degrading enzyme is activated on delivery of the CD inclusion complex formulation to a target tissue so as to: release borneol from the borneol CD inclusion complex; and release trans-cinnamaldehyde from the trans-cinnamaldehyde CD inclusion complex; and wherein the target tissue is a mammalian gastrointestinal (GI) tract.

27. The CD inclusion complex formulation of any one of claim 16-26, for use in treating a human.

28. The CD inclusion complex formulation of any one of claims 16-27, wherein the CD inclusion complex formulation is formulated for sustained release of: the nattokinase; the borneol from the borneol CD inclusion complex; and trans-cinnamaldehyde from the trans-cinnamaldehyde CD inclusion complex.