Complex of 7-deacetyl-forskolin and PVP

Abstract

The present invention relates to a complex of 7-deacetyl-forskolin (7-DAFSK) with polyvinylpyrrolidone (PVP). The present invention also relates to a method for preparing such a 7-DAFSK-PVP complex and to a pharmaceutical composition containing such a complex.

Description

Technical Field

[0001] The present invention relates to a complex of 7-deacetyl-forskolin (7-DAFSK) and polyvinylpyrrolidone (PVP), and a method for preparing such a complex.

Background Art

[0002] The diterpene forskolin is a substantially water-insoluble plant compound derived from Plectranthus barbatus (Coleus forskohlii). Forskolin is mainly focused on multi-purpose medical applications including the treatment of cardiovascular diseases, bronchial asthma, obesity, glaucoma, lung diseases, joint inflammation, and osteoarthritis / arthritis. Forskolin promotes chondrogenesis in joints and simultaneously prevents ossification and calcification. Furthermore, forskolin is being studied in stem cell research regarding differentiation and regression to pluripotency.

[0003] Forskolin activates adenylyl cyclase, a membrane-bound enzyme. Adenylyl cyclase converts adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP) and releases cAMP into the cytoplasmic matrix, increasing the intracellular concentration of cAMP in most cells and tissues (Metzger H. et al., 1981, Drug Research, pages 1248 - 50). cAMP acts as a second messenger in cell signaling and plays a role in activating many peptide hormones (protein kinases).

[0004] Forskolin is a diterpene having two α-hydroxy groups at the 1 and 9 positions, a β-hydroxy group at the 6 position, and a β-acetoxy group at the 7 position. These two α-hydroxy groups at the 1 and 9 positions are essential for binding to adenylate cyclase. Since forskolin has low solubility in water (0.01 mg / mL, WO2005025500A2), it requires different formulation prescriptions for use as a diagnostic or therapeutic agent. Such formulation prescriptions contain various solvents such as alcohol or DMSO, and these solvents are often not highly tolerable and cause side effects.

[0005] U.S. Patent No. 6346273 B1 considers a method for improving the water solubility of a pharmaceutically active substance with low water solubility, such as forskolin, by complex formation with a nonionic polymer such as polyvinylpyrrolidone (PVP). This document describes an improvement in the water solubility of forskolin.

[0006] By complexing forskolin with cyclodextrin, an improvement in the water solubility of forskolin is achieved. WO2005025500A2 describes a method for complexing forskolin with cyclodextrin in an aqueous medium.

[0007] WO2017103840A1 describes a water-soluble forskolin composition by complex formation with a water-soluble polymer such as PVP, among other things. After mixing forskolin with such a polymer, a starch solution is added, the mixture is heated, and then cooled and spray-dried.

[0008] WO2018127600A1 discloses a method for preparing a forskolin-cyclodextrin complex, which is based on a sintering process and enables an increase in the content of forskolin in the complex and an improvement in water solubility.

[0009] Another approach to improving the water solubility of forskolin is chemical derivatization. A forskolin derivative having higher water solubility compared to forskolin is 7-deacetylforskolin, which exhibits an effect similar to that of forskolin on adenylyl cyclase (Laurenza A. et al., Molecular Pharmacology 1987, 32(1), pp. 133-139, Pinto C. et al., Biochemical Pharmacology 2009, 78(1), pp. 62-69). However, deacetylation at the 7-position of forskolin can only improve water solubility to a limited extent (up to 1 mg / mL).

[0010] Therefore, there is a need for a form of a water-soluble improved forskolin derivative that is characterized by both high water solubility and thereby improved bioavailability and has a high forskolin derivative content.

[0011] For this reason, it is an object of the present invention to provide a form of 7-deacetyl-forskolin that has increased solubility in water and at the same time has a high content of 7-deacetyl-forskolin.

Summary of the Invention

Means for Solving the Problems

[0012] As described in the claims and embodiments of the present invention, the object of the present invention is solved by a complex of 7-deacetyl-forskolin (7-DAFSK) and polyvinylpyrrolidone (PVP).

[0013] The present invention discloses a complex of 7-deacetyl-forskolin (7-DAFSK) and polyvinylpyrrolidone (PVP). In one embodiment, the complex according to the present invention is characterized in that the average mass fraction of 7-DAFSK in the whole complex is in the range of 1 to 50 wt%, preferably 5, 10, 15, 20, or 25 wt%, more preferably 30 wt%, 35 wt%, 40 wt%, 45 wt% or more.

[0014] In one embodiment, the average molar ratio of 7-DAFSK to PVP in the complex is 0.1 or more, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60 or more, more preferably 3 or more.

[0015] In one embodiment, the complex according to the present invention is characterized in that PVP has an average molar mass of 1 to 40 kD. The PVP preferably has an average molar mass of 1 to 25 kD, more preferably about 2 to 3 kD, and most preferably about 2.5 kD.

[0016] In a further embodiment, the complex according to the present invention is characterized in that 7-DAFSK is derived from synthetic forskolin. In one embodiment, 7-DAFSK is present with a purity of at least 98.5%.

[0017] Surprisingly, it has been found that a particularly high mass fraction of 7-DAFSK in the 7-DAFSK-PVP complex can be obtained by heating a mixture of 7-DAFSK and PVP to a temperature above the glass transition temperature of the PVP used. In particular, the mixture is heated to a temperature of at least 20 °C, preferably at least 25 °C, 30 °C, 40 °C, 50 °C, 60 °C, 70 °C, 80 °C, 90 °C, 100 °C above the glass transition temperature of the PVP. In particular, the temperature is increased to a range of about 25 to 50 °C above the glass transition temperature of the PVP.

[0018] Accordingly, the present invention also provides a method for preparing a 7-DAFSK-PVP complex according to the present invention, characterized in that a mixture of 7-DAFSK and PVP is heated to a temperature higher than the glass transition temperature of the PVP used.

[0019] In one embodiment, the process according to the invention is characterized in that a solvent, preferably water, ethanol, methanol, acetone, ethyl methyl ketone, dichloromethane, and / or ethyl acetate, even more preferably water, ethanol, methanol, propanol, and / or dichloromethane, is added to the mixture of 7-DAFSK and PVP. Particularly preferably, water and / or ethanol or a mixture thereof is added to the mixture of 7-DAFSK and PVP.

[0020] In certain embodiments, the mixture according to the invention is kept at a temperature higher than the glass transition temperature of the PVP used for at least 5 minutes. In one embodiment, the method according to the invention comprises the following steps.

[0021] a) Preparing a mixture of 7-DAFSK and PVP and adding a solvent, preferably an alcohol, preferably ethanol, to the mixture; b) Adding water; c) Heating the mixture at least 5 minutes above the glass transition temperature of the PVP; d) Cooling the mixture and dissolving it in water; e) Optionally removing undissolved components by filtration.

[0022] In certain applications, the mixture is heated to at least 20, 25, 30, 35, 40, 45, 50, 55, 60 °C, 70 °C, 80 °C, 90 °C, 100 °C above the glass transition temperature of the PVP.

[0023] The application of the method according to the invention significantly improves the water solubility of 7-DAFSK and may contain a high mass fraction of 7-DAFSK, enabling an efficient and simple preparation of a 7-DAFSK-PVP complex that allows for beneficial applications of 7-DAFSK in diagnosis and / or treatment.

[0024] By using the complex of 7-DAFSK with PVP according to the present invention, the water solubility of 7-DAFSK can be improved. By using a high mass fraction of 7-DAFSK in the complex according to the present invention, the water solubility of 7-DAFSK can be improved without thereby inconveniently requiring a large amount of PVP. Formulation recipes using forskolin derivatives can thus also be assembled in a more material-saving way than before. By avoiding a large amount of PVP and its associated side effects, the present invention facilitates and improves the use of 7-DAFSK in diagnosis and / or treatment.

[0025] Accordingly, the present invention also provides a pharmaceutical composition containing the 7-DAFSK-PVP complex according to the present invention. In one embodiment, the pharmaceutical composition is characterized in that the composition contains 7-DAFSK at a concentration of at least 25 mg / L, 50 mg / L, 75 mg / L, 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, 500 mg / L, 600 mg / L, 800 mg / L, 1000 mg / L, 2000 mg / L, 3000 mg / L, or 4000 mg / L, preferably 4000 mg / L.

[0026] One aspect of the present invention relates to a pharmaceutical composition for oral, intravenous, or inhalation administration. In certain embodiments, the pharmaceutical composition according to the present invention is used in a treatment method. In another specific embodiment, the pharmaceutical composition according to the present invention is used for the treatment of cardiovascular diseases, bronchial asthma, obesity, glaucoma, lung diseases, joint inflammation, or osteoarthritis / arthritis.

[0027] One aspect of the present invention relates to the pharmaceutical composition according to the present invention for use as a medicament.

Brief Description of the Drawings

[0028]

Figure 1

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Mode for Carrying Out the Invention

[0029] 7-Deacetyl-forskolin (7-DAFSK) can be represented by the structural formula shown in FIG. 1. 7-DAFSK may exist in forms other than its molecular form, for example, in the form of salts, such as other forms such as alkali metal salts such as sodium salts or potassium salts. In the context of the present invention, the term "7-deacetyl-forskolin (7-DAFSK)" refers to both the molecular form shown by the structural formula in FIG. 1 and all other possible forms of salts of 7-DAFSK, preferably alkali metal salts, particularly preferably sodium salts or potassium salts.

[0030] Polyvinylpyrrolidone (PVP), also known as polyvidone or povidone, is a polymer of the compound vinylpyrrolidone. PVP is commercially available with various degrees of polymerization. The degree of polymerization is determined by the average molar mass of the polymer. As used herein, the term "average molar mass" refers to the mass average of the molar masses.

[0031] In the context of the present invention, PVP having an average molar mass of 1 to 40 kD is preferred because polymers of this size can still be sufficiently excreted by the kidneys and are not metabolized by the kidneys.

[0032] In one embodiment, the 7-DAFSK-PVP complex is characterized in that the PVP has an average molar mass of 1 to 60 kD, particularly 1 to 40 kD. Preferably, the PVP has an average molar mass of 1 to 25 kD, preferably about 2 to 9 kD, 2 to 8 kD, 2 to 7 kD, 2 to 6 kD, 2 to 5 kD, 2 to 4 kD, more preferably 2 to 3 kD, and most preferably about 2.5 kD.

[0033] As used herein, the term "about" refers to a specific value or a value deviating from a specific value by + / - 10%. The molecular weight of PVP can be obtained by viscosity measurement and is also defined by the K value determined by the viscosity average of the molar mass, instead of specifying the mass average of the molar mass. PVP having a mass average molecular weight of about 2.5 kD corresponds to PVP having a K value of about 12, which is denoted as "PVP K12" herein (K. Kolter et al., "Hot-Meld Extrusion with BASF Pharma Polymers", ISBN 978-3-00-039415-7, 2012). PVP having a weight average molecular weight of about 24 kD corresponds to PVP having a K value of about 25, which is denoted as "PVP K25" herein.

[0034] In the context of the present invention, the term "7-DAFSK-PVP complex" refers to a chemical product containing 7-DAFSK and PVP, and is characterized by the fact that a bond exists between 7-DAFSK and PVP. The term "complex" in no way limits the nature of the compound, but simply means that a bond exists between one or more 7-DAFSK molecules and one or more PVP molecules. The bond may be, for example, a covalent bond or, preferably, a non-covalent attachment of 7-DAFSK to PVP via, for example, van der Waals bonds or hydrogen bonds. It should be understood that the term "7-DAFSK-PVP" or "7-DAFSK-PVP complex" includes the chemical product as a whole and is not a single 7-DAFSK-PVP complex at the molecular level.

[0035] The terms "mass fraction" and "molar ratio" should always be understood as average values herein. These terms do not refer to each individual complex at the molecular level, but rather to the average value of the entire chemical product.

[0036] In the present invention, percentages (%) refer to weight percentages (wt%) in each case, unless otherwise indicated. In one embodiment, the average mass fraction of 7-DAFSK in the entire complex is 1 to 50 wt%, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 wt%, more preferably 30 wt%, 35 wt%, 40 wt%, 45 wt% or more. The mass fraction reflects, herein, the relative proportion of the mass of 7-DAFSK to the mass of the entire complex (7-DAFSK-PVP).

[0037] The ratio of 7-DAFSK in the 7-DAFSK-PVP complex can also be expressed as a molar ratio. For example, a 7-DAFSK-PVP complex consisting of 7-DAFSK of the molecule (molar mass: 368.5 g / mol) and PVP having an average molar mass of about 2.5 kD ("PVP K12"), a 7-DAFSK-PVP complex in which the average mass fraction of 7-DAFSK in the whole complex is 30 wt%, a 7-DAFSK-PVP complex in which the average molar ratio of 7-DAFSK to PVP in the complex is about 3. The average molar ratio reflects, in this specification, the ratio of the molar amount of 7-DAFSK to the molar amount of PVP.

[0038] In one embodiment of the present invention, the average molar ratio of 7-DAFSK to PVP in the complex is 0.1 or more, preferably 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more, more preferably 3 or more.

[0039] The ratio of 7-DAFSK in the whole complex (as well as the mass fraction in the same way as the molar ratio) should always be understood as the average ratio in this specification. A suitable method for measuring the average ratio of 7-DAFSK in the whole complex is high performance liquid chromatography (HPLC). Those skilled in the art are proficient in such measurement operations.

[0040] 7-DAFSK can be prepared from forskolin using a deacetylation reaction, for example, carbonate saponification, as described in specific examples of the present invention. Both natural and synthetic forskolin can be used as reactants. Natural forskolin typically exists as an enriched extract and is purified before use. In the context of the present invention, synthetic forskolin is commercially available in high purity and is preferably used as a reactant for 7-DAFSK synthesis.

[0041] Therefore, in one embodiment, the complex according to the present invention is characterized in that 7-DAFSK is derived from synthetic forskolin. In a preferred embodiment, 7-DAFSK is present with a purity of at least 98.5%.

[0042] The 7-DAFSK-PVP complex according to the present invention is characterized by improved water solubility compared to non-complexed 7-DAFSK-PVP, which is advantageous for pharmaceutical applications. Surprisingly, it has been found that the 7-DAFSK-PVP complex is readily available by synthesizing 7-DAFSK and PVP by heating the mixture to a temperature higher than the glass transition temperature of the PVP used. Surprisingly, this procedure also results in a high mass fraction of 7-DAFSK in the whole complex.

[0043] Accordingly, the present invention includes a process for preparing the 7-DAFSK-PVP complex according to the present invention, characterized in that a mixture of 7-DAFSK and PVP is heated to a temperature higher than the glass transition temperature of the PVP used.

[0044] As an amorphous substance, PVP does not have a melting point but exhibits a so-called glass transition temperature. The glass transition temperature varies, inter alia, depending on the degree of polymerization of PVP, i.e., the average molar mass (see Table 1).

[0045] [Table 1]

[0046] In this context, the "glass transition temperature of the PVP used" should be understood as the temperature at which the glass transition of the PVP in the mixture of the present invention with 7-DAFSK occurs. The glass transition temperature of the PVP used can be affected by the composition of the mixture, for example, when a solvent or water is added to the mixture. Methods for measuring the glass transition temperature are known to those skilled in the art. Preferably, the glass transition temperature can be measured according to the procedure of the corresponding DIN standard (Wampfler B., et al., Messunsicherheit in der Kunststoffanalytik - Ermittlung mit Ringversuchsdaten. Carl Hanser Verlag, Munich 2017, ISBN-10: 3446452869).

[0047] A particularly preferred embodiment of the process according to the present invention for the preparation of the 7-DAFSK-PVP complex is characterized in that the average molar mass of the PVP used is about 2.5 kD ("PVP K12") and the mixture is heated to a temperature above 93°C. Specifically, heating is carried out at a temperature of at least 25°C, particularly 30°C, 40°C, 45°C or more above the glass transition temperature of PVP K12.

[0048] Another preferred embodiment is characterized in that the average molar mass of the PVP used is about 9 kD ("PVP K17") and the mixture is heated to a temperature above 130°C. More specifically, the mixture is heated to a temperature of at least 25°C, particularly 30°C, 40°C, 45°C or more above the glass transition temperature of PVP K17.

[0049] Another preferred embodiment is characterized in that the average molar mass of the PVP used is about 24 kD ("PVP K25") and the mixture is heated to a temperature above 155°C. More specifically, the mixture is heated to a temperature of at least 25°C, particularly 30°C, 40°C, 45°C or more above the glass transition temperature of PVP K25.

[0050] Another preferred embodiment is characterized in that the average molar mass of the PVP used is about 40 kD (“PVP K30”) and the mixture is heated to a temperature above 175° C. More specifically, the mixture is heated to a temperature of at least 25° C., in particular 30° C., 40° C., 45° C. or more, above the glass transition temperature of PVP K30.

[0051] It has been found advantageous to add a solvent or a mixture of solvents to the mixture of 7-DAFSK and PVP according to the invention. Preferably, the mixture of 7-DAFSK and PVP is stirred like a paste in a small amount of solvent. The addition of the solvent can help to uniformly disperse 7-DAFSK and PVP and more 7-DAFSK can be attached to PVP. Suitable solvents are aqueous solvents, alcohol solvents or other organic solvents such as water, ethanol, methanol, propanol, acetone, ethyl methyl ketone, dichloromethane, and / or ethyl acetate.

[0052] In one embodiment, the process according to the invention for the preparation of the 7-DAFSK-PVP complex is characterized in that a solvent, preferably water, ethanol, methanol, propanol, acetone, ethyl methyl ketone, dichloromethane, and / or ethyl acetate, even more preferably water, ethanol, methanol, propanol, dichloromethane, and / or pyridine, is added to the mixture of 7-DAFSK and PVP. Particularly preferably, water and / or ethanol or a mixture thereof is added to the mixture of 7-DAFSK and PVP. The addition of water and / or ethanol may be simultaneous or sequential.

[0053] In the manufacturing process, it has been found useful to keep the mixture of 7-DAFSK and PVP at a temperature higher than the glass transition temperature of the PVP used for a certain period of time. Good results were obtained when the mixture was kept at a temperature above the glass transition temperature for at least 5 minutes.

[0054] Thus, in certain embodiments of the process according to the invention, the mixture of 7-DAFSK and PVP is maintained at a temperature higher than the glass transition temperature of the PVP used for at least 5 minutes.

[0055] In certain embodiments, the method according to the invention comprises the following steps carried out in sequence. a) Preparing a mixture of 7-DAFSK and PVP and adding a solvent, preferably an alcohol, preferably ethanol, to said mixture; b) Adding water; c) Heating said mixture at least 5 minutes above the glass transition temperature of said PVP; d) Cooling said mixture and dissolving it in water; e) Optionally removing undissolved components by filtration.

[0056] In one embodiment, the mixture of 7-DAFSK and PVP is prepared at a weight ratio of 2:1 to 1:4, preferably 2:1, 2:1.5, 1:1, 2:3, 1:2, 1:3, and 1:4, most preferably 1:1 or 1:2.

[0057] The application of the method according to the invention enables an efficient and simple preparation of the 7-DAFSK-PVP complex. The process according to the invention enables the preparation of 7-DAFSK-PVP complexes having a high mass fraction of 7-DAFSK. Conventional methods for preparing PVP complexes involve dissolving the components in a solvent or solvent mixture, which mixture is not heated above the glass transition temperature of PVP. In the specific examples described herein, the process of the invention resulted in an average mass fraction of 7-DAFSK in the 7-DAFSK-PVP complex that was 8 - 9% higher compared to conventional complex formation processes.

[0058] The high mass fraction of 7-DAFSK in the 7-DAFSK-PVP complex is particularly advantageous for pharmaceutical applications in diagnosis and / or treatment. By using the high mass fraction of 7-DAFSK in the complex according to the present invention, the water solubility of 7-DAFSK can be improved without inconveniently requiring a large amount of PVP. Formulation with forskolin derivatives can thus also be assembled in a more material-saving manner than before. By avoiding large amounts of adjuvant (PVP) and the associated side effects, the present invention facilitates and improves the use of 7-DAFSK in diagnosis and / or treatment.

[0059] The 7-DAFSK-PVP complex according to the present invention can be used for any dietary or medical purpose for which forskolin and / or 7-DAFSK is applicable. Accordingly, the present invention also relates to a pharmaceutical composition containing the 7-DAFSK-PVP complex of the present invention using 7-DAFSK as an active ingredient.

[0060] The term "pharmaceutical composition" as used herein relates to the 7-DAFSK-PVP complex according to the present invention using 7-DAFSK as an active ingredient, optionally in combination with additional ingredients and / or the 7-DAFSK-PVP complex.

[0061] The pharmaceutical composition according to the present invention may be present in various systemic and topical formulations. Non-limiting examples of systemic or topical formulations containing the 7-DAFSK-PVP complex of the present invention include oral, buccal, intralung, rectal, intrauterine, intradermal, topical, dermal, parenteral, intratumoral, intracranial, buccal, sublingual, nasal, subcutaneous, intravascular, intrathecal, inhalable, respirable, intra-articular, intracavitary, implantable, transdermal, iontophoretic, intraocular, intravaginal, optical, intravenous, intramuscular, intra-glandular, intra-organ, intralymphatic formulations, enteric coating, or sustained release formulations.

[0062] The composition may be administered once or multiple times a day. Preferred embodiments relate to pharmaceutical compositions according to the invention for oral, intravenous, or inhalation administration. Formulations for oral administration may be provided in individual units such as capsules, tablets, or lozenges containing the formulation as a powder or granules. Formulations for oral administration may be provided as a solution or suspension in an aqueous or non-aqueous medium, or as an emulsion.

[0063] Formulations for intravenous administration may be provided as a solution or suspension in an aqueous medium, for example, as a physiological saline solution. The specific dosage of the 7-DAFSK-PVP complex to obtain a therapeutic or prophylactic effect varies depending on the specific application, for example, the route of administration, the age, weight, and condition of the individual patient, the disease being treated, and the severity of the symptoms.

[0064] The dosage of 7-DAFSK-PVP in the composition according to the invention is preferably an effective dosage that can obtain a therapeutic or prophylactic effect without causing undesirable side effects, and the composition can be administered as a single dose or in multiple units.

[0065] In certain embodiments, the composition according to the invention is provided as a solution or aerosol containing 7-DAFSK at a concentration of at least 25 mg / L, preferably at least 50 mg / L, 75 mg / L, 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, 500 mg / L, 600 mg / L, 800 mg / L, or 1000 mg / L. In a preferred embodiment, the composition contains 7-DAFSK at a concentration of at least 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, or 4 mg / mL, most preferably 4 mg / mL.

[0066] In another specific embodiment, the composition according to the present invention is provided as a capsule or tablet containing 7-DAFSK in a pharmaceutically effective amount, for example, 10 mg to 2000 mg, preferably at least 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1000 mg, more preferably 500 mg.

[0067] In a specific embodiment, the pharmaceutical composition according to the present invention is used in a treatment method. Forskolin and 7-DAFSK are known to be non-specific cAMP stimulants. The 7-DAFSK-PVP complex according to the present invention can be used to treat any disease or disorder that can be treated with forskolin and / or 7-DAFSK. Non-limiting examples of such diseases include neurodegenerative diseases, Alzheimer's disease, motor dysfunction, acute and chronic cardiovascular diseases, lung diseases such as asthma, cystic fibrosis, vascular diseases associated with cystic fibrosis, bronchitis, chronic obstructive pulmonary disease (COPD), obesity, glaucoma, joint inflammation, osteoarthritis / arthritis, fibrotic degeneration such as idiopathic pulmonary fibrosis, post-traumatic pulmonary fibrosis, bronchopulmonary dysplasia (BPD), VPD or post-toxic liver diseases such as cirrhosis, and peripheral circulatory disorders including Raynaud's disease and scleroderma.

[0068] In a specific embodiment, the pharmaceutical composition according to the present invention is used for the treatment of cardiovascular diseases, bronchial asthma, obesity, glaucoma, lung diseases, joint inflammation, or osteoarthritis / arthritis.

[0069] Another aspect of the present invention relates to the pharmaceutical composition according to the present invention for use as a medicine. The present invention is further illustrated by the following examples and figures, but is not limited thereto.

Examples

[0070] The following examples describe the synthesis of 7 - deacetyl forskolin and the formation of its complex with PVP, in which PVPs with average molar masses of about 2.5 kD ("PVP K12") and about 24 kD ("PVP K25") were selected, and the ratio of the amount of 7 - deacetyl forskolin to PVP was varied. These examples further examine the characterization of the resulting complexes from the viewpoints of the 7 - deacetyl forskolin content, its water solubility, and its effect as a cAMP activator in the cAMP assay. These examples do not include detailed descriptions of conventional methods such as the implementation of content measurement by HPLC or the measurement of NMR spectra. Such methods are well known to those skilled in the art.

[0071] Example 1 Synthesis of 7 - deacetyl forskolin Synthetic forskolin from Mercachem (GMP - material), batch MAMA07 - 024 - 5, was used as a reactant for the synthesis of 7 - deacetyl - forskolin (7 - DAFSK). The deacetylation of forskolin was carried out via classical carbonate saponification.

[0072] The deacetylation was carried out according to the reaction scheme shown in Figure 1. First, in a 250 mL round - bottom flask, 200 mg of forskolin (synthetic forskolin, Mercachem, batch MAMA07 - 024 - 5) and 200 mg of calcined Na2CO3 were stirred in 10 mL of methanol at room temperature for 4 hours. Next, the reaction mixture was allowed to stand overnight (20 hours).

[0073] Next, the methanol was distilled off from the flask (Rotavapor) and dissolved in about 20 mL of CH2Cl2. This was treated with ultrasound for a short time (1 minute). The formed 7 - DAFSK was dissolved in CH2Cl2 and its salt was precipitated. The salt was filtered off (medium - speed filtration). Next, in a separatory funnel, the filtrate was washed with 2N HCl. The dichloromethane fraction was recovered and the solvent was distilled off using a rotavapor. The resulting white powder was analyzed by HPLC. A purity exceeding 99% was measured.

[0074] The structure of 7-DAFSK is 1 H NMR 13 The resulting 7-DAFSK was confirmed by C NMR. 1 H and 13 The C NMR spectrum is identical to the previously published spectrum of this compound (J. Org. Chem. 2006, 71, pp. 4619-4624), confirming the desired deacetylation of forskolin according to the reaction scheme shown in Figure 1.

[0075] The obtained 7-DAFSK was further characterized by HPLC. Deacetylforskolin and forskolin were eluted with a mobile phase of acetonitrile / water (55 / 45 v / v) at a flow rate of 0.6 mL / min (stationary phase: Nucleosil120 3C18, 250 x 4 mm). A calibration curve was prepared for the quantification of the 7-DAFSK content in the complex with PVP. Calibration solutions with 7-DAFSK concentrations of 0.014, 0.028, 0.056, and 0.56 mg / mL were used for calibration.

[0076] Example 2 Preparation of 7-DAFSK-PVP complex Since 7-deacetyl-forskolin itself is only moderately soluble in water, 7-DAFSK was complexed with PVP to improve its water solubility. The 7-DAFSK complex was prepared by the method of the present invention, hereafter referred to as the "heating method", and compared with conventional dissolution without heating (the "dissolution method"). 1. Preparation of 7-DAFSK-PVP Complex by Heating PVP Above Its Glass Transition Temperature ("Heat Treatment") 7-DAFSK-PVP K12 complex (complex of 7-DAFSK with PVP K12) For the formation of a complex of 7-deacetyl forskolin with PVP K12 ("Kollidon" 12PF, European Pharmacopoeia, United States Pharmacopoeia, Japanese Pharmacopoeia; BASF, average molar mass: about 2.5 kD), 20 mg of 7-DAFSK and 20 mg of PVP K12 were ground in a magnetic mortar, 300 μL of ethanol was added and stirred. After 10 minutes, 100 μL of H2O was added, stirred, and left standing for another 10 minutes. Next, in a drying oven, this solution was heated to 140 °C within 40 minutes and left in the drying oven for a total of 45 minutes. After cooling, 3 mL of H2O was added and stirred for 20 - 30 minutes. Next, this suspension was aspirated with a syringe and filtered through a 0.45 μm filter (hydrophilic). The mortar was rewashed with 3 mL of H2O, and this washing solution was also filtered through the said filter. The content of 7-DAFSK dissolved in 6 mL of H2O was measured by HPLC. For the measurement, the 7-DAFSK-PVP complex solution was diluted 1:10 using the current running medium. The mass fraction of 7-DAFSK in the entire 35 wt% complex was measured.

[0077] 7-DAFSK-PVP K25 complex (complex of 7-DAFSK with PVP K25) For the formation of a complex of 7-deacetyl forskolin with PVP K25 (FLUKA 81399, average molar mass: about 24 kD), 20 mg of 7-DAFSK and 20 mg of PVP K25 were ground in a magnetic mortar, 300 μL of ethanol was added and stirred. After 10 minutes, 100 μL of H2O was added, stirred, and left standing for another 10 minutes. Next, in a drying oven, this solution was heated to 185 °C within 40 minutes and left in the drying oven for a total of 45 minutes. After cooling, 3 mL of H2O was added and stirred for 20 - 30 minutes. Next, this suspension was aspirated with a syringe and filtered through a 0.45 μm filter (hydrophilic). The mortar was rewashed with 3 mL of H2O, and this washing solution was also filtered through the said filter. The content of 7-DAFSK dissolved in 6 mL of H2O was measured by HPLC. For the measurement, the 7-DAFSK-PVP 25 complex solution was diluted 1:10 using the current running medium. The mass fraction of 7-DAFSK in the entire 17 wt% complex was measured. 2. Preparation of 7-DAFSK-PVP Composite by Dissolution Process 7-DAFSK-PVP K12 Composite In a magnetic mortar, 20 mg of 7-DAFSK and 40 mg of PVP K12 were triturated, 300 μL of ethanol was added and stirred. After 10 minutes, 100 μL of H2O was added, stirred, and left standing for another 10 minutes. Next, this solution was left standing at room temperature for 45 minutes, 3 mL of H2O was added, and stirred for 20 - 30 minutes. Next, this suspension was sucked up with a syringe and filtered through a 0.45 μm filter (hydrophilic). The mortar was rewashed with 3 mL of H2O, and this washing solution was also filtered through the said filter. The content of 7-DAFSK dissolved in 6 mL of H2O was measured by HPLC. The mass fraction of 7-DAFSK in the whole 24 wt% composite was measured.

[0078] 7-DAFSK-PVP K25 Composite In a magnetic mortar, 20 mg of 7-DAFSK and 40 mg of PVP 25 were triturated, 300 μL of ethanol was added and stirred. After 10 minutes, 100 μL of H2O was added, stirred, and left standing for another 10 minutes. Next, this solution was left standing at room temperature for 45 minutes, 3 mL of H2O was added, and stirred for 20 - 30 minutes. Next, this suspension was sucked up with a syringe and filtered through a 0.45 μm filter (hydrophilic). The mortar was rewashed with 3 mL of H2O, and this washing solution was also filtered through the said filter. The content of 7-DAFSK dissolved in 6 mL of H2O was measured by HPLC. The mass fraction of 7-DAFSK in the whole 8 wt% composite was measured.

[0079] Example 3 Measurement of Water Solubility For the solubility test, each composite dried using a rotary evaporator was used. A specified amount of water was added, and then the composite was added until 7-DAFSK-PVP became excessive. This supersaturated solution was treated with ultrasonic waves, stirred, and slightly heated (water bath at 40 °C). Next, this suspension was filtered (0.45 μm filter), and the content of 7-DAFSK in this filtrate was measured by HPLC.

[0080] Example 4 Characteristic Evaluation of 7-DAFSK-PVP Composite Mass Fraction of 7-DAFSK in the Whole Composite Figure 2 shows a chart of the measured 7-DAFSK content in 7-DAFSK-PVP K12 and 7-DAFSK-PVP 25 composites where the ratio of 7-DAFSK to 20:20 PVP was selected. Here, it is distinguished between the heating method and the dissolution method. In all composites, a clear difference is observed between these two methods regarding the salt of 7-DAFSK. Using the heating method, a content 8 - 9% higher of the salt of 7-DAFSK can be obtained in the final composite. The 7-DAFSK-PVP K12 composite shows the highest content of 7-DAFSK (about 35 wt%).

[0081] To optimize the content of 7-DAFSK in the whole composite, the weight ratio of 7-DAFSK and PVP K12 used was varied (Figure 3). As can be seen from Figure 3, even when the ratio of 7-DAFSK / PVP K12 was varied, the amount that could be complexed with 7-DAFSK (20 mg) by this method was 50 - 60% or less of the materials used. It is clear that even though the amount of PVP 12 was increased, the final amount of complexed 7-DAFSK in the final composite did not change much. After preparation by heating, to confirm how much non-complexed 7-DAFSK would be in a dissolved state during the dissolution process, a control sample (containing only 7-DAFSK without PVP 12) treated in the same way as the composite sample was considered. Here, it is considered that less than one-fourth of the 7-DAFSK used could be in a dissolved state. Therefore, complex formation with PVP achieves an improvement in the solubility of 7-DAFSK.

[0082] Even by increasing the amount of solvent before heating, it was impossible to incorporate a larger amount of 7-DAFSK into the composite with PVP K12. Figure 4 shows that comparable results could be obtained despite using a very small amount of PVP.

[0083] The reported 7-DAFSK content in wt% was measured by HPLC. For calculation, it was assumed that the total amount of PVP would be in a dissolved state when dissolved in 6 mL of H2O. Water solubility Table 2 shows that a significant improvement in the water solubility of 7-DAFSK is possible by complex formation with PVP. The solubility of 7-DAFSK in the complex with PVP is up to three times higher than that of uncomplexed 7-DAFSK. In contrast to 7-DAFSK, forskolin hardly dissolves with PVP due to its steric structure and cannot form a complex with PVP.

[0084] Figure 5 shows that changing the DAFSK / PVP ratio does not significantly change its water solubility. The value varies between 2.5 mg / mL and 3.5 mg / mL. This is due to the fact that at each ratio, approximately the same proportion (50 - 60%) of 7-DAFSK in the complex is complexed, and each ratio does not significantly change the water solubility of the complex.

[0085]

Table 2

[0086] Example 5 Cell culture test: cAMP assay The cAMP assay was performed using HEK293 cells (human embryonic kidney cells, 1970) having prostaglandin receptor EP2 or EP4, or prostacyclin receptor IP. HEK293 cells were incubated with FCS and a radioactive ligand ( 3 H]adenine). The formation of cAMP ( 3 H]cAMP) was experimentally monitored.

[0087] The activation of adenylate cyclase (AC) by forskolin (dissolved in DMSO), as well as 7-DAFSK and 7-DAFSK-PVP dissolved in water, was compared. There were slight differences among the three forskolin derivatives in the activation of AC via the EP2 and EP4 receptors, and there was no significant difference in the activation of the IP receptor (Figure 6).

[0088] All three forskolin derivatives activate adenylate cyclase via the aforementioned receptors even in the presence of treprostinil. Treprostinil alone (control sample) does not show activation of AC. It is clear from Figure 6 that forskolin, 7-DAFSK, and 7-DAFSK-PVP enhanced the activity above the control. Therefore, these results suggest that 7-DAFSK-PVP is a suitable stimulant for adenylate cyclase.

[0089] The assay was performed as follows. Day 1: HEK293 cells stably expressing either the EP2, EP4, or IP receptor were grown in 2 mL of DMEM medium containing 10% FCS and 2 μCi 3 H]-adenine and cultured for 16 hours.

[0090] Day 2: The cells were stimulated with 30 μM treprostinil alone or in combination with a 30 μM forskolin DMSO solution, 100 μM 7-DAFSK aqueous solution, or a 7-DAFSK-PVP K12 complex aqueous solution containing 100 μM 7-DAFSK (in fresh medium, at room temperature for 30 minutes). Next, the cells were lysed with 2.5% perchloric acid (PCA) under ice-cold conditions for 30 minutes. This PCA extract was neutralized with KOH and 3 H]cAMP was separated by successive chromatography using DOWEX and alumina columns. Next, radioactivity was measured using a scintillator.

[0091] Radioactivity was measured using a fixed amount of PCA extract from each well. Example 6 Attempts to prepare forskolin-PVP complex by dissolution method In a magnetic crucible, 20 mg of FSK and 40 mg of PVP 25 were pulverized, 300 μL of ethanol (propanol as an alternative) was added and stirred. After 10 minutes, 100 μL of H2O was added, stirred, and left standing for another 10 minutes. Next, this solution was left standing at room temperature for 45 minutes, 3 mL of H2O was added, and stirred for 20 - 30 minutes. Next, this suspension was sucked up with a syringe and filtered through a 0.45 μm filter (hydrophilic). The crucible was rewashed with 3 mL of H2O, and this washing solution was also filtered through the filter. The content of FSK dissolved in 6 mL of H2O was measured by HPLC. The mass fraction of FSK in the whole complex less than 0.01 wt% was measured. FSK could not be detected in the filtrate by HPLC. In certain embodiments, the present invention may be as follows. [Embodiment 1] A complex of 7-deacetyl-forskolin (7-DAFSK) and polyvinylpyrrolidone (PVP). [Embodiment 2] The complex according to Embodiment 1, characterized in that PVP has an average molar mass relative to the weight-average molar mass of 1 - 40 kD, preferably 2 - 25 kD, particularly preferably about 2.5 kD. [Embodiment 3] The complex according to Embodiment 1 or 2, characterized in that 7-DAFSK is derived from synthetic forskolin. [Embodiment 4] The complex according to any one of Embodiments 1 - 3, characterized in that the average mass fraction of 7-DAFSK in the whole complex is in the range of 1 - 50 wt%, preferably 30 wt% or more. [Embodiment 5] A process for preparing the complex according to any one of Embodiments 1 - 4, characterized in that the mixture of 7-DAFSK and PVP is heated to a temperature above the glass transition temperature of the PVP used. [Aspect 6] The process according to aspect 5, characterized in that a solvent, preferably water, ethanol, methanol, acetone, ethyl methyl ketone, dichloromethane, and / or ethyl acetate, more preferably water, ethanol, methanol, propanol, and / or dichloromethane, even more preferably water and / or ethanol or a mixture thereof, is added to the mixture of 7-DAFSK and PVP. [Aspect 7] The process according to aspect 5 or 6, characterized in that the mixture is kept at a temperature above the glass transition temperature of the PVP used for at least 5 minutes. [Aspect 8] a) Preparing a mixture of 7-DAFSK and PVP, and adding a solvent, preferably an alcohol, preferably ethanol, to the mixture, b) Adding water or an aqueous solution, c) Heating the mixture at a temperature above the glass transition temperature of PVP for at least 5 minutes, d) Cooling the mixture and dissolving it in water or an aqueous solution, e) Optionally removing the undissolved components by filtration The method according to any one of aspects 5 to 7, comprising the above steps. [Aspect 9] A pharmaceutical composition comprising the complex according to any one of aspects 1 to 4. [Aspect 10] The pharmaceutical composition according to aspect 9, for use as a medicine. [Aspect 11] The pharmaceutical composition according to aspect 9, characterized in that the composition contains 7-DAFSK at a concentration of at least 25 mg / L, preferably at least 100 mg / mL, 250 mg / mL, 500 mg / mL, 1000 mg / mL, 2000 mg / mL, 3000 mg / mL, or 4000 mg / mL, particularly preferably 4000 mg / mL. [Aspect 12] The pharmaceutical composition according to aspect 9 or 11, for intravenous, oral, or inhalation administration. [Aspect 13] The pharmaceutical composition according to aspects 9 to 12, for use in a treatment method. [Aspect 14] The pharmaceutical composition according to any one of Aspects 9 to 13 for use in the treatment of cardiovascular diseases, bronchial asthma, obesity, glaucoma, lung diseases, joint inflammation, or osteoarthritis / arthritis.

Claims

1. A complex of 7-deacetyl-forskolin (7-DAFSK) and polyvinylpyrrolidone (PVP).

2. The complex according to claim 1, characterized in that the PVP has an average molar mass relative to the weight-average molar mass of 1 to 40 kDa, preferably 2 to 25 kDa, particularly preferably about 2.5 kDa.

3. The complex according to claim 1 or 2, characterized in that the 7-DAFSK is derived from synthetic forskolin.

4. The complex according to any one of claims 1 to 3, characterized in that the average mass fraction of 7-DAFSK in the whole complex is in the range of 1 to 50% by weight, preferably 30% by weight or more.

5. A process for the preparation of a complex according to any one of claims 1 to 4, characterized in that a mixture of 7-DAFSK and PVP is heated to a temperature above the glass transition temperature of the PVP used.

6. A solvent, preferably water, ethanol, methanol, acetone, ethyl methyl ketone, dichloromethane, and / or ethyl acetate, more preferably water, ethanol, methanol, propanol, and / or dichloromethane, even more preferably water and / or ethanol or a mixture thereof, is added to the mixture of 7-DAFSK and PVP, the process according to claim 5.

7. The process according to claim 5 or 6, characterized in that the mixture is kept at a temperature above the glass transition temperature of the PVP used for at least 5 minutes.

8. a) preparing a mixture of 7-DAFSK and PVP and adding a solvent, preferably an alcohol, preferably ethanol, to the mixture, b) adding water or an aqueous solution, c) heating the mixture at a temperature above the glass transition temperature of the PVP for at least 5 minutes, d) cooling the mixture and dissolving it in water or an aqueous solution, e) optionally removing the undissolved components by filtration The process according to any one of claims 5 to 7, comprising.

9. A pharmaceutical composition comprising the complex according to any one of claims 1 to 4.

10. The pharmaceutical composition according to claim 9 for use as a medicament.

11. The pharmaceutical composition according to claim 9, characterized in that the composition contains 7-DAFSK at a concentration of at least 25 mg / L, preferably at least 100 mg / mL, 250 mg / mL, 500 mg / mL, 1000 mg / mL, 2000 mg / mL, 3000 mg / mL, or 4000 mg / mL, particularly preferably 4000 mg / mL.

12. The pharmaceutical composition according to claim 9 or 11, for intravenous, oral, or inhalation administration.

13. The pharmaceutical composition according to any one of claims 9 to 12, for use in the treatment of cardiovascular diseases, bronchial asthma, obesity, glaucoma, lung diseases, joint inflammation, or osteoarthritis / arthritis.

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