Cancer treatment and metastasis inhibition
阅读说明：本技术 癌症治疗和转移抑制 (Cancer treatment and metastasis inhibition ) 是由 C.法尔顿 L.H.扬森 M.德扬格兹 于 2018-02-12 设计创作，主要内容包括：公开了通过至少减少电压门控钠通道电流的持续部分而不消除瞬态部分的作用来减少或预防表达VGSC的癌症中的转移行为的化合物和方法。(Compounds and methods are disclosed for reducing or preventing metastatic behavior in VGSC-expressing cancers by reducing at least the persistence portion of the voltage-gated sodium channel current without abrogating the effects of the transient portion.)
1. A compound of formula I or a pharmaceutically acceptable salt thereof
Wherein R1 is trifluoromethyl or trifluoromethoxy,
for use in a method of reducing or preventing metastatic behaviour and/or pain perception in a patient suffering from cancer.
2. The compound of claim 1 wherein R1 is trifluoromethoxy, i.e., 4- (pyrimidin-2-ylmethyl) -7- (4- (trifluoromethoxy) phenyl) -3, 4-dihydrobenzo [ f ] of the formula][1,4]Oxazazem
3. the compound of claim 1 wherein R1 is trifluoromethyl, i.e., 4- (pyrimidin-2-ylmethyl) -7- (4- (trifluoromethyl) phenyl) -3, 4-dihydrobenzo [ f ] of the formula][1,4]Oxazazem
4. the compound of any one of the preceding claims, wherein the cancer is a hematological cancer.
5. The compound of claim 4, wherein the cancer is leukemia or lymphoma.
6. A compound according to any one of claims 1 to 3, wherein the cancer is a solid tumour cancer.
7. The compound of claim 6, wherein the cancer is carcinoma, mesothelioma, sarcoma, melanoma, or neuroblastoma.
8. The compound of any one of claims 6 and 7, wherein the cancer is breast cancer.
9. The compound of any one of claims 6 and 7, wherein the cancer is colon cancer.
10. The compound of any one of claims 6 and 7, wherein the cancer is prostate cancer.
11. The compound of any one of claims 6 and 7, wherein the cancer is non-small cell lung cancer (NSCLC).
12. A compound according to any one of claims 6 and 7, wherein the cancer is pleural cancer (e.g. mesothelioma), cervical cancer, ovarian cancer, gastric cancer or neuroblastoma.
13. The compound of any one of the preceding claims, wherein the cancer is a cancer expressing voltage-gated sodium channels (VGSC).
14. The compound of any one of the preceding claims, wherein the cancer is stage 3,4 or 5.
15. The compound of any one of claims 1-12, wherein the cancer is not VGSC-expressing cancer.
16. The compound of claim 15, wherein the cancer is stage 1 or 2.
17. A compound according to any one of claims 1 to 14, wherein the use is in a method of reducing metastatic behaviour of cancer in a patient.
18. A compound according to any one of claims 1 to 16, wherein the use is in a method of preventing metastatic behaviour of cancer in a patient.
19. The compound of any one of the preceding claims for use in a method of reducing or preventing metastatic behaviour without killing cancer cells in a VGSC-expressing cancer.
20. The compound of any one of the preceding claims for use in a method of reducing or preventing metastatic behaviour without substantially affecting cancer cell proliferation in a VGSC-expressing cancer.
21. The compound of any one of the preceding claims for use in a method of reducing or preventing metastatic behavior in a VGSC-expressing cancer by reducing at least the effect of a persistent portion of VGSC current without abrogating a transient portion.
22. A compound according to any one of the preceding claims, wherein metastatic behaviour is reduced or prevented by:
(a) reducing the invasiveness of the cancer cells;
(b) reducing the motility of cancer cells, optionally under hypoxic but very aerobic conditions;
(c) reducing cancer cell expression of at least one VGSC, optionally under both normoxic and hypoxic conditions;
(d) increasing the adhesion of cancer cells;
(e) reducing the migratory capacity of cancer cells; or
(f) (ii) combinations of (a) and (b), (b) and (c), (a) - (c), or (a) - (e).
23. A compound according to any one of the preceding claims, wherein the compound is administered in a therapeutically effective dose, optionally at a dosage level corresponding to a range of 1 μmol-10 μmol.
24. The compound of any one of the preceding claims, wherein the compound is administered at a dose of about 1 mg to about 30 mg, optionally at a dose of about 1 mg to about 20 mg, and wherein the patient is an adult human patient, such as an adult human patient having a body weight in the range of about 50 to about 150 kg.
25. The compound of claim 24, wherein the compound is administered at a dose of about 3 mg, about 5 mg, about 6 mg, about 9 mg, about 10 mg, about 12 mg, about 15 mg, or about 19 mg.
26. The compound of any one of the preceding claims, wherein the compound is administered once daily, once every two days, once every three days, once every five days, once weekly, once biweekly, or once monthly, such as once daily.
27. The compound of claim 26, wherein the compound is administered for a period of at least 4 weeks, such as at least 8 weeks, such as at least 12 weeks, such as at least 24 weeks, such as at least 48 weeks or longer.
28. The compound of any one of claims 26 and 27, comprising administering a one-time prime booster dose of about 10 mg to about 100 mg of said compound.
29. A compound according to any one of the preceding claims, comprising administering the compound in a single initial booster dose of about 10 mg to about 100 mg, followed by a dose of about 1 mg to about 20 mg once daily for a period of at least 4 weeks, such as a daily dose of 3 mg or 6 mg.
30. The compound of any one of the preceding claims, wherein the compound is administered orally.
31. A method for reducing or preventing metastatic behaviour and/or pain perception in a patient suffering from cancer, which method comprises administering to said patient a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, wherein R1 is trifluoromethyl or trifluoromethoxy.
32. The method of claim 31, further comprising the features of any of claims 1-30.
Technical Field
The present invention relates to the treatment of cancer and in particular to the discovery of the treatment of all cancers that express voltage-gated sodium channels (VGSCs), such as, but not limited to, metastatic cancers, such as breast, ovarian, colon or prostate cancer.
Background
Metastatic disease accounts for more than 90% of all cancer-related deaths. The development of metastatic cancers (such as breast, colon and prostate cancers) is generally thought to involve 5 stages as follows:
1. occurrence, i.e. initial transformation of normal cells into cancer cells;
2. proliferation, i.e., increasing the number of cancer cells to form an increasing size primary tumor;
3. during the development or proliferation phase, transitioning from a state in which the cancer cells do not have the potential for metastatic behavior to a state in which the cancer cells have the potential for metastatic behavior;
4. cancer cells detach from the primary tumor, and these detached cells subsequently move toward the circulatory system to surrounding tissue areas within the same organ;
5. metastasis, i.e., the detached cells move through the circulation (blood or lymph) to other organs to produce secondary tumors in those other organs.
A significant change that occurs in cells and leads to the above phase 3 state transition is the expression of functional voltage-gated sodium channels (VGSCs). In humans, there are 9 different VGSC alpha subunits or "NaV" proteins (Nav1.1 to Nav1.9), and all have been found to be expressed on different types of cancer cells (Brackenbury, 2012; Rogeret al., 2015). In breast and colon cancer is usually Nav1.5 channel expression, and in the case of prostate cancer is usually Nav1.7 channel. VGSCs can be expressed in neonatal and/or adult forms. In the case of breast and colon cancer, the neonatal form of Nav1.5 channel (nNav1.5) expression. In the case of prostate cancer, neonatal splice variant expression (Diss) which is also Nav1.7et al., 2001). In the absence of such a channel, tumor cells have no invasive potential and therefore no metastatic behavior potential.
In some cases, the development stage involves the growth of cancer cells that have metastatic potential from the outset. Further, hematological cancers such as leukemia have inherent metastatic characteristics and can invade and accumulate in other organs like the liver or spleen (trends ski, 2015).
Attempts have been suggested to find therapies to prevent metastasis by one or more of preventing the expression of functional VGSCs, completely blocking the activity of the expressed functional VGSCs, or killing the cells. The present invention relates to different methods.
The current flows intermittently through the VGSC, that is, the current flows in pulses. As is well known, each pulse includes a transient (or peak) portion followed by a low level DC portion, referred to as late current or continuous current. The latter is promoted by hypoxia, which is well known to occur in growing tumors. VGSC activity increases invasiveness by promoting proton efflux and acidification of the space around the cell. VGSC also control pain sensations.
Eletlazine, 4- (pyrimidin-2-ylmethyl) -7- (4- (trifluoromethoxy) phenyl) -3, 4-dihydrobenzo [ f ] of formula Ia][1,4]Oxazazem-5(2H) -ketones:
and 4- (pyrimidin-2-ylmethyl) -7- (4- (trifluoromethyl) phenyl) -3, 4-dihydrobenzo [ f ] of the formula Ib][1,4]Oxazazem
both are known for the treatment of heart disease. It is further known that each of them differentially affects the amplitude of the transient and sustained portions of the VGSC current, the effect being a dose-dependent pattern. High doses of these drugs completely block VGSC current. The dose of these or any other drug having the effect of completely blocking the VGSC current in the heart tissue is lethal to the patientAs the heart requires these currents to function. It is further known that the compounds of the formulae Ia and Ib are cardiotonic sodium currents (l)NaL) (also known as sustained sodium current (INaP)) and is effective in the treatment of long QT syndrome, particularly long QT syndrome type 3 (LQT3), in humans, see US 2015/0038489A 1.
Summary of The Invention
The present invention is based, at least in part, on the following findings:
(i) inhibition of the sustained part of the Nav1.5 and Nav1.7 currents, inhibition of metastatic behaviour in breast and colon cancer and in prostate cancer, respectively;
(ii) it is not necessary to suppress the transient part of these currents to suppress the transfer behavior;
(iii) an appropriate dose of a compound of formula I as defined below will inhibit metastatic behaviour without preventing proliferation or destroying tumour cells; and
(iv) the inhibitory effect of the compounds of formula I on the sustained part of the current is greater in cells previously exposed to hypoxia, a state that occurs in growing tumors and that makes a key positive contribution to the metastatic process.
Accordingly, in a first aspect, the present invention relates to a compound of formula I below:
wherein R1 is trifluoromethoxy or trifluoromethyl, for use in a method of reducing or preventing metastatic behaviour and/or pain perception in a patient suffering from cancer. In one embodiment, the cancer is a cancer expressing voltage-gated sodium channels (VGSCs). In another embodiment, the cancer is not a VGSC-expressing cancer. For example, the patient may have cancer associated with risk of VGSC expression and/or metastatic behavior, but VGSC expression and/or metastatic behavior has not been determined.
In one embodiment of the invention, R1 in the compound of formula I is trifluoromethoxy, i.e. the compound of formula I is eleclazine, 4- (pyrimidin-2-ylmethyl) -7- (4- (trifluoromethoxy) group of formula Ia) Phenyl) -3, 4-dihydrobenzo [ f][1,4]Oxazazem
in another embodiment of the invention, R1 in the compound of formula I is trifluoromethyl, i.e. the compound of formula I is 4- (pyrimidin-2-ylmethyl) -7- (4- (trifluoromethyl) phenyl) -3, 4-dihydrobenzo [ f ] f of formula Ib][1,4]Oxazazem
in one embodiment of the invention, the cancer is a non-solid tumor cancer. In a particular embodiment, the cancer is leukemia. In another specific embodiment, the cancer is lymphoma.
In one embodiment of the invention, the cancer is a solid tumor cancer, such as a carcinoma, mesothelioma, sarcoma, melanoma, or neuroblastoma. In a particular embodiment, the cancer is breast cancer. In another particular embodiment, the cancer is colon cancer. In another particular embodiment, the cancer is prostate cancer. In another specific embodiment, the cancer is non-small cell lung cancer (NSCLC). In another specific embodiment, the cancer is cervical cancer. In another specific embodiment, the cancer is gastric cancer. In another specific embodiment, the cancer is neuroblastoma.
According to one embodiment of the invention, the cancer is
According to one embodiment of the invention, metastatic behaviour in cancer is inhibited or reduced by administering a compound of formula I (i.e. Ia or Ib) at an appropriate dose in a patient suffering from a cancer expressing voltage-gated sodium channels (VGSCs).
According to another embodiment of the invention, metastatic behaviour in cancer is inhibited or reduced by administering a compound of formula I at a suitable dose to inhibit or reduce the persistent part of the VGSC current without blocking, or at least not completely blocking, the transient part. Thus, metastasis in cancer can be inhibited or reduced in this manner without having to administer a lethal dose of drug.
According to another embodiment of the invention, which will be explained more fully below, the compound of formula I is administered at a dosage level that inhibits the sustained portion of the VGSC current without blocking or incompletely blocking the transient portion and without directly causing cell death. Therefore, tumors or metastases can be inhibited without causing death of cancer cells.
The fact that metastatic behaviour can be inhibited or reduced without causing cell death may be a significant advantage, as recent work has shown that treatment of cancer by killing cells may be counterproductive at least in some cases, in the sense that despite the short term benefit, cancer will still recur and proliferate. Thus, the present invention provides the possibility to inhibit or prevent metastatic behaviour without the potential problems that may arise from actually killing cancer cells.
According to one embodiment of the invention, metastatic behaviour is reduced or prevented by:
(a) reducing the invasiveness of the cancer cells;
(b) reducing the motility of cancer cells, optionally under hypoxic but very aerobic conditions;
(c) reducing cancer cell expression of at least one VGSC, optionally under both normoxic and hypoxic conditions;
(d) increasing the adhesion of cancer cells;
(e) reducing the migratory capacity of cancer cells; or
(f) (ii) combinations of (a) and (b), (b) and (c), (a) - (c), or (a) - (e).
The compounds are generally administered in therapeutically effective doses. Embodiments related to specific dosage regimens are described in more detail below. In a particular embodiment, the compound is administered in a dose or dosage regimen that provides a reduction or prevention of metastatic behaviour according to any one of (a) - (f) above.
According to another embodiment of the invention, the compounds of the formula I are used at dosage levels corresponding to the range from 1. mu. mol to 10. mu. mol.
These and other aspects and embodiments of the invention are further described below with reference to the experimental data set forth in the figures and examples.
Drawings
Figure 1 is a schematic of a timeline of cancer progression from primary tumorigenesis to secondary tumor (metastasis) formation.
FIG. 2 is a schematic representation of cellular processes occurring during the initiation and progression of cancer to metastasis.
Fig. 3(a) is a sketch illustrating the current through the VGSC, showing both the transient and sustained portions of the current, and also showing the current under both normoxic and hypoxic conditions.
FIG. 4 is a schematic view of a cell adhesion measuring device for individually measuring cell adhesion.
FIG. 5 is a schematic view of an apparatus for measuring lateral motility of cells. View (a) is a top plan view of a cell culture dish containing a semi-confluent cell layer; view (b) is a schematic cross-sectional side view of spreading cells; view (c) is a plan view of the spread cells at time t = 0, at which time the lesions have been created by the cell layer, and view (d) is a plan view of the spread cells at a later time (t = 24 hours) after the cells have moved and the wound has partially closed.
FIG. 6 is a schematic cross-sectional side view of an apparatus for measuring lateral migration of cells.
Fig. 7 is a schematic side sectional view of an apparatus for measuring cell invasiveness.
FIG. 8 is a graph showing the concentration-dependent effect of chemically induced hypoxia on single cell adhesion of human metastatic breast cancer MDA-MB-231 cells. Adhesion increased under low oxygen ("off-negative pressure" -DNP-decrease).
Fig. 9 is a graph showing the results of a trypan blue cell exclusion (i.e., cell viability) assay. MDA-MB-231 cells were treated with 20. mu.M eletlazine or 0.2% DMSO (negative solvent control) for 48 hours. Cell viability was almost 100% for both the treated and control groups, indicating that ELEClazine is non-toxic.
FIG. 10 shows the results of MTT (proliferation) assay. A. A standard curve. The total cell number (per well) increased linearly with the absorbance reading (570 nm). Background readings of 0.04 (empty wells) were subtracted from all data sets. B. Standardized data show proliferation of MDA-MB-231 cells over 48 hours under hypoxic and normoxic conditions. eletlazine and ranolazine were administered at a concentration of 10 μ M. DMSO (0.2%), TTX (10 μ M) and medium were negative controls. 2 mM TEA (K + channel blocker) was used as a positive control.
Fig. 11 shows the results of the wound healing assay. Motility index of hypoxic cells treated with 0.2% DMSO or 10. mu.M eleclazine for 48 hours. At all time points, eletlazine had reduced lateral motility.
FIG. 12 shows hypoxia (1% O) in vitro with 0.5. mu.M eletlazine and 20. mu.M tetrodotoxin (TTX)2) Effect of invasion of the lower MDA MB-231 cells. The boxplot shows the normalized number of MDA MB-231 cells that were challenged 16 hours after treatment with ELEClazine or TTX, compared to the control. Cells were preincubated with the corresponding treatment conditions for 24 hours before the assay started. For each condition, 12 fields from 4 individual inserts were evaluated, denoted P<0.001. 0.5. mu.M of eletlazine did not affect invasiveness, and 20. mu.M of TTX decreased (positive control).
FIG. 13 shows hypoxia (1% O) with 1. mu.M eletlazine and 1. mu.M ranolazine in vitro2) Effect of invasion of the lower MDA MB-231 cells. The box plot shows the number of MDA MB-231 cells that were challenged 16 hours after treatment with ELEClazine or ranolazine, compared to the control. Cells were preincubated with the corresponding treatment conditions for 24 hours before the assay started. The median and quartile distances are as follows: comparison: 175.0 (113& 255)；1 μM eleclazine 131.5 (100 &175) And 1. mu.M ranolazine 106.3 (181)&161). For each condition, 12 views from 8 individual inserts, denoted P<0.001, X represents P>0.05. ELEClazine and ranolazine (both 1. mu.M) are notable but are similarInhibiting the invasiveness.
FIG. 14 shows hypoxia (1% O) with 5. mu.M eletlazine and 5. mu.M ranolazine in vitro2) Effect of invasion of the lower MDA MB-231 cells. The box plot shows the number of MDA MB-231 cells that were challenged 16 hours after treatment with ELEClazine or ranolazine, compared to the control. Cells were preincubated with the corresponding treatment conditions for 24 hours before the assay started. For each condition, 12 views from 8 individual inserts, denoted P<0.001, X represents P>0.05. eletlazine and ranolazine (both 5 μ M) significantly inhibited invasiveness, but the effect of eletlazine was significantly greater.
FIG. 15 shows the effect of eletlazine (10. mu.M) on corrected total cellular fluorescence of MDA-MB-231 cells stained for nNav1.5 protein expression. eletlazine treatment reduced expression.
FIG. 16 shows the results of the treatment of normoxia and hypoxia (1% O)2) Next, the effect of eletlazine, ranolazine (both 5. mu.M) and TTX (0.1 and 10. mu.M) on the invasiveness of the human leukemia FLG29.1 cell line. All reagents (TTX was used as positive control) significantly inhibited invasiveness.
Detailed disclosure of the invention
The transfer behavior includes several phases, namely:
(a) detachment of cells from the tumor;
(b) the detached cells move into the surrounding tissue;
(c) moving through surrounding tissue towards the circulatory system; and
(d) move into the circulatory system (from which cells can eventually leave to form secondary tumors).
Thus, inhibiting or reducing the activity of cells at any one or more of these stages will help to at least reduce metastasis. As explained more fully below, the effect of a drug on each of these sub-stages can be determined by a number of experiments as follows, namely:
(a) testing the effect of the drug on cell adhesion;
(b) testing the effect of the drug on lateral motility of the cells;
(c) testing the effect of the drug on lateral migration of cells; and
(d) the effect of the drug on the invasiveness of the cells, i.e., the ability of the cells to move through the medium consumed by the cells, was tested.
Administration of the compounds of formula I at various dosage levels can increase the adhesion of cells and/or decrease one or more of lateral motility, lateral migration, and invasiveness of cells.
Thus, according to another aspect of the present invention, there is provided a compound, composition or other substance for use or intended to be used in an appropriate dose to inhibit or reduce the sustained portion of VGSC current in metastatic cancer cells, while leaving the transient portion unaffected or only partially reduced to inhibit or reduce metastasis, preferably without directly causing cell death.
Advantages resulting from the present invention include, in at least some aspects or forms, the following:
according to the present invention, breast, colon and prostate cancers (and other cancers in which VGSCs are or may become expressed as described herein) can be suppressed such that the patient may be able to tolerate such cancers without serious damage. As a result, the need for aggressive treatment of the patient to destroy cancer cells, such as by chemotherapy or radiation therapy, may be avoided. If the patient is suspected of having breast, colon or prostate cancer or other metastatic cancer, an immediate treatment with an appropriate dose of a compound of formula I can be administered to inhibit or prevent metastasis while awaiting a definitive test result. The dose required to achieve this only must be high enough to suppress a sustained portion of the VGSC current. A therapeutically acceptable dose of the compound of formula I will achieve the desired suppression of the sustained portion of these currents while leaving the transient portion substantially unaffected.
Referring to fig. 1, a
It has been determined that many human cancer cells (such as breast, colon, and prostate cancer cells) may not initially include any functional VGSC, and that unless such a pathway is expressed in a tumor, the tumor cells will not be invasive. However, in many such tumors, functional VGSCs will be expressed at some point, although initially there are no VGSCs. This triggers a change to a state in which the tumor may spread. Fig. 1 shows a situation in which the initial cell does not contain any functional VGSC, but at some point in
The above stages are represented diagrammatically in fig. 2, where
Certain cells 204 of the cancerous tumor 202 are shown detached from the tumor 203 and passing through the degraded region 202a of the basement membrane 202 into the
Cells 210 that have entered the bloodstream are shown to be carried within the bloodstream to region 211, where cells 212 are shown to have migrated outward through the blood vessel wall 207 towards another organ 213, such as a lymph gland or liver, where they may form a secondary tumor (not shown).
Reference numeral 214 denotes a quiescent cancer cell, which simply resides in or near the vessel wall 207.
As explained more fully below, the present invention provides therapies or means for preventing or reducing one or more metastatic behaviour of cancer cells occurring at each of said stages. In particular, the invention provides therapies or means for:
(a) increasing the adhesion of cells in the tumor so that they are less likely to detach; and/or
(b) Reducing the motility of cells that have become detached, so they are less likely to move and cross the basement membrane into the surrounding tissue; and/or
(c) Reducing invasiveness of cells that have entered surrounding tissue by reducing their ability to move through the tissue toward the circulatory system; and/or
(d) Reducing the ability of cells to migrate from the tissue into the circulatory system through the walls of the circulatory system.
It has been explained above that cancer cells that do not express functional VGSC do not have invasive behavior. Further, it is known that current passes through VGSC in pulses, each pulse comprising a transient or peak portion followed by a much lower level sustained or late portion. According to one aspect of the invention, one or more of the above metastatic behaviors are inhibited or reduced by inhibiting or reducing the persistent portion of the current while not eliminating the peak portion, thus making it possible to use drugs that preferentially reduce the persistent portion of the current.
Some of these drugs are known for the treatment of heart diseases such as cardiac arrhythmias or angina pectoris. In the case of treating the heart, it is crucial to ensure that the peak portion of the current is not eliminated, as this is necessary to maintain cardiac function and its rhythm. Thus, according to one aspect of the present invention, known drugs (such as the compounds of formula I) previously described for inhibiting or reducing the persistent part of the VGSC current without eliminating the peak part are used to inhibit or reduce metastatic behaviour in cancer, in particular breast, colon or prostate cancer.
The nature of the VGSC current will be further described with reference to fig. 3 (a).
Referring to fig. 3(a), a
Plot 304, plotted in dotted lines, shows the VGSC current pulse under low oxygen conditions. It can be seen that the
Definition of
"Voltage-gated sodium channels" or "VGSCs" are a known class of integral membrane proteins that form ion channels that conduct sodium ions (Na) through the plasma membrane of cells+). In humans, there are 9 genes (SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN8A, SCN9A, SCN10A, and SCN11A) that encode 9 different VGSC α subunits or "NaV" proteins (NaV 1.1-NaV1.9, respectively). Unless the context conflicts, the term as used herein may refer to any and all known VGSCs, including, but not limited to Nav1.5 (SCN5A) (neonatal or adult form), Nav1.6 (SCN8A), and Nav1.7 (SCN9A) (Fraser)et al., 2005; Djamgoz et al., 2011). Alternatively, Nav1.5 may be referred to herein as NAV-1.5.
As used herein, "treatment" of cancer includes, but is not limited to, reducing metastatic behavior of the cancer, preventing metastatic behavior of the cancer, reducing pain sensations, or any combination thereof.
By "therapeutically effective amount" or "therapeutically effective dose" is meant an amount or dose of a compound of formula I which, when administered to a patient suffering from cancer, results in a positive therapeutic response to the treatment of the patient, such as reducing metastatic behaviour of the cancer, preventing metastatic behaviour of the cancer, reducing pain, etc.
"reducing the metastatic behaviour of cancer" is intended to reduce any behaviour associated with detached cancer cells moving through the circulation (blood or lymph) to accumulate in other organs and/or to produce a secondary tumour or locally invade surrounding tissues. Typically, the patient is in
"preventing metastatic behaviour of cancer" is intended to refer to prophylactic treatment of a cancer patient at risk of, but not yet diagnosed with, metastatic disease, in order to prevent or reduce the risk of metastatic behaviour of cancer as described above. Typically, the patient is in
Hematologic cancers do not form metastases in the same way as solid tumor cancers, but are characterized by metastatic behavior because hematologic cancer cells can invade and accumulate in other organs. Thus, in the present disclosure, any embodiment relating to "preventing metastatic behaviour" or "reducing metastatic behaviour", particularly in the case of hematological cancers, may alternatively be denoted as "preventing aggressive behaviour" and "preventing aggressive behaviour".
Detailed description of the invention
As shown in this example 2, the compound of formula I (eleclazine) based on breast cancer cell tests had no effect on cell viability (20 μ M), no effect on cell proliferative activity (10 μ M), reduced lateral motility under hypoxic but normoxic conditions, significantly inhibited Matrigel invasiveness at clinically relevant concentrations (<10 μ M, concentration dependent), appeared to be more effective than the reference compound at least at some concentrations (5 μ M), and reduced expression of neonatal nav1.5 protein (10 μ M) under both normoxic and hypoxic conditions. Further, as shown in example 3, the compound of formula I (ELEClazine) has the anti-invasive effects of ELEClazine and ranolazine on human leukemia cell lines.
Compound (I)
The present invention relates to compounds of formula I, or pharmaceutically acceptable salts thereof, wherein R1 is trifluoromethyl or trifluoromethoxy, for use in a method of treating cancer in a patient suffering from cancer, inter alia, by reducing the metastatic behaviour of cancer, preventing the metastatic behaviour of cancer and reducing the perception of pain in cancer.
In some embodiments, R1 is trifluoromethyl and the compound is 4- (pyrimidin-2-ylmethyl) -7- (4- (trifluoromethoxy) phenyl) -3, 4-dihydrobenzo [ f][1,4]Oxazazem
In a separate and specific embodiment, it is preferred that the compound is not cytotoxic to cancer cells, does not substantially affect proliferation of cancer cells, and/or has the effect of at least reducing the sustained portion of the voltage-gated sodium channel current without eliminating the transient portion, at a therapeutically effective concentration, i.e., a concentration at which the compound reduces one or more metastatic behaviors of cancer cells. That is, the cancer cells themselves are not affected except for their growth and spread to surrounding tissues inhibited by tissue degradation via the VGSC mechanism. Proliferation itself is substantially unaffected.
Suitable salts of compounds of formula I (such as Ia and Ib) are described in US 2017/007617 a 1. In particular, such salts include pharmaceutically acceptable salts that are safe for administration to a patient and that maintain the biological effectiveness and properties of the compound. Pharmaceutically acceptable base addition salts can be prepared from inorganic and organic bases. By way of example only, salts derived from inorganic bases include sodium, potassium, lithium, ammonium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines. By way of example only, specific examples of suitable amines include isopropylamine, trimethylamine, diethylamine, tri (isopropyl) amine, tri (N-propyl) amine, ethanolamine, 2-dimethylaminoethanol, tromethamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, N-alkylglucamines, theobromine, purine, piperazine, piperidine, morpholine, N-ethylpiperidine, and the like.
Pharmaceutically acceptable acid addition salts may be prepared from inorganic and organic acids. Salts derived from inorganic acids include hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, and the like. Salts derived from organic acids include acetic, propionic, glycolic, pyruvic, oxalic, malic, malonic, succinic, maleic, fumaric, tartaric, citric, benzoic, cinnamic, mandelic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, salicylic, and the like.
According to one embodiment of the invention, the compound of formula I is selective for one or more VGSC, such as one, two, three, four, five, six, seven, eight or all of NAV-1.1 to 1.9. In some embodiments, the compound of formula I is selective for at least NAV-1.5, NAV-1.6, and NAV-1.7. In one embodiment of the invention, the compound of formula I is more selective for nNAV-1.5 than for adult NAV-1.5.
Therapeutic applications
Suitable patients include mammalian patients with cancer, such as humans, monkeys, rabbits, dogs, cats, cows, horses, pigs, mice, and rats. Preferably, the patient is a human patient, such as an adult human patient. Typically, such an adult human patient may have a body weight in the range of about 50 to about 150 kg, such as about 60 to about 100 kg, such as about 70 kg.
Generally, the cancer selected for treatment of the invention is a VGSC-expressing cancer or a cancer associated with a known risk of VGSC expression and thus with metastatic behavior.
In some embodiments, the cancer is a hematologic cancer, such as leukemia or lymphoma. In some embodiments, the cancer is a solid tumor cancer, such as a carcinoma, mesothelioma, sarcoma, or melanoma. In particular embodiments, the solid tumor cancer is breast cancer, colon cancer, prostate cancer, lung cancer (e.g., non-small cell lung cancer, NSCLC), pleural cancer (e.g., mesothelioma), cervical cancer, ovarian cancer, gastric cancer, or neuroblastoma. In a specific embodiment, the cancer is leukemia. In another specific embodiment, the cancer is breast cancer.
Table 1 below shows the associations found between some specific cancer forms and their VGSC expression.
TABLE 1
Cancer (carcinoma)
Subtype of VGSC
Breast cancer
Nav1.5
Cancer of colon
Nav1.5
Prostate cancer
Nav1.7 and/or Nav1.6
NSCLC
Nav1.7
Cervical cancer
Nav1.6
Stomach cancer
Nav1.7
Ovarian cancer
Nav1.5
Neuroblastoma
Nav1.5
Astrocytoma and astrocytoma
Nav1.5
Leukemia (leukemia)
Nav (undetermined subtype)
Melanoma (MEA)
Nav (undetermined subtype)
In some embodiments, the patient has a cancer that expresses VGSC. VGSC-expressing cancers can be identified, for example, by immunohistochemistry or analysis of a cancer cell-containing sample (such as a tumor biopsy or blood sample) obtained from the patient using a detectable monoclonal or polyclonal antibody specific for one or more VGSCs to detect expression of VGSCs on the cancer cells.
VGSC expressing cancer can be adult or neonatal form expression of Nav1.1-Nav1.9 in any one or more. In one embodiment, the cancer in adult and/or neonatal form expression of Nav1.5, Nav1.6 and Nav1.7 at least one. In a specific embodiment, the cancer in adult and/or neonatal form expression of Nav1.5, such as neonatal Nav1.5. In another specific embodiment, the cancer in adult or neonatal form (such as the neonatal form) expression of Nav1.6. In another embodiment, the cancer in adult or neonatal form (such as the neonatal form) expression of Nav1.7.
As described above, VGSC-expressing cancers are in
In one embodiment, the patient is in
In one embodiment, the cancer is stage 3. Patients with stage 3 cancer are generally not diagnosed with metastatic disease, but are at risk for metastatic behavior of the cancer, i.e., progression to stage 4 or 5. Thus, patients with stage 3 cancer may be treated according to the invention to prevent metastatic behaviour of the cancer.
In one embodiment, the cancer is stage 4. Patients with stage 4 cancer may not have been diagnosed with metastatic disease, but the cancer has progressed towards metastatic behavior. Thus, patients with stage 4 cancer may be treated according to the invention to reduce metastatic behavior of the cancer.
In one embodiment, the cancer is
In some embodiments, the patient may have cancer associated with risk of VGSC expression and/or metastatic behavior, but VGSC expression and/or metastatic behavior has not been determined. Cancers susceptible to metastatic behavior include, for example, leukemia, breast cancer, colon cancer, prostate cancer, lung cancer (e.g., non-small cell lung cancer, NSCLC), pleural cancer (e.g., mesothelioma), cervical cancer, and ovarian cancer (Roger)et al., 2015). For example, immunohistochemical analysis of a sample containing cancer cells (such as a tumor biopsy or blood sample obtained from a patient) may indicate that the tumor cells in the sample do not express one or more of the testsVGSC. Thus, the cancer may be at
In one embodiment, the cancer is
Patients suffering from cancer of any of
In one embodiment, the compound, when used in the methods of the invention, reduces or prevents metastatic behavior in VGSC-expressing cancers without killing cancer cells.
In one embodiment, the compounds, when used in the methods of the invention, reduce or prevent metastatic behavior in VGSC-expressing cancers without substantially affecting the proliferation of cancer cells.
In one embodiment, the compounds, when used in the methods of the invention, reduce or prevent metastatic behavior in VGSC-expressing cancers by reducing at least the effects of the persistent portion of VGSC current without abrogating the transient portion. Suitable assays for assessing the effect of compounds on VGSC current are known in the art (see, e.g., Rajamani @)et al., 2016)。
In other separate and specific embodiments, the compounds reduce or prevent metastatic behavior by:
(a) reducing the invasiveness of the cancer cells;
(b) reducing the motility of cancer cells, optionally under hypoxic but very aerobic conditions;
(c) reducing cancer cell expression of at least one VGSC, optionally under both normoxic and hypoxic conditions;
(d) increasing the adhesion of cancer cells;
(e) reducing the migratory capacity of cancer cells; or
(f) (ii) combinations of (a) and (b), (b) and (c), (a) - (c), or (a) - (e).
In one embodiment, the at least one VGSC comprises one, two, or all of Nav1.5, Nav1.6, and Nav1.7. In one embodiment, the at least one VGSC comprises or consists of neonatal nav 1.5.
In one embodiment, treating cancer cells with the compound results in cancer cells expressing at least one VGSC significantly less than a control (such as a predetermined control value), cancer cells not exposed to the compound, or cancer cells exposed to a reference compound. In one embodiment, treatment of cancer cells with a compound results in cancer cells treated with the compound having significantly lower invasiveness, motility, and/or migratory capacity than controls (such as predetermined control values), cancer cells not exposed to the compound, or cancer cells exposed to a selected reference compound.
Assays for evaluating (a) - (d) are known in the art and are described below and in the examples.
Administration mode
The compounds may be administered to a patient by any suitable route, including (but not limited to) oral, buccal, sublabial, sublingual, rectal, intravenous, subcutaneous, intradermal, intramuscular, transdermal and intranasal administration and/or direct administration to a tumor, such as a primary tumor. Preferably, the compounds are administered orally, for example as tablets or capsules. In some cases, the tablets or capsules may be formulated or coated so that the compound is not released until it reaches the desired destination (e.g., the stomach).
Sustained release systems may also be used, particularly to release the compound over an extended period of time.
The compounds are generally formulated with one or more pharmaceutically acceptable excipients, diluents or carriers according to methods well known in the art. For example, US 2017/0007617 a1 describes suitable formulations of compounds of formula I for intravenous administration.
Dosage regimen
For the intended purpose, the compounds are administered to the patient in therapeutically effective amounts and with a frequency and for a time period determined by a trained physician.
In one embodiment, a therapeutically effective dose is a dose that results in a plasma concentration (preferably steady state) of the compound of from about 0.01 μ M to about 10 μ M. Thus, in some embodiments, the compound is administered to achieve a steady state plasma concentration of about 0.01 μ M to about 10 μ M (such as about 1 μ M or 5 μ M).
In one embodiment, the compound is administered to a patient, such as an adult human patient, at a dose of about 1 mg to about 30 mg, such as 1 to about 15 mg, such as about 1 to about 10 mg, such as about 1 mg to about 5 mg, such as about 2 mg to about 4 mg. In another embodiment, the compound is administered to a patient, such as an adult human patient, at a dose of about 5 to about 15 mg, such as about 10 mg to about 15 mg, such as about 12 to about 14 mg. In another embodiment, the compound is administered to a patient, such as an adult human patient, at a dose of about 8 mg to about 13 mg.
In one embodiment, the compound is administered to a patient, such as an adult human patient, at a dose of about 1 mg, 3 mg, about 6 mg, about 9 mg, about 12 mg, about 15 mg, about 18 mg, about 21 mg, about 24 mg, about 27 mg, or about 30 mg.
In one embodiment, the compound is administered once daily, once every two days, once every three days, once every five days, once a week, once every two weeks, or once a month, such as once daily. Preferably, the compound is administered once daily, preferably orally (p.o), for maintenance therapy.
In one embodiment, the compound is administered as maintenance therapy for a period of at least 4 weeks, such as at least 8 weeks, such as at least 12 weeks, such as at least 24 weeks, such as at least 48 weeks or longer.
In one embodiment, a single initial booster dose of the compound of about 10 mg to about 100 mg, such as about 20 mg to about 80mg, such as about 30 mg to about 70 mg, such as about 40mg to about 60 mg, such as about 30 mg, about 50 mg, about 80mg, about 90 mg or about 95 mg is administered to a patient (such as an adult human patient) before maintenance therapy begins, e.g., one or two days before.
Without being limited by theory, according to current knowledge of the pharmacokinetics of eletlazine, after an initial booster dose of about 95 mg and in adultsThe desired concentration of the compound of formula Ia (eleclazine,451,83g/mol; PubChem CID: 71183216) the daily dose for maintenance therapy can be estimated as follows:
ELEClazine half-life (Tian)
2.5
5
10
15
Daily dosage (mg) to maintain concentration
19
9.5
4.7
3.2
Thus, in one embodiment, the compounds for use according to the invention are first administered orally (p.o.) as a one-time initial booster (or loading) dose of about 10 mg to about 100 mg, followed by oral administration of a dose of about 1 mg to about 20 mg, such as about 1 to about 15 mg, once daily for a period of at least 4 weeks.
In a particular embodiment, the compounds for use according to the invention are first administered orally (p.o.) as a one-time initial booster (or loading) dose of about 20 to about 100 mg, followed by oral administration of a dose of about 3 mg to about 20 mg, such as about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 12 mg, about 15 mg, about 19 mg or about 20 mg, once daily for a period of at least 4 weeks. In separate and specific embodiments, the initial loading dose is about 30 mg, about 50 mg, about 80mg and about 95 mg.
In a particular embodiment, the compounds for use according to the invention are first administered orally (p.o.) as a one-time initial booster (or loading) dose of about 20 to about 100 mg, followed by oral administration of a dose of about 3 mg to about 10 mg, such as about 3 mg, about 5 mg, about 6 mg, about 9 mg or about 10 m, once daily for a period of at least 4 weeks. In separate and specific embodiments, the initial loading dose is about 30 mg, about 50 mg, about 80mg and about 95 mg.
In a particular embodiment, the compounds for use according to the invention are first administered orally (p.o.) as a one-time initial booster (or loading) dose of about 30 mg to about 100 mg, followed by oral administration of a dose of about 3 mg once daily for a period of at least 4 weeks. In separate and specific embodiments, the initial loading dose is about 30 mg, about 50 mg, about 80mg and about 95 mg.
In a particular embodiment, the compounds for use according to the invention are first administered orally (p.o.) as a one-time initial booster (or loading) dose of about 30 mg to about 100 mg, followed by oral administration of a dose of about 5 mg once daily for a period of at least 4 weeks. In separate and specific embodiments, the initial loading dose is about 30 mg, about 50 mg, about 80mg and about 95 mg.
In a particular embodiment, the compounds for use according to the invention are first administered orally (p.o.) as a one-time initial booster (or loading) dose of about 30 mg to about 100 mg, followed by oral administration of a dose of about 6 mg once daily for a period of at least 4 weeks. In separate and specific embodiments, the initial loading dose is about 30 mg, about 50 mg, about 80mg and about 95 mg.
In a particular embodiment, the compounds for use according to the invention are first administered orally (p.o.) as a one-time initial booster (or loading) dose of about 30 mg to about 100 mg, followed by oral administration of a dose of about 9 mg once daily for a period of at least 4 weeks. In separate and specific embodiments, the initial loading dose is about 30 mg, about 50 mg, about 80mg and about 95 mg.
In a particular embodiment, the compounds for use according to the invention are first administered orally (p.o.) as a one-time initial booster (or loading) dose of about 30 mg to about 100 mg, followed by oral administration of a dose of about 10 mg once daily for a period of at least 4 weeks. In separate and specific embodiments, the initial loading dose is about 30 mg, about 50 mg, about 80mg and about 95 mg.
In a particular embodiment, the compounds for use according to the invention are first administered orally (p.o.) as a one-time initial booster (or loading) dose of about 30 mg to about 100 mg, followed by oral administration of a dose of about 19 mg once daily for a period of at least 4 weeks. In separate and specific embodiments, the initial loading dose is about 30 mg, about 50 mg, about 80mg and about 95 mg.
In a particular embodiment, the compounds for use according to the invention are first administered orally (p.o.) as a one-time initial booster (or loading) dose of about 30 mg to about 100 mg, followed by oral administration of a dose of about 20 mg once daily for a period of at least 4 weeks. In separate and specific embodiments, the initial loading dose is about 30 mg, about 50 mg, about 80mg and about 95 mg.
In another particular embodiment, the compounds for use according to the invention are first administered orally (p.o.) as a one-time initial booster (or loading) dose of about 30 mg, followed by oral administration of a dose of about 3 mg or about 6 mg once daily for a period of at least 4 weeks.
In another particular embodiment, the compounds for use according to the invention are first administered orally (p.o.) as a one-time initial booster (or loading) dose of about 48 mg, followed by oral administration of a dose of about 3 mg or about 6 mg once daily for a period of at least 4 weeks.
In one embodiment, the compounds for use according to the invention are administered at a dosage level corresponding to a range of from 1. mu. mol to 10. mu. mol, which corresponds to from about 0.45 mg to about 4.5 mg of the compound of formula Ia.
Measurement of
The following are non-limiting examples of assays for assessing the effect of the compounds of the invention on the metastatic behaviour or other properties of cancer cells.
Single cell adhesion assay
FIG. 4 shows a Palmeret al.(2008) Schematic representation of a Single Cell Adhesion Measurement Apparatus (SCAMA) described for the first time in the paper.
Human breast cancer cells from the MDA-MB-231 cell line at 2.5X 104The density of individual cells/ml was spread and left to stand in the
Using the
To mimic hypoxic conditions of cells, hypoxia was chemically induced by the addition of hydrogen peroxide (1-500 μ M) the last 24 hours prior to testing.
To test the reversibility of a given effect, the drug was washed away, fresh medium was added and the plates were incubated for a further 10 minutes before re-measurement. Each treatment was performed on cells from at least two dishes, at least 100 cells were measured per dish, and the experiment was repeated 3 times (using the corresponding controls).
Lateral motility assay
This assay is used to indicate "free" mobility of cancer cells during local spread. FIG. 5(a) is a top plan view of a cell culture dish 501 having a semi-confluent cell layer 502 on its surface, with cells in an aqueous medium 503.
To determine lateral motility, a "wound healing (" scratch ")" test was performed in which a-0.5 mm scratch 504 was prepared through the cell layer, as shown in fig. 5(b), which is a cross-sectional side view of the cell culture dish. During 24 hours after scratch formation, cells moved into the gap.
Fig. 5(c) and 5(d) are schematic plan views of the cell culture dish 501 at time t = 0 when the width of the scratch 504 is w0 and at time t = 24 hours when the width of the scratch 504 is w24, respectively.
Lateral migration assay
This assay is used to indicate the ability of cells to migrate when they are infiltrated inside/outside. FIG. 6 shows a schematic cross-sectional side view of a
The cells were allowed to migrate across the
The number of cells migrating to the underside of
Invasion assay
This measurement is an extension of the lateral migration measurement described above. To "invade", the cells need both: (i) move as in a lateral migration assay, and (ii) secrete proteolytic enzymes to digest their surroundings. Thus, the ability of cells to invade adjacent tissues by enzyme secretion was evaluated by using a layer of Matrigel ™ cassettes (BD Biosciences) dispersed across the porous membranes of Transwell inserts. A Matrigel ™ cassette consists of laminin, collagen IV, nidogen/enactin, and proteoglycans-a composition comparable to basement membrane proteins.
FIG. 7 is a schematic side cross-sectional view of an
Cells were spread overnight (12 hours) in a 1-5% FBS chemotactic gradient. The nutrient concentration in
The number of cells that invade the underside of the
Cell viability assay
Cells were plated at 5X104The density of individual cells/ml was seeded in 35 mm Falcon tissue culture dishes. After treatment with the given drug, the medium was removed and replaced with 800 μ l growth medium and 200 μ l 0.4% trypan blue (Sigma, Dorset, UK) and incubated for 10 minutes in an incubator. Trypan blue was removed and cells were washed once with 3 ml growth medium. For each treatment, cells from 30 random fields were counted at 100 x magnification. The number of dead cells stained blue was counted in each field. Data are expressed as the percentage of viable cells in the total number of cells in a given field of view. The percentages were averaged and the differences between control and treatment were compared from at least 3 independent experiments.
Cell growth (proliferation) assay
Cells were plated at 2X104Individual cells/ml were spread in 24-well plates (Becton-Dickinson) and allowed to stand overnight. The cells were then treated for the required incubation time (24 hours +) with media changes every 24 hours. At the end of the treatment, the medium was removed and the 3- [4, 5-dimethylthiazol-2-yl ] colorimeter was then carried out]-2, 5-Diphenyltetrazolium bromide (MTT) assay (Grimes)et al., 1995). Briefly, 0.1 ml MTT (5 mg/ml prepared in growth medium) and 0.4 ml growth medium were added to each well and the plates were incubated at 37 ℃ for 3-4 hours. The medium was then removed from the chamber and replaced with 0.5 ml dimethyl sulfoxide (DMSO) and 0.063 ml glycine buffer (0.1M glycine and 0.1M NaCl, pH 10.5). The absorbance at 570 nm was measured 15 minutes after the addition of glycine buffer. Results were calculated as the average of 9 replicates of each treatment from a single invaded well relative to control spectrophotometer readings.
Tissue culture
Experiments were performed on 4 strongly metastatic cell lines:
(i) human metastatic breast cancer MDA-MB-231,
(ii) human metastatic colon cancer SW620 cells,
(iii) human leukemia FLG29.1 cells, and
(iv) rat strongly metastatic prostate cancer Mat-LyLu.
Culturing of cells using known methods (e.g., Grimes)et al., 1995; Fraser et al., 2005)。
Normoxic and hypoxic conditions
In addition to the single cell adhesion test (discussed in the following paragraphs), the experiment was performed under either of the following conditions:
(i) normal normoxic conditions (95% oxygen, 5% carbon dioxide), or
(ii) After 24 hours, hypoxic pretreatment (2% O) was continued during the measurement period2，5% CO2，93% N2)。
In single cell adhesion experiments, hypoxia was chemically induced by the addition of hydrogen peroxide (1-500 μ M) for 24 hours.
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