Induction of caspase activity

Abstract

Embodiments are directed to a method of inducing caspase activity, the method comprising contacting a cell with a treating compound formed by alkoxylation of an initiator with an oxidizing agent.

Description

Technical Field

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

Background Art

[0002] Cancer is a group of diseases involving abnormal cell growth. Colorectal cancer, which may be referred to as colon cancer or bowel cancer, is a cancer resulting from uncontrolled cell growth in the colon or rectum.

[0003] Colorectal cancer is a commonly diagnosed malignancy. Treatments for colorectal cancer may include surgery, radiation therapy, and / or chemotherapy. However, new methods and / or new compositions that can be utilized for treatment are still needed.

Summary of the Invention

Problems to be Solved by the Invention

[0004] The present disclosure provides a method of inducing caspase activity, the method comprising contacting a cell with a treatment compound formed by alkoxylation of an initiator using an oxide.

[0005] The above summary of the present disclosure is not intended to describe each disclosed embodiment or all aspects of the present disclosure. The following description more particularly exemplifies illustrative embodiments. Throughout this application, guidance is provided through lists of examples, which may be used in various combinations. In each case, the listed lists merely serve as representative groups and should not be construed as exclusive lists.

Modes for Carrying Out the Invention

[0006] Without wishing to be bound by theory, one mechanism involved in the development of colorectal cancer is a mutation in the APC (adenomatous polyposis coli) gene that produces the APC protein. The APC protein is part of a protein-based destruction complex that helps prevent the accumulation of the β-catenin protein within cells. The APC protein and the β-catenin protein are part of one of the WNT (wingless / integrated) signaling pathways that transmit signals to cells via cell surface receptors. Generally, when a cell is stimulated by WNT, the destruction complex is inactivated, the β-catenin protein enters the nucleus, and binds to a transcription factor (TCF) that controls the transcription of genetic information. Genes involved in normal cell progression are activated, and this is a regulated process. In the absence of the APC protein, the β-catenin protein continuously accumulates to high levels, moves into the nucleus, binds to TCF, and then TCF binds to DNA and may 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] Methods for inducing caspase activity are disclosed herein. Advantageously, inducing caspase activity can induce apoptosis, i.e., induce cell death. In some applications, apoptosis is desirable compared to necrosis. By inducing caspase activity, several intracellular proteins may be degraded, leading to cell death. Cell death by apoptosis can, for example, have a desirable effect on 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 a numerical range by endpoints includes all numbers subsumed within that range; for example, the range of 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.

[0009] The method for inducing caspase activity disclosed herein includes contacting a cell with a treatment compound. As used herein, "treatment compound" refers to a compound that can be formed by the alkoxylation of an initiator using an oxide.

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

[0011] Embodiments of the present disclosure provide that the oxide can be selected from ethylene oxide, propylene oxide, butylene oxide, and combinations thereof.

[0012] One or more embodiments of the present disclosure provide that the treatment compound formed by the alkoxylation of an initiator using an oxide can be represented by the following formula I.

Chemical formula

[0013] An example of the treatment compound represented by Formula I is trimethylolpropane ethoxylate.

[0014] One or more embodiments of the present disclosure provide that the treatment compound may be represented by the following Formula II.

Chemical formula

[0015] An example of the treatment compound represented by Formula II is 4-arm poly(ethylene glycol).

[0016] One or more embodiments of the present disclosure provide that the treatment compound may be represented by the following Formula III.

Chemical formula

[0017] An example of the treatment 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 sub-ranges from 400 g / mol to 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 treatment compound can be prepared, for example, by alkoxylation of an initiator using an oxide, using known methods, equipment, and / or conditions that may vary depending on the different uses. The treatment compound can be commercially available.

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

[0021] The treatment compound can be utilized with a known treatment medium. For example, the treatment compound can be dissolved in a known therapeutic medium prior to 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 Medium), RPMI 1640, and McCoy's 5A, as well as combinations thereof. Some treatment media are commercially available.

[0022] The treatment compound can have a concentration in the treatment medium of from 0.001 millimolar (mM) to 75 mM. All individual values and subranges from 0.001 mM to 75 mM are included. For example, the 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 in the treatment medium to an upper limit of 75, 72, 70, 68, or 65 mM.

[0023] The cells can be contacted with an effective amount of a 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, for example, to induce caspase activity. Contacting the cells with an effective amount of the treatment compound can desirably provide treatment of a disease in which unwanted cells undergo cell death by apoptosis resulting from induction of caspase activity, for example, treatment of colorectal cancer. The effective amount can vary depending, among other considerations readily determinable by one of ordinary skill in the art, on the particular use, for example, in vitro or in vivo, the subject being treated, for example, the weight and age of the subject, the severity of the medical condition, and / or the method of administration. As used herein, a "subject" to be treated refers to any member of the animal kingdom, for example, a mammal including a human.

[0024] Embodiments of the present disclosure provide that the specific dosage can vary depending on the particular treatment compound utilized, the dosing regimen to be followed, the timing of administration, and / or the physical delivery system by which the treatment compound is carried. For example, an effective amount of the treatment compound can be contacted with the cells by a single administration or multiple administrations.

[0025] Embodiments of the present disclosure provide that the cells contacted with the treatment compound are cancerous cells. For example, the cells can be colorectal cancer cells. 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 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 a cell 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 described 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, determined, for example, by the 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 treatment compounds discussed herein can advantageously provide an improved, i.e., reduced, palliative effect compared to some other polymeric compounds utilized in cancer treatment. This reduced palliative effect can help provide an increased patient compliance compared to some other polymeric compounds associated with a relatively large palliative effect.

[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. The method can include administering a treatment compound to a mammal.

Example

[0031] In this example, various terms and names related to materials are used, including, for example, the following.

[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 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 containing calcium and magnesium (GIBCO 14040-133, obtained from ThermoFisher Scientific)

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

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

[0045] Initiation and maintenance of culture The initiation and maintenance of culture were performed as follows. The initiation and maintenance of culture were carried out according to the "Thawing, Propagating, and Cryopreserving Protocol" NCI-PBCF-HTB38 (HT-29) colorectal adenocarcinoma (ATCC® HTB-38™); February 27, 2012; version 1.6.

[0046] HT-29 (ATCC® HTB-38™) cells (approximately 1×10 per 1 mL) 6(Starting with) cells were seeded into a T-25 flask containing McCoy 5A and fetal bovine serum (10% (v / v)). Subsequently, HT-29 cells were grown using ATCC® 30-2007 (heated in a 37 °C water bath for at least 15 minutes). The cells were grown in a humidified incubator (SANYO INCT-16-CMT, MCO-19AIC (UV)) maintained at 37 °C and 5% CO2. Subsequently, the cells were rinsed with 1× Dulbecco's phosphate buffered saline and subcultured 1 to 3 times a week in a T-75 flask using 1× trypsin-EDTA, applied for less than 5 minutes, and the enzyme action of trypsin-EDTA was blocked by adding complete growth medium to the detached cells. Subsequently, when 80-90% confluence was reached, the cells were split at a split ratio range of 1:5 to 1:16. Subculture and growth proliferation activities such as passage number, confluence %, survival rate % (only on the experimental setting day), and cell morphology at all stages were recorded. The cells were maintained in the logarithmic growth phase.

[0047] Cell culture plating (Day 0) Cell 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 each of a 1:1 0.4% trypan blue dye (INVITROGEN T10282) and cell suspension in 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 having a viability of 90% or more) was plated into a sterile 96-well plate using a multi-channel pipette. 5000 - 6000 cells / well (40,000 - 48,000 cells / mL) per cell density were added to each well except for wells used as cell-free controls with "only saline", starting from column A to column H on the plate, and 125 μL of cell suspension of equal amounts was added to each well. The plates used for each of the two endpoints of apoptosis and cytotoxicity were a solid white plate and a transparent plate, respectively. The 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 at their respective target concentrations of trimethylolpropane ethoxylate, 4-arm poly(ethylene glycol), and glycerol ethoxylate in sterile saline. In this assay, based on solubility limits due to high molecular weight, adjustments of lower stock concentration preparations (w / v) were made as needed to produce either a solution or a pipettable suspension, or solubilization was achieved by incremental addition, continuous mixing, vortexing, sonication, or stirring of saline in small increments prior to use in the assay. If solubilization was required, saline was preheated to 37 °C and then mixed with trimethylolpropane ethoxylate, 4-arm poly(ethylene glycol), and / or glycerol ethoxylate. On the cell suspension plating day (day 0), 10 mL total volume 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. 30 mL total volume (w / v) was prepared for each set day (day 0) and stored at 4 °C until use.

[0050] Preparation of dosing solutions (day 0) The dosing solutions / suspensions of each test substance stock were prepared with McCoy's 5A and 1% fetal bovine serum, each totaling 15 mL. Various amounts of dosing stock were utilized to achieve dosing solutions / suspensions of 0.0015 - 60 mM. The dosing solutions / suspensions were prepared in a sterile reservoir and repeatedly mixed by pipetting until visible uniformity was achieved. Using a 2 mL volume sterile 96-deep well block, 2 mL of the dosing solution / suspension was added to each of the 6 replicate wells of the treatment group and to each of the 12 wells of a cell control of saline only and a "no cells" background correction control of saline only. 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 wells to be treated. The blocks were covered with sealing tape and the plate lids 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 at least 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 column A to column H. Using a multi-channel pipette, 100 μL of each dosing solution / suspension (from the block) was added to each well of a 96-well cell treatment plate starting from column A to column H (in the same order). To prevent well drying and maintain cell attachment and viability, all wells were aspirated and processed two columns at a time, and the pipette tips were changed for each column. All plates were placed in the incubator and allowed to be treated for 24 ± 2 hours or 48 ± 2 hours before collection.

[0052] Collection (Day 2 and Day 3) Apoptosis Apoptosis was evaluated as follows. Apoptosis was performed according to the "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 over approximately 60 minutes. The white plates were removed from the incubator (one by one), and the treatment medium was aspirated. Using a multi-channel 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, added to the reagent reservoir, and using a multi-channel pipette, 100 μL of the assay reagent mixture was added to each well of the 96-well plate. The plate(s) (protected from light using foil) was placed on a plate shaker and rotated at approximately 800 rpm for 5 minutes at room temperature. Then, the plate was incubated for an additional 25 minutes at room temperature before analysis. Luminescence was recorded for relative light units (RLU) of each plate on a FLUOstar Omega plate reader.

[0053] Cytotoxicity Cytotoxicity was evaluated as follows. As described above, the cytotoxicity reagent (5 mg / mL) was preheated to room temperature over approximately 30 minutes and then diluted in 1× Dulbecco's phosphate-buffered saline containing calcium and magnesium to a concentration of 0.675 mg / mL (final). The clear plates were removed from the incubator (one by one), and the treatment medium was aspirated. Using a multi-channel pipette, 200 μL of the cytotoxicity reagent (final) was added to each well of the 96-well plate, and then the plate was covered with sealing tape and incubated in a humidified incubator at 37°C for 4 hours. After incubation, the supernatant was aspirated, and dimethyl sulfoxide (200 μL) was added to each well. After repeated pipetting to mix thoroughly, the cell lysate was 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 The 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 バックグラウンド補正 ) of each well containing the test substance) / (average (RLU フォアグラウンド ) of 12 control wells with only physiological saline) = relative caspase activity (where RLU is relative light unit).

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

[0057] The 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 600-630 ) of each well containing the test substance) / (average (Abs 600-630 ) of 12 control wells with only physiological saline) = cell viability %.

[0062] Cell viability % of each well containing the test substance / 6 replicates = average cell viability %. The results for the various concentrations used are reported in Tables 2, 4, and 6.

Table 1

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

[0064] The data in Table 2 demonstrate that when cells were exposed to trimethylpropane ethoxylate at concentrations of 15 mM, 30 mM, and 60 mM, an appropriate survival rate, i.e., an average survival rate % of 50% or more for (Run 1, 2, 3, 4), was provided. [Table 3]

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

[0066] The data in Table 4 demonstrate that when cells were exposed to 4-arm poly(ethylene glycol) at concentrations of 1.5 mM, 3 mM, and 6 mM for 24 hours, an appropriate survival rate, i.e., an average survival rate % of 50% or more relative to the average survival rate % (Run 1, 2), was provided. [Table 5]

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

Table 6

[0068] The data in Table 6 demonstrate that 24 hours after cells were exposed to glycerol ethoxylate at concentrations of 15 mM and 30 mM, an appropriate survival rate, i.e., an average survival rate % (Run 1, 2, 3, 4) of 50% or more, was provided. (Aspect) (Aspect 1) A method of inducing caspase activity, the method comprising contacting a cell with a treatment compound formed by alkoxylation of an initiator using an oxide. (Aspect 2) The method according to aspect 1, wherein the initiator comprises a compound containing three or more available reactive hydroxyl groups, amine groups, or a combination thereof. (Aspect 3) The method according to aspect 1, wherein the initiator is selected from glycerol, diglycerol, triglycerol, hexaglycerol, tripentaerythritol, trimethylolpropane, sorbitol, ethylenediamine, triethylenetriamine, 2,2-bis(hydroxymethyl)-1,3-propanediol, ethanolamine, and combinations thereof. (Aspect 4) The method according to aspect 1, wherein the oxide is selected from ethylene oxide, propylene oxide, butylene oxide, and combinations thereof. (Aspect 5) The method according to aspect 1, wherein the treatment compound has a number average molecular weight of 400 to 40,000 g / mol. (Aspect 6) The method according to aspect 1, wherein the treatment compound has a concentration of 0.001 mmol to 75 mmol in the treatment medium. (Aspect 7) The method according to aspect 1, wherein the cell is a cancerous cell. (Aspect 8) The method according to aspect 1, wherein the caspase is an effector caspase. (Aspect 9) The method according to aspect 1, wherein the caspase is selected from caspase 3, caspase 6, caspase 7, or combinations thereof. (Aspect 10) The method according to aspect 1, further comprising inducing apoptosis.

Claims

**Claim 1** A preparation for the treatment of cancer, comprising physiological saline and a compound represented by the following formula I, formula II or formula III: 【Chemical 1】 where n is each independently from 1 to 303, 【Chemical Formula 2】 where n is each independently from 1 to 303, and a preparation comprising the same. **Claim 2** The preparation according to claim 1, wherein the compound is selected from trimethylpropane ethoxylate or glycerol ethoxylate. **Claim 3** The preparation according to claim 1 or 2, wherein the compound has a number average molecular weight of 400 to 40,000 g / mol. **Claim 4** The preparation according to any one of claims 1 to 3, wherein the cancer is colorectal cancer.

Images