Preparation of bioactive conjugates

Abstract

The present invention provides a method for preparing a bioactive conjugate, which comprises dissolving a mixture of a polypeptide component, such as alpha-lactalbumin or a fragment thereof, in powder form and solid oleic acid or a pharmaceutically acceptable salt thereof in an aqueous solvent containing a mixture of at least two, preferably three salts, the method being carried out at a moderate temperature, and the preparation does not require excessive heating, making it simple and efficient. [Selection diagram] None

Description

[Technical field]

[0001] The present invention relates to methods for preparing bioactive conjugates having therapeutic activity, particularly in the treatment of tumors or as antibacterial or antiviral agents.Furthermore, the present invention relates to methods for the treatment of tumors and cancer, particularly methods that selectively target tumor cells over healthy cells, and to novel conjugates and compositions for use in such methods. [Background technology]

[0002] There has been interest in the generation of complexes containing partially unfolded proteins and lipids. These proteins may have significantly different properties, especially biological properties, compared to their fully folded counterparts. The acquisition of novel beneficial functions by partial protein folding and fatty acid binding is a surprising phenomenon and may reflect an important general route of functional diversification of proteins through changes in their conformational state and the ligands they bind. Thus, in addition to alternative splicing of mRNA transcripts, post-translational modifications, and alterations in the tertiary structure of certain domains, partial folding of original native proteins has been recognized as a mechanism for the generation of functional diversity. This is due to the cellular response to unfolded proteins and lipid cofactors, which defines their altered properties. However, this response may be different, for example in tumor cells, which means that their therapeutic potential may arise. For the formation of stable moieties, unfolded proteins are often modified in some way and may be bound to cofactors, especially fatty acid cofactors. The complexes thus formed are stable and may give rise to therapeutic options.

[0003] HAMLET (human alpha-lactalbumin lethal to tumor cells) is one example of a novel family of tumoricidal molecules with important properties. Formed from partially folded alpha-lactalbumin and oleic acid as an essential component, HAMLET was discovered by chance during the study of the ability of human milk to prevent bacteria from binding to cells. Early in vitro experiments showed that HAMLET exhibited broad antitumor activity with high tumor selectivity, and later therapeutic studies confirmed the tumoricidal and relative selectivity of HAMLET for tumor tissue in vivo. In placebo-controlled clinical trials, topical HAMLET administration eliminated 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 was recently demonstrated in a mouse bladder cancer model, and in a rat glioblastoma xenograft model, treatment with HAMLET delayed tumor progression and increased survival, without evidence of cell death in healthy brain tissue. Thus, HAMLET appears to identify conserved death pathways in tumor cells, thereby distinguishing them from healthy differentiated cells.

[0004] Other complexes using horse lysozyme 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 may be cytotoxic when bound to appropriate cofactors.

[0005] Other work has 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 conjugates are prepared as described by Svensson et.al, (2000). Proc Natl Acad Sci USA 97, 4221-4226. Native alpha-lactalbumin was purified from human milk by hydrophobic interaction chromatography. The protein was developed with EDTA, subjected to ion exchange chromatography on a matrix pretreated with oleic acid, and eluted with high salt, in particular 1M NaCl, to obtain the bioactive conjugate. This type of procedure has been used to generate other bioactive conjugates, including BAMLET from bovine alpha-lactalbumin, which was formed from recombinant forms of alpha-lactalbumin, in particular those lacking cysteine ​​residues, as described in WO2010 / 079362.

[0007] An alternative preparation of such a bioactive complex is described in WO2010 / 131010. In this reference, BAMLET is prepared in a monophasic manner in which alpha-lactalbumin is reconstituted with phosphate-buffered saline (PBS) and sodium oleate added. 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 conjugates are prepared by dissolving previously lyophilized conjugates in PBS for use (see, for example, WO2010 / 079362).

[0009] It is therefore clear that complexes of this type are dependent on the presence of salt for their formation. The traditionally used phosphate buffered saline (PBS) contains a mixture of at least three, and sometimes four, salts: sodium chloride, disodium phosphate, and monopotassium phosphate, and may also contain potassium chloride.

[0010] Applicants have investigated the effect of salt mixtures used in the preparation of compounds and have surprisingly discovered that the exact nature of the salts used in the preparation can affect the activity of the product. This presentation shows that products can be identified and therefore those with a particular salt balance are unique 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 component in powder form and solid oleic acid or a pharma- ceutically 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, in particular the method being carried out at a suitable temperature.

[0012] The expression "suitable temperature" as used herein means a temperature below 50°C, e.g., 0-50°C, e.g., 10-40°C, more particularly 15-25°C, e.g., ambient temperature. Such temperatures are generally lower than the "melting temperature", the temperature at which the polypeptide unfolds or denatures. However, the applicants have discovered that bioactive complexes can be formed under these salt conditions.

[0013] Applicants have discovered that bioactive conjugates can be prepared by the simple dissolution method of the present invention that clearly exhibits a concentration-dependent response. The mixture may be warmed, for example, to below 50° C., for example, below 40° C. to achieve rapid dissolution, but without the need for extensive heating, such as boiling, as described for solutions, only that the appropriate salt balance is present in the aqueous solvent. Thus, in certain embodiments, the method is carried out at ambient temperature.

[0014] Dissolution may be aided by stirring, e.g., vortexing. If necessary, the solution may be filtered through a bacterial filter at this stage. Suitable filters include polyethersulfone membranes (PES) or Minisart® NML Cellulose acetate membranes.

[0015] Any such stirring process will be carried out for a period of time sufficient to ensure dissolution of the components in the salt solution, and while the exact timing may vary depending on factors such as the particular nature of the polypeptide used and the temperature to which the mixture is subjected, the timing will typically be quite short, such as 1-5 minutes, e.g., no more than 10 minutes, e.g., about 2 minutes.

[0016] In certain embodiments, the solvent further comprises a third salt, which is monosodium phosphate or monopotassium phosphate, in particular monopotassium phosphate. Such mixtures are found in conventional PBS solutions.

[0017] The method is therefore amenable to preparation in a variety of manufacturing and non-manufacturing environments.

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

[0019] Suitable "polypeptide elements" to be used in the methods of the invention include naturally occurring proteins, in particular alpha-lactalbumin, lysozyme or other proteins with membrane-perturbing activity, specific mutants of said naturally occurring proteins which are deficient in intramolecular binding, e.g. due to mutation of a cysteine ​​residue, or fragments of any of these proteins which are peptides of 50 amino acids or less.

[0020] The expression "variant" refers to a protein or polypeptide having the same biological function but differing in comparison with the original sequence from which the amino acid sequence is derived by the substitution of one or more amino acids in the sequence with other amino acids. Amino acid substitutions are considered to be "conservative" in that an amino acid is changed to a different amino acid having broadly similar properties. Non-conservative substitutions are those in which an amino acid is replaced by an amino acid of a different type.

[0021] A "conservative substitution" refers to the replacement of an amino acid with another amino acid of the same class, which classes are defined below. JPEG0007672122000001.jpg53159

[0022] As is well known to those of skill in the art, altering the primary structure of a peptide by conservative substitutions may not significantly alter the activity of the peptide, since the side chain of the amino acid inserted into the sequence may be able to form similar bonds and contacts as the side chain of the substituted amino acid, even when the substitution occurs in a region that is important in determining the three-dimensional structure of the peptide.

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

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

[0025] Determining the effect of substitutions (and indeed amino acid deletions or insertions) is well within the routine scope of one of skill in the art, who can readily determine whether a variant polypeptide retains the basic properties and activity of a basic protein. For example, in determining whether a variant of a polypeptide is within the scope of the present invention, one of skill in the art will determine whether a complex containing the variant retains the biological activity (e.g., tumor cell killing) of a complex formed with the unfolded form of the native protein, and which polypeptide has at least 60%, preferably 70%, more preferably 80%, and even more preferably 90%, 95%, 96%, 97%, 98%, 99% or 100% of the native protein.

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

[0027] The level of sequence identity is suitably determined using the BLASTP computer program 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 (accessed May 10, 2017).

[0028] In certain embodiments, the polypeptide element is a peptide, which may have as few as 50 amino acids, particularly 10 to 45 amino acids. Such conjugates are easy to prepare and have lower starting material costs. For example, peptides may be prepared using conventional methods for peptide production. The formed conjugates may be easy to handle and formulate for administration due to their small molecular weight.

[0029] It is suitably derived from a natural protein or a variant thereof. Suitable proteins are those identified as being active in such complexes, such as alpha-lactalbumin, beta-lactalbumin or lysozyme, but may be derived from membrane-perturbing proteins.

[0030] A membrane perturbing protein is a protein that has the ability to interact with the plasma membrane interface, in particular to cause a perturbation, such as tubulation, of the plasma membrane. Typically, the protein will become embedded in the plasma membrane. Examples of such proteins include coat complexes such as COPI, COPII (such as SAR1), HOPS / CORVET, SEA (Seh1-associated), clathrin complex BAR domain proteins such as endophilin, and ESCRT complexes that contain Snf7 domain subunits.

[0031] In particular, the peptides are derived from the alpha-helical domains of the naturally occurring proteins mentioned above, which may be determined using methods well known or conventional in the art.

[0032] Where the α-helical domain contains cysteine ​​residues, in some embodiments they may 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 alpha-lactalbumin, in particular a fragment of the alpha domain of alpha-lactalbumin. In certain embodiments, the peptide comprises the alpha1 (residues 1-40) or alpha2 (residues 81-123) of human alpha-lactalbumin, or an analog region of another alpha-lactalbumin, such as bovine alpha-lactalbumin.

[0034] The peptide suitably does not contain elements that allow folding and therefore suitably lacks amino acids that allow intramolecular bonds to form, such as cysteine ​​residues, particularly when the peptide is derived from a naturally occurring protein, in which case any cysteine ​​residues have been replaced with other amino acids, such as alanine.

[0035] Thus, in certain embodiments, the conjugate comprises α1 (residues 1-40) or α2 (residues 81-123) of human alpha-lactalbumin, in which cysteines have been replaced with other amino acids, such as alanine, to prevent intermolecular binding.

[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] Particular examples of such sequences are the sequences 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 may be truncated, for example by omitting the terminal alanine residue, resulting in the peptide of SEQ ID NO:6, of which SEQ ID NO:7 is a particular example. KQFTKXELSQLLKDIDGYGGIALPELIXTMFHTSGYDTQ (SEQ ID NO: 6) KQFTKAELSQLLKDIDGYGGIALPELIATMFHTSGYDTQ (SEQ ID NO: 7)

[0039] Such peptides are novel and together with the bioactive conjugates containing them form a further aspect of the present invention.

[0040] Other peptides may also be used in the conjugates and suitability may be tested by determining whether the conjugates containing fatty acid salts have cell killing activity 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 another embodiment, the polypeptide element is a naturally occurring protein of alpha-lactalbumin, in particular human, bovine, ovine or camel alpha-lactalbumin, or a synthetic form thereof. In particular the protein is bovine lactalbumin.

[0043] The term "biologically active" as used herein refers to a complex that has a biological activity that is different or stronger than the individual components. In particular, the complex can induce cell death, particularly in a tumor cell-selective manner, and / or have other therapeutic effects that are not found in natural proteins, including, for example, the monomeric form of alpha-lactalbumin.

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

[0045] In certain embodiments, a pharma- ceutically acceptable salt of oleic acid is used in the process. Suitable pharma- ceutically acceptable salts will be understood in the art.

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

[0047] Furthermore, salts, and particularly salts such as sodium oleate, have been found to have inherent tumoricidal activity, and therefore, its inclusion in the complex may result in enhanced activity.

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

[0049] In another embodiment, the second salt used in the methods of the invention is disodium phosphate.

[0050] In other specific embodiments, the third salt used in the methods of the invention is monopotassium phosphate.

[0051] The ratio of first salt:second salt used in the method of the invention is suitably 8:1 to 1:1, such as 5:1 to 2:1, in particular 4:1 to 3.5:1. The ratio of first salt:third salt may be 20:1 to 5:1, such as 15:1 to 10:1, in particular 12.5:1 to 11.5:1.

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

[0053] The ratio of oleic acid or oleate:peptide mixed in the method of the invention is suitably in the range of 20:1 to 1:1, but preferably an excess of oleate is present, for example an oleate:peptide ratio of about 5:1. Mixing may be carried out at a temperature of 0 to 50°C, conveniently 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, particularly with sterile water, for use. Such a procedure can be particularly appropriate when the polypeptide is a peptide rather than a protein. Applicants have discovered that proteins can return to their native folded state when subjected to such a process, such as lyophilization.

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

[0056] Thus, the conjugates of the second aspect of the invention may be formulated into useful pharmaceutical compositions by combining them with a pharma- ceutically acceptable carrier in conventional manner, and such compositions form a third aspect of the invention.

[0057] The compositions according to the third aspect of the invention are suitable pharmaceutical compositions in a form suitable for topical use, for example as a cream, ointment, gel, or aqueous or oily solution or suspension, which may contain pharma- ceutically acceptable carriers, filters and / or means generally known in the art.

[0058] The topical solution or cream suitably contains an emulsifier to bind the protein complex to the diluent or cream base.

[0059] The daily dose of the conjugate will vary, in accordance with standard clinical practice, depending on the patient, the nature of the condition being treated, etc. Generally, 2-200 mg / dose of the bioactive conjugate is used in each administration.

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

[0061] In particular, the complexes may be used to treat cancers such as human skin papillomas, human bladder cancer, and glioblastomas, in the latter case, administration may be by injection as is well known in the art.

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

[0063] The complex may be used for the prevention of cancer, in particular gastrointestinal cancer, as described in WO2014 / 023976 etc. In this case, the complex may be combined with a food product, such as a dairy product, such as yoghurt, for use as a functional food. Compositions of this type form a further aspect of the present invention.

[0064] Throughout this specification, from the detailed description to the claims, the terms "comprise" and "contain" and variations of these terms, such as "comprising" and "comprises," mean "including, but not limited to," and do not exclude other elements, integers, or steps. Furthermore, the singular includes the plural unless otherwise specified, and particularly where the indefinite article is used, the specification is understood to contemplate both the plural and the singular unless otherwise specified.

[0065] Preferred features of each aspect of the invention may be described in combination with any of the other aspects. Within the scope of this application, various aspects, embodiments, examples and variants are presented above, in the claims, and / or in the following detailed description and drawings, and it is expressly intended that each of their features may be taken independently or in any combination. That is, all embodiments and / or features of any embodiment may be combined in any way and / or may be combined insofar as such features are not incompatible. [Brief description of the drawings]

[0066] The invention will now be described more specifically, by way of example, with reference to the accompanying drawings in which: [Figure 1A] FIG. 1A shows the results of ATP Lite, Presto Blue and Trypan Blue tests obtained with a range of bioactive conjugates against tumor cells as described below. [Figure 1B] FIG. 1B shows the results of ATP Lite, Presto Blue and Trypan Blue tests obtained with a range of bioactive conjugates against tumor cells as described below. [Figure 1C] FIG. 1C shows the results of ATP Light, Presto Blue and Trypan Blue tests obtained with a range of bioactive conjugates against tumor cells as described below. [Diagram 2] FIG. 2 shows a comparison of similar results obtained with and without filtration of the solution. [Diagram 3] FIG. 3 is a diagram and photograph of the preparation of a complex containing bovine alpha-lactalbumin. [Figure 4] FIG. 4 shows the results of ATP light, presto blue and trypan blue tests on tumor cells administered with the resulting solution. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0067] Example 1 Generation of biologically acceptable conjugates A range of bioactive conjugates were prepared using the peptide of SEQ ID NO:7. Ac-KQFTKAELSQLLKDIDGYGGIALPELIATMFHTSGYDTQ-OH (SEQ ID NO: 7) It is a variant of a fragment of human alpha-lactalbumin.

[0068] Lyophilized peptide (700 μM) was added to the tubes along with sodium oleate flakes (3.5 mM), and each tube was then reconstituted with the required volume of either: 1) Phosphate buffered saline (NaCl 6.8 g / L), Na2HPO4 x 2H2O (4.8 g / L) and KH2PO4 (1.3 g / L) (pH 7.2) 2) NaCl solution (116mM) (pH7.01) 3) Na2HPO4 solution (31mM) (pH8.6) 4) KH2PO4 solution (9.56mM) (pH4.6) 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 is vortexed until the solution is clear.

[0069] The resulting complex was then freeze-dried under the following freeze-drying conditions: pressure below 1.2 mbar and temperature below -55°C.

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

[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 calf serum (FCS) at 37 °C and 5% CO2. For cell death assays, cells were cultured in 96-well plates (2 × 10 4 Cells were cultured overnight in a 500-well plate (Tecan Group Ltd). Cells were incubated at 37° C. in serum-free RPMI-1640 containing the bioactive conjugates obtained in Example 1 at doses corresponding to 7, 21 or 35 μM peptide. FCS was added after 1 h. Cell death was quantified 3 h after peptide-oleate treatment by three biological methods including: 1) assessment of cellular ATP levels using a luminescence based ATP Light Kit (Parkin Elmer), 2) Presto Blue fluorescent 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 in PBS had high activity and induced cell death in a concentration-dependent manner (Figure 1A). The complex prepared in PBS single salt only showed a significant reduction in activity (Figure 1B). However, as shown in Figure 1C, the mixture (6) maintained a reasonable level of tumor cell death activity in a concentration-dependent manner.

[0073] Example 3 Effect of filtration on the method The method of Example 1 was repeated twice using PBS solution (1), but now each solution was passed through chemically distinct filters, either a polyethersulfone membrane (12846445, VWR) or a Minisart® NML Cellulose acetate membrane (60810103, Sartorius). The biological effects of the products were tested as described in Example 2 in a side-by-side comparison with an unfiltered product. The results are shown in FIG.

[0074] No significant differences were observed in the biological activity of the conjugates as quantified by measurement of total cellular ATP levels, presto blue staining and trypan blue exclusion assays.

[0075] Example 4 Generating BAMLET Lyophilized bovine alpha-lactalbumin (700 μM) was added to a tube (3.5 mM) along with sodium oleate flakes. Phosphate-buffered saline (1 ml) was then added to the tube and vortexed for 1-2 min at room temperature. A clear solution was produced (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. Lyophilization of the complex eliminated the activity, although this effect was transient.

Claims

1. 1. A method for preparing a bioactive conjugate comprising the steps of: dissolving a mixture of a polypeptide in powder form and a solid pharma- ceutical acceptable salt of oleic acid 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 10 to 40° C. The method, wherein the polypeptide is a peptide of SEQ ID NO:

7.

2. 10. The method of claim 1, wherein the aqueous solvent further comprises a third salt which is monosodium phosphate or monopotassium phosphate.

3. 3. The method of claim 1 or 2, which is carried out at ambient temperature.

4. The method according to any one of claims 1 to 3, further comprising filtering the solution obtained by said dissolution.

5. The method of any one of claims 1 to 4, further comprising removing the solvent to obtain the complex in solid form.

6. The method of claim 5 , wherein the solvent is removed by lyophilization.

7. The method of any one of claims 1 to 6, wherein the first salt is sodium chloride.

8. The method of any one of claims 1 to 7, wherein the second salt is disodium phosphate.

9. The method of claim 2 , wherein the third salt is monopotassium phosphate.

10. The method according to claim 5 or 6, wherein the complex is reconstituted with sterile water immediately prior to use.

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