COMBINATION OF A CHEMOTHERAPEUTIC AGENT AND ALPHA-LACTOGLUBULIN-OLEIC ACID COMPLEX FOR CANCER THERAPY

Abstract

A first chemotherapeutic agent and a second chemotherapeutic agent for use in cancer therapy, wherein the second chemotherapeutic agent comprises a biologically active complex having anti-tumour activity, wherein the biologically active complex consists of: a peptide of at least 10 amino acids comprising an alpha-helical structure; and oleic acid or an oleate salt, in a ratio of at least 3 oleic acid or oleate salt molecules per peptide molecule. Either the first or second chemotherapeutic agent for use in cancer therapy, for use in conjunction with the other chemotherapeutic agent, pharmaceutical compositions related thereto, and method of treatment.

Claims

1. A first chemotherapeutic agent and a second chemotherapeutic agent for use in a combination cancer therapy, wherein the second chemotherapeutic agent comprises a biologically active complex having anti-tumour activity, wherein the biologically active complex consists of: a peptide of at least 10 amino acids comprising an alpha-helical structure; and oleic acid or an oleate salt, in a ratio of at least 3 oleic acid or oleate salt molecules per peptide molecule.

2. A first chemotherapeutic agent for use in treating cancer; wherein the agent is for administration to a subject to which a second chemotherapeutic agent will be administered, or has been administered, or is being administered; and wherein the second chemotherapeutic agent comprises a biologically active complex having anti-tumour activity, wherein the biologically active complex consists of: a peptide of at least 10 amino acids comprising an alpha-helical structure; and oleic acid or an oleate salt, in a ratio of at least 3 oleic acid or oleate salt molecules per peptide molecule.

3. A second chemotherapeutic agent for use in for use in treating cancer; wherein the agent is for administration to a subject to which a first chemotherapeutic agent will be administered, or has been administered, or is being administered; and wherein the second chemotherapeutic agent, wherein the second chemotherapeutic agent comprises a biologically active complex having anti-tumour activity, wherein the biologically active complex consists of: a peptide of at least 10 amino acids comprising an alpha-helical structure; and oleic acid or an oleate salt, in a ratio of at least 3 oleic acid or oleate salt molecules per peptide molecule.

4. A first chemotherapeutic agent and/or second chemotherapeutic agent for use according to any preceding claim, wherein the first chemotherapeutic agent is an intravesical chemotherapeutic agent, a topical chemotherapeutic agent, a DNA-interactive chemotherapeutic agent, a DNA-alkylator chemotherapeutic agent, and/or a DNA-crosslinker chemotherapeutic agent.

5. A first chemotherapeutic agent and/or second chemotherapeutic agent for use according to any preceding claim, wherein the first chemotherapeutic agent is selected from the group consisting of: atezolizumab, avelumab, Bacille Calmette-Guerin, bevacizumab, carbozantinib, cephalexin, ciprofloxacin, cisplatin, doxorubicin hydrochloride, durvalumab, eflornithine, epirubicin, erdafitinib, erlotinib, fenretinide, gemcitabine, gefitinib, lapatinib, mitomycin C, nivolumab, pazobanib, pembrolizumab, rapamycin, selenium, sorafenib, thiotepa, urocidin, valrubicin, and vicinium, preferably thiotepa, mitomycin C, Bacille Calmette-Guerin or epirubicin, more preferably mitomycin C or epirubicin.

6. A first chemotherapeutic agent and/or second chemotherapeutic agent for use according to any preceding claim, wherein the peptide of the biologically active complex is of a protein which has membrane perturbing activity.

7. A first chemotherapeutic agent and/or second chemotherapeutic agent for use according to any preceding claim, wherein the peptide of the biologically active complex, where present in the complex, has an increased conformational fluidity of three-dimensional structure as compared to the non-complexed peptide as indicated by an increased peak width on at least some .sup.1H NMR peaks of the complex as compared to the corresponding width of the peaks of a .sup.1H NMR of the non-complexed peptide.

8. A first chemotherapeutic agent and/or second chemotherapeutic agent for use according to any preceding claim, wherein the peptide of the biologically active complex lacks cysteine residues.

9. A first chemotherapeutic agent and/or second chemotherapeutic agent for use according to any preceding claim, wherein the peptide of the biologically active complex consists of up to 50 amino acids in length.

10. A first chemotherapeutic agent and/or second chemotherapeutic agent for use according to any preceding claim, wherein the peptide of the biologically active complex is alpha-lactalbumin or SAR-1, or a variant or fragment thereof, preferably an N-terminal fragment thereof.

11. A first chemotherapeutic agent and/or second chemotherapeutic agent for use according to any preceding claim, wherein the peptide of the biologically active complex comprises or consists of any of SEQ ID NOs: 1-4, or variants or fragments thereof. TABLE-US-00005 (SEQ ID NO: 1) KQFTKAELSQ LLKDIDGYGG IALPELIATM FHTSGYDTQ (SEQ ID NO: 2) MAGWDIFGWF RDVLASLGLW NKH (SEQ ID NO 3) KQFTKAELSQ LLKDI (SEQ ID NO 4) MAGWDIFGWF RDVLA.

12. A first chemotherapeutic agent and/or second chemotherapeutic agent for use according to any preceding claim, wherein the peptide of the biologically active complex consists of any of SEQ ID NOs: 1-4, preferably SEQ ID NO: 1.

13. A first chemotherapeutic agent and/or second chemotherapeutic agent for use according to any preceding claim, wherein the use is treatment or prevention of carcinomas, lymphomas or brain tumours, preferably treatment or prevention of cancer of the GI tract, mucosal cancer, bladder cancer, kidney cancer, lung cancer, glioblastomas and skin papilloma, more preferably treatment or prevention of bladder cancer.

14. A pharmaceutical composition comprising a first chemotherapeutic agent, a second chemotherapeutic agent, and a pharmaceutically acceptable carrier, excipient and/or adjuvant, wherein the second chemotherapeutic agent comprises a biologically active complex having anti-tumour activity, wherein the biologically active complex consists of: a peptide of at least 10 amino acids comprising an alpha-helical structure; and oleic acid or an oleate salt, in a ratio of at least 3 oleic acid or oleate salt molecules per peptide molecule.

15. A pharmaceutical composition according to claim 14 for use in cancer therapy.

16. A pharmaceutical composition according to claim 14 or 15, wherein the first chemotherapeutic agent is mitomycin, preferably mitomycin C.

17. A pharmaceutical composition according to claim 14 or 15, wherein the first chemotherapeutic agent is epirubicin.

18. A pharmaceutical composition according to any of claims 14 to 17, wherein the second chemotherapeutic agent comprises a biologically active complex having anti-tumour activity, wherein the biologically active complex consists of: a peptide having or comprising a peptide the sequence: TABLE-US-00006 KQFTKAELSQ LLKDIDGYGG IALPELIATM FHTSGYDTQ; and oleic acid or an oleate salt, in a ratio of at least 3 oleic acid or oleate salt molecules per peptide molecule.

19. A method of treating or preventing cancer, the method comprising administration to a subject in need thereof of a first chemotherapeutic agent in conjunction with a second chemotherapeutic agent, wherein the second chemotherapeutic agent comprises a biologically active complex having anti-tumour activity, wherein the biologically active complex consists of: a peptide of at least 10 amino acids comprising an alpha-helical structure; and oleic acid or an oleate salt, in a ratio of at least 3 oleic acid or oleate salt molecules per peptide molecule.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0061] One or more embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0062] FIG. 1 shows a schematic of a murine bladder tumor model for determining a dose-dependent therapeutic effect of alpha1-oleate: Bladder cancer was induced in C57BL/6 female mice by intra-vesical instillation of MB49 cells (2×10.sup.5 in 100 μl PBS). Mice were treated by intravesical instillation of alpha1-oleate (1.7 mM, 8.5 mM or 17 mM) on days 3, 5, 7, 9 and 11 and sacrificed on day 12. Sham treated mice received PBS.

[0063] FIG. 2 shows the dose dependent effect of alpha1-oleate, deduced from macroscopic inspection of gross bladder pathology.

[0064] FIG. 3 shows a comparison of bladder weight, bladder size and tumor area, data is presented as means±SEMs of two experiments (n=6+5 mice, * P<0.05, ** P<0.01 and *** P<0.001 compared to sham treated mice).

[0065] FIG. 4 shows accumulation of alpha1-oleate in tumor tissue: Alexa-Fluor 568-labeled alpha1-oleate was used to challenge tumor-bearing mice and to track the molecule in tumor tissue. (A) Scheme of intravesical Alexa-alpha1-oleate challenge. Intravesical instillations were performed on day 11 and the tissues were harvested after 6 hours. (B) Detection of Alexa-Fluor 568-labeled alpha1-oleate in tumor-bearing mice by confocal imaging of frozen tissue sections. Healthy mice challenged with labeled alpha1-oleate were used as controls. (C) Quantification of fluorescence intensity in (B). Means±SEMs of 5 images/dose.

[0066] FIG. 5 shows dose-dependent changes in gene expression in tumor-bearing mice: (A) Heat map showing a dose-dependent reduction in the number of regulated genes in treated mice. Red (left): upregulated genes, blue (right): down-regulated genes, cutoff fold change >2.0 compared to healthy bladder. (B) A dose-dependent reduction in the molecular mechanisms of cancer pathway genes. (C) Individual regulated genes in this pathway. (D) Principal component analysis of mRNA profiles in whole bladder tissue. Increasing doses of alpha1-oleate shifted the transcriptomic profiles from the tumor bearing, sham treated cluster towards the healthy bladder tissue cluster. (E) Scatter plot of probes comparing transcriptomic profiles in bladders from tumor-bearing mice to healthy mice (log.sub.2 signal intensity values). (F) Venn diagram of significantly regulated genes in the sham treated, and alpha1-oleate treated mice compared to healthy bladders.

[0067] FIG. 6 shows a schematic of the murine bladder tumor model: bladder cancer was induced in C57BL/6 female mice by intra-vesical instillation of MB49 cells (2×10.sup.5 in 100 μl PBS). Mice were treated by intravesical instillation of mitomycin C (25 μg; * 25 μg is half of the therapeutic dose in mice, 50 μg i.p. gives rise to systemic toxicity) or alpha1-oleate (8.5 mM) on days 3, 5, 7, 9 and 11 and sacrificed on day 12. Sham treated mice received PBS.

[0068] FIG. 7 shows bladder size (and any effect of tumor size on the macroscopic appearance of the bladders) after sacrifice of mice according to the model of FIG. 1, wherein the mice have been treated with sham, 8.5 mM alpha1-oleate or 25 μg mitomycin, healthy controls are also shown (all shown at the same magnification).

[0069] FIG. 8 shows the schematic of FIG. 1, modified to investigate the therapeutic effect of the alpha1-oleate and mitomycin C combination.

[0070] FIG. 9 shows bladder size (and any effect of tumor size on the macroscopic appearance of the bladders) after sacrifice of mice according to the model of FIG. 3, wherein the mice have been treated with sham, 1.7 mM alpha1-oleate, 25 μg mitomycin and 1.7 mM alpha1-oleate+25 μg mitomycin (all shown at the same magnification).

[0071] FIG. 10 shows study on bladder size (and any effect of tumor size on the macroscopic appearance of the bladders) after sacrifice of the 1.7 mM alpha1-oleate+25 μg mitomycin study of FIG. 4 in comparison with the bladder size (and any effect of tumor size on the macroscopic appearance of the bladders) of mice according to the same model but treated with 17 mM alpha1-oleate (all shown at the same magnification).

[0072] FIG. 11 shows a schematic of a murine bladder tumor model for determining a therapeutic effect of alpha1-oleate, mitomycin C (MMC) and the combination of the two: Bladder cancer was induced in C57BL/6 female mice by intra-vesical instillation of MB49 cells (2×10.sup.5 in 100 μl PBS). Mice were treated by intravesical instillation of alpha1-oleate (1.7 mM); MMC (25 μg); and alpha1-oleate (1.7 mM) and MMC (25 μg) on days 3, 5, 7, 9 and 11 and sacrificed on day 12. Sham treated mice received PBS.

[0073] FIG. 12 shows the effect of alpha1-oleate, MMC and alpha1-oleate and MMC, deduced from macroscopic inspection of gross bladder pathology.

[0074] FIG. 13 shows the effect of alpha1-oleate, MMC and alpha1-oleate and MMC, deduced from inspection of the bladder histology.

[0075] FIG. 14 shows a schematic of a murine bladder tumor model for determining a dose-dependent therapeutic effect of alpha1-oleate, mitomycin C (MMC) and the combination of the two: Bladder cancer was induced in C57BL/6 female mice by intra-vesical instillation of MB49 cells (2×10.sup.5 in 100 μl PBS). Mice were treated by intravesical instillation of alpha1-oleate (1.7 mM and 8.5 mM); MMC (25 μg); and alpha1-oleate (1.7 mM and 8.5 mM) and MMC (25 μg) on days 3, 5, 7, 9 and 11. The Sham group was sacrificed on day 12, treated groups were sacrificed in weeks 4 or 8. Sham treated mice received PBS.

[0076] FIG. 15 shows the dose dependent effect of alpha1-oleate, the effect of MMC and the dose dependent effect of alpha1-oleate and MMC, deduced from macroscopic inspection of gross bladder pathology.

[0077] FIG. 16 shows the dose dependent effect of alpha1-oleate, the effect of MMC and the dose dependent effect of alpha1-oleate and MMC, deduced from inspection of the bladder histology.

[0078] FIG. 17 shows a schematic of a murine bladder tumor model for determining a therapeutic effect of alpha1-oleate; mitomycin C (MMC); epirubicin (Epi); the combination of alpha1-oleate and MMC; and the combination of alpha1-oleate and Epi: Bladder cancer was induced in C57BL/6 female mice by intra-vesical instillation of MB49 cells (2×10.sup.5 in 100 μl PBS). Mice were treated by intravesical instillation of alpha1-oleate (1.7 mM); MMC (25 ug); Epi (25 ug); alpha1-oleate (1.7 mM) and MMC (25 ug); and alpha1-oleate (1.7 mM) and Epi (25 ug) on days 3, 5, 7, 9 and 11 and sacrificed on day 12. Sham treated mice received PBS.

[0079] FIG. 18 shows the effect of alpha1-oleate, Epi, and alpha1-oleate and Epi, deduced from macroscopic inspection of gross bladder pathology.

[0080] FIG. 19 shows a schematic similar to that shown in FIG. 17.

[0081] FIG. 20 shows the effect of alpha1-oleate, MMC, Epi, alpha1-oleate and MMC, and alpha1-oleate and Epi, deduced from macroscopic inspection of gross bladder pathology.

[0082] FIG. 21. Therapeutic efficacy of alpha1-oleate and Mitomycin C in a murine bladder cancer model.

[0083] (a) Schematic of the treatment model. Bladder cancer was induced in C57BL/6J female mice by intravesical instillation of MB49 cells (2×10.sup.5 in 50 μl PBS). The treatment group received alpha1-oleate (1.7 mM or 8.5 mM) or MMC (25 μg/mL) alone or in combination on days 3, 5, 7, 9 and 11. Sham treated mice received PBS and all mice were sacrificed on day 12. (b) Treatment effects deduced from macroscopic inspection of the bladders. (c, d, e) Pathology score, bladder weight, bladder size and tumor area (see also FIG. 20). Data is presented as means±SEMs of two experiments (n=5+6 mice per group, and analyzed by one-way ANOVA).

[0084] FIG. 22. Reduction in tumor size by alpha1-oleate and Mitomycin C, alone or in combination.

[0085] Tumor areas were compared between sham treated mice and mice receiving 1.7 mM of alpha1-oleate or 25 μg of MMC alone or in combination. (a) Sham treated mice show large tumors filling the bladder lumen (dotted line). (b-c) Alpha1-oleate (1.7 mM) and MMC treated mice show a reduction in tumor size. (d) A further reduction in tumor size in mice treated with a combination of alpha1-oleate (1.7 mM) and MMC (25 μg/mL), (e) Healthy bladders served as negative controls. Representative images are shown (n=6+5 mice per group). (n=5+6 mice per group, and analyzed by one-way ANOVA). The tumor area was identified in H&E-stained whole bladder sections and quantified using ImageJ.

[0086] FIG. 23. Therapeutic efficacy of alpha1-oleate and Epirubicin in a murine bladder cancer model.

[0087] (a) Schematic of the treatment model. The treatment group received alpha1-oleate by intravesical instillation (alpha1-oleate 1.7 mM or 8.5 mM) or Epirubicin (25 μg/mL) alone or in combination on days 3, 5, 7, 9 and 11. Sham treated mice received PBS and all mice were sacrificed on day 12. (b) Gross bladder pathology defined by macroscopic inspection. (c, d, e) Pathology score, bladder weight, bladder size and tumor area (see also FIG. 22). Data is presented as means±SEMs of two experiments (n=5+5 mice per group, and analyzed by one-way ANOVA).

[0088] FIG. 24. Reduction in tumor size by alpha1-oleate and Epirubicin therapy, alone or in combination. (a) Sham treated mice show large tumors filling the bladder lumen (dotted line). (b-c) Alpha1-oleate (1.7 mM or 8.5 mM) and Epirubicin treated mice show a reduction in tumor size. (d, e) Further reduction in tumor size in mice receiving a combination of alpha1-oleate (1.7 mM or 8.5 mM) and Epirubicin. The tumor area was identified in H&E-stained whole bladder sections and quantified using ImageJ.

[0089] FIG. 25. Prevention of tumor recurrence by a combination of alpha1-oleate and Mitomycin C.

[0090] The combination of alpha1 and Mitomycin C showed a prolonged protective effect, preventing tumor relapse for 4 weeks. (a) Effect of alpha1-oleate and MMC alone or in combination, defined by macroscopic inspection of the bladders (See also schematic in FIG. 19a). (c, d, e) Reduced pathology score, bladder weight, bladder size (p<0.001, see also FIG. 6). Data is presented as means±SEMs of two experiments (n=5+5 mice).

[0091] FIG. 26. Tumor parameters at long-term follow up of mice treated with alpha1-oleate or Mitomycin C.

[0092] (a) Quantification of pathology score, bladder size, bladder weight and tumor areas. (b-e) Tumor areas were compared after 4 weeks between mice receiving 1.7 mM and 8 mM of alpha1-oleate and 25 μg of MMC. (b, c) Evidence of tumor recurrence in mice receiving alpha1-oleate (1.7 mM) and MMC (25 μg) alone. (d-e) Prevention of tumor recurrence by the combination of alpha1-oleate 1.7 mM or 8.5 mM with Mitomycin C prevented tumor relapse. Tumors were not detected in H&E-stained whole bladder sections. Representative images are shown (n=5+5 mice per group).

[0093] FIG. 27. Tumor parameters at long-term follow up of mice treated with alpha1-oleate or Epirubicin.

[0094] (a) Quantification of pathology score, bladder size, bladder weight and tumor areas. (b-e) Tumor areas were compared after 4 weeks between mice receiving 1.7 mM and 8 mM of alpha1-oleate and 25 μg of Epirubicin. (b, c) Evidence of tumor recurrence in mice receiving alpha1-oleate (1.7 mM) and Epirubicin (25 μg) alone. (d-e) Prevention of tumor recurrence by the combination of alpha1-oleate 1.7 mM or 8.5 mM with Epirubicin prevented tumor relapse. Tumors were not detected in H&E-stained whole bladder sections. Representative images are shown (n=5+5 mice per group).

DETAILED DESCRIPTION

[0095] The present invention provides a novel molecular solution to targeting and killing tumor cells with greater efficacy and precision using a combination therapy. The inventors have shown that the effect of a biologically active complex according to the invention upon tumor cells is greater than just the direct anti-tumor properties of the complex. The complex also renders the tumor cells more susceptible to the effect of other chemotherapeutic agents. It is further noted that complexes according to the invention do not appear to affect healthy cells (see Example 2), suggesting that any enhanced effects of the further chemotherapeutic agents are limited to tumour cells. This enhancement may, for example, occur through disruption of the cell wall (such as by direct disruption of the lipid bilayer or through disruption of membrane-bound proteins, including ion channels) and/or through modulation of gene expression, particularly genes involved in the molecular mechanisms of the cancer pathway (see Example 3). In any event, the data collected for complexes according to the invention and other chemotherapeutic agents show a clear synergistic enhancement in therapeutic effect when comparing use individually (Example 4) and use in combination (Example 5).

Example 1—Dose Escalation Study of Alpha1-Oleate in Tumor-Bearing Mice

[0096] We performed a dose escalation study in the murine MB49 bladder cancer model (inoculation at day 0, FIG. 1). Mice in the treatment group received five intravesical instillations of alpha1-oleate on days 3, 5, 7, 9 and 11 and sham treated mice had PBS instilled at these time points. The sham treated mice developed palpable tumors that altered the macroscopic appearance of the bladders, compared to controls not receiving tumor cells. The tumors were growing rapidly, from the mucosa and the tumor mass gradually filled the bladder lumen, replacing functional bladder tissue.

[0097] Treated mice were administered increasing concentrations of alpha1-oleate (1.7, 8.5 or 17 mM in 100 μl, 5-6 mice per group, 2 experiments per dose). Bladders were harvested on day 12 and evaluated macroscopically (FIG. 2), bladder size, weight and tumor area were recorded (FIG. 3). Tumor growth was attenuated after treatment with 1.7 mM of alpha1-oleate, with a reduction in bladder weight, bladder size and tumor size (P<0.001 compared to sham treated mice, FIG. 3). A further, dose-dependent reduction in bladder weight, bladder size and tumor size was recorded in mice receiving 8.5 mM of alpha1-oleate (P<0.001) and at the highest concentration (17 mM) no macroscopically visible tumor tissue remained. The bladder size and weight in these mice did not differ from that in healthy control mice.

[0098] The results identify alpha1-oleate as a tumoricidal complex with therapeutic efficacy that increases with increasing doses of the compound, until most of the tumor is cleared.

Example 2—Accumulation of Alpha1-Oleate in Tumor Tissue

[0099] To examine if alpha1-oleate reaches tumor tissue, tumor-bearing mice were inoculated with Alexa-Fluor 568 labeled alpha1-oleate (day 11, n=2). Bladders were harvested six hours after instillation and frozen tissue sections were subjected to confocal imaging. The Alexa-Fluor labeled complex was shown to accumulate in tumor tissue, in a dose-dependent manner (1.7 versus 8.5 mM, 8.5 mM versus 17 mM) (FIGS. 4A-C) but Alexa-Fluor 568 alpha1-oleate was not detected in healthy mice subjected to the same procedure (FIGS. 4B and C). The results were confirmed by dot blot analysis of tumor-bearing mice treated with alpha1-oleate, using anti-alpha1 antibodies. The accumulation of alpha1-oleate in tumor tissue was confirmed by staining of frozen tissue sections with anti-alpha1 antibodies. Tissues from healthy mice exposed to alpha1-oleate were negative.

Example 3—Dose-Dependent Inhibition of Gene Expression in Tumor-Bearing Mice

[0100] To further characterize the tumor response to alpha1-oleate, total bladder RNA was subjected to whole-genome transcriptomic profiling. Gene expression was compared between the sham treated group and mice receiving increasing doses of alpha1-oleate. Healthy mice served as controls.

[0101] The transcriptomic data revealed major differences in gene expression between the sham treated and the alpha1-oleate treated mice. A dose-dependent reduction in the number of differentially expressed genes was observed, consistent with the drastic reduction in tumor size in the treated mice (fold change compared to healthy, see heat map in FIG. 5A). The molecular mechanisms of cancer pathway and comprising genes were deactivated in a dose-dependent manner, as shown by Ingenuity Pathway Analysis (IPA) (FIGS. 5B and C). By principal component analysis (PCA, FIG. 5D), the tumor-bearing, sham treated mice formed a distinct transcriptomic profile, which was clearly separated from the treated mice and the healthy controls. PCA1 (79.3% of variation) was dominated by differences between sham treated and healthy bladders. With increasing doses, the treated mice shifted towards the healthy expression profile. PCA2 (14.9% of variation) distinguished the untreated group from the alpha1-oleate treated mice. The conclusions were supported by scatter plot analysis of probe signal intensities (FIG. 5E), revealing a dose-dependent decrease in the number of differentially expressed transcripts between healthy control and treated mice, also illustrated in the Venn diagram (FIG. 5F).

Example 4—Examination of Individual Therapeutic Effect of Alpha1-Oleate and Mitomycin C

[0102] We performed another study in the murine MB49 bladder cancer model where mice in the treatment group received five intravesical instillations of 8.5 mM alpha1-oleate or 25 μg mitomycin C on days 3, 5, 7, 9 and 11 and sham treated mice had PBS instilled at these time points (FIG. 6).

[0103] Bladders were harvested on day 12 and evaluated (FIG. 7). Tumor growth was attenuated to a similar extent after treatment with 8.5 mM alpha1-oleate or 25 μg mitomycin, with a reduction in bladder weight, bladder size and tumor size compared to sham treated mice.

Example 5—Examination of Combined Therapeutic Effect of Alpha1-Oleate and Mitomycin

[0104] A modified version of the previous study was performed, wherein mice in the treatment group received five intravesical instillations of 1.7 mM alpha1-oleate, 25 μg mitomycin, or the combination of 1.7 mM alpha1-oleate+25 μg mitomycin and sham treated mice had PBS instilled at these time points (FIG. 8).

[0105] The results show that treatment with the combination of 1.7 mM alpha1-oleate+25 μg mitomycin produces bladders that are equivalent or almost equivalent to healthy bladders, unlike treatment with either the 1.7 mM alpha1-oleate or 25 μg mitomycin alone (FIG. 9). Furthermore, FIG. 10 shows that treatment with the combination is producing an effect on the tumors that appears equivalent with alpha1-oleate alone at 10 times the concentration (i.e. 17 mM alpha1-oleate).

Example 6—Examination of Combined Therapeutic Effect of Alpha1-Oleate and Mitomycin

[0106] A modified version of the previous study was performed, wherein mice in the treatment group received five intravesical instillations of 1.7 mM alpha1-oleate, 25 μg mitomycin, or the combination of 1.7 mM alpha1-oleate+25 μg mitomycin and sham treated mice had PBS instilled at these time points (FIG. 11).

[0107] The results show that treatment with the combination of 1.7 mM alpha1-oleate+25 μg mitomycin produces bladders that are equivalent or almost equivalent to healthy bladders, unlike treatment with either the 1.7 mM alpha1-oleate or 25 μg mitomycin alone (FIG. 12). Furthermore, FIG. 13 shows that treatment with the combination produces bladder histology that is equivalent to or almost equivalent to that seen in healthy bladders.

Example 7—Examination of Combined Therapeutic Effect of Alpha1-Oleate and Mitomycin

[0108] A modified version of the previous study was performed, wherein mice in the treatment group received five intravesical instillations of 1.7 or 8.5 mM alpha1-oleate, 25 μg mitomycin, or the combination of 1.7 or 8.5 mM alpha1-oleate+25 μg mitomycin and sham treated mice had PBS instilled at these time points (FIG. 14).

[0109] The results show that treatment with the combination of 1.7 mM alpha1-oleate+25 μg mitomycin produces bladders that are equivalent or almost equivalent to healthy bladders, unlike treatment with either the 1.7 mM alpha1-oleate or 25 μg mitomycin alone (FIG. 15). Furthermore, FIG. 15 shows that treatment with the combination is producing an effect on the tumors that appears equivalent with alpha1-oleate alone at 5 times the concentration (i.e. 8.5 mM alpha1-oleate). FIG. 16 shows that treatment with the combination produces bladder histology that is equivalent to or almost equivalent to that seen in healthy bladders. FIGS. 15 and 16 also show that the effects are maintained over extended time periods of 4 and 8 weeks from inoculation.

Example 8—Examination of Combined Therapeutic Effect of Alpha1-Oleate and Mitomycin or Epirubicin

[0110] A modified version of the previous study was performed, wherein mice in the treatment group received five intravesical instillations of 1.7 mM alpha1-oleate, 25 μg mitomycin, 25 μg epirubicin, the combination of 1.7 or mM alpha1-oleate+25 μg mitomycin or the combination of 1.7 or mM alpha1-oleate+25 μg epirubicin and sham treated mice had PBS instilled at these time points (FIG. 17).

[0111] The results show that treatment with the combination of 1.7 mM alpha1-oleate+25 μg epirubicin produces bladders that are equivalent or almost equivalent to those from mice treated with the combination of 1.7 mM alpha1-oleate+25 μg mitomycin and to healthy bladders, unlike treatment with either the 1.7 mM alpha1-oleate, 25 μg mitomycin or 25 μg epirubicin alone (FIG. 18). Furthermore, FIG. 18 shows that treatment with the combination of alpha1-oleate and epirubicin is producing an effect on the tumors that appears equivalent with alpha1-oleate alone at 5 times the concentration (i.e. 8.5 mM alpha1-oleate).

[0112] Materials and Methods

[0113] Chemicals and Antibodies

[0114] Oleic acid (Croda, batch number: 0001120439), poly-L-lysine solution (Sigma, Cat #RNBF4239), Alexa-Fluor 568 protein labeling kit (Thermo Scientific, Cat #A10238), ECL Plus detection reagent (GE healthcare, Cat #RPN2132), Richard-Allan Scientific Signature Series Hematoxylin and Eosin-Y (Thermo Scientific, Cat #7211 and 7111), DAPI (Sigma, Cat #D9542), anti-alpha lactalbumin (Mybiosource, Cat #MBS175270), monoclonal mouse anti-β-actin (Sigma-Aldrich, Cat #A2228), polyclonal rabbit anti-mouse IgG-HRP (Dako, Cat #P0260), polyclonal goat anti-rabbit IgG-HRP (Cell Signaling, Cat #7074), rabbit polyclonal anti-VEGF (Abcam, Cat #ab46154), mouse monoclonal anti-Ki-67 (BD Biosciences, Cat #556003), rabbit monoclonal anti-Cyclin D1 (Thermo Fisher, Cat #SC8396), goat anti-rabbit IgG Alexa-Fluor 488 (Thermo Fisher, Cat #A-11034), goat anti-mouse IgG Alexa 568 (Thermo Fisher, Cat #A-11004), DRAQ5 (Abcam, Cat #ab108410).

[0115] Peptide Synthesis and Complex Generation

[0116] Alpha1 was synthesized using Fmoc solid phase chemistry (Mimotopes). Alpha1 stock concentration was diluted in PBS and mixed with five times concentration of oleic acid in PBS to achieve the final alpha1-oleate complex. The alpha1 sequence is: Ac-KQFTKAELSQ LLKDIDGYGG IALPELIATM FHTSGYDTQ-OH.

[0117] Bladder Cancer Model

[0118] C57BL/6 female mice were bred at the Department of Laboratory Medicine, Lund University and used at ages from 7 to 12 weeks. For procedures, mice were anesthetized by intraperitoneal injection of a cocktail of ketamine (1.48 mg in 100 μl of NaCl, Intervet) and xylazine (0.22 mg in 100 μl of NaCl, Vetmedic). On day 0, the bladder was emptied and preconditioned by intravesical instillation of 100 μl poly-L-lysine solution (0.1 mg/ml) through a soft polyethylene catheter (Clay Adams) with an outer diameter of 0.61 mm. After 30 minutes, MB49 mouse bladder carcinoma cells (2×10.sup.5 in 100 μl PBS) were instilled. On days 3, 5, 7, 9 and 11, 100 μl of alpha1-oleate (alpha1: 1.7 mM, 8.5 mM or 17 mM, oleic acid: 8.5 mM, 42.5 mM or 85 mM, respectively) or PBS (sham treated controls) were instilled. Mice remained under anesthesia on preheated blocks with the catheter in place to prolong tumor exposure to the peptide-oleate complexes (approximately 1 hour). Groups of 5-6 mice for each treatment and control were sacrificed after 12 days, and bladders were imaged and processed for histology. Two independent experiments were performed.

[0119] Statistical Analysis

[0120] Results are presented as means±SEMs and groups are compared by one-way ANOVA. P values were calculated by Student's t test and one way analysis of variance followed by Bonferroni's post hoc testing using GraphPad Prism version 7 (GraphPad Software Inc.). P<0.05 was considered statistically significant. * P<0.05; ** P<0.01; *** P<0.001.

Example 9—Further Examination of Combination Therapies

[0121] Materials and Methods

[0122] Chemicals and Antibodies

[0123] Oleic acid (Croda, batch number: 0001120439), poly-L-lysine solution (Sigma, Cat #RNBF4239), Richard-Allan Scientific Signature Series Hematoxylin and Eosin-Y (Thermo Scientific, Cat #7211 and 7111), Epirubicin (Sigma-Aldrich, Cat #E9406) and Mitomycin Sigma-Aldrich, Cat #M0440).

[0124] Peptide Synthesis and Complex Generation

[0125] We have identified the N-terminal, alpha-helical domain of alpha-lactalbumin as a tumoricidal entity, which forms a complex with oleic acid (Ho J, Rydstrom A, Manimekalai MSS, Svanborg C, Grüber G. Low resolution solution structure of HAMLET and the importance of its alpha-domains in tumoricidal activity. PLoS One 2012; 7: e53051). For this study, we synthesized the 39 amino acid peptide (aa 1-39, Ac-KQFTKAELSQLLKDIDGYGGIALPELIATMFHTSGYDTQ-OH), using Fmoc solid phase chemistry with the purity of >95% (Polypeptide group, France). To form the alpha1-oleate complex, the alpha1 peptide was mixed with sodium oleate at a 1:5 molar ratio. The stock solution was further diluted in PBS to the appropriate concentration for each experiment.

[0126] Bladder Cancer Model

[0127] MB49 (RRID:CVCL_7076) cells were provided by Sara Mangsbo, Uppsala University, Sweden. MB49 bladder cancer was established as previously described (Mossberg A-K, Hou Y, Svensson M, Holmqvist B, Svanborg C. HAMLET treatment delays bladder cancer development. The Journal of urology 2010; 183: 1590-7). C57BL/6 female mice were bred at the Department of Laboratory Medicine, Lund University and used at ages from 7 to 12 weeks. For procedures, mice were anesthetized by intraperitoneal injection of a cocktail of ketamine (1.48 mg in 100 μl of 0.9% NaCl solution, Intervet) and xylazine (0.22 mg in 100 μl of 0.9% NaCl solution, Vetmedic). On day 0, the bladder was emptied and preconditioned by intravesical instillation of 100 μl of poly-L-lysine solution (0.1 mg/ml) through a soft polyethylene catheter (Clay Adams) with an outer diameter of 0.61 mm. After 30 minutes, MB49 mouse bladder carcinoma cells (2×10.sup.5 cells in 50 μl media) were instilled. On days 3, 5, 7, 9 and 11, mice were randomly assigned and instilled to the alpha1-oleate (1.7 mM or 8.5 mM), chemotherapeutic drugs (MMC 25 μg, epirubicin 25 μg), combination of alpha1-oleate with chemotherapeutic agent or sham PBS treatment group. The catheter was left in place for about one minute, but as the mice remained under anesthesia, the time to voiding (dwelling time) of the substance was 2-3 hours. Groups of 5-6 mice for each treatment and control were sacrificed at day 12. Follow up groups of 5-8 mice were sacrificed at week 4 and week 8. Bladders were imaged and processed for histology or RNA extraction. Two independent experiments were performed for each condition. All experiments were performed with mycoplasma-free cells.

[0128] Histology

[0129] Bladders were embedded in O.C.T. compound (VWR), and successive 5 μm sections were collected from the center of each bladder and placed on positively-charged microscope slides (Superfrost/Plus; Thermo Fisher Scientific). For hematoxylin-eosin (H&E) staining, Richard-Allan Scientific Signature Series Hematoxylin 7211 was used followed by Eosin-Y 7111 for counterstaining. Images was captured using the AX10 microscope (Carl Zeiss). The tumor circumferences were measured for analysis of the tumor area using ImageJ software.

[0130] Gene Expression Analysis

[0131] Frozen bladder tissue was pulverized using liquid nitrogen. (Tran T. Hien1, Ambite, I., Lam Yim Wan, Butler, D., Tran T. Hiep, Höglund, U., Babjuk, M. and Svanborg, C. Bladder cancer treatment without toxicity—A dose-escalation study of alpha1-oleate. International Journal of Cancer. in press). Total RNA was extracted (RNeasy Mini kit, Qiagen). 100 ng of total RNA was amplified using the GeneChip 3′IVT Express Kit and then fragmented. Next, labeled aRNA was hybridized onto Mouse Genome 430 PM array strips (Affymetrix) for 16 hours at 45° C., washed, stained (Applied Biosystems, ThermoFisher Scientific) and scanned using the GeneAtlas system (Affymetrix). All samples passed the internal quality controls included in the array strips (signal intensity by signal to noise ratio; hybridization and labeling controls; sample quality by GAPDH signal and 3′-5′ ratio <3). Transcriptomic data was normalized using Robust Multi Average implemented in the Transcriptome Analysis Console software (v.4.0.1.36, Applied Biosystems, ThermoFisher Scientific). Fold change was calculated by comparing tumor-bearing bladders or treated healthy bladders to untreated healthy bladder control tissue. Relative expression levels were analyzed and genes with an absolute fold change >2.0 were considered as differentially expressed. Heat-maps were constructed using the Gitools 2.1.1 software. Differentially expressed genes were functionally characterized using the Ingenuity Pathway Analysis (IPA, Qiagen) software.

[0132] Statistical Analysis

[0133] Results are presented as means±SEMs. p values were calculated by Student's t test or one-way ANOVA followed by Bonferroni's post hoc testing using Prism version 7 (GraphPad Software Inc.). p<0.05 was considered statistically significant. * p<0.05; ** p<0.01; *** p<0.001.

[0134] Results

[0135] Effects of Combination Therapy with Alpha1-Oleate and Mitomycin

[0136] The effects of alpha1-oleate and Mitomycin C were investigated in the murine MB49 bladder cancer model (for study design see FIG. 19A). Bladder cancer was established by intra-vesical instillation of MB49 cells and treatment was initiated on day 3. The treatment group received alpha1-oleate (1.7 mM or 8.5 mM), MMC (0.1 mL, 25 ug/dose) or a combination of alpha1-oleate (1.7 mM or 8.5 mM) with MMC (0.1 mL, 25 ug/dose). Instillations were performed on days 3, 5, 7, 9 and 11 and sham treated mice received PBS. Bladders were harvested at sacrifice on day 12 and tumor development was quantified as bladder weight, bladder size, tumor size and pathology score (FIG. 21c-f). The tumor area was quantified in H&E stained, whole-bladder tissue sections (FIG. 22).

[0137] The sham treated mice developed large tumors that filled the bladder lumen after 12 days (12/12 mice, FIG. 21b). Therapeutic efficacy of alpha1-oleate and Mitomycin C was demonstrated to the sham-treated group. Tumor progression was delayed by alpha1-oleate alone in a dose-dependent manner (p<0.01, FIG. 19c-f) and by Mitomycin C alone (0.1 mL, 25 ug/dose), (FIGS. 21 and 22).

[0138] The combined effect of Mitomycin and alpha1-oleate was examined by first inoculating the mice with alpha1-oleate (1.7 or 8.5 uM). After 2 hours, the mice received a second inoculation of Mitomycin (see FIG. 21a) and the procedure was repeated once daily on day 3, 5, 7, 9 and 11 followed by sacrifice on day 12. A strong synergistic effect was detected between the chemotherapeutic agents and alpha1-oleate (FIG. 21b). The therapeutic effect was further increased in mice receiving a combination of alpha1-oleate and Mitomycin C (p=0.03), most prominently in mice receiving the higher dose or alpha1-oleate (8.5 mM). No tumor tissue was detected in the treated mice by visual inspection at sacrifice and the bladder weight and bladder size did not differ from that in healthy bladders from control mice (p=0.99, FIGS. 21c and 21e). The reduction in tumor area was confirmed by microscopy after H&E staining. No tumor was detected in any of the treatment groups (p<0.001) (FIG. 20). As alpha1-oleate at a dose of 8.5 mM was efficient, a further increase of the therapeutic effect of MMC combination therapy was not detected at 12 days (p=0.99).

[0139] Effects of Combination Therapy with Alpha1-Oleate and Epirubicin

[0140] The mechanism of action of Epirubicin resembles that of Mitomycin, affecting DNA intercalation and arresting cellular growth. The two compounds also show a similar toxicity profile. Subsequent experiments therefore addressed the therapeutic efficacy of alpha1-oleate and Epirubicin alone and in combination (FIG. 23a). The combined effect of Epirubicin and alpha1-oleate was examined by first inoculating the mice with alpha1-oleate (1.7 or 8.5 uM). After 2 hours, the mice received a second inoculation of Epirubicin (see FIG. 23a) and the procedure was repeated once daily on day 3, 5, 7, 9 and 11 followed by sacrifice on day 12.

[0141] Tumor progression was delayed by Epirubicin (p<0.001 compared to sham treated controls), as defined by bladder weight, bladder size, tumor size and pathology score (FIG. 22c-f). The reduction in tumor area was confirmed by H&E staining of whole-bladder tissue sections. The therapeutic effect of Epirubicin alone was comparable to that of alpha1-oleate (1.7 mM, p=0.97). At the higher dose (8.5 mM), alpha1-oleate was more efficient (FIG. 24, p=0.004).

[0142] A strong synergistic effect was detected between Epirubicin and alpha1-oleate. No tumor tissue was detected in the treated mice by visual inspection at sacrifice and the bladder weight and bladder size did not differ from that in healthy bladders from control mice (FIG. 23c-e and FIG. 21c-e). The reduction in tumor area was confirmed by H&E staining, where no tumor tissue was detected (FIG. 24). A dose of 8.5 mM was more efficient than 1.7 mM (p=0.001) as stand-alone therapy but did not significantly increase the therapeutic effect of the MMC combination therapy, after 12 days (p=0.62).

[0143] The results suggest that alpha1-oleate treatment enhances the effects of the chemotherapeutic agents Mitomycin and Epirubicin, to the point of preventing tumor development.

[0144] Long-Term Follow Up

[0145] The duration of the therapeutic effect was evaluated by following the mice for a total of four weeks (FIG. 28). The sham treated group was not followed beyond 12 days, when they were sacrificed, due to the rapid growth of the tumor. Extended protection was observed in the treatment groups, however. After 4 weeks, mice receiving low dose of alpha1-oleate (1.7 mM), MMC or Epirubicin showed an increase in tumor growth. In the combination therapy groups however, mice remained protected with no evidence of tumor relapse (FIGS. 25 and 26). The combined effect of low dose alpha1-oleate (1.7 mM) and MMC or Epirubicin (25 uG/dose) was stronger than that of MMC or Epirubicin alone (p<0.001 compared to MMC or Epirubicin) but comparable to the higher dose of alpha1-oleate showing no significant differences (8.5 uM). Further protection was observed in mice treated with the combination of 8.5 mM of alpha1-oleate and Epirubicin (FIG. 27).