阅读说明：本技术 诱导胱天蛋白酶活性 (Induction of caspase Activity ) 是由 C·沃尔福-古普塔 A·斯科特 M·勒巴伦 D·威尔逊 S·L·乔丹 R·L·斯密特 R 于 2020-02-26 设计创作，主要内容包括：实施方案涉及诱导胱天蛋白酶活性的方法。该方法包括使细胞与通过使用氧化物将引发剂烷氧基化形成的治疗化合物接触。(Embodiments relate to methods of inducing caspase activity. The method includes contacting the cell with a therapeutic compound formed by alkoxylating an initiator using an oxide.)

1. A method of inducing caspase activity, the method comprising contacting a cell with a therapeutic compound formed by alkoxylating an initiator using an oxide.

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

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

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

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

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

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

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

9. 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 inducing apoptosis.

Technical Field

Embodiments of the present disclosure relate to methods of inducing caspase activity.

Background

Cancer is a group of diseases involving abnormal cell growth. Colorectal cancer, also known as colon cancer or bowel cancer, is a cancer caused by uncontrolled cell growth in the colon or rectum.

Colorectal cancer is a frequently diagnosed malignancy. Treatment of colorectal cancer may include surgery, radiation therapy, and/or chemotherapy. However, there remains a need for new methods and/or new compositions that are useful in therapy.

Disclosure of Invention

The present disclosure provides methods of inducing caspase activity, the methods comprising contacting a cell with a therapeutic compound formed by alkoxylating an initiator using an oxide.

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. Throughout this application, guidance is provided through lists of examples, which examples can be used in various combinations. In each case, the enumerated lists serve only as representative groups and should not be construed as exclusive lists.

Detailed Description

While not wishing to be bound by theory, one mechanism involved in the development of colorectal cancer is mutation of the APC (adenomatous polyposis coli) gene that produces the APC protein. APC proteins are part of a protein-based destruction complex that helps prevent the accumulation of β -catenin in cells. The APC protein and β -catenin are part of one of the WNT (Wingless/Integrated) signaling pathways that transmit signals to cells via cell surface receptors. In general, 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 cellular processes will be activated and this is a regulated process. Without the APC protein, β -catenin accumulates to high levels and migrates to the nucleus, binds to TCF, which in turn binds to DNA and activates transcription of proto-oncogenes. When proto-oncogenes are inappropriately expressed at high levels, they become oncogenes. Activated oncogenes lead to survival and proliferation of cells designated to undergo apoptosis, which may lead to individuals suffering from colorectal cancer.

Disclosed herein are methods of inducing caspase activity. Advantageously, inducing caspase activity may induce apoptosis, i.e. induce cell death. For many applications, apoptosis is desirable as compared to necrosis. Inducing caspase activity may provide for the degradation of a number of intracellular proteins to cause cell death. For example, cell death by apoptosis may be a desirable effect on colorectal cancer cells.

As used herein, unless otherwise specified, "a," "an," "the," "at least one," "a plurality," and "one or more" may be used interchangeably. The term "and/or" means one, one or more, or all of the listed items. The recitation of numerical ranges by endpoints includes all numbers subsumed within that range (e.g. 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

As disclosed herein, a method of inducing caspase activity comprises contacting a cell with a therapeutic compound. As used herein, "therapeutic compound" refers to a compound that can be formed by alkoxylating an initiator using an oxide.

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

Embodiments of the present disclosure provide that the oxide may be selected from the group consisting of ethylene oxide, propylene oxide, butylene oxide, and combinations thereof.

One or more embodiments of the present disclosure provide that therapeutic compounds formed by alkoxylating an initiator using an oxide can be represented by the following formula I:

where each n is independently from 1 to 303.

An example of a therapeutic compound represented by formula I is trimethylolpropane ethoxylate.

One or more embodiments of the present disclosure provide that the therapeutic compound may be represented by formula II below:

where each n is independently from 1 to 227.

An example of a therapeutic compound represented by formula II is 4-arm poly (ethylene glycol).

One or more embodiments of the present disclosure provide that the therapeutic compound may be represented by formula III below:

where each n is independently from 1 to 303.

An example of a therapeutic compound represented by formula III is glycerol ethoxylate.

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

The therapeutic compounds may be prepared using known methods, equipment and/or conditions, such as alkoxylating the initiator using an oxide, which may vary for different applications. Therapeutic compounds are commercially available.

As mentioned, as disclosed herein, a method of inducing caspase activity comprises contacting a cell with a therapeutic compound. One or more embodiments of the present disclosure provide that contacting a cell with a therapeutic compound occurs in vivo. One or more embodiments of the present disclosure provide for contacting a cell with a therapeutic compound to occur in vitro. The cells can be contacted with the therapeutic compound using a variety of different known methods, devices, and/or conditions. Various methods, devices, and/or conditions may be used for different applications.

The therapeutic compounds may be used with known therapeutic media. For example, the therapeutic 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 therapeutic compound and the therapeutic medium can be combined to form a solution. The solution may 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, combinations thereof, and the like. Many therapeutic media are commercially available.

The therapeutic compound may have a concentration of 0.001 millimolar (mM) to 75mM in the treatment medium. Including all individual values and subranges from 0.001 to 75 mM; for example, an effective concentration can be from a lower limit of 0.001, 0.005, 0.01, 0.1, or 1.0mM to an upper limit of 75, 72, 70, 68, or 65mM in the treatment medium.

The cells can be contacted with an effective amount of a therapeutic compound. As used herein, the term "effective amount" is used interchangeably with "therapeutically effective amount" and/or "therapeutic amount" and refers to an amount of a therapeutic compound sufficient to provide the intended use, e.g., induction of caspase activity. Contacting cells with an effective amount of a therapeutic compound can desirably provide treatment of a disease, such as colorectal cancer, in which undesired cells die as a result of apoptosis induced by caspase activity. An effective amount may vary depending on such considerations as the particular application (e.g., in vitro or in vivo), the subject being treated (e.g., the weight and age of the subject), the severity of the disease condition, and/or the mode of administration, and can be readily determined by one of ordinary skill in the art. As used herein, a "subject" being treated refers to any member of the animal kingdom, such as mammals, including humans.

Embodiments of the present disclosure provide that the specific dosage may vary depending on the specific therapeutic compound used, the dosing regimen to be followed, the time of administration, and/or the physical delivery system carrying the therapeutic compound. For example, an effective amount of a therapeutic compound can be contacted with a cell by a single administration or multiple administrations.

Embodiments of the present disclosure provide that the cell contacted with the therapeutic compound is a cancer cell. For example, the cell may be a colorectal cancer cell. Colorectal cancer cells may 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 therapeutic compound.

Although not intending to be bound by theory, caspases (which may be referred to as cysteine-aspartic proteases) are a family of cysteine proteases involved in apoptosis. There are two types of caspases: starting caspases, including caspases 2, 8, 9, 10, 11, 12, and effector caspases, including caspases 3, 6, 7. One or more embodiments of the present disclosure provide that contacting a cell with a therapeutic 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.

As described above, induction of caspase activity may advantageously induce apoptosis. The induced caspase activity may be determined by a variety of different known methods, devices, and/or conditions. For example, the induced caspase activity may be demonstrated by an average relative caspase activity greater than one (>1), e.g., for multiple experimental runs as determined by caspase-Glo 3/7assay 4.B. Standard protocols for cells in 96-well plates are available from Promega. As used herein, "relative caspase activity" may be used interchangeably with relative apoptosis.

As discussed herein, the use of a therapeutic compound may advantageously provide improved, i.e., reduced, laxative effects compared to some other polymeric compounds used in cancer therapy. This reduced laxative effect may help provide a desired increase in patient compliance compared to some other polymeric compounds associated with a relatively greater laxative effect.

One or more embodiments of the present disclosure provide a method of treating colorectal cancer. The method can include contacting the colorectal cancer cell with a therapeutic compound.

One or more embodiments of the present disclosure provide a method of treating cancer. The method may comprise administering a therapeutic compound to the mammal.

Examples

In embodiments, various terms and names of materials are used, including, for example, the following:

trimethylpropane ethoxylate (Mn 1014 g/mol; obtained from Sigma-Aldrich);

4-arm poly (ethylene glycol) (Mn 10,000 g/mol; obtained from Sigma-Aldrich);

glycerol ethoxylate (Mn 1000 g/mol; obtained from Sigma-Aldrich);

cells (human colon; colorectal adenocarcinoma; HT-29: (A))HTB-3); obtained from ATCC);

McCoy's 5A (growth medium; obtained from ThermoFisher Scientific);

fetal bovine serum (obtained from ATCC);

dulbecco's phosphate buffered saline (GIBCO 14190-144; available from ThermoFisher Scientific);

complete growth medium: (30-2007; obtained from ATCC);

trypsin-EDTA (GIBCO trypsin-EDTA (0.25%); catalog No. 25200056; available from ThermoFisher Scientific);

thiazole blue tetrazolium bromide (available from ThermoFisher Scientific);

dulbecco's phosphate buffered saline containing calcium and magnesium (GIBCO 14040-;

Caspase-Glo 3/7Assay (luminescence Assay; catalog number G8093; available from Promega);

dimethyl sulfoxide (Cat. No. 276855; obtained from Sigma-Aldrich).

Culture initiation and maintenance

Culture initiation and maintenance were performed as follows. Colonic adenocarcinoma according to the "thawing, propagation and cryopreservation protocol" NCI-PBCF-HTB38(HT-29) (ii)HTB-38^TM) (ii) a 2 month 27, 2012; version 1.6 was culture initiation and maintenance.

Initiating HT-29(HTB-38^TM) Cells (containing about 1X10 per mL)⁶Individual cells) and inoculated into T-25 flasks containing McCoy's 5A and fetal bovine serum (10% (v/v)). Then, use30-2007 (warming in a 37 ℃ water bath for at least 15 minutes) expanded HT-29 cells. Cells were maintained at 37 ℃ and 5% CO₂In a humidified incubator (SANYO INCT-16-CMT; MCO-19AIC (UV)). Then, cells were washed with 1X Dulbecco's phosphate buffered saline and subcultured 1 to 3 times per week in T-75 flasks using 1X trypsin-EDTA, applied<5 minutes; the enzymatic action of trypsin-EDTA was stopped by adding complete growth medium to the detached cells. Then, upon reaching 80% to 90% confluence, the cells were divided into the following split ratio ranges: 1:5 to 1: 16. Recording subculture and growth expansion activities, examplesSuch as passage number,% confluency,% survival (only on experimental set days) and cell morphology at all stages. Cells were maintained in log phase growth.

Cell culture plating (day 0)

Cell culture plating was performed as follows. Cell suspensions were collected from individual 80% to 90% confluent T-75 flasks using 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), in which 2 chambers of each slide were provided with 10 μ L of each of 1:1, 0.4% trypan blue dye (INVITROGEN T10282), and cell suspension. Cell counts and percent viability were averaged for both chambers of a single slide. Viable cells (defined as viability ≧ 90%) containing complete growth medium were then plated onto sterile 96-well plates using a multichannel pipettor. Per cell density, 5000 to 6000 cells per well (40,000 to 48,000 cells per ml) were added to each well, except for wells used as 'saline only' no cell control wells; starting from plate A to row H, an equal volume of 125. mu.L of cell suspension was added to each well. The plates for each of the 2 endpoints (apoptosis and cytotoxicity) were solid white and transparent plates, respectively. Cells were incubated 24 ± 2 hours to allow attachment.

Trimethylpropane ethoxylate/4-arm poly (ethylene glycol)/glycerol ethoxylate starting material preparation

Stock solutions were prepared in sterile saline at target concentrations for each of trimethylpropane ethoxylate, 4-arm poly (ethylene glycol) and glycerol ethoxylate. For the assay, depending on the solubility limit due to high molecular weight, the formulation is adjusted to a lower stock concentration (w/v) to generate a solution or a pipettable suspension if necessary, or solubilization is achieved by adding a small amount of saline, continuous mixing, vortexing, sonication, or stirring prior to use in the assay. The brine was preheated to 37 ℃ prior to mixing with trimethylpropane ethoxylate, 4-arm poly (ethylene glycol) and/or glycerol ethoxylate, if necessary for dissolution. On the day of cell suspension plating (day 0), the total volume prepared for each test substance was 10 mL.

Preparation of cytotoxic Agents

Thiazolylcarbamylium bromide was prepared at 5mg/mL in Dulbecco's phosphate buffered saline containing calcium and magnesium. A total volume (w/v) of 30mL was prepared for each set day (day 0) and stored at 4 ℃ until use.

Dosing solution preparation (day 0)

Dosing solutions/suspensions of stock solutions of each test substance were prepared in a total of 15mL of each of McCoy's 5A and 1% fetal bovine serum. Different amounts of dosing stock were used to obtain dosing solutions/suspensions from 0.0015 to 60 mM. The dosing solution/suspension was prepared in a sterile reservoir and mixed repeatedly with a pipette until visible homogeneity was achieved. Using a sterile 96 deep well block with a capacity of 2mL, 2mL of dosing solution/suspension was added to each of the 6 replicate wells for the treatment group, to each of the 12 wells for the saline cell only control and the saline only 'cell free' background correction control. The plates were built according to a semi-random statistical design. Each test substance is identified by a number and a color code for identifying the well to be treated. The blocks were covered with sealing tape, plate cover, and placed in a 4 ℃ laboratory refrigerator (Fischer Scientific, 135B 1; RFR-22-CMT) overnight.

Treatment (day 1)

All 96-deep well blocks containing the dosing solution/suspension were removed from the refrigerator and placed in a 37 ℃ bead bath for at least 30 minutes. Approximately 24 hours after plating, the plates were removed from the incubator and processed one at a time. All wells in the cell plate were aspirated using a 6-well aspiration device starting from row a to row H. Using a multichannel pipettor, add 100 μ Ι _ of dosing solution/suspension (from the block) to a 96-well cell treatment plate; starting from row a to row H (same order). All wells were aspirated and treated 2 rows at a time to prevent drying of the wells and to maintain cell attachment and viability; the pipette tip was changed for each row. All plates were placed in an incubator and pre-harvest treated for 24 + -2 or 48 + -2 hours.

Harvest (day 2 and day 3)

Apoptosis of cells

Assessment of apoptosis was performed as follows. Apoptosis was performed according to the "Caspase-Glo 3/7 Assay" 4.b. standard protocol (Promega) for cells in 96-well plates. The Caspase-Glo 3/7Assay module was preheated to room temperature for about 60 minutes. Remove (one at a time) the whiteboard from the incubator and aspirate the treatment medium. Using a multichannel pipettor, 100. mu.L of 1X Dulbecco's phosphate buffered saline was added to each well of a 96-well plate. Manually mixing and adding assay reagents (buffer and substrate) to the reagent reservoir; using a multichannel pipettor, 100 μ Ι _ of assay reagent mixture was added to each well of a 96-well plate. The plate (protected from light with foil) was placed on a plate shaker and allowed to spin at about 800rpm for 5 minutes at room temperature. The plates were then incubated at room temperature for an additional 25 minutes before analysis. Luminescence was recorded in Relative Light Units (RLU) for each Plate on a FLUOstar Omega Plate Reader.

Cytotoxicity

Evaluation of cytotoxicity was performed as follows. The cytotoxic agent as described previously (5mg/mL) was pre-warmed to room temperature for about 30 minutes and then diluted into calcium and magnesium in 1X Dulbecco phosphate buffered saline to provide a concentration of 0.675mg/mL (final). Remove the transparent plates from the incubator (one at a time) and aspirate the treatment medium. Using a multichannel pipettor, add 200 μ Ι _ of cytotoxic agent (final) to each well of a 96-well plate; the plates were then covered with sealing tape and incubated for 4 hours in a humidified 37 ℃ incubator. After incubation, the supernatant was aspirated and dimethylsulfoxide (200 μ L) was added to each well. After thorough mixing by repeated pipetting, the cell lysates were transferred to new clear 96-well plates and the absorbance at 600 and 630nm was quantified on a FLUOstar Omega plate reader.

Analysis of

The relative caspase activity was calculated as follows: (RLU)_{Prospect of})-(RLU_{Saline only 'cell free' control})＝(RLU_{Background correction})；

(of each well containing a test substance(RLU_{Background correction}) Averaged (RLU) of 12 saline only control wells_{Prospect of}) Relative caspase activity; where RLU is the relative light unit.

Relative caspase activity per 6 replicates per well containing the test substance-average relative caspase activity. The results for various concentrations used are reported in tables 1,3 and 5.

Cell viability was calculated as follows:

(Abs _{600 foreground})–(Abs _{600 saline only ` cell free ` control)}＝(Abs _{600 background correction})

(Abs _{630 foreground})–(Abs _{630 saline only ` cell free ` control}＝(Abs _{630 background correction})

(Abs _{600 background correction})–(Abs _{630 background correction})＝(Abs _600-630)

(of wells each containing a test substance (Abs)_600-630) )/(average of 12 saline-only control wells (Abs)_600-630))_＝% cell survival rate

The% cell viability per 6 replicates for each well containing test substance is the average% cell viability. The results for various concentrations used are reported in tables 2, 4 and 6.

TABLE 1

The data in table 1 demonstrate that the cells when exposed to trimethylpropane ethoxylates at concentrations of 15, 30 and 60mM provide advantageous relative caspase activities, i.e. average relative caspase activity >1, as shown by the respective average relative caspase activity (runs 1, 2, 3, 4) values.

TABLE 2

The data in table 2 demonstrate that the cells when exposed to trimethylpropane ethoxylate at concentrations of 15, 30 and 60mM provide sufficient survival after 24 hours, i.e. the average% survival for (runs 1, 2, 3, 4) is 50% or greater.

TABLE 3

The data in table 3 demonstrate that the cells when exposed to 4-arm poly (ethylene glycol) at concentrations of 1.5, 3 and 6mM provide advantageous relative caspase activities, i.e. average relative caspase activity >1, as shown by the respective average relative caspase activity (runs 1, 2) values.

TABLE 4

The data in table 4 demonstrate that the cells when exposed to 4-arm poly (ethylene glycol) at concentrations of 1.5, 3 and 6mM provide sufficient survival after 24 hours, i.e. the% mean survival for the% mean survival (runs 1, 2) is 50% or greater.

TABLE 5

The data in table 5 demonstrate that the cells when exposed to glycerol ethoxylates at concentrations of 15 and 30mM provide advantageous relative caspase activities, i.e. average relative caspase activity >1, as shown by the respective average relative caspase activity (runs 1, 2, 3, 4) values.

TABLE 6

The data in table 6 demonstrate that the cells when exposed to glycerol ethoxylate at concentrations of 15 and 30mM provide sufficient survival after 24 hours, i.e. for the average survival% (runs 1, 2, 3, 4) the average survival% is 50% or more.