ANTITUMOR DRUG COMPRISING BETA-CYCLODEXTRIN
20170296573 · 2017-10-19
Inventors
Cpc classification
A61K31/7004
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61K45/00
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K31/7004
HUMAN NECESSITIES
International classification
A61K45/06
HUMAN NECESSITIES
Abstract
The present invention relates to an antitumor drug comprising β-cyclodextrin or a derivative thereof. In addition, the present invention relates to β-cyclodextrin characterized by the combination use with another antitumor drug, an antitumor drug comprising the combination, a combination therapy with β-cyclodextrin and another antitumor drug for treating cancer or the like, etc.
Claims
1. A method for treating an antitumor agent, comprising administering β-cyclodextrin or its derivative, and 2-deoxyglucose to a patient in need thereof.
2. The method of claim 1 which further comprises administering one or more other antitumor agents.
3. The method of claim 2 wherein the other antitumor agents comprise an antitumor agent having apoptosis-inducing activity.
4. The method of claim 1 wherein the β-cyclodextrin or its derivative is administered at the same time as, prior to, or after administering 2-deoxyglucose.
5. The method of claim 1 wherein the β-cyclodextrin or its derivative is administered 1 to 2 hours after administering 2-deoxyglucose.
6. The method of claim 2 wherein the β-cyclodextrin or its derivative is administered at the same time as, prior to, or after administering the other antitumor agents.
7. The method of claim 1 wherein the β-cyclodextrin derivative is selected from the group consisting of methyl-β-cyclodextrin (MBCD), (2-hydroxypropyl)-β-cyclodextrin (HPBCD), carboxymethyl-β-cyclodextrin, carboxymethyl-ethyl-β-cyclodextrin, diethyl-β-cyclodextrin, dimethyl-β-cyclodextrin, glucosyl-β-cyclodextrin, hydroxybutenyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, maltosyl-β-cyclodextrin, random methyl-β-cyclodextrin, sulfobutylether-β-cyclodextrin, 2-selenium-bridged β-cyclodextrin, and 2-tellurium-bridged β-cyclodextrin.
8. The method of claim 1 wherein the antitumor agent having apoptosis-inducing activity is selected from the group consisting of an agent that can release Bak from Mcl-1 and/or Bcl-xL which are anti-apoptosis proteins, a Fas-related apoptosis-inducing ligand, and a TNF-related apoptosis-inducing ligand (TRAIL).
Description
BRIEF DESCRIPTION OF DRAWINGS
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DESCRIPTION OF EMBODIMENTS
[0051] β-Cyclodextrin (bCD) has a conical molecular structure composed of 7 linking sugar chains. In the present invention, β-cyclodextrin means β-cyclodextrin itself as well as its derivatives. The derivatives herein mean β-cyclodextrins having various substituents, including methyl-β-cyclodextrin (MBCD), (2-hydroxypropyl)-β-cyclodextrin (HPBCD), carboxymethyl-β-cyclodextrin, carboxymethyl-ethyl-β-cyclodextrin, diethyl-β-cyclodextrin, dimethyl-β-cyclodextrin, glucosyl-β-cyclodextrin, hydroxybutenyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, maltosyl-β-cyclodextrin, random methyl-β-cyclodextrin, sulfobutylether-β-cyclodextrin, 2-selenium-bridged β-cyclodextrin, and 2-tellurium-bridged β-cyclodextrin. Preferred β-cyclodextrins include methyl-β-cyclodextrin (MBCD), (2-hydroxypropyl)-β-cyclodextrin (HPBCD), hydroxybutenyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, random methyl-β-cyclodextrin, and sulfobutylether-β-cyclodextrin; more preferably, β-cyclodextrin, methyl-β-cyclodextrin (MBCD), and (2-hydroxypropyl)-β-cyclodextrin (HPBCD). Besides bCD, 2-hydroxypropyl-γ-cyclodextrin (HPGCD) which has a good property of holding cholesterol, can be used in the present invention.
[0052] bCD or its derivatives used herein can be administered orally or parenterally such as by injection and intravenously.
[0053] The dose of bCD is not limited as long as it can inhibit the signal transmission between PI3K and AKT, not seriously affecting patients. For example, bCD can be administered in a dose of 2 to 5000 mg, preferably 2 to 100 mg, per treatment.
[0054] The antitumor agent of the present invention that is used in combination with bCD includes 2DG as well as an antitumor agent having apoptosis-inducing action, for example, an agent that can release Bak from Mcl-1 and/or Bcl-xL which are anti-apoptosis proteins, specifically, A-385358, ABT-199, ABT-263 (Navitoclax), ABT-737, AT-101, GX15-070 (obatoclax), HA14-1, oblimersen, and the like, but not limited thereto. Besides, a Fas-related apoptosis-inducing ligand, a TNF-related apoptosis-inducing ligand (TRAIL) and the like can be also used herein, which include, for example, a TRAIL and a derivative thereof (e.g. AMG951), or an antibody that can activate a TRAIL receptor (e.g. mapatumumab, lexatumumab).
[0055] In addition, the antitumor agent used herein includes an agent that can induce apoptosis through a signal arising between endoplasmic reticulum and mitochondria, in combination with 2DG-bCD, for example, an inhibitor for HSP90 inhibitors such as gamitrinibs, PU24FCl, PU-H58, PU-H71, and shepherdin; endoplasmic reticulum stress agents; thapsigargin and a derivative thereof such as G-202.
[0056] 2DG used herein can be administered orally or parenterally using an injection or infusion.
[0057] The dose of 2DG is not limited unless it can seriously affect patients. For example, 2DG can be administered in a dose of 100 to 5000 mg, preferably 500 to 2000 mg, per treatment.
[0058] The other antitumor agent having apoptosis-inducing activity can be administered orally or parenterally using an injection or infusion, but it is preferable to administer the agent according to the administration route approved for the agent.
[0059] It is preferable that the dose of the other antitumor agent having apoptosis-inducing activity is decided according to the dose approved for the agent, and the dose may be suitably reduced to suppress the side effect of the antitumor agent having apoptosis-inducing activity.
[0060] In addition, 2DG may be administered with glucose whose dose is preferably the equal amount of 2DG.
[0061] The suppression of the pro-survival signal by bCD is limited in only a few hours after bCD is administered. Accordingly, in case of the combination therapy with another antitumor agent, it is necessary to adjust the timing of administering the other antitumor agent to meet the time period that AKT is inactive. In case of the combination therapy with a general antitumor agent that develops its effect shortly after the administration, it is preferable to administer the antitumor agent at the same time as the administration of bCD, or about 0 to about 2 hours later. On the contrary, in case of the combination therapy with an antitumor agent that slowly develops its effect, it is preferable to administer the antitumor agent before the administration of bCD. For example, in case of the combination therapy of 2DG and bCD, 2DG takes 1 to 2 hours to develop its effect, and bCD develops its effect in minutes. Thus, it is preferable to administer 2DG firstly, and then bCD 1 to 2 hours later.
[0062] Dosage forms used herein includes tablets, capsules, granules, powders, liquids, syrups, and suspensions as an oral formulation; and injections and suppositories as a parenteral formulation. These formulations can be prepared according to a conventional method. Namely, the preparations such as tablets, capsules, liquid for oral administration may be prepared by a conventional method. Tablets may be prepared by mixing the active ingredient(s) with conventional pharmaceutical carriers such as gelatin, starches, lactose, magnesium stearate, talc, gum arabic, and the like. Capsules may be prepared by mixing the active ingredient(s) with inert pharmaceutical fillers or diluents and filling hard gelatin capsules or soft capsules with the mixture. Oral liquid preparations such as syrups or elixirs are prepared by mixing the active ingredient(s) with sweetening agents (e.g. sucrose), preservatives (e.g. methylparaben, propylparaben), colorants, flavors, and the like. The preparations for parenteral administration may also be prepared by a conventional method, for example, by dissolving the active ingredient(s) of the present invention in a sterilized aqueous carrier, preferably water or a saline solution. Tablets and granules may be coated according to a well-known method. These formulations may include another ingredient having a therapeutic effect. The active ingredient(s) may be contained in 0.1-70% (w/w) per the preparation.
[0063] The tumor herein means malignant tumor such as cancer, benign tumor, or neoplastic disease, which also includes hyperplasia that can be treated through the apoptosis induction of the present invention. Specific diseases of the tumor in the present invention include, but not limited thereto unless the diseases are intracerebral tumor, for example, fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelioma, lymphangiosarcoma, lymphangioendothelioma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, gastric cancer, esophageal cancer, rectal cancer, pancreatic cancer, ovarian cancer, prostate cancer, uterine cancer, cancer of the head and neck, skin cancer, squamous cell carcinoma, sebaceous adenocarcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, Wilm's tumor, cervical cancer, testicular cancer, small cell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, retinoblastoma, leukemia, lymphoma, Kaposi sarcoma, endometrial hyperplasia, focal nodular hyperplasia, prostatic hyperplasia, and primary hyperaldosteronism.
EXAMPLE
[0064] The reagents, test methods, etc. used in the following examples are shown below.
Reagents
[0065] As β-cyclodextrin (bCD), methyl-β-cyclodextrin (MBCD) was used for in vitro tests, and (2-hydroxylpropyl)-β-cyclodextrin (HPBCD) was used for in vivo tests.
[0066] Anti-β-PI3K antibody was obtained from Santa Cruz (sc-12929), oligoclonal anti-PI3K antibody (6HCLC) was obtained from Pierce, anti-PI3K Class II antibody (D3Q5B) was obtained from CST, anti-cytochrome c antibodies were obtained from BD Pharmingen (Cat. 556433 for blots and Cat. 556432 for microscopy), and all other primary antibodies were purchased from Cell Signaling.
[0067] Secondary antibodies conjugated with HRP were purchased from GE Healthcare and Alexa Fluor-conjugated secondary antibodies were obtained from Life Technologies.
[0068] IGF1, EGF, insulin, propidium iodide and β-cyclodextrin were purchased from Wako.
[0069] ABT-263 was purchased from Chemietek.
[0070] 2-Deoxy-D-glucose, MBCD and HPBCD were purchased from Sigma.
[0071] Pan-caspase inhibitor z-VAD was purchased from Promega.
Cell Lines and Cell Culture
[0072] Renal cell carcinoma cell lines stably transfected with empty vector RCC4 or with vector encoding VHL were gifted from the Harada Laboratory (Kyoto University Hospital, Dept. of Anesthesia), and the cell lines UOK121 and UOK121+VHL stably transfected with VHL expression vector were gifted from Dr. Marston Linehan (Center for Cancer Research, Urologic Oncology Branch, NCI).
[0073] Panc-1 pancreatic cancer cells and A431 epidermoid carcinoma cells were also cultured in high glucose DMEM supplemented with 10% serum.
[0074] Panc-1 cells were gifted from Dr. Koji Yamada (Dept. of Bioscience and Biochemistry, Faculty of Agriculture, Kyusyu University, Japan) and A431 cells were gifted from Dr. Masaya Imoto (Dept. of Bioscience and Informatics, Faculty of Science and Technology, Keio University, Japan).
[0075] These cells and HeLa cells were all cultured in high glucose DMEM (4.5 g/ml) supplemented with 10% PBS.
[0076] The serum used herein was obtained from several different sources such as GE Health and Cosmo Bioscience.
Western Blot and Immunoprecipitation
[0077] We ran 20 μg of proteins per lane for western blots. We ran 8, 10, 12.5 and 15% gels depending on the size of proteins being detected by western blots. When there are two proteins having a similar size, gel is flowed in each protein to make western blot analysis about each protein. The immunoprecipitation was undergone by adding 200 μg of protein from solubilized cells and Protein G Sepharose or Protein A Sepharose (Sigma P3296/P9242) pre-conjugated with immunoprecipitation antibody to a buffer for immunoprecipitation, and slowly rotating the tube containing the sample buffer at 4° C. overnight. The buffer for immunoprecipitation comprises 20 mM Tris Ph 7.5, 1% Triton-X100, 150 mM NaCl, phosphatase inhibitor cocktail (Cell Signaling (#58709S)), and 10% glycerol. Next morning, the content in the tube was centrifuged. The precipitate was washed with the same buffer twice and dissolved in SDS buffer. The precipitate in the solution was separated with 15% SDS-PAGE or 12.5% SDS-PAGE and analyzed by Western blotting.
FACS Analysis
[0078] After apoptosis was chemically induced, cells were washed in PBS and re-incubated in regular medium and incubated overnight. The Cell Death Assay was performed the next morning using the Propidium Iodide Incorporation assay. Cells were analyzed with BD FACS Canto II or FACS Calibur II. Results were analyzed using FlowJo. These experiments were done in triplicate, and error bars indicate the standard deviations.
Live Cell Counts
[0079] Dead and live cells were counted by trypan-blue dye exclusion methods. These experiments were done in triplicate, and the error bars indicate the standard deviation.
Cytochrome c Release Assay
[0080] RCC4 cells grown and treated on glass cover slips were first fixed with 3.7% formaldehyde in PBS for 10 minutes. Then the cover slips were briefly exposed to 100% methanol kept in a −20° C. freezer. Cover slips were incubated with 10% serum in PBS before being stained with mouse anti-cytochrome c antibody overnight. Next morning, the cover slips were washed and blocked with 10% serum in PBS for 30 minutes. Then they were exposed to secondary anti-mouse antibody conjugated with AF488 for 30 minutes. They were washed again and exposed to propidium iodide (1 μg/mL in PBS) for 15 minutes to stain DNA in the nucleus before being washed and mounted on the glass slides. We used a Keyence BZ9000 microscope with a 100× objective lens for observation.
Mouse Xenografts
[0081] Twelve-week-old NSG mice (JAX™ Mice strain NOD. Cg-Prkdc.sup.scid I12rg.sup.tm1Wj1/SzJ obtained from Charles River, Japan) were engrafted with 5×10.sup.6 UOK121 cells in 0.2 ml 50% matrigel (Falcon 356234) s.c. in the lower left or right flank. Tumor-bearing mice were divided into four or five treatment groups (the first experiment and the second experiment, respectively, having at three mice to a group). They were treated orally with either 2 mg 2DG and 2 mg glucose in 0.2 ml PBS, or 2 mg HPBCD in 0.2 ml PBS. ABT-263 was initially administered orally (2 mg/kg ABT-263 in 10% ethanol, 30% polyethylene glycol 400 (Wako), and 60% Cremphore EL (Sigma). First, the 2DG/glucose mixture was administered, then two and half hours later, mice were treated with HPBCD, and thirty minutes later, mice were treated with ABT. Some mice were given all these reagents while others were given a subset of them or none at all. The first week, mice were treated twice. For the subsequent three weeks, mice were treated three times a week. Tumor size was measured three to four times a week by electronic calipers (volume=(length×width.sup.2)/2). A group of three mice were treated for each treatment condition, and the next day, tumor sizes were recorded. Error bars in the graph indicate the standard deviations. NSG mice without the UOK121 xenograft were also treated with the triple drug combination and their weights were recorded. Using blood from the tail vein, leukocyte, erythrocyte, and platelet in the blood were counted with Horiba Hematology Analyzer LC-152, and the blood glucose levels were measured with MediSafe Mini (Terumo, Japan).
Example 1
Effect of bCD (In Vitro)
[0082] Using HeLa cells which express both EGFR and IGF1R, the effects of bCD were tested about the decreases of EGF-stimulation and IGF1-stimulation to the cells. The bCD used herein was MBCD.
(Method)
[0083] (Test A) HeLa cells were incubated in serum-free medium with 0, 1.75, 3.5, and 7.0 mM bCD for 30 minutes, which were prepared in duplicate in each bCD concentration. Among the both duplicate sets, one set of the media was stimulated with 20 ng/mL IGF1 for 20 minutes, and the other set was used as its control group. The cells were harvested and analyzed by Western Blots for phosphor-serine AKT and AKT, in which the phosphorylation of AKT was used as an indicator of the AKT activation.
(Test B and Test C) HeLa cells were incubated in serum-free medium for 30 minutes with 10 mM bCD, and a control group (untreated) was also prepared. These cells were then challenged with 100 ng/mL EGF for 0, 2.5, and 5.0 minutes in Test B, and with 10 ng/mL IGF1 for 0, 5, and 10 minutes in Test C. The cells were harvested and analyzed by Western Blots about EGFR, IGF1R, ERK, PI3K, and AKT, as well as each phosphorylation thereof. Anti PI3K Class II antibody was used for the detection of PI3K.
(Result)
[0084] The result is shown in
[0085] The results in Test A showed that 7 mM bCD was enough to completely block the process that IGF1 generates signal to AKT (
[0086] In Tests B and C, HeLa cells untreated with bCD were activated undoubtedly in both the tests. The bCD-treated cells were clearly activated in any EGFR, IGF1R, and PI3K, but AKT activations were considerably diminished (the last lanes in
[0087] Thus, it is thought that bCD can interfere the signal transduction from PI3K to AKT.
(Discussion)
[0088] Most of RTKs activate two distinct signal transduction cascades: the RTK-Ras-ERK proliferation pathway and the RTK-PI3K-AKT pro-survival pathway.
[0089] At the same time, the RTK-Ras-ERK signals seem to be unaffected (the second boxes in
[0090] Thus, bCD disrupted the signal transduction between PI3K and AKT, and diminished PI3K-AKT pro-survival signals generated by these RTKs while leaving the Ras-ERK proliferation signals intact.
Example 2
Synergy effect of bCD and 2DG (1)
[0091] VHL-defective renal cancer cells such as RCC4 cells are less sensitive to 2DG-ABT largely because they express IGF1R. In order to see whether 2DG-ABT combined with bCD would increase its efficacy, it should be considered that 2DG stimulates AKT phosphorylation in many cancer cell lines. Thus, we first tested whether the dual treatment of 2DG with bCD would increase or decrease AKT phosphorylation in RCC4 cells.
[0092] Since it generally takes 1-2 hours for the effect of 2DG to become noticeable, whereas bCD works within 30 minutes as shown in Example 1, RCC4 cells in serum free media are first treated with 2DG for 2 hours, and in the last 30 minutes, the cells are also treated with bCD. The bCD used herein was MBCD.
(Method A)
[0093] RCC4 cells were incubated in serum free media for 2 hours with or without 10 mM 2DG. In the last hour, 10 mM bCD was added to each one subset of the cells treated/untreated with 2DG. Then 0-30 ng/ml IGF1 was added and the incubation continued for 5 minutes before the cells were harvested and analyzed by Western blotting.
(Result A)
[0094] The result is shown in
Example 3
Synergy Effect of bCD and 2DG (2)
[0095] In the above Example 2, the cells were incubated in serum-free medium and then stimulated with a particular growth factor, in order to test the effects of bCD on only a particular RTK. However, serum generally contains multiple growth factors as well as insulin that could activate multiple RTKs expressed in these cells. Thus, in order to test whether bCD still modulates the PI3K-AKT signals while they are continuously stimulated by serum, we treated RCC4 cells with bCD, with 2DG and with their combination, and examined the phosphorylation status of AKT. The bCD used herein was MBCD.
(Method B)
[0096] RCC4 cells were incubated with or without 10 mM 2DG for 2 hours in the presence of 10% serum. In the last 30 minutes, each one subset of cells treated/untreated with 2DG was exposed to 10 mM bCD before the cells were harvested and analyzed.
(Method C)
[0097] HeLa cells were incubated with 10 mM bCD for one hour in the presence of 10% serum before the cells were washed and re-incubated in medium containing 10% serum for the indicated period (˜120 minutes).
(Result)
[0098] The result of Method B is shown in
[0099] However, it did not take long for the AKT activities to come back when the cells were returned to media without bCD but containing 10% serum (
Example 4
Effect of bCD for IGF1-Induced Hypoglycemia (In Vivo)
[0100] Several studies had shown that when one of the bCD derivatives, hydroxypropyl-β-cyclodextrin (HPBCD) is injected into mice, the amount of bCD that remains in circulation four hours later would about 50% (J Inherit Metab Dis 2013, 36 (3): 491-498; Toxicol Pathol 2008, 36 (1): 30-42). Thus, to take advantage of bCD-induced absence of pro-survival signals for cancer therapies, apoptosis needs to be induced fairly quickly. In order to see whether bCD affects PI3K-AKT pathways in animals, we did the following test. The bCD used herein was HPBCD.
(Method D)
[0101] Five-hour starved mice were either pre-treated with 40 μg bCD for 30 minutes, or without. They were then injected with 100 ng IGF1. After thirty minutes, the blood glucose levels were measured by drawing blood from each mouse tail. The experiments were performed in triplicate samples and the error bars indicate the standard deviation. The mice were all about 20 g.
(Result)
[0102] The result is shown in
Example 5
Synergy Effect of bCD and 2DG for Promoting ABT-Induced Apoptosis
[0103] We tested if bCD in combination with 2DG-ABT can promote its apoptosis induction.
(Method E)
[0104] A subset of RCC4 cells was pre-incubated with 10 mM 2DG for 2 hours in the presence of 10% serum, another subset was pre-incubated with 10 mM bCD for 30 minutes in the presence of 10% serum, and yet the other subset was treated with both. And, a control group (Untreated) was also prepared. One hour after ABT addition indicated in
(Result)
[0105] The result is shown as a graph in
[0106] Using 1 μM ABT-263, the 2DG-bCD-ABT combination induced apoptosis in about 95% of RCC4 cells.
[0107] Using the data from
Example 6
Effect of 2DG-bCD-ABT Triple Combination Across a Broad Spectrum of Cancer Cells
[0108] First, we tested the effects of bCD on AKT pro-survival signals on several cancer cell lines in Test A. Regarding A431 epidermoid carcinoma cells, we also analyzed EGF, ERK1/2, PI3K, and each phosphorylated form thereof by Western Blotting in Test B. Next, we tested the apoptosis effect in combination with ABT in Test C. The bCD used herein was MBCD.
(Method)
[0109] (Test A) HeLa cervical cancer cells, UOK121 renal cancer cells, Panc-1 pancreatic cancer cells, and A431 squamous cancer cells were untreated, pre-treated with 10 mM 2DG, co-incubated with 10 mM bCD for the last 30 minutes, or both-treated with the 10 mM 2DG and the 10 mM bCD, in the presence of 25 mM glucose contained in the media. Each cell was harvested and analyzed by Western Blotting.
(Test B) The same samples of A431 cell lysates in Test A were analyzed for EGFR activation, ERK1/2 activation, and PI3K activation. For PI3K detection in A431 cells, anti-PI3K p85 oligoclonal antibody (6HCLC) was used.
(Test C) Untreated cells and cells treated with 2DG and bCD were prepared in the same manner as Test A. Cells incubated with 1 μM ABT for 2 hours and cells treated with all of the treatments of 2DG, bCD and ABT were also prepared. The cells were harvested and the live cells were counted by trypan-blue dye exclusion assay. Error bars indicate standard deviations from triplicate samples.
(Result)
[0110] As expected, bCD attenuated AKT phosphorylation in HeLa cervical cancer cells, UOK121 renal cancer cells, Panc-1 pancreatic cancer cells, and A431 squamous cancer cells. In all, bCD attenuated AKT pro-survival signals (
[0111] Test B was performed for evaluating the effect of bCD and/or 2DG for A431 squamous cancer cells that are known to overexpress EGFR among the cancer cells used in Test A, and the results thereof showed that EGFR, ERK1/2, and PI3K were all activated (
[0112] According to the result in Test C, the triple combination including ABT induced apoptosis in all the cell lines very effectively. Considering the results of Tests A and B, therefore it was suggested that the activity of ABT for inducing apoptosis can be enhanced by attenuating AKT pro-survival signals.
[0113] In the result of Panc-1 cells in Test C, there was not any noticeable difference between the double combination of 2DG/bCD and the triple combination including ABT, but it is thought to be because Panc-1 cells cannot take ABT intracellularly.
Example 7
Analysis of Mechanism of Apoptosis Induction With Triple Combination of 2DG-bCD-ABT
[0114] The results in the above examples show that the combination of 2DG and bCD can give a synergistic effect with overwhelming probability. In order to ensure the synergistic effect molecular-biologically, the following protocol of tests to investigate where in the cells the synergistic effect is observed was built. Firstly, proteins that precipitate with Bak were harvested and analyzed by Western Blots to identify when Mcl-1 and Bcl-xL are deleted from the Bak complex, and the microscopic examination was made to investigate when cytochrome c is released from mitochondria.
(Method)
[0115] (A and B) RCC4 cells were treated with 2DG, bCD, 2DG +bCD, or left untreated as done in Example 6. Approximately 20 μg of the whole cell lysates (WCL) were analyzed by western blotting. Bak- and Bcl-xL-bound proteins were immune-precipitated from approximately 200 μg of cell lysates and analyzed by western blotting.
(C) 3 μM ABT with or without the 20 μM pan-caspase inhibitor z-VAD was added to the cells pre-treated with the combination of 2DG and bCD over 2 hours, and analyzed by Western blotting for caspase 9. Cleaved caspase 9 was indicated by cC9.
(D) 200 μg from the last two samples were used to precipitate Bak-bound proteins and blotted for BcL-xL and Bak.
(E) RCC4 cells treated with 2DG-bCD (left panel), RCC4 cells treated with 2DG-bCD-ABT (middle panel), and RCC4 cells treated with 2DG-bCD-ABT in the presence of 20 μM pan-caspase inhibitor z-VAD (right panel) were immunostained using anti-cytochrome c antibody and GFP-conjugated anti-mouse antibody. Nuclei were stained red with propidium iodide.
[0116] The punctate green spots appearing in cells treated with both 2DG and bCD (left panel) represent mitochondria-localized cytochrome c, while the defused stains in both middle and right panels represent cytochrome c released from mitochondria. Note: cytochrome c release takes place only after the addition of ABT. The graphic illustration of the protocol for this and other experiments is found in
(Result)
[0117] The result is shown in
[0118] In contrast, ABT bound directly to Bcl-xL and caused the dissociation of the Bak-Bcl-xL complex (
[0119] Thus, the release of cytochrome c took place only after the addition of ABT, and it also took place in the presence of a caspase inhibitor, namely even in the absence of caspase activation (
[0120] We note that both the full release of cytochrome c and full caspase 9 activation were observed within 2 hours of ABT addition, and the apoptosis proceeded in the final step within 4 hours after first administering 2DG.
[0121] Considering these results, it has been found that both of 2DG and bCD are indispensable to sensitize cells to apoptosis caused by the release of Bak from Mcl-1 which is one of inhibitory factors of apoptosis. Namely, the synergistic effect of 2DG and bCD can release Mcl-1 from the Bak complex. In addition, it has been also found that when ABT is added, ABT binds Bcl-xL to delete Bcl-xL from the Bak complex, Bak is released from all the inhibitory factors to be activated, cytochrome c is released from mitochondria, and then apoptosis starts (see,
Example 8
Effect of 2DG-bCD-ABT Causing Tumor Regression In Vivo
(Method)
[0122] (A) UOK121 cells which were human-derived cancer cells were grafted into mice, and the treatment was begun on the 7th day according to the protocol (in vivo) in
(B) As an advanced assessment of (A), mice were divided into 5 treatment groups of untreated, or treated with 2DG-ABT, HPBCD, HPBCD-ABT, or 2DG-HPBCD-ABT, and they were treated 8 times from day 10 to day 30.
(Result)
[0123] Only in the group treated with the triple combination, tumor regression was observed (
[0124] The advanced assessment showed that only the mice treated with the triple combination responded to the treatment and tumors remained small (
Example 9
Effect of 2DG-bCD-TRAIL in Inducing Apoptosis in Pancreatic Cancer Cells
[0125] We hypothesized that 2DG-bCD can be combined with apoptosis inducers other than Bcl-2 antagonists, such as Fas and TNF-related apoptosis-inducing ligands (TRAIL), for efficient cancer therapy. There are reports suggesting that bCD does not interfere with death receptor activation of caspase 8, while bCD alone or together with 2DG clearly sensitizes mitochondria for induced cytochrome c release (Molecular and cellular biology 2002, 22 (1): 207-220). Thus, in type II cells, TRAIL may benefit from having mitochondria sensitized so that both the extrinsic and intrinsic pathways of apoptosis may be activated. In order to confirm the hypothesis, we did the following test.
(Method)
[0126] (A) Panc-1 cells were treated with the standard 2DG-bCD-TRAIL protocol (
(B) Western blots of Panc-1 cells left untreated, treated with TRAIL, treated with 2DG-bCD combination, and treated with the combination of 2DG-bCD-TRAIL as depicted in
(Result)
[0127] The result is shown in
[0128] However, when these cells were pre-treated with 2DG-bCD, the same 10 ng/ml TRAIL was enough to induce apoptosis in 90% of the cells. Thus, 2DG-bCD clearly sensitized Panc-1 cells for TRAIL-mediated apoptosis, suggesting that 2DG-bCD-TRAIL may be an effective treatment for pancreatic cancer.
Example 10
Effect of Different Kind of Cyclodextrin
[0129] We tested the effect of α-, β-, and γ-cyclodextrins on the activation of AKT and the apoptosis induction using 2DG-ABT.
(Method)
[0130] (Test A) UOK121 cells were incubated in 10% serum medium with 5 mM or 10 mM α-, β-, and γ-cyclodextrins for 45 minutes. The same medium without any cyclodextrin was also incubated as a control. The cells were harvested and analyzed by Western Blots for the phosphorylation of AKT and ERK1/2 as an indicator of the AKT and ERK1/2 activations.
(Test B) 10 mM 2DG was added to a medium of U0K121 cells in the presence of 25 mM glucose, and the medium was incubated for 1.5 hours. To the medium was added 10 mM α-, β-, or γ-cyclodextrin, and the medium was incubated for 30 minutes. Then, 0.3 μM ABT-263 was added thereto, and the medium was further incubated for 2 hours. Separately, a control that includes only α-, β-, or γ-cyclodextrin without 2DG or ABT-263, and another control that includes neither α-, β-, or γ-cyclodextrin, nor 2DG or ABT-263 were prepared. 2.5 hours after adding each cyclodextrin (i.e., 2 hours after adding ABT-263), all the cells were washed with the medium twice, and next day the live cells were counted by trypan-blue dye exclusion assay.
(Result)
[0131] The result is shown in