WO2017042706A1 - Pharmaceutical kit and use of erythropoietin and bruton's kinase inhibitor - Google Patents

Abstract

The subject matter of the invention is a pharmaceutical kit characterized in that it includes erythropoietin and Bruton's kinase inhibitor in the same package or in a separate package, and/or instruction for use. In addition, the subject matter of the invention is also erythropoietin and Bruton's kinase inhibitor for use as a drug. Furthermore, the subject matter of the invention is the use of erythropoietin and Bruton's kinase inhibitor for the manufacture of a drug used in the treatment of cancer.

Description

PHARMACEUTICAL KIT AND USE OF ERYTHROPOIETIN AND

BRUTON'S KINASE INHIBITOR

The subject matter of the invention is a pharmaceutical kit that includes erythropoietin and Bruton's kinase inhibitor for use as a drug, and the application of erythropoietin and Bruton's kinase inhibitor for the manufacture of a drug for the treatment of cancer, especially colon cancer.

Colon cancer is a significant problem, both from the medical and social point of view. Furthermore, the dynamically growing incidence rate of this cancer in Poland is higher than in other European countries. Therefore, there is a need to intensify research concerning early diagnostics and the treatment of this disease.

Patients with colon cancer are a relatively heterogeneous group, thus it is difficult to design a single effective treatment pattern.

There are certain protocols applied to oncological patients, with surgery being the basic element of treatment. The objective of surgery is to ensure colon tissues are free from neoplastic infiltration.

Chemotherapy is used as necessary adjuvant therapy after surgery. It is mostly based on fluoropyrimidine, 5- fluorouracil/ folic acid, capecitabine, and ocaliplatin. The treatment pattern is selected depending on the stage of cancer and the patient's clinical status. In accordance with current recommendations, the goal of cancer treatment is active therapy leading to the eradication of cancer cells from the body and ensuring optimal quality of life for the patient. It must be emphasized, however, that the currently used therapeutic patterns are definitely unsatisfactory.

This results from, among others, the fact that the progression of neoplastic lesions leads to the exhaustion of the body's compensation reserves, which causes many symptoms that make it difficult or even impossible for the patient to function in society.

Apart from the primary health condition - uncontrolled hyperplasia - anemia is a serious clinical problem. It occurs in approximately 60-90% of patients with cancer, and in nearly 30% the anemia is severe or life-threatening. Its degree of severity depends on the cancer type, clinical stage, disease duration, presence of complications, and methods and intensity of the applied treatment.

In the case of colon neoplastic lesions, the main reasons for anemia are chronic bleeding, impaired intestinal transit, and malabsorption.

According to cancer patients, the most serious problem, apart from pain (which can be properly treated) , is chronic exhaustion. It dramatically lowers comfort of living, and - what is even more important - reduces the efficacy of therapy (Alexiusdottir KK, Moller PH, Snaeb ornsson P, Jonasson L, Olafsdottir EJ, Bjornsson ES, Tryggvadottir L, Jonasson JG. Association of symptoms of colon cancer patients with tumor location and TNM tumor stage. Scand J

Gastroenterol.2012;47:795-801. The published in 2004 results of the ECAS (European Cancer Anemia Survey) observational study clearly demonstrated a close relationship between hemoglobin concentration and patient functioning.

The currently applied treatment of anemia occurring in the course of neoplastic diseases mainly involves the administration of red blood cell concentrate and supplementation of recombinant human erythropoietin (rhEpo) . Erythropoietin is a glycoprotein mainly produced by renal interstitial cells (80-90%) . Anemia and the degree of hypoxia are the main factors determining the amount of synthesized erythropoietin (Noguchi CT, Wang L, Rogers HM, Teng R, Jia Y. Survival and proliferative roles of erythropoietin beyond the erythroid lineage. Expert Rev Mol Med. 2008 Dec l;10:e36) .

The EPO gene is located on chromosome 7 and composed of five exons and four introns . The product of this gene's transcription is a chain originally composed of 193 amino acids. During the translation, 27 hydrophobic amino acids are detached, producing a chain of 166 amino acids. As a result of post-translation changes, the arginine residue is cleaved from the C-terminal, producing a chain of 165 amino acids with a molecular weight of approx. 30-34 kD (depending on the carbohydrate content) .

Apart from erythropoietin beta, erythropoietin alfa and darbepoetin alfa are also registered on the Polish pharmaceutical market. Erythropoietin alfa and erythropoietin beta are analogues of human erythropoietin obtained via genetic engineering methods using Chinese hamster ovary cell clones. The third compound, darbepoetin alfa, is a recombinant and modified derivative of human erythropoietin. That modification involves additional glycosylation and is characterized by approximately a 3-fold longer biological half-life than in the case of erythropoietin alfa and beta.

Erythropoietin beta is a highly purified glycoprotein with the amino acid and carbohydrate sequence identical to that of erythropoietin isolated from the urine of patients with anemia, i.e. endogenous erythropoietin. This compound has four carbohydrate chains, contrary to erythropoietin alfa (three carbohydrate chains) and darbepoetin alfa (five carbohydrate chains) . Erythropoietin beta activates the erythropoietin receptor weaker than erythropoietin alfa, but stronger than darbepoetin alfa. Its activity is characterized by lower activity in in vitro conditions, higher activity in in vivo conditions, and a longer biological half-life compared with erythropoietin alfa.

Pharmacokinetic studies carried out on healthy volunteers and patients with uremia showed that the biological half-life of erythropoietin beta administered intravenously (NeoRecormon) was 4 to 12 hours, and its volume of distribution was one to two times the volume of plasma. Similar results were obtained in experiments involving rats (healthy and with uremia) . After subcutaneous administration of erythropoietin beta to patients with uremia, prolonged absorption leads to a plateau of concentration in plasma, and maximum concentration is achieved on average after 12-28 hours. The biological half-life in the elimination phase is longer after subcutaneous than intravenous administration, and lasts on average 13-28 hours. The bioavailability of erythropoietin beta administered subcutaneously is 23-42% of that of intravenous administration.

Erythropoietin acts via the EpoR receptor, inducing its homodimerization leading to activation (through mutual phosphorylation) of two tyrosine kinases JAK2. Phosphorylation of this receptor, as well as eight tyrosine residues located in the cytoplasmic domain of the receptor, occurs. Further intracellular signal transduction pathway involves MAP kinases (activating differentiation and proliferation), STAT5 transcription factors (inhibiting apoptosis), protein kinase C (intensifying proliferation), and others .

Erythropoietin is widely used in the treatment of anemia, and it is responsible for maintaining an adequate number of erythrocytes in the cardiovascular system, thus ensuring proper tissue oxygenation. This drug promotes the survival, proliferation, and differentiation of erythropoiesis progenitors, and exhibits proangiogenic and anti-apoptotic activity . Recent studies show that while having a beneficial effect on hematological parameters, the use of erythropoietin to treat anemia in patients with hyperplastic lesions may have a serious adverse effect - promoting the neoplastic process. This effect is associated with, among others, the expression of erythropoietin and its receptor in certain tumor tissues. Their presence has been demonstrated in breast, cervical and endometrial, gastric, and colon cancer. Especially noteworthy is the fact that in areas of tumor hypoxia the expression of Epo and EpoR is particularly enhanced (Acs G, Acs P, Beckwith SM, Pitts RL, Clements E, Wong K, Verma A. Erythropoietin and erythropoietin receptor expression in human cancer. Cancer Res. 2001;61:3561-5, Jelkmann W, Bohlius J, Hallek M, Sytkowski AJ: The erythropoietin receptor in normal and cancer tissues. Crit Rev Oncol Hematol, 2008, 67, 39-61., Jelkmann W, Bohlius J, Hallek M, Sytkowski AJ: The erythropoietin receptor in normal and cancer tissues. Crit Rev Oncol Hematol, 2008, 67, 39-61) . The use of erythropoietin enables the possibility of additional activation of the erythropoietin receptor. This can lead to cancer progression and shorter life expectancy for patients receiving erythropoietin therapy. This was confirmed by studies carried out by Leyland-Jones et al . , which demonstrated that the use of rhEpo alpha in patients with metastatic breast cancer was the reason for the increased death rate in this group of patients due to disease progression and thrombotic incidents (Leyland-Jones B, Semiglazov V, Pawlicki M, Pienkowski T, Tjulandin S, Manikhas G, Makhson A, Roth A, Dodwell D, Baselga J, Biakhov M, Valuckas K, Voznyi E, Liu X, Vercammen E. Maintaining normal hemoglobin levels with epoetin alfa in mainly nonanemic patients with metastatic breast cancer receiving first-line chemotherapy: a survival study. J Clin Oncol. 2005 Sep 1;23(25) : 5960-72) . It was also proved that erythropoietin beta therapy in patients with head or neck cancer undergoing radiotherapy reduced the patient survival rate and intensified tumor progression (Henke M, Laszig R, Riibe C, Schafer U, Haase KD, Schilcher B, Mose S, Beer KT, Burger U, Dougherty C, Frommhold H. Erythropoietin to treat head and neck cancer patients with anaemia undergoing radiotherapy: randomised, double-blind, placebo-controlled trial. Lancet. 2003 Oct 18; 362 (9392) : 1255-60) . The results of three independent phase III clinical trials and two meta-analyses involving ten thousand and nearly fourteen thousand cancer patients, respectively, additionally confirmed the negative impact of erythropoietin on the survival of patients subjected to therapy using this drug.

In vitro studies on melanoma cells with EPOR gene knockout showed weaker phosphorylation of ERK kinase in response to erythropoietin, reduced cell proliferation, and a stronger chemotherapeutic effect of cisplatin (Kumar SM, Zhang G, Bastian BC, Arcasoy MO, Karande P, Pushpara an A, Acs G, Xu X. Erythropoietin receptor contributes to melanoma cell survival in vivo. Oncogene. 2012;31:1649-60) . These studies suggest that erythropoietin and its receptor may increase hypoxic cell viability in solid tumors, and as a result promote the selection of cells with reduced apoptotic potential and resistance to chemotherapy, and consequently intensify the neoplastic process.

In another in vitro study of human head and neck squamous cell carcinoma, the authors observed the activation of the tyrosine kinase pathway from the JAK group and transcription factors NF-kB and STAT in response to erythropoietin, which resulted in the inhibition of the apoptosis process (Lai S.Y., Childs E.E., Xi S. et al . Erythropoietin-mediated activation of JAK-STAT signaling contributes to cellular invasion in head and neck squamous cell carcinoma. Oncogene 2005; 24: 4442-4449) . This resulted in increased resistance to anticancer drugs, ionizing radiation, as well as increased proliferation, intensified migration and invasion of malignant cells.

2-cyano-N- (2, 5-dibromophenyl ) -3-hydroxy-2-butenamide

(further, LFM-A13) (Figur 1) is a low molecular weight active leflunomide metabolite designed and obtained with computer modeling techniques.

Figur 1

LFM-A13 is the first inhibitor of Bruton's tyrosine kinase (BTK) : a key signaling molecule of a receptor complex on the surface of B cells (US 6, 221 , 900B1 ) . BTK belongs to the Tec kinase family, a very numerous group of cytoplasmatic tyrosine kinases not directly connected with the receptor. It plays a significant role in immune system cell maturation: it is the key element of maturing B lymphocytes' intracellular signaling pathway. Apart from B lymphocytes, BTK is also present in monocytes, macrophages, neutrophils, mastocytes, and megakaryocytes, where it participates in the activation of signaling pathways responsible for processes connected with cell maturation and viability, production of cytokines, and cell degranulation . Bruton's kinase plays an important role in the development of cancers derived from B cells, activating anti-apoptotic pathways (US 6,221,900B1) . Its increased expression was also demonstrated in prostate cancer cells, where a high level of this kinase was positively correlated with cancer stage (Guo W, Liu R, Bhardwaj G, Yang JC, Changou C, Ma AH, Mazloom A, Chintapalli S, Xiao K, Xiao W, Kumaresan P, Sanchez E, Yeh CT, Evans CP, Patterson R, Lam KS, Kung HJ. Targeting Btk/Etk of prostate cancer cells by a novel dual inhibitor. Cell Death Dis. 2014 Sep 4;5:el409) . Blocking BTK activity by numerous inhibitors of this kinase, either those that have already been used in medicine (ibrutinib, sorafenib) or those that are currently being intensively studied (GDC0834, CGI-560, CGI-1746), is effective in inhibiting the neoplastic process. It must be emphasized that BTK inhibitors have already been applied in hyperplastic diseases of the hematopoietic system, but there is no data available concerning its effect on colon cancer cells. LFM-A13 is being intensively tested regarding the treatment of precursor B-cell lymphoblastic leukemia. The compound promotes apoptosis, has an antiproliferative effect, and increases cancer cell sensitivity to chemotherapy drugs. It also has a high safety profile, since high doses of LFM- A13 used in in vivo studies (20-100 mg/kg i.v.) do not cause any nephrotoxicity or changes in the blood picture (Uckun FM, Zheng Y, Cetkovic-Cvrl e M, Vassilev A, Lisowski E, Waurzyniak B, Chen H, Carpenter R, Chen CL . In vivo pharmacokinetic features, toxicity profile, and chemosensitizing activity of alpha-cyano-beta-hydroxy-beta- methyl-N- ( 2 , 5-dibromophenyl ) propenamide (LFM-A13), a novel antileukemic agent targeting Bruton's tyrosine kinase. Clin Cancer Res. 2002 May; 8 (5) : 1224-33) . Preliminary data on the high selectivity of this compound were not confirmed in subsequent studies, in which it was found that LFM-A13 also inhibits phosphoinositide 3-kinase (PI3K) and EPO-induced phosphorylation of erythropoietin receptor (EpoR) . This compound was proved to block Janus kinases (JAK2) binding to EpoR, thus breaking the intracellular signal transduction pathway (van den Akker E, van Dijk TB, Schmidt U, Felida L, Beug H, Lowenberg B, von Lindern M. The Btk inhibitor LFM-A13 is a potent inhibitor of Jak2 kinase activity. Biol Chem. 2004 May; 385 (5) : 409-13) .

Despite considerable progress in chemotherapy over recent years, designing an efficient anti-neoplastic therapy is a serious challenge for contemporary oncology. The cytostatic drugs used at present in colon cancer therapy cause many adverse effects, which reduce their therapeutic efficacy and dramatically lower patient quality of life. Looking for new therapeutic strategies combining anti-neoplastic activity with improvement of hematological parameters is an especially urgent task for researchers, doctors, and the pharmaceutical industry.

The subject matter of this invention is a pharmaceutical kit characterized in that it includes erythropoietin and Bruton's kinase inhibitor in the same package or in a separate package, and/or instruction for use.

Preferably, erythropoietin is selected from the group comprising erythropoietin alfa, erythropoietin beta and darbepoetin alfa, in particular erythropoietin beta and/or Bruton's kinase inhibitor, means in particular 2-cyano-N- ( 2 , 5-dibromophenyl ) -3-hydroxy-2-butenamide (LFM-A13) .

Preferably, erythropoietin and Bruton's kinase inhibitor are adapted to be administered simultaneously, separately, or sequentially .

Preferably, erythropoietin is adapted to be administered subcutaneously or intravenously, preferably subcutaneously, and Bruton's kinase inhibitor is adapted to be administered intraperitoneally .

Preferably, the kit includes a therapeutically effective amount of erythropoietin, in particular erythropoietin beta, to be administered 3 times a week, and a therapeutically effective amount of Bruton's kinase inhibitor, in particular 2-cyano-N- (2, 5-dibromophenyl) -3-hydroxy-2-butenamide (LFM- A13), to be administered 2 times a day.

Preferably, the therapeutically effective amount of erythropoietin beta to be administered 3 times a week is preferable 600 IU/kg per application, and the therapeutically effective amount of 2-cyano-N- ( 2 , 5-dibromophenyl ) -3-hydroxy- 2-butenamide (LFM-A13) to be administered 2 times a day is 10 mg/kg per application.

Preferably, the kit also includes at least one compound selected from the group comprising cytostatic drugs, preferably fluorouracil , fluoropyrimidine, and/or kinase inhibitors, preferably ibrutinib, sorafenib, AZD0530, GDC0834, CGI-560, CGI-1746.

The subject matter of the invention is also erythropoietin and Bruton's kinase inhibitor for use as a drug .

Preferably, erythropoietin is selected from the group comprising erythropoietin alfa, erythropoietin beta and darbepoetin alfa, in particular erythropoietin beta and/or Bruton's kinase inhibitor, meaning in particular 2-cyano-N- ( 2 , 5-dibromophenyl ) -3-hydroxy-2-butenamide (LFM-A13) .

Preferably, they are used in the treatment of neoplasms.

Preferably, the neoplasm shows high activity of Bruton's tyrosine kinase.

Preferably, the neoplasm showing high activity of

Bruton's tyrosine kinase comprises colon cancer and/or other solid tumors, in particular selected from the group comprising germ cell tumors, sarcomas, carcinomas, lymphomas, bone tumors, and nerve-derived tumors.

Preferably, a neoplasm with particularly high activity of

Bruton's kinase is selected from the group comprising hormone-dependent breast cancer in women, hormone-dependent prostate cancer, and lung adenocarcinoma.

Preferably, erythropoietin and Bruton's kinase inhibitor are administered at simultaneously, separately, or sequentially .

Preferably, erythropoietin is administered subcutaneously or intravenously, preferably subcutaneously, and Bruton's kinase inhibitor is administered intraperitoneally .

Preferably, the therapeutically effective amount of erythropoietin, in particular erythropoietin beta, is administered 3 times a week, and the therapeutically effective amount of Bruton's kinase inhibitor, in particular 2-cyano-N- (2, 5-dibromophenyl) -3-hydroxy-2-butenamide (LFM- A13), is administered 2 times a day. Preferably, the therapeutically effective amount of erythropoietin beta administered 3 times a week is 600 IU/kg per application, and the therapeutically effective amount of 2-cyano-N- (2, 5-dibromophenyl ) -3-hydroxy-2-butenamide (LFM- A13) administered 2 times a day is 10 mg/kg per application.

Preferably, additionally an effective amount of at least one compound, selected from the group comprising cytostatic drugs, preferably fluorouracil , fluoropyrimidine, and/or kinase inhibitors, preferably ibrutinib, sorafenib, AZD0530, GDC0834, CGI-560, CGI-1746 is used.

The subject matter of the invention is also the use of erythropoietin beta and Bruton's kinase inhibitor, in particular 2-cyano-N- (2, 5-dibromophenyl) -3-hydroxy-2- butenamide (LFM-A13), for the manufacture of a drug for the treatment of neoplasm.

Preferably, erythropoietin is selected from the group comprising erythropoietin alfa, erythropoietin beta and darbepoetin alfa, in particular erythropoietin beta, and/or Bruton's kinase inhibitor, meaning in particular 2-cyano-N- ( 2 , 5-dibromophenyl ) -3-hydroxy-2-butenamide (LFM-A13) .

Preferably, the neoplasm shows high activity of Bruton's tyrosine kinase.

Preferably, the neoplasm showing high activity of Bruton's tyrosine kinase comprises colon cancer, and/or other solid tumors, in particular selected from the group of germ cell tumors, sarcomas, carcinomas, lymphomas, bone tumors, and nerve-derived tumors .

Preferably, the neoplasm showing particularly high activity of Bruton's tyrosine kinase is selected from the group comprising hormone-dependent breast cancer in women, hormone-dependent prostate cancer, and lung adenocarcinoma.

Preferably, erythropoietin, in particular erythropoietin beta, and Bruton's kinase inhibitor, in particular 2-cyano-N- ( 2 , 5-dibromophenyl ) -3-hydroxy-2-butenamide (LFM-A13), are administered simultaneously, separately, or sequentially. Preferably, erythropoietin, in particular erythropoietin beta, is administered subcutaneously or intravenously, preferably subcutaneously, and Bruton's kinase inhibitor, in particular 2-cyano-N- (2, 5-dibromophenyl ) -3-hydroxy-2- butenamide (LFM-A13), is administered intraperitoneally .

Preferably, the therapeutically effective amount of erythropoietin, in particular erythropoietin beta, is administered 3 times a week, and the therapeutically effective amount of Bruton's kinase inhibitor, in particular 2-cyano-N- ( 2 , 5-dibromophenyl ) -3-hydroxy-2-butenamide (LFM- A13), is administered 2 times a day.

Preferably, the therapeutically effective amount of erythropoietin beta administered 3 times a week is 600 IU/kg per application, and the therapeutically effective amount of 2-cyano-N- ( 2 , 5-dibromophenyl ) -3-hydroxy-2-butenamide (LFM- A13) administered 2 times a day is 10 mg/kg per application.

Preferably, erythropoietin, in particular erythropoietin beta, and Bruton's kinase inhibitor, in particular 2-cyano-N- ( 2 , 5-dibromophenyl ) -3-hydroxy-2-butenamide (LFM-A13), are used in combination with at least one compound selected from the group comprising cytostatic drugs, preferably fluorouracil , fluoropyrimidine, and/or kinase inhibitors, preferably ibrutinib, sorafenib, AZD0530, GDC0834, CGI-560, CGI-1746.

The subject matter of the invention is a kit that uses erythropoietin, in particular recombinant erythropoietin beta, and Bruton's kinase inhibitor, in particular LFM-A13, as well as the use of erythropoietin, in particular erythropoietin beta and Bruton's kinase inhibitor, in particular LFM-A13, in the treatment of cancer, in particular colon cancer, and using them for the manufacture of a drug for neoplasm treatment.

Erythropoietin, in particular erythropoietin beta together with Bruton's kinase inhibitor, in particular LFM-A13, can be administered simultaneously, separately, or sequentially. A solution containing erythropoietin can be administered subcutaneously or intravenously. The preferred and most often used method of administration is subcutaneous injection, because intravenous administration is performed in patients undergoing hemodialysis via the arteriovenous fistula at the end of dialysis. It must be noted that the biological half- life in the elimination phase is longer after subcutaneous than intravenous administration, and lasts on average 13-28 hours .

LFM-A13 is a poorly water-soluble compound, thus the obtained suspension cannot be administered intravenously. The available data in the literature show that intraperitoneal injection is the most often applied method of LFM-A13 administration .

Preferably, erythropoietin, in particular erythropoietin beta, is administered subcutaneously 3 times a week, and Bruton's kinase inhibitor, in particular LFM-A13, is administered intraperitoneally 2 times a day; most preferably, erythropoietin beta is administered 3 times a week at an application of 600 IU/kg, and LFM-A13 is administered intraperitoneally 2 times a day at an application of 10 mg/kg.

The kit prepared in accordance with this invention, containing erythropoietin, in particular erythropoietin beta, and Bruton's kinase, in particular LFM-A13, can be applied in combination with other kinase inhibitors, for example ones such as ibrutinib and sorafenib already used in medicine, or ones that are being tested (AZD0530, GDC0834, CGI-560 or CGI- 1746), as well as fluorouracil or fluoropyrimidine, classic cytostatic drugs recommended in the treatment of colon cancer. Such combinations result in higher therapy efficacy (a hyper-additive effect) , with relatively weaker adverse effects .

The subject kit, including erythropoietin, in particular erythropoietin beta, and Bruton's kinase inhibitor, in particular LFM-A13, is useful in the therapy of colon cancer and other solid tumors.

The term "solid tumors" refers to malignant neoplastic lesions with a solid histological structure and focal proliferation. These include among others: germ cell tumors, sarcomas, carcinomas, lymphomas, bone tumors and nerve- derived tumors. In particular, the subject invention is effective in the case of neoplasms showing particularly high Bruton's tyrosine kinase activity, such as hormone-dependent breast cancer in women, hormone-dependent prostate cancer, and lung adenocarcinoma.

The revealed application of erythropoietin, in particular erythropoietin beta, with Bruton's kinase inhibitor, in particular LFM-A13, does not only significantly lower the speed of neoplastic cell growth, but it also unexpectedly leads to complete regression of low-stage neoplastic lesions.

Differences in tumor response to treatment significantly depend on the phase of the cell-division cycle of the neoplastic cells. It has been demonstrated that in solid tumors the number of neoplastic cells in the GO phase (resting phase) is much higher than in normal cells (Gasi ska A., Biesaga A. Nowe spojrzenie na proliferacje nowotworow. A new look at tumor proliferation. NOWOTWORY Journal of Oncology. 2010; 228-235) . The number even reaches 98% in the 769-P line, i.e. renal adenocarcinoma. Cancer cells in the quiescent phase (GO) are very often immune to cytostatic drugs. The Inventors have noticed that introducing them into subsequent cell cycle phases (S, M) may result in higher treatment responsivity . In studies using human renal adenocarcinoma cells (769-P, 768-0) and basal cell carcinoma cells (Caki-1) it was proved that erythropoietin, which is a growth factor, causes neoplastic cells to go from the GO phase to the cell division cycle, through Gl phase up to S (Miyake M, Goodison S, Lawton A, Zhang G, Gomes-Giacoia E, Rosser CJ. Erythropoietin is a JAK2 and ERK1/2 effector that can promote renal tumor cell proliferation under hypoxic conditions. J Hematol Oncol. 2013 Sep 3; 6: 65) .

It was unexpectedly noticed that intracellular pathways, partially activated after the administration of erythropoietin, were then inhibited as a result of administering LFM-A13, which in turn lead to limiting the proliferation of neoplastic cells. Combining a growth factor - such as erythropoietin beta - with LFM-A13 effectively inhibits the growth of tumor mass in mice with induced neoplasm lines DLD-1 and Ht-29. In the case of small tumors, combination therapy unexpectedly results in complete recession of hyperplastic lesions.

The inventors have noticed that erythropoietin significantly intensifies the anti-neoplastic activity of LFM-A13, probably by way of phosphorylation of intracellular pathways, which are the target of LFM-A13 activity only in the active form.

This combination is also effective in in vitro studies, in which the combined administration of erythropoietin beta with LFM-A13 results in the inhibition of proliferation, reducing the number of cells, reducing adhesion and impairment of cell divisions of both studied cell lines. The results of the studies clearly show that the subject invention, including in particular the presented application of the subject kit, displays high efficacy against colon cancer cells.

Apart from the above-mentioned cytostatic effect of the combination therapy, the subject invention reduces the anemia developing in tested animals, thus leading to an improvement in hematological parameters.

Another very important advantage of the subject invention is a reduction in adverse effects observed during classic therapy. Although LFM-A-13 monotherapy can cause hematological complications, including bleeding, simultaneous administration of erythropoietin considerably reduces this adverse effect.

Thus, the subject solution does not only have high anti^¬ neoplastic activity, but also significantly helps improve patient quality of life.

The implementation of the presented therapy based on combining the effects caused by erythropoietin and LFM-A13 creates a real chance of eradicating neoplastic cells, including in particular colon cancer cells, with relatively weak adverse effects.

Short description of figures:

Fig.l. Presents the effect of erythropoietin beta (Epo 1, 10, 100 IU) (A) , LFM-A13 (1, 10, 100 uM) ( B ) and their combined administration (C) on DLD-1 cell proliferation after 72 hours of incubation. The results are presented as mean values ± SD, n = 10-12.

Fig. 2. Presents the effect of erythropoietin beta (Epo 1, 10, 100 IU) (A) , LFM-A13 (1, 10, 100 uM) ( B ) and their combined administration (C) on Ht-29 cell proliferation after 72 hours of incubation. The results are presented as mean values ± SD, n = 10-12.

Fig. 3. Presents the effect of selected concentrations of erythropoietin beta (Epo 1, 10, 100 IU) , LFM-A13 (1, 10, 100 uM) and their combined administration on the number of DLD-1 cells after 24 (A) , 48 ( B ) , and 72 ( C ) hours of incubation. The results are presented as mean values ± SD, n = 10-12.

Fig. 4. Presents the effect of selected concentrations of erythropoietin beta (Epo 1, 10, 100 IU) , LFM-A13 (1, 10, 100 uM) and their combined administration on the number of Ht-29 cells after 24 (A) , 48 ( B ) , and 72 ( C ) hours of incubation. The results are presented as mean values ± SD, n = 10-12.

Fig. 5. Presents the effect of selected concentrations of erythropoietin beta (Epo 1, 10, 100 IU) , LFM-A13 (1, 10, 100 uM) and their combined administration on DLD-1 cell vitality after 24 (A) , 48 (B) , and 72 (C) hours of incubation. The results are presented as mean values ± SD, n = 10-12.

Fig. 6. Presents the effect of selected concentrations of erythropoietin beta (Epo 1, 10, 100 IU) , LFM-A13 (1, 10, 100 uM) and their combined administration on Ht-29 cell vitality after 24 (A) , 48 (B) , and 72 (C) hours of incubation. The results are presented as mean values ± SD, n = 10-12.

Fig. 7. Presents the effect of selected concentrations of erythropoietin beta (Epo 1, 10, 100 IU) , LFM-A13 (1, 10, 100 uM) and their combined administration on DLD-1 cell morphology after 24 hours of incubation.

Fig. 8. Presents the effect of selected concentrations of erythropoietin beta (Epo 1, 10, 100 IU) , LFM-A13 (1, 10, 100 uM) and their combined administration on Ht-29 cell morphology after 24 hours of incubation.

Fig. 9. Presents the effect of selected concentrations of erythropoietin beta (Epo 1, 10, 100 IU) , LFM-A13 (1, 10, 100 uM) and their combined administration on DLD-1 cell morphology after 48 hours of incubation.

Fig. 10. Presents the effect of selected concentrations of erythropoietin beta (Epo 1, 10, 100 IU) , LFM-A13 (1, 10, 100 uM) and their combined administration on Ht-29 cell morphology after 48 hours of incubation.

Fig. 11. Presents the effect of selected concentrations of erythropoietin beta (Epo 1, 10, 100 IU) , LFM-A13 (1, 10, 100 uM) and their combined administration on DLD-1 cell morphology after 72 hours of incubation.

Fig. 12. Presents the effect of selected concentrations of erythropoietin beta (Epo 1, 10, 100 IU) , LFM-A13 (1, 10, 100 uM) and their combined administration on Ht-29 cell morphology after 72 hours of incubation.

Fig. 13. Presents the colon tumor growth rate in Fox mice induced with DLD-1 and Ht-29 lines. 0 - beginning of observation, when the tumor is approx. 5x5 mm, 1 - after the first week, 2 - after the second week, 3 - after the third week, 4 - after the fourth week. The results are presented as mean values ± SD, n = 10,

Tumor volume (V) was calculated according to the method developed by Feldman et al . (Feldman JP, Goldwasser R, Mark S, Schwartz J, Orion I. A Mathematical Model For Tumor Volume Evaluation Using Two-Dimensions. JAQM 2009, 4, 455-462) using the following formula:

π 3

V= — f (length x width) 2

6

Fig. 14. Presents the effect of the combined activity of LFM-A13 (10 mg/kg b.m.) and erythropoietin beta (Epo, 600 IU/kg b.m.) on tumor volume in mice with induced colon cancer with DLD-1 (A) and Ht-29 (B) lines. 0 - before substance administration, 1 - after first week of substance administration, 2 - after second week of substance administration. The results are presented as mean values ± SD, n = 10.

Fig. 15. Presents the effect of LFM-A13 (10 mg/kg b.m.), erythropoietin beta (Epo, 600 IU/kg b.m.) and their combined administration on tumor volume in mice with induced colon cancer with DLD-1 (A) and Ht-29 (B) lines. 0 - before substance administration, 1 - after first week of substance administration, 2 - after second week of substance administration. The results are presented as mean values ± SD, n = 4-5.

Fig. 16. Presents the effect of LFM-A13 (10 mg/kg b.m.), erythropoietin beta (Epo, 600 IU/kg b.m.) and their combined administration on white blood cell count in Fox mice with induced colon cancer with DLD-1 (A) and Ht-29 (B) lines, after two weeks of substance administration. The results are presented as mean values ± SD, n = 10-12.

Fig. 17. Presents the effect of LFM-A13 (10 mg/kg b.m.), erythropoietin beta (Epo, 600 IU/kg b.m.) and their combined administration on erythrocytes in Fox mice with induced colon cancer with DLD-1 (A) and Ht-29 (B) lines, after two weeks of substance administration. The results are presented as mean values ± SD, n = 10-12.

Fig. 18. Presents the effect of LFM-A13 (10 mg/kg b.m.), erythropoietin beta (Epo, 600 IU/kg b.m.) and their combined administration on hemoglobin concentration in Fox mice with induced colon cancer with DLD-1 (A) and Ht-29 (B) lines, after two weeks of substance administration. The results are presented as mean values ± SD, n = 10-12.

Fig. 19. Presents the effect of LFM-Al 3 (10 mg/kg b.m.), erythropoietin beta (Epo, 600 IU/kg b.m.) and their combined administration on hematocrit value in Fox mice with induced colon cancer with DLD-1 (A) and Ht-29 (B) lines, after two weeks of substance administration. The results are presented as mean values ± SD, n = 10-12.

Fig. 20. Presents the effect of LFM-Al 3 (10 mg/kg b.m.), erythropoietin beta (Epo, 600 IU/kg b.m.) and their combined administration on red blood cell volume in Fox mice with induced colon cancer with DLD-1 (A) and Ht-29 (B) lines, after two weeks of substance administration. The results are presented as mean values ± SD, n = 10-12.

Fig. 21. Presents the effect of LFM-Al 3 (10 mg/kg b.m.), erythropoietin beta (Epo, 600 IU/kg b.m.) and their combined administration on mean hemoglobin mass per erythrocyte in Fox mice with induced colon cancer with DLD-1 (A) and Ht-29 (B) lines, after two weeks of substance administration. The results are presented as mean values ± SD, n = 10-12.

Fig. 22. Presents the effect of LFM-Al 3 (10 mg/kg b.m.), erythropoietin beta (Epo, 600 IU/kg b.m.) and their combined administration on mean corpuscular hemoglobin concentration in Fox mice with induced colon cancer with DLD-1 (A) and Ht- 29 (B) lines, after two weeks of substance administration. The results are presented as mean values ± SD, n = 10-12.

Fig. 23. Presents the effect of LFM-Al 3 (10 mg/kg b.m.), erythropoietin beta (Epo, 600 IU/kg b.m.) and their combined administration on platelet count in Fox mice with induced colon cancer with DLD-1 (A) and Ht-29 (B) lines, after two weeks of substance administration. The results are presented as mean values ± SD, n = 10-12.

Fig. 24. Presents the effect of LFM-A13 (10 mg/kg b.m.), erythropoietin beta (Epo, 600 IU/kg b.m.) and their combined administration on the mass of mice with induced colon cancer with DLD-1 (A) and Ht-29 (B) lines. The results are presented as mean values ± SD, n = 10-12. Below we present examples of implementation of the subject invention. The invention is not limited to these embodiments.

Embodiments

The compounds are commercially available. Erythropoietin beta (referred to as Epo in the examples) was purchased from the company Roche as NeoRecormon preparation, and LFM-A13 was purchased from the company Tocris.

The excipients used in the industrial production of the commercial preparation under the name NeoRecormon are necessary in the process of manufacturing the drug's form and ensure its stability during storage. The excipients present in NeoRecormon are:

Urea - its role is to increase the absorption/release of the medicinal substance, enabling obtaining better treatment results while lowering the drug dosage introduced into the body .

Sodium chloride, Sodium dihydrogen phosphate dihydrate, Disodium phosphate dodecahydrate - these are osmotically active and buffering agents that ensure maintaining stable storage conditions determined by the manufacturer for the preparation in the ready-to-use form.

Polysorbate 20 - it is a non-ionic hydrophilic excipient whose role is to enable introducing a poorly or very poorly water-soluble substance to a water solution by means of making an "oil-in-water " emulsion. Calcium chloride dihydrate - it is an acidity regulator and stabilizer, additionally serving as protection against excessive hydration of the active substance.

Glycine, L-leucine, L-isoleucine, L-threonine, L-glutamic acid, L-phenylalanine - these are amino acids necessary to maintain the right isoelectric properties of the polypeptide erythropoietin, and they serve as a buffer for the preparation solution.

Water for injections - it is necessary to dissolve the active substance and excipients and bring them into the form of the solution.

Benzyl alcohol - it serves as a preservative, necessary to keep the solution introduced into the living organism sterile and apyrogenic.

A. In vitro studies.

The first stage of research involved in vitro experiments using two lines of colon cancer, DLD-1 and Ht-29 (the DLD-1 and Ht-29 lines used in the research were obtained from the American Type Culture Collection and were continuous lines, but histopathologically they corresponded to colon cancer cells isolated from cancer patients) . Both lines are considered adhesive and quick-growing. Line DLD-1 contains the EpoR gene and protein, and histologically is the most similar to a primary tumor; while, line HT-29 is a negative control of the EpoR gene. Cell line DLD-1 was isolated from the epithelial tissue of colon cancer adenocarcinoma (stage C according to Dukes classification) of an adult man. It exhibits the presence of oncogenes myc, myb, ras, fos, sis, and p53; and the absence of oncogenes abl, ros, and src. Line Ht-29 was isolated from colon adenocarcinoma of a 44-year-old woman. In in vitro cultures it exhibits a morphology typical of epithelial cells, but it does not differentiate into forms making a brush border. A huge part of the Ht-29 cell population is goblet cells, so this line produces great amounts of intestinal mucus. Ht-29 line cells exhibit the presence of oncogenes myc, myb, ras, myb, fos, sis, and p53; and the absence of oncogenes abl, ros, and src.

Lines DLD-1 and Ht-29 were purchased from the American Type Culture Collection (ATCC) . Line DLD-1 was cultured in RPMI 1640 (ATCC) medium; and line Ht-29 in McCoy's 5A (ATCC) medium, including 10% FBS (ATCC) , 50 g/ml penicillin, and 50 g/ml streptomycin (ATCC) . The cells were cultured on "Falcon" plates (Becton Dickinson, USA) with a diameter of 100 mm in an incubator (Heraeus), in an atmosphere of 5% C02, with 95% humidity, and a temperature of 37 °C. The culture medium was replaced every 2-3 days. When the cells reached 70-80% confluence, they were rinsed with PBS, trypsinized with a phosphate buffer containing 0.25% trypsin, and moved to new plates. Cells between the 8th and 14th passage were used in the tests.

Example 1. The effect of erythropoietin beta on the proliferation of colon cancer cell lines DLD-1 and HT-29. The assessment was performed using the MTT method according to Mosmann (Plumb J. A., Milroy R., Kaye S. B.: Effects of the pH dependence of 3- ( 4 , 5-dimethylthiazol-2-yl ) - 2 , 5-diphenyl-tetrazolium bromide - formazan absorption on chemosensitivity determined by a novel tetrazolium-based assay. Cancer Res., 1989, 49: 4435-4440) involving the measurement of oxydo-reduction activity of mitochondria of live cells through the reduction of yellow water-soluble 3- (4, 5-dimethylthiazol-2-yl ) -2, 5-diphenyltetrazolium bromide (MTT) to purple insoluble formazan. The test was performed after 72 hours of adding erythropoietin to the culture medium. DLD-1 and Ht-29 line cells were placed on 24-well plates (1 X 100, 000/well) and incubated for 48 hours until complete confluence was achieved. The culture medium was replaced with a new one, including Epo at a concentration of 0 (control), 1, 10, and 100 IU/ml . After 72 hours of incubation with Epo, the medium was removed and the cells were rinsed with PBS. Then, 1 ml of MTT solution in PBS (5 mg/ml) was added to each group of cells, and incubated at 37 °C for 30 minutes. After thoroughly removing the MTT solution, 1 ml DMSO was added to each group of cells to induce lysis and dissolve formazan crystals, and 10 μΐ of Sorensen's phosphate buffer (0.1 mol/1 glycine, 0.1 mol/1 NaCl brought to pH 10.5 with a solution of 0.1 N NaOH) . Absorbance was measured in comparison with DMSO as the zero sample at a wavelength λ=570 nm, using the UVM 340 microplate reader (Asys, Germany) with MikroWin 2000 software. Results are presented as a percentage of the control sample value (control = 100%) . 72-hour incubation of DLD-1 neoplastic cells with Epo at a concentration of 100 IU/ml resulted in statistically significant inhibition of proliferation compared with cells incubated with Epo 1 IU/ml (p<0.05) (Fig. 1A) . 72-hour incubation of Ht-29 neoplastic cells with Epo at concentrations of 10 IU/ml and 100 IU/ml resulted in statistically significant inhibition of proliferation compared with the control (p<0.05 and p<0.001, respectively) . Incubation of cells with Epo 10 IU/ml resulted in statistically significant inhibition of proliferation compared with the proliferation of cells subject to the activity of Epo 1 IU/ml (p<0.01) . Statistically significant inhibition of proliferation of cells incubated with Epo 100 IU/ml was observed compared with cells subjected to the activity of Epo at concentrations of 10 IU/ml (p<0.001) and 1 IU/ml (p<0.001) (Fig. 2A) .

Example 2. The effect of LFM-A13 on the proliferation of colon cancer cell lines DLD-1 and HT-29. DLD-1 and Ht-29 cell lines were incubated with LFM-A13 at concentrations of 0 (control), 1, 10, and 100 μΜ for 72 hours. No statistically significant effect of LFM-A13 on the proliferation of DLD-1 cells was observed (Fig. IB) . However, the proliferation of Ht-29 cells after incubation with LFM- A13 at concentrations of 1 μΜ (p<0.05), 10 μΜ (p<0.001), and 100 μΜ (p<0.001) was statistically significantly inhibited compared with the control. The proliferation of cells incubated with LFM-A13 at a concentration of 100 μΜ was statistically significantly inhibited compared with cells subjected to the activity of LFM-A13 at concentrations of 1 IU/ml (p<0.001) and 10 IU/ml (p<0.001) (Fig. 2B) .

Example 3. The effect of the combined administration of erythropoietin beta and LFM-A13 on the proliferation of colon cancer cell lines DLD-1 and HT-29.

After 72-hour incubation of DLD-1 cells with Epo and LFM- A13 administered simultaneously, the proliferation of cells was statistically significantly inhibited compared with control (p<0.01) and LFM-A13 (p<0.001) . This test demonstrated the potentializing effect of combined administration of Epo and LFM-A13 compared with each of the compounds separately (Fig. 1C) . Similar results were obtained for the Ht-29 line: incubation of Ht-29 cells with Epo and LFM-A13 simultaneously caused statistically significant inhibition of cell proliferation compared with control (p<0.001), Epo (p<0.001), and LFM-A13 (p<0.001) . These experiments clearly show that combined administration of Epo and LFM-A13 is more effective in inhibiting cell proliferation of both studied lines of colon cancer cells compared with using these compounds separately (Fig. 2C) .

Example 4. The effect of selected concentrations of ythropoietin beta, LFM-A13, and their combined administration on the number of DLD-1 and Ht-29 cells after 24-hour incubation.

The number of cells was evaluated using a NucleoCounter® apparatus from ChemoMetec. It detects and intercepts signals emitted by propidium iodide (PI) penetrating the DNA of the nucleus. Administering Epo at concentrations of 10 and 100 IU caused a statistically significant increase in DLD-1 cells compared with the control (p<0.001, p<0.001, respectively) . LFM-A13 at a concentration of 100 μΜ reduced the number of cells compared with the control (p<0.05) . During the experiment it was demonstrated that the combined administration of erythropoietin beta and LFM-A13 (Epo 100 + LFM-A13 30; Epo 100 + LFM-A13 100) statistically significantly reduced the number of cells compared with control (p<0.05, p<0.05, respectively) and cells subject to the activity of Epo 100 alone (p<0.001, p<0.001, respectively) . Statistically significant reduction of the number of cells was also demonstrated after incubation with Epo 100 + LFM-A13 30 compared with the effect of LFM-A13 30 alone (p<0.01) (Fig. 3A) .

After 24-hour incubation of Ht-29 cells with Epo at a concentration of 100 IU, a reduction in the number of cells compared with control (p<0.001) was unexpectedly observed. A similar effect was observed after the administration of LFM- A13 at concentrations of 30 and 100 μΜ compared with control (p<0.01, p<0.01, respectively) . Similarly as in the case of the DLD-1 line, it was demonstrated that the combined administration of erythropoietin beta and LFM-A13 (Epo 100 + LFM-A13 30; Epo 100 + LFM-A13 100) statistically significantly reduced the number of Ht-29 cells compared with control (p<0.001, p<0.001, respectively) and cells subject to the activity of Epo 100 alone (p<0.01, p<0.001, respectively) . A significant reduction in the number of cells incubated with Epo 100 + LFM-A13 30 and Epo 100 + LFM-A13 100 was also observed compared with cells subject to the activity of LFM-A13 alone at concentrations of 30 and 100 μΜ (p<0.001; p<0.001, respectively) (Fig. 4A) .

Example 5. The effect of selected concentrations of erythropoietin beta, LFM-A13, and their combined administration on the number of DLD-1 and Ht-29 cells after 48-hour incubation.

Administering Epo at a concentration of 10 IU caused a statistically significant increase in DLD-1 cells compared with the control (p<0.001) . LFM-A13 at a concentration of 100 μΜ reduced the number of cells compared with the control (p<0.05) . The test results indicate that the combined administration of erythropoietin beta and LFM-A13 (Epo 100 + LFM-A13 30; Epo 100 + LFM-A13 100) statistically significantly reduced the number of cells compared with control (p<0.01, p<0.001, respectively) and cells subject to the activity of Epo 100 alone (p<0.001, p<0.001, respectively) . Statistically significant reduction of the number of cells was also demonstrated after incubation with Epo 100 + LFM-A13 30 compared with the effect of LFM-A13 100 alone (p<0.001) (Fig. 3B) .

After 48-hour incubation of Ht-29 cells with Epo at concentrations of 10 and 100 IU, LFM-A13 30 and 100 uM, as well as after their combined administration (Epo 10 + LFM-A13 30; Epo 10 + LFM-A13 100; Epo 100 + LFM-A13 30; Epo 100 + LFM-A13 100), a statistically significant reduction in the number of cells was observed compared with control (p<0.001, p<0.001, p<0.001, p<0.001, p<0.001, p<0.001, p<0.001, p<0.001, respectively) (Fig. 4B) . Moreover, a significant reduction in the number of cells incubated with Epo 10 + LFM- A13 100 was observed as compared with cells subject to the activity of LFM-A13 100 alone (p<0.01) . Example 6. The effect of selected concentrations of erythropoietin beta, LFM-A13, and their combined administration on the number of DLD-1 and Ht-29 cells after 72-hour incubation.

After 72-hour incubation of DLD-1 cells with Epo at concentrations of 10 and 100 IU, LFM-A13 30 and 100 uM, as well as after their combined administration (Epo 10 + LFM-A13 30; Epo 10 + LFM-A13 100; Epo 100 + LFM-A13 30; Epo 100 + LFM-A13 100), a statistically significant reduction in the number of cells was observed compared with control (p<0.001, p<0.001, p<0.001, p<0.001, p<0.001, p<0.001, p<0.001, p<0.001, respectively) . A significant reduction in the number of cells incubated with Epo 100 + LFM-A13 30 and Epo 100 + LFM-A13 100 was also observed compared with cells subject to the activity of LFM-A13 alone at concentrations of 30 and 100 μΜ (p<0.001; p<0.001, respectively) (Fig. 3C) . A similar effect was achieved after 72-hour incubation of

Ht-29 cells with Epo at concentrations of 10 and 100 IU, LFM- A13 100 μΜ, as well as after their combined administration (Epo 10 + LFM-A13 30; Epo 10 + LFM-A13 100; Epo 100 + LFM-A13 30; Epo 100 + LFM-A13 100) compared with control (p<0.001, p<0.001, p<0.001, p<0.001, p<0.001, p<0.001, p<0.001, respectively) . Combined administration of erythropoietin beta and LFM-A13 (Epo 100 + LFM-A13 30; Epo 100 + LFM-A13 100) statistically significantly reduced the number of cells compared with Epo 100 alone (p<0.05, p<0.01, respectively) . A similar effect was observed after the administration of Epo 100 + LFM-A13 30 compared with cells incubated with LFM-A13 30 μΜ (p<0.001) alone, and Epo 100 + LFM-A13 100 compared with cells subject to the activity of LFM-A13 100 μΜ alone (p<0.05) (Fig. 4C) . Example 7. The effect of selected concentrations of erythropoietin beta, LFM-A13, and their combined administration on the viability of DLD-1 and Ht-29 cells after 24-hour incubation.

After 24-hour incubation of DLD-1 (Fig. 5A) and Ht-29 (Fig. 6A) cells, no changes in cell viability were observed after the administration of the tested compounds. Example 8. The effect of selected concentrations of erythropoietin beta, LFM-A13, and their combined administration on the viability of DLD-1 and Ht-29 cells after 48-hour incubation. After 48-hour incubation of DLD-1 (Fig. 5B) and Ht-29

(Fig. 6B) cells, no changes in cell viability were observed after the administration of the tested compounds.

Example 9. The effect of selected concentrations of erythropoietin beta, LFM-A13, and their combined administration on the viability of DLD-1 and Ht-29 cells after 72-hour incubation.

After 72-hour incubation of DLD-1 cells, a reduction in the viability of cells subject to the activity of Epo 100, as well as Epo 100 + LFM-A13 100, was observed compared with control (p<0.05, p<0.01, respectively) . A significant reduction in the viability of cells incubated with Epo 100 + LFM-A13 100 was observed compared with cells subject to the activity of LFM-A13 100 alone (p<0.001) (Fig. 5C) .

In the Ht-29 line, 72-hour incubation with LFM-A13 100, Epo 10 + LFM-A13 100, Epo 100 + LFM-A13 30, Epo 100 + LFM-A13 100 caused a clear reduction in cell viability compared with control (p<0.001, p<0.001, p<0.001, p<0.001, respectively) . The test results indicate that the combined administration of erythropoietin beta and LFM-A13 (Epo 10 + LFM-A13 100) statistically significantly reduced cell viability compared with cells subject to the activity of Epo 10 alone (p<0.001) . A similar reduction in viability was observed after incubation of Ht-29 cells with Epo 100 + LFM-A13 30 and Epo 100 + LFM-A13 100 compared with cells subject to the activity of Epo 100 alone. Moreover, LFM-A13 at a concentration of 100 μΜ administered together with Epo 10 (Epo 10 + LFM-A13 100) and Epo 100 (Epo 100 + LFM-A13 100) reduced viability compared with cells incubated with LFM-A13 100 alone (p<0.001, p<0.001, respectively) (Fig. 6C) .

Example 10. Morphological observations of DLD-1 and Ht-29 cells . In order to assess the impact of erythropoietin beta, LFM-

A13, and both these compounds used together on morphological changes in DLD-1 and Ht-29, the cells were distributed on 24- well mesh plates. After 48-hour incubation, the medium was replaced with a new one, containing selected concentrations of the tested compounds. After 24, 48, and 72 hours of incubation, an image of the cells was recorded at a magnification of 1x200 using a Nikon Eclipse TS 100 phase- contrast microscope (Nikon, Japan) with a Nikon DS-Filc camera and NIS-Elements F software. The experiment was performed on confluent DLD-1 and Ht-29 cells incubated on a medium with 10% serum and different concentrations of the tested compounds for 24, 48, and 72 hours. Under the influence of erythropoietin beta and LFM-A13, the density of cell lines DLD-1 and Ht-29 in the culture as well as their adhesion decreased, and cell division was impaired. These changes were most observable in cells incubated with the tested compounds for 72 hours. The cell images are presented on Fig. 7 and 8 (24-hour incubation of lines DLD-1 and Ht-29, respectively), Fig. 9 and 10 (48-hour incubation of lines DLD-1 and Ht-29, respectively), and Fig. 11 and 12 (72-hour incubation of lines DLD-1 and Ht-29, respectively) .

B. In vivo studies.

The second stage of research was conducted using animals (Cby . Cg-Foxnlnu/ J female mice) (Jackson Laboratory, USA). This strain has defective development of the thymic epithelium (athymic) and a hair follicle defect (homozygous females) and is commonly used for studies of substances modulating tumor growth. All procedures were performed in accordance with the guidelines for animal experiments and the protocol approved by the Local Ethics Committee (resolution no. 31/2013) . The research, conducted in in vivo conditions, was carried out at the Center for Experimental Medicine of the Medical University of Bialystok. It is a modern unit that ensures a high standard of culture with SPF status of the breeding barrier. The animals are kept in overpressure conditions, with optimal temperature of 22°C, 55% air humidity, 15 exchanges per hour (speed does not exceed 0.3 m/s), and a 12/12 light day cycle. The rooms in which the animals are kept are monitored 24 hours a day. And both the animals (in accordance with FELASA recommendations) and the rooms in which they are kept are subject to inspections. The Center for Experimental Medicine has a Certificate of Compliance with the Principles of Good Laboratory Practice (GLP) .

For the preliminary studies, the mice were divided into two groups. The mice in the first group were injected subcutaneously on the dorsal side with 50 μΐ of suspension containing one hundred million DLD-1 cells in PBS; while the second group of mice with 50 μΐ of suspension containing one hundred million Ht-29 cells in PBS according to the method described by Shinohara et al . Line Ht-29 was a negative control of EpoR, while line DLD-1 was a positive control of EpoR.

After the first week, when the tumor reached a diameter of approx. 5 mm, i.e. according to the literature data that is a suitable size to conduct subsequent stages of research, erythropoietin administration was began at an application of 600 IU/kg, i.e. the therapeutic application used in humans three times a week. The moment of administering erythropoietin beta and LFM-A13 was designated as time 0. The animals received LFM-A13 intraperitoneally two times a day at an application of 10 mg/kg. The duration of compound administration was 2 weeks. The control group consisted of animals receiving 50 μΐ of PBS subcutaneously and 50 μΐ on 10% ethanol solution in PBS intraperitoneally. Before compound administration (time 0) as well as after the first (time 1) and second week (time 2) of substance administration, tumor size was assessed three-dimensionally using a digital caliper. Tumor volume was expressed in mm3 in accordance with the measurements described by Feldman et al . [Feldman JP, Goldwasser R, Mark S, Schwartz J, Orion I. A Mathematical Model For Tumor Volume Evaluation Using Two- Dimensions. JAQM 2009, 4, 455-462].

At the beginning of the in vivo experiment, the growth rate of tumor development in mice injected with DLD-1 and Ht- 29 lines was tested. Beginning with the second week of observation, much greater tumor growth was observed in the Ht-29 group. This difference remained for the duration of the experiment, i.e. 4 weeks (Fig. 13) .

In the group of animals with colon cancer induced with

DLD-1 line, tumor development was statistically significantly inhibited in animals receiving LFM-A13 + Epo both after the first and second week of the experiment compared with control (Fig. 14A) . Similarly, in animals with colon cancer induced with Ht-29 lines, tumor volume was statistically significantly reduced in the group of animals receiving both compounds simultaneously (Fig. 14B) .

The positive results motivated the researchers to repeat and develop consequent stages of research in the animal model described above. Tumor was again induced with lines DLD-1 and Ht-29, and then the animals were randomized into 4 groups within each line. This way, the following groups were obtained: control (con), LFM-A13, Epo, LFM-A13 + Epo . The animals received the tested compounds following the previous pattern. The tumor was measured before the beginning of administering the compounds and after the first and second week of the experiment.

In the animals injected with line DLD-1 in the first and second week, the development of tumor was significantly inhibited in the group receiving LFM-A13 + Epo compared with control (Fig. 15A) . In the animals injected with line Ht-29, inhibition of tumor development was only observed in the group receiving LFM-A13 alone in the second week of administration compared with control (Fig. 15B) .

Analysis of hematological parameters after two weeks of therapy showed a statistically significant increase in RBC in animals with Ht-29 receiving Epo, as well as LFM-A13 + Epo, compared with control; and an increase in RBC in the group receiving LFM-A13 + Epo compared with the group injected with LFM-A13 alone (Fig. 17B) . A significant increase of HGB and HCT was also observed in mice with tumor induced with the Ht- 29 line compared with control (p<0.05 and p<0.05, respectively) , and compared with the group receiving LFM-A13 alone (p<0.01, p<0.001, respectively) . It must be emphasized that these parameters (HGB, HCT) also improved in animals receiving Epo alone compared with control (p<0.01, p<0.001, respectively) (Fig. 18B, 19B) . In the case of mice injected with the DLD-1 line, no changes in HGB or HCT were observed, which was connected with lack of anemia development in all the studied groups (Fig. 18A, 19A) . In animals with neoplasm induced with DLD-1 and Ht-29 lines, MCV increased after treatment with Epo compared with control (p<0.001, p<0.01, respectively) . This parameter also increased in groups of animals receiving LFM-A13 + Epo compared with mice injected with LFM-A13 alone (p<0.001 in the DLD-1 group, p<0.01 in the Ht-29 group) and control animals with induced neoplasm with line Ht-29 (p<0.001) (Fig. 20A, 20B) . In DLD-1 mice, adding LFM-A13 to Epo caused a reduction in MCV compared with the group receiving Epo alone (p<0.05) (Fig. 20A) . MCHC was significantly reduced in the group of Ht-29 animals receiving LFM-A13 + Epo compared with control (p<0.01) and LFM-A13 (p<0.05) . Use of Epo alone also lowered MCHC (p<0.001) (Fig. 22B) . This parameter did not change in mice injected with the DLD-1 line (Fig. 22A) . No changes in the parameters of WBC (Fig. 16A, 16B), MCH (Fig. 21A, 21B), or PLT (Fig. 23A, 23B) were observed in mice with induced tumor with DLD-1 and Ht-29 lines .

Apart from hematological parameters, the animals' body weight was also monitored. In mice with neoplastic tissue of Ht-29 cells, a growth of body weight was only observed in the group that received Epo alone for 2 weeks (p<0.05) (Fig. 24B) . While in animals with DLD-1 neoplasm, no significant changes in body weight were observed in any of the analyzed groups (Fig. 24A) .

Statistical analysis

The obtained results were statistically analyzed. The

Shapiro-Wilk test was used to assess characteristics consistent with normal distribution; Student's t-test was used for comparisons between two groups; and the Mann-Whitney test was used for features inconsistent with the distribution. For comparisons of more than two groups, analysis of variance with Bonferroni post hoc test or the Kruskal-Wallis test were used. Student's t-test for pairs and Wilcoxon signed-rank test were used to analyze measurements in groups at time intervals. A level of p < 0.05 was considered statistically significant in the calculations. The calculations were done using the SPSS statistical package.

Claims

Claims

1. A pharmaceutical kit characterized in that it includes erythropoietin and Bruton' s kinase inhibitor as well as in the same package or in a separate package, and/or instruction for use ,

2. The kit according to claim 1, characterized in that erythropoietin is selected from the group comprising erythropoietin alfa, erythropoietin beta and darbepoetin alfa, in particular erythropoietin beta and/or Bruton' s kinase inhibitor means in particular 2-cyano-N- ( 2 , 5-dib.rom.oph.eriy.l ) -3- hydroxy-2-butenamide (LFM-A13) .

3. The kit according to one of the preceding claims, characterized in that erythropoietin and Bruton' s kinase inhibitor are adapted to be administered simultaneously, separately, or sequentially.

4. The kit according to one of the preceding claims, characterized in that erythropoietin is adapted to be administered subcutaneously or intravenously, preferably subcutaneously, and Bruton' s kinase inhibitor is adapted to be administered intraperitoneally .

5. The kit according to one of the preceding claims, characterized in that it includes a therapeutically effective amount of erythropoietin, in particular erythropoietin beta, to be administered 3 times a week, and a therapeutically effective amount of Bruton' s kinase inhibitor, in particular 2-cyano-N- (2, 5-dibromophenyl ) -3-hydroxy-2-butenamide (LFM- A13), to be adm.inist.ered 2 tim.es a day.

6. The kit according to one of the preceding claims, characterized in that the therapeutically effective amount of erythropoietin beta to be administered 3 times a week is preferably 600 lU/kg per application, and the therapeutically effective amount of 2-cyano-N- ( 2 , 5-dibromophen.yl ) -3-hydroxy-2- butenamide (LFM-A13) to be administered 2 times a day is 10 mg/kg per application.

7. The kit according to one of the preceding claims, characterized in that it additionally includes at least one compound selected from the group comprising cytostatic drugs, preferably fluorouracil , fluoropyrimidine, and/or kinase inhibitors, preferably ibrutinib, sorafenib, AZD0530, GDC0834, CGI-560, CGI-1746.

8. Erythropoietin and Bruton's kinase inhibitor for use as a drug .

9. The erythropoietin and Bruton's kinase inhibitor, for use in accordance with claim 8, characterized in that erythropoietin is selected from. the group comprising erythropoietin alfa, erythropoietin beta and darbepoetin alfa, in particular erythropoietin beta and/or Bruton' s kinase inhibitor means in particular 2-cyano-N- (2, 5-dibromophenyl ) -3- hydroxy-2-but.enamide (LFM-A13) .

10. The erythropoietin and Bruton's kinase inhibitor, for use in accordance with claim 8 or 9, characterized in that are used in the treatment of neoplasm.

11. The erythropoietin and Bruton's kinase inhibitor, for use in accordance with one of the preceding claims 8 - 10, characterized in that the neoplasm shows high activity of Bruton's tyrosine kinase.

12. The erythropoietin and Bruton's kinase inhibitor, for use in accordance with one of the preceding claims 8 - 11, characterized in that the neoplasm showing high activity of Bruton's tyrosine kinase comprises colon cancer and/or other solid tumors, in particular selected from the group comprising germ cell tumors, sarcomas, carcinomas, lymphomas, bone tumors and nerve-derived tumors.

13. The erythropoietin and Bruton' s kinase inhibitor, for use in accordance with one of the preceding claims 8 - 12, characterized in that the neoplasm, showing particularly high activity of Bruton' s tyrosine kinase is selected from the group comprising hormone-dependent breast cancer in women, hormone-dependent prostate cancer, and lung adenocarcinoma.

14. The erythropoietin and Bruton' s kinase inhibitor, for use in accordance with one of the preceding claims 8 - 13, characterized in that erythropoietin and Bruton' s kinase inhibitor are administered simultaneously, separately, or sequentially .

15. The erythropoietin and Bruton' s kinase inhibitor, for use in accordance with one of the preceding claims 8 - 14, characterized in that erythropoietin is administered subcutaneously or intravenously, preferably subcutaneously, and Bruton' s kinase inhibitor is administered intraperitoneally .

16. The erythropoietin and Bruton' s kinase inhibitor, for use in accordance with one of the preceding claims 8 - 15, characterized in that the therapeutically effective amount of erythropoietin, in particular erythropoietin beta, is administered 3 times a week, and the therapeutically effective amount of Bruton' s kinase inhibitor, in particular 2-cyano-N- ( 2 , 5-dibromophenyl ) -3-hydroxy-2-butenamide (LFM-A13) , is adm.inist.ered 2 times a day.

17. The erythropoietin and Bruton' s kinase inhibitor, for use in accordance with one of the preceding claims 8 - 16, characterized in that the therapeutically effective amount of erythropoietin beta adm.inist.ered 3 times a week is 600 IU/kg per application, and the therapeutically effective amount of 2-cyano-N- (2, 5-dibromophenyl) -3-hydroxy-2-butenamide (LFM-A13) administered 2 times a day is 10 mg/kg per application.

18. The erythropoietin and Bruton' s kinase inhibitor, for use in accordance with one of the preceding claims 8 - 17, characterized in that additionally an effective amount of at least one compound, selected from the group comprising cytostatic drugs, preferably fluorourac.il, fluoropyrimidine, and/or kinase inhibitors, preferably ibrutinib, sorafenib, AZD0530, GDC0834, CGI-560, CGI-1746 is used.

19. The use of erythropoietin beta and Bruton' s kinase inhibitor, in particular 2-cyano-N- (2, 5-di.bromophen.yl ) -3- hydroxy-2-butenamide (LFM-A13), for the manufacture of a drug for the treatment of a neoplasm.

20. The use according to claim 19, characterized in that erythropoietin is selected from the group comprising erythropoietin, alfa, erythropoietin beta and darbepoetin alfa, in particular erythropoietin beta and/or Bruton' s kinase inhibitor means in particular 2-cyano-N- (2, 5-dibromophenyl ) -3- hydroxy-2-butenamide (LFM-A13) .

21. The use according to one of the preceding claims 19-20, characterized in that the neoplasm shows high activity of Bruton ' s tyrosine kinase.

22. The use according to one of the preceding claims 19 -

21, characterized in that the neoplasm showing high activity of Bruton' s tyrosine kinase comprises colon cancer and/or other solid tumors, in particular selected from the group comprising germ cell tumors, sarcomas, carcinomas, lymphomas, bone tumors, and nerve-derived tumors.

23. The use according to one of the preceding claims 19 -

22, characterized in that the neoplasm showing particularly high activity of Bruton' s tyrosine kinase is selected from the group comprising hormone-dependent breast cancer in women, hormone-dependent prostate cancer, and lung adenocarcinoma.

24. The use according to one of the preceding claims 19 -

23, characterized in that erythropoietin, in particular erythropoietin beta, and Bruton' s kinase inhibitor, in particular 2-cyano-N- (2, 5-dibromophenyl ) -3-hydroxy-2- bu.tenami.de (LF -A13) , are administered simultaneously, separately, or sequentially.

25. The use according to one of the preceding claims 19 -

24, characterized in that erythropoietin, in particular erythropoietin beta, is administered subcutaneous l.y or intravenously, preferably subcutaneously, and Bruton' s kinase inhibitor, in particular 2-cyano-N- (2, 5-dibromophenyl) -3- hydroxy-2-butenamide (LFM-A13) , is administered intraperi tonea11y .

26. The use according to one of the preceding claims 19 -

25, characterized in that the therapeutically effective amount of erythropoietin, in particular erythropoietin beta, is administered 3 times a week, and the therapeutically effective amount of Bruton' s kinase inhibitor, in particular 2-cyano-N- (2 , 5-dibromophenyl ) -3-hydroxy-2-butenamide (LFM-A13) , is administered 2 times a day.

27. The use according to one of the preceding claims 19 - 26, characterized in that, the therapeutically effective amount of erythropoietin beta administered 3 times a week is 600 lU/kg per application, and the therapeutically effective amount of 2-cyano-N- (2, 5-dibromophenyl) -3-hydroxy-2-butenamide (LFM-A13) adm.inist.ered 2 times a day is 10 mg/kg per application .

28. The use according to one of the preceding claims 19 - 27, characterized in that erythropoietin, in particular erythropoietin beta, and Bruton' s kinase inhibitor, in particular 2-cyano-N- (2, 5-dibromophenyl ) -3-hydroxy-2- butenamide (LFM-A13), are used in combination with at least one compound selected from the group comprising cytostatic drugs, preferably fluorouracil , fluoropyrimidine, and/or kinase inhibitors, preferably ibrutinib, sorafenib, AZD0530, GDC0834, CGI-560, CGI-1746.