INHIBITATION OF ACID LIPASE FOR CANCER THERAPY

Abstract

The invention relates to new cancer treatment strategies based on the inhibition of acid lipase. In addition, the invention relates to methods for discovering new active substances suitable for cancer therapy.

Claims

1. A method for finding inhibitors of acid lipase that are suitable for treatment of cancers, comprising the steps of: (a) providing an inhibitor of acid lipase; (b) incubating cancer cells of a cancer cell line with the inhibitor of acid lipase and ascertaining a property of the cancer cells; (c) incubating cancer cells of the same cancer cell line as in step (b) in the absence of the inhibitor of acid lipase and ascertaining the same property of the cancer cells as in step (b) under the same conditions as in step (b); (d) comparing the ascertained property of the cancer cells according to steps (b) and (c).

2. The method as claimed in claim 1 for finding inhibitors of acid lipase that are suitable for adjuvant treatment of cancers, wherein step (b) is carried out in the presence of (i) a cell-toxic substance, (ii) a substance having an antitumor effect, or (iii) immune cells directed against cancer cells; and wherein step (c) is carried out in the presence of the same substance or the same immune cells as in step (b).

3. The method as claimed in claim 1, wherein the property of the cancer cells that is ascertained is the survivability thereof

4. The method as claimed in claim 2, wherein: the (i) cell-toxic substance is selected from the group consisting of taxol, docetaxel, cisplatin, carboplatin, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracenedione, mitoxantrone, mithramycin, actinomycin, d,1-dihydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin; preferably taxol; the (ii) substance having an antitumor effect is selected from the group consisting of targeted active antitumor ingredients, active ingredients of anti-hormone therapy and immunostimulatory antibodies; or the (iii) immune cells directed against cancer cells are modified cytotoxic T cells.

5. The method as claimed in claim 1, wherein step (a) comprises the substeps of: (a.sub.1) providing multiple test substances; (a.sub.2) screening the test substances for their inhibitory effect on the enzymatic activity of acid lipase; (a.sub.3) selecting at least one screened test substance, the inhibitory effect of which is stronger than the inhibitory effect of at least one other screened test substance, and providing this selected test substance as an inhibitor of acid lipase.

6. A use of an inhibitor of acid lipase, preferably Lalistat or one of its physiologically acceptable salts, for production of a drug for treatment of a cancer, preferably triple-negative mammary carcinoma or tamoxifen-resistant hormone receptor-positive breast cancer, wherein the drug preferably contains a scavenger receptor B inhibitor, preferably BLT-1 (CAS No.: 321673-30-7), and/or a CD36 inhibitor.

7. The use as claimed in claim 6, wherein: the treatment increases the apoptosis inducibility of the cancer cells; and/or the treatment reduces the proliferation of the cancer cells; and/or the treatment reduces the mesenchymal character and/or stem-cell character of the cancer cells; and/or the treatment increases the immunogenicity of the cancer cells; and/or the treatment (i) reduces or prevents the metastasis of the cancer cells and/or (ii) metastases-forming circulating tumor cells and/or cell clusters are killed and/or the formation of metastases therefrom is prevented; and/or the cancer cells to be treated are tumor stem cells.

8. The use as claimed in claim 6 or 7, wherein the treatment is effected as an adjuvant treatment in addition to a treatment (i) with a cell-toxic substance, (ii) with a substance having an antitumor effect or (iii) with immune cells directed against cancer cells.

9. The use as claimed in claim 8, wherein: the (i) cell-toxic substance is selected from the group consisting of taxol, docetaxel, cisplatin, carboplatin, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracenedione, mitoxantrone, mithramycin, actinomycin, d,1-dihydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin; preferably taxol; the (ii) substance having an antitumor effect is selected from the group consisting of targeted active antitumor ingredients, active ingredients of anti-hormone therapy and immunostimulatory antibodies; or the (iii) immune cells directed against cancer cells are modified cytotoxic T cells.

10. A pharmaceutical composition comprising an inhibitor of acid lipase, preferably Lalistat or one of its physiologically acceptable salts, and also (i) a cell-toxic substance, (ii) a substance having an antitumor effect or (iii) immune cells directed against cancer cells.

11. The composition as claimed in claim 10, wherein: the (i) cell-toxic substance is selected from the group consisting of taxol, docetaxel, cisplatin, carboplatin, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracenedione, mitoxantrone, mithramycin, actinomycin, d,1-dihydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin; preferably taxol; the (ii) substance having an antitumor effect is selected from the group consisting of targeted active antitumor ingredients, active ingredients of anti-hormone therapy and immunostimulatory antibodies; or the (iii) immune cells directed against cancer cells are modified cytotoxic T cells.

12. The composition as claimed in claim 10, which is prepared for treatment of a cancer, preferably prepared for treatment of triple-negative mammary carcinoma or tamoxifen-resistant hormone receptor-positive breast cancer.

13. The composition as claimed in claim 10 for use in the treatment of a cancer, preferably for use in the treatment of triple-negative mammary carcinoma or tamoxifen-resistant hormone receptor-positive breast cancer.

14. A use of the composition as claimed in claim 10 for production of a drug for treatment of a cancer, preferably for production of a drug for treatment of triple-negative mammary carcinoma or tamoxifen-resistant hormone receptor-positive breast cancer.

Description

EXAMPLE 1

[0104] MDA-MB-231 cancer cells were incubated with Lalistat 2 (selective LAL inhibitor) or the formulation control (mock).

[0105] FIG. 1 shows the effects of acid lipase-inhibiting substances on cell proliferation in TNBC MDA-MB-231 cancer cells. As is evident from FIG. 1, the inhibition of the cellular lipase by means of Lalistat in MDA-MB-231 cancer cells leads to a distinct inhibition of cell proliferation compared to the formulation control.

[0106] The data depicted in FIG. 1 prove that, especially in the case of aggressive triple-negative breast cancer (TNBC), the use of Lalistat to pharmacologically address acid lipase has a high therapeutic potential to inhibit the cell growth of cancer cells.

EXAMPLE 2

[0107] MDA-MB-231 cancer cells were incubated with Lalistat 2 (selective LAL inhibitor) or the formulation control (mock). For the formulation control, paclitaxel alone was used with the same concentration of DMSO as for Lalistat 2. 24 hours after seeding, the cancer cells were incubated for 3 days in 15% FCS-containing RPMI medium having concentrations of Lalistat descending from 50 μM or the corresponding DMSO concentration as control. Subsequently, CellTiterGlo® (Promega) was used to quantify the number of vital cancer cells.

[0108] PANC-1 cancer cells and MDA-MB-231 cancer cells were incubated with Lalistat 2 (selective LAL inhibitor) or the formulation control (mock) and paclitaxel (taxol). For the formulation control, paclitaxel alone was used with the same concentration of DMSO as for Lalistat 2. In a comparative experiment, the cancer cells were incubated with simvastatin (statin, HMG-CoA reductase inhibitor) or formulation control (mock) and paclitaxel (taxol) under otherwise identical conditions. 24 hours after seeding, the cancer cells were incubated for 6 days in 15% FCS-containing RPMI medium containing 50 μM Lalistat or 0.5 μM simvastatin and also a paclitaxel concentration of 80 nM to 40 μM. Subsequently, the viability of the cancer cells (cell viability) was determined using CellTiter-Glo® (Promega), a dose-effect curve was created and the ICso was calculated.

[0109] FIG. 2 shows the effects of acid lipase-inhibiting substances on the toxicity of paclitaxel in TNBC (A, B) MDA-MB-231 cancer cells and in pancreatic ductal adenocarcinoma (C) PANC-1 cancer cells. As is evident from FIG. 2, the inhibition of the cellular lipase by means of Lalistat/paclitaxel leads, in contrast to simvastatin/paclitaxel, to a reduction in the IC.sub.so value by approx. a factor of 4 in MDA-MB-231 cancer cells and approx. a factor of 3 in PANC-1 cancer cells compared to the formulation control. In contrast to simvastatin, Lalistat therefore distinctly increases the induction of cell death by paclitaxel, presumably via apoptosis.

[0110] Whereas statins for adjuvant hormone therapy of estrogen receptor-positive (ER-positive) breast cancer have been successfully tested in initial therapeutic approaches, no improvements in overall survival (OS) have been observed in initial studies for triple-negative breast cancer (TNBC) under statin treatment.

[0111] The data depicted in FIG. 2 prove that, especially in the case of aggressive triple-negative breast cancer (TNBC) or pancreatic ductal adenocarcinoma, the use of Lalistat in synergistic cancer therapies to pharmacologically address acid lipase has a significantly higher therapeutic potential than the inhibition of HMG-CoA reductase using statins.

EXAMPLE 3

[0112] 24 hours after seeding, MDA-MB-231 cancer cells were incubated for 6 days in 15% FCS-containing RPMI medium containing 50 μM Lalistat or formulation control (0.05% DMSO). Subsequently, the relative expression of the (A-C) tumor stem cell markers ALDH1, CD44 and the ratio CD44/CD24 and of the (D) mesenchymal marker vimentin was analyzed by means of quantitative PCR. The values were normalized to the housekeepers GAPDH or HRPT1. The target was amplified in a singleplex assay using “SYBR Green”.

[0113] FIG. 3 shows the relative change in expression of tumor stem cell markers and vimentin in MDA-MB-231 cancer cells after 6 days of treatment with Lalistat. The results of two independent experiments with three biological replicates in each case are depicted. Significance difference compared to formulation control: ALDH: 0.0026 (GAPDH), 0.0004 (HRPT1); CD44: >0.0001 (GAPDH/HRPT1); CD44/CD24: <0.01

[0114] As evidenced by the data in FIG. 3, the incubation of cancer cells with Lalistat leads to changes in epigenetics. What was demonstrated was a reduction in the expression of (tumor) stem cell markers and mesenchymal marker. As a result of the treatment with Lalistat, the cancer cells thus lose their (tumor) stem cell or mesenchymal character and thereby their tumorigenicity and malignancy. Since cell-endogenous defense mechanisms are reactivated in the course of re-differentiation, what can be expected in addition to the described apoptosis inducibility is, for example, also a higher immunogenicity of the cancer cells, which can be combined synergistically with immunotherapy. The inventors were able to demonstrate this for the first time via reduced PD-L1 expression on cancer cells after Lalistat treatment.

[0115] Further preferred embodiments (“AFs”) are the following: [0116] AF 1: A method for finding inhibitors of acid lipase that are suitable for treatment of cancers, comprising the steps of [0117] (a) providing an inhibitor of acid lipase; [0118] (b) incubating cancer cells of a cancer cell line with the inhibitor of acid lipase and ascertaining a property of the cancer cells; [0119] (c) incubating cancer cells of the same cancer cell line as in step (b) in the absence of the inhibitor of acid lipase and ascertaining the same property of the cancer cells as in step (b) under the same conditions as in step (b); [0120] (d) comparing the ascertained property of the cancer cells according to steps (b) and (c). [0121] AF2: The method according to AF1 for finding inhibitors of acid lipase that are suitable for adjuvant treatment of cancers, wherein step (b) is carried out in the presence of (i) a cell-toxic substance, (ii) a substance having an antitumor effect or (iii) immune cells directed against cancer cells; and wherein step (c) is carried out in the presence of the same substance or the same immune cells as in step (b). [0122] AF3: The method according to AF 1 or AF2, wherein the property of the cancer cells that is ascertained is the survivability thereof. [0123] AF4: The method according to AF2 or AF3, wherein [0124] the (i) cell-toxic substance is selected from the group consisting of taxol, docetaxel, cisplatin, carboplatin, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracenedione, mitoxantrone, mithramycin, actinomycin, d, 1-dihydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin; preferably taxol; [0125] the (ii) substance having an antitumor effect is selected from the group consisting of targeted active antitumor ingredients, active ingredients of anti-hormone therapy and immunostimulatory antibodies; or [0126] the (iii) immune cells directed against cancer cells are modified cytotoxic T cells. [0127] AF5: The method according to any of the preceding AFs, wherein step (a) comprises the substeps of [0128] (a.sub.1) providing multiple test substances; [0129] (a.sub.2) screening the test substances for their inhibitory effect on the enzymatic activity of acid lipase; [0130] (a.sub.3) selecting at least one screened test substance, the inhibitory effect of which is stronger than the inhibitory effect of at least one other screened test substance, and providing this selected test substance as an inhibitor of acid lipase. [0131] AF6: The use of an inhibitor of acid lipase, preferably Lalistat or one of its physiologically acceptable salts, for production of a drug for treatment of a cancer. [0132] AF7: The use according to AF6, wherein the treatment is effected as an adjuvant treatment in addition to a treatment (i) with a cell-toxic substance, (ii) with a substance having an antitumor effect or (iii) with immune cells directed against cancer cells. [0133] AF8: The use according to AF7, wherein [0134] the (i) cell-toxic substance is selected from the group consisting of taxol, docetaxel, cisplatin, carboplatin, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracenedione, mitoxantrone, mithramycin, actinomycin, d, 1-dihydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin; preferably taxol; [0135] the (ii) substance having an antitumor effect is selected from the group consisting of targeted active antitumor ingredients, active ingredients of anti-hormone therapy and immunostimulatory antibodies; or [0136] the (iii) immune cells directed against cancer cells are modified cytotoxic T cells. [0137] AF9: A pharmaceutical composition comprising an inhibitor of acid lipase, preferably Lalistat or one of its physiologically acceptable salts, and also (i) a cell-toxic substance, (ii) a substance having an antitumor effect or (iii) immune cells directed against cancer cells. [0138] AF10: The composition according to AF9, wherein [0139] the (i) cell-toxic substance is selected from the group consisting of taxol, docetaxel, cisplatin, carboplatin, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracenedione, mitoxantrone, mithramycin, actinomycin, d, 1-dihydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin; preferably taxol; [0140] the (ii) substance having an antitumor effect is selected from the group consisting of targeted active antitumor ingredients, active ingredients of anti-hormone therapy and immunostimulatory antibodies; or [0141] the (iii) immune cells directed against cancer cells are modified cytotoxic T cells.