WO2024018334A1 - Combination comprising a ggt1 inhibitor and a cysltr1 antagonist and use thereof in the treatment of invasive stages of cancer - Google Patents

Combination comprising a ggt1 inhibitor and a cysltr1 antagonist and use thereof in the treatment of invasive stages of cancer Download PDF

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WO2024018334A1
WO2024018334A1 PCT/IB2023/057151 IB2023057151W WO2024018334A1 WO 2024018334 A1 WO2024018334 A1 WO 2024018334A1 IB 2023057151 W IB2023057151 W IB 2023057151W WO 2024018334 A1 WO2024018334 A1 WO 2024018334A1
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ggt1
cells
cysltrl
colorectal cancer
invasive
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Katarzyna Ewa SOBIERAJSKA
Wojciech Michał CISZEWSKI
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Uniwersytet Medyczny w Łodzi
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/655—Azo (—N=N—), diazo (=N2), azoxy (>N—O—N< or N(=O)—N<), azido (—N3) or diazoamino (—N=N—N<) compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis

Definitions

  • Combination comprising a GGT1 inhibitor and a CysLTRl antagonist and use thereof in the treatment of invasive stages of cancer.
  • the subject of the present invention is a combination comprising a GGT1 inhibitor and a CysLTRl antagonist and the use thereof in the treatment of invasive stages of cancer by inhibiting their invasive abilities (abilities to form metastases).
  • Leukotrienes known as lipid mediators of inflammation, are delivered from the 5- lipoxygenase-catalyzed oxygenation of arachidonic acid and are secreted by the ABCC1 (MRP1) transporter into the extracellular matrix (ECM) as a leukotriene C4 (LTC4).
  • LTC4 binds preferentially to the cysteinyl leukotriene receptor 2 (CysLTR2). Moreover, it can be converted to leukotriene D4 (LTD4) by gamma-glutamyltransferase 1 (GGT1), an extracellular enzyme anchored to the plasma membrane of cells.
  • LTD4 preferentially binds to the cysteinyl leukotriene receptor 1 (CysLTRl) located in the cancer cell membrane.
  • CysLTRl cysteinyl leukotriene receptor 1
  • the present patent application demonstrates that leukotriene is responsible for an increase in the invasiveness of colorectal cancer. Moreover, it was observed (Fig. 1) that the level of secreted leukotriene is higher in colorectal cancer cell lines isolated from invasive tumor stages, and that the extracellular addition of leukotriene to colorectal cancer cell lines isolated from pre-invasive tumor stages increases their invasive capacity.
  • the present patent application demonstrates for the first time that inhibition of the GGT1 protein activity leads to the differentiation of colorectal cancer cells. It was observed (Fig. 2) that the inhibition of the GGT1 protein activity is accompanied by an increase of the mucin 2 (MUC2) level, which is a marker of differentiation of colorectal cancer cells [6,7], A similar effect was observed when the GGT1 protein level was reduced by silencing the GGT1 gene expression (Fig. 3). Moreover, it was observed (Fig. 3) that an increase in the GGT1 level correlates with the degree of invasiveness of colorectal cancer, i.e., the GGT1 level increases in the colorectal cell lines isolated from the advanced stages of tumor development.
  • MUC2 mucin 2
  • the present patent application demonstrates that inhibition of CysLTRl using its antagonists inhibits the invasive abilities of colorectal cancer. It should be emphasized that the applied doses of antagonists did not cause an increase in MUC2 level, i.e., they did not induce an increase in the differentiation of colorectal cancer cells (fig. 4).
  • the present patent application demonstrates, for the first time, that the simultaneous inhibition of the GGT1 protein activity and the inhibition of CysLTRl simultaneously leads to differentiation of the cells and inhibition of the invasive abilities of the colorectal cancer cells characterized by an increased ability to metastasize (fig. 5).
  • GGT1 protein inhibitors such as acivicin (ACV) or 6-diazo-5-oxo-L-nor-leucine and CysLTRl antagonists such as montelukast or zafirlukast in cancer therapies is known in the art.
  • Applications EP3328827 and US5695751 disclose prodrugs such as acivicin and 6- diazo-5-oxo-L-nor-leucine for cancer treatment.
  • Applications EP2990056 and EP3585809 disclose CysLTRl antagonists for use in the treatment of cancer, including montelukast and zafirlukast.
  • EP2945644 discloses the use of acivicin in treating colorectal cancer and, more specifically, for preventing metastasis or recurrence of the disease.
  • EP3923936 describes the use of a pharmaceutical composition comprising zafirlukast for the treatment of tumors such as colorectal cancer.
  • a combination comprising a GGT1 protein inhibitor and a CysLTRl antagonist has not yet been described in the prior art.
  • the mentioned inhibitors and antagonists are only applicable as cytotoxic agents. Blocking the GGT1 protein activity by inhibiting the conversion of LTC4 to LTD4 reduces the ability of the latter to induce the invasive capacity of cancer cells.
  • the inventors showed that the use of GGT1 inhibitors, acivicin or 6-diazo-5-oxo-L-nor-leucine, resulted in an increase in MUC2, which is a marker of colorectal cancer cell differentiation. Furthermore, they also showed that inhibiting the conversion of LTC4 to LTD4 by reducing the GGT1 protein level utilizing siRNA increases MUC2, which promotes cell differentiation. It follows directly from this that any factor leading to reduction the level or activity of the GGT1 protein will also induce the differentiation of cancer cells.
  • GGT1 inhibitors acivicin or 6-diazo-5-oxo-L-nor-leucine
  • CysLTRl antagonists montelukast or zafirlukast
  • the application of the combination comprising the GGT1 inhibitor and the CysLTRl antagonist causes: (a) on the one hand, inhibition of the pathway responsible for inducing the cell invasiveness (by blocking the interaction of LTD4 and CysLTRl), (b) on the other hand, a stimulation of cell differentiation, i.e., cell transformation to a less advanced stage (by increasing the LTC4 level, and consequently stimulating the LTC4- CysLTR2 interaction).
  • the subject of the invention is a combination comprising a gamma-glutamyltransferase 1 (GGT1) protein inhibitor and a cysteinyl type 1 receptor (CysLTRl) antagonist.
  • GTT1 gamma-glutamyltransferase 1
  • CysLTRl cysteinyl type 1 receptor
  • the GGT1 protein inhibitor is selected from the group comprising: acivicin, 6-diazo- 5-oxo-L-nor-leucine, azaserine, OU749, GGsTop, GGTI-DU40 and its analogues, 26S -amino- 4-azi do-butanoic acid, y-phosphono monoester of glutamate and its analogues, y-phosphono diester of glutamate and its analogues, L-ABBA and its analogues, glutathione-analogous peptidyl phosphorus esters, phospho-glutathione ACPB analogues.
  • CysLTRl antagonist is selected from the group comprising: montelukast, zafirlukast and pranlukast.
  • the GGT1 protein inhibitor is selected from acivicin and 6-diazo-5-oxo-L- norleucine
  • the CysLTRl antagonist is selected from montelukast and zafirlukast.
  • Further subject of the invention is the combination according to the invention for use in the treatment of invasive stages of cancer.
  • the cancer is colorectal cancer.
  • treatment is a supportive treatment in chemotherapy or radiotherapy.
  • Fig. 1 The effect of leukotriene on the invasiveness of colorectal cancer cells.
  • Fig. 2 The effect of GGT1 inhibitors on modulating the differentiation process of colorectal cancer cells and their invasion abilities.
  • Colorectal cancer cells isolated from patients with different colon cancer invasive stages were treated with IpM of GGT1 inhibitors: ACV (acivicin) or DON (6-diazo-5-oxo-L-norleucine) for 48 hours, followed by a determination of (A) the level of the mucin 2 (MUC2) which is a marker of differentiation of colorectal cancer cells, (B) the ability of the cells to invade.
  • Fig. 3 The effect of reducing the GGT1 protein level on the differentiation of colorectal cancer cells.
  • GGT1 was downregulated with siRNA in colon cancer cells with different invasiveness properties, followed by a determination of the level of mucin 2 (MUC2), which is a marker of differentiation of colorectal cancer cells.
  • MUC2 mucin 2
  • Fig. 4 The effect of CysLTRl antagonists on modulating colon cancer cells invasion abilities and their differentiation.
  • Colorectal cancer cells isolated from patients with different colon cancer invasive stages were treated with lOpM of CysLTRl antagonists: MON (montelukast) or ZAF (zafirlukast) for 48 hours, followed by a determination of (A) the ability of the cells to invade, (B) the level of the mucin 2 (MUC2) which is a marker of differentiation of colorectal cancer cells.
  • Fig. 5 The effect of the simultaneous use of the GGT1 inhibitors and the CysLTRl antagonists on modulating the differentiation process of colorectal cancer cells and their invasion abilities.
  • Colorectal cancer cells isolated from patients with different colon cancer invasive stages were treated with IpM of ACV (acivicin) alone, or in combination with lOpM of MON (montelukast) or ZAF (zafirlukast); or IpM of DON (6-diazo-5-oxo-L-norleucine) alone, or in combination with lOpM of MON (montelukast) or ZAF (zafirlukast) for 48 hours, followed by a determination of (A) the level of the mucin 2 (MUC2) which is a marker of differentiation of colorectal cancer cells, (B) the ability of the cells to invade.
  • the cells lines were cultured as a monolayer and maintained in DMEM with GlutaMAXTM supplemented with 10% (v/v) fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 pg/mL) in an incubator under standard conditions (temperature of 37°C, CO2 concentration of 5%, relative humidity of 85%),). Cells were cultured in a logarithmic growth phase and preventing them from reaching of confluence. The protein level in the cell lines was estimated by the Western Blot technique.
  • siRNA interfering RNA
  • Xfect® Reagent from TakaraBio according to the manufacturer's instructions.
  • the leukotriene level was analyzed by the commercially available ELISA assay (Leukotriene C4 ELISA Kit) according to the manufacturer's instructions.
  • the invasive potential of the studied cell lines was determined based on an analysis of the ability of the cells to transmigrate to a chemoattractant (20% FBS) through a Matrigel-covered membrane with 8.0 pm pores.
  • the ability of the cells to differentiate was determined based on investigating the changes in the level of MUC2 (a marker of differentiation of colorectal cancer cells) by immunofluorescence staining.
  • the cells were washed with PBS, fixed (4% formaldehyde in a PHEM buffer, 20 min at RT), permeabilized (0.1% Triton X-100 in a PHEM buffer, 5 min at 4°C), and then labelled with antibodies (incubated with mucin-2-recognizing antibodies, 2h at 37°C; then with secondary antibodies conjugated to the Alexa488 fluorescent dye for Ih at RT).
  • the staining was visualized with EVOS FLoid Imaging Station and analyzed using Imaged software.
  • Example 1 Testing the effect of leukotriene on invasive abilities induction in colorectal cancer cells.
  • the cells were seeded onto a 6-well plate, and after a 24-hour incubation, the culture medium was changed to a fresh one. After a 48-hour culture under standard conditions (temperature of 37°C, CO2 concentration of 5%, relative humidity of 85%), the level of the leukotriene secreted into the medium was measured by the ELISA assay.
  • LoVo cells isolated from a metastatic colorectal cancer (Dukes' D Class), secreted about 8 times more leukotriene than the SW620 cells, which had been isolated from the lymph node (Dukes' C Class), as shown in Fig. 1A.
  • the effect of leukotriene on inducing the invasiveness in the SW620 cells was determined.
  • Example 2 Testing the effect of the inhibition of the GGT1 protein activity on the differentiation of colorectal cancer cells.
  • the cells were seeded onto a 6-well plate, and after a 24-hour incubation, the culture medium was changed to a fresh one. Then, cells were treated with IpM of acivicin or 6-diazo- 5-oxo-L-norleucine and after a 48-hour of incubation, an immunocytochemical assay of the level of MUC2 was performed as described above. The results are shown in Fig. 2A.
  • GGT1 inhibition by acivicin resulted in a 4- and 6,5-fold increase in MUC2 for SW620 and LoVo, respectively.
  • 6-diazo-5-oxo-L-norleucine caused a 2.7- and 11.1-fold increase in MUC2 for SW620 and LoVo, respectively.
  • Example 3 Testing the effect of the inhibition of the GGT1 protein activity on the invasiveness of colorectal cancer cells.
  • the cells were seeded onto a 6-well plate, and after a 24-hour incubation, the culture medium was changed to a fresh one. Then, cells were treated with IpM of acivicin or 6-diazo- 5-oxo-L-norleucine, and after a 48-hour of incubation, the invasion potential of studied cell lines was estimated, as described above. The results are shown in Fig. 2B. None of the tested inhibitors affected the invasive potential of the studied cells.
  • Example 4 Testing the effect of reducing the GGT1 protein level on the differentiation of colorectal cancer cells.
  • the GGT1 protein level was determined in colorectal cancer cells isolated from patients with different colon cancer invasive stages: SW620 (a low invasiveness potential) and LoVo (a high invasiveness potential). It was observed (Fig. 3 A) that the GGT1 protein level was higher in the cells with a high invasiveness potential (LoVo).
  • the cells were seeded onto a 6-well plate, and after a 24-hour incubation, the culture medium was changed to a fresh one and the GGT1 gene expression was silenced for 48 hours by the small interfering RNA technique as described above.
  • the silencing efficiency of GGT1 was about 80% (Fig. 3B).
  • the MUC2 level was determined immunocytochemically, as described above.
  • the GGT1 downregulation increased the MUC2 level by 3.7-fold and 11.3-fold in SW620 and LoVo, respectively (Fig. 3C).
  • Example 5 Testing the effect of the CysLTRl antagonists on the invasiveness of colorectal cancer cells.
  • the cells were seeded onto a 6-well plate, and after a 24-hour incubation, the culture medium was changed to a fresh one. Then, cells were treated with 10 pM of CysLTRl antagonists: montelukast or zafirlukast and after a 48-hour of incubation the invasion potential of studied cell lines was estimated, as described above. The results are shown in Fig. 4A. Both antagonists showed a similar effect against LoVo, resulting in a nearly 8-fold decrease in the invasion abilities of cells. None of the tested inhibitors affected the invasive potential of SW620 cells.
  • Example 6 Testing the effect of the CysLTRl antagonists on the differentiation of colorectal cancer cells.
  • the cells were seeded onto a 6-well plate, and after a 24-hour incubation, the culture medium was changed to a fresh one. Then, cells were treated with 10 pM of CysLTRl antagonists: montelukast or zafirlukast and after a 48-hour of incubation, an immunocytochemical assay of the level of MUC2 was performed, as described above. The results are shown in Fig. 4B. None of the tested antagonists affected the MUC2 level in the studied cell lines.
  • Example 7 Testing the effect of the simultaneous use of the GGT1 protein inhibitors and the CysLTRl leukotriene receptor antagonists on the differentiation of colorectal cancer cells and on the inhibition of the invasiveness of colorectal cancer cells.
  • the cells were seeded onto a 6-well plate, and after a 24-hour incubation, the culture medium was changed to a fresh one. Then, cells were treated with IpM of acivicin (GGT1 inhibitor) alone or in combination with a 10 pM of CysLTRl antagonist, montelukast or zafirlukast; or with IpM of 6-diazo-5-oxo-L-norleucine (GGT-1 inhibitor) alone or in combination with a 10 pM of CysLTRl antagonist, montelukast or zafirlukast. After a 48-hour incubation, an immunocytochemical assay of the level of MUC2 and the invasion potential of studied cell lines were estimated, as described above. The results are shown in Fig. 5.
  • the mixture of the GGT1 protein inhibitor - acivicin together with the CysLTRl antagonist: montelukast or zafirlukast was observed to cause the level of MUC2 in the treated cells to increase to the level which was observed in the case of the use of acivicin alone.
  • the CysLTRl antagonist montelukast or zafirlukast
  • a 2.7- and 4.5-fold reduction in the invasive potential of the cells were observed for the combination of acivicin with montelukast and zafirlukast, respectively.
  • no change in the effect on the invasive potential of SW620 cells was observed after using the combination of acivicin with montelukast or zafirlukast.
  • acivicin alone did not affect the invasive potential of the cells.
  • a similar level of change as for the combination of acivicin with montelukast or zafirlukast was observed for the mixture of another GGT1 protein inhibitor, 6-diazo-5-oxo-L-nor-leucine together with the CysLTRl antagonist montelukast or zafirlukast.

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Abstract

The subject of the present invention is a combination comprising a GGTI protein inhibitor and a CysLTRl receptor antagonist and use thereof in the treatment of invasive stages of cancer. The GGTI protein inhibitor is preferably acivicin or 6-diazo-5-oxo-L-norleucine; the CysLTRl receptor antagonist is preferably montelukast or zafirlukast.

Description

Combination comprising a GGT1 inhibitor and a CysLTRl antagonist and use thereof in the treatment of invasive stages of cancer.
Description of the invention
The subject of the present invention is a combination comprising a GGT1 inhibitor and a CysLTRl antagonist and the use thereof in the treatment of invasive stages of cancer by inhibiting their invasive abilities (abilities to form metastases).
Leukotrienes, known as lipid mediators of inflammation, are delivered from the 5- lipoxygenase-catalyzed oxygenation of arachidonic acid and are secreted by the ABCC1 (MRP1) transporter into the extracellular matrix (ECM) as a leukotriene C4 (LTC4). LTC4 binds preferentially to the cysteinyl leukotriene receptor 2 (CysLTR2). Moreover, it can be converted to leukotriene D4 (LTD4) by gamma-glutamyltransferase 1 (GGT1), an extracellular enzyme anchored to the plasma membrane of cells. LTD4 preferentially binds to the cysteinyl leukotriene receptor 1 (CysLTRl) located in the cancer cell membrane. Previous epidemiological studies on the development of colorectal cancer have shown a relationship between the level of cysteinyl receptor expression and the prognosis and predictable survival of the patient. In particular, an increased CysLTRl level is observed in patients with advanced cancer, and an elevated CysLTR2 level characterizes morphologically differentiated cancers observed in the pre-invasive stages of tumor development [1,2]. At the same time, it should be mentioned that morphologically differentiated cancers respond more effectively to the chemotherapeutic agents used in anti -cancer therapy [3,4], Therefore, the prognosis for patients with elevated CysLTRl levels is worse than for patients with elevated CysLTR2 levels. Moreover, activation of CysLTR2 has been found to lead to the differentiation of colorectal cancer cells. This data suggests that the balance between these two receptors is essential for tumor progression and the course of the disease [5], It also seems that preventing the dedifferentiation of tumor cells or inducing their re-differentiation will increase the percentage of patients cured. [3,4],
The present patent application demonstrates that leukotriene is responsible for an increase in the invasiveness of colorectal cancer. Moreover, it was observed (Fig. 1) that the level of secreted leukotriene is higher in colorectal cancer cell lines isolated from invasive tumor stages, and that the extracellular addition of leukotriene to colorectal cancer cell lines isolated from pre-invasive tumor stages increases their invasive capacity.
The present patent application demonstrates for the first time that inhibition of the GGT1 protein activity leads to the differentiation of colorectal cancer cells. It was observed (Fig. 2) that the inhibition of the GGT1 protein activity is accompanied by an increase of the mucin 2 (MUC2) level, which is a marker of differentiation of colorectal cancer cells [6,7], A similar effect was observed when the GGT1 protein level was reduced by silencing the GGT1 gene expression (Fig. 3). Moreover, it was observed (Fig. 3) that an increase in the GGT1 level correlates with the degree of invasiveness of colorectal cancer, i.e., the GGT1 level increases in the colorectal cell lines isolated from the advanced stages of tumor development. As a result, more effective conversion of LTC4 to LTD4 occurs in the invasive cells, and thus LTD4 can bind preferentially to CysLTRl. When GGT1 is inhibited, the conversion of LTC4 to LTD4 is blocked and the free LTC4 pool can preferentially bind to the CysLTR2 receptor. It should be emphasized that the investigated doses of GGT1 inhibitors did not inhibit the invasive capacity of colorectal cancer (fig. 2).
The present patent application demonstrates that inhibition of CysLTRl using its antagonists inhibits the invasive abilities of colorectal cancer. It should be emphasized that the applied doses of antagonists did not cause an increase in MUC2 level, i.e., they did not induce an increase in the differentiation of colorectal cancer cells (fig. 4).
The present patent application demonstrates, for the first time, that the simultaneous inhibition of the GGT1 protein activity and the inhibition of CysLTRl simultaneously leads to differentiation of the cells and inhibition of the invasive abilities of the colorectal cancer cells characterized by an increased ability to metastasize (fig. 5).
The use of GGT1 protein inhibitors, such as acivicin (ACV) or 6-diazo-5-oxo-L-nor-leucine and CysLTRl antagonists such as montelukast or zafirlukast in cancer therapies is known in the art. Applications EP3328827 and US5695751 disclose prodrugs such as acivicin and 6- diazo-5-oxo-L-nor-leucine for cancer treatment. Applications EP2990056 and EP3585809 disclose CysLTRl antagonists for use in the treatment of cancer, including montelukast and zafirlukast. EP2945644 discloses the use of acivicin in treating colorectal cancer and, more specifically, for preventing metastasis or recurrence of the disease. EP3923936 describes the use of a pharmaceutical composition comprising zafirlukast for the treatment of tumors such as colorectal cancer. However, a combination comprising a GGT1 protein inhibitor and a CysLTRl antagonist has not yet been described in the prior art. Moreover, the mentioned inhibitors and antagonists are only applicable as cytotoxic agents. Blocking the GGT1 protein activity by inhibiting the conversion of LTC4 to LTD4 reduces the ability of the latter to induce the invasive capacity of cancer cells. Simultaneous use of the CysLTRl antagonist will prevent the remaining LTD4 pool from binding to the receptor, thus blocking the path of metastasis. On the other hand, an increase of LTC4 level will result in an increased possibility of binding this form of leukotriene to the CysLTR2 receptor. Therefore, it will cause re-differentiation of tumor cells, thus reverting it to earlier stages of development more sensitive to the effects of standard chemotherapy.
In their studies on colorectal cancer cell lines, the inventors showed that the use of GGT1 inhibitors, acivicin or 6-diazo-5-oxo-L-nor-leucine, resulted in an increase in MUC2, which is a marker of colorectal cancer cell differentiation. Furthermore, they also showed that inhibiting the conversion of LTC4 to LTD4 by reducing the GGT1 protein level utilizing siRNA increases MUC2, which promotes cell differentiation. It follows directly from this that any factor leading to reduction the level or activity of the GGT1 protein will also induce the differentiation of cancer cells. In conclusion, it has been shown that the inhibition of the GGT1 protein activity, resulting in an increase in the LTC4 concentration, leads to the differentiation of tumor cells and the transition of the tumor to a pre-invasive state (stimulation of the CysLTR2-dependent pathway) [2,5,8], In addition, it was shown that using CysLTRl antagonists (montelukast or zafirlukast) decreased the invasive capacity of cells. At the same time, it should be noted that the antagonists in the doses used did not exhibit anti-proliferative properties against colorectal cancer cells. Therefore, the observed inhibition of invasiveness is not a consequence of the anti -proliferative or cytotoxic effect of the compounds used. Based on the above data, it was found that each of these compounds can lead to the inhibition of the invasive capacity of cells.
Moreover, in cells with a higher invasive potential, the simultaneous use of GGT1 inhibitors (acivicin or 6-diazo-5-oxo-L-nor-leucine) and CysLTRl antagonists (montelukast or zafirlukast) leads to both an increase of MUC2, therefore the induction of cell differentiation, as well as the inhibition of the invasive abilities of colorectal cancer cells. What is important, in addition to acivicin and 6-diazo-5-oxo-L-nor-leucine such compounds as: azaserine, OU749, GGsTop, GGTI-DU40 and its analogues, 2 (S) -ami no-4-azi do-butanoic acid, y-phosphono monoester of glutamate and its analogues, y-phosphono diester of glutamate and its analogues, L-ABBA and its analogues, glutathione-analogous peptidyl phosphorus esters, phosphoglutathione ACPB analogues, and other compounds of such function described in the state of art can be used, whilst as CysLTRl antagonists, in addition to montelukast or zafirlukast, also pranlukast can be used as well as other compounds of such function disclosed in the state of the art. It should be noted that using only an inhibitor or only an antagonist leads to only induction of differentiation or only to inhibition of invasiveness, respectively. In addition, it should be emphasized that such a solution is not obvious, and it is possible only in the case of cancer cells that are characterized by an increased ability to secrete leukotriene. Cells that meet these criteria are cells in the invasive stages of tumor development. They secrete more LTC4, which is converted to LTD4 by GGT1 protein. Subsequently, LTD4 binds to CysLTRl, increasing the ability of cells to invade. Thus, the application of the combination comprising the GGT1 inhibitor and the CysLTRl antagonist causes: (a) on the one hand, inhibition of the pathway responsible for inducing the cell invasiveness (by blocking the interaction of LTD4 and CysLTRl), (b) on the other hand, a stimulation of cell differentiation, i.e., cell transformation to a less advanced stage (by increasing the LTC4 level, and consequently stimulating the LTC4- CysLTR2 interaction).
The subject of the invention is a combination comprising a gamma-glutamyltransferase 1 (GGT1) protein inhibitor and a cysteinyl type 1 receptor (CysLTRl) antagonist.
Preferably the GGT1 protein inhibitor is selected from the group comprising: acivicin, 6-diazo- 5-oxo-L-nor-leucine, azaserine, OU749, GGsTop, GGTI-DU40 and its analogues, 26S -amino- 4-azi do-butanoic acid, y-phosphono monoester of glutamate and its analogues, y-phosphono diester of glutamate and its analogues, L-ABBA and its analogues, glutathione-analogous peptidyl phosphorus esters, phospho-glutathione ACPB analogues.
Preferably the CysLTRl antagonist is selected from the group comprising: montelukast, zafirlukast and pranlukast.
Preferably the GGT1 protein inhibitor is selected from acivicin and 6-diazo-5-oxo-L- norleucine, and the CysLTRl antagonist is selected from montelukast and zafirlukast. Further subject of the invention is the combination according to the invention for use in the treatment of invasive stages of cancer.
Preferably the cancer is colorectal cancer.
Preferably treatment is a supportive treatment in chemotherapy or radiotherapy.
Preferred features of the present invention are illustrated by the following drawings that supplement the information provided in the embodiments.
Description of the drawings
Fig. 1 The effect of leukotriene on the invasiveness of colorectal cancer cells. (A) Colorectal cancer cells isolated from patients with different colon cancer invasive stages were grown in a culture vessel for 48 hours, and then the level of leukotriene secreted by the cells into the medium was determined by the ELISA assay. The graphs display mean ± S.D. (n=4). (B) SW620 cells were stimulated with increasing leukotriene concentrations for 48 hours, and then the ability of the cells to invade was determined. The graphs display mean ± S.D. (n=3).
Fig. 2 The effect of GGT1 inhibitors on modulating the differentiation process of colorectal cancer cells and their invasion abilities. Colorectal cancer cells isolated from patients with different colon cancer invasive stages were treated with IpM of GGT1 inhibitors: ACV (acivicin) or DON (6-diazo-5-oxo-L-norleucine) for 48 hours, followed by a determination of (A) the level of the mucin 2 (MUC2) which is a marker of differentiation of colorectal cancer cells, (B) the ability of the cells to invade. The graphs display mean ± S.D. (n=3).
Fig. 3 The effect of reducing the GGT1 protein level on the differentiation of colorectal cancer cells. (A) The GGT1 protein level was determined in colorectal cancer cells isolated from patients with different colon cancer invasive stages by the Western Blot techniques. The protein level was normalized to GAPDH. The graphs display mean ± S.D. (n=3). (B) The effectiveness of GGT1 silencing in LoVo cells was confirmed by the WB assay. The GGT1 protein level was normalized to GAPDH. The graphs display mean ± S.D. (n=3). (C) GGT1 was downregulated with siRNA in colon cancer cells with different invasiveness properties, followed by a determination of the level of mucin 2 (MUC2), which is a marker of differentiation of colorectal cancer cells. The graphs display mean ± S.D. (n=3).
Fig. 4 The effect of CysLTRl antagonists on modulating colon cancer cells invasion abilities and their differentiation. Colorectal cancer cells isolated from patients with different colon cancer invasive stages were treated with lOpM of CysLTRl antagonists: MON (montelukast) or ZAF (zafirlukast) for 48 hours, followed by a determination of (A) the ability of the cells to invade, (B) the level of the mucin 2 (MUC2) which is a marker of differentiation of colorectal cancer cells. The graphs display mean ± S.D. (n=3).
Fig. 5 The effect of the simultaneous use of the GGT1 inhibitors and the CysLTRl antagonists on modulating the differentiation process of colorectal cancer cells and their invasion abilities. Colorectal cancer cells isolated from patients with different colon cancer invasive stages were treated with IpM of ACV (acivicin) alone, or in combination with lOpM of MON (montelukast) or ZAF (zafirlukast); or IpM of DON (6-diazo-5-oxo-L-norleucine) alone, or in combination with lOpM of MON (montelukast) or ZAF (zafirlukast) for 48 hours, followed by a determination of (A) the level of the mucin 2 (MUC2) which is a marker of differentiation of colorectal cancer cells, (B) the ability of the cells to invade. The graphs display mean ± S.D. (n=3).
EXAMPLES
General procedures used
In the described examples, an analysis was performed using cell lines isolated from various stages of colorectal cancer development: SW620 (a model of cells with a lower invasive potential) and LoVo (a model of cells with a higher invasive potential). The cells lines were cultured as a monolayer and maintained in DMEM with GlutaMAX™ supplemented with 10% (v/v) fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 pg/mL) in an incubator under standard conditions (temperature of 37°C, CO2 concentration of 5%, relative humidity of 85%),). Cells were cultured in a logarithmic growth phase and preventing them from reaching of confluence. The protein level in the cell lines was estimated by the Western Blot technique. Silencing the GGT1 gene expression and consequently reducing the GGT1 protein level was performed by the interfering RNA (siRNA) technique using the Xfect® Reagent from TakaraBio according to the manufacturer's instructions. The leukotriene level was analyzed by the commercially available ELISA assay (Leukotriene C4 ELISA Kit) according to the manufacturer's instructions. Moreover, the invasive potential of the studied cell lines was determined based on an analysis of the ability of the cells to transmigrate to a chemoattractant (20% FBS) through a Matrigel-covered membrane with 8.0 pm pores. The ability of the cells to differentiate was determined based on investigating the changes in the level of MUC2 (a marker of differentiation of colorectal cancer cells) by immunofluorescence staining. The cells were washed with PBS, fixed (4% formaldehyde in a PHEM buffer, 20 min at RT), permeabilized (0.1% Triton X-100 in a PHEM buffer, 5 min at 4°C), and then labelled with antibodies (incubated with mucin-2-recognizing antibodies, 2h at 37°C; then with secondary antibodies conjugated to the Alexa488 fluorescent dye for Ih at RT). The staining was visualized with EVOS FLoid Imaging Station and analyzed using Imaged software.
Example 1. Testing the effect of leukotriene on invasive abilities induction in colorectal cancer cells.
The cells were seeded onto a 6-well plate, and after a 24-hour incubation, the culture medium was changed to a fresh one. After a 48-hour culture under standard conditions (temperature of 37°C, CO2 concentration of 5%, relative humidity of 85%), the level of the leukotriene secreted into the medium was measured by the ELISA assay. LoVo cells, isolated from a metastatic colorectal cancer (Dukes' D Class), secreted about 8 times more leukotriene than the SW620 cells, which had been isolated from the lymph node (Dukes' C Class), as shown in Fig. 1A. Next, the effect of leukotriene on inducing the invasiveness in the SW620 cells was determined. Cells were treated with increasing doses of leukotriene (0.5, 1, and 2nM) for 48- hour. Then, the invasive abilities of the cells were determined as described above. It was observed that leukotriene stimulated the invasion abilities of colon cancer cells in a concentration-dependent manner, as shown in Fig. IB.
Example 2, Testing the effect of the inhibition of the GGT1 protein activity on the differentiation of colorectal cancer cells. The cells were seeded onto a 6-well plate, and after a 24-hour incubation, the culture medium was changed to a fresh one. Then, cells were treated with IpM of acivicin or 6-diazo- 5-oxo-L-norleucine and after a 48-hour of incubation, an immunocytochemical assay of the level of MUC2 was performed as described above. The results are shown in Fig. 2A. GGT1 inhibition by acivicin resulted in a 4- and 6,5-fold increase in MUC2 for SW620 and LoVo, respectively. 6-diazo-5-oxo-L-norleucine caused a 2.7- and 11.1-fold increase in MUC2 for SW620 and LoVo, respectively.
Example 3, Testing the effect of the inhibition of the GGT1 protein activity on the invasiveness of colorectal cancer cells.
The cells were seeded onto a 6-well plate, and after a 24-hour incubation, the culture medium was changed to a fresh one. Then, cells were treated with IpM of acivicin or 6-diazo- 5-oxo-L-norleucine, and after a 48-hour of incubation, the invasion potential of studied cell lines was estimated, as described above. The results are shown in Fig. 2B. None of the tested inhibitors affected the invasive potential of the studied cells.
Example 4, Testing the effect of reducing the GGT1 protein level on the differentiation of colorectal cancer cells.
The GGT1 protein level was determined in colorectal cancer cells isolated from patients with different colon cancer invasive stages: SW620 (a low invasiveness potential) and LoVo (a high invasiveness potential). It was observed (Fig. 3 A) that the GGT1 protein level was higher in the cells with a high invasiveness potential (LoVo).
It was also checked whether a reduction in the GGT1 protein level would affect the differentiation of colorectal cancer cells. The cells were seeded onto a 6-well plate, and after a 24-hour incubation, the culture medium was changed to a fresh one and the GGT1 gene expression was silenced for 48 hours by the small interfering RNA technique as described above. The silencing efficiency of GGT1 was about 80% (Fig. 3B).Then, the MUC2 level was determined immunocytochemically, as described above. The GGT1 downregulation increased the MUC2 level by 3.7-fold and 11.3-fold in SW620 and LoVo, respectively (Fig. 3C).
Example 5, Testing the effect of the CysLTRl antagonists on the invasiveness of colorectal cancer cells.
The cells were seeded onto a 6-well plate, and after a 24-hour incubation, the culture medium was changed to a fresh one. Then, cells were treated with 10 pM of CysLTRl antagonists: montelukast or zafirlukast and after a 48-hour of incubation the invasion potential of studied cell lines was estimated, as described above. The results are shown in Fig. 4A. Both antagonists showed a similar effect against LoVo, resulting in a nearly 8-fold decrease in the invasion abilities of cells. None of the tested inhibitors affected the invasive potential of SW620 cells.
Example 6, Testing the effect of the CysLTRl antagonists on the differentiation of colorectal cancer cells.
The cells were seeded onto a 6-well plate, and after a 24-hour incubation, the culture medium was changed to a fresh one. Then, cells were treated with 10 pM of CysLTRl antagonists: montelukast or zafirlukast and after a 48-hour of incubation, an immunocytochemical assay of the level of MUC2 was performed, as described above. The results are shown in Fig. 4B. None of the tested antagonists affected the MUC2 level in the studied cell lines.
Example 7, Testing the effect of the simultaneous use of the GGT1 protein inhibitors and the CysLTRl leukotriene receptor antagonists on the differentiation of colorectal cancer cells and on the inhibition of the invasiveness of colorectal cancer cells.
The cells were seeded onto a 6-well plate, and after a 24-hour incubation, the culture medium was changed to a fresh one. Then, cells were treated with IpM of acivicin (GGT1 inhibitor) alone or in combination with a 10 pM of CysLTRl antagonist, montelukast or zafirlukast; or with IpM of 6-diazo-5-oxo-L-norleucine (GGT-1 inhibitor) alone or in combination with a 10 pM of CysLTRl antagonist, montelukast or zafirlukast. After a 48-hour incubation, an immunocytochemical assay of the level of MUC2 and the invasion potential of studied cell lines were estimated, as described above. The results are shown in Fig. 5.
The mixture of the GGT1 protein inhibitor - acivicin together with the CysLTRl antagonist: montelukast or zafirlukast was observed to cause the level of MUC2 in the treated cells to increase to the level which was observed in the case of the use of acivicin alone. Moreover, in the case of the LoVo cells, a 2.7- and 4.5-fold reduction in the invasive potential of the cells were observed for the combination of acivicin with montelukast and zafirlukast, respectively. However, no change in the effect on the invasive potential of SW620 cells was observed after using the combination of acivicin with montelukast or zafirlukast. Moreover, acivicin alone did not affect the invasive potential of the cells. A similar level of change as for the combination of acivicin with montelukast or zafirlukast was observed for the mixture of another GGT1 protein inhibitor, 6-diazo-5-oxo-L-nor-leucine together with the CysLTRl antagonist montelukast or zafirlukast.
References:
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Claims

Claims A combination comprising a gamma-glutamyltransferase 1 (GGT1) protein inhibitor and a cysteinyl type 1 receptor (CysLTRl) antagonist. The combination according to claim 1 characterised in that the GGT1 protein inhibitor is selected from the group comprising: acivicin, 6-diazo-5-oxo-L-nor-leucine, azaserine, OU749, GGsTop, GGTI-DU40 and its analogues, 26S -amino-4-azido- butanoic acid, y-phosphono monoester of glutamate and its analogues, y-phosphono diester of glutamate and its analogues, L-ABBA and its analogues, glutathione- analogous peptidyl phosphorus esters, phospho-glutathione ACPB analogues. The combination according to claim 1 or 2 characterized in that the CysLTRl antagonist is selected from the group comprising: montelukast, zafirlukast and pranlukast. The combination according to claim 1 or 2 or 3, characterized in that the GGT1 protein inhibitor is selected from acivicin and 6-diazo-5-oxo-L-norleucine, and the CysLTRl antagonist is selected from montelukast and zafirlukast. The combination as defined in any one of claims 1-4 for use in the treatment of invasive stages of cancer. The combination according to claim 5, characterized in that the cancer is colorectal cancer. The combination according to claim 5, characterized in that the treatment is a supportive treatment in chemotherapy or radiotherapy.
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