Patent Application
20250082763
The present invention belongs to the medical field, and in particular, the present invention relates to drug combinations for the treatment of triple negative breast cancer (TNBC).
Breast cancer has become the most common female malignant tumor in the world, its incidence rate and mortality rate are the first among female tumors, and thus it seriously threatens and endangers women's physical and mental health. Different types of breast cancer have significantly different biological characteristics and clinical manifestations. At present, immunohistochemical methods are mainly used in clinical practice. According to the detection results of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER-2), breast cancer is divided into four molecular subtypes: luminal A (ER-positive or PR-positive, HER-2-negative, low Ki67 expression), luminal B (ER-positive or PR-positive, HER-2-positive), HER-2 overexpression (ER- and PR-negative, HER-2 positive, high expression of most Ki67) and basal-like (ER-, PR-, and HER-2 negative). Triple negative breast cancer (TNBC), as a subtype of breast cancer with the highest degree of malignancy, is characterized by negative ER, PR and HER-2, together with high recurrence rate, high early metastasis rate, strong invasion and poor prognosis.
At present, chemotherapy is one of the main ways to treat breast cancer. Cyclophosphamide, doxorubicin and 5-fluorouracil (5-FU) have been recognized as first-line chemotherapy drugs for the treatment of advanced breast cancer. With the emergence of paclitaxel and other medicaments, more and more clinicians believe that paclitaxel and cisplatin can also be used as first-line chemotherapy options. However, the therapeutic efficacy of these chemotherapy drugs still needs to be further improved.
Androgen receptor (AR) belongs to the nuclear receptor family and is a type of ligand-dependent transcription factor. The abnormal regulation of AR signaling pathway plays an important role in the occurrence and development of prostate cancer. It has been shown that castration-resistant prostate cancer (CRPC) still depends on the action of AR. Proteolytic targeting chimeras (PROTACs) have attracted widespread attention as small molecules that can induce degradation of target proteins.
PROTACs, as bifunctional molecules, include a small molecule compound that can bind to the protein of interest (POI), a linker introduced at its suitable position, and a small molecule compound which can bind to E3 ubiquitin ligases. The obtained small molecule probe can simultaneously bind to the target protein and E3 ubiquitin ligases, thereby promoting the ubiquitination of the target protein, by which the protein can be recognized and degraded by the proteasome. Using the PROTACs strategy, proteolysis-targeting chimeras capable of recognizing/binding androgen receptors (AR PROTACs) were prepared, which can regulate the levels of androgen receptors by the intracellular ubiquitin-proteasome system (UPS), and induce the degradation of androgen receptors, and thus achieve the effect of treating related diseases regulated by androgen receptors such as prostate cancer.
However, it has not been reported that AR PROTACs is used to treat TNBC.
An object of the present invention is to provide the use of an AR degrader in the manufacture of medicaments for the prevention and/or treatment of TNBC.
Another object of the present invention is to provide the use of an AR degrader, in combination with targeted drugs (including PI3Kα inhibitors and PARP inhibitors), immunotherapeutic drugs or chemotherapeutic drugs, in the manufacture of medicaments for the prevention and/or treatment of TNBC.
The present invention specifically provides the use of an AR degrader in the manufacture of medicaments for the prevention and/or treatment of TNBC.
The present invention also provides the use of an AR degrader, in combination with targeted drugs, in the manufacture of medicaments for the prevention and/or treatment of TNBC.
Further, the targeted drug is a PI3K inhibitor.
Further, the PI3K inhibitor is a PI3Kα inhibitor.
Further, the PI3K inhibitor is selected from the group consisting of following compounds, or optical isomers thereof, or tautomers thereof, or salts thereof, or prodrugs thereof, or hydrates thereof, or solvates thereof: Alpelisib, GDC-0077, TAK-117, AZD-8186, IPI-549, Idelalisib, Buparlisib, Pilaralisib, Copanlisib, PX-866, Paxalisib, Duvelisib, Umbralisib, Taselisib, Perifosine, Buparlisib, Dactolisib, CUDC-907, and Voxtalisib.
Further, the targeted drug is a PARP inhibitor.
Further, the PARP inhibitor is selected from the group consisting of Olaparib, Rupapani, Talazoparib, Niraparib, Pamiparib, or Fluzopanib.
Further, the targeted drug is Alpelisib, and the molar ratio of the AR degrader to Alpelisib is 1:27-3:1;
The present invention also provides an AR degrader, in combination with immunotherapeutic drugs, for use in the manufacture of medicaments for preventing and/or treating TNBC.
The present invention also an AR degrader, in combination with chemotherapeutic drugs, for use in the manufacture of medicaments for preventing and/or treating TNBC.
Further, the chemotherapeutic drug is Paclitaxel or 5-Fluorouracil.
Further, the chemotherapeutic drug is Paclitaxel, and the molar ratio of the AR degrader to Paclitaxel is 20:1-5000:1;
Further, the protein degradation agent targeting androgen receptor is a PROTAC bifunctional chimeric molecule represented by formula (I), or an optical isomer thereof, a tautomer thereof, or a pharmaceutically acceptable salt thereof:
Further, said U is a CRBN E3 ligase ligand with the following structure:
Further, the structure of said U is selected from the group consisting of:
Further, the PROTAC bifunctional chimeric molecule is selected from the group consisting of ARV-110, ARV-766, HP518, AC0176, GT20029, ASN-1780, ARD-2128, and ARD-2585.
Further, the structure of said PROTAC bifunctional chimeric molecule is as represented by formula (II):
Further, the structure of said PROTAC bifunctional chimeric molecule is selected from the group consisting of:
Further, said triple negative breast cancer (TNBC) is AR-positive TNBC.
In the present invention, it is first discovered that the AR degrader can effectively prevent and/or treat TNBC (including AR-positive TNBC), and has good prospects in the manufacture of medicaments for preventing and/or treating TNBC. In the present invention, it is first disclosed that the AR degrader, in combination with targeted drugs (including PI3Kα inhibitors and PARP inhibitors), immunotherapeutic drugs or chemotherapeutic drugs, can effectively prevent and/or treat TNBC, and has good prospects in the manufacture of medicaments for preventing and/or treating TNBC.
For the definition of the terms used in the present invention: unless indicated otherwise, the initial definition provided for the group or the term herein is applicable to those in the whole specification; for terms not specifically defined herein, according to the disclosure content and the context, the term will have their common meaning as understood by one of ordinary skill in the art to which this invention pertains.
In the present invention, “aromatic ring” denotes all-carbon monocyclic group or fused polycyclic group with conjugated 7L electron system, such as phenyl and naphthyl. Said aromatic ring can be fused to other cyclic moieties (including saturated and unsaturated rings), but cannot contain heteroatoms such as nitrogen, oxygen, or sulfur. At the same time, the point linking to the parent must be on the carbon in the ring having the conjugated 7L electron system.
“Heteroaromatic ring” refers to a heteroaromatic group containing one or more heteroatoms. The heteroatoms, as used herein, include oxygen, sulfur, and nitrogen. For example, furanyl, thienyl, pyridinyl, pyrazolyl, pyrrolyl, N-alkylpyrrolyl, pyrimidyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl ring can be fused to aryl, heterocyclic group, or cycloalkyl ring, wherein the ring linked to the parent structure is a heteroaryl ring.
In the present invention, the AR degrader refers to the AR degradation agent.
“Pharmaceutically acceptable” means certain carriers, vehicles, diluents, excipients, and/or formed salts are usually chemically or physically compatible with other ingredients constituting certain pharmaceutical dosage forms, as well as physiologically compatible with the recipient.
“Salt” means acid and/or basic salt which is formed by reaction of compound or its stereoisomer with inorganic and/or organic acid and/or base, and also includes zwitterionic salts (inner salts), and further includes quaternary ammonium salts, such as alkylammonium salt. These salts can be directly obtained during the final isolation and purification of a compound. The salts can also be obtained by mixing the compound or its stereoisomers with a certain amount of acid or base appropriately (for example, in equivalent). These salts may form a precipitate in the solution, and be collected by filtration, or recovered after evaporation of the solvent, or obtained by freeze-drying after reaction in an aqueous medium. The salt in the present invention may be compounds' hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate.
“Solvate” means a solvate formed by the compound of the present invention and a solvent, wherein the solvent includes (but is not limited to) water, ethanol, methanol, isopropanol, propanediol, tetrahydrofuran, and dichloromethane.
Obviously, based on the above content of the present invention, according to the common technical knowledge and the conventional means in the field, other various modifications, alternations, or changes can further be made, without department from the above basic technical spirits.
With reference to the following specific examples, the above content of the present invention is further illustrated. But it should not be construed that the scope of the above subject matter of the present invention is limited to the following examples. The techniques realized based on the above content of the present invention are all within the scope of the present invention.
The starting materials and equipment used in the present invention are all known products and can be obtained by purchasing those commercially available.
Triple negative breast cancer MDA-MB-453 cells in logarithmic growth stage were inoculated into a 96-well plate at a concentration of 4×103 cells/well, and then incubated in an incubator at 37° C., 5% CO2, and saturated humidity for 24 h or 48 h. To the negative control well, was added RPMI 1640 cell medium containing 10% fetal bovine serum in the same volume as the drug. After cultured for 24 h or 48 h, to the 96-well plate, were added AR PROTACs compounds 162, 177, 230, 259, 279, 281, 282, 325, 357, 400, ARV-110, ARV-766 and AR inhibitor HC-1119, respectively. The concentrations of compounds 177, 230, 259, 279, 281, 282, 325, 357, 400, ARV-110, and ARV-766 are 0 μM, 0.002 μM, 0.005 μM, 0.014 μM, 0.041 μM, 0.123 μM, 0.370 μM, 1.111 μM, 3.333 μM or 10 μM; while the concentrations of HC-1119 are 0 μM, 1.23 μM, 3.70 μM, 11.11 μM, 33.33 μM or 100 μM.
After addition of the drugs and co-incubation for 7-10 days, 10 μL of CCK-8 was added to each well, and then the plate was further cultured for 2 h. The absorbance value (OD) of each well was measured using a microplate reader at a wavelength of 450 nm. The survival rate was calculated using Prism software, the relative survival rate=(OD value of drug well/OD value of control well)×100%. Alternatively, 100 μL of medium was removed from each well, and then 50 μL of CTG was added. The plate was shaken in the dark for 2 min, allowed to stand for 10 min, and then measured using a microplate reader in Luminescence mode. Prism software was used to calculate the survival rate, the relative survival rate=(the RLU value of drug well/the RLU value of control well)×100%.
(1) IC50 values of compounds 177, 230, 259, 279, 281, 282, 325, 357, 400, ARV-110, and ARV-766 were obtained against MDA-MB-453 cells.
IC50 values of compounds 177, 230, 259, 279, 281, 282, 325, 357, 400, ARV-110, and ARV-766 against MDA-MB-453 cells are shown in Table 1, and the judgment criteria were as follows: when IC50 value≤2 μM, it was expressed as A, while when IC50 value >2 μM, it was expressed as B. As shown in Table 1, compounds 177, 230, 259, 279, 281, 282, 325, 357, 400, ARV-110, and ARV-766 could inhibit the proliferation of MDA-MB-453 cells to varying degrees, and could be used to effectively treat TNBC.
(2) IC50 value of HC-1119 against MDA-MB-453 cells
The IC50 value of HC-1119 against MDA-MB-453 cells is shown in Table 1, and the judgment criteria were as follows: when IC50 value≤2 μM, it was expressed as A, while when IC50 value>2 μM, it was expressed as B. As shown in Table 1, HC-1119 has a weak inhibitory effect on the proliferation of MDA-MB-453 cells, indicating that the effect of AR inhibitor HC-1119 on TNBC was significantly weaker than that of AR PROTACs compounds 177, 230, 259, 279, 281, 282, 325, 357, 400, ARV-110, and ARV-766.
For MDA-MB-453, MFM-223, SUM159PT, MDA-MB-468, and HCC1937, 4,000, 3,000, 1,000, 3,000, and 3,000 cells were plated per well in 96-well microplates (Corning Inc), respectively. Cells were treated with serial dilutions of tested compounds with the maximum working concentration of 10 μM. Cell survival was measured 5 days later using a CellTiter-Glo luminescent cell viability assay kit (Promega Corporation). Luminescence was recorded by FLUOstar Omega microplate reader (BMG Labtech). Half maximal inhibitory concentration (IC50) was determined with GraphPad Prism using baseline correction (by normalizing to dimethyl sulfoxide (DMSO) control)
As shown in Table 2, AR PROTACs potently inhibited AR+ TNBC cell growth (MDA-MB-453, SUM159PT and MFM-223), but showed very minor effect on AR-TNBC (MDA-MB-468 and HCC1937), and AR inhibitor HC-1119 failed to show anti-proliferation activity in these cell lines. Compound 279 has exhibited superior anti-proliferation activity compared to other AR PROTACs in our AR-positive TNBC studies, indicating its potential as a first- and best-in-class AR PROTAC for AR-positive TNBC treatment.
Cells were plated in 12-well microplates and incubated at 37° C. overnight for attachment. Cells were then treated for 24 hours with serial dilutions of tested compounds with the maximum working concentration of 1 μM. Cell lysate were subjected to the western blotting assay against AR antibody.
Primary antibodies: Androgen Receptor (D6F11) XP® Rabbit mAb; Anti-GAPDH Rabbit mAb; Secondary antibody: Goat Anti-Rabbit IgG H&L (HRP)
As shown in
Triple negative breast cancer MDA-MB-453 cells in logarithmic growth stage were inoculated into a 96-well plate at a concentration of 4×103 cells/well, and then incubated in an incubator at 37° C., 5% CO2, and saturated humidity for 24 h. To the negative control wells, were added RPMI 1640 cell culture medium containing 10% fetal bovine serum in the same volume as the drug. After cultured for 24 h, to the 96-well plate, were sequentially added AR PROTACs compounds 279, 281, 282, ARV-110, ARV-766 and PI3K inhibitor Alpelisib, respectively.
The concentrations of compounds 279, 281, 282, ARV-110, and ARV-766 were: 0 μM, 0.002 μM, 0.005 μM, 0.014 μM, 0.041 μM, 0.123 μM, 0.370 μM, 1.111 μM, 3.333 μM or 10 μM; the concentration of Alpelisib was: 0 μM, 0.123 μM, 0.370 μM, 1.111 μM, 3.333 μM or 10 μM.
After addition of the drugs and co-incubation for 7 days, 10 μL of CCK-8 was added to each well, and then the plate was further cultured for 2 h. The absorbance value (OD) of each well was measured using a microplate reader at a wavelength of 450 nm. The survival rate was calculated using Prism software, the relative survival rate=(OD value of drug well/OD value of control well)×100%. Alternatively, 100 μL of medium was removed from each well, and then 50 μL of CTG was added. The plate was shaken in the dark for 2 min, allowed to stand for 10 min, and then measured using a microplate reader in Luminescence mode. Prism software was used to calculate the survival rate, the relative survival rate=(the RLU value of drug well/the RLU value of control well)×100%. Then, the experimental results were determined according to the drug Combination Index (CI), with the following criteria: when CI<1, it was indicated that the drug combination had played a synergistic effect.
The results for the combination of compounds 279, 281, 282, ARV-110, ARV-766 with Alpelisib are shown in
Triple negative breast cancer MDA-MB-453 cells in logarithmic growth stage were inoculated into a 96-well plate at a concentration of 4×103 cells/well, and then incubated in an incubator at 37° C., 5% CO2, and saturated humidity for 24 h. To the negative control wells, were added RPMI 1640 cell culture medium containing 10% fetal bovine serum in the same volume as the drug. After cultured for 24 h, to the 96-well plate, were sequentially added AR PROTACs compounds 279, 281, 282, ARV-110 and PI3K inhibitor Duvelisib, respectively.
The concentrations of compounds 279, 281, 282, and ARV-110 were: 0 μM, 0.002 μM, 0.005 μM, 0.014 μM, 0.041 μM, 0.123 μM, 0.370 μM, 1.111 μM, 3.333 μM or 10 μM; the concentration of Duvelisib was: 0 μM, 1.23 μM, 3.70 μM, 11.11 μM, 33.33 μM or 100 μM.
After adding the drugs and co-incubating for 7-10 days, 10 μL of CCK-8 was added to each well, and then the plate was further cultured for 2 h. The absorbance value (OD) of each well was measured using a microplate reader at a wavelength of 450 nm. The survival rate was calculated using Prism software, the relative survival rate=(OD value of drug well/OD value of control well)×100%. Alternatively, 100 μL of medium was removed from each well, and then 50 μL of CTG was added. The plate was shaken in the dark for 2 min, allowed to stand for 10 min, and then measured using a microplate reader in Luminescence mode. Prism software was used to calculate the survival rate, the relative survival rate=(the RLU value of drug well/the RLU value of control well)×100%. Then, the experimental results were determined according to the drug Combination Index (CI), with the following criteria: when CI<1, it was indicated that the drug combination had played a synergistic effect.
The results for the combination of compounds 279, 281, 282, ARV-110 with Duvelisib are shown in
Triple negative breast cancer MDA-MB-453 cells in logarithmic growth stage were inoculated into a 96-well plate at a concentration of 4×103 cells/well, and then incubated in an incubator at 37° C., 5% CO2, and saturated humidity for 24 h. To the negative control wells, were added RPMI 1640 cell culture medium containing 10% fetal bovine serum in the same volume as the drug. After cultured for 24 h, to the 96-well plate, were sequentially added AR PROTACs compounds 279, 281, 282, ARV-110, ARV-766 and PI3K inhibitor Idelalisib, respectively.
The concentrations of compounds 279, 281, 282, ARV-110, and ARV-766 were: 0 μM, 0.002 μM, 0.005 μM, 0.014 μM, 0.041 μM, 0.123 μM, 0.370 μM, 1.111 μM, 3.333 μM or 10 μM; the concentration of Idelalisib was: 0 μM, 1.23 μM, 3.70 μM, 11.11 μM, 33.33 μM or 100 μM.
After adding the drugs and co-incubating for 7-10 days, 10 μL of CCK-8 was added to each well, and then the plate was further cultured for 2 h. The absorbance value (OD) of each well was measured using a microplate reader at a wavelength of 450 nm. The survival rate was calculated using Prism software, the relative survival rate=(OD value of drug well/OD value of control well)×100%. Alternatively, 100 μL of medium was removed from each well, and then 50 μL of CTG was added. The plate was shaken in the dark for 2 min, allowed to stand for 10 min, and then measured using a microplate reader in Luminescence mode. Prism software was used to calculate the survival rate, the relative survival rate=(the RLU value of drug well/the RLU value of control well)×100%. Then, the experimental results were determined according to the drug Combination Index (CI), with the following criteria: when CI<1, it was indicated that the drug combination had played a synergistic effect.
The results for the combination of compounds 279, 281, 282, ARV-110, ARV-766 with Idelalisib are shown in
Triple negative breast cancer MDA-MB-453 cells in logarithmic growth stage were inoculated into a 96-well plate at a concentration of 4×103 cells/well, and then incubated in an incubator at 37° C., 5% CO2, and saturated humidity for 24 h. To the negative control wells, were added RPMI 1640 cell culture medium containing 10% fetal bovine serum in the same volume as the drug. After cultured for 24 h, to the 96-well plate, were sequentially added AR PROTACs compounds 279, 281, 282, ARV-110 and chemotherapeutic drug Paclitaxel, respectively.
The concentrations of compounds 279, 281, 282, and ARV-110 were: 0 μM, 0.002 μM, 0.005 μM, 0.014 μM, 0.041 μM, 0.123 μM, 0.370 μM, 1.111 μM, 3.333 μM or 10 μM; the concentration of Paclitaxel was: 0 nM, 0.025 nM, 0.074 nM, 0.222 nM, 0.667 nM or 2 nM.
After adding the drugs and co-incubating for 7 days, 10 μL of CCK-8 was added to each well, and then the plate was further cultured for 2 h. The absorbance value (OD) of each well was measured using a microplate reader at a wavelength of 450 nm. The survival rate was calculated using Prism software, the relative survival rate=(OD value of drug well/OD value of control well)×100%. Alternatively, 100 μL of medium was removed from each well, and then 50 μL of CTG was added. The plate was shaken in the dark for 2 min, allowed to stand for 10 min, and then measured using a microplate reader in Luminescence mode. Prism software was used to calculate the survival rate, the relative survival rate=(the RLU value of drug well/the RLU value of control well)×100%. Then, the experimental results were determined according to the drug Combination Index (CI), with the following criteria: when CI<1, it was indicated that the drug combination had played a synergistic effect.
The results for the combination of compounds 279, 281, 282, ARV-110 with Paclitaxel are shown in
In summary, in the present invention, it was first discovered that the AR degrader could effectively prevent and/or treat TNBC (including AR-positive TNBC), and has good prospects in the manufacture of medicaments for preventing and/or treating TNBC. In the present invention, it was first disclosed that the AR degrader, in combination with targeted drugs (including PI3Kα inhibitors and PARP inhibitors), immunotherapeutic drugs or chemotherapeutic drugs, could effectively prevent and/or treat TNBC, and has good prospects in the manufacture of medicaments for preventing and/or treating TNBC.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202210536761.3 | May 2022 | CN | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2023/094686 | May 2023 | WO |
| Child | 18950818 | US |
