Preparation of Bioactive Complexes

Abstract

Bioactive conjugates having therapeutic activity, particularly in the treatment of tumors or as antibacterial or antiviral agents, and methods for their preparation are provided. The method comprises dissolving a mixture of a polypeptide component in powder form and solid oleic acid or a pharmaceutically acceptable salt thereof in an aqueous solvent containing at least two salts, the first salt being sodium chloride or potassium chloride and the second salt being disodium phosphate or monopotassium phosphate, the dissolution being carried out at a suitable temperature below 50° C. The polypeptide component is preferably alpha-lactalbumin, which has membrane-perturbing activity.

Description

Technical Field

[0001] The present invention relates to a method for preparing a bioactive complex having therapeutic activity, particularly as a tumor treatment or an antibacterial or antiviral agent. Further, the present invention relates to methods for treating tumors and cancers, particularly methods for selectively targeting tumor cells over healthy cells, and novel complexes and compositions for use in those methods.

Background Art

[0002] Interest has been shown in the generation of complexes containing proteins and lipids that are not partially folded. These proteins can have significantly different properties, particularly biological properties, compared to the corresponding fully folded proteins. The acquisition of new beneficial functions through partial protein folding and fatty acid binding is a surprising phenomenon and may reflect an important general pathway for the functional diversification of proteins through changes in their conformational states and bound ligands. Thus, in addition to alternative splicing of mRNA transcripts, post-translational modifications, and changes in the tertiary structure of specific domains, partial folding of the original native protein has been recognized as a mechanism for generating functional diversity. This is due to the cellular response to unfolded proteins and lipid cofactors, which defines their altered properties. However, this response can be different, for example, in tumor cells, which means that it can give rise to their therapeutic potential. For the formation of stable moieties, unfolded proteins are often modified in some way and can be bound to cofactors, particularly fatty acid cofactors. The complexes thus formed are stable and can give rise to therapeutic options.

[0003] HAMLET (human α-lactalbumin made lethal to tumor cells) is an example of a novel family of antitumor molecules with important properties. HAMLET, formed from partially folded α-lactalbumin and oleic acid as an essential component, was serendipitously discovered during studies of the ability of human milk to prevent bacteria from binding to cells. Initial in vitro experiments showed that HAMLET exhibits broad antitumor activity with high tumor selectivity, and subsequent therapeutic studies confirmed the antitumor and relative selectivity of HAMLET against tumor tissue in vivo. In placebo-controlled clinical trials, local administration of HAMLET removed or reduced the size of skin papillomas, and local injection of HAMLET in patients with bladder cancer caused rapid death of tumor cells but did not kill healthy tissue surrounding the tumor. The therapeutic efficacy of HAMLET in bladder cancer has recently been demonstrated in a mouse model of bladder cancer, and treatment with HAMLET in a rat glioblastoma xenograft model delayed tumor progression and increased survival without signs of cell death in healthy brain tissue. Thus, HAMLET appears to identify death pathways conserved in tumor cells, thereby allowing them to be distinguished from healthy differentiated cells.

[0004] Other complexes using umarizyme and oleic acid have also been found to cause cell death (Vukojevic et al. Langmuir, 2010, 26(18) 14782-14787), and it has been proposed that folded proteins can exhibit cytotoxicity when bound to appropriate cofactors.

[0005] Other studies have focused on the use of peptide fragments of these proteins that can be used (see, for example, European Patent No. 2643010 and UK Patent Application No. 1621752.3).

[0006] Classically, these types of complexes are prepared as described in Svensson et.al, (2000). Proc Natl Acad Sci U. S. A. 97, 4221-4226. Native α-lactalbumin was purified from human milk by hydrophobic interaction chromatography. The protein was subjected to ion exchange chromatography on a matrix developed with EDTA and pretreated with oleic acid, and eluted with high concentration salts, particularly 1M NaCl, to obtain the bioactive complex. This type of procedure has been used to generate other bioactive complexes including BAMLET from bovine α-lactalbumin and is formed from recombinant forms of α-lactalbumin, particularly those without cysteine residues as described in WO2010 / 079362.

[0007] An alternative preparation of such bioactive complexes is described in WO2010 / 131010. In this reference, BAMLET is prepared in a single-phase manner in which α-lactalbumin is reconstituted with phosphate buffered saline (PBS) and added sodium oleate. The mixture is then heated to a temperature of 60 °C or higher to obtain the active complex. This method has the advantage of being simplified for implementation and can even be performed in situ in a clinical situation using a kit.

[0008] In other references, bioactive complexes are prepared by dissolving pre-lyophilized complexes in PBS for use (see for example WO2010 / 079362).

[0009] Thus, it is clear that this type of complex is dependent on the presence of salts in their production. Phosphate buffered saline (PBS), which has been used previously, contains a mixture of at least three and sometimes four salts. These are sodium chloride, disodium phosphate and monopotassium phosphate, and may also contain potassium chloride.

[0010] The present applicants investigated the influence of salt mixtures used in the preparation of compounds and surprisingly discovered that the exact nature of the salts used in the production can affect the activity of the product. This presentation shows that the products can be identified and thus those with a specific salt balance are the only products.

Summary of the Invention

[0011] According to the present invention, there is provided a method for preparing a bioactive complex, comprising dissolving a mixture of a polypeptide element in powder form and solid oleic acid or a pharmaceutically acceptable salt thereof in an aqueous solvent containing at least two salts, wherein the first salt is sodium chloride or potassium chloride, the second salt is disodium phosphate or monopotassium phosphate, and in particular the method is carried out at a suitable temperature.

[0012] As used herein, the expression "suitable temperature" means a temperature of 50 °C or lower, for example 0 to 50 °C, for example 10 to 40 °C, more specifically 15 to 25 °C, for example the outside air temperature. Such a temperature is generally lower than the "melting temperature", which is the temperature at which the polypeptide unfolds or denatures. However, the present applicants have discovered that a bioactive complex can be formed under the conditions of those salts.

[0013] The present applicants have discovered that the bioactive complex can be prepared by the simple dissolution method of the present invention, which clearly shows a concentration-dependent reaction. The mixture may be warmed to 40 °C or lower, for example 50 °C or lower, for example to achieve rapid dissolution, but does not require extensive heating such as boiling as described for the solution, as long as an appropriate salt balance is present in the aqueous solvent. Thus, in certain embodiments, the method is carried out at the outside air temperature.

[0014] Dissolution can be promoted by stirring, for example by vortexing. If necessary, the solution can be filtered at this stage through a bacterial filtration filter. Suitable filters include polyethersulfone membranes (PES) or Minisart® NML cellulose acetate membranes.

[0015] Any such stirring process will be carried out for a period sufficient to ensure dissolution of the components in the salt solution. The exact timing may vary depending on factors such as the specific nature of the polypeptide used and the temperature at which the mixture is placed, but the timing will typically be a very short time, for example as little as 10 minutes, or as short as 1 - 5 minutes such as about 2 minutes.

[0016] In certain embodiments, the solvent further comprises a third salt, which is sodium monophosphate or potassium monophosphate, particularly potassium monophosphate. Such mixtures are found in conventional PBS solutions.

[0017] Accordingly, this method is easy to prepare in various manufacturing and non - manufacturing environments.

[0018] As used herein, the term "polypeptide" includes peptides including proteins and long - chain peptides.

[0019] Suitable "polypeptide components" used in the method of the present invention include natural proteins, particularly α - lactalbumin, lysozyme or other proteins having activity to perturb membranes, such as specific variants of said natural proteins lacking intramolecular bonds due to mutations in cysteine residues, or fragments of any of those proteins in peptides of 50 amino acids or less.

[0020] The expression "variant" means a protein or polypeptide having the same biological function but having one or more amino acids in the sequence substituted with other amino acids, different from the original sequence from which it is derived. Amino acid substitutions are said to be "conservative" when an amino acid is changed to a different amino acid having broadly similar properties. A non - conservative substitution is when an amino acid is substituted with a different type of amino acid.

[0021] ​"Conservative substitution" means the substitution of an amino acid by another amino acid of the same class, which class is defined below. TIFF0007705160000001.tif38137

[0022] As is well known to those skilled in the art, changing the primary structure of a peptide by conservative substitution may not significantly alter the activity of the peptide, since the side chain of the inserted amino acid can form similar bonds and contacts as the side chain of the substituted amino acid. This can be said even when the substitution occurs in a region important for determining the three-dimensional structure of the peptide.

[0023] Non-conservative substitutions may be possible as long as they do not interfere with the function of the polypeptide of the DNA binding domain.

[0024] Broadly speaking, there are few non-conservative substitutions that do not change the biological activity of a polypeptide.

[0025] Determining the effect of substitutions (and indeed amino acid deletions or insertions) is entirely within the routine of those skilled in the art, who can readily determine whether a mutant polypeptide retains the basic characteristics and activity of the native protein. For example, when determining whether a mutant of a polypeptide is within the scope of the present invention, those skilled in the art will determine whether a complex containing the mutant retains the biological activity (e.g., tumor cell death) of a complex formed by the unfolded form of the native protein, and the polypeptide has at least 60%, preferably 70%, more preferably 80%, still more preferably 90%, 95%, 96%, 97%, 98%, 99% or is 100% of the native protein.

[0026] A mutant of a polypeptide may comprise or consist essentially of an amino acid sequence having at least 70% identity, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98% or 99% identity, to the sequence of a native protein such as an α-lactalbumin or lysozyme sequence.

[0027] The level of sequence identity is appropriately determined using the BLASTP computer program along with the native protein sequence as the original sequence. This means that the native protein sequence forms the comparison sequence for determining the percentage of identity. The BLAST software is publicly accessible at http: / / blast.ncbi.nlm.nih.gov / Blast.cgi (accessible on May 10, 2017).

[0028] In certain embodiments, the polypeptide element is a peptide having as few as 50 amino acids, particularly 10 - 45 amino acids. Such complexes are easy to prepare and have a lower cost of starting materials. For example, the peptide can be prepared using conventional methods for peptide production. The formed complex can be easily formulated for handling and administration due to its small molecular weight.

[0029] It is appropriately derived from a native protein or a variant thereof. Suitable proteins are those identified as being active in such complexes such as α - lactalbumin, β - lactalbumin, or lysozyme, but can be derived from membrane - perturbing proteins.

[0030] A membrane - perturbing protein is a protein having the ability to interact with the cell membrane interface, particularly the ability to cause perturbations such as tubulation of the cell membrane. Typically, the protein will become embedded in the cell membrane. Examples of such proteins include coat complexes such as COPI, COPII (SAR1, etc.), HOPS / CORVET, SEA (Seh1 - related), clathrin complexes such as endophilin, BAR domain proteins, and ESCRT complexes including the Snf7 domain subunit.

[0031] In particular, the peptide is derived from the α - helix domain of the above - mentioned native protein. The α - helix domain of the protein can be determined using methods well - known in the art or conventional methods.

[0032] When the α-helix domain contains cysteine residues, in some embodiments, they can be mutated to different amino acid residues such as alanine residues to avoid intermolecular disulfide bonds.

[0033] In certain embodiments, the peptide is a fragment of α-lactalbumin, particularly a fragment of the α-domain of α-lactalbumin. In certain embodiments, the peptide comprises α1 (residues 1-40) or α2 (residues 81-123) of human α-lactalbumin, or an analog region of other α-lactalbumins such as bovine α-lactalbumin.

[0034] The peptide does not appropriately contain elements that cause folding and thus appropriately lacks amino acids that cause intramolecular bonds such as cysteine residues. In particular, when the peptide is derived from a natural protein, any cysteine residues are substituted with other amino acids such as alanine.

[0035] Thus, in certain embodiments, the complex comprises α1 (residues 1-40) or α2 (residues 81-123) of human α-lactalbumin, and cysteine is substituted with other amino acids such as alanine to prevent intermolecular bonding.

[0036] Thus, the peptide may be SEQ ID NO: 1 or SEQ ID NO: 2, KQFTKXELSQLLKDIDGYGGIALPELIXTMFHTSGYDTQA (SEQ ID NO: 1) LDDDITDDIMXAKKILDIKGIDYWLAHKALXTEKLEQWLXEKL (SEQ ID NO: 2) X is an amino acid residue other than cysteine.

[0037] A specific example of such a sequence is the sequence of SEQ ID NO: 3 or SEQ ID NO: 4. KQFTKAELSQLLKDIDGYGGIALPELIATMFHTSGYDTQA (SEQ ID NO: 3) LDDDITDDIMAAKKILDIKGIDYWLAHKALATEKLEQWLAEKL (SEQ ID NO: 4)

[0038] In some cases, the peptide of SEQ ID NO: 1 can be truncated, for example, by omitting the terminal alanine residue, thereby resulting in the peptide of SEQ ID NO: 6, and SEQ ID NO: 7 is a specific example thereof. KQFTKXELSQLLKDIDGYGGIALPELIXTMFHTSGYDTQ (SEQ ID NO: 6) KQFTKAELSQLLKDIDGYGGIALPELIATMFHTSGYDTQ (SEQ ID NO: 7)

[0039] Such peptides are novel and form further aspects of the invention together with the bioactive complexes containing them.

[0040] Other peptides may also be used in the complex, and the compatibility can be tested by determining whether the complex containing the fatty acid salt has the activity to kill cells using, for example, the methods described below.

[0041] In other embodiments, the peptide is derived from a COPII family protein such as SAR1. A specific example of such a peptide is the peptide of SEQ ID NO: 5. MAGWDIFGWF RDVLASLGLW NKH (SEQ ID NO: 5)

[0042] In other embodiments, the polypeptide element is a natural protein of α-lactalbumin such as human, bovine, ovine or camel α-lactalbumin or a synthetic form thereof. In particular, the protein is bovine lactalbumin.

[0043] As used herein, the term "bioactivity" means a complex having biological activity, the activity of which is different from or stronger than that of each of its components. In particular, the complex can induce cell death, especially selectively induce cell death in tumor cells, and / or other therapeutic effects can be obtained, such as antibacterial or antiviral effects not seen in natural proteins including monomeric forms of α-lactalbumin.

[0044] In particular, the oleic acid used in the method of the present invention is C18:1 oleic acid with the chemical formula CH3(CH2)7CH=CH(CH2)7COOH or CH3(CH2)7CH=CH(CH2)7COO.

[0045] In certain embodiments, pharmaceutically acceptable salts of oleic acid are used in the process. Suitable pharmaceutically acceptable salts will be understood in the art.

[0046] The use of salts, especially water-soluble salts of oleic acid, fatty acids or lipids, means that the preparation method is facilitated because an aqueous solution can be formed for application to, for example, an ion exchange column. Suitable water-soluble salts are alkali or alkaline earth metal salts such as sodium or potassium salts.

[0047] Furthermore, it has been discovered that salts and especially salts such as sodium oleate originally have an antitumor effect. Therefore, its inclusion in the complex can result in increased activity.

[0048] In certain embodiments, the first salt used in the method of the present invention is sodium chloride.

[0049] In other embodiments, the second salt used in the method of the present invention is disodium phosphate.

[0050] In other specific embodiments, the third salt used in the method of the present invention is potassium dihydrogen phosphate.

[0051] The ratio of the first salt:the second salt used in the method of the present invention is suitably from 8:1 to 1:1, for example from 5:1 to 2:1, especially from 4:1 to 3.5:1. The ratio of the first salt:the third salt is from 20:1 to 5:1, for example from 15:1 to 10:1, and may especially be from 12.5:1 to 11.5:1.

[0052] In certain embodiments, the ratio of the first salt:the second salt:the third salt is from 13 - 12:4 - 3:1.

[0053] The ratio of oleic acid or oleate:peptide mixed in the method of the present invention is suitably within the range of 20:1 to 1:1, but preferably an excess of oleate is present, for example the ratio of oleate:peptide is about 5:1. The mixing can be carried out at a temperature of 0 - 50 °C and conveniently can be carried out at ambient temperature and atmospheric pressure.

[0054] If necessary, the product of the process of the present invention can be solidified, for example by lyophilization, for storage or formulation. It can then be reconstituted, especially using sterile water, for use. Such procedures can be particularly appropriate when the polypeptide is rather a peptide than a protein. The Applicants have found that proteins can return to their native folded state when subjected to treatments such as such lyophilization.

[0055] That problem can be alleviated by stabilizing the polypeptide in an unfolded state during the preparation method, for example by lowering the pH of the solution to, for example, 4 or less, or by adding a calcium chelating agent such as EDTA to the solvent. In a second aspect, the present invention provides a complex obtainable by the method of the first aspect.

[0056] Accordingly, the complexes of the second aspect of the present invention can be formulated into useful pharmaceutical compositions by combining them with a pharmaceutically acceptable carrier in a conventional manner. Such compositions form the third aspect of the present invention.

[0057] The composition according to the third aspect of the present invention is a suitable pharmaceutical composition in a form suitable for topical use, such as a cream, ointment, gel, or aqueous or oily solution or suspension. These may include pharmaceutically acceptable commonly known carriers, filters and / or means.

[0058] Topical solutions or creams appropriately contain an emulsifier to incorporate the protein complex with a diluent or cream base.

[0059] The daily dose of the complex varies and depends on the patient, the nature of the condition being treated, etc., according to standard clinical practice. Generally, 2 - 200 mg / dose of the bioactive complex is used per administration.

[0060] In a further aspect of the present invention, there is provided a method for treating cancer comprising administering the bioactive complex as described above to a patient in need thereof.

[0061] In particular, the complex can be used to treat cancers such as human cutaneous papilloma, human bladder cancer, and glioblastoma. In the latter case, administration can be made by infusion well known in the art.

[0062] The present invention provides the bioactive complex as described above for use in treatment, particularly in the treatment of cancer.

[0063] The complex can be used for the prevention of cancer, particularly gastrointestinal cancers as described in WO2014 / 023976 etc. In this case, the complex can be combined with foods such as dairy products like yogurt for use as a functional food. This type of composition forms a further aspect of the present invention.

[0064] Throughout the entire description from the detailed description to the claims, the terms "comprise" and "contain" and variations thereof such as, for example, "comprising" and "comprises" mean "including but not limited to" and do not exclude other components, integers or steps. Further, the singular form includes the plural form unless otherwise specified, and particularly when the indefinite article is used, in the specification, it is understood that the plural and singular forms are considered as such unless otherwise specified.

[0065] Preferred features of each aspect of the present invention may be described in combination with any of the other aspects. Within the scope of this application, various aspects, embodiments, examples and variations are presented in the foregoing, the claims, and / or the following detailed description and drawings, and in particular, it is explicitly intended that their respective features be taken independently or any be combined. That is, all embodiments and / or features of any embodiments can be combined in any way and / or can be combined as long as such features are compatible.

Brief Description of the Drawings

[0066] The present invention will be specifically described by way of examples with reference to the following attached drawings.

Figure 1A

Figure 1B

Figure 1C

Figure 2

Figure 3

Figure 4

Mode for Carrying Out the Invention

[0067] (Example 1) Generation of Biologically Acceptable Complexes Biologically active complexes within a predetermined range were prepared using the peptide of SEQ ID NO: 7. Ac-KQFTKAELSQLLKDIDGYGGIALPELIATMFHTSGYDTQ-OH (SEQ ID NO: 7) This is a mutant of a fragment of human α-lactalbumin.

[0068] The freeze-dried peptide (700 μM) was added to a tube together with sodium oleate flakes (3.5 mM). Subsequently, each tube was reconstituted with any of the following required volumes. 1) Phosphate-buffered saline (NaCl 6.8 g / L), Na2HPO4×2H2O (4. 8 g / L) and KH2PO4 (1.3 g / L) (pH 7.2) 2) NaCl solution (116 mM) (pH 7.01) 3) Na2HPO4 solution (31 mM) (pH 8.6) 4) KH2PO4 solution (9.56 mM) (pH 4.6) 5) Mixture of (2) and (4) (pH 4.63) 6) Mixture of (2) and (3) (pH 8.37) 7) Mixture of (3) and (4) (pH 7.29) Each mixture was vortexed until the solution became clear.

[0069] Subsequently, the obtained complex was freeze-dried. The freeze-drying conditions were a pressure of less than 1.2 mbar and a temperature of less than -55°C.

[0070] Each tube was stored at -20°C or below and reconstituted by adding 30 mL of sterile water immediately before use.

[0071] (Example 2) Cell death assay Human lung cancer cells (A549, ATCC) were cultured in RPMI-1640 containing non-essential amino acids (1 :100), 1 mM sodium pyruvate, 50 μg / ml gentamicin and 5 - 10% fetal bovine serum (FCS) at 37°C and 5% CO2. For the cell death test, cells were cultured overnight in a 96-well plate (2×10 4 / well, Tecan Group Ltd). The cells were incubated at 37°C in serum-free RPMI-1640 containing the bioactive complex obtained in Example 1 at a dose corresponding to 7, 21 or 35 μM of the peptide. FCS was added after 1 hour. Cell death was quantified 3 hours after peptide-oleate treatment by three biological methods including 1) evaluation of the ATP level of cells using luminescence based on the ATP Lite kit (Parkin Elmer), 2) PrestoBlue fluorescence staining (Invitrogen, A13262) and 3) trypan blue exclusion assay. Fluorescence and luminescence were measured using a microplate reader (InfiniteF200, Tecan).

[0072] The results are shown in Figure 1. The complex prepared with PBS had high activity and caused cell death in a concentration-dependent manner (Figure 1A). The complex prepared with only the single salt of PBS showed a significant reduction in activity (Figure 1B). However, as shown in Figure 1C, the above mixture (6) maintained a reasonable level of tumor cell death activity in a concentration-dependent manner.

[0073] (Example 3) Effect of filtration in the method The method of Example 1 was repeated twice using PBS solution (1), where each solution was passed through chemically different filters, either a polyethersulfone membrane (12846445, VWR) or a Minisart® NML cellulose acetate membrane (60810103, Sartorius). The biological effect of the product was tested as described in Example 2 by comparing it side by side with the unfiltered product. The results are shown in Figure 2.

[0074] No significant differences were observed in the biological activity of the complex quantified by measurements of total cellular ATP levels, PrestoBlue staining, and trypan blue exclusion assays.

[0075] (Example 4) Generation of BAMLET Lyophilized bovine α-lactalbumin (700 μM) was added to a tube (3.5 mM) together with sodium oleate flakes. Subsequently, phosphate buffered saline (1 ml) was added to the tube and vortexed at room temperature for 1 - 2 minutes. A clear solution was generated (Figure 3A).

[0076] The resulting solution was tested using the assay described in Example 2. The results are shown in Figure 4. It is clear that the solution is biologically active and kills A549 lung cancer cells in a concentration-dependent manner. This effect is transient, but lyophilization of the complex removed the activity.

Claims

1. comprising the peptide of SEQ ID NO: 7 and sodium oleate, wherein the peptide of SEQ ID NO: 7 in powder form and the solid sodium oleate are dissolved in an aqueous solvent containing at least two salts, the first salt of the aqueous solvent is sodium chloride or potassium chloride, and the second salt is disodium phosphate or monopotassium phosphate, a bioactive complex.

2. The peptide of SEQ ID NO:

7.

3. A method for preparing a bioactive complex, comprising dissolving a mixture of a polypeptide element in powder form and solid oleic acid or a pharmaceutically acceptable salt thereof in an aqueous solvent containing at least two salts, the first salt is sodium chloride or potassium chloride, and the second salt is disodium phosphate or monopotassium phosphate, the dissolution is carried out at a suitable temperature of 50 °C or lower, the polypeptide element is the peptide of SEQ ID NO: 7, a method.

4. The method according to claim 3, wherein the aqueous solvent further comprises a third salt which is monosodium phosphate or monopotassium phosphate.

5. The method according to claim 3 or 4, further comprising filtering the solution obtained by the dissolution.

6. The method according to any one of claims 3 to 5, further comprising removing the solvent to obtain the solid complex.

7. The method according to claim 6, wherein the solvent is removed by lyophilization.

8. The method according to any one of claims 3 to 7, wherein the first salt is sodium chloride.

9. The method according to any one of claims 3 to 8, wherein the second salt is disodium phosphate.

10. The method according to claim 4, wherein the third salt is monopotassium phosphate.

11. The method according to claim 6 or 7, wherein the complex is reconstituted with sterile water immediately before use.

12. A pharmaceutical composition comprising the bioactive complex according to claim 1.

13. A functional food composition comprising the bioactive complex according to claim 1.

14. A pharmaceutical composition for treating or preventing cancer, comprising an effective amount of the bioactive complex according to claim 1 or the composition according to claim 12 or 13.

Images