Combination of a MAPK/ERK pathway inhibitor and a glycosaminoglycan for the treatment of cancer

Abstract

A negatively charged glycosaminoglycan is provided for use as a medicament for the treatment of cancer. A combined administration of a negatively charged glycosaminoglycan is provided in which the glycosaminoglycan is characterised by the absence of the terminal pentasaccharide of Heparin, and an inhibitor of the MAPK/ERK pathway. A combined administration of a glycosaminoglycan and a MAPK/ERK pathway inhibitor is provided as a medicament for the treatment of cancer types that exhibit a resistance towards a single MAPK/ERK pathway inhibitor treatment.

Claims

1. A method of treatment of cancer in a subject comprising: administering to the subject a combined administration of: (1) a negatively charged glycosaminoglycan, wherein said negatively charged glycosaminoglycan is sulfated and characterised by the absence of the terminal pentasaccharide of Heparin, and (2) an inhibitor of the MAPK/ERK pathway, wherein the cancer comprises cancerous cells that are resistant to, or are at elevated risk of developing resistance to an inhibitor of the MAPK/ERK pathway, or a combination thereof.

2. The method according to claim 1, wherein the degree of sulfation of said negatively charged glycosaminoglycan is >1.0.

3. The method according to claim 1, wherein said negatively charged glycosaminoglycan is characterised by the absence of a pentasaccharide GlcNAc/NS(6S)-GlcA-GlcNS(3S,6S)-IdoA(2S)-GlcNS(6S).

4. The method according to claim 1, wherein said negatively charged glycosaminoglycan exhibits an average molecular weight of about 5000 to about 12000 daltons.

5. The method according to claim 1, wherein the inhibitor of the MAPK/ERK pathway for the combined administration is a MEK Inhibitor.

6. The method according to claim 5, wherein the MEK inhibitor is Selumetinib.

7. The method according to claim 1, wherein the MEK inhibitor is selected from the group consisting of Trametinib (GSK1120212), Cobimetinib or XL518, Binimetinib (MEK162), PD-325901, CI-1040, PD035901, and TAK-733.

8. The method according to claim 1, wherein the inhibitor of the MAPK/ERK pathway for combined administration is a Raf Inhibitor.

9. The method according to claim 1, wherein the cancer comprises cancerous cells that exhibit the presence of one or more ErbB-family proteins on the cell surface.

10. The method according to claim 1, wherein the cancer comprises cancerous cells that exhibit increased expression (up-regulation) of at least one of one or more ErbB-family proteins or increased ErbB signalling compared to an appropriate (non-cancerous) control cell.

11. The method according to claim 10, wherein the up-regulated ErbB-family protein is Her1 (EGFR, ErbB1), Her2 (Neu, ErbB2), Her3 (ErbB3), or Her4 (ErbB4).

12. The method according to claim 1 further comprising: allowing sufficient time for the combined administration that forms a composition to treat the cancer, the cancer being one of: leukaemia, lymphoma, melanoma, non-small cell lung cancer, glioma, hepatocellular (liver) carcinoma, glioblastoma, carcinoma of the thyroid, bile duct, bone, gastric, brain/CNS, head and neck, hepatic, stomach, prostate, breast, renal, testicular, ovarian, skin, cervical, lung, muscle, neuronal, oesophageal, bladder, lung, uterine, vulval, endometrial, kidney, colorectal, pancreatic, pleural/peritoneal membranes, salivary gland, and epidermoid tumours and haematological malignancies, and metastases of any of the aforementioned.

13. The method of claim 1 wherein the cancer is a tumour associated with the MAPK/ERK (Ras-Raf-MEK-ERK) pathway or which is dependent alone, or in part, on the biological activity of the MAPK/ERK (Ras-Raf-MEK-ERK) pathway.

14. The method of claim 1 wherein the administration is local administration of said negatively charged glycosaminoglycan in proximity to a tumour of the cancer.

15. The method of claim 14 wherein the local administration is by injection, transmucosaL or transdermal.

16. The method of claim 1 wherein the administration is sequential of the negatively charged glycosaminoglycan and the inhibitor of the MAPK/ERK pathway, or vice versa.

17. A method of treatment of cancer, comprising the combined administration of (1) a negatively charged sulfated polysaccharide, wherein said sulfated polysaccharide is characterised by the absence of the terminal pentasaccharide of Heparin, and (2) an inhibitor of the MAPK/ERK pathway, and wherein the cancer comprises cancerous cells that are resistant to, or are at elevated risk of developing resistance to an inhibitor of the MAPK/ERK pathway, or a combination thereof.

18. The method according to claim 17, wherein the negatively charged sulfated polysaccharide is pentosan polysulfate (PPS).

19. The method according to claim 17, wherein the negatively charged sulfated polysaccharide is dextran sulfate (DXS).

20. The method according to claim 17, wherein the cancer comprises cancerous cells that exhibit the presence of one or more ErbB-family proteins on the cell surface.

Description

SHORT DESCRIPTION OF THE FIGURE

(1) FIG. 1: Schematic illustration of a growth factor dependent escape mechanism leading to a MEK inhibitor resistance in cancerous cells.

(2) FIG. 2: Schematic illustration of the benefit of an additional administration of a negatively charged glycosaminoglycan to prevent the development of a MEK inhibitor resistance in cancerous cells

DETAILED DESCRIPTION OF THE FIGURE

(3) FIG. 1 illustrates of a growth factor dependent escape mechanism leading to an MEK inhibitor resistance in cancerous cells. In the MAPK/ERK pathway activation of a membrane bound RTK initiates a Ras tyrosine kinase chain leading to the activation of downstream transcription factors in support of cell proliferation. In this process platelets are involved. MEK inhibitors such as Selumetinib inhibit the phosphorylation and hereby also cell proliferation. However, tumours or cancerous cells treated with Selumetinib rapidly develop a resistance to the inhibitor. The resistance is likely caused by the fact that unphosphorylated ERK activates another tyrosine kinase chain, the depicted MAP3K1 pathway, which leads to a de novo synthesis of RTKs belonging to the HER family, e.g. ERBB3. The HER RTKs are activated by growth factors leading to MAP3K1 pathway signalling that enables the cell to bypass the compromised Ras signalling chain.

(4) In the aforementioned escape mechanism, platelets docking in the provision of growth factors play a role, as can be illustrated the following steps: 1 A platelet docks to the platelet receptor on the membrane of a growth committed cell and thereupon is activated. In the course of this process, the contents of its alpha-granules are released. 2 These contain among other substances also growth factors, which now are available to dock to Receptor Tyrosine Kinases (RTK) 3 Thereupon the RTK activate Ras in the canonical Ras-Raf-MEK-ERK pathway (left). This pathway is a main signaling cascade in cell proliferation, reaching as an activation cascade MEK, which function it is to phosphorylate and activate ERK. Therapeutic approaches to treat cancer interfere with the pathway by providing MEK inhibitors blocking the phosphorylation of ERK and thereby stopping further signalling towards cell proliferation. MEK inhibitors have proven as a promising anti-cancer agent promoting apoptosis of tumour cells and preventing their cell proliferation. 4 However, in the clinical situation often the development of resistance to MEK inhibitors is observed. In particular, unphosphorylated ERK can upregulate and/or activate the MAP3K1 phosphokinase. 5 The MAP3K1 phosphokinase dependent pathway (left) can among other functions also induce nuclear synthesis of m-RNA for other Receptor Tyrosine Kinases, such as e.g. ERBB3. 6 Through the activation of the MAP3K1 phosphokinase synthesis of mRNA (such as ERBB3) is promoted. 7 ERBB3 and similar Receptor Tyrosine Kinases may upon reaction with growth factors released from activated platelet start as an alternative route a MAP3K1 dependent pathway to promote cell proliferation. By bypassing the Ras-Raf-MEK-ERK pathway, the cell has thus developed a resistance against the MEK inhibitor and can continue proliferation.

(5) As described herein, negatively charged glycosaminoglycans may inhibit the adherence of platelets to platelet receptors expressed on the cell surface of growth committed cells, thus also inhibiting the escape route via an activation of MAP3K1. Thereby a resistance towards the MEK inhibitor may be abolished.

(6) FIG. 2 illustrates the effect of a combination of a MEK-inhibitor treatment together with the administration of a negatively charged glycosaminoglycan.

(7) A negatively charged glycosaminoglycan prevents the platelet from docking to the cell and thus the provision of growth factors. Due to the absence of available growth factors, the alternative signalling route via MAP3K1 cannot be activated and the development of a resistance towards a MEK inhibitory treatment is impeded.

(8) The MEK inhibitor thus retains its anticancer and antiproliferative effectivity due a combined administration of the negatively charged glycosaminoglycan, which acts upstream of the RTK's in the chain of biological processes leading to tumour growth.

(9) Such a combined treatment is particularly useful in the treatment of metastases. 90 percent of patients dying of cancer do so because of metastases. These are daughter tumours, typically arising in other tissues than the primary tumour tissue. At the onset of a metastases, when the tumour is at a single cell stage, either circulating in the blood or dwelling in a metastatic niche or if the tumour is still smaller than some millimetres in diameter, the combined treatment of a negatively charged glycosaminoglycan and a MEK inhibitor is particularly efficient. The treatment therefore also presents new opportunities to selectively prevent metastases.

EXAMPLES

(10) The invention is further described by the following examples. These are not intended to limit the scope of the invention.

Example 1

(11) The experiment is performed on cancerous cells that have developed a resistance against the MEK inhibitor Selumetinib. As described in Little et al. 2011 in detail cancerous cells that have developed a resistance against the MEK inhibitor Selumetinib are generated by growing colorectal cancer cell lines harboring mutations in BRAF (COL0205 and HT29 lines) or KRAS (HCT116 and LoVo lines) in the presence of increasing concentrations of AZD6244 (Selumetinib) without clonal selection until they grow apparently normally in 1 μm, 2 μM or 4 μM of the drug.

(12) For each of the cancerous cells lines that have developed a resistance against the MEK inhibitor Selumetinib the following steps are performed:

(13) Three populations of cells are cultivated in the presence of the inhibitor under serum-free conditions.

(14) For a first population of the cells PPS is added to the cell culture dishes, while for a second population, i.e. the control cells, no PPS is added.

(15) For a third population of the cells DXS is added to the cell culture dishes.

(16) Each day all cell populations are co-incubated with platelets for 30 minutes, which are subsequently washed away.

(17) Control cells in the growth cycle express platelet receptors on their surface. Platelets adhere to the control cells and release growth factors from their alpha granules. The control cells will take up the factors and proceed through the cell cycle, even in the presence of the MEK inhibitor.

(18) For the first and third population of cells, the PPS and DXS prevents the platelet from adhering to the cancerous cells, therefore no platelet-derived growth factors are released and proliferation is impeded.

(19) While control cancerous cells continue to grow and divide, even in the presence of a MEK inhibitor, the cell populations which are incubated in the presence of the MEK inhibitor and PPS or DXS perish.

Example 2

(20) The second experiment is performed as described for Example 1 except that cells are cultivated in the presence of the MEK inhibitor CI-1040 (PD184352).

(21) As described in Little et al. 2011 in detail cancerous cells that have developed a resistance against the MEK inhibitor PD184352 are generated by growing colorectal cancer cell lines harboring mutations in BRAF (COL0205) or KRAS (HCT116) in the presence of increasing concentrations of AZD6244 (Selumetinib) without clonal selection until they grow apparently normally in 1 μm, 2 μM or 4 of the drug.

(22) For each of the cancerous cells lines that have developed a resistance against the MEK inhibitor CI-1040 (PD184352) the cultivation steps as in Example 1 are performed, except that the cells are cultivated in the presence of the MEK inhibitor CI-1040 (PD184352).

(23) While control cancerous cells continue to grow and divide, even in the presence of the MEK inhibitor CI-1040 (PD184352), the cell populations which are incubated in the presence of the MEK inhibitor CI-1040 (PD184352) and the PPS or DXS perish.

Example 3

(24) The third experiment is performed as described for Example 1 except that cells that have developed a resistance against the MEK inhibitor trametinib (GSK1120212) are used and cultivated in the presence of the MEK inhibitor trametinib.

(25) As described in Vujic et al. 2014 in detail cancerous cells that have developed a resistance against the MEK inhibitor trametinib are generated by growing Human NRAS mutant melanoma cell lines DO4 and MM415 in the presence of increased concentrations of trametinib (GSK1120212) over a period of approximately 6 months.

(26) For each of the cancerous cell lines that have developed a resistance against the MEK inhibitor trametinib the cultivation steps as in Example 1 are performed, except that the cells are cultivated in the presence of the MEK inhibitor trametinib.

(27) While control cancerous cells continue to grow and divide, even in the presence of the the MEK inhibitor trametinib, the cell populations which are incubated in the presence of the MEK inhibitor trametinib and the PPS or DXS perish.

Example 4

(28) The fourth experiment is performed as described for Example 1 except that cells that have developed a resistance against the Raf inhibitor vemurafenib are used and cultivated in the presence of the Raf inhibitor vemurafenib.

(29) As described in Sandri et al. 2016 in detail cancerous cells that have developed a resistance against the Raf inhibitor vemurafenib are generated by growing melanoma cell line SK-MEL-28 carrying the BRAF.sup.V600E mutation in the presence of 0.5-0.6 μM vemurafenib for 4-6 weeks and subsequently isolating clonal colonies.

(30) For each of the cancerous cells lines that have developed a resistance against the Raf inhibitor vemurafenib the cultivation steps as in Example 1 are performed, except that the cells are cultivated in the presence of the Raf inhibitor vemurafenib.

(31) While control cancerous cells continue to grow and divide, even in the presence of the the Raf inhibitor vemurafenib, the cell populations which are incubated in the presence of the Raf inhibitor vemurafenib and the PPS or DXS perish.

Example 5

(32) The fifth experiment is performed as described for Example 1 except that cells that have developed a resistance against the Raf inhibitor sorafenib are used and cultivated in the presence of the Raf inhibitor sorafenib.

(33) As described in Chen et al. 2011 in detail cancerous cells that have developed a resistance against the Raf inhibitor sorafenib are generated by growing the human Hepatocellular carcinoma (HCC) Huh7 in a long term exposure to sorafenib.

(34) For each of the cancerous cells lines that have developed a resistance against the Raf inhibitor sorafenib the cultivation steps as in Example 1 are performed, except that the cells are cultivated in the presence of the Raf inhibitor sorafenib.

(35) While control cancerous cells continue to grow and divide, even in the presence of the the Raf inhibitor sorafenib, the cell populations which are incubated in the presence of the Raf inhibitor sorafenib and the PPS or DXS perish.

Example 6

(36) The sixth experiment is performed as described for Example 1 except that cells that have developed a resistance against the Raf inhibitor dabrafenib are used and cultivated in the presence of the Raf inhibitor dabrafenib.

(37) As described in Caparorali et al. 2011 in detail cancerous cells that have developed a resistance against the Raf inhibitor dabrafenib are generated by growing the human melanoma cell line A375 in gradually increasing concentrations of dabrafenib (from 1 nM up to 1.5 μM) over a period of 4 months and subsequently maintaining the dabrafenib-resistance cell lines in CM supplemented with 1.5 μM dabrafenib.

(38) For each of the cancerous cells lines that have developed a resistance against the Raf inhibitor dabrafenib the cultivation steps as in Example 1 are performed, except that the cells are cultivated in the presence of the Raf inhibitor dabrafenib.

(39) While control cancerous cells continue to grow and divide, even in the presence of the the Raf inhibitor dabrafenib, the cell populations which are incubated in the presence of the Raf inhibitor dabrafenib and the PPS or DXS perish.

Example 7

(40) The seventh experiment is performed as described for Example 1, except that the cancerous cells are resistant against an RTK inhibitor and the three populations of cells are cultivated in the presence of the RTK inhibitor.

(41) While control cancerous cells continue to grow and divide, even in the presence of the RTK inhibitor, the cell populations incubated in the additional presence of PPS or DXS perish.

REFERENCES

(42) Anderson N G., Mailer J. L., Tonks N. K., Sturgill T. W., Requirement for integration of signals from two distinct phosphorylation pathways for activation of MAP kinase, Nature, 343:651-653 (1990). Caporali et al. Targeting the PI3K/AKT/mTOR pathway overcomes the stimulating effect of dabrafenib on the invasive behavior of melanoma cells with acquired resistance to the BRAF inhibitor, International Journal of Oncology 49: 1164-1174 (2016). Chapman, P. B. et al. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N. Engl. J. Med. 364:2507-2516 (2011). Chen et al. Activation of Phosphatidylinositol 3-Kinase/Akt Signaling Pathway Mediates Acquired Resistance to Sorafenib in Hepatocellular Carcinoma Cells, The Journal of Pharmacology and Experimental Therapeutics, 337:155-161. Cole et al. Synthetic Heparan Sulfate Oligosaccharides Inhibit Endothelial Cell Functions Essential for Angiogenesis, PLoS One, 5(7):e11644 Dooley A. J. et al. Intermittent dosing with vemurafenib in BRAF V600E-mutant melanoma: Review of case series, Therapeutic Advances in Medical Oncology, 6(6): 262-266 (2014). Eroglu Z., Ribas A., Combination therapy with BRAF and MEK inhibitors for melanoma: latest evidence and place in therapy, Therapeutic Advances in Medical Oncology, 8(1): 48-56 (2016). Flaherty, K. T. et al. Inhibition of mutated, activated BRAF in metastatic melanoma. N. Engl. J. Med. 363:809-819 (2010). Grimaldi M. et al., Combined BRAF and MEK Inhibition with Vemurafenib and Cobimetinib for Patients with Advanced Melanoma, European Oncology & Haematology, 13:1-5 (2017). Harter K., Levine M., Henderson O., Anticoagulation Drug Therapy: A Review, Western Journal of Emergency Medicine. 16:1 (2015). Hauschild, A. et al. Dabrafenib in BRAF-mutated metastatic melanoma: a multicentre, open-label, phase 3 randomised controlled trial. Lancet 380:358-365 (2012). Holderfield M., Deuker M. M., McCormick F., McMahon M., Targeting RAF kinases for cancer therapy: BRAF mutated melanoma and beyond, Nat Rev Cancer, 14:455-467 (2014). Hoshino R., Chatani Y., Yamori T., Tsuruo T., Oka H., Yoshida O., Shimada Y., Ari-i S., Wada H., Fujimoto J., Kohno M., Constitutive activation of the 41-/43-kDa mitogen-activated protein kinase signalling pathway in human tumours, Oncogene, 18:813-822 (1999). Little et al. Amplification of Driving Oncogene, KRAS or BRAF, underpins resistance acquired to MEK1/2 inhibitors in colorectal cancer cells, Science Signalling 4 (166) ra17 (2011). Lugowska I., Kosela-Paterczyk H., Kozak K., Rutkowski P., Trametinib: a MeK inhibitor for management of metastatic melanoma, OncoTargets and Therapy, 8:2251-2259 (2015). McCubrey J. A, Steelman L. S., Chappell W. H., Abrams S. L., Wong E. W. T., Chang F., Lehmann B., David M. T., Milella M., Tafuri A., Stivala F., Libra M., Evangelisti J. B. C., Martelli A. M., Franklin R. A., Roles of the Raf/MEK/ERK pathway in cell growth, malignant transformation and drug resistance, Biochimica et Biophysica Acta (BBA)—Molecular Cell Research, 1773:1263-1284 (2007). Milhavet O., Gary D. S., Mattson M. P., RNA interference in biology and medicine, Pharmacol Rev., 55:629-48 (2003). Modery-Pawlowski, C. L., Master, A. M., Pan, V., Howard, G., & Gupta, A. S., A Platelet-Mimetic Paradigm for Metastasis-Targeted Nanomedicine Platforms. Biomacromolecules, 14(3):910-919 (2013). Opalinska J. B., Gewirtz A. M., Therapeutic potential of antisense nucleic acid molecules, Sci STKE, 206:pe47 (2003). Peyssonnaux C., Eychène A., The Raf/MEK/ERK pathway: new concepts of activation, Biology of the Cell, 93: 53-62 (2001). Poulikakos P. I. et al., Resistance to MEK Inhibitors: Should We Co-Target Upstream?, Science Signalling 4(166): pe16 (2011). Rhomberg A. J., Ernst S., Sasisekharan R., and Biemann K., Mass spectrometric and capillary electrophoretic investigation of the enzymatic degradation of heparin-like glycosaminoglycans, Proc. Natl. Acad. Sci., 95:4176-4181 (1998). Rosen N. et al. Tumour adaptation and resistance to RAF inhibitors, Nature Medicine, 19:11 (2013) Sandri S. et al. Vemurafenib resistance increases melanoma invasiveness and modulates the tumor microenvironment by MMP-2 upregulation, Pharmacological Research 111:523-533 (2016). Schwartz, C. F., Kilgore, K. S., Homeister, J. W., Levy, B. A., Lucchesi, B. R., & Bolling, S. F., Increased rat cardiac allograft survival by the glycosaminoglycan pentosan polysulfate. Journal of Surgical Research, 86: 24-28 (1999). Sivaraman, V. S., Wang, H., Nuovo, G. J., & Malbon, C. C. Hyperexpression of mitogen-activated protein kinase in human breast cancer. Journal of Clinical Investigation, 99:1478-1483 (1997). Takagi, S., Sato, S., Oh-hara, T., Takami, M., Koike, S., Mishima, Y., Hatake K., Fujita, N., Platelets Promote Tumour Growth and Metastasis via Direct Interaction between Aggrus/Podoplanin and CLEC-2. PLoS ONE, 8(8):e73609 (2013). Turnbull J. E., Hopwood J. J., Gallagher J. T., Strategy for rapid sequencing of heparan sulfate and heparin saccharides, Proc. Natl. Acad. Sci., 96:2698-2703 (1999). Vujic et al. Metformin and trametinib have synergistic effects on cell viability and tumor growth in NRAS mutant cancer, Oncotarget (November 2014). Wang, Z., Zhang, F., Dordick, J. S., & Linhardt, R. J. Molecular Mass Characterization of Glycosaminoglycans with Different Degrees of Sulfation in Bioengineered Heparin Process by Size Exclusion Chromatography. Current Analytical Chemistry, 8(4), 506-511 (2012). Zhao, Y., Adjei, A. A., The clinical development of MEK inhibitors. Nat. Rev. Clin. Oncol. 11:385-400 (2014).