Patent Description:
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 from uncontrolled cell growth in the colon or rectum.

<CIT> describes methods for and of treating, ameliorating or preventing colorectal cancer (CRC) in humans using polyethylene glycol (PEG) or a PEG block-copolymer such as Pluronic® F68. The use of PEG derived molecules as vehicles or carriers for other active compounds is described by <NPL>) and <NPL>). <CIT> discloses the use of amphiphilic block copolymers for treating and preventing cancer, and in particular by reducing the proliferation rate of cancer cells. Preferred block copolymers comprises a central hydrophobic chain, preferably a polypropylene oxide chain, two which at least two hydrophilic side chains, preferably polyethylene oxide chains, are connected. <NPL>) describes the use of Tween <NUM> to induce apoptosis in cancer cells.

Colorectal cancer is a commonly diagnosed malignancy. Treatments for colorectal cancer can include surgery, radiation therapy, and/or chemotherapy. However, there remains a need for new methods and/or new compositions that may be utilized for treatment.

The present disclosure provides in vitro methods of inducing caspase activity, the method comprising contacting a cell with a treatment compound formed by alkoxylation of an initiator using an oxide. The present disclosure provides a treatment compound for use in a method of treating colorectal cancer.

While not wishing to be bound to theory, one mechanism involved in the development of colorectal cancer is the 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 to prevent the accumulation of the β-catenin protein in a cell. The APC protein and the β-catenin protein are part of one of the WNT (Wingless/Integrated) signaling transduction pathways that pass signals into a cell through cell surface receptors. In general, when cells are stimulated by WNT, the destruction complex is deactivated, and β-catenin protein will enter the nucleus and bind to the transcription factor (TCF) which controls the transcription of genetic information. The genes involved in regular cell progression will be activated and it is a regulated process. Without the APC protein, β-catenin protein will continuously accumulate to high levels and translocate into the nucleus, bind to the TCF, which can then bind to DNA, and 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 to survive and proliferate instead, which can lead to the development of colorectal cancer, in an individual.

Methods of inducing caspase activity are disclosed herein. Advantageously, inducing caspase activity can incite apoptosis, i.e. induce cell death. For a number of applications, apoptosis is desirable, as compared to necrosis. Inducing caspase activity can provide for degradation of a number of intracellular proteins to result in cell death. Cell death by apoptosis can be a desirable effect on colorectal cancer cells, for instance.

As used herein, "a", "an", "the", "at least one", "a number of", and "one or more" may be used interchangeably unless indicated otherwise. The term "and/or" means one, one or more, or all of the listed items. The recitations of numerical ranges by endpoints include all numbers subsumed within that range, e.g., <NUM> to <NUM> includes <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, etc..

The references to the methods of treatment by therapy or surgery in this description are to be interpreted as references to compounds, pharmaceutical compositions and medicaments of the present invention for use in those methods.

The methods of inducing caspase activity, as disclosed herein, include contacting a cell in vitro with a treatment compound. A treatment compound for use in a method of treating colorectal cancer is also disclosed herein. As used herein, "treatment compound" refers to compounds that may formed by alkoxylation of an initiator using an oxide.

Embodiments of the present disclosure provide that the initiator includes compounds containing three or more reactive available 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, triethyleneamine, <NUM>,<NUM> bis(hydroxymethyl)-<NUM>,<NUM>-propanediol, ethanolamine, and combinations thereof.

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

One or more embodiments of the present disclosure provide that the treatment compound formed by alkoxylation of an initiator using an oxide may be represented by the following Formula I:
<CHM>
where each n is independently from <NUM> to <NUM>.

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

One or more embodiments of the present disclosure provide that the treatment compound may be represented by the following Formula II:
<CHM>
where each n is independently from <NUM> to <NUM>.

An example of the treatment compound represented by Formula II is <NUM>-arm poly(ethylene glycol).

One or more embodiments of the present disclosure provide that the treatment compound may be represented by the following Formula III:
<CHM>
where each n is independently from <NUM> to <NUM>.

An example of the treatment compound represented by Formula III is glycerol ethoxylate.

Embodiments of the present disclosure provide that the treatment compound has a number average molecular weight (Mn) from <NUM> to <NUM>,<NUM>/mol. All individual values and subranges from <NUM> to <NUM>,<NUM>/mol are included; for example, the treatment compound can have Mn from a lower limit of <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>/mol to an upper limit of <NUM>,<NUM>, <NUM>,<NUM>, <NUM>,<NUM>, <NUM>,<NUM>, or <NUM>,<NUM>/mol.

The treatment compound may be prepared, e.g., alkoxylation of the initiator using the oxide, using known methods, equipment, and/or conditions, which may vary for different applications. The treatment compound may be obtained commercially.

As mentioned, methods of inducing caspase activity, as disclosed herein, include contacting a cell with the treatment compound in vitro. The cell may be contacted with the treatment compound by utilizing a number of different known methods, equipment, and/or conditions. Various methods, equipment, and/or conditions may be utilized for different applications.

The treatment compound may be utilized with a known treatment medium. For instance, the treatment compound may be dissolved, to provide an effective amount, in a known treatment medium prior to contacting the cell. One or more embodiments provide that the treatment compound and the treatment medium may be combined to form a solution. The solution may be a homogeneous solution. Examples of treatment mediums include, but are not limited to, DMEM (Dulbecco's Modified Eagle Medium), RPMI <NUM>, and McCoy's 5A, and combinations thereof, among others. A number of treatment mediums are commercially available.

The treatment compound can have a <NUM> millimolar (mM) to <NUM> concentration in the treatment medium. All individual values and subranges from <NUM> to <NUM> are included; for example, the effective concentration can be from a lower limit of <NUM>, <NUM>, <NUM>, <NUM> or <NUM> to an upper limit of <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> of the treatment compound in the treatment medium.

The cell may be contacted with an effective amount of the treatment compound. As used herein, the term "effective amount", which may be used interchangeably with "therapeutic effective amount" and/or "therapeutic amount", refers to an amount of the treatment compound that is sufficient to provide the intended application, e.g., induce caspase activity. Contacting the cell with an effective amount of the treatment compound may desirably provide a disease treatment, e.g., a colorectal cancer treatment, where undesirable cells are subject to cell death by apoptosis that results from inducing caspase activity. The effective amount may vary depending upon 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 manner of administration, among other considerations, which can readily be determined by one of ordinary skill in the art. As used herein, a "subject" that is treated refers to any member of the animal kingdom, e.g., mammals, including humans.

Embodiments of the present disclosure provide that specific doses may vary depending on the particular treatment compound utilized, the dosing regimen to be followed, timing of administration, and/or the physical delivery system in which the treatment compound is carried. For instance, the effective amount of the treatment compound may be contacted with the cell by a single dosing or by multiple dosings.

Embodiments of the present disclosure provide that the cell that is contacted with the treatment compound is a colorectal cancer cell. Colorectal cancer cells may also be referred to as colon cancer cells, bowel cancer cells, and/or colorectal adenocarcinoma cells. One or more embodiments of the present disclosure provide that additional cells, i.e. non-cancerous cells, may be contacted with the treatment compound.

While not intending to be bound by theory, caspases, which may 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, which include caspase <NUM>,<NUM>,<NUM>,<NUM>,<NUM>,<NUM>, and effector caspases, which include caspase <NUM>,<NUM>,<NUM>. One or more embodiments of the present disclosure provide that contacting a cell with a treatment compound induces an effector caspase activity. One or more embodiments of the present disclosure provide that the caspase is selected from caspase <NUM>, caspase <NUM>, caspase <NUM>, or combinations thereof.

As mentioned, inducing caspase activity can advantageously incite apoptosis. Induced caspase activity may be determined by a number of different known methods, equipment, and/or conditions. For instance, induced caspase activity may be evidenced by an average relative caspase activity greater than one ( ><NUM>), e.g., for a number of experimental runs, as determined by Caspase-Glo <NUM>/<NUM> Assay <NUM>. Standard Protocol for Cells in a <NUM>-Well Plate, available from Promega. As used herein, "relative caspase activity" can be utilized interchangeably with relative apoptosis.

Utilizing the treatment compound, as discussed herein, may advantageously provide an improved, i.e. reduced, laxative effect, as compared to some other polymeric compounds utilized for cancer treatment. This reduced laxative effect may help to provide a desirable increase with patient compliance, as compared to some other polymeric compounds associated with a relatively greater laxative effect.

One or more embodiments of the present disclosure provide a treatment compound for use in a method of treating colorectal cancer. The method can include contacting a colorectal cancer cell with the treatment compound. The method can include administering the treatment compound to a mammal.

In the Examples, various terms and designations for materials are used including, for instance, the following:.

Culture initiation and maintenance was performed as follows. Culture initiation and maintenance was performed in accordance with "Thawing, Propagating, and Cryopreserving Protocol" NCI-PBCF-HTB38 (HT-<NUM>) Colon Adenocarcinoma (ATCC®HTB-<NUM>™); February <NUM>, <NUM>; Version <NUM>.

HT-<NUM> (ATCC® HTB-<NUM>™) cells (which contained approximately <NUM> × <NUM><NUM> cells per mL) were initiated and seeded into a T-<NUM> flask containing McCoy's 5A and fetal bovine serum (<NUM>% (v/v)). Then, ATCC® <NUM>-<NUM> (warmed in <NUM>° C water bath for at least <NUM> minutes) was used to expand the HT-<NUM> cells. The cells were grown in a humidified incubator (SANYO INCT-<NUM>-CMT; MCO-19AIC (UV)) maintained at <NUM>° C and <NUM>% CO<NUM>. Then, the cells were rinsed with 1X Dulbecco's Phosphate-Buffered Saline and sub-cultured in T-<NUM> flasks <NUM> to <NUM> times per week using 1X Trypsin-EDTA, applied for < <NUM> minutes; enzymatic action of the trypsin-EDTA was stopped by adding complete growth medium to the detached cells. Then, upon reaching <NUM> to <NUM>% confluency the cells were split into the following split ratio ranges: <NUM>:<NUM> to <NUM>:<NUM>. Subculture and growth expansion activities were recorded, such as passage number, % confluency, % viability (only on experimental set-up day), and cell morphology throughout all phases. The cells were maintained in log-phase growth.

Cell culture plating was performed as follows. A cell suspension from a single <NUM> to <NUM>% confluent T-<NUM> flask was harvested 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-<NUM>-CMT) in which <NUM> chambers of each slide were provided with <NUM>µL each of <NUM>:<NUM>, <NUM>% trypan-blue dye (INVITROGEN T10282) and cell suspension. Cell counts and percent viability were averaged from both chambers of a single slide. Then, viable cells (defined as viability ≥ <NUM>%) containing complete growth medium were plated onto sterile <NUM>-well plates using a multi-channel pipette. Per cell density, between <NUM> and <NUM> cells per well (<NUM>,<NUM> to <NUM>,<NUM> cells per mL) were added to each well, except for wells that were utilized as 'saline only' no cell control wells; equal volumes each of <NUM>µL of cell suspension were added per well beginning with row A to H on the plate. The plates used for each of <NUM> endpoints, apoptosis and cytotoxicity, were solid white plates and clear plates, respectively. The cells were incubated for <NUM> ± <NUM> hours to allow attachment.

Stock solutions were prepared at respective target concentrations of trimethylpropane ethoxylate, <NUM>-arm poly(ethylene glycol), and glycerol ethoxylate in sterile saline. For the assays, based on the solubility limits due to high molecular weight, adjustments to lower stock concentration preparations (w/v) to generate either a solution or a pipettable suspension were made if necessary, or solubilization was achieved by adding small increments of saline, continuous mixing, vortexing, sonicating, or stirring prior to use in assay. If necessary for solubilization, the saline was pre-heated to <NUM>° C prior to mixing with the trimethylpropane ethoxylate, <NUM>-arm poly(ethylene glycol), and/or glycerol ethoxylate. Total volumes of <NUM> were prepared per tested substance on the day of cell suspension plating (Day <NUM>).

Thiazolyl blue tetrazolium bromide was prepared at <NUM>/mL in Dulbecco's Phosphate-Buffered Saline with calcium and magnesium. Total volumes of <NUM> were prepared (w/v) per set-up day (Day <NUM>) and stored at <NUM> until use.

Dosing solutions/suspensions of each test substance stock were prepared in a total of <NUM> each of McCoy's 5A and <NUM>% fetal bovine serum. Various amounts of dosing stock were utilized to achieve dosing solutions/suspensions from <NUM> to <NUM>. The dosing solutions/suspensions were prepared in sterile reservoirs and repeatedly mixed with a pipette until visible uniformity was achieved. Using a <NUM> capacity sterile <NUM>-deep well block, <NUM> of dosing solution/suspension was added to each of <NUM> replicate wells for the treatment groups and each of <NUM> wells for the saline only cell controls and saline only 'no cell' background correction controls. The plates were established following a semi-randomized statistical design. Each test substance was identified numerically and via a color code used for identifying wells to be treated. The blocks were covered with sealing tape, plate lid and placed into a <NUM> lab refrigerator (Fischer Scientific, 135B1; RFR-<NUM>-CMT) overnight.

All <NUM>-deep well blocks containing dosing solutions/suspensions were removed from the refrigerator and placed in a <NUM> bead bath for a minimum of <NUM> minutes. Approximately <NUM> hours after plating, well plates were removed from the incubator and treated one at a time. All wells from a cell plate were aspirated using a <NUM>-well aspirating device starting with row A to H. Using a multi-channel pipette, <NUM>µL each of dosing solution/suspension (from the blocks) was added to each well of the <NUM>-well cell treatment plate; starting from row A to row H (same order). All wells were aspirated and treated <NUM> rows at a time to prevent well drying and maintain cell attachment and viability; pipette tips were changed per row. All plates were placed into the incubator and allowed to treat for <NUM> ± <NUM> or <NUM> ± <NUM> hours prior to harvest.

Assessment of apoptosis was performed as follows. Apoptosis was performed in accordance with "Caspase-Glo <NUM>/<NUM> Assay" <NUM>. Standard Protocol for Cells in a <NUM>-Well Plate (Promega). The Caspase-Glo <NUM>/<NUM> Assay components were pre-warmed to room temperature for approximately <NUM> minutes. White plates were removed (one at a time) from the incubator and the treatment medium was aspirated. Using a multi-channel pipette, <NUM>µL of 1X Dulbecco's Phosphate-Buffered Saline was added to each well of the <NUM>-well plate. The Assay reagents (buffer and substrate) were manually mixed and added to a reagent reservoir; using a multi-channel pipette, <NUM>µL of the Assay reagent mixture was added to each well of the <NUM>-well plate. The plate(s) (protected with foil from light) were placed on a plate shaker and allowed to rotate for <NUM> minutes at approximately <NUM> rpm at room temperature. The plates were then incubated at room temperature for an additional <NUM> minutes prior to analysis. Luminescence was recorded in terms of Relative Light Units (RLU) for each plate on a FLUOstar Omega Plate Reader.

Assessment of cytotoxicity was performed as follows. Cytotoxicity reagent (<NUM>/mL), as previously described, was pre-warmed to room temperature for approximately <NUM> minutes and then diluted into 1X Dulbecco's Phosphate-Buffered Saline with calcium and magnesium to provide a concentration of <NUM>/mL (final). Clear plates were removed from the incubator (one at a time) and the treatment medium was aspirated. Using a multi-channel pipette, <NUM>µL of the cytotoxicity reagent (final) was added to each well of the <NUM>-well plate; then the plates were covered with sealing tape and incubated in a humidified <NUM> incubator for <NUM> hours. Following incubation, the supernatant was aspirated and dimethyl sulfoxide (<NUM>µL) was added to each well. Following thorough mixing by repeat pipetting, the cell lysate was transferred to a new clear <NUM>-well plate and absorbance was quantified at <NUM> and <NUM> on a FLUOstar Omega Plate Reader.

Relative caspase activity of each test substance containing well / <NUM> replicates = Average relative caspase activity. The results are reported in Tables <NUM>, <NUM>, and <NUM> for the various utilized concentrations.

Cell viability was calculated as follows:
<MAT>
<MAT>
<MAT> <MAT>.

% Cell Viability of each test substance containing well / <NUM> replicates = Average % Cell Viability. The results are reported in Table <NUM>, <NUM>, and <NUM> for the various utilized concentrations.

The data of Table <NUM> illustrate that advantageous relative caspase activities, i.e. average relative caspase activity ><NUM>, were provided when cells were exposed to <NUM>, <NUM>, and <NUM> concentrations of trimethylpropane ethoxylate, as indicted by the respective average relative caspase activity (Runs <NUM>, <NUM>, <NUM>, <NUM>) values.

The data of Table <NUM> illustrate that adequate viability, i.e. average viability % of <NUM>% or greater for (Runs <NUM>, <NUM>, <NUM>, <NUM>), were provided after <NUM> hours when cells were exposed to <NUM>, <NUM>, and <NUM> concentrations of trimethylpropane ethoxylate.

The data of Table <NUM> illustrate that advantageous relative caspase activities, i.e. average relative caspase activity ><NUM>, were provided when cells were exposed to <NUM>, <NUM>, and <NUM> concentrations of <NUM>-arm poly(ethylene glycol), as indicted by the respective average relative caspase activity (Runs <NUM>, <NUM>) values.

The data of Table <NUM> illustrate that adequate viability, i.e. average viability % of <NUM>% or greater for average viability % (Runs <NUM>, <NUM>), were provided after <NUM> hours when cells were exposed to <NUM>, <NUM>, and <NUM> concentrations of <NUM>-arm poly(ethylene glycol).

The data of Table <NUM> illustrate that advantageous relative caspase activities, i.e. average relative caspase activity ><NUM>, were provided when cells were exposed to <NUM> and <NUM> concentrations of glycerol ethoxylate, as indicted by the respective average relative caspase activity (Runs <NUM>, <NUM>, <NUM>, <NUM>) values.

Claim 1:
A treatment compound for use in a method of treating colorectal cancer, wherein the treatment compound is represented by
(i) Formula I:
<CHM>
where each n is independently from <NUM> to <NUM>;
(ii) Formula II:
<CHM>
where each n is independently from <NUM> to <NUM>; or
(iii) Formula III:
<CHM>
where each n is independently from <NUM> to <NUM>