Anandamide cyclodextrin inclusion complex vehicle

Abstract

The oral cyclodextrin inclusion complex formulation of the present invention comprises an anandamide cyclodextrin inclusion complex and an enzyme having cyclodextrin degrading activity capable of digesting cyclodextrin, and upon delivery of the formulation to a target tissue, the enzyme is activated and anandamide is released from the cyclodextrin cavity. In an alternative embodiment, the cyclodextrin inclusion complex formulation is a time release formulation and treats or prevents nitric oxide deficiency or a disease that can be treated or prevented by increasing endogenous nitric oxide levels in a mammal.

Description

[Technical field]

[0001] The present invention relates to the field of biochemical constructs for the delivery of lipid based cannabinoid receptor agonists as inclusions within cyclodextrins in formulations that contain enzymes with cyclodextrin degrading activity. [Background technology]

[0002] Cyclodextrins are non-reducing cyclic glucose oligosaccharides, often the products of starch degradation catalyzed by cyclomaltodextrin glucanotransferase (EC 2.4.1.19; CGTase). Cyclodextrins can have a variety of structures (see Saenger et al., Chem. Rev. 98 (1998) 1787-1802), including three common cyclodextrins (α-, β-, and γ-cyclodextrin, respectively) with six, seven, or eight D-glucopyranosyl residues linked in the ring by α-1,4 glycosidic bonds. The truncated cone shape of cyclodextrins forms a cavity or lumen, the diameter of which varies with the number of glucose units. The dimensions of selected cyclodextrin (CD) structures are shown in Table 1. Larger cyclodextrins such as cyclomaltononaose (δ-CD) and cyclomaltodecaose (ε-CD) are also possible, as are various cyclodextrin-based supramolecular structures (see Zhang and Ma, Adv Drug Deliv Rev. 2013 Aug;65(9):1215-33).

[0003] [Table 1]

[0004] Cyclodextrins are generally amphiphilic, with the wider edge of the cavity exhibiting 2-OH and 3-OH groups, and the narrower edge exhibiting 6-OH groups. These hydrophilic hydroxyl groups are therefore on the outside of the cavity, while the inner surface is generally hydrophobic and lined with anomeric oxygen atoms and C3-H and C5-H hydrogen atoms. In aqueous solution, this hydrophobic cavity may contain water molecules, e.g., about three (α-CD), seven (β-CD) or nine (γ-CD) water molecules, which have low retention but are relatively easily displaceable due to their low entropy. Thus, cyclodextrins that would otherwise be hydrophilic may bind and retain one or more molecules of suitable size within or partially within the CD cavity to form cyclodextrin inclusion complexes or complexes. For example, non-polar aliphatic and aromatic compounds, including drugs such as lipophilic drugs, may be bound to increase the water solubility of normally hydrophobic compounds or to minimize undesirable properties such as odor or taste of 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 are used in a variety of sustained release drug preparations, for example for inclusion complexes of medicinal compounds with hydrophobic cyclodextrin derivatives (U.S. Pat. No. 4,869,904).

[0005] Cyclodextrins can be chemically modified in various ways, for example, to change the inclusion specificity, physical and chemical properties of cyclodextrins. The hydroxyl groups of CDs can be derivatized, for example. For example, two modified CDs are used in many 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. Further CD derivatives include sugammadex or Org-25969, in which the 6-hydroxy group of γ-CD is replaced with a carboxythioacetate ether bond, and hydroxybutenyl-β-CD. Alternative forms of cyclodextrin include 2,6-di-O-methyl-β-CD (DIMEB), 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), randomly methylated-β-cyclodextrin (RAMEB), sulfobutylether-β-cyclodextrin (SBE-β-CD), and sulfobutylether-γ-cyclodextrin (SBEγCD), sulfobutylated β-cyclodextrin sodium salt, sulfobutylated β-cyclodextrin sodium salt, (2-hydroxypropyl )-α-cyclodextrin, (2-hydroxypropyl)-β-cyclodextrin, (2-hydroxypropyl)-γ-cyclodextrin, DIMEB-50 heptakis(2,6-di-O-methyl)-β-cyclodextrin, TRIMEB heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin, methyl-β-cyclodextrin, octakis(6-deoxy-6-iodo)-γ-cyclodextrin, and octakis(6-deoxy-6-bromo)-γ-cyclodextrin. Although such CDs have been developed to have favorable pharmacological and toxicological profiles, following administration, and especially after parenteral administration, residual CDs may perturb the pharmacokinetic properties of drugs, including coadministered drugs (see Stella and He, Toxicol Pathol January 2008 vol. 36 no. 1 30-42).

[0006] Cyclodextrins are susceptible to enzymatic digestion in various ways. For example, γ-CD is relatively easily hydrolyzed by α-amylase, whereas α-cyclodextrin is resistant to hydrolysis. CD-based therapeutics generally rely on the activity of endogenous amylase to digest CD. However, amylase activity varies widely from patient to patient. For example, patients with pancreatic insufficiency, cystic fibrosis, celiac disease, or Crohn's disease may lack normal amounts of amylase. Similarly, patients, especially elderly patients, may lack gastric acid production and therefore be unable to create the low pH conditions in the duodenum required to adequately trigger the release of pancreatic amylase. Similar effects may result from the general increased use of antacids, histamine 2 blockers, proton pump inhibitors, or alternative acid blockers.

[0007] Various microbial cyclodextrin digestive 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), and neopullulanase (EC 3.2.1.135). These have been reported to have hydrolytic activity against CD and possibly against additional substrates such as pullulan and starch. Cyclodextrinase (CDase) can catalyze the hydrolysis of CD to form linear α-1,4-linked oligosaccharides, thereby releasing the material from the CD inclusion complex. CDase from Bacillus macerans was reported in 1968, and since then, CDases from many bacteria have been characterized, including enzymes from Bacillus spp., Thermoanaerobacter ethanolicus strain 39E, Flavobacterium spp., and Klebsiella oxytoca strain M5a1. Archaeal CDases from Archaeoglobus fulgidus, Thermococcus sp. B1001, Thermococcus sp. CL1, Thermophilum pendens, and Pyrococcus furiosus have been characterized, and the structure of a CDase from Flavobacterium sp. has been characterized (see Sun et al., Archaea, Volume 2015 (2015), Article ID 397924, which reports the identification of a gene encoding a cyclodextrinase from Thermococcus kodakaraensis KOD1).

[0008] Anandamide is a lipid cannabinoid receptor ligand and was the first compound identified as an endocannabinoid (also known as (5Z,8Z,11Z,14Z)-N-(2-hydroxyethyl)icosa-5,8,11,14-tetraenamide, N-arachidonoylethanolamine, or arachidonoylethanolamide). Anandamide analogues in cyclodextrin inclusion complexes have been described as useful for the treatment of intraocular hypertension (WO 1996 / 001558 A1). Anandamide is a CB 1 It is a partial agonist of the CB receptor. 2It is a weak partial agonist of the vanilloid receptor VR1 (also known as transient receptor potential cation channel subfamily V member 1, or TrpV1) and a partial agonist of the GPR 55 (Reggio PH. Endocannabinoid binding to the cannabinoid receptors: what is known and what remains unknown. Curr Med Chem. 2010;17(14):1468-1486; Roberts LA, Christie MJ, Connor M. Anandamide is a partial agonist at native vanilloid receptors in acutely isolated mouse trigeminal sensory neurons. Br J Pharmacol. 2002 Oct;137(4):421-8). CB 1 and C.B. 2 The binding affinity of anandamide and its analogues to the CB 1 It has been shown that CB 1 K for i is less than 100 nM, and CB 2 K for i exceeds 1000 nMK (Lin, S., Khanolkar, AD, Fan, P., Goutopoulos, A., Qin, C., Papahadjis, D., & Makriyannis, A. (1998). Novel Analogues of Arachidonylethanolamide (Anandamide): Affinities for the CB1 and CB2 Cannabinoid Receptors and Metabolic Stability. Journal of Medicinal Chemistry, 41(27), 5353-5361).

[0009] Nitric oxide free radicals are involved in a wide range of physiological signaling functions, and we now know that there is a complex relationship between the endocannabinoid system and nitrergic signaling (Christopher Lipina, Harinder S. Hundal, The endocannabinoid system: 'NO' longer anonymous in the control of nitrergic signalling?, Journal of Molecular Cell Biology, Volume 9, Issue 2, April 2017, Pages 91-103). Nitric oxide is physiologically synthesized by the nitric oxide synthase family, which converts L-arginine to L-citrulline and nitric oxide, which is then recycled to provide L-arginine. The fractional NO concentration in exhaled breath (FE NO ) measurement has been used as a quantitative, non-invasive method to measure airway inflammation. For example, methods and devices have been described that measure NO in body fluids by measuring a salivary nitric oxide test object containing nitrite as a precursor and biomarker of nitric oxide, with the salivary nitric oxide test object concentration range detectable at 25->400 umol / L nitrite (e.g., using visually distinguishable colorimetric subranges: 0-25, 25-100, 100-200, 200-350, and >400 umol / L nitrite) (U.S. Patent No. 9,759,716). NO concentrations in body fluids have been suggested to be useful as an indicator of physiological anandamide levels (U.S. Patent Application Publication No. 20190265258). A relatively wide range of perivascular NO concentrations has been reported under control conditions, e.g., values ​​ranging from approximately 200 to 1,000 nM (Chen K, Pittman RN, Popel AS. Nitric oxide in the vasculature: where does it come from and where does it go? A quantitative perspective. Antioxid Redox Signal. 2008;10(7):1185-1198. doi:10.1089 / ars.2007.1959).

[0010] Citrulline has been described as useful in treating a variety of conditions associated with NO deficiency (US Patent Publication No. 20010056068). Similarly, L-arginine and other nitric oxide donors have been described as useful in treating conditions involving NO signaling (EP 0441119A2, US 5595970 and US 5508045). [Prior art documents] [Patent documents]

[0011] [Patent Document 1] U.S. Patent No. 4,869,904 [Patent Document 2] International Application Publication No. 1996 / 001558A1 [Patent Document 3] U.S. Patent No. 9,759,716 [Patent Document 4] U.S. Patent Application Publication No. 20190265258 [Patent Document 5] US Patent Application Publication No. 20010056068 [Patent Document 6] European Patent No. 0441119A2 [Patent Document 7] U.S. Patent No. 5,595,970 [Patent Document 8] U.S. Patent No. 5,508,045 [Patent Document 9] U.S. Patent No. 4,538,920 [Patent Document 10] U.S. Patent No. 8,100,295 [Patent Document 11] U.S. Patent No. 8,308,340 [Patent Document 12] U.S. Patent No. 8,875,947 [Patent Document 13] U.S. Patent No. 8,499,976 [Patent Document 14] International Application Publication No. 2007041266 [Patent Document 15] International Application Publication No. 2000021842 [Patent Document 16] U.S. Patent Application Publication No. 20090029020 [Patent Document 17] U.S. Patent Application Publication No. 20090214446 [Patent Document 18] U.S. Patent No. 5,070,081 [Patent Document 19] U.S. Patent No. 5,552,378 [Patent Document 20] U.S. Patent No. 5,674,854 [Patent Document 21] U.S. Patent No. 8,658,692 [Patent Document 22] US Patent Application Publication No. 20140094433 [Patent Document 23] US Patent Application Publication No. 20130022652 [Non-patent literature]

[0012] [Non-Patent Document 1] Chaudhary & Patel, IJPSR, 2013, Vol. 4(1): 68-76 [Non-Patent Document 2] Carneiro et al., 2019, Int. J. Mol. Sci. 2019, 20, 642 [Non-Patent Document 3] Sharma et al., Pharm Dev Technol. 2013 May-Jun;18(3):560-9 [Non-Patent Document 4] “Remington's Pharmaceutical Sciences” (20th edition), ed. A. Gennaro, 2000, Mack Publishing Company, Easton, PA. DISCLOSURE OF THEINVENTION

[0013] Cyclodextrin inclusion complex delivery vehicles are provided with anandamide or anandamide analog as guest molecule.These formulations are provided for use in increasing endogenous nitric oxide levels, and thus for treating conditions where increasing endogenous NO levels has therapeutic or prophylactic benefits, such as conditions associated with NO deficiency, conditions characterized by anxiety, or erectile dysfunction.The formulations can be used, for example, to increase the measurable NO levels of a subject, or to provide erectile effects to male subjects.In some formulations, these effects may be sustained, for example, to achieve sustained NO levels.In addition to anandamide and anandamide analogs, the formulations may contain additional active agents, such as NO generators including citrulline and / or arginine, optionally in the form of inclusion complex guest molecules.

[0014] The cyclodextrin inclusion complex may comprise a biologically acceptable carrier, whereby the guest molecule is included within the carrier and stably held by the cyclodextrin. The vehicle may also contain an enzyme with cyclodextrin degrading activity capable of digesting the cyclodextrin holding the guest molecule. The enzyme may be formulated such that upon delivery of the vehicle to a target, the cyclodextrin degrading activity is activated and the guest molecule is released from the cavity of the cyclodextrin.

[0015] In alternative embodiments of the delivery vehicle, the enzyme may be co-formulated with a cyclodextrin inclusion complex, or the enzyme may be included in the delivery vehicle together with a cyclodextrin inclusion complex. When the enzyme is included in the delivery vehicle together with a cyclodextrin inclusion complex, the delivery vehicle may further include a biochemically acceptable carrier for the enzyme.

[0016] The enzyme may be, for example, an amylase, a cyclodextrinase, a maltogenic amylase, or a neopullulanase. The amylase may be, for example, a mammalian salivary amylase, a pancreatic amylase, or an amylase of fungal or bacterial origin. The cyclodextrinase may be, for example, a microbial cyclodextrinase.

[0017] The cyclodextrin may be a CD derivative, such as, for example, a hydrophobic alkylated cyclodextrin or a mixed methylated / ethylated cyclodextrin.

[0018] The ratio of cyclodextrin to guest molecule may be, for example, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, or 1:5, although a wide range of alternatives are possible, including non-integer ratios.

[0019] The cyclodextrin may be, for example, α, β or γ-cyclodextrin, but again, a wide range of alternative CD structures can be used. The biologically acceptable carrier may be a pharma- ceutical acceptable carrier. The delivery vehicle may be formulated to provide sustained release of the guest molecule and / or other active agent. Thus, the present invention provides alternative embodiments in which the CD delivery vehicle can be formulated and used as a medicament.

[0020] Methods are provided for treating patients having nitric oxide deficiency or disorders that can be treated or prevented by increasing endogenous nitric oxide levels in a mammalian subject. Also provided are methods for providing erectile benefits in male subjects, such as men over the age of 50, 60, or 70, or male subjects with symptoms of erectile dysfunction. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Anandamide has the following structure:

[0022] [ka]

[0023] A variety of anandamide analogs are known, including various analogs having the structure of formula (I).

[0024] [ka]

[0025] During the ceremony, x is an integer from 1 to 6; y is an integer from 1 to 6; z is an integer from 1 to 6; R 1 and R 2 are each independently H, an alkyl group having 1 to 6 carbon atoms, or (CH 2 ) w -R 3 is selected from the group consisting of w is an integer from 0 to 6; R 3 is CH 3 , OH, SH, F, Cl, Br, I, C≡CH, C≡N, a carbocycle having 3 to 7 carbons, and a heterocycle having 3 to 7 carbons and at least one heteroatom selected from N, O, and S; R 1 and / or R 2 may be bonded to N' or O' to form a heterocycle having 3 to 7 atoms.

[0026] Anandamide analogs for use in the formulations of the invention may be characterized by receptor binding activity, e.g., CB 1 Partial agonist of the receptor, CB 2 Weak partial agonist of the receptor, partial agonist of the vanilloid receptor VR1 and / or GPR 55 It is an agonist ligand for the CB receptor. 1 and C.B. 2 The binding affinity of anandamide analogues to the CB 1 It may be characterized by a preferential affinity for binding, e.g., CB 1K for i is less than 100 nM, and CB 2 K for i exceeds 1000nM.

[0027] The formulations of the present invention may be used to increase physiological NO levels, for example, to treat or prevent nitric oxide deficiency or diseases that can be treated or prevented by increasing endogenous nitric oxide levels in mammals. Nitric oxide deficiency is believed to contribute to the pathogenesis of cardiovascular disease, including hypertension and cardiovascular disease (e.g., atherosclerosis, restenosis). In human female patients, NO deficiency is believed to be involved in pathologies such as preeclampsia, premature labor, cervical incompetence, recurrent miscarriage, dysmenorrhea, infertility, hot flashes, cardiovascular disease, urinary incontinence, and cognitive impairment. Similarly, NO deficiency is involved in cardiovascular disease, hypertension, impotence, and osteoporosis, especially in older men. Thus, provided herein are treatments for nitric oxide-related disorders and diseases, such as, for example, hypertension, cardiovascular disease (e.g., atherosclerosis, restenosis), osteoporosis, pre-eclampsia, preterm labor, dysmenorrhea, cervical dystonia, urinary incontinence, male impotence, female infertility, and the like.

[0028] Selected embodiments of the formulations of the invention include citrulline or a citrulline analog, such as, for example, D,L-citrulline, L-citrulline, L-citrulline monoacetate, L-citrulline hydrochloride, L-citrulline methyl ester, L-citrulline ethyl ester, L-citrulline-n-hexyl ester, L-citrulline (benzoylmethyl) ester, α-N-benzoyl-L-citrulline methyl ester, N-Boc-L-citrulline, or N1-2,4-dinitrophenyl-D,L-citrulline. In alternative embodiments, the citrulline analog may have the structure of formula (II):

[0029] [ka]

[0030] During the ceremony, R 1 is hydrogen, alkyl having 1 to 10 carbon atoms, alkenyl having 1 to 10 carbon atoms, aryl, -CH 2 ) 1-3 (C=O)aryl, ω-hydroxyalkyl, or ω-methoxyalkyl; R 2 and R 3 are each independently selected from hydrogen, alkyl having 1 to 10 carbon atoms, aryl, acetyl, benzoyl, and tert-butoxycarbonyl.

[0031] Specific examples of formulations include cyclodextrin inclusion complex formulations prepared by co-formulating anandamide or an analogue thereof as a cyclodextrin inclusion in combination with a CD degrading enzyme.

[0032] In selected embodiments, the enzyme contained in the vehicle may be formulated such that upon delivery of the vehicle to the target, the cyclodextrin degrading activity is activated, releasing the guest molecule from the cyclodextrin cavity. Activation of the enzyme may be achieved in the drug, e.g., in a dry form, such as an oral capsule or tablet, in which the enzyme is mixed, or may remain inactive until activated by moisture in the host's gastrointestinal tract. Similarly, a wide range of time-release matrices and formulations are known and may be adapted for use in the CD delivery vehicle to tailor the appropriate activation of the CD degrading enzyme upon delivery to the target.

[0033] In various embodiments, the CD delivery vehicle may include the enzyme co-formulated in the cyclodextrin inclusion complex, as described above, or the enzyme may be included in the delivery vehicle together with the cyclodextrin inclusion complex. If included together, the delivery vehicle may include, for example, a biochemically acceptable carrier for the enzyme, separate from the carrier for the CD inclusion complex. For example, the delivery vehicle may include separate compartments for the CD inclusion complex and the CD degrading enzyme, such that the delivery vehicle is composed of a compartment for the CD inclusion complex and a compartment for the CD degrading enzyme linked thereto. A mechanism may be provided for the combined release of the CD inclusion complex and the CD degrading enzyme from their respective compartments in the delivery vehicle. For example, the delivery vehicle may be a syringe with such separate compartments, ejected by a common ejection mechanism, such as a mechanism for discharging aliquots of the CD inclusion complex and the CD degrading enzyme by moving pistons in each compartment in concert, thereby mixing the enzyme and the inclusion complex and activating the release of the guest molecule from the CD by the enzyme. This type of vehicle can be used, for example, to dispense topical creams or other surfactant formulations. A wide variety of delivery vehicles of this type can be adapted from devices known for dispensing two-part compositions such as epoxy resins, two-part pharmaceuticals or dental preparations, for example, as described in U.S. Pat. No. 4,538,920, U.S. Pat. No. 8,100,295, U.S. Pat. No. 8,308,340, U.S. Pat. No. 8,875,947, U.S. Pat. No. 8,499,976, and WO 2007041266 and WO 2000021842.

[0034] There are a wide variety of techniques available for preparing CD inclusion complexes, such as those described in Chaudhary & Patel, IJPSR, 2013, Vol. 4(1): 68-76; Carneiro et al., 2019, Int. J. Mol. Sci. 2019, 20, 642, US Patent Application Publication No. 20090029020, US Patent Application Publication No. 20090214446, US Patent No. 5,070,081, US Patent No. 5,552,378, US Patent No. 5,674,854, and US Patent No. 8,658,692. A common technique is known as the kneading method, in which CDs are mixed with water or aqueous alcohol to form a paste. The bioactive molecule may then be added to this paste and kneaded for a certain period of time. The kneaded mixture may then be dried and, if necessary, passed through a sieve. Alternatively, the slurry method involves mixing the bioactive molecule with cyclodextrin, adding an appropriate amount of water to the mixture, typically with vigorous mixing, until a paste or slurry is formed, continuing mixing for an appropriate period of time, e.g., 15 minutes, while adding more water if necessary to maintain the consistency of the paste or slurry, to form the inclusion complex, and drying the final product. Other ingredients, such as emulsifiers, can facilitate the formation of the inclusion complex, for example, a process involving dry mixing the cyclodextrin with an emulsifier (e.g., pectin), mixing and stirring the dry mixture of cyclodextrin and emulsifier with a solvent, e.g., water, in a reactor, adding the guest molecule and stirring (e.g., for about 5-8 hours), optionally cooling the reaction mixture with stirring, and emulsifying the mixture and then drying the cyclodextrin inclusion complex to form a powder. Other known techniques for preparing CD inclusions include freeze-drying, microwave irradiation and supercritical fluid antisolvent techniques.

[0035] The CD delivery vehicles of the present invention can be provided alone or in combination with other compounds (e.g., nucleic acid molecules, small molecules, peptides or peptide analogs) in the presence of a carrier, e.g., liposomes, adjuvants, or any pharma- ceutically or biologically acceptable carrier. Selected embodiments include the agent in a form suitable for administration to an animal host, such as a mammal, e.g., a human. As used herein, a "pharma-ceutically acceptable carrier" or "excipient" includes any and all solvents, dispersion media, coatings, antibacterial agents, antifungal agents, and absorption delaying agents, etc., that are physiologically compatible. The carrier may be compatible in any suitable mode of administration, including topical, subcutaneous, intradermal, intravenous, parenteral, intraperitoneal, intramuscular, sublingual, inhalation, intratumoral, or oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable aqueous solutions or dispersions. The use of such media and agents for pharma-ceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the biologically active compound, its use in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0036] Conventional pharmaceutical practice may be employed to provide a formulation or composition suitable for administration of the delivery vehicle to a subject. Suitable routes of administration may be employed, such as parenteral, intravenous, intradermal, subcutaneous, intramuscular, intracranial, intraorbital, ocular, intraventricular, intracapsular, intraspinal, intrathecal, intracapsular, intraperitoneal, intranasal, inhalation, aerosol, topical, intratumoral, sublingual or oral administration. Therapeutic formulations may be in the form of liquid solutions or suspensions, for oral administration the formulations may be in the form of tablets or capsules, for intranasal formulations in the form of powders, nose drops or aerosols, and for sublingual formulations in the form of drops, aerosols or tablets.

[0037] Cyclodextrin degrading or digestive enzymes may be formulated, for example, for oral delivery, as enteric enzyme formulations, such as submicron particle formulations prepared by emulsion solvent evaporation (Sharma et al., Pharm Dev Technol. 2013 May-Jun;18(3):560-9), etc. Similarly, the delivery vehicle may be formulated as a hydrogel (see US Patent Publication No. 20140094433), or as a medicated gum (see US Patent Publication No. 20130022652).

[0038] Methods known in the art for making formulations are described, for example, in "Remington's Pharmaceutical Sciences" (20th edition), ed. A. Gennaro, 2000, Mack Publishing Company, Easton, PA. Formulations for parenteral administration may contain, for example, excipients, sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes. Biocompatible, biodegradable lactide polymers, lactide / glycolide copolymers, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, such as lactose, or may be aqueous solutions, such as polyoxyethylene-9-lauryl ether, glycocholic acid, or deoxycholic acid, or may be oily solutions for administration in the form of nose drops or as a gel.

[0039] The pharmaceutical composition of the present invention may be in any form that allows 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, but are not limited to, oral, topical, parenteral, sublingual, rectal, vaginal, and intranasal. The term parenteral as used herein includes subcutaneous injection, intravenous, intramuscular, epidural, intrasternal injection, or infusion techniques. The pharmaceutical composition of the present invention is formulated so that the active ingredient contained therein is bioavailable upon administration of the composition to a patient. The composition administered to a patient may take the form of one or more dosage units, for example, a single tablet, capsule, or cachet may be a single dosage unit, and a container of the compound in aerosol form may hold multiple dosage units.

[0040] Materials used in the preparation of pharmaceutical compositions should be pharma- ceutical pure and non-toxic in the amounts used. The compositions of the present invention may contain one or more compounds (active ingredients) that are known to have a particular desired effect. It will be clear to those skilled in the art that the optimal dose of the active ingredient in a pharmaceutical composition depends on a variety of factors. Relevant factors include, but are not limited to, the type of subject (e.g., human), the particular form of the active ingredient, the method of administration, and the composition used.

[0041] Generally, pharmaceutical compositions comprise the delivery vehicle of the present invention described herein mixed with one or more carriers.Carriers can be particulate, in which case compositions are in the form of, for example, tablets or powders.Carriers can be liquid, in which case compositions are in the form of, for example, oral syrups or injectable liquids.Furthermore, carriers can be gaseous, and can provide aerosol compositions that are useful, for example, for inhalation administration.

[0042] When intended for oral administration, the compositions are preferably in solid or liquid form, which forms considered herein as solid or liquid include semi-solid, semi-liquid, suspension and gel forms.

[0043] As a solid composition for oral administration, the composition may be formulated into a form of a powder, granules, compressed tablets, pills, capsules, cachets, chewing gum, wafers, lozenges, etc. Such solid compositions will typically include one or more inert diluents or edible carriers. Additionally, one or more of the following adjuvants may be included: binders such as syrup, acacia, sorbitol, polyvinylpyrrolidone, carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, tragacanth or gelatin, and mixtures thereof; excipients such as starch, lactose or dextrin; 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, mannitol, 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, and the like; wetting agents such as sodium lauryl sulfate; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; flavoring agents such as peppermint, methyl salicylate, or orange flavor; and coloring agents.

[0044] When the composition is in the form of a capsule, for example, a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or fatty oil.

[0045] The compositions may be in the form of liquids, such as elixirs, syrups, solutions, aqueous or oily emulsions or suspensions, or dry powders that can be reconstituted with water and / or other liquid media before use. The liquids may be for oral administration or for delivery by injection, to name just two examples. When intended for oral administration, preferred compositions contain, in addition to the compounds of the invention, one or more of sweeteners, thickeners, preservatives (e.g., alkyl p-hydroxybenzoates), dyes / colorants, and flavor enhancers (flavorings). Compositions intended for administration by injection may contain one or more of surfactants, preservatives (e.g., alkyl p-hydroxybenzoates), wetting agents, dispersing agents, suspending agents (e.g., sorbitol, glucose or other sugar syrups), buffers, stabilizers, and isotonic agents. The emulsifier may be selected from lecithin or sorbitol monooleate.

[0046] Liquid pharmaceutical compositions of the present invention, whether in solution, suspension or other similar form, may contain one or more of the following adjuvants: sterile diluents such as water for injection, saline, preferably saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono- or diglycerides that can serve as solvents or suspending media, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and agents for adjusting isotonicity such as sodium chloride or dextrose. Parenteral preparations can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Saline is the preferred adjuvant. Pharmaceutical compositions for injection are preferably sterilized.

[0047] The pharmaceutical composition may be intended for topical administration, in which case the carrier may suitably consist of a solution, emulsion, ointment, cream or gel base. The base may, for example, comprise one or more of the following: petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, diluents such as water and alcohol, as well as emulsifiers and stabilizers. Thickening agents may be present in pharmaceutical compositions for topical administration. If intended for transdermal administration, the composition may comprise a transdermal patch or iontophoresis device. Topical formulations may contain a concentration of the biologically active compound of about 0.1 to about 25% w / v (weight per unit volume).

[0048] The composition may be for rectal administration, for example in the form of a suppository that melts in the rectum and releases the drug. The composition for rectal administration may contain an oily base as a suitable non-irritating excipient. Such bases include, but are not limited to, lanolin, cocoa butter and polyethylene glycol. Low melting waxes are preferred for the preparation of suppositories, and mixtures of fatty acid glycerides and / or cocoa butter are suitable waxes. The wax may be melted and stirred to disperse the aminocyclohexyl ether compound homogeneously therein. The molten homogeneous mixture is then poured into suitable sized molds, allowed to cool, and thereby solidify.

[0049] The composition may contain various materials that modify the physical form of solid or liquid dosage units.For example, the composition may contain materials that form a coating shell around the active ingredient.The materials that form the coating shell are typically inert and may be selected from, for example, sugar, shellac and other enteric coating agents.Alternatively, the active ingredient may be enclosed in a gelatin capsule or cachet.

[0050] The pharmaceutical composition of the present invention may consist of a gaseous dosage unit, for example, in the form of an aerosol. The term aerosol is used to refer to a variety of systems, from colloidal to systems consisting of pressurized packages. Delivery may be by liquefied or compressed gas, or by a suitable pump system to dispense the active ingredient. Aerosols of the compounds of the present invention may be delivered in single-phase, two-phase, or three-phase systems to deliver the active ingredient. Aerosol delivery includes the necessary containers, activators, valves, subcontainers, etc., which may be combined to form a kit.

[0051] The biologically active compounds may be in the form of a free base or in the form of a pharma- ceutically acceptable salt, such as hydrochloride, sulfate, phosphate, citrate, fumarate, methanesulfonate, acetate, tartrate, maleate, lactate, mandelate, salicylate, succinate, and other salts known in the art. An appropriate salt will be selected to enhance the bioavailability or stability of the compound in the appropriate mode of use (e.g., oral or parenteral administration route).

[0052] Compositions intended for administration by injection can be prepared by combining the delivery vehicle of the present invention with water and preferably a buffer 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. A surfactant is a compound that non-covalently interacts with the aminocyclohexyl ether compound to facilitate dissolution or homogeneous suspension of the aminocyclohexyl ether compound in the aqueous delivery system. Since the aminocyclohexyl ether compound according to the present invention may be hydrophobic, a surfactant is desirably present in the aqueous composition of the present invention. Other carriers for injection include, but are not limited to, sterile peroxide-free ethyl oleate, dehydrated alcohol, propylene glycol, and mixtures thereof.

[0053] Suitable pharmaceutical adjuvants for injection solutions include stabilizers, solubilizers, buffers and viscosity adjusters. Examples of these adjuvants include, for example, ethanol, ethylenediaminetetraacetic acid (EDTA), tartrate buffer, citrate buffer, and high molecular weight polyethylene oxide viscosity adjuster. These pharmaceutical preparations may be injected intramuscularly, epidurally, intraperitoneally, or intravenously.

[0054] The present invention also provides a kit with a pharmaceutical composition comprising one or more delivery vehicles. The kit also includes instructions for use of the pharmaceutical. Preferably, the commercial package includes one or more unit doses of the pharmaceutical composition. For example, such a unit dose may be sufficient for preparation of an intravenous injection. It will be clear to those skilled in the art that light-sensitive and / or air-sensitive compounds may require special packaging and / or formulation. For example, the packaging may be opaque to light and / or sealed from contact with the atmosphere and / or formulated with suitable coatings or excipients.

[0055] An "effective amount" of a CD inclusion complex delivery vehicle according to the present invention includes a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" refers to an amount effective at a dosage and for a period of time necessary to achieve a desired therapeutic result. The therapeutically effective amount of a delivery vehicle will vary according to factors such as the individual's medical condition, age, sex, and weight, and the ability of the compound to elicit a desired response in the individual. Dosage regimens may be adjusted to obtain an optimal therapeutic response. A therapeutically effective amount may also be one in which any toxic or adverse 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 a dosage and for a period of time necessary to achieve a desired prophylactic result. Typically, a prophylactic dose is used in subjects prior to or at an early stage of disease onset, such that the prophylactically effective amount will be less than the therapeutically effective amount. For a particular subject, the timing and dosage of treatment may be adjusted over time (e.g., timing may be daily, every other day, weekly, monthly) according to the needs of the individual and the professional judgment of the person administering or supervising the administration of the composition.

[0056] In selected embodiments, the present invention provides compositions or medicaments comprising one or more biologically active molecules selected from a biologically active compound or a solvate, a pharma- ceutically acceptable salt, an ester, an amide, a complex, a chelate, a stereoisomer, a mixture of stereoisomers, a geometric isomer, a crystalline or amorphous form, a metabolite, a metabolic precursor or a prodrug thereof (including isolated enantiomers, diastereoisomers and geometric isomers thereof), and mixtures thereof, in combination with a pharma- ceutically acceptable carrier, diluent or excipient, and further provides methods for the preparation of such compositions or medicaments.

[0057] Although various embodiments of the present invention are disclosed herein, many adaptations and modifications can be made within the scope of the present invention according to the common general knowledge of those skilled in the art. Such modifications include the substitution of known equivalents in any of the aspects of the present invention to achieve the same result in substantially the same way. Numeric ranges include the numerical values ​​that define the range. The term "comprising" is used herein as an open-ended term substantially equivalent to the phrase "including, but not limited to," and the term "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 two or more such things.

[0058] The citation of a reference herein is not an admission that such reference is prior art to the present invention. Priority documents and all publications, including but not limited to patents and patent applications, cited herein are incorporated herein by reference. All documents cited or referenced in the documents cited herein, as well as manufacturer's instructions, descriptions, product specifications and product sheets for products mentioned herein or in documents incorporated herein by reference, are hereby incorporated herein by reference and may be used in the practice of the present invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated herein by reference and to the same extent as if fully set forth herein. The present invention includes all embodiments and variations substantially as described above and with reference to the examples and drawings.

[0059] In some embodiments, the present invention excludes steps involving medical or surgical treatment. EXAMPLES

[0060] Exemplary formulations that have been tested in subjects are shown below.

[0061] Anandamide with a molecular weight of 347 was prepared and formulated in 1:2 gamma-cyclodextrin to give 11.5 wt% anandamide in 80% anandamide oil. The final concentration of anandamide was approximately 9.2 wt%. Alternative anandamide CD inclusion complex formulations were similarly prepared to give 7.7 wt% anandamide and 8.8 wt% anandamide.

[0062] Capsules were prepared from the anandamide inclusion complex formulation as follows. 42mg capsule Anandamide 9.2% CD inclusion complex 465mg, net anandamide 42mg E4M Cellulose 30mg (hydroxypropyl methylcellulose, delivers 1-2 hour release) Amylase 4mg Calcium laurate (flow agent) 5mg 10mg capsule Anandamide 7.7% CD inclusion complex 130mg E4M Cellulose 40mg Serine 335mg Amylase 5mg Sodium stearyl fumarate (SSF - flow agent) 2mg 5 mg capsule Anandamide 7.7% CD inclusion complex 65mg Serine 400mg Amylase 5mg 2 mg of SSF 25mg capsule Amylase-containing anandamide 7.7% CD inclusion complex 325mg E4M Cellulose 30mg Serine 230mg 2 mg of SSF

[0063] Example 1 A male subject was treated with one 42 mg capsule in the morning and one 10 mg capsule in the evening. The subject reported that this treatment completely neutralized stress and improved sleep. After a period of time, the subject continued with a dose of two 10 mg capsules in the morning and no evening treatment, resulting in continued stress reduction.

[0064] The subject then began taking two 25 mg capsules in the morning and experienced a remarkable increase in blood flow during erections. The subject had been taking sildenafil in various doses ranging from 10 to 25 mg for many years. In conjunction with the administration of the 25 mg Anandamide CD formulation, the subject realized that the 10 mg of sildenafil produced more pronounced results than the 25 mg dose taken prior to supplementation with Anandamide CD. The subject realized that the Anandamide CD formulation had a noticeable cumulative effect over time, improving blood flow with long-term treatment.

[0065] The subject then changed his dosage to one 25 mg capsule in the morning and one 10 mg capsule in the afternoon. In previous salivary NO testing using commercially available nitrite test strips (described in U.S. Patent No. 9,759,716), the subject had never reached above "Depleted" or "Low" on the NO color indicator strip. However, at this stage of anandamide CD treatment, the NO test strips were reading well above "Low" on the color scale, reaching color intensity above "Optimal."

[0066] Example 2 This example relates to the use of two alternative formulations enriched with arginine, a nitric oxide synthase substrate, and citrulline, which aids in the sustained production of arginine. 10mg sustained release anandamide (SR) Anandamide 8.5% CD inclusion complex containing amylase as a substrate 120mg K250 Cellulose 72mg (sustained release 10-12 hours) Citrulline 120mg (no amino acids) Arginine 100mg (no amino acids) Calcium laurate 5mg 10mg Rapid Release Anandamide (QR) Anandamide 8.5% CD inclusion complex containing amylase as a substrate 120mg E4M Cellulose 15mg (sustained release 10-12 hours) Citrulline 160mg (no amino acids) Arginine 120mg (no amino acids) Calcium laurate 5mg

[0067] The subject in Example 1 determined that the combination of these formulations allowed for controlled administration with bolus and / or extended release regimens. This combination therapy provided more convincing therapeutic results in terms of NO production and concentration, and erectile ability. The subject took 2 SR capsules and 1 QR capsule in the morning, 1 QR capsule in the afternoon, and 2 SR capsules at approximately 6:00 p.m., resulting in a painful erection after stimulation at approximately 9:30 p.m. that night.

[0068] Example 3 Subject EL self-administered a daily dose of 500 mg of the following formulation for approximately 4 months: 500mg capsule Anandamide 7.8% CD inclusion complex 160mg, net anandamide 12.5mg Citrulline 300mg E4M Cellulose 20mg (hydroxypropyl methylcellulose, delivers 1-2 hour release) Amylase 5mg Calcium laurate (flow agent) 15mg

[0069] Subject EL experienced a significant reduction in anxiety and stress, as well as much improved sleep and improved VO 2 The subjects reported a 2 point increase in Max (measured by Garmin device), a significant increase in lung capacity, a decrease in heart rate at high intensity, and improved running performance (estimated 10-15% improvement). This example demonstrates that the use of a cyclodextrin (CD) inclusion complex formulation improves cardiovascular function in human subjects.

Claims

A cyclodextrin (CD) inclusion complex preparation for use in the treatment of erectile dysfunction in humans, comprising: An inclusion complex preparation of CD, which contains anandamide as a guest molecule in the CD and a co-formulated cyclodextrin-degrading enzyme, and further contains an effective amount of citrulline or a citrulline analog. **Claim 2** The CD inclusion complex preparation according to claim 1, further comprising a sustained-release agent. **Claim 3** The CD inclusion complex preparation according to claim 2, wherein the sustained-release agent is hydroxypropyl methylcellulose. **Claim 4** The CD inclusion complex preparation according to claim 1, wherein the cyclodextrin-degrading enzyme is amylase, cyclodextrinase, microbial cyclodextrinase, maltose-forming amylase, or neopullulanase. **Claim 5** The CD inclusion complex preparation according to claim 4, wherein the amylase is mammalian salivary amylase, mammalian pancreatic amylase, or microbial amylase. **Claim 6** The CD inclusion complex preparation according to claim 1, further comprising a pharmaceutically acceptable carrier. **Claim 7** The CD inclusion complex preparation according to claim 6, wherein the pharmaceutically acceptable carrier is calcium laurate. **Claim 8** The enzyme is formulated such that the cyclodextrin-degrading activity is activated upon delivery of the preparation to the target tissue to release the guest molecule from the cyclodextrin. The CD inclusion complex preparation according to claim 1, wherein the preparation is formulated for oral use and the target tissue is the human gastrointestinal (GI) tract. **Claim 9** The CD inclusion complex preparation according to claim 1, wherein the cyclodextrin comprises α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin. **Claim 10** The CD inclusion complex preparation according to claim 1, wherein the cyclodextrin is γ-cyclodextrin. **Claim 11** The CD inclusion complex preparation according to claim 1, wherein the cyclodextrin is a mixed methylated / ethylated cyclodextrin or a hydrophobic alkylated cyclodextrin. **Claim 12** The CD inclusion complex preparation according to claim 1, wherein the preparation further comprises one or more additional CD guest molecules. **Claim 13** The CD inclusion complex preparation according to claim 1, wherein the ratio of the cyclodextrin to the anandamide is from 5:1 to 1:

5. **Claim 14** The CD inclusion complex preparation according to claim 1, wherein the preparation is formulated such that the anandamide is continuously released.