阅读说明：本技术 用于诱导胱天蛋白酶活性的聚乙二醇衍生物 (Polyethylene glycol derivatives for inducing 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下式表示的治疗化合物接触,其中R～(1)选自氢或具有1至约16个碳原子的烷基；R～(2)选自羟基、甲苯磺酸酯基、式OR～(3)的烷氧基,其中R～(3)选自具有1至约16个碳原子的烷基,或式OCOR～(4)的酯基,其中R～(4)为具有1至约16个碳原子的烷基,且n为4至46,000,条件是当R～(1)为氢且R～(2)为羟基时治疗化合物的数均分子量为10,100至2,000,0000g/mol。(Embodiments relate to methods of inducing caspase activity. The method comprises contacting the cell with a therapeutic compound represented by the formula wherein R 1 Selected from hydrogen or alkyl groups having from 1 to about 16 carbon atoms; r 2 Selected from hydroxy, tosylate, formula OR 3 Alkoxy of (2), wherein R 3 Selected from alkyl groups having 1 to about 16 carbon atoms, or formula OCOR 4 Ester group of (2), wherein R 4 Is an alkyl group having from 1 to about 16 carbon atoms, and n is from 4 to 46,000, with the proviso that when R is 1 Is hydrogen and R 2 The number average molecular weight of the therapeutic compound in the case of a hydroxyl group is from 10,100 to 2,000,0000 g/mol.)

1. A method of inducing caspase activity, the method comprising contacting a cell with a therapeutic compound represented by the formula:

wherein R is¹Selected from hydrogen or alkyl groups having from 1 to about 16 carbon atoms; r²Selected from hydroxy, tosylate, formula OR³Alkoxy of (2), wherein R³Selected from alkyl groups having 1 to about 16 carbon atoms, or formula OCOR⁴Ester group of (2), wherein R⁴Is an alkyl group having from 1 to about 16 carbon atoms, and n is from 4 to 46,000, with the proviso that when R is¹Is hydrogen and R²In the case of a hydroxyl group, the therapeutic compound has a number average molecular weight of 10,100 to 2,000,0000 g/mol.

2. The method of claim 1, wherein the therapeutic compound has a number average molecular weight of 200 to 2,000,000 g/mol.

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

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

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

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

7. 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 a method of inducing caspase activity, the method comprising contacting a cell with a therapeutic compound represented by the formula:

wherein R is¹Selected from hydrogen or alkyl groups having from 1 to about 16 carbon atoms; r²Selected from hydroxy, tosylate, formula OR³Alkoxy of (2), wherein R³Selected from alkyl groups having 1 to about 16 carbon atoms, or formula OCOR⁴Ester group of (2), wherein R⁴Is an alkyl group having from 1 to about 16 carbon atoms, and n is from 4 to 46,000, with the proviso that when R is¹Is hydrogen and R²In the case of hydroxyl, the therapeutic compound has a number average molecular weight of 10,100 to 2,000,0000 g/mol.

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 intending 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 includes contacting a cell, e.g., a plurality of cells, with a therapeutic compound. As used herein, "therapeutic compound" refers to a compound that can be represented by formula I below:

wherein R is¹Selected from hydrogen or alkyl groups having from 1 to about 16 carbon atoms; r²Selected from hydroxy, tosylate, formula OR³Alkoxy of (2), wherein R³Selected from alkyl groups having 1 to about 16 carbon atoms, or formula OCOR⁴Ester group of (2), wherein R⁴Is an alkyl group having from 1 to about 16 carbon atoms, and n is from 4 to 46,000, with the proviso that when R is¹Is hydrogen and R²In the case of hydroxyl, the therapeutic compound has a number average molecular weight of 10,100 to 2,000,0000 g/mol.

As mentioned above, R¹、R³And R⁴Each of which may be an alkyl group having from 1 to about 16 carbon atoms. For example, R¹、R³And R⁴The alkyl groups of (a) may each independently comprise 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 carbon atoms. R¹、R³And R⁴Each alkyl group of (a) may independently be a saturated or unsaturated alkyl group. R¹、R³And R⁴Each alkyl group of (a) may independently be a linear or branched alkyl group.

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

wherein n is 4 to 46,000. The therapeutic compound represented by formula II may be referred to as poly (ethylene glycol) methyl ether.

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

wherein n is 4 to 46,000. The therapeutic compound represented by formula III may be referred to as poly (ethylene glycol) methyl ether tosylate.

Embodiments of the present disclosure provide that the therapeutic compound has a number average molecular weight (Mn) of 200 to 2,000,000 g/mol. Includes all individual values and subranges from 200 to 2,000,0000 g/mol; for example, the therapeutic compound may have a lower limit of from 200, 300, 400, or 500g/mol to 2,000,0000; 1,750,0000, respectively; 1,500,0000, respectively; 1,250,0000, respectively; or 1,000,0000 g/mol.

Embodiments of the disclosure provide that when R¹Is hydrogen and R²The therapeutic compound represented by formula I has a number average molecular weight of 10,100 to 2,000,0000g/mol when it is a hydroxyl group of the therapeutic compound. Includes all individual values and subranges from 10,100 to 2,000,0000 g/mol; for example, when R is¹Is hydrogen and R²When the hydroxy group of the therapeutic compound represented by formula I, the therapeutic compound can have a molecular weight of from 10,100; 10,500; 11,000; 15,000; 20,000; 35,000; 50,000; 75,000; or a lower limit of 100,000g/mol to 2,000,0000; 1,750,0000, respectively; 1,500,0000, respectively; 1,250,0000, respectively; or 1,000,0000 g/mol.

Therapeutic compounds can be prepared using known methods, equipment, and/or conditions, which can 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, induced Caspase activity may be demonstrated by an average relative Caspase activity greater than one (>1), e.g., as determined by Caspase-Glo 3/7Assay 4.b. standard protocol for cells in 96-well plates available from Promega for multiple experimental runs. 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:

poly (ethylene glycol) methyl ether (Mn 2000 g/mol; obtained from Sigma-Aldrich);

poly (ethylene glycol) methyl ether (Mn 5000 g/mol; obtained from Sigma-Aldrich);

poly (ethylene glycol) methyl ether tosylate (Mn 2000 g/mol; obtained from Sigma-Aldrich);

poly (ethylene glycol) methyl ether tosylate (Mn 5000 g/mol; obtained from Sigma-Aldrich);

POLYOX N12K (polyethylene glycol, Mn 1,000,000 g/mol; from Dupont);

POLYOX VSR N750 (polyethylene glycol, Mn 300,000 g/mol; from Dupont);

POLYOX VSR N3000 (polyethylene glycol, Mn 400,000 g/mol; from Dupont);

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, the cells were washed with 1XDulbecco phosphate buffered saline and subcultured 1 to 3 times per week in a T-75 flask using 1 Xtrypsin-EDTA, applied ≤ 5 min; 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. Subculture and growth expansion activities were recorded, e.g.passage number,% confluence,% survival (only on experimental set-up days) and cells at all stagesForm is shown. 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.

Preparation of poly (ethylene glycol) methyl ether/poly (ethylene glycol) methyl ether tosylate/polyethylene glycol stock solution

Stock solutions were prepared in sterile saline at target concentrations of poly (ethylene glycol) methyl ether, poly (ethylene glycol) methyl ether tosylate and polyethylene glycol. 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. If dissolution is desired, the brine is preheated to 37 ℃ prior to mixing with the poly (ethylene glycol) methyl ether, poly (ethylene glycol) methyl ether tosylate and/or polyethylene glycol. On the day of cell suspension plating (day 0), the total volume prepared for each test substance was 10 mL.

Cytotoxic agentsPreparation of

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

Relative caspase Activity (please provide definition for this)

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

(each containing a test substanceOf wells (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 the various concentrations used are reported in tables 1, 3, 5, 7, 9, 11 and 13.

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 the various concentrations used are reported in tables 2, 4, 6, 8, 10, 12 and 14.

TABLE 1

The data in table 1 demonstrate that the cells when exposed to poly (ethylene glycol) methyl ether with Mn of 2000g/mol 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) values.

TABLE 2

The data in table 2 demonstrate that the cells when exposed to poly (ethylene glycol) methyl ether at concentrations of 15, 30 and 60mM with Mn of 2000g/mol provide sufficient survival after 24 hours, i.e. the% mean survival for the% mean survival (runs 1,2, 3) is 70% or more.

TABLE 3

The data in table 3 demonstrate that the cells when exposed to poly (ethylene glycol) methyl ether with Mn of 5000g/mol 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 poly (ethylene glycol) methyl ether with Mn of 5000g/mol 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 70% or more.

TABLE 5

The data in table 5 demonstrate that the cells when exposed to poly (ethylene glycol) methyl ether mesylate at concentrations of 15 and 30mM with Mn of 2000g/mol 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 a concentration of 15 and 30mM of poly (ethylene glycol) methyl ether mesylate, Mn 2000g/mol, provided sufficient survival after 24 hours, i.e., the% mean survival was 70% or greater for the% mean survival (runs 1,2, 3, 4).

TABLE 7

The data in table 7 demonstrate that cells when exposed to poly (ethylene glycol) methyl ether mesylate at concentrations of 1.5, 3 and 6mM with Mn of 5000g/mol 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 8

The data in Table 8 demonstrate that the cells when exposed to poly (ethylene glycol) methyl ether mesylate at concentrations of 1.5, 3 and 6mM with Mn of 5000g/mol provide sufficient survival after 24 hours, i.e., the% mean survival was 70% or greater for the% mean survival (runs 1, 2).

TABLE 9

The data in table 9 demonstrate that cells when exposed to polyethylene glycol with Mn of 1,000,000g/mol at concentrations of 0.0015, 0.003 and 0.006mM provide advantageous relative caspase activities, i.e. average relative caspase activity >1, as shown by the average relative caspase activity (run 1) values.

Watch 10

The data in table 10 demonstrate that cells exposed to 0.0015, 0.003, and 0.006mM concentrations of polyethylene glycol with Mn of 1,000,000g/mol provided sufficient survival after 24 hours, i.e., the% mean survival was 70% or greater for the% mean survival (run 1).

TABLE 11

The data in table 11 demonstrate that cells exposed to polyethylene glycol with Mn of 300,000g/mol at concentrations of 0.003 and 0.006mM provide advantageous relative caspase activities, i.e., average relative caspase activity >1, as shown by the average relative caspase activity (run 1) values.

TABLE 12

The data in Table 12 demonstrate that the cells when exposed to polyethylene glycol with Mn of 300,000g/mol at concentrations of 0.003 and 0.006mM provide sufficient survival after 24 hours, i.e., the% mean survival is 70% or greater for the% mean survival (run 1).

Watch 13

The data in table 13 demonstrate that cells when exposed to polyethylene glycol with Mn of 400,000g/mol at concentrations of 0.0015 and 0.006mM provide advantageous relative caspase activities, i.e., average relative caspase activity >1, as shown by the average relative caspase activity (run 1) values.

TABLE 14

The data in Table 14 demonstrate that the cells when exposed to polyethylene glycol with Mn of 400,000g/mol at concentrations of 0.0015 and 0.006mM provide sufficient survival after 24 hours, i.e., the% mean survival for the% mean survival (run 1) is 70% or greater.