GB2574845A

GB2574845A - Therapeutically active complexes

Info

Publication number: GB2574845A
Application number: GB201810108A
Authority: GB
Inventors: Svanborg Catharina; Shing Ho Chin
Original assignee: Hamlet Pharma AB
Current assignee: Hamlet Pharma AB
Priority date: 2018-06-20
Filing date: 2018-06-20
Publication date: 2019-12-25
Also published as: GB201810108D0

Abstract

A peptide which comprises a N-terminal fragment of SEQ ID NO 1 of up to 19 amino acids in length. This peptide could for example have the amino acid sequence of SEQ ID NO 2 (Sar1a1) or SEQ ID NO 6 (Sar1a2). Both SEQ ID NO 2 and SEQ ID NO 6 are demonstrated to kill human lung carcinoma cells (A549, ATCC) in cell death assays. Also disclosed is a biologically active complex comprising a peptide which comprises a truncated form of SEQ ID NO 1 of up to 19 amino acids in length in combination with oleic acid or a salt thereof. The complexes may be uses in therapy or the treatment of cancer. The oleate salt may be water soluble and may be an alkali metal salt such as sodium oleate or potassium oleate. MAGWDIFGWFRDVLASLGLWNKH (SEQ ID NO 1) MAGWDIFGWFRDVLA (SEQ ID NO 2) WFRDVLASLGLWNKH (SEQ ID NO 6)

Description

THERAPEUTICALLY ACTIVE COMPLEXES

TECHNICAL FIELD

The present invention relates to a class of peptides which have therapeutic activity, in particular as anti-cancer or anti-tumour agents. Methods for preparing these peptides, as well as pharmaceutical compositions containing them form a further aspect of the invention.

BACKGROUND

Novel cancer treatments should ideally combine efficacy with selectivity for the targeted tumor and new, targeted therapies act with greater precision. Tissue toxicity and side effects are still the norm, however, and the notion of new, tumor specific mechanisms of cell death is justly regarded with skepticism. Yet, recent investigations into the tumoricidal effects of certain protein-lipid complexes suggest that tumor cells may share conserved mechanisms of cell death that distinguish them from normal, differentiated cells. These protein-lipid complexes insert into lipid bilayers and trigger cell death by perturbing the membrane structure of tumor cells. The subsequent internalization and inhibition of critical cellular functions distinguishes tumor cells from healthy differentiated cells and as a result, the tumor cells are killed while normal, differentiated cells survive.

These properties identify protein-lipid complexes as interesting drug candidates, with broad tumoricidal activity and documented tumor specificity. The feasibility of this approach is illustrated by HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells), which was discovered serendipitously, in a fraction of human milk. HAMLET kills many different tumor cells with rapid kinetics and shows therapeutic efficacy in animal models of colon cancer, glioblastoma and bladder cancer. Investigator-driven clinical trials have demonstrated that HAMLET is active topically, against skin papillomas and induces shedding of dead tumor cells into the urine of patients with bladder cancer.

Alpha-lactalbumin is the most abundant protein in human milk, essential for the survival of lactating mammals, due to its role as a substrate specifier in the lactose synthase complex. HAMLET is formed by partial unfolding of globular alpha-lactalbumin and binding of deprotonated oleic acid, with a stoichiometry of 1/4-8.

A number of peptides derived from alpha-lactalbumin have also been found to have therapeutic effects in their own right (see for example WO2012/069836).

The applicants have previously identified specific alpha-lactalbumin peptide domains as the functional ligands for tumor cell recognition and death. Shared peptide reactivity among tumor cells from different tissues suggests that the alpha-helical peptide is recognized by tumor cell membranes in the context of oleic acid and that this interaction triggers a conserved death response in cancer cells and established cancers, in vivo. Thus, complexes comprising these peptides show broad tumoricidal activity, as exemplified by work done with the known complexes based upon alpha-helical domains of alphalactalbumin. However, the applicants have surprising found that these effects can be generalized to other alpha-domain peptides with membrane perturbing activity.

As described in co-pending International Patent Application No. PCT/IB2017/058140 a new, general mechanism by which alpha-helical peptides can target and kill tumor cells has been determined. The applicants found that membrane interactive peptide-domains form oleate complexes with broad tumoricidal activity. This concept is exemplified by the N-terminal alpha helices of alpha-lactalbumin, which invades tumor cells and accumulates in nuclear speckles, where it suppresses transcription through a direct effect on the speckle constituents SC-35, PKC and Pol II. This gain of function was reproduced for Sari in the COPII family, where the alpha-helical, membrane-integrating peptide gained tumoricidal activity, when mixed with oleate. The beta sheet domains of these proteins, in contrast, were sorted to the lysosomes for degradation. Synthetic alpha 1 peptide formed therapeutic oleate complexes that reduced tumor load in a murine bladder cancer model. These findings suggested that tumor cells recognize alpha-helical peptide motifs in the context of oleate and respond by activating a conserved mechanism of tumor cell death.

The peptides were derived from proteins with a membrane perturbing activity. In particular, the peptides were from 20-40 amino acids in length. They include an alpha peptide domain derived from the SARI protein of SEQ ID NO 1.

MAGWDIFGWF RDVLASLGLW NKH (SEQ ID NO 1)

As used herein the expression 'alpha-helical domain' refers to a motif in the secondary structure of the peptide in which a right-hand coiled or spiral conformation (helix) is formed, in which every backbone N-H group donates a hydrogen bond to the backbone C=O group of the amino acid located three or four residues earlier along the peptide sequence.

The applicants have now found that highly active complexes may be derived from smaller peptide fragments.

SUMMARY OF THE INVENTION

According to the present invention, there is provided a biologically active complex comprising a peptide which comprises a truncated form of SEQ ID NO 1 of up to 19 amino acids in length, and oleic acid or a salt thereof.

The truncated sequence of SEQ ID No 1 is up to 19 amino acids in length, for example up to 18 amino acids, or up to 15 amino acids in length. Typically, the peptide will be from 10-19 amino acids in length. Suitably, the peptide is an N-terminal fragment of SEQ ID NO 1.

A particular example of such a peptide is a peptide of SEQ ID NO 2.

MAGWDIFGWF RDVLA (SEQ ID NO 2)

The applicants have found that complexes may be formed with peptides comprising truncated forms of SEQ ID NO 1, whereas complexes made using truncated forms of other peptides known to produce active complexes, such as truncated forms of alpha peptides derived from alphalactalbumin, as described for example in EP-B-2643010, have reduced activity. The fact that peptides such as SEQ ID NO 2 produce complexes having such activity is therefore unexpected.

The use of shorter peptides may be desirable, both from the point of view of cost and complexity of production, and also from the point of view of reliability and consistency of product.

If desired however, the peptide may comprise small numbers of additional amino acid residues not obtained from SEQ ID NO 1, for example up to 6 amino acids, such as up to 3 amino acids, for example 1 or 2 additional amino acids, may be attached at N and/or C terminal of the peptide, if convenient, for example for expression or purification purposes, as would be understood in the art.

The complexes of the invention further comprise oleic acid or a salt thereof. In particular, the complex further comprises a water soluble oleate salt. Particular examples of suitable salts may include alkali or alkaline earth metal salts. In a particular embodiment, the salt is an alkali metal salt such as a sodium- or potassium salt.

According to a further aspect of the present invention there is provided a method for preparing a biologically active complex as described above. Said method may comprise combining together peptide as defined above; with oleic acid or a salt thereof, under conditions in which they form a biologically active complex.

Typically, the preparation may be carried out simply by mixing together a suitable peptide and oleic acid or a salt thereof, for example in a solution such as an aqueous solution. The ratio of oleate: peptide added to the mixture is suitably in the range of from 20:1 to 1 to 1, but preferably an excess of oleate is present, for instance in a ratio of oleate:peptide of about 5:1. The mixing can be carried out at a temperature of from 0-50°C, conveniently at ambient temperature and pressure. This simple preparation method provides a particular advantage for the use of such peptides in the complexes. The methods can be carried out in situ, when required for treatment. Thus kits may be provided comprising peptides and salts for mixing immediately prior to administration.

Such kits, and reagents for use in the kits form a further aspect of the invention. Peptides are suitably synthetic peptides although they may be prepared by recombinant DNA technology.

Peptides useful in forming the complexes of the invention are novel and these form yet a further aspect of the invention.

The complex of the invention can be used in the treatment of cancer. For this purpose, the complex is suitably formulated as a pharmaceutical composition.

Thus, complexes as described above and/or oleate salts also as described above, may be formulated into useful pharmaceutical compositions by combining them with pharmaceutically acceptable carriers in the conventional manner. Such compositions form a further aspect of the invention.

The compositions in accordance with this aspect of invention are suitably pharmaceutical compositions in a form suitable for topical use, for example as creams, ointments, gels, or aqueous or oily solutions or suspensions. These may include the commonly known carriers, fillers and/or expedients, which are pharmaceutically acceptable.

Topical solutions or creams suitably contain an emulsifying agent for the protein complex together with a diluent or cream base.

The daily dose of the complex varies and is dependent on the patient, the nature of the condition being treated etc. in accordance with normal clinical practice. As a general rule from 2 to 200 mg/dose of the biologically active complex is used for each administration.

In a further aspect of the invention, there is provided a method for treating cancer which comprises administering to a patient in need thereof, a biologically active complex as described above.

In particular, the complex may be used to treat cancers such as human skin papillomas, human bladder cancer, colon cancer, kidney cancer, lung cancer and glioblastomas. In the latter case, administration may be by infusion as is known in the art.

The invention further provides the biologically active complex as defined above for use in therapy, in particular in the treatment of cancer.

Throughout the description and claims of this specification, the words comprise and contain and variations of the words, for example comprising and comprises, mean including but not limited to, and do not exclude other components, integers or steps. Moreover the singular encompasses the plural unless the context otherwise requires: in particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Preferred features of each aspect of the invention may be as described in connection with any of the other aspects. Within the scope of this application it is expressly intended that the various aspects, embodiments, examples and alternatives set out in the preceding paragraphs, in the claims and/or in the following description and drawings, and in particular the individual features thereof, may be taken independently or in any combination. That is, all embodiments and/or features of any embodiment can be combined in any way and/or combination, unless such features are incompatible.

BRIEF DESCRIPTION OF THE DRAWINGS

One or more embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

Figure 1 is a schematic diagram mapping the truncated peptides tested with the longer active peptides from which they are derived;

Figure 2 shows the results obtained in a cell death assay using complexes obtained using the peptides of Figure 1, where (A) shows the results of an ATPIite assay and (B) shows the results of a Prestoblue assay:

Figure 3 is a graph showing the average of 3 repeats of the cell death assay carried out on the complexes of the invention and complexes comprising the alpha-1 peptide of alphalactalbumin;

Figure 4 shows the results complexes of the invention on membrane perturbation using a giant unilamellar vesicle model.

DETAILED DESCRIPTION

Example 1

Peptide synthesis

The peptides to individual domain of alpha lactalbumin were commercially synthesized using the mild Fmoc chemistry method (Mimotopes, Melbourne, Australia). For biotinylated peptides, an aminohexanoic acid (Ahx) spacer was added to ensure adequate separation between the biotin and the peptide moiety. The sequences for the peptides are set out in the following table 1

Table 1

llllllllllllllll	Sequence	SEQ liillll
alphal-pl	KQFTKAELSQLLKDI	3
alphal-p2	LLKDIDGYGGIALPE	4
al-plp2	KQFTKAELSQLLKDIDGYGGIALPE	5
sarla-1	MAGWDIFGWFRDVLA	2
sarla-2	WFRDVLASLGLWNKH	6
Alpha-1	KQFTKAELSQLLKDIDGYGGIALPELIATMFHTSGYDTQ	7

Of these, peptides of SEQ ID No 3-5 represent fragments of the known active alpha peptide of alphalactalbumin, which is comprised in SEQ ID NO 7. Similarly, peptides of SEQ ID no 2 and 6 were fragments of the known active peptide of SEQ ID NO 4. This is illustrated schematically in Figure 1.

Complex Preparation

5mg of sodium oleate was dissolved in 1 ml of PBS to give a 16 mM clear solution, which was mixed with peptides in a ratio of oleate:peptide of 5:1.

Cellular assays

Human lung carcinoma cells (A549, ATCC) were cultured in RPMI-1640 with non-essential amino acids (1:100), 1 mM sodium pyruvate, 50 pg/ml Gentamicin and 5-10% fetal calf serum (FCS) at 37 °C, 5 % CO2. For cell death experiment, cells were grown on 96-well plate (2xl0⁴/well, Tecan Group Ltd) overnight. Cells were incubated with peptide-oleate complexes in serum-free RPMI-1640 at 37 °C. FCS was added after 1 hour. After 3, 7 and 12 hours treatment cell death was quantified by two biochemical methods: Cell viability was quantified by Presto Blue fluorescence (Invitrogen, A13262) and cellular ATP levels using luminescence based ATPIite™ kit (Perkin Elmer). Fluorescence and luminescence was measured using a microplate reader (Infinite F200, Tecan).

The results are illustrated in Figure 2. It is clear from this result, that the peptide of SEQ ID NO 2 produced efficiacy similar to that obtainable using the favoured alpha-peptide of alpha-lactalbumin, in spite of the fact that it is considerably shorter. This result was confirmed when the average of 3 trials was compared as shown in Figure 3.

The complex of the invention was shown to produce membrane perturbation using a giant unilamellar vesicle model.

Coverslips were cleaned with IM NaOH and plasma etched for 1 min using BD-20 laboratory corona treater (Electro Technic Products Inc., USA) to render the surface clean and hydrophilic. A thin film of 1% solution of molten agarose was made on the coverslip to provide a safe reaction bed for the giant unilamellar vesicles (GUVs) to form. 10 mM solution of Phosphatidyl Choline and O.lmM l,2-dipalmitoyl-sn-glycero-3phosphoethanolamine-N-(cap biotinyl) (DPPE-biotin) in chloroform was labelled using 1 to 25 concentrations of Img/ml of Rhodamine. GUVs from this solution were formed onto the coverslips using a syringe through an air-blow dispersion method. Syke-Moore chambers were sonicated in IM Sodium Hydroxide and rinsed with Milli-Q water. The coverslips were locked in the chambers and the GUVs were mobilized and rehydrated using 200 mM of Sucrose solution for 25 minutes. Further, the GUVs were collected and allowed to settle in 200 mM of glucose solution for 60 minutes. Alexa-633 labelled and unlabeled peptidesoleate complex in the ratio of 1:4 was added to the solution in the observation chambers and monitored chronologically for at most 90 minutes. The GUVs were washed with PBS and monitored again to visualize co-localization.

The results are shown in Figure 4.

Claims

1. A biologically active complex comprising a peptide which comprises a truncated form of SEQ ID NO 1 of up to 19 amino acids in length,

MAGWDIFGWF RDVLASLGLW NKH (SEQ ID NO 1) and oleic acid or a salt thereof.

2. A biologically active complex according to claim 1 wherein the peptide is of SEQ ID NO 2

MAGWDIFGWF RDVLA (SEQ ID NO 2).

3. A biologically active complex according to any one of the preceding claims which comprises a water soluble oleate salt.

4. A biologically active complex according to claim 3 wherein the salt is an alkali metal salt such as a sodium- or potassium oleate.

5. A method for preparing a biologically active complex according to any one of the preceding claims which comprises combining together a peptide as defined in claim 1 or claim 2, with oleic acid or a salt thereof, under conditions in which they form a biologically active complex.

6. A kit comprising a peptide as defined in claim 1 or claim 2 and oleic acid or a salt thereof.

7. A peptide which comprises a N-terminal fragment of SEQ ID NO 1 of up to 19 amino acids in length.

8. A peptide according to claim 7 which is of SEQ ID NO 2.

9. A pharmaceutical composition comprising a biologically acceptable complex according to any one of claims 1 to 4 in combination with a pharmaceutically acceptable carrier or excipient.

10. A method for treating cancer which comprises administering to a patient in need thereof, an effective amount of a biologically active complex according to claim 1 or claim 2, or a pharmaceutical composition according to claim 9.

11. A method according to claim 102 wherein the cancer is a human skin papilloma, human bladder cancer, kidney cancer, lung cancer and glioblastomas.

12. A biologically active complex as defined in any one of claim 1 or claim 2 for use in

15 therapy, in particular in the treatment of cancer.