Treating COVID-19

Abstract

Provided herein are compositions useful for the treatment of COVID-19.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 235,906, filed August 23, 2021, which is incorporated herein by reference in its entirety. [Background technology]

[0002] COVID-19 is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Symptoms of COVID-19 include fever, cough, fatigue, difficulty breathing, and loss of smell or taste. Some patients develop severe symptoms such as difficulty breathing, respiratory failure, shock, and multiple organ dysfunction, which can lead to death. Additional treatments for patients with COVID-19 are needed. Summary of the Invention

[0003] In certain embodiments, disclosed herein is a composition of bazedoxifene or a pharmaceutically acceptable salt thereof and sulfobutylether beta-cyclodextrin. In certain embodiments, disclosed herein is a liquid pharmaceutical formulation comprising bazedoxifene or a pharmaceutically acceptable salt thereof, sulfobutylether beta-cyclodextrin, and further comprising at least one pharmaceutically acceptable solvent. In certain embodiments, disclosed herein is a solid pharmaceutical formulation comprising bazedoxifene or a pharmaceutically acceptable salt thereof, sulfobutylether beta-cyclodextrin, and further comprising at least one pharmaceutically acceptable excipient. In certain embodiments, disclosed herein is an inhalable pharmaceutical formulation comprising bazedoxifene or a pharmaceutically acceptable salt thereof, sulfobutylether beta-cyclodextrin, and further comprising at least one pharmaceutically acceptable solvent and at least one pharmaceutically acceptable propellant and / or aerosol-forming gas. In some embodiments, the molar ratio of bazedoxifene to sulfobutylether beta-cyclodextrin is at least about 1:1. In some embodiments, the molar ratio of bazedoxifene to sulfobutyl ether beta-cyclodextrin is at least about 1:5.

[0004] In certain aspects, disclosed herein is a method of treating a viral infection caused by a coronavirus, comprising administering (in a therapeutically effective dose) to a subject in need of treatment for a viral infection the composition disclosed herein, or the pharmaceutical formulation or a pharma- ceutically acceptable salt thereof, and sulfobutyl ether beta-cyclodextrin. In some embodiments, the composition is administered by inhalation, intranasally, orally, or intraocularly. In some embodiments, the composition is administered by a nebulizer, nasal spray, oral formulation, or eye drops. In some embodiments, the composition is administered intravenously.

[0005] In certain aspects, disclosed herein is a method of treating an infection caused by the SARS-CoV-2 virus, comprising administering to a subject in need of treatment of the infection the composition or pharmaceutical formulation disclosed herein. In some embodiments, the composition is administered by inhalation, intranasally, orally, or intraocularly. In some embodiments, the composition is administered by nebulizer, nasal spray, oral formulation, or eye drops. In some embodiments, the composition is administered intravenously.

[0006] In certain aspects, disclosed herein is a method of cancer treatment, comprising administering to a subject in need of cancer treatment (a therapeutically effective dose) the pharmaceutical formulation disclosed herein. In some embodiments, the pharmaceutical formulation is administered by inhalation, intranasally, orally, or intraocularly. In some embodiments, the pharmaceutical formulation is administered by nebulizer, nasal spray, liquid formulation, or eye drops. In some embodiments, the pharmaceutical formulation is administered intravenously. In some embodiments, the cancer is breast cancer or pancreatic cancer.

[0007] In certain aspects, disclosed herein is a method for treating osteoporosis, comprising administering to a subject in need of osteoporosis treatment (a therapeutically effective dose) the pharmaceutical composition disclosed herein. In some embodiments, the osteoporosis is postmenopausal osteoporosis.

[0008] In certain embodiments, disclosed herein are salts of bazedoxifene with sulfobutyl ether beta-cyclodextrin.

[0009] In certain embodiments, compositions are disclosed herein for use as a medicament. In certain embodiments, compositions are disclosed herein for use in treating a disease selected from a viral infection caused by a coronavirus, a SARS-CoV-2 infection, cancer, and osteoporosis.

[0010] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief description of the drawings]

[0011] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings, in which: [Figure 1] FIG. 1 depicts solubility isotherms for bazedoxifene acetate with selected cyclodextrins and shows the interpolated fitted line. [Diagram 2] FIG. 1 shows solubility isotherms for bazedoxifene acetate with selected cyclodextrins and an interpolated fitted straight line. [Diagram 3] FIG. 1 depicts solubility isotherms for bazedoxifene acetate with selected cyclodextrins, with polynomial interpolation fitted lines. [Figure 4] FIG. 1 depicts UV-Vis spectra showing aggregation of bazedoxifene acetate in saline. [Diagram 5] FIG. 1 shows the UV-Vis spectrum of HPBCD encapsulation of bazedoxifene acetate diluted 1000-fold. [Figure 6] FIG. 1 shows the UV-Vis spectrum of gamma-CD encapsulation of 1000-fold diluted bazedoxifene acetate. [Figure 7] FIG. 1 shows the UV-Vis spectrum of SBECD encapsulation of 1000-fold diluted bazedoxifene acetate. [Figure 8] FIG. 1 shows the UV-Vis spectrum of beta-CD encapsulation of 1000-fold diluted bazedoxifene acetate. [Figure 9] FIG. 1 shows powder X-ray diffraction patterns of BAZ (top), SBECD (middle) and the BAZ:SBECD complex (bottom). [Figure 10] FIG. 1 depicts differential scanning calorimetry thermograms of BAZ (middle), SBECD (top) and the BAZ:SBECD complex (bottom). [Figure 11] FIG. 1 shows the structure of BAZ and the atomic numbering used to evaluate the NMR spectrum. [Figure 12] FIG. 1 depicts the 1H NMR spectrum of BAZ (600 MHz, 298 K, MeOD:D2O 3:4 (v / v)) with integrations and full assignments. [Figure 13] FIG. 13 depicts the DEPT and HSQC spectra of BAZ with complete assignments. [Figure 14] FIG. 1 shows the structure of SBECD and the atomic numbering used in evaluating the NMR spectrum. [Figure 15] FIG. 13 depicts the 1H NMR spectrum (600 MHz, 298 K, DO) of the BAZ:SBECD complex with full assignments. [Figure 16] FIG. 13 shows the DEPT and HSQC spectrum (600 MHz, 298 K, DO) of the BAZ:SBECD complex with full assignments. [Figure 17] FIG. 1 shows the structure of the BAZ:SBECD complex based on NMR studies. [Figure 18] FIG. 1 shows powder X-ray diffraction patterns of BAZ (top), the BAZ:GCD complex (middle), and GCD (bottom). [Figure 19] FIG. 1 depicts differential scanning calorimetry thermograms of BAZ (top), GCD (middle) and the BAZ:GCD complex (bottom). [Figure 20] FIG. 1 shows the structure and atom numbering of GCD. [Figure 21] FIG. 1 depicts the H NMR spectrum (600 MHz, 298 K, DO) of BAZ:GCD 1:1 with partial assignments. [Figure 22] FIG. 13. Extended 1H spectrum of BAZ:GCD 1:1 showing A) the aromatic region of BAZ and B) the core region of GCD. [Diagram 23] FIG. 1 shows the structure of BAZ:GCD 1:1. [Figure 24] FIG. 13 depicts the H NMR spectrum of the BAZ:GCD complex (600 MHz, 298, DO:DMSO-d6 6:1 (v / v)) with partial assignments. [Diagram 25] FIG. 13 depicts an extended 1H spectrum (600 MHz, 298, DO:DMSO-d6 6:1 (v / v)) of the BAZ:GCD complex showing A) the aromatic region of BAZ and B) the core region of GCD with peak assignments. [Figure 26] FIG. 1 shows the mean plasma concentration time profiles (linear scale) for Cohort B. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Bazedoxifene belongs to a group of drugs designated as selective estrogen receptor modulators (SERMs). It has affinity for the estrogen receptor but exhibits tissue-selective estrogenic effects and is used for the prevention of osteoporosis in postmenopausal women and for the treatment of moderate to severe vasomotor symptoms associated with menopause (e.g., hot flashes, sweating). Bazedoxifene has estrogen agonist effects on bone and the cardiovascular system, but estrogen antagonist effects on breast and uterine tissues. This differential activity is crucial not only to obtain the beneficial effects of estrogen on bone in reducing resorption and obtaining a turning point, but also to eliminate the potentially deleterious effects of estrogen-induced stimulation of breast and uterine tissues. For example, bazedoxifene has been studied for the treatment of oncological diseases such as breast, colon, pancreatic, gastric and endometrial cancers.

[0013] There are several serious challenges that hinder the exploitation of bazedoxifene, such as the drug's poor solubility in aqueous systems (0.000564 mg / mL, drugbank.ca) and the resulting poor biological activity. There remains a need for the development of solubilization systems for bazedoxifene and its derivatives and analogues that can be used in pharmaceutical formulations to enable their use in the treatment of COVID-19 disease by targeted delivery to the lungs and brain.

[0014] compound In certain aspects, described herein are pharmaceutical compositions of the compound bazedoxifene or a pharma- ceutical acceptable salt thereof that have significantly increased solubility and improved biological efficacy as compared to the compound bazedoxifene or a pharma- ceutical acceptable salt thereof.

[0015] [ka]

[0016] In a first aspect of the present invention, there is provided a pharmaceutical composition of the general compound bazedoxifene and sulfobutyl ether beta-cyclodextrin.

[0017] Pharmaceutical Compositions Without being bound by theory, in certain embodiments, the interaction of bazedoxifene or its pharma- ceutically acceptable salt with cyclodextrin is significantly different depending on the type of cyclodextrin. The interaction with hydroxypropyl-beta-cyclodextrin to dissolve bazedoxifene or its salt has been shown to be unsuitable for pharmaceutical use due to the formation of aggregates with low bioavailability, which are known to reduce biological efficiency and thus reduce the reproducibility of quality in pharmaceutical products. Beta-cyclodextrin forms a complex with bazedoxifene compound or its pharma- ceutically acceptable salt, but is unstable. The cavity of alpha-cyclodextrin is too small to include bazedoxifene compound or its pharma- ceutically acceptable salt. The use of substituted gamma-cyclodextrin did not sufficiently increase the solubility of bazedoxifene compound or its pharma- ceutically acceptable salt. Thus, the use of cyclodextrins to improve the solubility of the compound bazedoxifene or its pharma- ceutically acceptable salts did not appear to lead to compositions with the desired properties for pharmaceutical use. Sulfobutyl ether beta-cyclodextrin (SBECD) exhibited desirable properties for practical use in pharmaceutical formulations, which may include significantly increased solubility without reduced biological activity due to unwanted formation of aggregates.

[0018] In some embodiments, sulfobutylether beta-cyclodextrin forms a stable salt with bazedoxifene. In some embodiments, the sulfo groups of sulfobutylether beta-cyclodextrin form salts with the OH and O groups of bazedoxifene.

[0019] In some embodiments, the compositions described herein form an encapsulation / salt for bazedoxifene or a salt thereof with sulfobutyl ether beta-cyclodextrin.

[0020] In some embodiments, the molar ratio of bazedoxifene or its salt to sulfobutyl ether beta-cyclodextrin in the pharmaceutical formulation is within the range of 1:1 to 1:20, 1:1 to 1:15, 1:1 to 1:10, 1:1 to 1:9, 1:1 to 1:8, 1:1 to 1:7, 1:1 to 1:6, 1:1 to 1:5, 1:1 to 1:4, 1:1 to 1:3, or 1:1 to 1:2. In some embodiments, the molar ratio of bazedoxifene or its salt to sulfobutyl ether beta-cyclodextrin is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, about 1:15, about 1:16, about 1:17, about 1:18, about 1:19 or about 1:20.

[0021] In some embodiments, the concentration of bazedoxifene or a salt thereof is at least about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 12.5 mg / mL, about 15 mg / mL, about 17.5 mg / mL, about 20 mg / mL, or greater than about 20 mg / mL. In some embodiments, the concentration of bazedoxifene or a salt thereof is about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 12.5 mg / mL, about 15 mg / mL, about 17.5 mg / mL, about less than or equal to about 20 mg / mL.

[0022] Pharmaceutically acceptable salts of bazedoxifene are salts with pharma- ceutically acceptable acids, such as hydrochloride, chloride, hydrobromide, bromide, hydroiodide, iodide, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, phosphonate, sulfate, hydrogen sulfate, sulfite, acetate, phenylacetate, trifluoroacetate, acrylate, ascorbate, benzoate, chlorobenzoate, methylbenzoate, methoxybenzoate, dinitrobenzoate, hydroxybenzoate, acetoxybenzoate, naphthalene-2-benzoate, isobutyrate, phenylbutyrate, hydroxybutyrate, butyn-1,4-dioate, hexyn-1,6-dioate, caprate, caprylate, cinnamate, and the like. The salts may include, for example, citrate, formate, fumarate, glycolate, heptanoate, hippurate, lactate, malate, maleate, hydroxymaleate, malonate, mandelate, mesylate, nicotinate, isonicotinate, oxalate, phthalate, terephthalate, propionate, phenylpropionate, salicylate, sebacate, suberate, sulfonate, benzenesulfonate, p-bromophenylsulfonate, chlorobenzenesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate, methanesulfonate, p-toluenesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, xylenesulfonate, and tartrate salts.

[0023] In some embodiments, the pharma- ceutically acceptable salt of bazedoxifene is bazedoxifene acetate.

[0024] In some embodiments, the sulfobutyl ether beta-cyclodextrin is preferably a beta-cyclodextrin substituted with 6 to 7 (preferably 6.5) sulfobutyl ether groups. In some embodiments, the sulfobutyl ether beta-cyclodextrin may be a beta-cyclodextrin substituted with about 1, 2, 3, 4, 5, 6, 7 or more sulfobutyl ether groups. In some embodiments, the sulfobutyl ether groups may be attached to any position of the beta-cyclodextrin core. In some embodiments, the counter cation of the sulfo group is an alkali metal. In some embodiments, the counter cation of the sulfo group is sodium or potassium, or an ammonium cation.

[0025] In some embodiments, pharmaceutical compositions of the invention are prepared by combining a solution of the corresponding cyclodextrin with a solution or suspension of bazedoxifene or a pharma- ceutically acceptable salt thereof.

[0026] In some embodiments, the pharmaceutical compositions of the present invention are prepared by combining a solution of the corresponding cyclodextrin with solid bazedoxifene or a pharma- ceutically acceptable salt thereof, in some embodiments, sonication is applied to accelerate and complete the dissolution of bazedoxifene and its salts.

[0027] In some embodiments, the pharmaceutical compositions of the present invention are prepared by mechanical processing (machining) of a mixture of solid bazedoxifene or a pharma- ceutically acceptable salt thereof and a solid corresponding cyclodextrin. In some embodiments, the mechanical processing comprises grinding or milling the mixture. In some embodiments, the mechanical processing comprises grinding or milling at at least 600 rpm. In some embodiments, the mechanical processing comprises grinding for at least 10 minutes.

[0028] In some embodiments, the pharmaceutical compositions of the present invention may be formulated into solid or liquid pharmaceutical formulations.

[0029] Liquid pharmaceutical formulations may include, for example, solutions, injectable solutions, infusion solutions, inhalation solutions, and nebulizable solutions. Liquid pharmaceutical formulations include pharmaceutical compositions containing bazedoxifene and sulfobutyl ether beta-cyclodextrin, and at least one pharma- ceutically acceptable solvent, and optionally at least one pharma- ceutically acceptable excipient.

[0030] Pharmaceutically acceptable solvents may preferably include water, saline, phosphate buffered saline, and pharma- ceutically acceptable buffers.

[0031] Solid pharmaceutical formulations may include, for example, tablets, dragees, hard capsules, soft capsules, implantable preparations, ointments, gels, suppositories, etc. Solid pharmaceutical formulations include pharmaceutical compositions comprising bazedoxifene or a salt thereof and sulfobutyl ether beta-cyclodextrin, and at least one pharma- ceutically acceptable excipient.

[0032] Pharmaceutically acceptable excipients include anti-adherents, binders, coating agents, coloring agents, disintegrants, fillers, flavoring agents, glidants, lubricants, preservatives, adsorbents, sweeteners, and vehicles.Suitable excipients are known to those skilled in the art and can be selected by those skilled in the art based on the specific formulation and intended use.Lists of suitable excipients are available in pharmacopoeias and databases such as https: / / www.accessdata.fda.gov / scripts / cder / iig / index.cfm.

[0033] In some embodiments, the pharmaceutical formulation is a formulation for inhalation or intranasal administration.

[0034] Pharmaceutically acceptable propellant gases commonly used in pharmaceutical aerosols include chlorofluorocarbons, fluorocarbons (e.g., trichloromonofluoromethane, dichlorodifluoromethane), hydrocarbons (e.g., propane, butane, isobutane), hydrochlorofluorocarbons and hydrofluorocarbons, inert gases (e.g., nitrogen, NO 2 , CO 2 ), air, as well as oxygen. In some embodiments, the propellant gas is maintained in the dispensing device under pressure greater than atmospheric pressure.

[0035] In some embodiments, the pharma- ceutically acceptable aerosol-forming gas comprises air or an inert gas such as nitrogen.

[0036] In some embodiments, the compositions described herein are administered by nebulizer. The nebulized solution is dispersed in air to form an aerosol, and the nebulizer generates very fine droplets suitable for inhalation into the lungs. Nebulizers typically use compressed air, ultrasound, or vibrating mesh to create a mist of droplets, and may also have baffles to remove larger droplets from the mist by collision. For this purpose, various nebulizers are available, such as ultrasonic nebulizers, jet nebulizers, and breath-actuated nebulizers. In use, a mouthpiece or mask is typically attached to the patient to aid in the delivery of the nebulized solution.

[0037] In some embodiments, the compositions described herein are delivered intranasally. Intranasal administration of compounds offers several advantages over traditional surgical, intravenous or oral routes for administration across the blood-brain barrier (BBB). Intranasal administration to the olfactory region avoids gastrointestinal destruction, such as destruction of drugs by liver enzymes, and hepatic first-pass metabolism, making more drugs cost-effective, rapid and predictably bioavailable compared to oral administration. Intranasal administration can provide ease, convenience and safety. Intranasal drug administration is generally painless (considering that pain may be a subjective measure that varies from patient to patient) and generally immediate and easily available to all patients without the need for aseptic technique, intravenous catheters or other invasive devices. Intranasal administration can rapidly achieve therapeutic drug concentration in the brain and spinal cord.

[0038] In some embodiments, the compositions described herein are delivered intravenously.

[0039] In some embodiments, the compositions of the present invention allow for the development of liquid or oral formulations with improved bioavailability, thereby reducing the required dosage of the active ingredient and reducing the amount of ballast material that must be ingested with the active ingredient.

[0040] Treatment method In some embodiments, the composition comprising bazedoxifene or a pharma- ceutically acceptable salt thereof and sulfobutylether beta-cyclodextrin is suitable for therapeutic or prophylactic use. In some embodiments, the composition described herein is suitable for use in the treatment of viral diseases. In some embodiments, the composition described herein is suitable for use in the treatment of diseases caused by coronaviruses. In some embodiments, the composition described herein is suitable for use in the treatment of infectious diseases caused by the SARS-CoV-19 virus or a viral strain derived therefrom, i.e., the treatment of COVID-19 disease.

[0041] In some embodiments, bazedoxifene and its analogs prevent cytokine storm, a common complication of COVID-19 disease. In some embodiments, bazedoxifene and its analogs prevent ARDS associated with COVID-19. In some embodiments, the effect is due to its anti-inflammatory activity and anti-IL6 signaling. In some embodiments, bazedoxifene exhibits direct antiviral activity against SARS-CoV-2.

[0042] In some embodiments, inhalation or intranasal administration of the pharmaceutical compositions described herein is suitable for the treatment of COVID-19, as it allows for the direct delivery of the active ingredients to the infected tissues, most typically the lungs or the brain and central nervous system. In some embodiments, intravenous administration of the pharmaceutical compositions described herein is suitable for the treatment of COVID-19.

[0043] In some embodiments, the compositions described herein are useful for the treatment of cancer or osteoporosis, in line with the known therapeutic activity of bazedoxifene. In some embodiments, the compositions described herein are useful for the treatment of breast cancer and pancreatic cancer. In some embodiments, the compositions described herein are useful for the treatment of osteoporosis. In some embodiments, the compositions are in a formulation that is delivered intraocularly. In some embodiments, the compositions are delivered as eye drops.

[0044] definition Unless otherwise defined, all technical terms, notations, and other scientific and technical terms or terminology used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some cases, terms having commonly understood meanings are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not necessarily be understood as representing a substantial departure from what is commonly understood in the art.

[0045] Throughout the application, various embodiments may be presented in a range format. It should be noted that the range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Thus, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numerical values ​​within that range, such as 1, 2, 3, 4, 5, and 6. This is true regardless of the breadth of the range.

[0046] As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a sample" includes a plurality of samples, including mixtures thereof.

[0047] The terms "determining," "measuring," "evaluating," "assessing," "assaying," and "analyzing" are often used interchangeably herein to refer to forms of measurement. The terms include determining whether an element is present or not (e.g., detecting). These words can include quantitative, qualitative, or both quantitative and qualitative determinations. Assessment can be relative or absolute. "Detecting the presence of" can include determining whether something is present or absent as well as determining the amount of something present, depending on the context.

[0048] The terms "subject", "individual", or "patient" are often used interchangeably herein. A "subject" may be a biological entity that contains expressed genetic material. The biological entity may be, for example, a plant, an animal, or a microorganism, including bacteria, viruses, fungi, and protozoa. A subject may be tissues, cells, and progeny of a biological entity obtained in vivo or cultured in vitro. A subject may be a mammal. A mammal may be a human. A subject may be diagnosed with or suspected of being at risk for a disease. In some cases, a subject may not necessarily be diagnosed with or suspected of being at risk for the disease.

[0049] The term "in vitro" is used to describe events that occur in a container that holds laboratory reagents separate from the biological source from which the material is obtained. In vitro assays can include cell-based assays in which living or dead cells are used. In vitro assays can also include cell-free assays in which no intact cells are used.

[0050] As used herein, the term "about" and a number refers to the number plus or minus 10%. The term "about" and a range refers to a range from the lower limit minus 10% to the upper limit plus 10%.

[0051] As used herein, the term "treatment" or "treating" is used in reference to a pharmaceutical or other intervention regimen to obtain a beneficial or desired result in a recipient. Beneficial or desired results include, but are not limited to, therapeutic benefit and / or prophylactic benefit. Therapeutic benefit may refer to the eradication or amelioration of symptoms or the underlying disease being treated. Therapeutic benefit may also be achieved by eradication or amelioration of one or more of the physiological symptoms associated with the underlying disease, such that an improvement is observed in the subject, although the subject may still suffer from the underlying disease. Prophylactic effects include delaying, preventing or eliminating the appearance of a disease or condition, delaying or eliminating the manifestation of a symptom of a disease or condition, slowing, stopping or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, subjects at risk of a particular disease or who complain of one or more of the physiological symptoms of a disease may be treated even if they have not been diagnosed with the disease.

[0052] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. EXAMPLES

[0053] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[0054] Example 1: Materials and Methods Phase solubility test: Isothermal solubility assays were performed according to Higuchi-Connors in purified water at room temperature, where CD solutions of individual concentrations were weighed and an excess of bazedoxifene acetate was added (see also Advances in Analytical Chemistry and Instrumentation ed. CN Reilly, Whiley, New York, 1965, vol. 4, pp. 117-212). After an equilibration time of 24 hours at 25 ± 3 °C (with a magnetic stirrer at 500 rpm), the dissolved bazedoxifene concentration was determined by HPLC after filtration through a syringe filter with a polyethylene sulfone membrane of nominal pore size of 0.45 μm.

[0055] X-ray powder diffraction: Powder X-ray diffraction patterns were recorded on an X'pert Pro MDP (PANalytical Bv, The Netherlands) X-ray diffractometer using Cu K-α radiation and a Ni metal filter.

[0056] Differential scanning calorimetry: Measurements were performed using a modulated DSC2920 instrument (TA Instruments, Delaware, USA). Samples (1–5 mg) were measured in sealed Al pans at a heating rate of 10 K / min. Pure In metal standards were applied for temperature and enthalpy calibration of the DSC instrument.

[0057] NMR: 1 All H and 2D NMR experiments were performed on a 600 MHz Varian DDR NMR spectrometer equipped with a 5 mm inverse detection gradient (IDPFG) probe at 298 K. Standard pulse sequences and processing routines available in VnmrJ 4.1 were used. 1 H chemical shift value (δ) is D 2 The residual HOD peak (δ = 4.7900 ppm) present in O was referenced to. From 16 scans with a relaxation delay of 2 s. 1 H spectra were recorded.

[0058] 2D rotating frame Overhauser enhanced spectroscopy (ROESY) spectra were recorded from 8 scans / increment with a relaxation delay of 2 s and 512 increments, and the spin lock time was set to 300 ms.

[0059] Edited heteronuclear single quantum coherence (DEPT and HSQC) spectra were recorded with distortion-free enhancement by polarization transfer from 4 scans / increment with 256 increments and a relaxation delay of 1 s.

[0060] Example 2: Phase solubility studies The solubility of compositions of bazedoxifene acetate with various cyclodextrins was investigated. Figure 1 shows the most relevant results obtained for compositions with beta-cyclodextrin (BCD), gamma-cyclodextrin (GCD), hydroxypropyl-beta-cyclodextrin (HPBCD) and sulfobutylether beta-cyclodextrin (SBECD) by fitted interpolated lines. Figure 2 shows the same data by linear interpolation fitting. The linear interpolation fitted data are summarized in Table 1. Figure 3 shows the same data by polynomial interpolation fitting, and the polynomial interpolation fitted data are summarized in Table 2. BAZ is the abbreviation for bazedoxifene acetate.

[0061] [Table 1]

[0062] [Table 2]

[0063] The results of the cyclodextrin tests indicated that sufficient BCD concentrations were not achieved due to poor solubility in aqueous media. HPBCD resulted in compositions with solubility isotherms corresponding to a fourth order polynomial, indicating the formation of unwanted aggregates that negatively interfered with the solubilization and dispersion of either the bazedoxifene acetate or its cyclodextrin complexes that may have formed. The solubility isotherms obtained using GCD and SBECD allowed for close to linear fitting, indicating minimal, if any, aggregate formation.

[0064] The aggregation behavior of HPBCD alone has been observed by some authors in the academic literature, e.g., Sa Couto AR, Ryzhakov A, Loftsson T. Materials (Basel). 2018; 11(10): 1971, doi: 10.3390 / ma11101971. The observed aggregates of HPBCD were measured to be in the size range of 80-800 nm.

[0065] The unwanted aggregation effect of HPBCD encapsulation implied that as the cyclodextrin concentration increased, the proportion of cyclodextrin units that did not complex or dissolve the drug may have also increased, which may have led to the addition of additional cyclodextrin units to the already formed aggregates of cyclodextrin-drug complexes. This phenomenon indicated that formulating a dosage form with HPBCD may result in the formation and release of a mixture of different aggregates with significantly different pharmacokinetic properties.

[0066] Further experiments were performed to examine the effect of bazedoxifene-cyclodextrin compositions.

[0067] Due to the poor solubility of bazedoxifene in aqueous media, DMSO was used as part of standard procedures in biological studies to prepare its stock solutions. In view of the above, bazedoxifene acetate drug stock solutions (4.8 mg / mL) were prepared and proportional portions were diluted with saline (11.2 μL to 1000 μL). The drug concentration was 10 mmol / mL. The samples were left at room temperature for 4 hours. UV-visible absorbance spectra of the solutions were measured before and after filtration (using a 0.22 μm disk filter). After filtration, there was a sharp drop in the absorbance features corresponding to the drug (Figure 4), suggesting the formation of unwanted aggregates that were removed by filtration. This suggests that the limited efficacy of the drug observed in medical applications may be due to its strong aggregation.

[0068] To test the hypothesis that compositions of HPBCD and bazedoxifene exhibited increased aggregation, while compositions of GCD or SBECD with bazedoxifene did not exhibit this unwanted aggregation effect, the solubility and aggregation of bazedoxifene cyclodextrin encapsulation was investigated. 80 mg of bazedoxifene acetate and 600 mg of cyclodextrin (selected from BCD, HPBCD, GCD and SBECD) were weighed into a 15 mL test tube and 5.4 mL of distilled water was added. The formulation was placed in an ultrasonic bath at high temperature (40°C) for 6 hours. After cooling to room temperature, UV-Vis spectra were measured for the unfiltered solution and for the solution after filtration through a 0.22 μm disk filter.

[0069] It was observed that bazedoxifene acetate encapsulation with HPBCD (Figure 5) had a lower throughput through the filters used than that observed for BAZ encapsulation with GCD (Figure 6) and BAZ encapsulation with SBECD (Figure 7). In the case of BCD, only UV-visible absorbance data from the filtered solution was measured, since the unfiltered solution formed a slurry (Figure 8).

[0070] Solubility studies of bazedoxifene acetate using binding isotherms showed that the bazedoxifene GCD composition had lower solubility than the HPBCD composition, but also revealed significant unwanted aggregation of the bazedoxifene HPBCD complex in biological systems. These aggregates may be large enough to significantly reduce the efficacy of the drug by reducing the amount of drug that can be absorbed by cells or possibly its longevity in the blood before being blocked by macrophages (Hoshyar N, Gray S, Han H, Bao G. The effect of nanoparticle size on in vivo pharmacokinetics and cellular interaction. Nanomedicine (Lond). 2016; 11 (6): 673-692. DOI: 10.2217 / nnm.16.5). In contrast, appropriately selected cyclodextrin formulations may have had significantly increased efficacy due to a significantly smaller particle size. It may therefore be concluded that the biological potency of bazedoxifene compositions having an HPBCD may be significantly less than the biological potency of compositions having a GCD or an SBECD.

[0071] Example 3: Solubility of Bazedoxifene Acetate in Gamma-Cyclodextrin in Water 11.9 mg of bazedoxifene acetate was weighed into a 1.5 mL microtube. 1.2 mL of 40 mg / mL GCD in distilled water was added to the microtube. The solution was sonicated at 40°C for 6 hours. After dissolution of bazedoxifene acetate, aliquots of the solution were diluted with 40 mg / mL GCD in distilled water in a ratio of 1:10 and 1:100. Thus, the resulting three solutions had bazedoxifene concentrations of 9.9 mg / mL, 0.99 mg / mL, and 0.099 mg / mL. The GCD concentration remained constant in all solutions, specifically 40 mg / mL.

[0072] An aliquot of the solution was filtered through a 0.22 μm disk filter, and comparison of the unfiltered and filtered solutions by absorbance measurements at 300 nm (the characteristic maximum absorbance wavelength for bazedoxifene) showed that 97% of the bazedoxifene acetate was dissolved.

[0073] Example 4: Solubility of Bazedoxifene Acetate in Gamma-Cyclodextrin in Saline 6 mg of bazedoxifene acetate was weighed into a 1.5 mL microtube. 1.5 mL of 40 mg / mL gamma-cyclodextrin in saline was added to the microtube. The solution was sonicated at 40° C. for 6 hours. After dissolution of the bazedoxifene acetate, aliquots of the solution were diluted with 40 mg / mL GCD in saline at a ratio of 1:10 and 1:100. Thus, the resulting three solutions had bazedoxifene concentrations of 4 mg / mL, 0.4 mg / mL, and 0.04 mg / mL. The gamma-cyclodextrin concentration remained constant in all solutions, specifically 40 mg / mL.

[0074] An aliquot of the solution was filtered through a 0.22 μm disk filter, and comparison of the unfiltered and filtered solutions by absorbance measurement at 300 nm showed that 92% of the bazedoxifene acetate was dissolved.

[0075] Example 5: Solubility of Bazedoxifene Acetate in Gamma-Cyclodextrin in Buffer Solutions 3.2 mg of bazedoxifene acetate was weighed into a 1.5 mL microtube. 1.2 mL of 40 mg / mL GCD in phosphate buffered saline was added to the microtube. The solution was sonicated at 40°C for 6 hours. After dissolution of the bazedoxifene acetate, aliquots of the solution were diluted with 40 mg / mL GCD in phosphate buffered saline in ratios of 1:10 and 1:100. Thus, the resulting three solutions had bazedoxifene concentrations of 9.9 mg / mL, 0.99 mg / mL, and 0.099 mg / mL. The GCD concentration remained constant in all solutions, specifically 40 mg / mL.

[0076] An aliquot of the solution was filtered through a 0.22 μm disk filter, and comparison of the unfiltered and filtered solutions by absorbance measurement at 300 nm showed that 95% of the bazedoxifene acetate was dissolved.

[0077] Example 6: Aqueous solubility of bazedoxifene acetate in gamma-cyclodextrin prepared by milling 50 mg of bazedoxifene acetate, 250 mg of GCD and 10 g of grinding balls were weighed into a 100 mL steel grinding vessel. The resulting mixture was milled for 20 minutes at a grinding speed setting of 1000 rpm and a grinding temperature of 37°C.

[0078] 20 mg of the ground mixture was weighed into a microtube and combined with 0.5 mL of distilled water. The sample was then sonicated at 45°C for 5 minutes.

[0079] An aliquot of the sonicated solution was filtered through a 0.22 μm disk filter. Comparison of the unfiltered and filtered solutions by absorbance measurement at 300 nm indicated that 99% of the starting bazedoxifene had dissolved.

[0080] Example 7: Study of the interactions between bazedoxifene acetate and cyclodextrins (CDs) NMR experiments were used to investigate the solubility and interactions between bazedoxifene acetate (BAZ) and cyclodextrin (CD). 2D NMR and 13 The assignment of BAZ was performed by using C NMR experiments. Sample preparation was performed by dissolving 5 mg of BAZ in 700 μL of deuterated solvent. The composition of the prepared solution is listed in Table 3. In the case of 1:1 BAZ:SBECD, the mixture was filtered before transfer to the NMR tube. All samples were measured using standard 5 mm glass NMR sample tubes.

[0081] A feasibility study was conducted on the suitability of broadly available, especially orally available, cyclodextrins for the encapsulation of bazedoxifene acetate (BAZ).Sulfobutyl ether beta-cyclodextrin (SBECD) and gamma-cyclodextrin (GCD) were selected as the best cyclodextrins for solubilization of BAZ.

[0082] The BAZ:SBECD complex was prepared as follows: 11.0 g of SBECD (corresponding to an actual weight of 11.76 g with a moisture content of 6.5 wt %) was dissolved in 44 mL of Millipore Synergy purified water on a dry basis. 0.90 g of BAZ was dissolved in the medium. This SBECD / BAZ ratio ensured an excess of SBECD of approximately 30% compared to the equilibrium ratio, which was within the range of industrial availability and was made available in previous experience developing compositions of cyclodextrins with other active pharmaceutical agents. Instead of the usual 2 hours of stirring at room temperature (with a magnetic stirrer at 500 rpm), the mixture became a clear solution only after stirring overnight, since the BAZ used was not in micronized form. After clarification filtration through a PES membrane with a nominal pore size of 0.45 μm, the solution was frozen, lyophilized, and then ground into a fine powder.

[0083] Compositions of 1:1 (mol / mol) BAZ:SBECD were prepared by combining equimolar amounts of solid bazedoxifene acetate and solid SBECD in the deuterated solvent or solvent mixtures specified in Table 3. The resulting solutions were filtered before transfer to NMR tubes. All samples were run using standard 5 mm glass NMR sample tubes.

[0084] [Table 3]

[0085] The BAZ:GCD complex was prepared as follows: 19.2 g GCD (corresponding to an actual weight of 21.3 g due to 10.0 w% moisture) and 0.78 g BAZ were weighed into a porcelain mortar on a dry basis. The powder mixture was homogenized dry and then wetted with 10 mL purified water. The resulting paste was kneaded for 15 min (during which time additional purified water was added twice at 1.0 mL each to allow kneading) until the viscosity of the material did not allow further kneading (hardening was observed). The GCD:BAZ ratio utilized ensured an excess of GCD of approximately 30% compared to the equilibrium ratio, which is within the range of amounts utilized in industry and available in previous experience developing compositions of cyclodextrins with other active pharmaceutical agents. The wet paste was subjected to P for 48 h in vacuum. 2 O 5 It was then dried and ground into a fine powder.

[0086] A 1:1 (mol / mol) BAZ:GCD composition was prepared by combining equimolar amounts of solid bazedoxifene acetate and solid GCD in the deuterated solvent mixtures specified in Table 4. The dissolved mixture was filtered before transfer to the NMR tube. All samples were run using standard 5 mm glass NMR sample tubes.

[0087] [Table 4]

[0088] Example 8: BAZ:SBCD Instrumentation Analysis X-ray powder diffraction of BAZ:SBECD Powder X-ray diffraction (PXRD) patterns of the starting material and the resulting binary BAZ:SBECD composition were recorded and are shown in Figure 9. The PXRD data indicated that the starting drug substance BAZ was a crystalline material, while SBECD was amorphous. The PXRD profile of the BAZ:SBECD composition also indicated an amorphous structure, suggesting that the BAZ was molecularly dispersed within the cyclodextrin matrix and that a complex had formed between the two components.

[0089] Differential scanning calorimetry of BAZ:SBECD Differential Scanning Calorimetry (DSC): DSC thermograms of the starting material and the resulting binary BAZ:SBECD composition were recorded (Figure 10). Thermal analysis showed that the peak melting enthalpy of crystalline BAZ was at 180.4°C. Amorphous SBECD did not have such a characteristic phase transition in the temperature range tested, only a broad endothermic effect due to loss of moisture was observed. The DSC profile of the BAZ:SBECD composition also showed an amorphous structure, which, similar to the results of the PXRD analysis, suggested the absence of crystalline BAZ in the sample. These results provided further evidence that a complex had formed between the two constituents.

[0090] Nuclear Magnetic Resonance (NMR) Study of BAZ:SBECD 2D NMR and 13 Combined with C NMR experiments 1 NMR spectral assignments of BAZ were made using 1 H NMR spectroscopy. The structure and atom numbering system of BAZ referred to herein is shown in Figure 11. Figure 12 shows the structure of BAZ with integrals and complete assignments. 1The H NMR spectrum is shown. Figure 13 shows the fully assigned DEPT and HSQC spectra of BAZ. To map the intramolecular interactions of BAZ, 2D ROESY experiments were performed using the same set of parameters used to study the complex. The observed cross peaks were easily distinguishable from those due to host-guest type interactions.

[0091] Since SBECD was not a single isomeric compound, but rather a randomly substituted derivative of BCD, a complex mixture of isomers with various degrees of substitution (DS) and substitution patterns was present. Thus, the assignment of NMR spectra collected from samples containing SBECD can be difficult due to the presence of overlapping signals. In the case of SBECD, this isomeric heterogeneity was further complicated by the fact that the proton NMR resonances of some of the sulfobutyl side chains resonated at the same frequency as those corresponding to the core region of the CD unit (containing signals of the glucopyranose unit in addition to the anomeric protons). As a result, it was not possible to determine the sites of interaction at the atomic level using conventional NMR spectroscopy with the materials used. The structure and atom numbering system of SBECD referred to herein are shown in Figure 14.

[0092] Figure 15 shows the fully assigned BAZ:SBECD complex. 1 The H NMR spectrum is shown. 1H At δ = 6.5-7.5 ppm, the aromatic protons of BAZ (7, 8, 13, 14, 17, 18, 20) were seen. It was easily recognized that these signals were significantly broadened compared to those observed in the spectrum of pure BAZ, indicating some interaction with SBECD in solution. 1HAnomeric protons of the CD units were generally observed between δ = 5.0 and 5.5 ppm. In the case of randomly substituted CD derivatives, the NMR signals corresponding to the two anomers are separated into the substituted type (1's) corresponding to glucopyranose units with a side chain at at least one position, and the unsubstituted type (1'us) representing glucopyranose units without side chains. Duplication of this signal can also be characteristically observed for the rest of the glucose unit signals (as can be seen in the DEPT and HSQC spectra presented in Figure 16). The signals corresponding to the CD core region are located at δ 1H = 3.5-4.3 ppm, which included all the protons of the glucose units (2', 3', 4', 5', and 6') except for the one at the anomeric site (1 and 1'). The signals corresponding to the side chain protons of SBECD closest to the CD cavity (α) were also observed in the same region. The signals corresponding to the middle methylene units of the side chains (β and γ) completely overlapped with the signals of the azepane moieties of BAZ (1 and 2) at δ. 1H = 1.5-2.0 ppm, while the terminal side chain methylene protons adjacent to the sulfo group (δ) are observed in a separate region at δ 1H = 3.0 ppm. Further resolved resonance signals of BAZ (5, 3, and 23) are 1H = 4.4, δ 1H = 3.3 and δ 1H = 2.4. 1H The peak at = 4.79 ppm was the HDO signal from the solvent.

[0093] Fully attributed, 1 The DEPT and HSQC spectra of the BAZ:SBECD complex with the H spectrum on the x-axis are shown in FIG.

[0094] Compared to the DEPT and HSQC spectra of BAZ, the number of distinguishable signals in the spectrum of BAZ:SBECD was clearly reduced. Notably, in the case of BAZ alone, there were resolved signals corresponding to a set of seven chemically nonequivalent aromatic protons (7, 8, 13, 14, 17, 18, and 20), whereas in the case of the BAZ:SBECD complex only five resolved cross-peaks in the same region of the spectrum could be identified. 13 Only two of the signals (13 and 20) could be confidently assigned based on the C chemical shift values, because of the prominent 13 C chemical shift changes did not generally occur upon complexation or solvent change. In contrast, the higher δ 13C The aromatic protons of BAZ (presumably bearing 8) were found to be upfield (to lower frequencies) and overlapping with other peaks (it should also be emphasized that differences in the solvent composition of the samples can substantially affect the observed resonances).

[0095] Taking the above findings together, it may be concluded that BAZ was able to form a host-guest complex with SBECD. A plausible structure of this complex was constructed based on 2D ROESY analysis, as shown in Figure 17.

[0096] Example 9: BAZ:GCD Instrumentation Analysis X-ray powder diffraction of BAZ:GCD Powder X-ray diffraction (PXRD) patterns of the starting material and the resulting binary BAZ:GCD composition were recorded and are shown in Figure 18. The PXRD data indicates that the starting drug substances BAZ and GCD are both crystalline materials. The profile of the BAZ:GCD composition is also crystalline, but the PXRD profile is significantly different compared to that of either starting material, suggesting that a complex had formed between the two components.

[0097] Differential scanning calorimetry of BAZ:GCD Differential scanning calorimetry (DSC) thermograms were recorded for the starting material and the resulting binary BAZ:GCD composition (Figure 19). Thermal analysis showed a peak melting enthalpy of crystalline BAZ at 180.4°C. GCD does not have such a characteristic phase transition in the temperature range tested, with a broad endotherm due to loss of moisture observed around 100°C and features indicative of thermal decomposition observed at temperatures above about 250°C. The DSC profile of the BAZ:GCD composition suggests that the BAZ is molecularly dispersed within the GCD matrix, as no crystalline BAZ is observed in the sample. Crucially, the observation that no crystalline BAZ is present in the binary composition provides further evidence that a complex has formed between the two constituents.

[0098] Nuclear Magnetic Resonance (NMR) Study of BAZ:GCD 2D NMR and 13 Combined with C NMR experiments 1 NMR spectral assignments of BAZ were made using 1 H NMR spectroscopy. The structure and atom numbering system of BAZ referred to herein is shown in Figure 11. Figures 12 and 13 show the fully assigned structure of BAZ. 1 The H- and DEPT and HSQC spectra are shown. The structure and numbering system of GCD referred to herein is shown in FIG.

[0099] In this experiment, equimolar amounts of BAZ and GCD were weighed together, and D 2 FIG. 21 shows the partially assigned BAZ:GCD 1:1 1 The H NMR spectrum is shown.

[0100] The signal corresponding to the core region of the native CD is δ 1H = 3.5-4.0 ppm and contains all the protons of the glucopyranose ring in addition to those at the anomeric carbon atom. Signals corresponding to protons attached to the anomeric carbon atom appear in a separate region of the spectrum, δ 1H = 5.0 to 5.2 ppm.1 Another well-resolved set of signals in the high frequency region of the H NMR spectrum corresponded to the aromatic region of BAZ. The assignments of the two sets of signals were determined as shown in Figure 22.

[0101] According to these findings, BAZ could penetrate the cavity of GCD to form an inclusion complex with the proposed structure depicted in Figure 23 / A, as obtained from 2D ROESY data analysis.

[0102] The simultaneous presence of ROESY cross peaks between 3' and 13 / 14 and between 6' and 13 / 14 suggested that a second type of BAZ:GCD complex may have been formed with the proposed structure depicted in Figure 23 / B, where the B ring of BAZ may have penetrated the cavity of GCD, possibly through the first ring. To create a more realistic picture of the proposed structures of these inclusion complexes, the complexes were represented using a 3D model of BAZ sourced from the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / compound / Bazedoxifene). See Figure 23.

[0103] Examination of the interaction between BAZ and GCD in the prepared complexes In this experiment, evidence of inclusion was examined by dissolving previously prepared BAZ:GCD complexes in deuterated solvents and using NMR spectroscopy in the same manner as for the model complexes.

[0104] The prepared BAZ:GCD complex 1 H NMR spectra were collected as shown in Figure 24 with partial assignments. Expanded sections of the aromatic region of the BAZ and the core region of the GCD were observed as shown in Figures 25A and 25B, respectively, with peak assignments.

[0105] In addition to the previously observed interaction between the methyl protons of BAZ (23) and the internal protons of GCD (3', 5', and 6'), a clear cross peak between the interaromatic methylene unit of BAZ (10) and the internal protons of GCD confirmed the presence of a structure similar to that depicted in Figure 23A. Furthermore, an interaction between this methylene unit of BAZ (10) and the external protons of GCD (2' and 4') was detected, which also suggested the formation of an external complex, although no other part of BAZ was observed to be associated with such an interaction.

[0106] Taken together these findings, NMR spectroscopic examination of the prepared BAZ:GCD complex supports a host-guest complex having the proposed structure depicted in FIG.

[0107] Example 9: Antiviral Testing - Cytotoxicity and Mean Viral Titer cytotoxicity The toxicity of the test substances was measured in cultures of Vero-E6 cells (ATCC CRL-1586) in 96-well microtiter plates (2 × 10 cells per well). 4 1) at 37°C and 5% CO 2 The cells were cultured for 24 h at 4°C for 1 h. After that, the test substances were added to the cells at concentrations ranging from 0 to 15.2 μg / μL, and the cells thus treated were then incubated for another 48 h. After that, the cell viability was determined using the Cell Counting Kit-8 kit (Dojindo Molecular Technologies, Munich, Germany) strictly according to the manufacturer's instructions.

[0108] Mean viral titer Antiviral activity was tested using confluent cultures of Vero-E6 cells (ATCC CRL-1586) in 96-well microtiter plates (2 × 10 cells per well). 4 1) at 37°C and 5% CO 2The cells were cultured for 24 h at 4°C for 1 h. The medium was then aspirated and replaced with fresh medium containing the test substances in the concentration range of 0–3.8 μg / μL. At the same time, the cells were infected with the SARS-CoV-2 virus (strain SARS-CoV-2 / human / Czech Republic / 951 / 2020) at a multiplicity of infection of 0.1. The thus treated and infected cells were incubated for 48 h at 37°C and 5% CO2. The medium was then aspirated from the culture wells and the virus titer was determined by plaque titration (Stefanik et al., Microorganisms 2021, 9(3), 471).

[0109] [Table 5]

[0110] Example 10: Bazedoxifene Sample Preparation Freeze-dried products 0.6 g of bazedoxifene acetate and 7 g of SBECD were dissolved in 80-120 g of distilled water. The mixture of compounds was stirred at 500 rpm for 120 min while being heated to 44-60 °C with a heated magnetic stirrer. After mixing, the solution was lyophilized to obtain a dry product with a moisture content of 2-10%. The lyophilized product was analyzed by DSC, mass spectrometry (MS) and NMR spectroscopy.

[0111] Nebulizer solution concentrate 0.6 g of bazedoxifene acetate and 7 g of SBECD were dissolved in 100 g of distilled water. The compound mixture was stirred at 500 rpm for 120 min while being heated to 44-60 °C with a heated magnetic stirrer. The final concentration of bazedoxifene in the product is 5 mg / mL. The product is stored in glass vials housed in a paper box in a refrigerator under monitored temperature (2-8 °C, data logger).

[0112] Example 11: Bazedoxifene Administration to Rats The objective of the study was to evaluate the safety of the test formulation, bazedoxifene acetate with SBECD, following a single intravenous (iv) and single intranasal (in) dose in rats.

[0113] The study was carried out on 52 rats (26 males and 26 females). The animals were divided into two treatment groups (G1 and G2) and two control groups (C1 and C2).

[0114] Treatment group G1 included 10 males and 10 females. Each rat received a single intravenous dose of 0.5 mg / kg bazedoxifene acetate in SBECD. Treatment group G2 included 10 males and 10 females. Each rat received a single intranasal dose of 0.5 mg / kg bazedoxifene acetate in SBECD. Control group C1 included 3 males and 3 females. Each rat received a single intravenous dose of saline (NaCl 0.9%). Control group C2 included 3 males and 3 females. Each rat received a single intranasal dose of saline (NaCl 0.9%).

[0115] During the acclimatization and testing period, the animals were fed ad libitum with a standard pelleted diet Altromin (Germany). The quality of the food was monitored and the corresponding certificates were available in the archives of the test facility IPHYS CAS.

[0116] The animals were housed under conventional laboratory conditions (Building HII, room no. 022). Room temperature was 20-24°C and the relative humidity of the air was 30-70%. The room was monitored and ventilated. The lighting regime was 12 h light and 12 h dark. Food and water containers were changed and disinfected at least twice a week. Safe (Germany) was used as bedding and was changed at least twice a week.

[0117] No clinical signs of toxicity were observed in animals of both treatment groups after a single intravenous and intranasal dose of bazedoxifene at 0.5 mg / kg under the test conditions used. Neither a single intravenous nor intranasal dose of bazedoxifene at 0.5 mg / kg caused pathological lesions in the lungs and trachea of ​​rats.

[0118] No clinical signs of toxicity were observed in two control groups of animals treated intravenously and intranasally with saline under the conditions used in this study.

[0119] All animals survived until scheduled necropsy (Isoflurin anesthesia) 7 days after dosing (day 8) and underwent gross necropsy. All gross pathological changes were recorded. Lung tissues (L and R posterior lobes) and trachea were retained for further histopathological examination.

[0120] Example 12: Phase I Clinical Trial for Bazedoxifene The safety, tolerability and pharmacokinetics of a novel inhaled form of bazedoxifene acetate (BAZE-X1) were evaluated in a Phase 1 randomized, double-blind, placebo-controlled, single-dose study of BAZE-X1 in healthy volunteers. For preparation of the concentrated formulation, a sterile solution of bazedoxifene acetate with SBECD (sulfobutyl ether beta cyclodextrin) in water for injection (WFI) was used. The concentrated formulation is shown in Table 6.

[0121] [Table 6]

[0122] In the first cohort (completed in September 2021), eight healthy volunteers (six in the nebulized formulation of bazedoxifene acetate at the 1.13 mg dose level and two in the placebo) were enrolled and completed the study. Of these eight subjects, only one male was enrolled and treated with BAZE-X1. The mean age in the group treated with BAZE-X1 was 32.2 years and the mean age in the group receiving placebo was 33.5 years.

[0123] After analysis of the data from this cohort of eight subjects, no safety issues were found. All safety assessments performed did not indicate any safety concerns. No adverse events occurred during the study.

[0124] Analysis of the pharmacokinetic data showed that the observed plasma levels (pg / mL) and AUC values ​​(h.pg / mL) of bazedoxifene were very low, mostly near the limit of quantification. In the first measurement (after 10 minutes), the time to maximum plasma concentration was observed. The AUC curve in the first 10-30 minutes dropped steeply, predicting that the drug was not retained in the lung tissue and was absorbed very rapidly into the plasma in healthy volunteers. The elimination half-life was shorter (mean=21 hours) and the CL / F values ​​were slightly higher compared to the oral and intravenous data, indicating a more rapid elimination after administration by inhalation. The plasma concentrations over time and the pharmacokinetic parameters are shown in Figure 26 and Tables 7 and 8.

[0125] [Table 7]

[0126] [Table 8]

[0127] Example 13: Phase II Clinical Trial of Safety and Efficacy of Bazedoxifene and BECD This is a Phase 2 study to evaluate the safety and tolerability of bazedoxifene concentrated solution for nebulization (BAZE-X1) in patients hospitalized with moderate to severe COVID-19 pneumonia. The study consists of two parts: Part A (open-label, non-randomized) and Part B (randomized, double-blind).

[0128] The duration of treatment in both parts (A and B) of the study will be a minimum of 3 days (thus assuming 9 doses) and a maximum of 5 days (thus assuming 15 doses) depending on the length of hospital stay and the patient's condition at the discretion of the investigator. A booster dose (applicable only to Part B) will be equivalent to three doses of 1.13 mg of bazedoxifene acetate or placebo.

[0129] Part A (open label, non-randomized) Part A will enroll a total of 10 patients and the IMP to be tested will be BAZE-X1. All 10 patients will receive one dose level of bazedoxifene acetate 1.13 mg (equivalent to 1 mg bazedoxifene) three times daily according to the dosing schedule described in Section 1.8.1.1.

[0130] Part B (randomized, double-blind) Part B will begin with Cohort 1 and will enroll 8 patients. Patients within each cohort will be randomized between active treatment (6 patients) and placebo (2 patients).

[0131] In Cohort 1, one dose level of 1.13 mg bazedoxifene acetate (equivalent to 1 mg bazedoxifene) or placebo administered three times daily will be tested. If three doses of 1.13 mg bazedoxifene acetate or placebo are not available on day 1, patients enrolled in Part B may receive a single booster dose of 3.39 mg bazedoxifene acetate or placebo on day 1. Patients who receive the booster dose will continue on the standard treatment schedule (1.13 mg bazedoxifene acetate or placebo three times daily) on day 2. IMP will be administered according to the dosing regimen described in Section 1.8.1.2.

[0132] After completion of Cohort 1, safety data will be evaluated by the established DSMB. Additional cohorts, each with a dose escalation, will be included in the study. The DSMB will participate in defining the dose escalation for the next cohort based on the interim analysis of the Cohort 1 results. Additional cohorts will be conducted in the study protocol via substantive amendments after receiving approval from the RA and EC.

[0133] First endpoint The primary endpoint will be the safety and tolerability at a single dose level of BAZE-X1 administered three times daily to patients with COVID-19 pneumonia, as measured by the occurrence and spectrum of all adverse events.

[0134] Secondary endpoint The secondary endpoints were the efficacy of BAZE-X1 in preventing cytokine storm as measured by changes in leukocyte, lymphocyte, neutrophil counts and plasma concentrations of CRP, D / dimer, ferritin and IL-6 at EoT compared to baseline (D1), the efficacy of the antiviral effect of BAZE-X1 as measured by changes in blood N (nucleocapsid) antigen concentrations at EoT compared to baseline (D1), calculated from baseline (D1) to discharge or final follow-up (D28). Efficacy of BAZE-X1 in patients with COVID-19 pneumonia measured by the number of days free of respiratory (invasive / non-invasive mechanical ventilation) and cardiovascular (infusion of vasopressors / inotropes at any dose) support within 28 days after the study period calculated from baseline (D1) to discharge or last follow-up (D28), and the number of days on supplemental oxygen therapy (facemask ≤ 15 L / min) within 28 days after the study period calculated from baseline (D1) to discharge or last follow-up (D28). Efficacy of BAZE-X1 measured by number of days in HFNC (high flow nasal cannula device) within 28 days after the study period calculated from baseline (D1) to hospital discharge or last follow-up (D28) compared to baseline (D1) efficacy of BAZE-X1 measured by time to hospital discharge within 28 days after the study period calculated from baseline (D1) to hospital discharge or last follow-up (D28) in patients receiving oxygen (supplemental oxygen therapy or HFNC) at baseline (D1) measured by change in oxygen flow (L / min) at EoT and D28 Efficacy of E-X1 included in-hospital mortality at D28, and efficacy measured by change in patient clinical status (using a 7-point ordinal scale) at baseline (D1), EoT and D28 (1-death; 2-hospitalization under invasive mechanical ventilation or ECMO; 3-hospitalization under non-invasive ventilation or high-flow oxygen device; 4-hospitalization requiring supplemental oxygen; 5-hospitalization not requiring supplemental oxygen / requiring continuing medical care (COVID-19 related or other); 6-hospitalization not requiring supplemental oxygen / no longer requiring continuing medical care; 7-not hospitalized).

[0135] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be used in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered therein.

Claims

1. A composition of bazedoxifene or a pharmaceutically acceptable salt thereof and sulfobutyl ether beta-cyclodextrin.

2. A liquid pharmaceutical formulation comprising bazedoxifene or a pharmaceutically acceptable salt thereof, sulfobutyl ether beta-cyclodextrin, and at least one pharmaceutically acceptable solvent.

3. A solid pharmaceutical formulation comprising bazedoxifene or a pharmaceutically acceptable salt thereof, sulfobutyl ether beta-cyclodextrin, and at least one pharmaceutically acceptable excipient.

4. An inhalable pharmaceutical formulation comprising bazedoxifene or a pharmaceutically acceptable salt thereof, sulfobutyl ether beta-cyclodextrin, at least one pharmaceutically acceptable solvent, and at least one pharmaceutically acceptable propellant and / or aerosol-forming gas.

5. 5. The pharmaceutical formulation of any one of claims 2 to 4, wherein the molar ratio of the bazedoxifene to the sulfobutyl ether beta-cyclodextrin is at least about 1:

1.

6. 6. The pharmaceutical formulation of claim 5, wherein the molar ratio of said bazedoxifene to said sulfobutyl ether beta-cyclodextrin is at least about 1:

5.

7. 10. The composition of claim 1 for use in a method for the treatment of a viral infection caused by a coronavirus, said method comprising administering to a subject in need of such treatment a therapeutically effective dose of said composition.

8. A pharmaceutical formulation according to any one of claims 2 to 4 for use in a method for the treatment of a viral infection caused by a coronavirus, said method comprising the step of administering the pharmaceutical formulation in a therapeutically effective dose to a subject in need of such treatment.

9. The composition described in claim 7, wherein the viral infection is caused by the SARS-CoV-2 virus.

10. The pharmaceutical preparation of claim 8, wherein the viral infection is caused by the SARS-CoV-2 virus.

11. The composition of claim 1 for use in a method for treating cancer or osteoporosis, the method comprising administering the composition in a therapeutically effective dose to a subject in need of such treatment.

12. A pharmaceutical formulation according to any one of claims 2 to 4 for use in a method for treating cancer or osteoporosis, said method comprising the step of administering the pharmaceutical formulation in a therapeutically effective dose to a subject in need of such treatment.

13. The composition described in claim 11, wherein the osteoporosis is postmenopausal osteoporosis.

14. The pharmaceutical formulation of claim 12, wherein the osteoporosis is postmenopausal osteoporosis.

15. The composition of claim 7 or 11, wherein the composition is administered by inhalation, intranasally, orally, or intraocularly.

16. The pharmaceutical formulation of claim 8, wherein the pharmaceutical formulation is administered by inhalation, intranasally, orally, or intraocularly.

17. The pharmaceutical formulation of claim 12, wherein the pharmaceutical formulation is administered by inhalation, intranasally, orally, or intraocularly.

18. The composition of claim 7 or 11, wherein the composition is administered by a nebulizer, nasal spray, oral formulation, or eye drops.

19. The pharmaceutical formulation of claim 8, wherein the pharmaceutical formulation is administered by a nebulizer, nasal spray, oral formulation, or eye drops.

20. The pharmaceutical formulation of claim 12, wherein the pharmaceutical formulation is administered by a nebulizer, nasal spray, oral formulation, or eye drops.

21. The composition described in claim 7 or 11, wherein the composition is administered intravenously.

22. The pharmaceutical formulation of claim 8, wherein the pharmaceutical formulation is administered intravenously.

23. The pharmaceutical formulation of claim 12, wherein the pharmaceutical formulation is administered intravenously.

24. A salt of bazedoxifene with sulfobutyl ether beta-cyclodextrin.

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