KR101988124B1 - Pharmaceutical compositions for targeting cancer associated fibroblast - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for screening cancer associated fibroblasts, which comprises Bortezomib and Panobinostat as an active ingredient, wherein, through the composition according to the present invention, cancer - The effect of inhibiting the proliferation or activity of cancer associated fibroblasts (CAFs), which plays an important role in the development, progression and metastasis of cancer in the cells constituting the microenvironment, has been specifically confirmed, and the bortezomib (Bortezomib) ) And panobinostat (Panobinostat), the inhibition of growth of various cancer cells can be effectively used as an anticancer agent.

Description

[0001] The present invention relates to a pharmaceutical composition for cancer-associated fibroblast

The present invention relates to a pharmaceutical composition targeting cancer-associated fibroblasts, and more particularly, to a composition comprising Bortezomib and Panobinostat as an active ingredient, and cancer-associated fibroblasts cancer-associated fibroblasts as a target for inhibiting the proliferation or activity of the cancer-associated fibroblasts.

Cancer is one of the most serious diseases facing humanity, and massive research is underway to treat cancer. Although various anti-cancer drugs have been developed to inhibit the proliferation of cancer cells in cancer therapy, in particular, in the medical treatment of cancer, these drugs inhibit not only cancer cells but also normal cell proliferation, thereby suppressing nausea, vomiting, Inhibition, renal disorder, neurological disorder, and the like. As a method of reducing such side effects, attempts have been recently made to specifically transmit cancer drugs to cancer cells or cancer tissues. By the specific transfer of the anticancer agent, the anticancer agent can be prevented from reaching the normal cell, the side effect can be reduced, and the dosage of the anticancer agent can be reduced, which is economically advantageous.

Transformed tumor cells according to the anticancer agent can be reconstituted by surrounding the tumor cell environment, including recruitment of fibroblasts, immune cell migration, substrate remodeling, and vascular network development, Cancer - Build up a microscopic environment. Continuous crosstalk between direct cell contact or tumor cells mediated through secretory factors such as cytokines, chemokines, and growth factors and their surroundings has made tumors more adaptable to their environment.

Recent studies have shown that cancerous environments, such as blood vessels, extracellular matrix, and fibroblasts, play an important role in cancer development and progression. Although normal fibroblasts inhibit tumor development, cancer associated fibroblast (CAF) induces tumor metastasis and invasion by extensive tissue regeneration through secretion of proteolytic enzymes, deposition of extracellular matrix, and vascular network development . Tumor epileptic pressure composed of CAFs induces drug resistance by providing a protective environment for tumor cells by limiting access to drug intake to cancer cells that provide indirect drug resistance. In addition, adhesion of cancer cells to ECM via integrins has been reported to inhibit apoptosis and activate survival signaling pathways including PI3K-AKT, ERK, and NF-kB pathways.

Bortezomib (BTZ), on the other hand, is the first proteasome inhibitor to destroy multiple cell signaling pathways and induce tumor cell death in preclinical and clinical studies. Although BTZ has shown a significant effect on tumors, Have been reported to have some limitations, including endogenous or acquired resistance responses and peripheral neuropathy. To overcome the limitations of BTZ, combination therapy with BTZ and panobinostat (PST) was approved by the US FDA for the treatment of multiple myeloma in 2014.

PST is a potent pan-histone deacetylase inhibitor (HDACi) that mediates the up-regulation of p21, inhibits cell proliferation and induction of apoptosis, and modulates the acetylation status of major tumor suppressor proteins or cancer genes, It is used as a unique anti-proliferative agent. PST has been used in several clinical trials for the treatment of various cancers such as leukemia, lymphoma, neuroendocrine tumors, renal cell carcinoma, non-small cell lung cancer, breast cancer, prostate cancer, colorectal cancer and thyroid cancer, And the synergistic action of inhibitors and HDACis.

The importance of the environment surrounding the cancer has begun to be emphasized, and a new treatment method targeting the surrounding environment, not the cancer cell itself, is being considered. Particularly, CAF that secretes various physiologically active substances and has a deep influence on the onset and progression of cancer has recently been attracting attention (Korean Patent No. 10-1585947). However, the history of full-scale research is still only several decades, and CAF secretion Although some effects have been confirmed in the cancer treatment targeting a physiologically active substance, the achievement of a cancer treatment method targeting CAF itself has not yet been proved.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made in view of the above problems, and it is an object of the present invention to provide cancer-associated fibroblasts (CAFs) which play an important role in the development, progression and metastasis of cancer among cells constituting cancer- The inventors of the present invention completed the present invention based on the finding that a drug library was screened to identify drugs capable of inhibiting CAF growth.

It is an object of the present invention to provide a pharmaceutical composition for screening cancer associated fibroblasts, which comprises Bortezomib and Panobinostat as an active ingredient.

Another object of the present invention is to provide a pharmaceutical combination preparation for the treatment of cancer associated fibroblast target comprising Bortezomib and Panobinostat as an active ingredient.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention provides a pharmaceutical composition for cancer-associated fibroblasts, which comprises Bortezomib and Panobinostat as an active ingredient.

In one embodiment of the present invention, the bortezomib may be represented by the following formula (1).

[Chemical Formula 1]

In another embodiment of the present invention, the panobinostat may be represented by the following formula (2).

(2)

In another embodiment of the present invention, the composition may inhibit the proliferation or activity of cancer-associated fibroblasts.

In another embodiment of the present invention, the cancer associated fibroblasts may be selected from the group consisting of esophageal cancer-associated fibroblasts, gastric cancer-associated fibroblasts, and breast cancer-associated fibroblasts.

In another embodiment of the present invention, the composition can induce cleavage and activity of caspase-3.

In another embodiment of the present invention, the composition may induce cleavage of PARP (poly (ADP-ribose) polymerase).

In addition, the present invention provides a pharmaceutical combination preparation for cancer-associated fibroblast target treatment comprising Bortezomib and Panobinostat as an active ingredient.

In another embodiment of the present invention, the Bortezomib and the Panobinostat can be administered simultaneously, separately or sequentially.

The present invention also provides a method for inhibiting the proliferation or activity of cancer-associated fibroblasts, comprising the step of administering the composition to a subject.

In addition, the present invention provides a use of the composition for inhibiting proliferation or activity of cancer-associated fibroblasts.

The pharmaceutical composition for cancer-associated fibroblasts according to the present invention comprises Bortezomib and Panobinostat as an active ingredient, wherein the bortezomib or the panovinostat (Panobinostat) inhibits the proliferation of cancer-associated fibroblast proliferation which constitutes the cancer-microenvironment which is a cause of drug resistance, and it is confirmed that synergistic effect is remarkably high when the two drugs are co-administered as compared with the single administration.

Therefore, it can be used not only as a therapeutic agent for various carcinomas, but also in the development of new drugs that overcome drug resistance.

FIG. 1 shows the results of screening of drugs targeting cancer-associated fibroblasts (CAFs) and their cell viability, and (a) high-throughput screening of various drugs targeting cancer-associated fibroblasts (CAFs) The results of the screening show the results of (b) cell viability by treatment with bortezomib, and (c) cell viability following treatment with panobinostat.
FIG. 2 is a graph showing the results of drug efficacy of BTZ and PST on cancer-associated fibroblasts (CAFs) alone or in combination with (a) human esophagus cancer CAF # 8, (b) gastric cancer CAF # 14 (left), PST alone (middle), BTZ and PST combination (right) treatment for gastric cancer CAF # 36, and (d) gastric cancer CAF # It shows the CI value for confirmation.
FIG. 3 is a graph showing the results of flow cytometry of cell death through apoptosis of BTZ and PST according to the present invention, wherein (a) ECAF (D) ECAF # 12, (e) SCAF # 32, and (f) SCAF # 36 as a result of measurement of the flow cells in SCAF # And (g) SCAF # 32 and (h) SCAF # 36, respectively. The result of the combined treatment of BTZ and PTS against cell cycle and cell death was shown.
FIG. 4 is a graph showing the results of confirming the increase of caspase-3 activity by BTZ and PST according to the present invention alone or in combination, wherein (a) Western blotting results in SCAF # 14 by BTZ and PST alone or in combination treatment , (b) the cut strength of caspase-3, (c) the break strength of PARP, (d) the Western blot in SCAF # 32, (H) caspase-3 cleavage strength, and (i) cleavage strength of PARP, as shown in (g) Western blotting in SACF # 36.
FIG. 5 is a graph showing the results of animal experiments performed with BTZ and PST alone or in combination with (a) a representative photograph of a sarcoma tumor from a xenograft model using cancer cells alone or with cancer cells having MMTV-CAF, (b) Tumor volume in a 1: 1 ratio in mouse breast cancer cells alone or in a xenograft model of MMTV-CAF.
FIG. 6 is a photograph showing the results of (a) a representative photograph of a sarcoma tumor, (b) a tumor volume during drug treatment, (c) a cut caspase-3 microscope according to the drug treatment, 21 days after treatment of the control PBS, BTZ, PST and BTZ + (D) the percentage of caspase-3 positive cells cut out, and (e) the mean percentage of fibroblasts in tumor sections.
FIG. 7 shows the results of drug efficacy of the drug alone or in combination with (a) 168FARN mouse breast cancer or (b) mouse MMTV-CAF cells in an animal test by BTZ and PST alone or in combination.

Hereinafter, the present invention will be described in detail.

Conventional anticancer drugs are effective in the early stage of drug administration for the purpose of removing only cancer cells themselves. However, there is a problem that drug resistance occurs, and drug resistance is caused by the fact that cancer cells establish a special cancer- To this end, the inventors sought to identify drugs capable of inhibiting cancer-associated fibroblasts (CAFs), which play an important role in the development, progression and metastasis of cancer in cells constituting the cancer-microenvironment .

Thus, in one embodiment of the present invention, high-throughput screening (HTS) is performed to select potential drugs that target CAFs, thereby selecting candidates Bortezomib (BTZ) and panovinostat Panobinostat, PST) (see Example 2), and the synergistic effect on the inhibitory effect of CAFs upon the combination treatment of Bortezomib and Panobinostat was confirmed (see Example 3) .

Further, it was confirmed whether BTZ and PST treatment were caused by apoptosis through inhibition of CAF, and it was concretely confirmed that the apoptosis was mediated by caspase-3 and PARP cleavage (see Examples 4 and 5) ).

In addition, the combined administration effect of BTZ and PST in vivo was specifically confirmed through a mouse xenograft model (see Example 6).

The above results show that Bortezomib and Panobinostat inhibit cancer-associated fibroblast cells, and exhibit synergistic effects when the two drugs are administered in combination. Therefore, they can be used as therapeutic agents for various carcinomas .

Accordingly, the present invention provides a pharmaceutical composition for screening cancer-associated fibroblasts, which comprises Bortezomib and Panobinostat as an active ingredient.

Cancer-associated fibroblast (CAF) ', which is an object of inhibition according to the present invention, refers to α-SMA (α-smooth muscle actin) -containing fibroblasts present in and / or around cancer lesions, Is found in various cancers such as colon cancer, lung cancer, prostate cancer, breast cancer, gastric cancer, cholangiocarcinoma, and basal cell carcinoma.

The cancer associated with the CAF of the present invention is not particularly limited and includes cancer of the brain, head and neck cancer, breast cancer, lung cancer, esophageal cancer, gastric cancer, duodenal cancer, appendix cancer, colon cancer, rectal cancer, liver cancer, pancreatic cancer, Pancreatic cancer, rectal cancer, uterine cancer, ovarian cancer, vulvar cancer, vaginal cancer, skin cancer, and the like. Or CAF is generally associated with cancer but, as long as similar properties are present, malignant solid tumors other than cancer, such as fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, , Lymphangiosarcoma, synovial sarcoma, chondrosarcoma, osteosarcoma, and the like are also included in the scope of the present invention, and it is more preferable to be an esophageal cancer, stomach cancer or breast cancer, but the present invention is not limited thereto.

Bortezomib, which is an active ingredient of the composition of the present invention, is a proteasome antagonist and has anti-cancer effect by inhibiting proteas in cancer cells.

Panobinostat, another active ingredient of the composition of the present invention, exhibits an anti-cancer effect by inhibiting HDAC associated with the treatment of a cell proliferative disease, and can be represented by the following formula (2).

The pharmaceutical composition according to the present invention contains bortezomib and panobinostat as an active ingredient and may also include a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are those conventionally used in the field of application and include, but are not limited to, saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, And may further contain other conventional additives such as antioxidants and buffers as needed. It may also be formulated into injectable formulations, pills, capsules, granules, or tablets such as aqueous solutions, suspensions, emulsions and the like by additionally adding diluents, dispersants, surfactants, binders, lubricants and the like. Suitable pharmaceutically acceptable carriers and formulations can be suitably formulated according to the respective ingredients using the methods disclosed in Remington's reference. The pharmaceutical composition of the present invention is not particularly limited to a formulation, but may be formulated into an injection, an inhalant, an external preparation for skin, or an oral ingestion agent.

The pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenous, subcutaneous, skin, nasal, or airway) according to the desired method, The type of administration, the route of administration, and the time, but may be suitably selected by those skilled in the art.

The composition according to the invention is administered in a pharmaceutically effective amount. In the present invention, " pharmaceutically effective amount " means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dosage level is determined depending on the type of disease, severity, , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the composition according to the present invention may vary depending on the age, sex, and body weight of the patient. In general, 0.001 to 150 mg, preferably 0.01 to 100 mg, It may be administered one to three times a day. However, the dosage may be varied depending on the route of administration, the severity of obesity, sex, weight, age, etc. Therefore, the dosage is not limited to the scope of the present invention by any means.

In another aspect of the present invention, there is provided a pharmaceutical combination preparation for cancer-associated fibroblast target treatment comprising Bortezomib and Panobinostat as an active ingredient .

The present invention relates to the combination of Bortezomib and Panobinostat, which are active ingredients of the above pharmaceutical combination, to inhibit the proliferation or activity of cancer-associated fibroblast cells compared with the administration of bortezomib or panovinostat alone Is characterized by markedly improving the inhibitory efficacy and may be administered simultaneously, separately, or sequentially, to enhance the therapeutic effect. At this time, it is important to take all of the above factors into consideration and administer an amount capable of achieving the maximum effect in a minimal amount without side effects, which can be easily determined by those skilled in the art. Specifically, the effective amount of the pharmaceutical combination according to the present invention may vary depending on the age, sex, condition, body weight, absorbency, inactivation rate and excretion rate of the active ingredient in the body, type of disease,

In another embodiment of the present invention, the present invention provides a method for inhibiting proliferation or activity inhibition of cancer-associated fibroblasts comprising administering the pharmaceutical composition to an individual.

As used herein, the term "individual" means a subject in need of a method for preventing, controlling or treating a disease, and more specifically, a human or non-human primate, a mouse, a rat, , Horses, and cows.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Example]

Example 1. Experimental Preparation and Experimental Method

1-1. Human fibroblast culture

The human fibroblasts used in the present invention were obtained by thoroughly examining tumor specimens from patients who underwent surgery at Sungkyunkwan University School of Medicine Samsung Medical Center and obtaining representative samples of tumor tissues and distal normal tissues from known pathologists . The obtained tissues were washed twice with PBS containing antibiotics, finely chopped and finely chopped. Twenty milliliters of DEME / F12 (1: 1) containing collagenase / ) ≪ / RTI > medium (Welgene). Subsequently, the dissociated samples were centrifuged, washed twice with PBS and then cultured in DMEM / F12 (1: 1) medium supplemented with 10% FBS (Welgene) and 1% antibiotic (Life Technologies).

1-2. Reagents and Antibodies

The Ez-cytox cell survival assay kit (Ez-cytox cell, Bortezomib, PS-341, Carfilzomib, PR-171 and Panobinostat, LBH589) was purchased from ApexBio The viability assay kit was purchased from DaeillLab, Annexin-V staining kit was purchased from BD Bioscience, and the cell cycle analysis kit was provided from Abcam (Cat: ab139418). (Cat: 9532, Cell Signaling), cleaved PARP (Cat: 9546, Cell Signaling), caspase-3 (Cat: SC-7148, Santa Cruz), cleaved caspase-3 Signaling) and alpha-tubulin antibody (Cat: SC-8035, Santa Cruz) were used for immunoblotting.

1-3. High-throughput screening

In order to carry out high-throughput screening for screening candidate drug candidates targeting cancer-associated fibroblasts (CAFs) according to the present invention, nine types of CAF cell lines (human esophagus cancer-related fiber # 14, # 32, # 36, # 41, and # 42) were used as test samples. Each of the above CAFs was dispensed into 384-well plates per well and treated for 6 days for drug screening in a clinical trial or a drug library containing FDA-approved drugs.

The drug efficacy was measured using an ATPlite assay kit (Cat: 6016949, Perkin Elmer) and EnVision (Perkin Elmer) to measure cellular ATP levels. IC ₅₀ and AUCs (Areas under concentration response curves) To calculate from raw data. In addition, drug candidates were selected by selecting the lowest AUC value.

1-4. Fibroblast survival and proliferation assay

To confirm the in vitro drug efficacy, CAF was inoculated in a 96-well plate at 10,000 cells per well. The following day, the cells were washed with PBS and incubated in serum-free medium for 6 hours, then washed with 1% FBS and 1% The drug candidate substance dissolved in DMEM / F12 medium was treated three times. At this time, the inhibitory effect of the drug on the viability of CAF was evaluated by treatment with an EZ-cytox cell viability assay kit (Cat: EZ-100, DaeilLab) for 48 hours.

Drug efficacy was assessed according to the combination index (CI). The combination index (CI) was quantified by compusyn software (ComboSyn, Inc., Paramus, NJ, USA) based on the Chou Talalay method, The index (CI) evaluation is as follows.

CI = 1: additive effect;

CI > 1: antagonism;

CI <1: synergy.

1-5. Western blot analysis &lt; RTI ID = 0.0 &gt;

Cells were harvested and resuspended in RIPA lysis buffer containing 50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% Na-Doc, 0.1% SDS and protease inhibitor (Roche) Min. &Lt; / RTI &gt; After centrifugation at 13,000 xg, the supernatant was used for immunoblotting to detect PARP, cleaved PARP, caspase-3, cleaved caspase-3 and alpha-tubulin.

1-6. Cell cycle and apoptosis analysis

For cell cycle analysis, the cells were washed with PBS, fixed with 70% ethanol at 4 ° C overnight, and fixed cells were incubated with a propidium iodide and a cell cycle assay kit (RNase A) containing RNaseA Cell cycle analysis kit, Cat: ab139418, Abcam) and incubated at 37 ° C for 30 min. Thereafter, the stained cells were subjected to flow cytometry to record the DNA content of the cells.

Next, apoptosis was analyzed using BD Annexin V-APC apoptosis detection kit (Cat: 556547, BD Biosciences) according to the manufacturer's instructions. More specifically, the obtained cells were washed twice with cold PBS, resuspended in Annexin-V binding buffer, and cells were stained with Annexin-V conjugated with APC for 30 minutes at room temperature in the dark , Flow cytometry was performed as soon as possible (within 1 hour).

1-7. Evaluation of in vivo drug efficacy

in vivo drug efficacy evaluation in the case of, we used a mouse xenograft model, and more specifically, was to remove the MMTV-CAF from Tg (MMTV-PyMT) mouse breast cancer cell model, this time, the separation of the MMTV-CAF are human fibroblasts . Then, in order to confirm the tumor promoting effect of CAFs after culturing in DMEM medium supplemented with 10% serum and 1% antibiotic, 5 × 10 ⁵ 168FARN cells or 5 × 10 ⁵ cells were injected on the side of BALB / c-nude mouse (OrientBio) 5 × 10 ⁵ MMTV-CAF were subcutaneously injected and tumor size was measured using a caliper.

Next, 5 × 10 ⁵ 168FARN cells were subcutaneously inoculated into BALB / c-nude mice with 5 × 10 ⁵ MMTV-CAF to confirm the tumor suppressive effect of the drug on xenotransplantation, and 10 days after xenotransplantation , Mice were randomly assigned to one of four groups to treat the drug via the intraperitoneal route (0.5 mg / kg BTZ, 12.5 mg / kg PST, or 0.25 mg / kg BTZ + 6.25 mg / kg PST) Three times per week.

At this time, the diameter of the tumor was checked three times a week using a caliper, the volume of the tumor was measured according to the following equation (1), and the difference in tumor volume was evaluated with a two tailed student t-test.

[Equation 1]

The volume of the tumor (V) = (4/3)? X (a / 2) x (b / 2) ²

The mice were then sacrificed when the mean tumor volume reached 1,000 mm ³ and the tumor tissue was paraffinized, and immunostaining for cleaved caspase-3 was performed on the tumor tissue to verify drug efficacy. At this time, all animal tests were conducted with the approval of Institutional Animal Care, Samsung Medical Center, Sungkyunkwan University.

1-8. Histological analysis and immunohistochemistry

The mice were dissected and the tumors were separated and fixed with 10% neutral buffered formalin for 48 hours. Tissue sections were stained with H & E for histopathological diagnosis. Next, the slide was deparaffinized and antigen retrieval was performed in Tris-EDTA buffer (pH 9.25) for 15 minutes to proceed with immunostaining. The peroxidase activity and nonspecific binding were then blocked and the slides were incubated with anti-cleaved caspase-3 antibody (Cat: 9661, Cell Signaling) for 1 hour followed by anti-rabbit antibody (Cat: 31190 , Thermoscientific) for 45 minutes in a wet chamber. Slides were made with AEC solution (Cat: sk-4205, Vector), dehydrated by counterstaining with hematoxylin, and then mounted with mounting media (Cat: 008010, Life technology) Respectively.

Example 2. Screening for cancer-associated fibroblast (CAF) target drug and inhibition of CAF cell viability

High-throughput screening (HTS) was performed as described in Examples 1-3 above to select potential drugs targeting CAFs, and cell viability for candidate substances was determined by measuring ATP levels ATPlite assay kit and the AUC value as a proxy for cell number and growth and the IC ₅₀ value for each drug were calculated (see FIG. 1A and Table 1).

[Table 1]

Based on the lowest AUC and IC ₅₀ values, various CAF target drug candidates were selected, including Bortezomib (BTZ), and Carfilzomib (CFZ), according to Table 1 and FIG.

Next, five CAFs (one type of esophageal cancer-associated fibroblast ECAF # 8, and four types of gastric cancer-associated fibroblast SCAF # 14, # 32, and # 36 Bortezomib (BTZ), Carfilzomib (CFZ), and Panobinostat (PST) were treated with various concentrations for 48 hours to confirm cell viability.

As a result, as shown in FIG. 1B to FIG. 1D, it was confirmed that the treatment of candidate drug substances BTZ, PST, and CFZ resulted in a decrease in cell viability in a dose dependent manner, wherein ECAF # 8, SCAF # 14, SCAF # 32, SCAF # 36, and # 39 in the SCAF IC ₅₀ values for each of BTZ is 2.64. IC ₅₀ values for PST were 91.11, 45.03, 136.83, 132.24, and 70.56 nM, respectively, and IC ₅₀ values for CFZ were 12.33, 6.68, 7.30, 5.43, and 3.03 nM .

Based on the above results, it was confirmed that BTZ, PST, and CFZ are potentially targets of CAFs in vitro .

Example 3 Confirmation of CAZ Survival Synergistic Effect by Combined Treatment of BTZ and PST

BTZ and PST were treated alone or in combination with four types of CAFs in order to confirm synergistic effects of inhibition of CAFs by combination treatment of BTZ and PST.

As a result, as shown in Figs. 2A to 2D, it was confirmed that the combined treatment of BTZ and PST significantly reduced cell viability in all cell lines as compared to single treatment. More specifically, the combined treatment of BTZ and PST in SCAF # 14, SCAF # 32 and SCAF # 36 resulted in an improvement of more than 50% in sub-lethal dosage compared to single treatment, and ECAF # 8 And the quasi - lethal dose was less than 50%.

Next, in order to evaluate the reduced cell viability and the IC ₅₀ decrease due to the combination treatment, the intermediate drug effect analysis according to the above Example 1-4 was carried out to determine the combination of the synergistic effect or the quantitative degree of the drug interaction with the antagonistic action Index (CI) was calculated.

As a result, as shown in FIG. 2E, the CI value of each cell line was 1.0 or less, confirming that the combined use of BTZ and PST showed a synergistic effect.

At this time, when BTZ and PST were treated at 5 different concentrations using a combination ratio of 1: 25, synergy was observed in SCAF # 14 and SCAF # 32 (CI value 0.2 to 0.5), SCAF # 36 Value 0.5 to 0.7), and a moderate synergy effect at ECAF # 8.

Example 4. Confirmation of synergistic effect on induction of CAF apoptosis by BTZ and PST treatment

In order to determine whether the inhibition of cell viability of different types of CAF by BTZ and PST treatment was due to apoptosis, three cell line types ECAF # 12, SCAF # 32, and SCAF # 36 were treated with 16 nM Treatment of either BTZ or 400 nM PST alone and 8 nM of BTZ and 200 nM of PST in combination was carried out for 24 hours. Each of the treated cells was stained with Annexin V-apc / propidium iodide (PI) and subjected to cell death by apoptosis through flow cytometry Respectively.

As a result, as shown in FIGS. 3A to 3C, the drug concentration used in the combination treatment used half of the drug concentration in the single treatment drug treatment, but when BTZ and PST were combined with the CAF cell line, And it was confirmed that the cell death inducing effect was superior.

As shown in FIGS. 3D to 3F, when BTZ and PST were used in combination, cell death was more than 10% in ECAF # 12, 19% in SCAF # 36 and 5% in SCAF # 32, And the cell death rate was high.

On the other hand, it has been reported that BTZ induces G2 / M inhibition in several human cancer cell lines. In order to confirm whether BTZ induces cell cycle arrest in CAF, SCAF # 32 and SCAF # 36 cell lines were treated with 2, 4, Of BTZ or 50, 100, 200 nM of PST alone and BTZ and PST of 1 nM + 25 nM, 2 nM + 50 nM and 4 nM + 100 nM, respectively, for 16 hours. After this, fixed and permeable cells were stained with PI and their DNA content was analyzed by flow cytometry.

As a result, as shown in Figs. 3G and 3H, unlike the human cancer cell line, no BTZ or PST alone drug and G2 / M stopping by BTZ and PST combination treatment were observed in the CAF cell line. At this time, when PST alone treatment was performed with ECAF # 32, the ratio of cells in G0 / G1 was slightly increased independently of the drug concentration, and concomitant drug treatment was observed in both SCAF # 32 and SCAF # Gt; G0 / G1 &lt; / RTI &gt; step, indicating a &lt; RTI ID = 0.0 &gt;

Example 5. Confirmation of caspase-3 activity increase by BTZ and PST treatment

In contrast to BTZ or PST single drug treatment according to the above example, it was confirmed that the combined treatment of BTZ and PST induced the synergistic effect of apoptosis. Thus, it was confirmed whether or not the increase in the induction of apoptosis was due to caspase activity Western blotting analysis was performed to detect the cleavage of PARP (poly (ADP-ribose) polymerase) in SCAF # 14, SCAF # 32 and SCAF # 36, and cleavage of SCAF # 14, SCAF # 32 and SCAF # # 36 examined caspase-3 cleavage.

As a result, as shown in FIGS. 4A to 4C, treatment of BTZ or PST alone with SCNF # 14 resulted in a relatively high concentration of 16 nM of BTZ or 400 nM of PST, but also of caspase-3 and PARP But only to a slight increase in cutting. On the other hand, it was confirmed that caspase-3 and PARP cleavage was strongly induced by treatment with 8 nM BTZ and 200 nM PST, which are concentrations of each half of the above-mentioned drug concentrations.

In addition, as shown in Figs. 4d to 4f, almost no cleavage of caspase-3 was observed when treating BTZ and PST drugs alone in SCAF # 32, and only cleaved PARP from BTZ (12 nM) , But it was confirmed that caspase-3 and PARP strongly cleaved in the cells were detected only by treatment with BTN at a low concentration of 3 nM and PST at a concentration of 70 nM when treated with SCZF # 32 using BTZ and PST drugs I could.

Furthermore, as shown in Figs. 4G to 4I, when treating BTZ and PST alone with SCAF # 36, cleavage of caspase-3 and PARP is induced by BTZ alone, but BTZ and PST are used in combination And the cleavage of caspase-3 and PARP was significantly enhanced in the case of treatment.

Based on the above results, it was found that cell death by the combination treatment of BTZ and PST was mediated through caspase-3 mediated PARP cleavage.

Example 6. Confirmation of synergistic effect of tumor suppression in animal model by BTZ and PST combination administration

To confirm the combined efficacy of BTZ and PST in vivo , antitumor efficacy was evaluated using a mouse xenograft model.

More specifically, the mouse breast cancer cell line 168FARN alone or 168FARN + MMTV-CAF was inoculated on the nude mouse side to confirm the importance of the tumor microenvironment.

As a result, as shown in FIGS. 5A and 5B, when the 168FARN and CAF cells (average 168FARN + CAF tumor volume: 933 mm ³ vs. 168 FARN average tumor volume: 116 mm ³ ) were inoculated, tumor formation was improved And it was confirmed that CAF contributes to tumor formation improvement.

Next, the antitumor effect according to the combination of BTZ and PST targeting CAFs was evaluated with a mouse xenograft model using a mouse breast cancer cell line 168FARN containing mouse CAFs. More specifically, mice were injected with 168FARN cells at a ratio of 1: 1 with mouse CAF, and one of the 4 groups was randomly assigned and the control PBS, BTZ, PST, or BTZ and PST combination drugs were administered by intraperitoneal injection Treated three times a week.

As a result, as shown in FIGS. 6A and 6B, it was confirmed that the drug-treated mice significantly inhibited tumor growth as compared with the PBS-treated mice in the control group. As compared with the single drug treatment, Formation was markedly suppressed. At this time, no significant volume difference was found in the mice.

In addition, as shown in FIGS. 6C and 6D, when caspase-3 was cleaved by immunostaining, cell death was slightly increased in BTZ or PST-treated mice compared with PBS treated mice, And PST-treated mice showed a strong increase in cell death.

In addition, as shown in FIG. 6E, there was no significant change in the mean percentage of fibroblasts in tumors treated with BTZ, and the treatment with PST or BTZ and PST showed somewhat higher percentage of fibroblasts in the tumor than in PBS treated tumors Respectively.

On the other hand, 168FARN cells or MMTV-CAF cells were plated and treated with BTZ, PST, and BTZ for 2 days to confirm whether single or combination treatment of BTZ and PST showed cell death in 168FARN mouse breast cancer cells and mouse CAF cells And PST were used in combination.

As a result, as shown in FIGS. 7A and 7B, since single or combination treatment of BTZ and PST induced cell death in both 168FARN mouse breast cancer cells and mouse CAF cells in vitro , May be explained by the different susceptibility of each cell line.

These results suggest that BTZ and PST targeting various types of tumor compartments such as cancer cells and CAF may weaken tumor growth in vivo as a result of increased cell death in xenograft tumor tissues and the combined treatment of BTZ and PST It was confirmed that the efficacy of each drug can be improved.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (15)

A test tube composition for inhibiting cancer associated fibroblasts, comprising Bortezomib and Panobinostat as an active ingredient,
Wherein said cancer associated fibroblasts are selected from the group consisting of esophageal cancer-associated fibroblasts, gastric cancer-associated fibroblasts, and breast cancer-associated fibroblasts. The composition of claim 1, wherein the Bortezomib is represented by the following formula (1).
[Chemical Formula 1]

The composition according to claim 1, wherein the panobinostat is represented by the following formula (2).
(2)

delete delete 2. The composition of claim 1, wherein said composition induces cleavage and activity of caspase-3.
The composition of claim 1, wherein the composition induces cleavage of PARP (Poly (ADP-ribose) polymerase). delete delete delete delete delete delete delete delete

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