Inducing caspase activity

Abstract

To provide methods of inducing caspase activity.SOLUTION: The method comprises contacting a cell with a treatment compound formed by alkoxylation of an initiator using an oxide. The treatment compound is exemplified by Formula III below. In the formula, n is independently 1 to 303.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Embodiments of the present disclosure are directed to methods of inducing caspase activity. [Background technology]

[0002] Cancer is a group of diseases involving abnormal cell growth. Colorectal cancer, which may also be called colon cancer or intestinal cancer, is cancer that results from uncontrolled cell growth in the colon or rectum.

[0003] Colorectal cancer is a commonly diagnosed malignant tumor.The treatment for colorectal cancer can include surgery, radiotherapy and / or chemotherapy.However, there is still a need for new methods and / or new compositions that can be used for treatment. Summary of the Invention [Problem to be solved by the invention]

[0004] The present disclosure provides a method of inducing caspase activity, comprising contacting a cell with a treating compound formed by alkoxylation of an initiator with an oxidizing agent.

[0005] The above summary of the present disclosure is not intended to describe each disclosed embodiment or every implementation of the present disclosure. The following description more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves merely as a representative group and should not be interpreted as an exclusive list. DETAILED DESCRIPTION OF THE INVENTION

[0006] Without wishing to be bound by theory, one mechanism involved in the development of colorectal cancer is mutation of the APC (adenomatous polyposis coli) gene, which produces the APC protein. The APC protein is part of a protein-based destruction complex that helps prevent the accumulation of β-catenin protein within cells. The APC and β-catenin proteins are part of the WNT (wingless / integrated) signaling pathway, which transmits signals to cells via cell surface receptors. Generally, when cells are stimulated by WNT, the destruction complex is inactivated, and β-catenin enters the nucleus and binds to transcription factors (TCFs) that control the transcription of genetic information. Genes involved in normal cell progression are activated, a regulated process. In the absence of the APC protein, β-catenin protein continuously accumulates at high levels, translocates to the nucleus, and binds to TCFs. TCFs then bind to DNA and can activate the transcription of proto-oncogenes. When proto-oncogenes are inappropriately expressed at high levels, they become oncogenes. Activated oncogenes can cause cells designated for apoptosis in an individual to survive and instead proliferate, which can lead to the development of colorectal cancer.

[0007] Disclosed herein is a method for inducing caspase activity. Advantageously, inducing caspase activity can induce apoptosis, i.e., induce cell death. In some applications, apoptosis is more desirable than necrosis. Inducing caspase activity can degrade some intracellular proteins, leading to cell death. Cell death by apoptosis can have a desirable effect on, for example, colorectal cancer cells.

[0008] As used herein, "a," "an," "the," "at least one," "some," and "one or more" can be used interchangeably unless otherwise indicated. The term "and / or" means one, more than one, or all of the listed items. The recitation of numerical ranges by endpoints includes all numbers subsumed within that range, for example, 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.

[0009] The methods of inducing caspase activity disclosed herein include contacting a cell with a treatment compound. As used herein, "treatment compound" refers to a compound that can be formed by alkoxylation of an initiator with an oxidizing agent.

[0010]

[0010] Embodiments of the present disclosure provide that the initiator comprises a compound containing three or more available reactive hydroxyl groups, amine groups, or a combination thereof. One or more embodiments provide that the initiator can be selected from glycerol, diglycerol, triglycerol, hexaglycerol, tripentaerythritol, trimethylolpropane, sorbitol, ethylenediamine, triethyleneamine, 2,2-bis(hydroxymethyl)-1,3-propanediol, ethanolamine, and combinations thereof.

[0011] An embodiment of the present disclosure provides that the oxide may be selected from ethylene oxide, propylene oxide, butylene oxide, and combinations thereof.

[0012] One or more embodiments of the present disclosure provide that the treating compound formed by alkoxylation of an initiator with an oxide can be represented by Formula I below: [ka] In the formula, each n is independently 1 to 303.

[0013] An example of a treating compound represented by Formula I is trimethylolpropane ethoxylate.

[0014] One or more embodiments of the present disclosure provide that the treating compound can be represented by the following Formula II: [ka] In the formula, each n is independently 1 to 227.

[0015] An example of a treating compound represented by Formula II is a four-arm poly(ethylene glycol).

[0016] One or more embodiments of the present disclosure provide that the treating compound can be represented by the following Formula III: [ka] In the formula, each n is independently 1 to 303.

[0017] An example of a treating compound represented by formula III is glycerol ethoxylate.

[0018] Embodiments of the present disclosure provide that the treatment compound has a number average molecular weight (Mn) of 400 to 40,000 g / mol. All individual values and subranges between 400 g / mol and 40,000 g / mol are included, for example, the treatment compound can have an Mn from a lower limit of 400, 450, 500, 600, 700, 800, 900, or 1000 g / mol to an upper limit of 40,000, 30,000, 20,000, 15,000, or 10,000 g / mol.

[0019] The treated compounds can be prepared using known methods, equipment, and / or conditions, which may vary for different applications, for example, by alkoxylation of an initiator with an oxide. The treated compounds can be commercially available.

[0020] As described above, the method of inducing caspase activity disclosed herein includes contacting cells with a treatment compound. One or more embodiments of the present disclosure provide that contacting cells with the treatment compound occurs in vivo. One or more embodiments of the present disclosure provide that contacting cells with the treatment compound occurs in vitro. Cells can be contacted with the treatment compound by utilizing several different known methods, devices, and / or conditions. Various methods, devices, and / or conditions can be utilized for different applications.

[0021] The treatment compound can be used with a known treatment medium. For example, the treatment compound can be dissolved in a known treatment medium before contacting the cells to provide an effective amount. One or more embodiments provide that the treatment compound and the treatment medium can be combined to form a solution. The solution can be a homogeneous solution. Examples of treatment media include, but are not limited to, DMEM (Dulbecco's Modified Eagle's Medium), RPMI 1640, and McCoy's 5A, among others, and combinations thereof. Several treatment media are commercially available.

[0022] The treatment compound can have a concentration in the treatment medium from 0.001 millimolar (mM) to 75 mM, including all individual values and subranges between 0.001 mM and 75 mM, for example, an effective concentration of the treatment compound can range from a lower limit of 0.001, 0.005, 0.01, 0.1, or 1.0 mM to an upper limit of 75, 72, 70, 68, or 65 mM in the treatment medium.

[0023] The cells may be contacted with an effective amount of the treatment compound. As used herein, the term "effective amount," which may be used interchangeably with "therapeutically effective amount" and / or "therapeutic amount," refers to an amount of the treatment compound sufficient to provide the intended use, e.g., induce caspase activity. Contacting the cells with an effective amount of the treatment compound can desirably provide disease treatment, e.g., colorectal cancer treatment, in which undesirable cells undergo apoptotic cell death due to the induction of caspase activity. The effective amount may vary depending on the particular use, e.g., in vitro or in vivo, the subject being treated, e.g., the subject's weight and age, the severity of the condition, and / or the method of administration, among other considerations that can be easily determined by those skilled in the art. As used herein, the "subject" being treated refers to any member of the animal kingdom, e.g., a mammal, including a human.

[0024] Embodiments of the present disclosure provide that the specific dose may vary depending on the particular treatment compound utilized, the administration regimen followed, the timing of administration, and / or the physical delivery system the treatment compound is delivered in. For example, an effective amount of the treatment compound may be contacted with the cells via a single administration or multiple administrations.

[0025] The present disclosure provides that the cell that is contacted with the treatment compound is a cancerous cell.For example, the cell can be a colorectal cancer cell.Colorectal cancer cells can also be referred to as colon cancer cells, intestinal cancer cells, and / or colorectal adenocarcinoma cells.One or more embodiments of the present disclosure provide that additional cells, i.e., non-cancerous cells, can be contacted with the treatment compound.

[0026] Without intending to be bound by theory, caspases, which may also be referred to as cysteine-aspartic acid proteases, are a family of cysteine proteases involved in apoptosis. There are two types of caspases: initiator caspases, including caspases 2, 8, 9, 10, 11, and 12, and effector caspases, including caspases 3, 6, and 7. One or more embodiments of the present disclosure provide that contacting cells with a treatment compound induces effector caspase activity. One or more embodiments of the present disclosure provide that the caspase is selected from caspase 3, caspase 6, caspase 7, or a combination thereof.

[0027] As mentioned above, inducing caspase activity can advantageously induce apoptosis. Induced caspase activity can be determined by several different known methods, instruments, and / or conditions. For example, induced caspase activity can be demonstrated by an average relative caspase activity greater than 1, as determined by, for example, Caspase-Glo 3 / 7 Assay 4.B. Standard Protocol for Cells in a 96-well Plate, available from Promega. As used herein, "relative caspase activity" can be used interchangeably with relative apoptosis.

[0028] Utilizing the treating compounds discussed herein may advantageously provide improved, i.e., reduced, laxative effects compared to some other polymeric compounds utilized in cancer treatment, which may help provide a desirable increase in patient compliance compared to some other polymeric compounds associated with greater laxative effects.

[0029] One or more embodiments of the present disclosure provide a method of treating colorectal cancer. The method can include contacting colorectal cancer cells with a treatment compound.

[0030] One or more embodiments of the present disclosure provide a method of treating cancer, which can include administering a treatment compound to a mammal. [Example]

[0031] In the present examples, various terms and names for materials are used, including, for example:

[0032] Trimethylpropane ethoxylate (Mn: 1014 g / mol, obtained from Sigma-Aldrich)

[0033] 4-arm poly(ethylene glycol) (Mn: 10,000 g / mol, obtained from Sigma-Aldrich)

[0034] Glycerol ethoxylate (Mn: 1000 g / mol, obtained from Sigma-Aldrich)

[0035] Cells (human colon, colorectal adenocarcinoma, HT-29 (ATCC® HTB-3), obtained from ATCC)

[0036] McCoy's 5A (growth medium, obtained from ThermoFisher Scientific)

[0037] Fetal bovine serum (obtained from ATCC)

[0038] Dulbecco's phosphate-buffered saline (GIBCO 14190-144, obtained from ThermoFisher Scientific)

[0039] Complete Growth Medium (ATCC® 30-2007, obtained from ATCC)

[0040] Trypsin-EDTA (GIBCO Trypsin-EDTA (0.25%), catalog number 25200056, obtained from ThermoFisher Scientific)

[0041] Thiazolyl blue tetrazolium bromide (obtained from ThermoFisher Scientific)

[0042] Dulbecco's phosphate-buffered saline with calcium and magnesium (GIBCO 14040-133, obtained from ThermoFisher Scientific)

[0043] Caspase-Glo 3 / 7 Assay (luminescent assay, catalog number G8093, obtained from Promega)

[0044] Dimethyl sulfoxide (catalog number 276855, obtained from Sigma-Aldrich)

[0045] Culture initiation and maintenance Cultures were initiated and maintained as follows: Cultures were initiated and maintained according to the "Thawing, Propagating, and Cryopreserving Protocol" NCI-PBCF-HTB38 (HT-29) Colon Adenocarcinoma (ATCC® HTB-38™); February 27, 2012; Version 1.6.

[0046] HT-29 (ATCC® HTB-38™) cells (approximately 1 x 10 per mL) 6HT-29 cells were seeded into T-25 flasks containing McCoy's 5A and fetal bovine serum (10% (v / v)). HT-29 cells were then propagated using ATCC® 30-2007 (warmed in a 37°C water bath for at least 15 minutes). Cells were grown in a humidified incubator (SANYO INCT-16-CMT, MCO-19AIC(UV)) maintained at 37°C and 5% CO2. Cells were then rinsed with 1x Dulbecco's phosphate-buffered saline and subcultured 1-3 times per week in T-75 flasks using 1x trypsin-EDTA (applied for less than 5 minutes). The enzymatic action of trypsin-EDTA was blocked by adding complete growth medium to the detached cells. Upon reaching 80-90% confluency, cells were split at a split ratio ranging from 1:5 to 1:16. Subculture and growth activity, including passage number, % confluency, % viability (experimental setup day only), and cell morphology at all phases, were recorded. Cells were maintained in logarithmic growth phase.

[0047] Cell culture plating (day 0) Cell culture plating was performed as follows. Cell suspensions from a single 80-90% confluent T-75 flask were collected with trypsin-EDTA and complete growth medium. To obtain cell concentration and viability, cell counts were obtained using a COUNTESS automated cell counter (INVITROGEN C10227, CNTR-7-CMT) with 10 µL of 1:1 0.4% trypan blue dye (INVITROGEN T10282) and cell suspension in each of the two chambers of each slide. Cell counts and viability were averaged from both chambers of a single slide. Then, complete growth medium containing viable cells (defined as ≥90% viability) was plated into a sterile 96-well plate using a multichannel pipette. A cell density of 5,000–6,000 cells / well (40,000–48,000 cells / mL) was added to each well, except for wells that served as "saline only" cell-free controls. An equal volume of 125 μL of cell suspension was added per well, starting from row A through row H on the plate. The plates used for each of the two endpoints, apoptosis and cytotoxicity, were solid white and clear plates, respectively. Cells were incubated for 24 ± 2 hours to allow attachment.

[0048] Preparation of trimethylpropane ethoxylate / 4-arm poly(ethylene glycol) / glycerol ethoxylate stock Stock solutions were prepared in sterile saline at the target concentrations of trimethylolpropane ethoxylate, four-arm poly(ethylene glycol), and glycerol ethoxylate. Based on the solubility limits of their high molecular weights, lower stock concentration preparations (w / v) were prepared as needed to produce either solutions or pipettable suspensions, or solubilization was achieved by incremental addition of saline, continuous mixing, vortexing, sonication, or agitation prior to use in the assay. When necessary for solubilization, saline was preheated to 37°C before mixing with trimethylolpropane ethoxylate, four-arm poly(ethylene glycol), and / or glycerol ethoxylate. On the day of cell suspension plating (day 0), a total volume of 10 mL was prepared for each test substance.

[0049] Preparation of cytotoxic reagents Thiazolyl blue tetrazolium bromide was prepared at 5 mg / mL in Dulbecco's phosphate-buffered saline containing calcium and magnesium. A total volume of 30 mL (w / v) was prepared on each day (day 0) and stored at 4°C until use.

[0050] Preparation of dosing solutions (Day 0) Dosing solutions / suspensions of each test substance stock were prepared in a total of 15 mL each in McCoy's 5A and 1% fetal bovine serum. Varying volumes of dosing stock were utilized to achieve dosing solutions / suspensions ranging from 0.0015 to 60 mM. Dosing solutions / suspensions were prepared in sterile reservoirs and mixed repeatedly with a pipette until visible homogeneity was achieved. Using a sterile 96-deep-well block with a 2 mL capacity, 2 mL of dosing solution / suspension was added to each of six replicate wells for the treatment group, as well as to each of 12 wells for the saline-only cell control and the saline-only "no cells" background correction control. The plates were established according to a semi-randomized statistical design. Each test substance was identified numerically and by a color code used to identify the treated well. The block was covered with sealing tape, the plate lid, and placed in a 4°C laboratory refrigerator (Fischer Scientific, 135B1, RFR-22-CMT) overnight.

[0051] Treatment (Day 1) All 96-deep-well blocks containing the dosing solutions / suspensions were removed from the refrigerator and placed in a 37°C bead bath for a minimum of 30 minutes. Approximately 24 hours after plating, the well plates were removed from the incubator and processed one by one. All wells of the cell plate were aspirated using a 6-well aspiration device, starting from row A through row H. Using a multichannel pipette, 100 μL of each dosing solution / suspension (from the block) was added to each well of the 96-well cell treatment plate, starting from row A through row H (in the same order). To prevent wells from drying out and to maintain cell attachment and viability, all wells were aspirated and processed two rows at a time, with pipette tips changed between rows. All plates were placed in the incubator and allowed to process for 24 ± 2 or 48 ± 2 hours before harvesting.

[0052] Collection (Days 2 and 3) apoptosis Apoptosis was assessed as follows: Apoptosis was assessed according to "Caspase-Glo 3 / 7 Assay" 4.B. Standard Protocol for Cells in a 96-Well Plate (Promega). The Caspase-Glo 3 / 7 Assay components were preheated to room temperature for approximately 60 minutes. The white plates were removed from the incubator (one at a time), and the treatment medium was aspirated. Using a multichannel pipette, 100 μL of 1× Dulbecco's phosphate-buffered saline was added to each well of the 96-well plate. The assay reagents (buffer and substrate) were manually mixed and added to the reagent reservoir, and using a multichannel pipette, 100 μL of the assay reagent mixture was added to each well of the 96-well plate. The plate(s) (protected from light with foil) were placed on a plate shaker and rotated at approximately 800 rpm for 5 minutes at room temperature. The plates were then incubated for an additional 25 minutes at room temperature before analysis. Luminescence was recorded in terms of relative light units (RLU) for each plate on a FLUOstar Omega plate reader.

[0053] cytotoxicity Cytotoxicity assessment was performed as follows. As described above, the cytotoxicity reagent (5 mg / mL) was prewarmed to room temperature for approximately 30 minutes and then diluted in 1x Dulbecco's phosphate-buffered saline containing calcium and magnesium to a final concentration of 0.675 mg / mL. The clear plates were removed from the incubator (one at a time), and the treatment medium was aspirated. Using a multichannel pipette, 200 μL of the cytotoxicity reagent (final) was added to each well of the 96-well plate. The plate was then covered with sealing tape and incubated in a humidified incubator at 37°C for 4 hours. After incubation, the supernatant was aspirated, and 200 μL of dimethyl sulfoxide was added to each well. After thorough mixing by repeated pipetting, the cell lysates were transferred to a new clear 96-well plate and quantified by absorbance at 600 nm and 630 nm on a FLUOstar Omega plate reader.

[0054] analysis Relative caspase activity was calculated as follows: (RLU フォアグラウンド )-(RLU 生理食塩水のみの「細胞なし」対照 )=(RLU バックグラウンド補正 ),

[0055] (RLU) of each test substance-containing well バックグラウンド補正 )) / (mean (RLU) of 12 saline-only control wells フォアグラウンド ) = relative caspase activity (where RLU is relative light units).

[0056] Relative caspase activity of each test substance-containing well / 6 replicates = average relative caspase activity. Results for the various concentrations utilized are reported in Tables 1, 3, and 5.

[0057] Cell viability was calculated as follows:

[0058] (Abs 600 フォアグラウンド )-(Abs 600 生理食塩水のみの「細胞なし」対照) =(Abs 600 バックグラウンド補正 )

[0059] (Abs 630 フォアグラウンド )-(Abs 630 生理食塩水のみの「細胞なし」対照) =(Abs 630 バックグラウンド補正 )

[0060] (Abs 600 バックグラウンド補正 )-(Abs 630 バックグラウンド補正) =(Abs 600-630 )

[0061] (Abs of each test substance-containing well) 600-630 )) / (mean of 12 saline-only control wells (Abs 600-630 )=cell viability%

[0062] % cell viability of each test substance-containing well / 6 replicates = mean % cell viability. Results for the various concentrations utilized are reported in Tables 2, 4, and 6. [Table 1]

[0063] The data in Table 1 demonstrate that exposure of cells to concentrations of 15 mM, 30 mM, and 60 mM trimethylpropane ethoxylate provided favorable relative caspase activities, i.e., average relative caspase activities greater than 1, as indicated by the respective mean relative caspase activity (Runs 1, 2, 3, 4) values. [Table 2]

[0064] The data in Table 2 illustrate that exposure of cells to concentrations of 15 mM, 30 mM, and 60 mM trimethylpropane ethoxylate provided adequate viability, i.e., average % viability of 50% or more (Runs 1, 2, 3, 4). [Table 3]

[0065] The data in Table 3 demonstrate that exposure of cells to 1.5 mM, 3 mM, and 6 mM concentrations of 4-arm poly(ethylene glycol) provided favorable relative caspase activities, i.e., average relative caspase activities greater than 1, as indicated by the respective mean relative caspase activity (Run 1, 2) values. [Table 4]

[0066] The data in Table 4 illustrate that 24 hours after exposure of cells to 4-arm poly(ethylene glycol) at concentrations of 1.5 mM, 3 mM, and 6 mM provided adequate viability, i.e., average viability % of 50% or more relative to the mean viability % (Runs 1, 2). [Table 5]

[0067] The data in Table 5 demonstrate that exposure of cells to glycerol ethoxylate at concentrations of 15 mM and 30 mM provided favorable relative caspase activities, i.e., average relative caspase activities greater than 1, as indicated by the respective mean relative caspase activity (Runs 1, 2, 3, 4) values. [Table 6]

[0068] The data in Table 6 illustrate that after 24 hours of exposure of cells to glycerol ethoxylate at concentrations of 15 mM and 30 mM provided adequate viability, i.e., average viability % of more than 50% for the mean viability % (Runs 1, 2, 3, 4).

Claims

1. A method of inducing caspase activity, comprising contacting a cell with a treating compound formed by alkoxylation of an initiator with an oxidizing agent.

2. The method of claim 1 , wherein the initiator comprises a compound containing three or more available reactive hydroxyl groups, amine groups, or a combination thereof.

3. 2. The method of claim 1, wherein the initiator is selected from glycerol, diglycerol, triglycerol, hexaglycerol, tripentaerythritol, trimethylolpropane, sorbitol, ethylenediamine, triethyleneamine, 2,2 bis(hydroxymethyl)-1,3-propanediol, ethanolamine, and combinations thereof.

4. The method of claim 1 , wherein the oxide is selected from ethylene oxide, propylene oxide, butylene oxide, and combinations thereof.

5. The method of claim 1, wherein the treating compound has a number average molecular weight of 400 to 40,000 g / mol.

6. 10. The method of claim 1, wherein the treatment compound has a concentration in the treatment medium of 0.001 millimolar to 75 millimolar.

7. The method of claim 1 , wherein the cell is a cancerous cell.

8. The method of claim 1 , wherein the caspase is an effector caspase.

9. 2. The method of claim 1, wherein the caspase is selected from caspase 3, caspase 6, caspase 7, or a combination thereof.

10. The method of claim 1 further comprising inducing apoptosis.

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