Cellular uptake

Abstract

To provide cellular uptake.SOLUTION: The invention provides a bile acid or a pharmaceutically acceptable salt thereof for use in a method for treating the human or animal body, the method comprises administering to the human or animal body a therapeutic compound, where: a. the bile acid is chenodeoxycholic acid or deoxycholic acid; b. optionally, the bile acid is employed in conjunction with EDTA; c. in the method, the bile acid or salt thereof and EDTA are used to enable or enhance intracellular uptake of the therapeutic compound.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Technical Field The present invention relates to a method for introducing substances into living cells, particularly intestinal cells. The method is applicable to the delivery of therapeutic compounds (including macromolecules such as proteins) into cells and can be used in the treatment of diseases involving the use of therapeutic compounds to exert a therapeutic effect after entering the intracellular environment. [Background technology]

[0002] Background technology The following discussion of the background art is intended solely to facilitate an understanding of the present invention and is not intended to constitute an admission or acknowledgement that any of the material cited is or was ever part of the common general knowledge as of the priority date of this application.

[0003] Because uptake of therapeutic compounds into cells is axiomatic for medical applications, there is great scientific interest in facilitating this uptake. Although various approaches have been widely explored, challenges exist within these approaches, meaning that there remains a significant need for improved methods to achieve this goal.

[0004] Therefore, living cells have many specific mechanisms for controlling the uptake of substances into their interior, mostly through the action of conformationally selective receptors embedded in their surface membranes. However, in general, cellular structures are designed to exclude hydrophilic molecules from the interior of the cell, since the phospholipid membrane surrounding all mammalian cells is an essentially hydrophobic barrier that prevents the passage of water-soluble molecules. This is especially true for macromolecules, and a large part of recent pharmaceutical research has been devoted to promoting the entry of macromolecules into cells.

[0005] Intracellular uptake can obviously be useful for the introduction of therapeutic compounds that exert beneficial effects inside cells, for example, in cases such as gene therapy or enzyme replacement therapy. Gene therapy treats diseases through the production of therapeutic proteins inside cells. Therefore, for nucleic acid molecules used in gene therapy, the target site is mostly located inside the cell, in the cytoplasm or nucleus, and therefore the nucleic acid molecule must cross the plasma membrane to reach the target site. However, most genetic molecules are large and charged, which makes it difficult for them to cross the plasma membrane on their own, so an appropriate gene delivery system is required for efficient cellular uptake. Synthetic or non-viral gene delivery systems can avoid some of the problems associated with viral vectors, such as non-specific inflammation and unexpected immune responses. Furthermore, non-viral vectors are advantageous in terms of simplicity of use and ease of mass production. However, their relatively low efficiency is a major drawback of non-viral vectors, and efforts to solve this problem continue.

[0006] One of the various methods developed to date for the introduction of substances into living cells is to reduce the polarity or hydrophilicity of therapeutic compounds, for example, by complexing them with lipophilic moieties. Thus, electrostatic binding of DNA to cationic lipids, such as lipofectin, can enhance DNA uptake (Felgner PL (1991) Cationic liposome-mediated transfection with lipofectin). TM (Reagent. Methods Mol Biol 7:81-9). However, such methods suffer from inefficiency, and in the case of cationic lipids, high levels of cytotoxicity can be observed depending on the cell type and reagent density, making them technically undesirable for in vivo use. Problems of stability and lack of reproducibility also arise. For certain protein molecules, a similar effect can be achieved by chemically conjugating the protein with long-chain hydrocarbons, but such procedures can increase the immunogenicity of the protein involved.

[0007] Another approach that has been considered, particularly when the therapeutic compound is a macromolecule, is to encapsulate the therapeutic compound inside a specific carrier (e.g., liposome), which can then be easily taken up by phagocytes. Such technology has been used with some success as a potential treatment method for Gaucher's disease (genetic deficiency of glucocerebrosidase), in which this enzyme is incorporated into liposomes and delivered to cells that lack the ability to degrade glucosylceramide (Belchetz PE, Crawley JC, Braidman IP, Gregoriadis G (1977) Treatment of Gaucher's disease with liposome-entrapped glucocerebroside: beta-glucosidase. Lancet. 16; 2 (8029): 116-7).Recently, beta-galactosidase (Umezawa F,Eto Y,Tokoro T,Ito F,Maekawa K.Enzyme replacement with liposomes containing beta-galactosidase from Charonia lumpas in murine globoid cell leukodystrophy(twitcher)Biochem Biophys Res Commun.1985 Mar 15;127(2):663-7) and transglutaminase (Aufenvenne K,Fernando Larcher,Ingrid Hausser,Blanca Duarte,Vinzenz Oji,Heike Nikolenko,Marcela Del Rio,Margitta Dathe and Heiko Traupe(2013)Topical Enzyme-Replacement Therapy Restores Transglutaminase 1 Activity and Corrects Architecture of Transglutaminase-1-Deficient Skin Grafts The American Journal of Human Genetics 93,620-630,October Similar studies have been conducted using vesicles (Schmidt et al., 2013). This method is optimal for cells that exhibit significant phagocytic activity. For other cell types, specific targeting can be facilitated by incorporating polyethylene glycol-lipids and target-specific antibodies into the outer membrane. This can ensure vesicle binding to the outer surface of the membrane, but does not guarantee internalization.

[0008] Another approach, which is also applicable when the therapeutic compound is a protein, is to conjugate the protein of interest to a ligand that can specifically interact with a receptor molecule that promotes internalization of the peptide. An example of this approach is an immunotoxin in which tumor internalization of ricin A chain is enhanced by linking it to an antibody molecule that targets the tumor-specific antibody (ML Grossbard, JG Gribben, AS Freedman, JM Lambert, J Kinsella, SN Rabinowe, L Eliseo, JA Taylor, WA Blattler and CL Epstein (1993) Adjuvant Immunotoxin Therapy With Anti-B4-Blocked Ricin After Autologous Bone Marrow Transplantation for Patients With B-Cell Non-Hodgkin's Lymphoma Blood 81 2263-2271; and Antignani A and FitzGerald D. (2013) Immunotoxins: The Role of the Toxin Toxins 5 1486-1502). Abrin has been used as an immunotoxin for the same purpose (Gadadhar S, Karande AA (2013) Abrin Immunotoxin: Targeted Cytotoxicity and Intracellular Trafficking Pathway. PLoS ONE 8(3):e58304). However, this method is only applicable to highly potent drugs because the ratio of macromolecule to antibody is approximately 1:1.

[0009] Regarding other possible approaches, some approaches have started from the point that several pathways have been characterized that allow small molecules (such as protein degradation products formed by proteolysis) to pass through intestinal cells in a selective manner. Thus, approaches have been considered in which therapeutic compounds such as macromolecules can be conjugated to such small molecules, which can then be recognized by the relevant receptor, so that the entire conjugate can then be pulled through the membrane. These pathways that use specific receptors on the cell surface include vitamin uptake pathways for vitamin B12 or biotin (using dipeptidyl receptors), and pathways involving receptors for conjugated bile salts. In the case of dipeptidyl receptors, receptor density can be affected by environmental conditions and the total daily requirement of these vitamins in terms of quantity is low, so they act relatively slowly, limiting the amount of substance delivered. In contrast, bile salt receptors can internalize tens of grams of material daily. Therefore, they have great potential as a delivery mechanism, and approaches have been described in which compounds (to be delivered) are attached to bile salts, with enhanced cellular uptake of the resulting conjugates. Although limited success has been claimed with certain peptides (see, e.g., U.S. Pat. No. 7,153,930), the molecules attached to bile salts are generally small, low-molecular-weight entities. In this regard, it is generally believed that bile salts must be in a monomeric form (i.e., without additional bile salts associated) to bind to the relevant receptor, and that therapeutic compounds must be covalently bound to the bile salt. Indeed, given that binding between bile salts and their receptors occurs through noncovalent interactions, a single bile salt molecule would generally be expected to be too small to interact with therapeutic compounds via noncovalent interactions. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] U.S. Patent No. 7,153,930

Non-licensed literature

[0011] [Non-licensed document 1] Felgner PL(1991)Cationic liposome-mediated transfection with lipofectinTM reagent.Methods Mol Biol 7:81-9 [Non-licensed document 2] Belchetz PE, Crawley JC, Braidman IP, Gregoriadis G (1977) Treatment of Gaucher's disease with liposome-entrapped glucocerebroside:beta-glucosidase. Lancet.16;2(8029):116-7 [Non-licensed document 3] Umezawa F,Eto Y,Tokoro T,Ito F,Maekawa K.Enzyme replacement with liposomes containing beta-galactosidase from Charonia lumpas in murine globoid cell leukodystrophy(twitcher)Biochem Biophys Res Commun.1985 Mar 15;127(2):663-7

Non-licensed Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Summary of the Invention

[0012] The present invention relates to a novel approach for facilitating the cellular uptake of compounds. The invention is based on the surprising finding that certain bile acids and / or pharmaceutically acceptable salts thereof (also referred to herein as "bile acid(s) / salt(s)") can interact with receptors on the cell surface. (Interestingly, this effect is observed only for two specific unconjugated bile acids / salts, namely, chenodeoxycholic acid and deoxycholic acid and their salts—not for conjugated bile acids / salts, or indeed for other unconjugated bile acids / salts.) In this regard, bile salts, particularly in micellar form, can interact with more than one receptor at a time, and the resulting aggregation of receptors on the membrane surface can be expected to induce membrane invagination and internalization (i.e., cellular uptake of substances from outside the cell) (e.g., of receptors and micelles, among others). The present invention relates to the utilization of this newly discovered pathway to facilitate the uptake of substances into cells, particularly via a process that stimulates vesiculation.

[0013] Preferably, the methods of the present invention described herein use receptor-mediated internalization of bile acids or their salts via clathrin-coated pits to enable or enhance the cellular uptake of therapeutic compounds. Thus, the mechanism by which bile acids / salts interact with cell surface receptors can trigger an internalization process, which can be utilized to enable or enhance the cellular uptake of therapeutic compounds, even macromolecular compounds. In this regard, in one aspect of the present invention, cellular uptake of therapeutic compounds is possible by simultaneously positioning the therapeutic compound and the bile acid / salt in close proximity to the cell surface. Thus, in a preferred embodiment of the methods of the present invention, receptor-mediated internalization of bile acids or their salts via clathrin-coated pits is used to enable or enhance the cellular uptake of one or more therapeutic compounds positioned around the cell (but not conjugated to a bile acid / salt). The aforementioned process is concentration-dependent, and despite the fact that bile acids or their salts are generally used at relatively high concentrations, they can be effective at concentrations that are not toxic to cells. Fluorescence studies have shown that bile acids or their salts enable or enhance the uptake of even large (macromolecular) therapeutic compounds, which are then visible in intracellular vacuoles. Studies have also shown that the inclusion of a clathrin inhibitor blocks the intracellular absorption-enhancing effect of bile acids or their salts, indicating that the mechanism of action of bile acids or their salts is clathrin-mediated endocytosis.

[0014] It has also been found that EDTA can further enhance protein uptake stimulated by chenodeoxycholate. Unexpectedly, while EDTA alone has no effect on uptake at non-toxic concentrations, EDTA can enhance the stimulation seen with chenodeoxycholate, and in some cases, combining these two agents results in uptake even when the two agents are used individually at the same concentrations and no uptake is observed. Thus, it appears that EDTA can cooperate with chenodeoxycholate to enhance protein uptake into enterocytes. This phenomenon is unlikely to be simply caused by chelating activity, since other agents known to exhibit chelating activity like EDTA (e.g., DTPA, EGTA, ortho-phenanthroline) do not exhibit the same effect as EDTA. A further feature of the present invention is that a formulation containing a combination of chenodeoxycholate and EDTA can be used to enhance protein uptake by enterocytes, and this combination can provide a more effective means of cellular uptake than chenodeoxycholate alone. In this way, uptake and passage through the enterocyte barrier is possible, thereby introducing macromolecules into the body as a result of administration via the oral route. The concentration of EDTA on the surface of cells where cellular uptake is to be enabled or enhanced can range from 0.1 mg / ml to 10 mg / ml, preferably 0.2 mg / ml to 10 mg / ml.

[0015] Prior to the present invention, it was believed that bile acids / salts were not known to be capable of assisting the cellular uptake of macromolecular therapeutic compounds. Rather, it was generally assumed that uptake would not occur unless bile salts were in a monomeric form covalently linked to the therapeutic compound. It was also believed that it was not known that receptors could bind to and be activated by either of two specific bile salts, chenodeoxycholate and deoxycholate. Accordingly, the scientific literature has understood that such unconjugated bile salts are taken up into the body from the gastrointestinal lumen primarily by passive diffusion across the cell membrane of enterocytes (e.g., Trauner et al. (Physiol Rev, Vol 83, April 2003); Stamp et al. (An Overview of Bile-Acid Synthesis, Chemistry and Function, Issues in Toxicology, Bile Acids: Toxicology and Bioactivity, Royal Society of Chemistry, 2008); Michael W. King, PhD (Bile Acid Synthesis and Utilization, 1996-2014 (C) themedicalbiochemistrypage.org) and Dawson et al (J Lipid Res. 2009 Dec;50(12):2340-2357), Dawson & Karpen (Journal of Lipid Research, Volume 56, 2015, pages 1085-1099 - see in particular Figure 1, which shows that receptor-mediated uptake of unconjugated chenodeoxycholate into the small intestine appears to be unknown), Ferrebee et al (Acta Pharmaceutica Sinica B 2015;5(2):129-134), and Ninomiya et al (Biochimica et Biophysica Acta 1634(2003)116-125)). In contrast, conjugated tauro- or glycol-bile acids and salts are said to be required for active transport mechanisms—for example, the uptake of conjugated bile acids / salts has previously been described as operating via specific receptors—and although these receptors may be capable of recognizing unconjugated bile salts, their affinity suggests that conjugated bile salts are the most important. Therefore, while receptors that bind and are activated by two specific unconjugated bile acids / salts (chenodeoxycholate and deoxycholate) in the context described herein are believed to be unreported in the literature as not binding and / or being activated by either (i) conjugated bile acids / salts or (ii) other unconjugated bile acids / salts.

[0016] In this regard, as background for the bile acids / salts used in the present invention, bile acids as a general class are one of a wide variety of compounds that have been previously considered for their potential use as agents that can help promote the absorption of therapeutic compounds across cell barriers. For example, the types of compounds that have been described for this purpose include chelating agents (such as EDTA or EGTA); nonionic surfactants (such as polyoxyethylene ethers, pt-octylphenol polyoxyethylene, nonylphenoxy-polyoxyethylene, and polyoxyethylene sorbitan esters); anionic agents (such as cholesterol derivatives (including bile acids)); cationic agents (such as acylcarnitines, acylcholines, lauroylcholines, cetylpyridinium chloride, and cationic phospholipids); and other agents (such as α-galactosidase, β-mannanase, sodium caprate, sodium salicylate, n-dodecyl-β-D-maltopyranoside, N,N,N-trimethylchitosan chloride (TMC), cyclodextrins, and NO-donating compounds).

[0017] The use of bile salts as absorption enhancers has recently been reviewed by Moghimipour et al. (Absorption-Enhancing Effects of Bile Salts, Molecules 2015, 20, 14451-14473). As evident from Table 1 of this paper, the focus is on conjugated bile acids (i.e., bile acids with a glycosylated or tauro group at the C-24 position). This may be due in part to the fact that conjugated salts have been reported to have superior emulsifying properties compared to unconjugated bile salts. For example, with regard to oral drug delivery, several mixed results have been reported for sodium taurodeoxycholate, along with some positive results for sodium glycolate (see pages 14457 and 14458 of Moghimipour et al.).

[0018] Regarding possible mechanisms of action that suggest how bile acids may enhance absorption, these mechanisms include damaging / opening tight junctions between epithelial cells to facilitate paracellular transport (e.g., via binding Ca2+ in the intercellular space, disrupting hemidesmosomes, interacting with filamentous actin, and / or forming reverse micelles), and protease inhibition. Several papers on pulmonary and oral administration routes have described that bile salts can enhance paracellular transport in a concentration-dependent manner, while certain conjugated bile salts, especially at high concentrations, may also promote the uptake of a certain proportion of material into cells (Nicolazzo et al., Journal of Controlled Release 105 (2005) 1-15; Hoogstraate et al., Journal of Controlled Release 40 (1996) 211-221; Hussain et al., Journal of Controlled Release 94 (2004) 15-24; and Dodla et al., Asian J Pharm Clin Res, Vol. 6, Issue 3, 2013, pp. 39-47). However, this is suggested to be due to disruption / disruption of cell membranes, for example, by lipid extraction (which is not a desirable approach for delivering therapeutic compounds into cells). Thus, despite potential adverse effects on membranes (e.g., due to disruption of important cellular structures and / or functions) and the fact that a significant proportion of compounds in these cases take the intracellular route, disrupting membranes in this manner may allow the penetration of toxic or otherwise undesirable substances that may be present near the disrupted area. Furthermore, the high concentrations of conjugated bile acids that need to be used before such adverse effects are observed to any significant extent are generally too high from a toxicological standpoint, in any case.

[0019] As described above, the present invention is based on the surprising finding that two unconjugated bile acids / salts, namely, chenodeoxycholic acid and deoxycholic acid, and their salts, can facilitate the uptake of substances into cells by interacting with receptors on the cell membrane (while other bile salts, including more commonly conjugated bile salts, cannot). Therefore, according to the present invention, bile acids / salts can be used to induce the uptake of therapeutic compounds (such as macromolecules), thereby assisting their delivery to intracellular compartments, such as the cytoplasm or nucleus. Furthermore, this effect is concentration-dependent; bile acids / salts are generally used at relatively high concentrations, but are effective at concentrations that are not toxic to cells. Studies have shown that bile acids / salts enable or enhance the uptake of even large (macromolecular) therapeutic compounds, which are then visible in intracellular vacuoles. The inclusion of a clathrin inhibitor has also been shown to block the intracellular uptake-enhancing effect of bile acids / salts, indicating that the mechanism of action is clathrin-mediated endocytosis.

[0020] Accordingly, the present invention provides a bile acid, or a pharmaceutically acceptable salt thereof, for use in a method of treatment of the human or animal body, said method comprising the step of administering to the human or animal body a therapeutic compound together with a bile acid, wherein said bile acid is chenodeoxycholic acid or deoxycholic acid.

[0021] Preferably, bile acids or their salts are used to enable or enhance cellular uptake of the therapeutic compound. Optionally, the bile salts are used in conjunction with EDTA.

[0022] The present invention also provides a method of treating the human or animal body, comprising administering to the human or animal body a therapeutic compound together with a bile acid, wherein the bile acid is chenodeoxycholic acid or deoxycholic acid, and the bile acid or a pharmaceutically acceptable salt thereof is present in an amount sufficient to enable or enhance cellular uptake of the therapeutic compound, optionally using the bile acid salt in combination with EDTA.

[0023] The present invention also provides the use of a therapeutic compound and a bile acid or its salt in the manufacture of a medicament for use in a method for treating a human or animal body in need of the therapeutic compound.Preferably, the bile acid or its salt is present in an amount that allows or enhances the cellular uptake of the therapeutic compound.Optionally, the bile acid or its salt is used in combination with EDTA.

[0024] The present invention also provides a pharmaceutical or therapeutic composition comprising (a) a bile acid or a pharmaceutically acceptable salt thereof, wherein the bile acid is chenodeoxycholic acid or deoxycholic acid; and (b) a therapeutic compound. Preferably, the composition also comprises one or more additional compounds selected from fusogenic lipids, cell-penetrating peptides, lysosomotropic agents, membrane-disrupting peptides, membrane-disrupting polymers, photochemical internalization agents, and agents that alter intracellular vesicle trafficking, and optionally, the bile salt is used in combination with EDTA for this purpose.

[0025] Desirably, in any formulation of the present invention, the bile acid or salt thereof and therapeutic compound are encapsulated by a coating that (a) limits the dissolution of said bile acid or salt thereof and therapeutic compound under aqueous conditions at pH 7.4, but removes the limit on dissolution of said bile acid or salt thereof and therapeutic compound at a pH below 7.4, and / or (b) includes one or more moieties capable of binding to said target cell population in the body.

[0026] As mentioned above, the treatment methods of the present invention described herein involve administering a therapeutic compound to the human or animal body, and in this regard, it is of course intended, as a general matter, that said therapeutic compound then provide the therapeutic effect upon which the treatment method is based. [Brief explanation of the drawings]

[0027] BRIEF DESCRIPTION OF THE DRAWINGS Further features of the present invention will be described in more detail in the following description of several non-limiting embodiments of the invention. This description is for illustrative purposes only and should not be understood as limiting the above summary, disclosure, or description of the invention. This description will be made with reference to the accompanying drawings, in which: [Figure 1] Figure 1 shows photomicrographs of Caco-2 cells after incubation with medium containing (i) FITC-insulin alone (at a concentration of 100 μg / ml), (ii) FITC-insulin with sodium chenodeoxycholate (1 mg / ml), and (iii) FITC-insulin with sodium chenodeoxycholate (2 mg / ml), demonstrating that little uptake occurs in cells treated with insulin alone, but that the presence of chenodeoxycholate enhances uptake in a concentration-dependent manner. [Figure 2] FIG. 2 shows the relative uptake of fluorescent albumin in Caco-2 cells at different time points in (i) the absence of chenodeoxycholate and propyl gallate, (ii) the presence of chenodeoxycholate but in the absence of propyl gallate, and (iii) the presence of low, medium, and high concentrations of chenodeoxycholate and propyl gallate (both). [Figure 3] FIG. 3 shows the effect of different bile salt ranges on the uptake of fluorescent albumin into Caco-2 cells. [Figure 4]Figure 4 shows the results for FITC-BSA uptake for cells adherent to microplate wells demonstrating that chlorpromazine (both high and low concentrations) inhibits uptake stimulated by all concentrations of bile salts. [Figure 5] FIG. 5 shows the results for FITC-BSA uptake demonstrating that for cells in suspension, chlorpromazine (both high and low concentrations) inhibits uptake stimulated by all concentrations of bile salts. [Figure 6] FIG. 6 shows that uptake is significantly enhanced when chenodeoxycholate and EDTA are combined. [Figure 7] FIG. 7 shows that chenodeoxycholate enhances the uptake of hGH into cells in a dose-dependent manner similar to that observed for the uptake of other proteins (such as insulin, BSA, and casein). [Figure 8] FIG. 8 shows that protein uptake into IEC6 cells was enhanced by the presence of chenodeoxycholate in a manner similar to that observed for Caco-2 cells, confirming that this is a phenomenon normally common to enterocytes. [Figure 9] FIG. 9 shows that the enhancement of BSA uptake by chenodeoxycholate is augmented by EDTA in a dose-dependent manner in IEC6 cells. DETAILED DESCRIPTION OF THE INVENTION

[0028] Description of the embodiment For convenience, the following section provides a general overview of various meanings of terms used herein. This discussion is followed by a discussion of general aspects relating to the compositions, pharmaceutical uses, and methods of the present invention, followed by specific examples demonstrating the nature of various embodiments of the invention and the manner in which they may be used. definition

[0029] The meanings of certain terms and phrases used in the specification, examples, and appended claims are provided below. In the event of an apparent discrepancy between the usage of a term in the art and the definition of a term provided herein, the definition provided herein shall control.

[0030] Those skilled in the art will recognize that the invention described herein is capable of variations and modifications other than those specifically described. The invention includes all such variations and modifications. Also, the invention includes all steps, features, formulations, and compounds referred to or depicted herein, individually or collectively, including any and all combinations of steps or features, or any two or more steps or features.

[0031] Each document, reference, patent application, or patent cited herein is expressly incorporated herein by reference in its entirety, meaning that the reader should read and consider these documents to be part of this specification. It is for the sole purpose of brevity that each document, reference, patent application, or patent cited herein is not repeated herein. However, the cited materials and the information contained therein should not be understood to be general knowledge.

[0032] Manufacturer's instructions, manuals, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein are incorporated by reference herein and may be used in the practice of this invention.

[0033] The present invention is not to be limited in scope by any specific embodiments described herein. These embodiments are intended to be exemplary only. Functionally equivalent products, formulations, and methods are clearly within the scope of the invention described herein.

[0034] Except in the operating examples or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein shall be understood to be modified in all instances by the term "about," which, when used in connection with percentages, can mean ±1%.

[0035] The invention described herein can encompass one or more ranges of values ​​(e.g., size, concentration, etc.). A range of values ​​will be understood to include all values ​​within the range (including the values ​​defining the range) and values ​​near said range (where said values ​​are adjacent to the values ​​defining the boundaries of said range, thereby achieving the same or substantially the same results). For example, one skilled in the art will understand that a 10% variation of the upper or lower limit of a range would be entirely appropriate and encompassed by the present invention. More specifically, a variation of the upper or lower limit of a range would be 5% or a variation generally recognized in the art, whichever is greater.

[0036] In this application, the use of the singular includes the plural unless specifically indicated otherwise. In this application, the use of "or" means "and / or" unless specifically stated otherwise. Furthermore, the use of the term "including" and other forms such as "includes" and "included" does not imply limitation. Also, unless specifically indicated otherwise, terms such as "element" or "component" encompass both elements and components that contain one unit and elements and components that contain more than one subunit. Additionally, when the term "moiety" is used, it can include a portion of a component or the entire component.

[0037] Throughout this specification, unless the context requires otherwise, the term "comprise" or variations thereof (such as "comprises" or "comprising") will be understood to mean the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[0038] The terms "reduce," "decreased," "reduction," "reducing," or "inhibit" are all used generally herein to mean a statistically significant amount of reduction. To avoid confusion, however, "decreased," "reduction," or "reducing" or "inhibit" means a reduction of at least 10% (e.g., or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%), or at least more than about 60%, or at least about 70%, or at least about 80% reduction) compared to a baseline level (e.g., in the absence of an agent).

[0039] The terms "increased," "increase," or "enhance," or "activate" are all used generally herein to mean an increase by a statistically significant amount; for the avoidance of any doubt, the terms "increased," "increase," or "enhance," or "activate" mean an increase of at least 10% compared to baseline levels (e.g., in the absence of agent) (e.g., at least about 20%, or at least about 30%, or at least about 40%, or at least about 50% compared to baseline levels), or at least about 60%, or at least about 70%, or at least about 80%, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold increase, or any increase between 2-fold and 10-fold or more).

[0040] As used herein, the term "administering" refers to the placement of a composition within a subject by a method or route that localizes at least a portion of the composition at a desired site to achieve a desired effect. The compounds or compositions described herein can be administered by any suitable route known in the art, including, but not limited to, oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, respiratory (aerosol), pulmonary, intranasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, the compounds are administered parenterally or by other methods that allow delivery to a target site.

[0041] Other definitions for selected terms used herein may be found within the detailed description of the invention and may be applied throughout. Unless otherwise defined, all other scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention belongs.

[0042] Aspects of the present invention will now be discussed with reference to the following non-limiting descriptions and examples. Embodiment

[0043] Accordingly, the present invention provides a bile acid, or a pharmaceutically acceptable salt thereof, for use in a method of treatment of the human or animal body, said method comprising the step of administering to the human or animal body a therapeutic compound together with a bile acid, wherein said bile acid is chenodeoxycholic acid or deoxycholic acid.

[0044] In this regard, the bile acid is preferably chenodeoxycholic acid. It is also preferred to use the bile acid in the form of a salt. Therefore, preferably, the bile acid or its pharmaceutically acceptable salt is a salt of chenodeoxycholic acid or deoxycholic acid, more preferably a salt of chenodeoxycholic acid.

[0045] The pharmaceutically acceptable salt of bile acid is typically a salt with a pharmaceutically acceptable base. Pharmaceutically acceptable bases include alkali metals (e.g., sodium or potassium), alkaline earth metals (e.g., calcium or magnesium), hydroxides, and organic bases (e.g., alkylamines, aralkylamines, and heterocyclic amines). Alkali metals, particularly sodium, are preferred. Therefore, most preferably, the bile acid or its pharmaceutically acceptable salt is sodium chenodeoxycholate.

[0046] It is possible for more than one of the bile acids and / or salts to be present (e.g., two, three, or more, preferably two or three, more preferably two), but typically only one is present.

[0047] The bile acids or salts thereof for use in the methods of the present invention are preferably contained in a pharmaceutical composition.

[0048] The therapeutic compound for use according to the present invention can be any compound that has a therapeutic effect inside the human or animal body (including inside one or more cells in the human or animal body). In this regard, generally, reference herein to a therapeutic compound for use according to the present invention is intended to encompass the possibility of using more than one therapeutic compound (such as two, three, or more therapeutic compounds). However, typically, reference to a therapeutic compound preferably refers to only one therapeutic compound.

[0049] As mentioned above, the effect underlying the present invention is sufficiently robust that even the cellular uptake of large macromolecules is possible. Considering that it may be difficult to enable and / or enhance the uptake of such physically large molecules into cells, the effectiveness of the present invention is particularly useful in this context. Therefore, preferably, the therapeutic compound for use according to the present invention is a macromolecule. In this regard, the therapeutic compound preferably has a molecular weight of approximately 1000 Da or more (for example, 2000 Da or more, or 3000 Da or more, etc.).

[0050] The therapeutic compound is preferably, but not necessarily, a peptide, more preferably a polypeptide, and even more preferably a protein. Examples of suitable therapeutic compounds include insulin, calcitonin, human serum albumin, growth hormone, growth hormone-releasing factor, galanin, parathyroid hormone, peptide YY, oxyntomodulin, blood clotting proteins (such as kinogen, prothrombin, fibrinogen, factor VII, factor VIII, or factor IX), erythropoietin and EPO mimetics, colony-stimulating factors (including GCSF and GMCSF), platelet-derived growth factor, epidermal growth factor, fibroblast growth factor, Transforming growth factors; GLP-1, GLP-2; GLP-1 analogs and fusion proteins, GIP, glucagon; exendins; leptin; GAGs; cytokines; insulin-like growth factors; bone and cartilage induction factors; neurotrophic factors; interleukins (IL-I, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12); interferons (interferon gamma, interferon-1a, interferon-alfa, TNF-alpha; TNF-beta; TGF-beta; cholera toxin A and B fragment; E. coli enterotoxin A and B fragment; secretin; enzymes (including histone deacetylase, superoxide dismutase, catalase, adenosine deaminase, thymidine kinase, cytosine deaminase, proteases, lipases, carbohydrases, nucleotidases, polymerases, kinases, and phosphatases); transport or binding proteins, particularly those that bind and / or transport vitamins, metal ions, amino acids, or lipids or lipoproteins (such as cholesterol ester transfer protein, phospholipid transfer protein, HDL binding protein, etc.); connective tissue proteins (such as collagen, elastin, or fibronectin); muscle proteins (such as actin, myosin, dystrophin, or mini-dystrophin); nerve, liver, heart, or adipocyte proteins; cytotoxic proteins; cytochromes; proteins that enable cells to replicate, grow, or differentiate;These include, but are not limited to, signaling molecules (such as intracellular or extracellular signaling proteins (e.g., hormones)); trophic factors (such as BDNF, CNTF5, NGF, IGF, GMF, aFGF, bFGF, VEGF, NT3, T3, and HARP); apolipoproteins; antibody molecules, antibody fragments, single-domain antibodies; soluble forms of receptors (such as T cell receptors and receptors for cytokines, interferons, or chemokines); proteins or peptides containing antigenic epitopes and fragments; and albumin fusion proteins, derivatives, conjugates, and sequence variants of any of the above. These and other proteins may be derived from human, plant, animal, bacterial, or fungal sources and may be extracted from natural sources, prepared recombinantly by fermentation, or chemically synthesized. In a preferred embodiment, the therapeutic compound is a peptide selected from insulin, calcitonin, growth hormone, growth hormone-releasing factor, galanin, parathyroid hormone, peptide YY, oxyntomodulin, erythropoietin, colony-stimulating factor, platelet-derived growth factor, epidermal growth factor, fibroblast growth factor, transforming growth factor, GLP-1, GLP-2, GIP, glucagon, exendin, leptin, neurotrophic factor, insulin-like growth factor, chondrogenic factor, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, interferon-gamma, interferon-1a, interferon-alpha, conjugates of any of the foregoing, fusion proteins comprising any of the foregoing, and combinations thereof;

[0051] Therapeutic compounds for use in the present invention are preferably comprised in a pharmaceutical composition.

[0052] It is possible to administer the bile acid / salt and the therapeutic compound (and / or, if present, any additional agents for use in the method) separately. Preferably, however, the aforementioned methods of the present invention comprise administering a composition comprising (both) the bile acid / salt and the therapeutic compound. Furthermore, when one or more additional agents are used in combination with the bile acid / salt in the method, the aforementioned composition also preferably comprises one or more of the aforementioned one or more additional agents, typically all of the aforementioned one or more additional agents.

[0053] In the methods of the present invention, the upper limit of the desired concentration of bile acids / salts on the surface of cells into which therapeutic compound uptake is enabled or enhanced can be determined by the level at which the bile acids / salts are not toxic to the (target) cell type and / or surrounding cells in vivo. Thus, in the methods of the present invention, the concentration of bile acids / salts on the surface of cells into which intracellular uptake is enabled or enhanced must be below a level that would be toxic to the (target) cell type and / or surrounding cells.

[0054] In the methods of the present invention, the lower limit of the desired bile acid / salt concentration on the surface of cells (at which uptake of therapeutic compounds into the cells becomes possible or enhanced) can preferably correspond to the critical micelle concentration (CMC) (i.e., the concentration above which bile acids / salts form micelles). The exact CMC may vary depending on, among other factors, temperature, pressure, and the presence and concentration of other agents (particularly surfactants and electrolytes), and can be determined experimentally. Thus, in the methods of the present invention, the concentration of bile acid / salt on the surface of cells at which intracellular uptake becomes possible or enhanced preferably exceeds the CMC of the bile acid / salt (in that particular environment).

[0055] Generally, in the methods of the present invention, the concentration of bile acids / salts on the surface of the cells into which uptake of a therapeutic compound is to be enabled or enhanced is preferably in the range of 0.1 mg / ml to 500 mg / ml, preferably 0.5 mg / ml to 100 mg / ml, more preferably 1 mg / ml to 50 mg / ml, and even more preferably 2 mg / ml to 40 mg / ml (such as 5 mg / ml to 20 mg / ml).

[0056] The content of bile acid / salt in the pharmaceutical composition to be administered to patient is preferably selected so as to achieve such concentration around the cell that is desired to be taken up.This may vary depending on, for example, the mode of administration and the nature and size of target site.Therefore, those skilled in the art can adapt the concentration.

[0057] In one embodiment, the amount of bile acid / salt can be 0.1 mg to 500 mg, preferably 0.5 mg to 100 mg, more preferably 1 mg to 50 mg, and even more preferably 2 mg to 40 mg (e.g., 5 mg to 20 mg) per cubic centimeter of target cells (e.g., tumor cells). These amounts are particularly useful when the bile acid or its salt and the therapeutic compound are contained in a single composition, which is intended for administration into the bloodstream (e.g., intravenously) in a form that will release the bile acid or its salt and the therapeutic compound to a specific location in the body (e.g., the acidic environment of tumor cells).

[0058] In another embodiment, the amount of bile acid / salt in the composition can be at least 0.1 mg, preferably at least 1 mg, more preferably at least 10 mg, and even more preferably at least 20 or at least 50 mg. The amount of bile acid / salt in the composition can be up to 1 g, preferably up to 500 mg, more preferably up to 200 mg, and even more preferably up to 100 mg. Typical amounts are 50-100 mg (e.g., 60-80 mg, or approximately 70 mg). These amounts are particularly useful when the bile acid / salt and therapeutic compound are included in an oral composition intended to achieve uptake of the therapeutic compound and bile acid / salt into enterocytes (e.g., by preventing release of the contents into the stomach via an enteric coating), because when the contents of the composition are released into the intestine, the therapeutic compound and bile acid / salt will typically become dispersed primarily in a localized aqueous solution of several milliliters (e.g., up to approximately 5 milliliters).

[0059] The amount of therapeutic compound for use in accordance with the present invention may depend on the underlying disease or condition, the nature and size of the target cell population, the type and severity of the disease, the therapeutic compound, the age, weight, and condition of the patient, and the mode and frequency of administration. Typical dosage levels of therapeutic compounds are 0.01 to 100 mg / kg (e.g., 0.1 to 10 mg / kg), although one of skill in the art would be able to select an appropriate amount.

[0060] The ratio of bile acid / salt to therapeutic compound to be administered in combination according to the present invention is preferably 100:1 to 1:1 by weight, more preferably 70:1 to 3:2, even more preferably 50:1 to 2:1, more preferably 30:1 to 4:1, and most preferably 20:1 to 5:1 (e.g., 15:1 to 10:1, etc.).

[0061] As described above, the methods of the present invention involve the use of bile acids / salts to enable or enhance cellular uptake of therapeutic compounds, preferably receptor-mediated internalization of bile acids or their salts via clathrin-coated pits. In this regard, in one aspect, a therapeutic compound can be internalized by simultaneously positioning it in sufficient proximity to the cell surface with the bile acid / salt. However, in a second (non-mutually exclusive) aspect, a therapeutic compound may be internalized by directly associating with the bile acid / salt (e.g., in the form of a micelle) via a non-covalent interaction. While the following embodiments relate to both aspects, it will be apparent that in some cases a given embodiment will be particularly relevant to the second of these two aspects of the invention.

[0062] Thus, in a preferred embodiment of the method of the present invention, bile acids / salts are used in combination with one or more additional agents.

[0063] In one embodiment, the one or more additional agents are: a) stabilize (against dissociation) the micellar form of bile acids or salts thereof; and / or b) enabling or enhancing the formation of micellar forms of bile acids or their salts; Contains one or more drugs.

[0064] Suitable agents for these uses include those that are or contain hydrophobic entities. Because an aqueous environment would likely expose the hydrophobic entities to water, the hydrophobic entities can be incorporated into micelles, thereby making disruption of the micelles energetically less likely in an aqueous environment. In one embodiment, the therapeutic compound itself may, at least to some extent, help stabilize and / or enable or enhance the formation of the micellar form, for example, when the therapeutic compound contains a hydrophobic moiety (such as a lipid tail composed of long hydrocarbon chains). In this embodiment, the inclusion of an additional agent may not be necessary.

[0065] However, in any event, it is generally advantageous to include one or more additional (separate) agents that stabilize and / or enable or enhance the formation of micellar morphology, as this can help facilitate the internalization process. Agents that can generally be used in this regard include any agent with a hydrophobic moiety, such as a long (e.g., C4 or greater, C5 or greater, or C6 or greater, e.g., up to C30, C20, or C12) hydrocarbyl chain. Examples include fatty acids or steroids, such as cholesterol (i.e., the result of disruption of lipid structures in foods), and hydrophobic antioxidants, such as aromatic alcohols, including propyl gallate and butylated hydroxyanisole. Propyl gallate is particularly preferred.

[0066] In this regard, in embodiments of the invention in which the bile acid / salt and therapeutic compound are contained in a single composition and said composition is a solid, the amount of said one or more additional (separate) agents (preferably propyl gallate) that stabilize and / or enable or enhance the formation of a micellar form, if present, is preferably at least 1 mg (such as at least 10 mg or at least 20 mg). Said amount can be up to 150 mg (such as up to 100 mg or up to 50 mg). A typical amount is 20-50 mg (such as 30-40 mg).

[0067] The size of the micelles is not particularly limited, but the micelles will have an aggregation size of at least 2 molecules, preferably at least 3 molecules, and more preferably at least 4 molecules. The aggregation size is generally 30 molecules or less, preferably 20 molecules or less, more preferably 10 molecules or less, and typically 8 molecules or less. An average aggregation size of approximately 5 to 7 molecules (e.g., approximately 6 molecules) is particularly preferred.

[0068] In some embodiments, the one or more additional agents for use in the present invention may include a pH adjusting agent (such as a carbonate or bicarbonate), which can help improve the solubility of bile acids / salts thereof, for example, by raising the pH from 7.5 to 9.

[0069] According to the present invention, bile acid / salt and therapeutic compound are used in a composition.There is no particular limitation on the administration method of bile acid / salt and therapeutic compound, provided that they are administered so that both bile acid / salt and therapeutic compound are simultaneously present in the vicinity of cells where uptake is desired and at a sufficiently high concentration.Bile acid / salt and therapeutic compound can be administered separately, simultaneously, or as part of a single composition.As mentioned above, preferably, bile acid / salt and therapeutic compound are combined into a single composition before administration.In this regard, bile acid / salt can be simply mixed with therapeutic compound.

[0070] In the methods of the present invention, cellular uptake is believed to occur via clathrin-mediated endocytosis, i.e., via the formation of clathrin-coated pits. Thus, as reported in the Examples below, uptake was found to be inhibited by chlorpromazine (an inhibitor of clathrin-coated pit formation), but not by nystatin (vesicles) or amiloride (macropinocytosis). Pit formation is believed to be a receptor-mediated process in which bile acids / salts specifically bind to cell surface receptors.

[0071] Thus, the present invention is generally applicable to any method of treatment requiring intracellular delivery of a therapeutic compound. Thus, the discovery of this novel effect of bile acids / salts opens up new therapies, for example, for treatments where it was previously not practical and / or economically feasible to effectively deliver a given therapeutic compound intracellularly to a target cell population within a patient.

[0072] Regarding the administration mode to be used in the method of the present invention, this administration mode should be selected so that therapeutic compound and bile acid / salt are delivered to target cell population before contacting any other cells.One way to do this is to simply administer bile acid / salt and therapeutic compound directly to relevant location.Therefore, if location is skin, it can be administered using topical preparation, or if location is nasal cavity or lung, it can be administered using nebulizer or inhaler.It can be delivered to the location inside the body by injection or keyhole type delivery / release.

[0073] However, the target cell population does not need to be present at the administration site. Thus, the bile acids / salts and therapeutic compounds can be formulated in one or more compositions that delay contact with surrounding cells until the composition(s) reach the target cell population. This allows for high concentrations of bile acids / salts and therapeutic compounds on the surface of the target cells while also avoiding elevated concentrations elsewhere in the body.

[0074] In this regard, bile acids / salts and therapeutic compounds can be administered parenterally, for example, by intravenous injection. A preferred example of this administration mode is an embodiment of the present invention in which the bile acids / salts and therapeutic compounds are encapsulated by a coating that (a) restricts the dissolution of the bile acids / salts and therapeutic compounds under aqueous conditions of pH 7.4 (pH 7.4 is physiological pH), but releases the control of the dissolution of the bile acids or their salts and therapeutic compounds below pH 7.4, and / or (b) contains one or more moieties that can bind to target cell populations in the body. The coating can be designed, for example, to become permeable or destructible under certain circumstances (e.g., within a certain pH range). This approach can be used, for example, in the treatment of cancer, by encapsulating the therapeutic compounds and bile acids / salts in a coating that releases its cargo in the acidic microenvironment of tumor tissue (e.g., below pH 7.4). For example, the coating can be designed to release the therapeutic compound and bile acid or salt thereof at approximately pH 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, or 7.3, or indeed any subrange based on any or all values ​​within the range of pH 5.0-7.3. The exact pH will vary depending on the tumor. It may be appropriate to test the pH in the environment of the tumor cells prior to treatment to determine which formulation is most desirable. Potential formulations that can be used in this regard include micronized pellets that (a) can be formulated to limit dissolution of the pellet but dissolve in such an acidic microenvironment, and / or (b) comprise a coating that includes one or more molecules or moieties that can bind to receptors present on the target cell population (e.g., cancer cells) (in this regard, it may also be advantageous if release of the composition from within the coating can be triggered / facilitated when the molecule reaches the target site and the relevant moiety binds to the intended receptor).In one embodiment, cancer cells can be targeted by using bile acids / salts and therapeutic compounds in combination with DOPE and PEG, where the acid-sensitive PEG is broken down in the acidic cancer environment, exposing the bile acids / salts and therapeutic compounds to the target cells.

[0075] Alternatively, bile acids / salts and therapeutic compounds may be administered orally. For example, if the intended site of cellular uptake of a therapeutic compound is the cells of the small intestine, the therapeutic compound can be safely passed through the stomach by encapsulating it inside an enteric-coated capsule, tablet, or other device that resists dissolution at the low pH found in the stomach but disintegrates at higher pHs, releasing the compound into the small intestine (e.g., the duodenum, jejunum, or ileum). This prevents dissolution of the bile acids / salts and therapeutic compound in the stomach (at concentrations too low for effective cellular uptake) and / or degradation of one or more of the components (particularly the therapeutic compound). Pharmaceutical compositions for use according to the present invention in this regard preferably have an enteric coating that is permeable at a pH of 3 to 7, more preferably 4 to 6.5, and most preferably 5 to 6. Suitable enteric coatings are known in the art.

[0076] The fate of a therapeutic compound after intracellular uptake can depend on factors including the properties of the therapeutic compound itself, as well as the properties of any other compounds that are incorporated into the cell together with the therapeutic compound, and the properties of the incorporated cell.For example, some cells have the inherent function of transporting materials incorporated into vesicles across cells (e.g., barrier cells such as endothelial cells in the blood-brain barrier) for subsequent release.In some situations, this can certainly serve a useful function.However, in situations where a therapeutic compound is intended to have a therapeutic effect within the cell, this represents a type of situation in which it may be particularly advantageous to take additional measures to help promote this effect.

[0077] Thus, as mentioned above, in the methods of the present invention, bile acids or salts thereof may preferably be used in combination with one or more additional agents. In this regard, in preferred embodiments, said one or more additional agents include one or more agents that act to increase the likelihood that the therapeutic compound will successfully exert its therapeutic effect in cells (where uptake occurs) by one or more of the following mechanisms: a) inhibition of subsequent release of a therapeutic compound from a cell after the therapeutic compound has been taken up; b) protection of therapeutic compounds from the acidic pH of endosomes / lysosomes; c) protection of the therapeutic compound from one or more digestive enzymes of the lysosome; d) promoting penetration of the endosomal barrier (facilitating endosomal escape); e) increasing the stability of therapeutic compounds in the cytosol; f) facilitating penetration of the nuclear membrane (if the intended site of action is the nucleus (e.g., for plasmid DNA delivery)); and g) Altered intracellular vesicle transport.

[0078] For example, the one or more additional agents may include one or more agents selected from the following: a) Fusogenic lipids (e.g., cationic liposomes and / or neutral helper lipids). When the therapeutic compound is a nucleic acid, cationic liposomes can be advantageously used to form a complex with the nucleic acid, thereby facilitating gene transfection. As for neutral helper lipids, agents such as DOPE (dioleoylphosphatidylethanolamine) can be advantageously used. DOPE can mediate fusion between liposomes and endosomal membranes after endocytosis, and can therefore be used to enhance, for example, the expression of complexed genes. When the neutral helper lipid is DOPE, it is preferably in a reverse hexagonal form. DOPE can also be stimulated to form a reverse hexagonal form in situ by lowering the pH. In some embodiments, this can be achieved by including an appropriate pH adjuster. Additionally, anionic lipids (such as phosphatidic acid or cholesteryl hemisuccinate) can be used as fusogenic lipids, optionally in combination with a neutral helper lipid such as DOPE, especially when the therapeutic compound has a positive charge. In this regard, cell-penetrating peptides can also be used. b) Cell-penetrating peptides, preferably having 2 to 12 amino acids (such as octaarginine, nonaarginine, or octalysine), and / or arginine-rich cell-penetrating peptides, which may be advantageously used in combination with other agents selected from one or more additional compounds described herein, particularly in combination with a fusogenic lipid such as DOPE. c) Lysosomotropic agents (e.g., chloroquine). d) Membrane disruptive peptides (e.g., peptides INF7, H5WYG, 43E (composed of three LAEL amino acid sequence units), and histidine 10). e) Membrane disruptive polymers (e.g., polyethyleneimine). f) Photochemical internalization agents (e.g., fluorescent labels such as fluorescein isothiocyanate or compounds containing same). g) Agents that alter intracellular vesicle trafficking, for example, by targeting factors that mediate the formation of transport vesicles (such as guanine nucleotide exchange factor 1 (GBF1), which mediates transport vesicle formation by recruiting COPI coat proteins to cargo-binding receptor proteins found at the membranes of the Golgi; inhibition of GBF1 activity induces the retrograde transport of secretory proteins from the Golgi to the ER; retrograde trafficking from the Golgi to the ER triggers activation of the unfolded protein response, ultimately leading to apoptosis). An example of such an agent is Brefeldin A.

[0079] If present, the one or more additional agents should be administered in combination with the bile acid / salt and therapeutic compound—the agents can be administered separately, simultaneously, or as part of a single composition. Preferably, the bile acid / salt and therapeutic compound and the one or more additional agents are all administered as part of the same composition. In one embodiment, the one or more additional agents (or active portion(s) thereof) can be chemically attached to the therapeutic compound (e.g., covalently or non-covalently conjugated). This can help ensure that the one or more additional agents (or active portion(s) thereof) are correctly incorporated into the cell along with the therapeutic compound (i.e., where intended to exert their effect). This approach is particularly preferred when photochemical internalization is used to promote efficacy.

[0080] Furthermore, although cell-penetrating peptides are listed above as a possible alternative for one or more of the aforementioned additional agents, it is worth noting that such peptide agents have also previously been described for use in enhancing the uptake of compounds into cells.Because bile acids or their salts already perform their function in the present invention, it is generally not necessary to include a cell-penetrating peptide solely for this purpose, and therefore, in some embodiments, it is preferable not to include such a peptide agent.However, there may be instances where it may be advantageous to include a cell-penetrating peptide to provide a beneficial effect once the therapeutic compound is taken up into cells and / or to further enhance the uptake of the therapeutic compound into cells.

[0081] Typically, only one of the one or more additional agents listed above will be used, although in some embodiments it may be preferable to include two, three, four, or even more agents.

[0082] Regarding the method of administering bile acids / salts and / or therapeutic compounds according to the present invention, they are typically formulated (each) for administration with a pharmaceutically acceptable carrier or diluent. The bile acids / salts and / or therapeutic compounds can be administered in various dosage forms. Thus, they can be administered orally, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders, or granules. They can also be administered parenterally (either subcutaneously, intravenously, intramuscularly, intrasternally, transdermally, or by infusion techniques). They can also be administered as suppositories. They can also be administered via buccal, sublingual, rectal, topical, oral, nasal, or pulmonary routes, either as aerosol sprays or drops.

[0083] For example, solid oral forms may contain, together with the active compound, diluents (e.g., lactose, dextrose, saccharose, cellulose, corn starch, or potato starch); lubricants (e.g., silica, talc, stearic acid, magnesium stearate or calcium stearate, and / or polyethylene glycol); binders (e.g., starch, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, or polyvinylpyrrolidone); disaggregating agents (e.g., starch, alginic acid, alginate, or sodium starch glycolate); effervescent mixtures; dyes; sweeteners; mucolytic agents; wetting agents (e.g., lecithin, polysorbate, lauryl sulfate, etc.); and non-toxic, pharmacologically inactive substances generally used in pharmaceutical formulations. Such pharmaceutical preparations can be produced in a known manner (e.g., by mixing, granulating, tableting, sugar-coating, or film-coating processes).

[0084] Composition can be liquid.Possible liquid composition includes solution, suspension and dispersion.The liquid preparation for oral administration can be syrup, emulsion and suspension.Syrup can contain as carrier, for example, sucrose or sucrose containing glycerin and / or mannitol and / or sorbitol.

[0085] Suspensions and emulsions may contain as a carrier, for example, natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. Suspensions or solutions for intramuscular injection may contain, together with the active compound, a pharmaceutically acceptable carrier (for example, sterile water, olive oil, ethyl oleate, glycols (for example, propylene glycol)), and, if necessary, an appropriate amount of lidocaine hydrochloride.

[0086] The solutions for injection or infusion may contain as carrier, for example, sterile water or preferably they may be in the form of aqueous sterile, isotonic saline solutions.

[0087] In a preferred embodiment of the present invention, bile acids / salts and therapeutic compounds are provided as a composition for the introduction of a therapeutic compound (preferably a macromolecule) into cells, wherein said therapeutic compound is mixed in solution with a bile salt, preferably a chenodeoxycholate (such as sodium chenodeoxycholate), optionally said bile salt being used in conjunction with EDTA to this effect.

[0088] In another preferred embodiment of the present invention, bile acid / salt, EDTA, and therapeutic compound are provided as a solid dry powder containing a mixture of components that form a solution in body fluids after administration.Therefore, in this embodiment, the present invention provides a (dry) solid composition containing a therapeutic compound and a bile salt (preferably a chenodeoxycholate, such as sodium chenodeoxycholate), formulated as a capsule or pellet, for introducing a therapeutic compound (preferably a macromolecule) into cells, and optionally, the bile salt is used in conjunction with EDTA for this purpose.The dry solid can be prepared, for example, by mixing the individual components together in appropriate ratios to form a dry powder, or by first dissolving all the components of the formulation in a liquid medium, and then drying the solution by any known method, such as evaporation, vacuum drying, or freeze-drying.

[0089] In another preferred embodiment, the present invention provides a composition for the introduction of a therapeutic compound (typically a macromolecule) into cells, wherein the therapeutic compound and a bile acid / salt are combined, for example in the form of a paste, ointment, or gel for topical application, optionally using the aforementioned bile salts in conjunction with EDTA to this effect.

[0090] Additional excipients may be included in the above compositions, such as one or more antioxidants, preservatives, solubility aids, pH adjusters, and / or taste-masking agents (for oral or nasal administration).

[0091] Preferred agents for use in the compositions of the invention when the therapeutic compound and bile acid / salt are present in solid form are (i) fumed silica (aerosol), which may be used in an amount of, for example, 0.01 to 100 mg, preferably 0.1 to 10 mg, typically 0.5 to 5 mg; (ii) sodium starch glycolate, which may be used in an amount of, for example, 0.1 to 500 mg, preferably 1 to 100 mg, typically 5 to 50 mg; (iii) one or more glidants, and / or (iv) one or more disintegrants.

[0092] Preferably, bile acids or their salts are used to enable or enhance cellular uptake of the therapeutic compound. Optionally, the bile salts are used in conjunction with EDTA.

[0093] The present invention also provides a method of treating the human or animal body, comprising administering to the human or animal body a therapeutic compound together with a bile acid, wherein the bile acid is chenodeoxycholic acid or deoxycholic acid, and the bile acid or a pharmaceutically acceptable salt thereof is present in an amount sufficient to enable or enhance cellular uptake of the therapeutic compound, optionally using the bile acid salt in combination with EDTA.

[0094] The present invention also provides the use of a therapeutic compound and a bile acid or its salt in the manufacture of a medicament for use in a method for treating a human or animal body in need of the therapeutic compound.Preferably, the bile acid or its salt is present in an amount that allows or enhances the cellular uptake of the therapeutic compound.Optionally, the bile acid or its salt is used in combination with EDTA.

[0095] The present invention also provides a pharmaceutical or therapeutic composition comprising (a) a bile acid or a pharmaceutically acceptable salt thereof, wherein the bile acid is chenodeoxycholic acid or deoxycholic acid; and (b) a therapeutic compound. Preferably, the composition also comprises one or more additional compounds selected from fusogenic lipids, cell-penetrating peptides, lysosomotropic agents, membrane-disrupting peptides, membrane-disrupting polymers, photochemical internalization agents, and agents that alter intracellular vesicle trafficking, and optionally, the bile salt is used in combination with EDTA for this purpose.

[0096] Desirably, in any formulation of the present invention, the bile acid or salt thereof and therapeutic compound are encapsulated by a coating that (a) limits the dissolution of said bile acid or salt thereof and therapeutic compound under aqueous conditions at pH 7.4, but removes the limit on dissolution of said bile acid or salt thereof and therapeutic compound at a pH below 7.4, and / or (b) includes one or more moieties capable of binding to said target cell population in the body.

[0097] In a first further preferred embodiment, the present invention provides a bile acid or a pharmaceutically acceptable salt thereof for use in a method of treating the human or animal body, said method comprising administering a therapeutic compound to the human or animal body, wherein: a) the bile acid or a pharmaceutically acceptable salt thereof is a salt of chenodeoxycholic acid, preferably sodium chenodeoxycholate; b) the bile acid / salt and the therapeutic compound are contained in the same pharmaceutical composition; c) the composition is encapsulated by a coating that (a) limits dissolution of the bile acid or salt thereof and therapeutic compound under aqueous conditions at pH 7.4 but removes the limit on dissolution of the bile acid or salt thereof and therapeutic compound at a pH below 7.4, and / or (b) includes one or more moieties capable of binding to the target cell population in the body; d) The aforementioned compositions are preferably in liquid form, more preferably in a form suitable for intravenous administration.

[0098] In a particularly preferred aspect of the first further preferred embodiment described above, the bile acid / salt is formulated in the composition in combination with DOPE and PEG such that the DOPE and / or PEG degrades to release the therapeutic compound when exposed to a pH below 7.4, preferably below 7.0, more preferably below 6.5.

[0099] In a second further preferred embodiment, the present invention provides a bile acid or a pharmaceutically acceptable salt thereof for use in a method of treating the human or animal body, said method comprising administering a therapeutic compound to the human or animal body, wherein: a) the bile acid or its pharmaceutically acceptable salt is a salt of chenodeoxycholic acid, preferably sodium chenodeoxycholate, and if necessary, the bile salt is used in combination with EDTA; b) using a bile acid or salt thereof in the aforementioned method to enable or enhance cellular uptake of the therapeutic compound in the intestine (preferably, receptor-mediated internalization of the bile acid / salt occurs via clathrin-coated pits), typically in the small intestine; c) the therapeutic compound is a protein, e.g., a protein containing a hydrophobic moiety, which, when in a micellar form, can be non-covalently conjugated to chenodeoxycholate; d) The method comprises oral administration of a pharmaceutical composition comprising a bile salt, a protein, and propyl gallate.

[0100] Suitable proteins for use in this embodiment include those listed above and / or used in the Examples.

[0101] Also for this second more preferred embodiment, the composition is preferably formulated in a device (preferably an enteric-coated formulation, such as an enteric capsule) that resists dissolution at pHs found in the stomach but disintegrates at pHs found in the small intestine. For example, the composition can be formulated in an enteric-coated capsule that becomes permeable at a pH in the range of 5-6.

[0102] In a third further preferred embodiment, the present invention provides a bile acid or a pharmaceutically acceptable salt thereof for use in a method of treating the human or animal body, said method comprising administering a therapeutic compound to the human or animal body, wherein: a) the bile acid or its pharmaceutically acceptable salt is a salt of chenodeoxycholic acid, preferably sodium chenodeoxycholate, and if necessary, the bile salt is used in combination with EDTA; b) the bile acid / salt and the therapeutic compound are contained in the same pharmaceutical composition; c) the pharmaceutical composition is suitable for topical administration; d) The method includes applying a composition to the skin.

[0103] Generally, the present invention relates to the treatment of humans, although in one embodiment, the present invention relates to the treatment of non-human animals. Thus, subjects that may utilize the present invention include, by way of example, humans, mammals, companion animals, and birds (with humans being most preferred).

[0104] The following examples illustrate the present invention, but do not limit the invention in any way.

[0105] The following examples are intended to illustrate the present invention and are not to be construed as limiting the present invention. [Example]

[0106] Example Example 1 Caco-2 cells (passage 51) were cultured at a density of 1 x 10 cells / ml in DMEM (supplemented with 10% FBS) on plastic cover slips in the bottom of 1 ml wells in 24-well cluster plates for 3 days. 0.5 ml of medium was removed from each well and replaced with medium containing (i) FITC-insulin only (concentration 100 μg / ml), (ii) FITC-insulin with sodium chenodeoxycholate (1 mg / ml), or (iii) FITC-insulin with sodium chenodeoxycholate (2 mg / ml). The cells were incubated for half an hour at 37°C in 5% CO2. The supernatant was then removed and replaced with 0.5 ml of 4% paraformaldehyde solution and incubated for 15 minutes at room temperature. The paraformaldehyde was then removed, and the wells were washed three times with phosphate-buffered saline. The cover slips were then mounted with mounting medium and observed under a confocal microscope. Images are shown in Figure 1. As can be seen from the micrographs, uptake was minimal in cells treated with insulin alone, but the presence of chenodeoxycholate enhanced uptake in a concentration-dependent manner, and at high concentrations of bile salts, fluorescent material was clearly visible in intracellular vacuoles. Example 2

[0107] Caco-2 cells (passage 51) were cultured at a density of 2 x 10 cells / ml in DMEM (supplemented with 10% FBS) in the bottom of 0.2 ml wells of a 96-well cluster plate for 4 days, with medium changes as needed. 0.2 ml of medium was removed from each well and cultured in the following wells: (i) FITC-albumin (bovine) only (concentration 100 μg / ml) in row A; (ii) FITC-albumin 100 μg / ml with sodium chenodeoxycholate (high concentration: 1.33 mg / ml) in row B; and (iii) FITC-albumin with sodium chenodeoxycholate / propyl gallate solution (0.66 and 0.33 mg / ml, respectively - total solids 1 mg / ml) in row C. (iv) FITC-albumin 100 μg / ml with sodium chenodeoxycholate / propyl gallate solution (1.0 and 0.5 mg / ml, respectively—1.5 mg / ml total solids) in row D, and (v) FITC-albumin 100 μg / ml with sodium chenodeoxycholate / propyl gallate solution (1.33 and 0.66 mg / ml, respectively—2 mg / ml total solids) in row E. Cells were incubated at 37°C in 5% CO for 10 minutes, then the supernatant was removed from rows 1, 2, and 3 and gently washed three times with phosphate-buffered saline. Plates were immediately read on a Spectramax fluorescent plate reader (excitation wavelength 494 nm, cutoff 515 nm, emission 515 nm). The plates were then further incubated for 10 minutes at 37°C in 5% CO2, and the supernatants from wells 4, 5, and 6 were removed by washing in PBS before being read on a plate reader as described above. The plates were further incubated for 10 minutes, and then the supernatants from wells 7, 8, and 9 were removed, the wells were washed, and read as described above. The readouts showing the amount of fluorescent substance taken up by the cells for each group at each time point are shown in Figure 2, where "low," "medium," and "high" refer to chenodeoxycholate concentrations of 0.66, 1.0, and 0.33 mg / ml, respectively. The results are shown in Figure 2.As can be seen, the presence of chenodeoxycholate alone enhances cellular uptake of fluorescent albumin, but when propyl gallate is included together with bile salts, the rate of uptake is faster and reaches a maximum much earlier (10 min compared to 30 min for chenodeoxycholate alone). Example 3

[0108] Caco-2 cells (passage 52) were cultured at a density of 2 x 10 cells / ml in DMEM (supplemented with 10% FBS) in the bottom of 0.2 ml wells of a 96-well cluster plate for 4 days, with medium changes as needed. 0.2 ml of medium was removed from each well and replaced with medium containing FITC-albumin (bovine) alone (100 μg / ml) or FITC-albumin 100 μg / ml with various bile salts at 2 μg / ml. Cells were incubated at 37°C in 5% CO for 30 minutes, after which the supernatant was removed from the wells, washed gently three times with phosphate-buffered saline (PBS), and then filled with 0.2 ml of PBS. Plates were read using a Spectramax fluorescent plate reader (excitation wavelength 494 nm, cutoff 515 nm, emission 515 nm). The bile salts tested (all in their sodium salt form) were chenodeoxycholate, deoxycholate, cholate, glycodeoxycholate, glycochenodeoxycholate, glucocholate, taurocholate, taurochenodeoxycholate, and taurodeoxycholate. Fluorescence readings obtained after subtraction of the background from cells incubated in medium alone are shown in Figure 3. Visual inspection of cells under a microscope in this and repeat experiments confirmed that where fluorescence was observed above background, it was localized intracellularly, often in individual vacuoles. Uptake into cells was evident after incubation with chenodeoxycholate and deoxycholate, whereas uptake was not induced by cholate or taurocholate. Subsequent studies demonstrated that no uptake of labeled proteins was observed with any naturally conjugated bile salt (i.e., tauro or glycoderivatives) at concentrations both above and below the toxic limit. Example 4

[0109] Caco-2 cells (passage 56) were cultured at a density of 0.5 x 10 cells / ml in DMEM (supplemented with 10% FBS) in the bottom of 0.2 ml wells of a 96-well cluster plate for 4 days, with medium changes as needed. 0.2 ml of medium was removed from each well and replaced with 50 μl of medium lacking FBS but containing different concentrations of chlorpromazine. Incubation continued for 10 minutes, and then 50 μl of medium containing FITC-albumin (bovine) alone (concentration 200 μg / ml) or FITC-albumin with chenodeoxycholate (200 μg / ml) (no FBS) was added at various concentrations. The cells were incubated for an additional 20 minutes at 37°C in 5% CO2, after which the supernatant was removed from the wells, gently washed three times with FBS-free medium, and then filled with 0.2 ml of medium (again, no FBS). Otherwise, FITC-albumin uptake into cells was assessed visually under a fluorescence microscope and scored according to fluorescence intensity. As can be seen in the table below, which shows fluorescence scores based on microscopic images, a concentration-dependent inhibition of uptake was observed upon preincubation with chlorpromazine (an inhibitor of clathrin-coated pit formation). This contrasts with the results of similar experiments in which preincubation with either amiloride (an inhibitor of macropinocytosis) or nystatin (a caveolin inhibitor) did not result in a decrease in uptake. [Table 1] symbol +++ High level of fluorescence ++ Medium level fluorescence + Low level fluorescence □ Very low / sporadic levels of fluorescence - No fluorescence Example 5

[0110] Caco-2 cells (passage 57) were cultured at a density of 0.5 x 10 cells / ml in DMEM (supplemented with 10% FBS) in the bottom of 0.2 ml wells of a 96-well cluster plate for 4 days, with medium changes as needed. 0.2 ml of medium was removed from each well and replaced with 50 μl of medium (lacking FBS) containing different concentrations of sodium ursodeoxycholate. Incubation continued for 10 minutes, and 50 μl of medium containing FITC-albumin (bovine) alone (at a concentration of 100 μg / ml) or FITC-albumin with various concentrations of chenodeoxycholate (minus FBS) was added. The cells were incubated for an additional 20 minutes at 37°C in 5% CO2, after which the supernatant was removed from the wells, gently washed three times with FBS-free medium, and then filled with 0.2 ml of medium (minus FBS). Otherwise, FITC-albumin uptake into cells was assessed visually under a fluorescence microscope and scored according to fluorescence intensity using the same grading scheme as in Example 4. As can be seen in the table below, preincubation with ursodeoxycholate inhibited uptake in a concentration-dependent manner. [Table 2] Example 6

[0111] Caco-2 cells (passages 59 and 60) were cultured at a density of 0.5 x 10 cells / ml in DMEM (supplemented with 10% FBS) in the bottom of 0.2 ml wells of a 96-well cluster plate for 4 days, with medium changes as needed. 0.2 ml of medium was removed from each well and replaced with 50 μl of medium (without FBS) containing either FITC-albumin (bovine) or FITC-casein (at a concentration of 100 μg / ml) alone, supplemented with various concentrations of chenodeoxycholate. The cells were incubated for an additional 20 minutes at 37°C in 5% CO2, after which the supernatant was removed from the wells, gently washed three times with FBS-free medium, and then filled with 0.2 ml of medium (again, without FBS). Otherwise, FITC-albumin uptake into the cells was assessed visually under a fluorescence microscope and scored according to fluorescence intensity using the same grading scheme as in Example 4. The experiment was repeated on two separate occasions on different days. As can be seen in the table below, similar degrees of uptake were observed for both BSA and casein, indicating that uptake was not specific to a particular protein. [Table 3-1] [Table 3-2] Example 7

[0112] Caco-2 cells (passage 60) were cultured at a density of 0.5 x 10 cells / ml in DMEM (supplemented with 10% FBS) in the bottom of 0.2 ml wells of a 96-well cluster plate for 4 days, with medium changes as needed. 0.2 ml of medium was removed from each well and replaced with 50 μl of medium (without FBS) containing 200 μg / ml of FITC-albumin (bovine) alone (100 μg / ml), or 200 μg / ml of FITC-albumin supplemented with various concentrations of chenodeoxycholate alone, chenodeoxycholate:propyl gallate (2:1 wt:wt), taurocholate alone, or taurocholate:propyl gallate (2:1 wt:wt). The cells were incubated for an additional 20 minutes at 37°C in 5% CO2, after which the supernatant was removed from the wells, gently washed three times with FBS-free medium, and then filled with 0.2 ml of medium (again without FBS). Otherwise, FITC-albumin uptake into the cells was assessed visually under a fluorescence microscope and scored according to fluorescence intensity using the same grading scheme as in Example 4 (nd = not performed / not tested). As can be seen from the table below, the presence of propyl gallate appears to enhance chenodeoxycholate-mediated uptake, as at a bile salt concentration of 1.0 mg / ml, a higher level of uptake was observed with the cheno / PG combination than with cheno alone. In contrast, no uptake was observed for taurocholate, either in the presence or absence of PG. The effect of PG may be to promote and enhance the formation of bile salt micelles. Chenodeoxycholate has a low CMC and will form micelles at the low concentrations tested here. However, enhanced micelle formation increases protein uptake, as expected, following an uptake mechanism in which a receptor-mediated process recognizes bile salts in micellar form. However, bile salt micelle formation alone is not sufficient for this uptake to occur, as no uptake is observed in the presence of taurocholate, even in the presence of PG. [Table 4-1] [Table 4-2] Example 8

[0113] Caco-2 cells (passage 60) were cultured at a density of 0.5 x 10 cells / ml in DMEM (supplemented with 10% FBS) in the bottom of 0.2 ml wells of a 96-well cluster plate for 4 days, with medium changes as needed. 0.2 ml of medium was removed from each well and replaced with 50 μl of medium (FBS-free) containing FITC-albumin (bovine) supplemented with chenodeoxycholate at a concentration of 0.5 mg / ml, either alone or in combination with sodium EDTA or orthophenanthroline at a concentration of 5 mg / ml. The cells were incubated for an additional 25 minutes at 37°C in 5% CO2, after which the supernatant was removed from the wells, gently washed three times with FBS-free medium, and then filled with 0.2 ml of medium (again, FBS-free). Otherwise, FITC-albumin uptake into the cells was assessed visually under a fluorescence microscope and scored according to fluorescence intensity using the same grading scheme as in Example 4. From the table below, it can be seen that at the concentrations used, neither chenodeoxycholate nor EDTA alone was able to enhance protein uptake. However, when combined, significant uptake was observed, indicating that EDTA can cooperate with chenodeoxycholate in stimulating protein uptake by enterocytes. However, another chelating agent, o-phenanthroline, does not have this effect. [Table 5] Example 9

[0114] The experiment described in Example 8 was repeated, except that the chenodeoxycholate concentrations were 0, 0.5, and 1 mg / ml, and EDTA, DTPA, and EGTA were used at a concentration of 1 mg / ml. The incubation time was 30 minutes. As can be seen in the table below, the addition of EDTA exceeded the enhancement of uptake observed with chenodeoxycholate alone, but not with DTPA or EGTA. Similar findings were observed when chenodeoxycholate was added first, incubated for 20 minutes, and then replaced with EDTA for 10 minutes. [Table 6] Example 10

[0115] Caco-2 cells (passage 61) were cultured at 0.5 × 10 5 Cells were cultured at a density of 1000 cells / ml in DMEM (supplemented with 10% FBS) in the bottom of 0.2 ml wells of a 96-well black cluster plate for 4 days, with medium changes as needed. 0.2 ml of medium was removed from each well and replaced with 50 μl of medium (without FBS) containing different concentrations of chlorpromazine. After 20 minutes, 100 μl of FITC-albumin (bovine) alone (concentration 100 μg / ml) or FITC-albumin containing different concentrations of chenodeoxycholate was added. The cells were incubated for another 20 minutes at 37°C in 5% CO2, after which the supernatant was removed from the wells, gently washed three times with FBS-free medium, and then filled with 0.2 ml of medium (again without FBS). Otherwise, FITC-albumin uptake into the cells was measured using a TECAM plate reader (excitation wavelength 492 nm, emission wavelength 525 nm). The FITC-BSA uptake results, shown in the chart in Figure 4, demonstrate that chlorpromazine (at both high and low concentrations) inhibits uptake stimulated by all concentrations of bile salts, confirming the experimental results described in Example 4, in which preincubation with chlorpromazine (an inhibitor of clathrin-coated pit formation) inhibited uptake in a concentration-dependent manner. Example 11

[0116] Caco-2 cells (passage 61) were cultured at 0.5 × 10 5 The cells were cultured in DMEM (supplemented with 10% FBS) in culture flasks at a density of 1.3 × 10 cells / ml for 4 days, with medium changes as needed. Cells were then trypsinized, suspended in FBS-free medium, and washed by centrifugation to a final cell concentration of 1.3 × 10 cells. 6 A suspension containing 0.4 ml of cells / ml was obtained. 0.4 ml of the suspension was dispensed into each of eleven 1.5 ml plastic Eppendorf vials, and 40 μl of medium (without FBS) containing different concentrations of chlorpromazine was added to each vial, followed by incubation at 37°C for 20 minutes. 400 μl of FITC-albumin (bovine) alone (concentration 100 μg / ml) or FITC-albumin containing different concentrations of chenodeoxycholate was then added. The suspension was incubated for another 20 minutes at 37°C, and the cells were then gently washed three times by centrifugation using FBS-free medium. The pellet was then resuspended in 600 μl of medium (again without FBS), and 200 μl was transferred to each of three wells of a black 98-well microplate. FITC-albumin uptake into the cells was then measured using a Spectramax The uptake was measured on a Gemini fluorescent plate reader (excitation wavelength 492 nm, emission wavelength 525 nm). The FITC-BSA uptake results, shown in the chart in Figure 5, demonstrate that chlorpromazine (at both high and low concentrations) inhibits uptake stimulated by all concentrations of bile salts. This confirms the findings shown in Experiment 10 and demonstrates that measurements of uptake from cells in suspension yield results very similar to experiments performed with cells adhered to plastic surfaces. Example 12

[0117] Caco-2 cells (passage 61) were cultured at 0.5 × 10 5The cells were cultured in DMEM (supplemented with 10% FBS) in culture flasks at a density of 1 × 10 cells / ml for 4 days, with medium changes as needed. Cells were then trypsinized, suspended in FBS-free medium, and washed by centrifugation to a final cell concentration of 1 × 10 cells. 6 A suspension containing 100 cells / ml was obtained. 1 ml of the suspension was dispensed into a 1.5 ml plastic Eppendorf vial, and the cells were centrifuged in culture medium (without FBS) and suspended to a volume of 100 μl. Next, 1 ml of FITC-albumin (bovine) alone (concentration 100 μg / ml) or FITC-albumin containing chenodeoxycholate and EDTA alone or in combination at a concentration of 1 mg / ml was added. The suspension was incubated for an additional 15 minutes at 37°C, and the cells were then gently washed three times by centrifugation using FBS-free culture medium. The pellet was then resuspended in 600 μl of culture medium (again without FBS), and 200 μl was transferred to each of three wells of a black 98-well microplate. FITC-albumin uptake into the cells was then measured using a Spectramax Gemini fluorescence plate reader (excitation wavelength 492 nm, emission wavelength 525 nm). In this experiment, the concentrations of the two agents (chenodeoxycholate and EDTA) individually were too low to enhance protein uptake within the time frame tested, however, the results shown in Figure 6 demonstrate that the combination of the two agents highly significantly enhanced uptake. Example 13

[0118] Caco-2 cells (passage 61) were cultured at 0.5 × 10 5 The cells were cultured in DMEM (supplemented with 10% FBS) in culture flasks at a density of 1 × 10 cells / ml for 4 days, with medium changes as needed. Cells were then trypsinized, suspended in FBS-free medium, and washed by centrifugation to a final cell concentration of 1 × 10 cells. 6A suspension containing 100 cells / ml was obtained. 1 ml of the suspension was dispensed into a 1.5 ml plastic Eppendorf vial, and the cells were centrifuged in culture medium (without FBS) and suspended in a volume of 100 μl. 1 ml of FITC-hGH alone (concentration 100 μg / ml) or FITC-hGH containing different concentrations of chenodeoxycholate was then added. The suspension was incubated at 37°C for another 15 minutes, and the cells were then gently washed three times by centrifugation using FBS-free culture medium. The pellet was then resuspended in 600 μl of culture medium (again without FBS), and 200 μl was transferred to each of three wells of a black 98-well microplate. FITC-hGH uptake into the cells was then measured using a Spectramax Gemini fluorescence plate reader (excitation wavelength 492 nm, emission wavelength 525 nm). The data presented in Figure 7 demonstrate that chenodeoxycholate enhances the uptake of hGH into cells in a dose-dependent manner similar to that observed for the uptake of other proteins (such as insulin, BSA, and casein). Example 14

[0119] Human intestinal IEC6 cell line cells (passage 48) were cultured at 0.5 × 10 5 The cells were cultured in DMEM (supplemented with 10% FBS) in culture flasks at a density of 1 × 10 cells / ml for 4 days, with medium changes as needed. Cells were then trypsinized, suspended in FBS-free medium, and washed by centrifugation to a final cell concentration of 1 × 10 cells. 6A suspension containing 100 cells / ml was obtained. 1 ml of the suspension was dispensed into a 1.5 ml plastic Eppendorf vial, and the cells were centrifuged in culture medium (without FBS) and suspended in a volume of 100 μl. Then, 1 ml of FITC-albumin (bovine) alone (concentration 100 μg / ml) or FITC-albumin containing different concentrations of chenodeoxycholate alone was added. The suspension was incubated for another 15 minutes at 37°C, and the cells were then gently washed three times by centrifugation using FBS-free culture medium. The pellet was then resuspended in 600 μl of culture medium (again without FBS), and 200 μl was transferred to each of three wells of a black 98-well microplate. The uptake of FITC-albumin into the cells was then measured using a Spectramax Gemini fluorescence plate reader (excitation wavelength 492 nm, emission wavelength 525 nm). The results shown in Figure 8 demonstrate that protein uptake in IEC6 cells is enhanced by the presence of chenodeoxycholate in a manner similar to that observed in Caco-2 cells, confirming that this is a phenomenon common to enterocytes in general. Example 15

[0120] Human intestinal IEC6 cell line cells (passage 48) were cultured at 0.5 × 10 5 The cells were cultured in DMEM (supplemented with 10% FBS) in culture flasks at a density of 1 × 10 cells / ml for 4 days, with medium changes as needed. Cells were then trypsinized, suspended in FBS-free medium, and washed by centrifugation to a final cell concentration of 1 × 10 cells. 6A suspension containing 100 cells / ml was obtained. 1 ml of the suspension was dispensed into a 1.5 ml plastic Eppendorf vial, and the cells were centrifuged in medium (without FBS) and suspended in a volume of 100 μl. Then, 1 ml of FITC-albumin (bovine) alone (concentration 100 μg / ml) or FITC-albumin containing 0.5 mg / ml chenodeoxycholate and different concentrations of EDTA was added. The suspension was incubated for another 15 minutes at 37°C, and the cells were then gently washed three times by centrifugation using FBS-free medium. The pellet was then resuspended in 600 μl of medium (again without FBS), and 200 μl was transferred to each of three wells of a black 98-well microplate. The uptake of FITC-albumin into the cells was then measured using a Spectramax Gemini fluorescence plate reader (excitation wavelength 492 nm, emission wavelength 525 nm). The results shown in Figure 9 demonstrate that the enhancement of BSA uptake by chenodeoxycholate is augmented by EDTA in a dose-dependent manner in IEC6 cells. The present invention provides, for example, the following items. (Item 1) 1. A bile acid or a pharmaceutically acceptable salt thereof for use in a method of treatment of the human or animal body, said method comprising administering to said human or animal body a therapeutic compound, wherein: a) the bile acid is chenodeoxycholic acid or deoxycholic acid; b) A bile acid or a pharmaceutically acceptable salt thereof, wherein said bile acid or salt thereof is used in said method to enable or enhance cellular uptake of said therapeutic compound. (Item 2) 2. The bile acid or salt thereof according to item 1, for use in the method defined in item 1, wherein the bile acid or salt thereof is used in combination with EDTA. (Item 3) 3. The bile acid or salt thereof according to item 1 or 2, for use in a method as defined in item 1 or 2, wherein said therapeutic compound is a macromolecule. (Item 4) 3. The bile acid or salt thereof according to item 1 or 2, for use in a method as defined in item 1 or 2, wherein said therapeutic compound is a protein. (Item 5) 5. The bile acid or salt thereof according to any one of items 1 to 4, for use in the method defined in any one of items 1 to 4, wherein the bile acid or the pharmaceutically acceptable salt thereof is chenodeoxycholate, preferably sodium chenodeoxycholate. (Item 6) 10. The bile acid or salt thereof according to any one of the preceding items, for use in the method defined in any one of the preceding items, wherein the concentration of the bile acid or salt thereof on the surface of the cells at which cellular uptake is enabled or enhanced is between 2 mg / ml and 40 mg / ml. (Item 7) Item 2.6. The bile acid or salt thereof according to any one of Items 2 to 6, for use in the method according to any one of Items 2.6, wherein the concentration of the EDTA on the surface of the cells at which intracellular uptake is enabled or enhanced is 0.1 mg / ml to 10 mg / ml. (Item 8) Item 2.6. The bile acid or salt thereof according to any one of Items 2 to 6, for use in the method according to any one of Items 2.6, wherein the concentration of the EDTA on the surface of the cells at which intracellular uptake is enabled or enhanced is 0.2 mg / ml to 10 mg / ml. (Item 9) 10. The bile acid or salt thereof of any one of the preceding items for use in the method defined in any one of the preceding items, wherein the method uses receptor-mediated internalization of the bile acid or salt thereof via clathrin-coated pits to enable or enhance cellular uptake of the therapeutic compound. (Item 10) 10. The bile acid or salt thereof of any one of the preceding items for use in the method as defined in any one of the preceding items, wherein the bile acid or salt thereof is in micellar form. (Item 11) 10. The bile acid or salt thereof of any one of the preceding items for use in a method as defined in any one of the preceding items, wherein the bile acid or salt thereof is used in combination with one or more additional agents. (Item 12) The use of the bile acid or salt thereof, wherein the bile acid or salt thereof is in a micellar form and the one or more further agents are a) stabilizing the micellar form of the bile acid or salt thereof; and / or b) enabling or enhancing the formation of said micellar form; 12. A bile acid or salt thereof according to item 11, for use in a method as defined in item 11, comprising one or more agents. (Item 13) 13. The bile acid or salt thereof according to item 11 or 12, for use in the method defined in item 11 or 12, wherein the one or more agents comprise propyl gallate. (Item 14) 14. The bile acid or salt thereof according to item 11, 12 or 13, for use in the method as defined in item 11, 12 or 13, wherein the one or more further agents comprise one or more agents selected from: a) a fusogenic lipid, preferably selected from cationic liposomes, neutral helper lipids such as DOPE, and anionic lipids such as phosphatidic acid; b) cell-penetrating peptides, preferably cell-penetrating peptides having 2 to 12 amino acids (such as octaarginine, nonaarginine, and octalysine); c) a lysosomotropic agent, preferably chloroquine; d) a membrane disruptive peptide, preferably INF7, H5WYG, 43E, or histidine 10; e) a membrane disruptive polymer, preferably polyethyleneimine; f) a photochemical internalization agent, preferably fluorescein isothiocyanate or a compound containing same; and / or g) Agents that alter intracellular vesicle trafficking, preferably Brefeldin A. (Item 15) 10. The bile acid or salt thereof of any one of the preceding items for use in the method defined in any one of the preceding items, wherein the therapeutic compound is a nucleic acid polymer and the method of treatment is gene therapy. (Item 16) 10. The bile acid or salt thereof of any one of the preceding items, for use in the method defined in any one of the preceding items, wherein said therapeutic compound comprises a hydrophobic moiety. (Item 17) 10. The bile acid or salt thereof of any one of the preceding items for use in the method defined in any one of the preceding items, wherein the bile acid or salt thereof is used in the form of a micelle non-covalently conjugated to the therapeutic compound to enable or enhance cellular uptake of the therapeutic compound. (Item 18) 10. The bile acid or salt thereof of any one of the preceding items for use in the method defined in any one of the preceding items, wherein the bile acid or salt thereof is used to enable or enhance cellular uptake of one or more therapeutic compounds placed in the vicinity of the cell. (Item 19) 10. The bile acid or salt thereof of any one of the preceding items for use in the method defined in any one of the preceding items, wherein the method comprises administering a composition comprising the bile acid or salt thereof and the therapeutic compound, and where one or more additional agents are used in the method in combination with the bile acid or salt thereof, preferably also comprising one or more of the one or more additional agents. (Item 20) 20. The bile acid or salt thereof according to item 19, for use in the method defined in item 16, wherein the weight ratio of bile acid or salt thereof to the therapeutic compound in the composition is from 20:1 to 5:1. (Item 21) 21. The bile acid or salt thereof according to item 19 or 20, for use in the method defined in item 19 or 20, wherein the composition is in the form of a solution, suspension or dispersion. (Item 22) 21. The bile acid or salt thereof according to item 19 or 20, for use in the method defined in item 19 or 20, wherein the composition is in the form of a powder, the powder being contained in a capsule or pellet. (Item 23) 21. The bile acid or salt thereof according to item 19 or 20, for use in a method as defined in item 19 or 20, wherein the composition is in the form of a paste, ointment, or gel. (Item 24) 22. The bile acid or salt thereof according to item 21, for use in the method defined in item 21, wherein the composition is for intravenous administration, the bile acid or salt thereof and therapeutic compound in the composition are encapsulated by a coating that restricts dissolution of the bile acid or salt thereof and therapeutic compound, and the coating (a) removes the restriction on dissolution of the bile acid or salt thereof and therapeutic compound at a pH below 7.4, and / or (b) comprises one or more moieties that can bind to a target cell population in the body. (Item 25) 25. A therapeutic compound as defined in any one of items 1 to 24 for use in a method as defined in any one of items 1 to 24. (Item 26) 25. Use of a bile acid or salt thereof as defined in any one of items 1 to 24 in the manufacture of a medicament for use in a method as defined in any one of items 1 to 24. (Item 27) 25. Use of a therapeutic compound as defined in any one of items 1 to 24 in the manufacture of a medicament for use in a method as defined in any one of items 1 to 24. (Item 28) 25. A method for treating the human or animal body, wherein the method is as defined in any one of items 1 to 24. (Item 29) 1. A pharmaceutical composition comprising: a) a bile acid or a pharmaceutically acceptable salt thereof, wherein the bile acid is chenodeoxycholic acid or deoxycholic acid; b) a therapeutic compound; and c) one or more additional compounds selected from fusogenic lipids, cell-penetrating peptides, lysosomotropic agents, membrane-disrupting peptides, membrane-disrupting polymers, photochemical internalization agents, and agents that alter intracellular vesicle trafficking. A pharmaceutical composition comprising: (Item 30) 30. The pharmaceutical composition according to item 29, wherein the bile salt is used in combination with EDTA. (Item 31) 1. A pharmaceutical composition comprising: a) a bile acid or a pharmaceutically acceptable salt thereof, wherein the bile acid is chenodeoxycholic acid or deoxycholic acid; and b) therapeutic compound Including, c) The bile acid or salt thereof and therapeutic compound are encapsulated by a coating that (a) limits dissolution of the bile acid or salt thereof and therapeutic compound under aqueous conditions at pH 7.4 but removes the limit on dissolution of the bile acid or salt thereof and therapeutic compound at a pH below 7.4, and / or (b) comprises one or more moieties capable of binding to a target cell population in the body. (Item 32) 32. The pharmaceutical composition according to item 31, wherein the bile salt is used in combination with EDTA. (Item 33) 33. The composition according to item 32, wherein the composition further comprises one or more additional agents as defined in any one of items 11 to 13. (Item 34) 34. The composition according to any one of items 29 to 33, wherein the bile acid or a pharmaceutically acceptable salt thereof is a chenodeoxycholate, preferably sodium chenodeoxycholate. (Item 35) 35. The composition according to any one of items 29 to 34, wherein the therapeutic compound is a macromolecule. (Item 36) 36. The composition of any one of items 29 to 35, wherein the therapeutic compound is a protein. (Item 37) 37. The composition according to any one of items 29 to 36, wherein the ratio of the bile acid or salt thereof to the therapeutic compound is 20:1 to 5:1. (Item 38) 38. The composition according to any one of items 29 to 37, wherein the concentration of the EDTA on the surface of the cells at which cellular uptake is enabled or enhanced is between 0.1 mg / ml and 10 mg / ml. (Item 39) 39. The composition according to any one of items 29 to 38, wherein the concentration of the EDTA on the surface of the cells at which cellular uptake is enabled or enhanced is between 0.2 mg / ml and 10 mg / ml.

Claims

[Claim 1] The invention described in the specification.

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