Patent Grant
9040662
The Sequence Listing submitted Nov. 21, 2014 as a text file named “21650—0024U2_revised_sequence_listing.txt,” created on Nov. 21, 2014 and having a size of 17,889 bytes, is hereby incorporated by reference pursuant to 37 C.F.R. §1.52(e)(5).
The ability to kill cells provides a powerful therapeutic approach to treatment of disease or infection. Bacteria and fungi are becoming resistant to many of the currently available antimicrobial or antimycotic therapeutic compounds. Cancer cells frequently acquire mutations that enhance resistance to standard treatments. Principal among these mechanisms of drug resistance is abnormal expression of members of the B cell lymphoma-2 (Bcl-2) family (Oltersdorf et al., 2005). The Bcl-2 family consists of more than twenty anti- and pro-apoptotic members that modulate the balance between life and death. Tumors expressing high levels of anti-apoptotic proteins, such as Bcl-2, Mcl-1 or Bcl-xl, can be resistant to the effects of chemotherapeutics (Oltersdorf et al., 2005). This is accomplished, in part, by inhibition of the pro-apoptotic Bcl-2 family members, such as Bax, first identified as a protein that interacts with Bcl-2 (Oltvai et al., 1993). The association of Bax with mitochondria is linked to the release of cytochrome c and other death-mediators from mitochondrial reserves (Eskes et al., 1998).
Bax is a 21 kD protein of 192 amino acids, comprised of nine alpha helices (Suzuki et al., 2000). Under non-apoptotic conditions, Bax predominantly resides in the cytosol, with a small percentage of the protein localized to the mitochondria (Boohaker et al., 2011; Kaufmann et al., 2003; Putcha et al., 1999).
Despite advances in understanding the physiology and pathophysiology of cancer and/or aberrant cell growth, there is still a scarcity of compounds that are efficacious and safe in the treatment of cancer and/or aberrant cell growth. Therapeutics that are effective against bacteria and fungi are needed as these microorganisms grow more resistant to current therapies. These needs and other needs are satisfied by the present invention.
Disclosed herein is a method of permeabilizing membranes of cells. Such membranes may be outer membranes, cell membranes or interior cellular membranes. The cells may be individual cells or cells in a subject. Methods may comprise administering to at least a cell an effective amount of a C-terminal Bax peptide (CT20p peptide) or a composition comprising an effective amount of a CT20p peptide.
Disclosed herein is a method of killing cancer cells in a subject comprising administering to at least one cell of a subject an effective amount of CT20p peptide or a composition comprising an effective amount of CT20p peptide. As used herein, CT20, CT20p peptide or CT20p peptide refers to a peptide comprising the last 20 amino acids of the Bax C-terminus.
Disclosed herein is a method of killing microbial cells, including but not limited to bacteria and fungi, whether on a surface, present in a colony or in a subject, comprising administering to at least one cell of a subject an effective amount of a CT20p peptide or a composition comprising an effective amount of a CT20p peptide.
Disclosed herein is a method of permeabilizing membranes of cells. Such membranes may be outer membranes, cell membranes or interior cellular membranes. The cells may be individual cells or cells in a subject. The method comprises (i) administering an effective amount of a CT20p peptide, and (ii) forming at least one pore in a membrane of at least one cell, wherein the peptide or a composition comprising an effective amount of a CT20p peptide destabilizes at least one membrane, facilitates ion exchange, and/or causes a sequestered molecule to be released.
Disclosed herein is a method of killing cancer cells in a subject comprising (i) administering to a subject an effective amount of a CT20p peptide, (ii) permeabilizing at least one membrane in a cell of the subject, and (iii) inducing cell death.
Disclosed herein is a method of killing bacterial or fungal cells in a subject comprising (i) administering to a subject an effective amount of a CT20p peptide, (ii) permeabilizing at least one membrane in a cell of the subject, and (iii) inducing cell death.
Disclosed herein is a composition for permeabilizing membranes on or in cells comprising a CT20p peptide.
Disclosed herein is a composition for killing cells comprising a CT20p peptide.
Disclosed herein is a composition for permeabilizing lipid membranes in cells comprising a CT20p peptide and one or more therapeutic agents, including but not limited to antibiotics, antimycotis, and anti-cancer drugs.
Disclosed herein is a composition for killing cells comprising a CT20p peptide and one or more therapeutic agents, including but not limited to antibiotics, antimycotis, and anticancer drugs.
The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects and together with the description serve to explain the principles of the invention.
Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or can be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
The present invention comprises methods and compositions for disrupting the cellular membranes of cells. The cells may be procaryotes or eucaryotes, and may be found as individual cells, in colonies, on or within multi-celled organisms such as plants or animals. For example, microbial cells may be found on the surface of an animal, such as a human, or within the animal. Cancer cells may be found in a subject, such as a plant, human or animal.
Mitochondria play a central role in regulating both apoptotic and non-apoptotic or necrotic pathways by selectively releasing cell death promoting factors. The determination as to whether or not an apoptotic or non-apoptotic cell death pathway will be activated is dependent on the type of death mediator released from the mitochondria.
Most current anti-cancer drugs only trigger the apoptotic pathway. Defects in the apoptotic machinery can contribute to tumor formation and resistance to treatment, creating a need to identify anti-cancer agents that kill cells by novel mechanisms. The development of a new therapeutic agent that induces cell death via a non-apoptotic mechanism and can be used in combination with standards drugs that induce apoptosis will have significant impact on the treatment of drug resistance cancers and could greatly improve overall patient outcomes. As described herein, the death-inducing properties of the C-terminal (CT) of the alpha-9 helix of Bax, an amphipathic domain with putative membrane binding properties, were examined.
To examine the cytotoxic potential of the CT domain of Bax, a peptide (CT20) was generated. The CT20p peptide permeabilized mitochondrial-like lipid vesicles and caused cell death. The cell death pathway triggered by the CT20p peptide was independent of effector caspases and resistant to Bcl-2 over-expression. This indicates that the CT20p peptide can be used in combinatorial therapies to sensitize drug-resistant cancer cells to treatment. Other properties of the CT20 Bax peptide, show that the CT20p peptide causes cell membrane permeability, and such a peptide may be combined with antimicrobial therapeutics or therapies to cause cell death in microorganisms, such as gram-positive and gram-negative bacteria, other types of bacteria, fungi and other microorganisms and infectious agents.
Disclosed herein are compositions comprising a CT20p peptide.
1. Compositions for Permeabilizing Membranes in Cells
Disclosed herein is a composition for permeabilizing membranes in cells comprising a CT20p peptide. The cells may be individual cells, or cells that are on or in a subject. In an aspect, the cells are eukaryotic or prokaryotic cells, including but not limited to bacteria and fungi. In an aspect, the cells are in a subject. In an aspect, the cells are on a surface, which may be inert or may be the surface of a subject. In an aspect, the cells are cancer cells or transformed cells. In an aspect, the cancer cell can be a cell from any type of cancer including, but not limited to, cancer of the head and neck cancer, esophagus, stomach, pancreas, kidney, bladder, bone, brain, and cervix. In an aspect, the cancer is breast cancer. In an aspect, the cancer is colorectal cancer. In an aspect, the cancer is lung cancer. In an aspect, the cancer is a drug resistant cancer. In an aspect, the cancer cell is a drug resistant cancer cell. In an aspect, a disclosed composition comprising a truncated Bax peptide is administered directly into a tumor.
In an aspect, a disclosed composition for permeabilizing membranes in cells forms one or more pores in the membranes of the cells. In an aspect, the membrane may be an outer membrane, a cellular membrane or an organelle membrane. In an aspect, the membrane is a mitochondrial membrane. In an aspect, the one or more pores are formed in a mitochondrial membrane of a cancer cell.
In an aspect, a disclosed composition for permeabilizing membranes in cells destabilizes a cellular membrane. In an aspect, a disclosed composition facilitates ion exchange. In an aspect, a disclosed composition causes a sequestered molecule to be released. In an aspect, a disclosed composition destabilizes a cellular membrane, facilitates ion exchange, and causes a sequestered molecule to be released. In an aspect, a disclosed composition destabilizes a cellular membrane and facilitates ion exchange. In an aspect, a disclosed composition destabilizes a cellular membrane and causes a sequestered molecule to be released. In an aspect, a disclosed composition facilitates ion exchange and causes a sequestered molecule to be released.
In an aspect, a disclosed composition for permeabilizing membranes in cells comprising a CT20p peptide induces cell death. In an aspect, the cell death mimics necrosis. In an aspect, the cell death occurs independent of endogenous Bax activity. In an aspect, the cell death occurs independent of endogenous caspase activity. In an aspect, the cell death is resistant to Bcl-2 over-expression.
In an aspect, a disclosed composition for permeabilizing membranes in cells comprising a CT20p peptide induces cell death, wherein (i) the cell death mimics necrosis, (ii) the cell death occurs independent of endogenous Bax activity, (iii) the cell death occurs independent of endogenous caspase activity, or (iv) the cell death is resistant to Bcl-2 over-expression, or (v) the cell death exhibits a combination thereof.
In an aspect, a pore-forming composition may comprise an amount of a CT20p peptide so that pores comprising from two to ten peptides, from two to eight peptides, from four to ten peptides, from five, six, seven, eight, nine, ten or more peptides, can be formed.
In an aspect, a disclosed composition for permeabilizing membranes in cells comprises a CT20p peptide comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and/or SEQ ID NO: 4. For example, in an aspect, a disclosed CT20p peptide is VTIFVAGVLTASLTIWKKMG (SEQ ID NO: 1). In an aspect, a disclosed CT20p peptide is VTIFVAGVLTASLTIWEEMG (SEQ ID NO: 2). In an aspect, a disclosed CT20p peptide is VTIFVAGVLTASLTIWLLMG (SEQ ID NO: 3). In an aspect, a disclosed CT20p peptide is VTIFVAGVLTASLTIWRRMG (SEQ ID NO: 4). In an aspect, a disclosed composition for permeabilizing membranes in cells comprises one or more CT20 Bax peptides, wherein the one or more CT20 Bax peptides comprise SEQ ID NO: 1, SEQ NO: 2, SEQ ID NO: 3; or SEQ ID NO: 4.
In an aspect, a CT20p peptide of a disclosed composition for permeabilizing membranes in cells is encapsulated in polymeric nanoparticles. In an aspect, the nanoparticles are aminated. In an aspect, the nanoparticles are carboxylated.
In an aspect, a disclosed composition for permeabilizing membranes in cells comprising a CT20p peptide further comprises one or more therapeutic compounds, such as one or more antimicrobial compounds, one or more antibacterial compounds, one or more antifungal compounds, or one or more anti-cancer drugs, or a combination thereof. In an aspect, the one or more anti-cancer drugs comprise cisplatin. In an aspect, the one or more anti-cancer drugs induce apoptosis. In an aspect, a disclosed composition comprising a CT20p peptide further comprises one or more chemotherapeutic drugs. In an aspect, a disclosed composition comprising a CT20p peptide further comprises one or more radiosensitizers.
In an aspect, a disclosed composition for permeabilizing membranes in cells comprising a CT20p peptide further comprises (i) one or more anti-cancer drugs, (ii) one or more chemotherapeutic drugs, and (iii) one or more radiosensitizers. In an aspect, a disclosed composition further comprises one or more anti-cancer drugs and one or more chemotherapeutic drugs. In an aspect, a disclosed composition further comprises one or more anti-cancer drugs and one or more radiosensitizers. In an aspect, a disclosed composition further comprises one or more chemotherapeutic drugs and one or more radiosensitizers.
In an aspect, a disclosed composition for permeabilizing membranes in cells is administered to a subject. In an aspect, the subject is a mammal. In an aspect, the mammal is a primate. In an aspect, the mammal is a human. In an aspect, the human is a patient.
In an aspect, a disclosed composition for permeabilizing membranes in cells comprising a CT20p peptide is administered to a subject at least two times. In an aspect, a disclosed composition is administered to the subject two or more times. In an aspect, a disclosed composition is administered at routine or regular intervals. For example, in an aspect, a disclosed composition is administered to the subject one time per day, or two times per day, or three or more times per day. In an aspect, a disclosed composition is administered to the subject daily, or one time per week, or two times per week, or three or more times per week, etc. In an aspect, a disclosed composition is administered to the subject weekly, or every other week, or every third week, or every fourth week, etc. In an aspect, a disclosed composition is administered to the subject monthly, or every other month, or every third month, or every fourth month, etc. In an aspect, the repeated administration of a disclosed composition occurs over a pre-determined or definite duration of time. In an aspect, the repeated administration of a disclosed composition occurs over an indefinite period of time.
In an aspect, following the administration of a disclosed composition for permeabilizing membranes in cells comprising a CT20p peptide, the cells are sensitized to treatment. In an aspect, following the administration of a disclosed composition comprising a CT20p peptide, a subject is sensitized to treatment. In an aspect, an increased sensitivity or a reduced sensitivity to a treatment, such as a therapeutic treatment, is measured according to one or more methods as known in the art for the particular treatment. In an aspect, methods of measuring sensitivity to a treatment include, but not limited to, cell proliferation assays and cell death assays. In an aspect, the sensitivity of a cell or a subject to treatment can be measured or determined by comparing the sensitivity of a cell or a subject following administration of a disclosed composition comprising a CT20p peptide to the sensitivity of a cell or subject that has not been administered a disclosed composition comprising a CT20p peptide.
For example, in an aspect, following the administration of a disclosed composition for permeabilizing membranes in cells comprising a CT20p peptide, the cell is 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, or greater, more sensitive to treatment than a cell that has not been administered a disclosed composition comprising a CT20p peptide. In an aspect, following the administration of a disclosed composition comprising a CT20p peptide the cell is 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, or greater, less resistant to treatment than a cell that has not been administered a disclosed composition comprising a CT20p peptide. The determination of a cell's or a subject's sensitivity or resistance is routine in the art and within the skill of an ordinary clinician and/or researcher.
In an aspect, the determination of a cell's or a subject's sensitivity or resistance to treatment can be monitored. For example, in an aspect, data regarding sensitivity or resistance can be acquired periodically, such as every week, every other week, every month, every other month, every 3 months, 6 months, 9 months, or every year, every other year, every 5 years, every 10 years for the life of the subject, for example, a human subject or patient with cancer and/or aberrant cell growth. In an aspect, data regarding sensitivity or resistance can be acquired at various rather than at periodic times. In an aspect, treatment for a subject can be modified based on data regarding a cell's or a subject's sensitivity or resistance to treatment. For example, in an aspect, the treatment can modified by changing the dose of a disclosed compositions, the route of administration of a disclosed compositions, the frequency of administration of a disclosed composition, etc.
Disclosed herein is a composition for permeabilizing lipid membranes in cells comprising a CT20p peptide and one or more anti-cancer drugs.
2. Compositions for Killing Cells
Disclosed herein is a composition for killing cells comprising a CT20p peptide. In an aspect, the cells are eukaryotic or prokaryotic cells, including but not limited to bacteria and fungi. In an aspect, the cells are in a subject. In an aspect, the cells are on a surface, which may be inert or may be the surface of a subject. In an aspect, the cells are cancer cells or transformed cells. In an aspect, the cancer cell can be a cell from any type of cancer including, but not limited to, cancer of the head and neck cancer, esophagus, stomach, pancreas, kidney, bladder, bone, brain, and cervix. In an aspect, the cancer is breast cancer. In an aspect, the cancer is colorectal cancer. In an aspect, the cancer is lung cancer. In an aspect, the cancer is a drug resistant cancer. In an aspect, the cancer cell is a drug resistant cancer cell. In an aspect, a disclosed composition comprising a CT20p peptide is administered directly into a tumor.
In an aspect, a disclosed composition for killing cells forms one or more pores in the membranes of the cells. In an aspect, the membrane may be an outer membrane, a cellular membrane or an organelle membrane. In an aspect, the membrane is a mitochondrial membrane. In an aspect, the one or more pores are formed in a mitochondrial membrane. In an aspect, the one or more pores are formed in a mitochondrial membrane of a cancer cell.
In an aspect, a disclosed composition for killing cells destabilizes a cellular membrane. In an aspect, a disclosed composition facilitates ion exchange. In an aspect, a disclosed composition causes a sequestered molecule to be released. In an aspect, a disclosed composition destabilizes a cellular membrane, facilitates ion exchange, and causes a sequestered molecule to be released. In an aspect, a disclosed composition destabilizes a cellular membrane and facilitates ion exchange. In an aspect, a disclosed composition destabilizes a cellular membrane and causes a sequestered molecule to be released. In an aspect, a disclosed composition facilitates ion exchange and causes a sequestered molecule to be released.
In an aspect, a disclosed composition for killing cells comprising a CT20p peptide induces cell death. In an aspect, the cell death mimics necrosis. In an aspect, the cell death occurs independent of endogenous Bax activity. In an aspect, the cell death occurs independent of endogenous caspase activity. In an aspect, the cell death is resistant to Bcl-2 over-expression.
In an aspect, a disclosed composition for killing cells comprising a CT20p peptide induces cell death, wherein (i) the cell death mimics necrosis, (ii) the cell death occurs independent of endogenous Bax activity, (iii) the cell death occurs independent of endogenous caspase activity, or (iv) the cell death is resistant to Bcl-2 over-expression, or (v) the cell death exhibits a combination thereof.
In an aspect, a composition for killing cells comprises a pore-forming composition which may comprise an amount of a CT20p peptide so that pores comprising from two to ten peptides, from two to eight peptides, from four to ten peptides, from five, six, seven, eight, nine, ten or more peptides, can be formed.
In an aspect, a disclosed composition for killing cells comprises a CT20p peptide comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and/or SEQ ID NO: 4. For example, in an aspect, a disclosed CT20p peptide is VTIFVAGVLTASLTIWKKMG (SEQ ID NO: 1). In an aspect, a disclosed CT20p peptide is VTIFVAGVLTASLTIWEEMG (SEQ ID NO: 2). In an aspect, a disclosed CT20p peptide is VTIFVAGVLTASLTIWLLMG (SEQ ID NO: 3). In an aspect, a disclosed CT20p peptide is VTIFVAGVLTASLTIWRRMG (SEQ ID NO: 4). In an aspect, a disclosed composition for killing cells comprises one or more CT20 Bax peptides, wherein the one or more CT20 Bax peptides comprise SEQ ID NO: 1, SEQ NO: 2, SEQ ID NO: 3; or SEQ ID NO: 4.
In an aspect, a CT20p peptide of a disclosed composition for killing cells is encapsulated in polymeric nanoparticles. In an aspect, the nanoparticles are aminated. In an aspect, the nanoparticles are carboxylated.
In an aspect, a disclosed composition for killing cells comprising a CT20p peptide further comprises one or more therapeutic compounds, such as one or more antimicrobial compounds, one or more antibacterial compounds, one or more antifungal compounds, or one or more anti-cancer drugs, or a combination thereof. In an aspect, the one or more anti-cancer drugs comprise cisplatin. In an aspect, the one or more anti-cancer drugs induce apoptosis. In an aspect, a disclosed composition for killing cells comprising a CT20p peptide further comprises one or more chemotherapeutic drugs. In an aspect, a disclosed composition for killing cells comprising a CT20p peptide further comprises one or more radiosensitizers.
In an aspect, a disclosed composition for killing cells comprising a CT20p peptide further comprises (i) one or more anti-cancer drugs, (ii) one or more chemotherapeutic drugs, and (iii) one or more radiosensitizers. In an aspect, a disclosed composition for killing cells further comprises one or more anti-cancer drugs and one or more chemotherapeutic drugs. In an aspect, a disclosed composition for killing cells further comprises one or more anti-cancer drugs and one or more radiosensitizers. In an aspect, a disclosed composition for killing cells further comprises one or more chemotherapeutic drugs and one or more radiosensitizers.
In an aspect, a disclosed composition for killing cells is administered to a subject. In an aspect, the subject is a mammal. In an aspect, the mammal is a primate. In an aspect, the mammal is a human. In an aspect, the human is a patient.
In an aspect, a disclosed composition for killing cells comprising a CT20p peptide is administered to a subject at least two times. In an aspect, a disclosed composition is administered to the subject two or more times. In an aspect, a disclosed composition is administered at routine or regular intervals. For example, in an aspect, a disclosed composition is administered to the subject one time per day, or two times per day, or three or more times per day. In an aspect, a disclosed composition is administered to the subject daily, or one time per week, or two times per week, or three or more times per week, etc. In an aspect, a disclosed composition is administered to the subject weekly, or every other week, or every third week, or every fourth week, etc. In an aspect, a disclosed composition is administered to the subject monthly, or every other month, or every third month, or every fourth month, etc. In an aspect, the repeated administration of a disclosed composition occurs over a pre-determined or definite duration of time. In an aspect, the repeated administration of a disclosed composition occurs over an indefinite period of time.
In an aspect, following the administration of a disclosed composition for killing cells comprising a CT20p peptide, the cells are sensitized to treatment. In an aspect, following the administration of a disclosed composition for killing cells comprising a CT20p peptide, a subject is sensitized to treatment. In an aspect, an increased sensitivity or a reduced sensitivity to a treatment, such as a therapeutic treatment, is measured according to one or more methods as known in the art for the particular treatment. In an aspect, methods of measuring sensitivity to a treatment include, but not limited to, cell proliferation assays and cell death assays. In an aspect, the sensitivity of a cell or a subject to treatment can be measured or determined by comparing the sensitivity of a cell or a subject following administration of a disclosed composition for killing cells comprising a CT20p peptide to the sensitivity of a cell or subject that has not been administered a disclosed composition for killing cells comprising a CT20p peptide.
For example, in an aspect, following the administration of a disclosed composition for killing cells comprising a CT20p peptide, the cell is 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, or greater, more sensitive to treatment than a cell that has not been administered a disclosed composition for killing cells comprising a CT20p peptide. In an aspect, following the administration of a disclosed composition for killing cells comprising a CT20p peptide, the cell is 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, or greater, less resistant to treatment than a cell that has not been administered a disclosed composition for killing cells comprising a CT20p peptide. The determination of a cell's or a subject's sensitivity or resistance is routine in the art and within the skill of an ordinary clinician and/or researcher.
In an aspect, the determination of a cell's or a subject's sensitivity or resistance to treatment can be monitored. For example, in an aspect, data regarding sensitivity or resistance can be acquired periodically, such as every week, every other week, every month, every other month, every 3 months, 6 months, 9 months, or every year, every other year, every 5 years, every 10 years for the life of the subject, for example, a human subject or patient with cancer and/or aberrant cell growth. In an aspect, data regarding sensitivity or resistance can be acquired at various rather than at periodic times. In an aspect, treatment for a subject can be modified based on data regarding a cell's or a subject's sensitivity or resistance to treatment. For example, in an aspect, the treatment can modified by changing the dose of a disclosed compositions, the route of administration of a disclosed compositions, the frequency of administration of a disclosed composition, etc.
Disclosed herein is a composition for killing cells comprising a CT20p peptide and one or more anti-cancer drugs.
3. Pharmaceutical Compositions
In an aspect, the invention relates to pharmaceutical compositions comprising a disclosed composition for permeabilizing membranes in cells. In an aspect, the invention relates to pharmaceutical compositions comprising a disclosed composition for killing cells. In an aspect, the disclosed compositions for permeabilizing membranes and cells and for killing cells comprise a CT20 Bax peptide. In an aspect, a pharmaceutical composition can be provided comprising a therapeutically effective amount of at least one disclosed composition and a pharmaceutically acceptable carrier.
1. Permeabilizing Membranes of Cells
Disclosed herein are methods of permeabilizing membranes of cells. The cells may be individual cells, or cells that are on or in a subject. In an aspect, the cells are eukaryotic or prokaryotic cells, including but not limited to bacteria and fungi.
In an aspect, disclosed herein is a method of permeabilizing membranes of cells in a subject comprising administering to at least a cell a CT20p peptide. In an aspect, disclosed herein is a method of permeabilizing membranes of cells in a subject comprising administering to at least a cell a composition comprising an effective amount of a CT20p peptide. In an aspect, the membrane may be an outer membrane, a cellular membrane or an organelle membrane. In an aspect the cell is a bacterial cell. In an aspect, the cell is a fungal cell. In an aspect, the cell is a microbial cell. In an aspect, the cell is a cancer cell or a transformed cell. In an aspect, the cancer cell can be a cell from any type of cancer including, but not limited to, cancer of the head and neck cancer, esophagus, stomach, pancreas, kidney, bladder, bone, brain, and cervix. In an aspect, the cancer is breast cancer. In an aspect, the cancer is colorectal cancer. In an aspect, the cancer is lung cancer. In an aspect, the cancer is a drug resistant cancer. In an aspect, the cancer cell is a drug resistant cancer cell. In an aspect, a CT20p peptide or a composition comprising an effective amount of a CT20p peptide is administered directly into a tumor.
In an aspect of a disclosed a method of permeabilizing membranes of cells, a CT20p peptide or a composition comprising an effective amount of a CT20p peptide forms at least one pore in the membrane of the cell. In an aspect, a CT20p peptide or a composition comprising an effective amount of a CT20p peptide forms two or more pores in the membrane of the cell. In an aspect, cell is a cancer cell. In an aspect, the one or more pores are formed in a mitochondrial membrane. In an aspect, the one or more pores are formed in a mitochondrial membrane of a cancer cell.
In an aspect of a disclosed method of permeabilizing membranes of cells in a subject, a CT20p peptide or a composition comprising an effective amount of a CT20p peptide destabilizes a cellular membrane, facilitates ion exchange, and causes a sequestered molecule to be released. In an aspect, a CT20p peptide or a composition comprising an effective amount of a CT20p peptide destabilizes a cellular membrane. In an aspect, a CT20p peptide or a composition comprising an effective amount of a CT20p peptide facilitates ion exchange, exchange. In an aspect, a CT20p peptide or a composition comprising an effective amount of a CT20p peptide causes a sequestered molecule to be released. In an aspect, a CT20p peptide or a composition comprising an effective amount of a CT20p peptide destabilizes a cellular membrane and facilitates ion exchange. In an aspect, a CT20p peptide or a composition comprising an effective amount of a CT20p peptide destabilizes a cellular membrane and causes a sequestered molecule to be released. In an aspect, a CT20p peptide or a composition comprising an effective amount of a CT20p peptide facilitates ion exchange and causes a sequestered molecule to be released.
In an aspect, following the administration of a CT20p peptide or a composition comprising an effective amount of a CT20p peptide, the cells are sensitized to treatment. In an aspect, following the administration of CT20p peptide or a composition comprising an effective amount of a CT20p peptide, a subject is sensitized to treatment. In an aspect, an increased sensitivity or a reduced sensitivity to a treatment, such as a therapeutic treatment, is measured according to one or more methods as known in the art for the particular treatment. In an aspect, methods of measuring sensitivity to a treatment include, but not limited to, cell proliferation assays and cell death assays. In an aspect, the sensitivity of a cell or a subject to treatment can be measured or determined by comparing the sensitivity of a cell or a subject following administration of a CT20p peptide or a composition comprising an effective amount of a CT20p peptide to the sensitivity of a cell or subject that has not been administered a CT20p peptide or a composition comprising an effective amount of a CT20p peptide.
For example, in an aspect, following the administration of a CT20p peptide or a composition comprising an effective amount of a CT20p peptide, the cell is 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, or greater, more sensitive to treatment than a cell that has not been administered a CT20p peptide or a disclosed composition comprising an effective amount of a CT20p peptide. In an aspect, following the administration of a CT20p peptide or a composition comprising an effective amount of a CT20p peptide, the cell is 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, or greater, less resistant to treatment than a cell that has not been administered a CT20p peptide or a disclosed composition comprising an effective amount of a CT20p peptide. The determination of a cell's or a subject's sensitivity or resistance is routine in the art and within the skill of an ordinary clinician and/or researcher.
In an aspect, the determination of a cell's or a subject's sensitivity or resistance to treatment can be monitored. For example, in an aspect, data regarding sensitivity or resistance can be acquired periodically, such as every week, every other week, every month, every other month, every 3 months, 6 months, 9 months, or every year, every other year, every 5 years, every 10 years for the life of the subject, for example, a human subject or patient with cancer and/or aberrant cell growth. In an aspect, data regarding sensitivity or resistance can be acquired at various rather than at periodic times. In an aspect, treatment for a subject can be modified based on data regarding a cell's or a subject's sensitivity or resistance to treatment. For example, in an aspect, the treatment can modified by changing the dose of a disclosed compositions, the route of administration of a disclosed compositions, the frequency of administration of a disclosed composition, etc.
In an aspect, a disclosed method of permeabilizing membranes of cells in a subject further comprises repeating the administration a CT20p peptide or a composition comprising an effective amount of a CT20p peptide. In an aspect, a CT20p peptide or a composition comprising an effective amount of a CT20p peptide is administered to the subject at least two times. In an aspect, a CT20p peptide or a composition comprising an effective amount of a CT20p peptide is administered to the subject two or more times. In an aspect, a CT20p peptide or a composition comprising an effective amount of a CT20p peptide administered at routine or regular intervals. For example, in an aspect, a CT20p peptide or a composition comprising an effective amount of a CT20p peptide is administered to the subject one time per day, or two times per day, or three or more times per day. In an aspect, a CT20p peptide or a composition comprising an effective amount of a CT20p peptide is administered to the subject daily, or one time per week, or two times per week, or three or more times per week, etc. In an aspect, a CT20p peptide or a composition comprising an effective amount of a CT20p peptide is administered to the subject weekly, or every other week, or every third week, or every fourth week, etc. In an aspect, a CT20p peptide or a composition comprising an effective amount of a CT20p peptide is administered to the subject monthly, or every other month, or every third month, or every fourth month, etc. In an aspect, the repeated administration of a CT20p peptide or a composition comprising an effective amount of a CT20p peptide occurs over a pre-determined or definite duration of time. In an aspect, the repeated administration of a CT20p peptide or a composition comprising an effective amount of a CT20p peptide occurs over an indefinite period of time.
In an aspect, a disclosed method of permeabilizing membranes of cells in a subject further comprises inducing cell death. In an aspect, cell death mimics necrosis. In an aspect, cell death occurs independent of endogenous Bax activity. In an aspect, cell death occurs independent of endogenous caspase activity. In an aspect, cell death is resistant to Bcl-2 over-expression.
In an aspect, a disclosed method of permeabilizing membranes of cells in a subject comprising administering a CT20p peptide or a composition comprising an effective amount of a CT20p peptide further comprises inducing cell death. Cell death may include, but is not limited to wherein (i) cell death mimics necrosis, (ii) cell death occurs independent of endogenous Bax activity, (iii) cell death occurs independent of endogenous caspase activity, or (iv) cell death is resistant to Bcl-2 over-expression, or (v) cell death exhibits a combination thereof.
In an aspect, a CT20p peptide or a composition comprising an effective amount of a CT20p peptide of a disclosed method of permeabilizing cell membranes comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and/or SEQ ID NO: 4. For example, in an aspect, a CT20p peptide is VTIFVAGVLTASLTIWKKIVIG (SEQ ID NO: 1). In an aspect, a CT20p peptide is VTIFVAGVLTASLTIWEEMG (SEQ ID NO: 2). In an aspect, a CT20p peptide is VTIFVAGVLTASLTIWLLMG (SEQ ID NO: 3). In an aspect, a CT20p peptide is VTIFVAGVLTASLTIWRRMG (SEQ ID NO: 4). In an aspect, a disclosed composition comprising an effective amount of a CT20p peptide comprises one or more CT20 Bax peptides, wherein the one or more CT20 Bax peptides comprise SEQ ID NO: 1, SEQ NO: 2, SEQ ID NO: 3; or SEQ ID NO: 4, or two or more of each. In an aspect, a CT20p peptide of a disclosed method of permeabilizing membranes of cells in a subject is encapsulated in polymeric nanoparticles. In an aspect, the nanoparticles are aminated. In an aspect, the nanoparticles are carboxylated.
In an aspect, a disclosed method of permeabilizing membranes comprising administering a CT20p peptide or a composition comprising an effective amount of a CT20p peptide further comprises administering one or more anti-cancer drugs. In an aspect, the one or more anti-cancer drugs comprise cisplatin. In an aspect, the one or more anti-cancer drugs induce apoptosis. In an aspect, a disclosed method of permeabilizing membranes comprising administering a CT20p peptide or a composition comprising an effective amount of a CT20p peptide further comprises administering one or more chemotherapeutic drugs. In an aspect, a disclosed method of permeabilizing membranes comprising administering a CT20p peptide or a composition comprising an effective amount of a CT20p peptide further comprises administering one or more radiosensitizers.
In an aspect, a disclosed method of permeabilizing membranes comprising administering a CT20p peptide or a composition comprising an effective amount of a CT20p peptide further comprises administering (i) one or more anti-cancer drugs, (ii) one or more chemotherapeutic drugs, and (iii) one or more radiosensitizers. In an aspect, a disclosed method further comprises administering one or more anti-cancer drugs and one or more chemotherapeutic drugs. In an aspect, a disclosed method further comprises administering one or more anti-cancer drugs and one or more radiosensitizers. In an aspect, a disclosed method further comprises administering one or more chemotherapeutic drugs and one or more radiosensitizers.
In an aspect of a disclosed method of permeabilizing membranes of cells, which may be in or on a subject, the subject is a mammal. In an aspect, the mammal is a primate. In an aspect, the mammal is a human. In an aspect, the human is a patient.
In an aspect, disclosed herein is a method of permeabilizing membranes of cells in a subject comprising (i) administering to a subject an effective amount of a CT20p peptide or a composition comprising an effective amount of a CT20p peptide, and (ii) forming at least one pore in a membrane of at least one cell of the subject, wherein the peptide destabilizes at least one membrane, facilitates ion exchange, and/or causes a sequestered molecule to be released.
2. Killing Cancer Cells
Disclosed herein are methods of killing cancer cells.
In an aspect, disclosed herein is a method of killing cancer cells in a subject comprising administering to at least one cell of a subject an effective amount of a CT20p peptide. In an aspect, disclosed herein is a method of killing cancer cells in a subject comprising administering to at least one cell of a subject a composition comprising an effective amount of a CT20p peptide. In an aspect, the cell is a cancer cell or a transformed cell. In an aspect, the cancer cell can be a cell from any type of cancer including, but not limited to, cancer of the head and neck cancer, esophagus, stomach, pancreas, kidney, bladder, bone, brain, and cervix. In an aspect, the cancer is breast cancer. In an aspect, the cancer is colorectal cancer. In an aspect, the cancer is lung cancer. In an aspect, the cancer is a drug resistant cancer. In an aspect, the cancer cell is a drug resistant cancer cell. In an aspect, a CT20p peptide or a composition comprising an effective amount of a CT20p peptide is administered directly into a tumor.
In an aspect, a disclosed method of killing cancer cells in a subject further comprises permeabilizing at least one membrane in the cell. In an aspect, the membrane is a mitochondrial membrane. In an aspect, the one or more pores are formed in a mitochondrial membrane. In an aspect, the one or more pores are formed in a mitochondrial membrane of a cancer cell.
In an aspect, permeabilizing at least one membrane in the cell comprises destabilizing a cellular membrane, facilitating ion exchange, and causing a sequestered molecule to be released. In an aspect, permeabilizing at least one membrane in the cell comprises destabilizes a cellular membrane. In an aspect, permeabilizing at least one membrane in the cell comprises facilitating ion exchange. In an aspect, permeabilizing at least one membrane in the cell comprises causing a sequestered molecule to be released. In an aspect, permeabilizing at least one membrane in the cell comprises destabilizing a cellular membrane and facilitating ion exchange. In an aspect, permeabilizing at least one membrane in the cell comprises destabilizing a cellular membrane and causing a sequestered molecule to be released. In an aspect, permeabilizing at least one membrane in the cell comprises facilitating ion exchange and causing a sequestered molecule to be released.
In an aspect of a disclosed method of killing cancer cells in a subject, the cancer cells are sensitized to treatment. In an aspect of a disclosed method of killing cancer cells in a subject, the subject is sensitized to treatment. In an aspect, an increased sensitivity or a reduced sensitivity to a treatment, such as a therapeutic treatment, is measured according to one or more methods as known in the art for the particular treatment. In an aspect, methods of measuring sensitivity to a treatment include, but not limited to, cell proliferation assays and cell death assays. In an aspect, the sensitivity of a cell or a subject to treatment can be measured or determined by comparing the sensitivity of a cell or a subject following administration of a CT20p peptide or a composition comprising a CT20p peptide to the sensitivity of a cell or subject that has not been administered a CT20p peptide or a composition comprising a CT20p peptide.
For example, in an aspect, following the administration of a CT20p peptide or a composition comprising a CT20p peptide, the cell is 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, or greater, more sensitive to treatment than a cell that has not been administered a CT20p peptide or a composition comprising a CT20p peptide. In an aspect, following the administration of a CT20p peptide or a composition comprising a CT20p peptide, the cell is 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, or greater, less resistant to treatment than a cell that has not been administered a CT20p peptide or a composition comprising a CT20p peptide. The determination of a cell's or a subject's sensitivity or resistance is routine in the art and within the skill of an ordinary clinician and/or researcher.
In an aspect, the determination of a cell's or a subject's sensitivity or resistance to treatment can be monitored. For example, in an aspect, data regarding sensitivity or resistance can be acquired periodically, such as every week, every other week, every month, every other month, every 3 months, 6 months, 9 months, or every year, every other year, every 5 years, every 10 years for the life of the subject, for example, a human subject or patient with cancer and/or aberrant cell growth. In an aspect, data regarding sensitivity or resistance can be acquired at various rather than periodic times. In an aspect, treatment for a subject can be modified based on data regarding a cell's or a subject's sensitivity or resistance to treatment.
In an aspect, a disclosed method of killing cancer cells in a subject further comprises repeating the administration of a CT20p peptide or a composition comprising an effective amount of a CT20p peptide. In an aspect, a CT20p peptide or a composition comprising an effective amount of a CT20p peptide is administered to the subject at least two times. In an aspect, a CT20p peptide or a composition comprising an effective amount of a CT20p peptide is administered to the subject two or more times. In an aspect, a CT20p peptide or a composition comprising an effective amount of a CT20p peptide is administered at routine or regular intervals. For example, in an aspect, a CT20p peptide or a composition comprising an effective amount of a CT20p peptide is administered to the subject one time per day, or two times per day, or three or more times per day. In an aspect, a CT20p peptide or a composition comprising an effective amount of a CT20p peptide is administered to the subject daily, or one time per week, or two times per week, or three or more times per week, etc. In an aspect, a CT20p peptide or a composition comprising an effective amount of a CT20p peptide is administered to the subject weekly, or every other week, or every third week, or every fourth week, etc. In an aspect, a CT20p peptide or a composition comprising an effective amount of a CT20p peptide is administered to the subject monthly, or every other month, or every third month, or every fourth month, etc. In an aspect, the repeated administration of a CT20p peptide or a composition comprising an effective amount of a CT20p peptide occurs over a pre-determined or definite duration of time. In an aspect, the repeated administration of a CT20p peptide or a composition comprising an effective amount of a CT20p peptide occurs over an indefinite period of time.
In an aspect, a disclosed method of killing cancer cells in a subject further comprises inducing cell death. In an aspect, cell death mimics necrosis. In an aspect, cell death occurs independent of endogenous Bax activity. In an aspect, cell death occurs independent of endogenous caspase activity. In an aspect, cell death is resistant to Bcl-2 over-expression. In an aspect, a disclosed method of killing cancer cells induces cell death, wherein (i) cell death mimics necrosis, (ii) cell death occurs independent of endogenous Bax activity, (iii) cell death occurs independent of endogenous caspase activity, or (iv) cell death is resistant to Bcl-2 over-expression, or (v) cell death exhibits a combination thereof.
In an aspect, a CT20p peptide of a disclosed method of killing cells comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and/or SEQ ID NO: 4. For example, in an aspect, a C-terminal truncated Bax is VTIFVAGVLTASLTIWKKMG (SEQ ID NO: 1). In an aspect, a CT20p peptide is VTIFVAGVLTASLTIWEEMG (SEQ ID NO: 2). In an aspect, a CT20p peptide is VTIFVAGVLTASLTIWLLMG (SEQ ID NO: 3). In an aspect, a CT20p peptide is VTIFVAGVLTASLTIWRRMG (SEQ ID NO: 4). In an aspect, a disclosed composition comprising an effective amount of a CT20p peptide comprises one or more CT20 Bax peptides, wherein the one or more CT20 Bax peptides comprise SEQ ID NO: 1, SEQ NO: 2, SEQ ID NO: 3; or SEQ ID NO: 4.
In an aspect, a CT20p peptide of a disclosed method of killing cancer cells in a subject is encapsulated in polymeric nanoparticles. In an aspect, the nanoparticles are aminated. In an aspect, the nanoparticles are carboxylated.
In an aspect, a disclosed method for killing cancer cells comprising administering a CT20p peptide or a composition comprising an effective amount of a CT20p peptide further comprises administering one or more anti-cancer drugs. In an aspect, the one or more anticancer drugs comprise cisplatin. In an aspect, the one or more anti-cancer drugs induce apoptosis. In an aspect, a disclosed method for killing cancer cells comprising administering a CT20p peptide or a composition comprising an effective amount of a CT20p peptide further comprises administering one or more chemotherapeutic drugs. In an aspect, a disclosed method for killing cancer cells comprising administering a CT20p peptide or a composition comprising an effective amount of a CT20p peptide further comprises administering one or more radiosensitizers.
In an aspect, a disclosed method for killing cancer cells comprising administering a CT20p peptide or a composition comprising an effective amount of a CT20p peptide further comprises administering (i) one or more anti-cancer drugs, (ii) one or more chemotherapeutic drugs, and (iii) one or more radiosensitizers. In an aspect, a disclosed method for killing cancer cells further comprises administering one or more anti-cancer drugs and one or more chemotherapeutic drugs. In an aspect, a disclosed method for killing cancer cells further comprises administering one or more anti-cancer drugs and one or more radiosensitizers. In an aspect, a disclosed method for killing cancer cells further comprises administering one or more chemotherapeutic drugs and one or more radiosensitizers.
In an aspect of a disclosed method of killing cancer cells in a subject, the subject is a mammal. In an aspect, the mammal is a primate. In an aspect, the mammal is a human. In an aspect, the human is a patient.
Disclosed herein is a method of killing cancer cells in a subject comprising (i) administering to a subject an effective amount of a CT20p peptide, (ii) permeabilizing at least one membrane in a cell of the subject, and (iii) inducing cell death.
3. Killing Cancer Cells
Disclosed herein are methods of killing microbial cells.
In an aspect, disclosed herein is a method of killing microbial cells in a subject comprising administering to at least one cell of a subject an effective amount of a CT20p peptide. In an aspect, disclosed herein is a method of killing microbial cells in a subject comprising administering to at least one cell of a subject a composition comprising an effective amount of a CT20p peptide. In an aspect, the cell is a microbial cell, including eukaryotic and prokaryotic cells, such as bacteria and/or fungi. In an aspect, the cell is a drug resistant microbial cell. In an aspect, a CT20p peptide or a composition comprising an effective amount of a CT20p peptide is administered directly.
In an aspect, a disclosed method of killing microbial cells in a subject further comprises permeabilizing at least one membrane in the cell. In an aspect, the membrane is a mitochondrial membrane. In an aspect, the one or more pores are formed in a mitochondrial membrane. In an aspect, the one or more pores are formed in a mitochondrial membrane of a microbial cell.
In an aspect, permeabilizing at least one membrane in the cell comprises destabilizing a cellular membrane, facilitating ion exchange, and causing a sequestered molecule to be released. In an aspect, permeabilizing at least one membrane in the cell comprises destabilizes a cellular membrane. In an aspect, permeabilizing at least one membrane in the cell comprises facilitating ion exchange. In an aspect, permeabilizing at least one membrane in the cell comprises causing a sequestered molecule to be released. In an aspect, permeabilizing at least one membrane in the cell comprises destabilizing a cellular membrane and facilitating ion exchange. In an aspect, permeabilizing at least one membrane in the cell comprises destabilizing a cellular membrane and causing a sequestered molecule to be released. In an aspect, permeabilizing at least one membrane in the cell comprises facilitating ion exchange and causing a sequestered molecule to be released.
In an aspect of a disclosed method of killing microbial cells in a subject, the microbial cells are sensitized to treatment. In an aspect of a disclosed method of killing microbial cells in a subject, the subject is sensitized to treatment. In an aspect, an increased sensitivity or a reduced sensitivity to a treatment, such as a therapeutic treatment, is measured according to one or more methods as known in the art for the particular treatment. In an aspect, methods of measuring sensitivity to a treatment include, but not limited to, cell proliferation assays and cell death assays. In an aspect, the sensitivity of a cell or a subject to treatment can be measured or determined by comparing the sensitivity of a cell or a subject following administration of a CT20p peptide or a composition comprising a CT20p peptide to the sensitivity of a cell or subject that has not been administered a CT20p peptide or a composition comprising a CT20p peptide.
For example, in an aspect, following the administration of a CT20p peptide or a composition comprising a CT20p peptide, the cell is 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, or greater, more sensitive to treatment than a cell that has not been administered a CT20p peptide or a composition comprising a CT20p peptide. In an aspect, following the administration of a CT20p peptide or a composition comprising a CT20p peptide, the cell is 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, or greater, less resistant to treatment than a cell that has not been administered a CT20p peptide or a composition comprising a CT20p peptide. The determination of a cell's or a subject's sensitivity or resistance is routine in the art and within the skill of an ordinary clinician and/or researcher.
In an aspect, the determination of a cell's or a subject's sensitivity or resistance to treatment can be monitored. For example, in an aspect, data regarding sensitivity or resistance can be acquired periodically, such as every week, every other week, every month, every other month, every 3 months, 6 months, 9 months, or every year, every other year, every 5 years, every 10 years for the life of the subject, for example, a human subject. In an aspect, data regarding sensitivity or resistance can be acquired at various rather than periodic times. In an aspect, treatment for a subject can be modified based on data regarding a cell's or a subject's sensitivity or resistance to treatment.
In an aspect, a disclosed method of killing microbial cells in a subject further comprises repeating the administration of a CT20p peptide or a composition comprising an effective amount of a CT20p peptide. In an aspect, a CT20p peptide or a composition comprising an effective amount of a CT20p peptide is administered to the subject at least two times. In an aspect, a CT20p peptide or a composition comprising an effective amount of a CT20p peptide is administered to the subject two or more times. In an aspect, a CT20p peptide or a composition comprising an effective amount of a CT20p peptide is administered at routine or regular intervals. For example, in an aspect, a CT20p peptide or a composition comprising an effective amount of a CT20p peptide is administered to the subject one time per day, or two times per day, or three or more times per day. In an aspect, a CT20p peptide or a composition comprising an effective amount of a CT20p peptide is administered to the subject daily, or one time per week, or two times per week, or three or more times per week, etc. In an aspect, a CT20p peptide or a composition comprising an effective amount of a CT20p peptide is administered to the subject weekly, or every other week, or every third week, or every fourth week, etc. In an aspect, a CT20p peptide or a composition comprising an effective amount of a CT20p peptide is administered to the subject monthly, or every other month, or every third month, or every fourth month, etc. In an aspect, the repeated administration of a CT20p peptide or a composition comprising an effective amount of a CT20p peptide occurs over a pre-determined or definite duration of time. In an aspect, the repeated administration of a CT20p peptide or a composition comprising an effective amount of a CT20p peptide occurs over an indefinite period of time.
In an aspect, a disclosed method of killing microbial cells in a subject further comprises inducing cell death. In an aspect, cell death mimics necrosis. In an aspect, cell death occurs independent of endogenous Bax activity. In an aspect, cell death occurs independently of endogenous caspase activity. In an aspect, cell death is resistant to Bcl-2 over-expression. In an aspect, a disclosed method of killing cells induces cell death, wherein (i) cell death mimics necrosis, (ii) cell death occurs independent of endogenous Bax activity, (iii) cell death occurs independent of endogenous caspase activity, or (iv) cell death is resistant to Bcl-2 over-expression, or (v) cell death exhibits a combination thereof.
In an aspect, a CT20p peptide of a disclosed method of killing cells comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and/or SEQ ID NO: 4. For example, in an aspect, a C-terminal truncated Bax is VTIFVAGVLTASLTIWKKMG (SEQ ID NO: 1). In an aspect, a CT20p peptide is VTIFVAGVLTASLTIWEEMG (SEQ ID NO: 2). In an aspect, a CT20p peptide is VTIFVAGVLTASLTIWLLMG (SEQ ID NO: 3). In an aspect, a CT20p peptide is VTIFVAGVLTASLTIWRRMG (SEQ ID NO: 4). In an aspect, a disclosed composition comprising an effective amount of a CT20p peptide comprises one or more CT20 Bax peptides, wherein the one or more CT20 Bax peptides comprise SEQ ID NO: 1, SEQ NO: 2, SEQ ID NO: 3; or SEQ ID NO: 4.
In an aspect, a CT20p peptide of a disclosed method of killing microbial cells in a subject is encapsulated in polymeric nanoparticles. In an aspect, the nanoparticles are aminated. In an aspect, the nanoparticles are carboxylated.
In an aspect, a disclosed method for killing microbial cells comprising administering a CT20p peptide or a composition comprising an effective amount of a CT20p peptide further comprises administering one or more anti-microbial drugs. In an aspect, a disclosed method for killing microbial cells comprising administering a CT20p peptide or a composition comprising an effective amount of a CT20p peptide further comprises administering one or more chemotherapeutic drugs. In an aspect, a disclosed method for killing cancer cells comprising administering a CT20p peptide or a composition comprising an effective amount of a CT20p peptide further comprises administering one or more antimicrobial therapeutics or therapies.
In an aspect of a disclosed method of killing microbial cells in or on a subject, the subject is a mammal. In an aspect, the mammal is a primate. In an aspect, the mammal is a human. In an aspect, the human is a patient.
Disclosed herein is a method of killing microbial cells in a subject comprising (i) administering to a subject an effective amount of a CT20p peptide, (ii) permeabilizing at least one membrane in a cell of the subject, and (iii) inducing cell death.
4. Other Uses
Also disclosed herein are uses of a disclosed composition as an investigational and/or research tool in the development and standardization of in vitro and in vivo test systems for evaluation in laboratory animals such as cats, dogs, rabbits, monkeys, rats and mice, as part of the search for (i) new therapeutic approaches for permeabilizing membranes of cells and for killing cancer or microbial cells as well as (ii) the evaluation of the permeabilization of membranes of cells and for the killing of cancer or microbial cells. In an aspect, the search for new therapeutic approaches and the evaluation of new therapeutic approaches involves a subject, such as a human subject or human patient.
Methods of killing microbial organisms on an inert surface.
The present invention comprises methods of treating a surface to render it antimicrobial, comprising contacting a CT20p peptide composition comprising CT20p peptide to a surface. The peptides may be air-dried and remain on the surface or may be affixed to the surface by binding to other proteins or binding-partners on the surface, or may be attached by adhesive compounds or other compounds known for attaching proteins to a surface, or the peptides may remain in solution in a liquid or semisolid or other form that contacts the surface. The CT20p peptide composition may comprise SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and/or SEQ ID NO: 4, or a combination of two or more of SEQ ID NOs 1-4. When a surface treated with a CT20p peptide composition comprising one or more CT20p peptides is then contacted by microbial organisms, it is believed that the cellular membranes of the microbial organisms are permeabilized and microbial death may occur.
Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.
As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.
The word “or” as used herein means any one member of a particular list and also includes any combination of members of that list.
Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.
As used herein, the amino acid abbreviations are conventional one letter codes for the amino acids and are expressed as follows: A, alanine; B, asparagine or aspartic acid; C, cysteine; D aspartic acid; E, glutamate, glutamic acid; F, phenylalanine; G, glycine; H histidine; I isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine; Z, glutamine or glutamic acid.
“Peptide” as used herein refers to any peptide, oligopeptide, polypeptide, gene product, expression product, or protein. For example, a peptide can be a fragment of a full-length protein, such as, for example, the CT20 Bax peptide. A peptide is comprised of consecutive amino acids. The term “peptide” encompasses naturally occurring or synthetic molecules.
In general, the biological activity or biological action of a peptide refers to any function exhibited or performed by the peptide that is ascribed to the naturally occurring form of the peptide as measured or observed in vivo (i.e., in the natural physiological environment of the protein) or in vitro (i.e., under laboratory conditions). For example, a biological activity of the CT20p peptide is the cytotoxic activity of the CT20 Bax peptide.
The term “enzyme” as used herein refers to any peptide that catalyzes a chemical reaction of other substances without itself being destroyed or altered upon completion of the reaction. Typically, a peptide having enzymatic activity catalyzes the formation of one or more products from one or more substrates. Such peptides can have any type of enzymatic activity including, without limitation, the enzymatic activity or enzymatic activities associated with enzymes such as those disclosed herein.
References in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.
A weight percent (wt. %) of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included.
As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or can not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.
As used herein, the terms “transformation” and “transfection” mean the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell including introduction of a nucleic acid to the chromosomal DNA of said cell. The art is familiar with various compositions, methods, techniques, etc. used to effect the introduction of a nucleic acid into a recipient cell. The art is familiar with such compositions, methods, techniques, etc for both eukaryotic and prokaryotic cells. The art is familiar with such compositions, methods, techniques, etc. for the optimization of the introduction and expression of a nucleic acid into and within a recipient cell.
As used herein, “a CT20p peptide” may refer to one peptide or may refer one or more peptides, such as molar concentrations of the peptide, as would be found in a composition. Those skilled in the art understand where an individual peptide is intended and where a molar, or smaller or larger amount, of many of the same peptide are intended.
As used herein, the term “subject” refers to the target of administration, e.g., an animal. Thus, the subject of the herein disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. Alternatively, the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. In one aspect, the subject is a patient. A patient refers to a subject afflicted with a disease or disorder, such as, for example, cancer and/or aberrant cell growth. The term “patient” includes human and veterinary subjects. In some aspects of the disclosed methods, the subject has been diagnosed with a need for treatment for cancer and/or aberrant cell growth.
Therapeutic agents may include antimicrobial agents, such as antibiotics or antimycotic compounds, including but not limited to, active agents such as antifungal agents, antibacterial agents, anti-viral agents and antiparasitic agents, and metals. An antimicrobial agent may comprise a substance, compound or molecule, that kills or inhibits the growth of microorganisms such as bacteria, fungi, or protozoans. Antimicrobial agents may either kill microbes (microbiocidal) or prevent the growth of microbes (microbiostatic). Disinfectants are antimicrobial substances used on non-living objects or outside the body. Anitmicrobial agents include those obtained from natural sources, such as Beta-lactam antibiotics (such as penicillins, cephalosporins), and protein synthesis inhibitors (such as aminoglycosides, macrolides, tetracyclines, chloramphenicol, polypeptides), and those from synthetic sources such as sulphonamides, cotrimoxazole, quinolones, anti-fungals, anti-cancer drugs, antimalarials, anti-tuberculosis drugs, anti-leprotics, and anti-protozoals.
Examples of antimicrobial agents that can be used in the present invention include, but are not limited to, isoniazid, ethambutol, pyrazinamide, streptomycin, clofazimine, rifabutin, fluoroquinolones, ofloxacin, sparfloxacin, rifampin, azithromycin, clarithromycin, dapsone, tetracycline, erythromycin, ciprofloxacin, doxycycline, ainpicillin, amphotericin B, ketoconazole, fluconazole, pyrimethamine, sulfadiazine, clindamycin, lincomycin, pentamidine, atovaquone, paromomycin, diclazaril, acyclovir, trifluorouridine, foscarnet, penicillin, gentamicin, ganciclovir, iatroconazole, miconazole, Zn-pyrithione, heavy metals including, but not limited to, gold, platinum, silver, zinc and copper, and their combined forms including, salts, such as chloride, bromide, iodide and periodate, and complexes with carriers, and other forms. As used herein, the term metal includes all metal salts or metal compounds, including, but not limited to, metal chlorides, metal phosphates, metal sulfates, metal iodides or metal bromides. The active form of some metal salts is the ionic form. Other antimicrobial agents include, but are not limited to, polyene antifungals, Amphotericin B, Candicidin, Filipin, Hamycin, Natamycin, Nystatin, Rimocidin, Imidazoles, Bifonazole, Butoconazole, Clotrimazole, Econazole, Fenticonazole, Isoconazole, Ketoconazole, Miconazole, Omoconazole, Oxiconazole, Sertaconazole, Sulconazole, Tioconazole, Triazoles, Albaconazole, Fluconazole, Isavuconazole, Itraconazole, Posaconazole, Ravuconazole, Terconazole, Voriconazole, Thiazoles, Abafungin, Allylamines, Amorolfin, Butenafine, Naftifine, Terbinafine, Echinocandins, Anidulafungin, Caspofungin, Micafungin.
Examples of microbial organisms that may be treated by the present invention include, but are not limited to, pathogenic fungi including, but not limited to, Candida species, Aspergillus species, Cryptococcus species, histoplasa species, stachybotrus species, and pneumacystus species; bacteria including but not limited to:
Acetobacter aurantius
Calymmatobacterium granulomatis
Acinetobacter baumannii
Campylobacter
Actinomyces israelii
Campylobacter coli
Agrobacterium radiobacter
Campylobacter fetus
Agrobacterium tumefaciens
Campylobacter jejuni
Azorhizobium caulinodans
Campylobacter pylori
Azotobacter vinelandii
Chlamydia
Anaplasma
Chlamydia trachomatis
Anaplasma phagocytophilum
Chlamydophila
Acetobacter aurantius
Chlamydophila pneumoniae
Bacillus
Chlamydophila psittaci
Bacillus anthracis
Clostridium
Bacillus brevis
Clostridium botulinum
Bacillus cereus
Clostridium difficile
Bacillus fusiformis
Clostridium perfringens
Bacillus licheniformis
Clostridium tetani
Bacillus megaterium
Corynebacterium
Bacillus mycoides
Corynebacterium diphtheriae
Bacillus stearothermophilus
Corynebacterium fusiforme
Bacillus subtilis
Coxiella burnetii
Bacteroides
Ehrlichia chaffeensis
Bacteroides fragilis
Enterobacter cloacae
Bacteroides gingivalis
Enterococcus
Bacteroides melaninogenicus
Enterococcus avium
Bartonella
Enterococcus durans
Bartonella henselae
Enterococcus faecalis
Bartonella quintana
Enterococcus faecium
Bordetella
Enterococcus galllinarum
Bordetella bronchiseptica
Enterococcus maloratus
Bordetella pertussis
Escherichia coli
Borrelia burgdorferi
Francisella tularensis
Brucella
Fusobacterium nucleatum
Brucella abortus
Haemophilus
Brucella melitensis
Haemophilus ducreyi
Gardnerella vaginalis
Haemophilus influenzae
Klebsiella pneumoniae
Haemophilus parainfluenzae
Lactobacillus
Haemophilus pertussis
Lactobacillus acidophilus
Haemophilus vaginalis
Lactobacillus casei
Helicobacter pylori
Lactococcus lactis
Neisseria
Legionella pneumophila
Neisseria gonorrhoeae
Listeria monocytogenes
Neisseria meningitidis
Lactobacillus Bulgaricus
Rhizobium radiobacter
Mycoplasma
Rickettsia
Mycoplasma fermentans
Rickettsia prowazekii
Mycoplasma genitalium
Rickettsia psittaci
Mycoplasma hominis
Rickettsia quintana
Mycoplasma penetrans
Rickettsia rickettsii
Mycoplasma pneumoniae
Rickettsia trachomae
Methanobacterium extroquens
Rochalimaea
Microbacterium multiforme
Rochalimaea henselae
Micrococcus luteus
Rochalimaea quintana
Moraxella catarrhalis
Rothia dentocariosa
Mycobacterium
Rhizobium radiobacter
Mycobacterium avium
Salmonella
Mycobacterium bovis
Salmonella enteritidis
Mycobacterium diphtheriae
Salmonella typhi
Mycobacterium intracellulare
Salmonella typhimurium
Mycobacterium leprae
Serratia marcescens
Mycobacterium lepraemurium
Shigella dysenteriae
Mycobacterium phlei
Staphylococcus
Mycobacterium smegmatis
Staphylococcus aureus
Mycobacterium tuberculosis
Staphylococcus epidermidis
Pasteurella
Stenotrophomonas maltophilia
Pasteurella multocida
Streptococcus
Pasteurella tularensis
Streptococcus agalactiae
Peptostreptococcus
Streptococcus avium
Porphyromonas gingivalis
Streptococcus bovis
Pseudomonas aeruginosa
Streptococcus cricetus
Streptococcus mitior
Streptococcus faceium
Streptococcus mitis
Streptococcus faecalis
Streptococcus mutans
Streptococcus ferus
Streptococcus oralis
Streptococcus gallinarum
Streptococcus pneumoniae
Streptococcus lactis
Streptococcus pyogenes
Streptococcus sanguis
Streptococcus rattus
Streptococcus sobrinus
Streptococcus salivarius
Wolbachia
Treponema
Vibrio parahaemolyticus
Treponema pallidum
Vibrio vulnificus
Treponema denticola
Yersinia
Vibrio
Yersinia enterocolitica
Vibrio cholerae
Yersinia pestis
Vibrio comma
Yersinia pseudotuberculosis
The terms “treating”, “treatment”, “therapy”, and “therapeutic treatment” as used herein refer to curative therapy, prophylactic therapy, or preventative therapy. As used herein, the terms refers to the medical management of a subject or a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder, such as, for example, cancer or a tumor. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. In various aspects, the term covers any treatment of a subject, including a mammal (e.g., a human), and includes: (i) preventing the disease from occurring in a subject that can be predisposed to the disease but has not yet been diagnosed as having it; (ii) inhibiting the disease, i.e., arresting its development; or (iii) relieving the disease, i.e., causing regression of the disease. In an aspect, the disease, pathological condition, or disorder is cancer, such as, for example, breast cancer, lung cancer, colorectal, liver cancer, or pancreatic cancer.
As used herein, the term “prevent” or “preventing” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed.
As used herein, the term “diagnosed” means having been subjected to a physical examination by a person of skill, for example, a physician or a researcher, and found to have a condition that can be diagnosed or treated by compositions or methods disclosed herein. For example, “diagnosed with cancer” means having been subjected to a physical examination by a person of skill, for example, a physician or a researcher, and found to have a condition that can be diagnosed or treated by a compound or composition that alleviates or ameliorates cancer and/or aberrant cell growth.
As used herein, the phrase “identified to be in need of treatment for a disorder,” or the like, refers to selection of a subject based upon need for treatment of the disorder. For example, a subject can be identified as having a need for treatment of a disorder (e.g., a disorder related to cancer and/or aberrant cell growth) based upon an earlier diagnosis by a person of skill and thereafter subjected to treatment for the disorder. It is contemplated that the identification can, in one aspect, be performed by a person different from the person making the diagnosis. It is also contemplated, in a further aspect, that the administration can be performed by one who subsequently performed the administration.
As used herein, the terms “administering” and “administration” refer to any method of providing a peptide (such as a CT20 Bax peptide), or a composition (such as a composition comprising a CT20 Bax peptide), or pharmaceutical preparation (such as a preparation comprising a CT20p peptide or a composition comprising a CT20 Bax peptide) to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, intracardiac administration, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intraarterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. In various aspects, a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. In further various aspects, a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition.
The term “contacting” as used herein refers to bringing a disclosed composition or peptide or pharmaceutical preparation and a cell, target receptor, or other biological entity together in such a manner that the compound can affect the activity of the target (e.g., receptor, transcription factor, cell, etc.), either directly; i.e., by interacting with the target itself, or indirectly; i.e., by interacting with another molecule, co-factor, factor, or protein on which the activity of the target is dependent.
As used herein, the term “determining” can refer to measuring or ascertaining a quantity or an amount or a change in expression and/or activity level, e.g., of a nucleotide or transcript or polypeptide. For example, determining the amount of a disclosed transcript or polypeptide in a sample as used herein can refer to the steps that the skilled person would take to measure or ascertain some quantifiable value of the transcript or polypeptide in the sample. The art is familiar with the ways to measure an amount of the disclosed nucleotides, transcripts, polypeptides, etc.
In an aspect, “determining” as used herein can refer to measuring or ascertaining the level of cell death or cell survival, for example, following administration of a CT20p peptide or a composition comprising an effective amount of a CT20p peptide. Methods of measuring or ascertaining cell survival and cell death are known to the art and include, but are not limited to, histochemical staining (e.g., TUNEL), cell proliferation assay, cell death assays, morphological examination, etc. In an aspect, the size of a tumor can be measured non-invasively through ultrasound.
As used herein, the term “level” refers to the amount of a target molecule in a sample, e.g., a sample from a subject. The amount of the molecule can be determined by any method known in the art and will depend in part on the nature of the molecule (i.e., gene, mRNA, cDNA, protein, enzyme, etc.). The art is familiar with quantification methods for nucleotides (e.g., genes, cDNA, mRNA, etc) as well as proteins, polypeptides, enzymes, etc. It is understood that the amount or level of a molecule in a sample need not be determined in absolute terms, but can be determined in relative terms (e.g., when compare to a control or a sham or an untreated sample).
As used herein, the terms “effective amount” and “amount effective” refer to an amount that is sufficient to achieve the desired result or to have an effect on an undesired condition. For example, in an aspect, an effective amount of a CT20p peptide is an amount that permeabilizes cell membranes and/or kills cells without causing extraneous damage to surrounding non-cancerous cells. For example, a “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side affects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors well known in the medical arts.
By “modulate” is meant to alter, by increase or decrease. As used herein, a “modulator” can mean a composition that can either increase or decrease the expression level or activity level of a gene or gene product such as a peptide. Modulation in expression or activity does not have to be complete. For example, expression or activity can be modulated by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or any percentage in between as compared to a control cell wherein the expression or activity of a gene or gene product has not been modulated by a composition.
As used herein, “EC50,” is intended to refer to the concentration or dose of a substance (e.g., a CT20p peptide or a disclosed composition comprising a CT20p peptide) that is required for 50% enhancement or activation of a biological process, or component of a process, including a protein, subunit, organelle, ribonucleoprotein, etc. EC50 also refers to the concentration or dose of a substance that is required for 50% enhancement or activation in vivo, as further defined elsewhere herein. Alternatively, EC50 can refer to the concentration or dose of compound that provokes a response halfway between the baseline and maximum response. The response can be measured in an in vitro or in vivo system as is convenient and appropriate for the biological response of interest. For example, the response can be measured in vitro using cultured cancer cells or in an ex vivo organ culture system with isolated cancer cells, e.g., pancreatic cancer cells, breast cancer cells, liver cancer cells, lung cancer cells, colorectal cancer cells, etc.). Alternatively, the response can be measured in vivo using an appropriate research model such as rodent, including mice and rats. The mouse or rat can be an inbred strain with phenotypic characteristics of interest such as, for example, cancer and/or aberrant cell growth. As appropriate, the response can be measured in a transgenic or knockout mouse or rat wherein a gene or genes has been introduced or knocked-out, as appropriate, to replicate a disease process.
As used herein, “IC50,” is intended to refer to the concentration or dose of a substance (e.g., a CT20p peptide or a disclosed composition comprising a CT20p peptide) that is required for 50% inhibition or diminution of a biological process, or component of a process, including a protein, subunit, organelle, ribonucleoprotein, etc. IC50 also refers to the concentration or dose of a substance that is required for 50% inhibition or diminution in vivo, as further defined elsewhere herein. Alternatively, IC50 also refers to the half maximal (50%) inhibitory concentration (IC) or inhibitory dose of a substance. The response can be measured in an in vitro or in vivo system as is convenient and appropriate for the biological response of interest. For example, the response can be measured in vitro using cultured cancer cells or in an ex vivo organ culture system with isolated cancer cells (e.g., breast cancer cells, pancreatic cancer cells, liver cancer cells, lung cancer cells, colorectal cancer cells, etc.). Alternatively, the response can be measured in vivo using an appropriate research model such as rodent, including mice and rats. The mouse or rat can be an inbred strain with phenotypic characteristics of interest such as, for example, cancer and/or aberrant cell growth. As appropriate, the response can be measured in a transgenic or knockout mouse or rat wherein a gene or genes has been introduced or knocked-out, as appropriate, to replicate a disease process.
The term “pharmaceutically acceptable” describes a material that is not biologically or otherwise undesirable, i.e., without causing an unacceptable level of undesirable biological effects or interacting in a deleterious manner. As used herein, the term “pharmaceutically acceptable carrier” refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Suitable inert carriers can include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers.
As used herein, the term “cancer” refers to a proliferative disorder or disease caused or characterized by the proliferation of cells which have lost susceptibility to normal growth control. The term “cancer” includes tumors and any other proliferative disorders. Cancers of the same tissue type originate in the same tissue, and may be divided into different subtypes based on their biological characteristics. Cancer includes, but is not limited to, melanoma, leukemia, astocytoma, glioblastoma, lymphoma, glioma, Hodgkins lymphoma, and chronic lymphocyte leukemia. Cancer also includes, but is not limited to, cancer of the brain, bone, pancreas, lung, liver, breast, thyroid, ovary, uterus, testis, pituitary, kidney, stomach, esophagus, anus, and rectum.
As used herein, the term “sensitizing” refers to an increased sensitivity of a cell or a subject to a treatment, such as a therapeutic treatment. The term “sensitizing” also refers to a reduction or decrease in the resistance of a cancer cell or a subject with cancer in responding to a therapeutic treatment. An increased sensitivity or a reduced sensitivity to a therapeutic treatment is measured according to a known method in the art for the particular treatment and methods including, but not limited to, cell proliferation assays and cell death assays. The sensitivity or resistance may also be measured in a subject by measuring the tumor size reduction over a period of time, such as, for example, every 1 to 3 to 6 month for a human subject and every 2 to 4 to 6 weeks for non-human subject (e.g., mouse or rat). The sensitivity of a cell or a subject to treatment can be measured or determined by comparing the sensitivity of a cell or a subject following administration of a CT20p peptide or a composition comprising an effective amount of a CT20p peptide to the sensitivity of a cell or subject that has not been administered a CT20p peptide or a composition comprising an effective amount of a CT20p peptide.
As used herein, the term “anti-cancer” or “anti-neoplastic” drug refers to one or more drugs that can be used in conjunction with a CT20p peptide or a composition comprising an effective amount of a CT20p peptide to treat cancer and/or aberrant cell growth. Examples of anti-cancer drugs or anti-neoplastic drugs include, but are not limited to, the following: Acivicin; Aclarubicin; Acodazole Hydrochloride; AcrQnine; Adozelesin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride; Decitabine; Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin; Edatrexate; Eflomithine Hydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium; Etanidazole; Ethiodized Oil I 131; Etoposide; Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil; Fluorocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Gold Au 198; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-n1; Interferon Alfa-n3; Interferon Beta-1a; Interferon Gamma 1 b; Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole; Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine; Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium; Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofurin; Riboprine; Rogletimide; Safingol; Safingol Hydrochloride; Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin; Spirogermanium Hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Strontium Chloride Sr 89; Sulofenur; Talisomycin; Taxane; Taxoid; Tecogalan Sodium; Tegafur; Teloxantrone Hydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Tiazofurin; Tirapazamine; Topotecan Hydrochloride; Toremifene Citrate; Trestolone Acetate; Triciribine Phosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide; Verteporfin; Vinblastine Sulfate; Vincristine Sulfate; Vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate; Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; Zorubicin Hydrochloride.
Other anti-neoplastic compounds include: 20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; atrsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen, antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocannycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulant peptides; insulin-like growth factor-1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; 4-ipomeanol; irinotecan; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotrophin; monophosphoryl lipid A+myobacterium cell wall sk; mopidamol; multiple drug resistance genie inhibitor; multiple tumor suppressor 1-based therapy; mustard anticancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel analogues; paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum-triamine complex; porfimer sodium; porfiromycin; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitors; microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide; rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single chain antigen binding protein; sizofuran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-cell division inhibitors; stipiamide; stromelysin inhibitors; sulfmosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfin; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thalidomide; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene dichloride; topotecan; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector system, erythrocyte gene therapy; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; zinostatin stimalamer.
As used herein, radiosensitizers make a cancer cell more likely to be damaged. Radiosensitizers enhance the sensitivity of cancer cells and/or a tumor to ionizing radiation, thereby increasing the efficacy of radiotherapy. Examples of radiosensitizers include gemcitabine, 5-fluorouracil, pentoxifylline, and vinorelbine.
The majority of chemotherapeutic drugs can be divided in to: alkylating agents (e.g., cisplatin, carboplatin, oxaliplatin, mechloethamine, cyclophosphamide, chlorambucil, anti-metabolites (e.g., azathioprine, mercaptopurine), anthracyclines, plant alkaloids and terpenoids (e.g., vinca alkaloids (e.g., vincristine, vinblastine, vinorelbine, vindesine, and podophyllotoxin) and taxanes (e.g., paclitaxel and docetaxel), topoisomerase inhibitors (e.g., irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, and teniposide), monoclonal antibodies (e.g., trastuzumab, cetuximab, rituximab, bevacizumab), other antitumour agents (e.g., dactinomycin), and hormonal therapy (e.g., steroids such as dexamethasone), finasteride, aromatase inhibitors, and gonadotropin-releasing hormone agonists).
Disclosed are the components to be used to prepare a composition of the invention as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds can not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules including the compounds are discussed, specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the compositions of the invention. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the methods of the invention.
All patents, patent applications, and other scientific or technical writings referred to anywhere herein are incorporated by reference in their entirety. The invention illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of, and “consisting of can be replaced with either of the other two terms, while retaining their ordinary meanings. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by embodiments, optional features, modification and variation of the concepts herein disclosed can be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C. or is at ambient temperature, and pressure is at or near atmospheric.
1. General Experiments
a. Establishment of Cell Lines
The Flp-In T-REx-293 cell line (Invitrogen) stably expressed the lacZ-Zeocin fusion gene and Tet repressor. The 293 line was maintained in Dulbecco's Modified Eagle Medium (DMEM), 10% fetal bovine serum (FBS) (tetracycline-reduced), 2 mM L-glutamine and 1% Penicillin-Streptomycin. The HCT-116 Bax−/− and Bax+/+ colorectal cancer cell lines (Zhang et al., 2000) were maintained in McCoy's 5A media, 10% FBS, and 1% Penicillin-Streptomycin. The breast cancer cell lines, MCF-7 and MCB-MD-231 (ATCC), were maintained in DMEM, 10% FBS, and 1% Penicillin-Streptomycin. MCF-7 cells were supplemented with 1% L-Glutamine every 15 days. Early passages of all cell lines were frozen as stocks at time of receipt. Cell lines were used at less than 10 passages from stocks.
b. Generation of the CT20 Bax Peptide
The CT20p peptide (Ac-VITFVAGVLTASLTIWKKMG-NH2) (Biopeptide Co., Inc.) (SEQ ID NO: 53) was commercially synthesized at >98% purity.
C. Generation of Plasmids for Mutagenesis and Transfection
For inducible expression of Bax, the Flp-In T-REx System (Invitrogen) was utilized according to the manufacturer's protocol. Briefly, PCR-directed mutagenesis of K189/K190 was performed using HA-tagged primer sets (Table 1). Bax constructs were amplified from pEGFP-Bax, digested with EcoRV, and cloned into the plasmid pcDNA5/FRT/TO, which undergoes DNA recombination at the Flp Recombination Target (FRT) site when co-expressed with the Flp recombinase pOG44 plasmid. Constructs were confirmed by sequencing. Fugene transfection reagent (Roche) was used to co-transfect plasmids at a ratio of 9:1. Stable Flp-In T-REx expression cell lines were selected for Blasticidin resistance (10 μg/mL), Hygromycin resistance (100 μg/mL) and Zeocin sensitivity (200 μg/mL). Bax expression was induced with 1 μg/mL tetracycline. Cells were assayed after 24 hours of induction. See Table 1 for sequences and primers used herein.
To generate the Destabilization Domain (DD)-tagged Bax CT (amino acids 173-192) fusion proteins with K189/K190 (wild-type) or EE, LL, and RR mutations, primers (Table 1) were annealed and ligated into the ProteoTuner vector (Clontech) digested by EcoRI and BamHI. Generation of DD-tagged, full-length WT Bax was previously described (Boohaker et al., 2011). The ProteoTuner IRES2 system (Clontech) also had the marker protein GFP downstream to the internal ribosome entry sequence (IRES) and was translated independently of the DD-tagged protein. Cells were transiently transfected using the TransIT-LT1 transfection reagent (Minis) for 24 hours and microscopically assayed for GFP expression. Expression of DD-tagged proteins was induced for 4-5 hours by adding 500 nM of Shield (Clontech).
MDA-MB-231 cells were transiently transfected with pcDNA-Bcl2 (or as control pEGFP (Clontech)) using the TransIT-LT1 transfection reagent (Minis). To assess transfection efficiency, cells were assayed microscopically for EGFP expression. To assess Bcl-2 expression, cells lysates were immunoblotted.
d. Detection of Mitochondrial Translocation
Mitochondrial and cytosolic proteins were isolated using a mitochondrial enrichment kit (Pierce). Western blots were run using 12-15% SDS-PAGE gels and PVDF membranes and probed with the following primary antibodies: 16B12 anti-HA mouse monoclonal (Covance) for HA-tagged Bax; 631073Anti-DD monoclonal (Clontech) for DD-Bax; N-20 (Santa Cruz) for endogenous Bax; Ab-2 (Fitzgerald) for prohibitin; C20 (MAPK) (Santa Cruz) for p38 MAP kinase; and rabbit polyclonal for Bcl-2 (Santa Cruz). The appropriate secondary antibodies conjugated to horseradish peroxidase (HRP) was then used and visualized with enhanced chemiluminescence kit (Pierce). Molecular weight markers (SeeBlue Plus 2 (Invitrogen)) were used to approximate the position of protein bands in blots.
e. Co-Localization of Mitochondrial Proteins
To determine whether expression of DD-tagged Bax C-terminal peptides co-localized with mitochondria, Bax+/+ HCT-116 cells and Bax−/− HCT-116 cells were cultured for 24 hours on coverslips pretreated with laminin. The DD-Bax C-terminal WT peptide and DD-Bax C-terminal EE, LL, and RR mutations constructs were transfected into the cells using Minis LT-1 reagent according manufacturer's protocol. After 24 hours, expression of the transfected peptides was induced by the addition of shield 1 for 4 hrs. Cells were fixed with 2% w/v formaldehyde/PBS for 15 minutes and permeabilized using 0.05% Triton X-100/PBS for 15 minutes. After washing, cells were incubated with primary antibodies HSP60 (H-300, Santa Cruz) and DD monoclonal antibody for 1 hour at room temperature, which was followed by incubation with secondary anti-rabbit-Cy3 (81-6115, Invitrogen) and anti-mouse-Texas red (715076020, Jackson Immunoresearch) for 30 minutes. After the final wash, cells were mounted with gel/mount medium (Mol, Biomeda) and images were acquired with UltraView (PerkinElmer) microscopy with a plan-apochromat 63×/1.4 oil objective. The scanned images were processed and Pearson's correlation coefficients determined using Velocity Version 5.5 (Perkin Elmer).
f. Generation and Expression of EGFP-CT20p Peptide Fusion Constructs
To generate the EGFP-CT20p peptide fusion proteins, primers incorporating the CT (amino acids 173-192) of Bax with mutations targeting K189/K190 (Table 1) were used to amplify EGFP from the template pEGFP (Clontech). The PCR insert was cloned into pcDNA5/FRT/TO as previously above. HCT-116 cells were transiently transfected using the TransIT-LT1 transfection reagent (Minis) and cells assayed microscopically for EGFP expression up to 12 hours later.
g. Confocal Imaging of Live Cells
Images were acquired through a PerkinElmer UltraView spinning disc confocal system with AxioObserver.Z1 stand (Carl Zeiss) in a humidity and temperature-controlled chamber (LiveCell) with cells cultured on MatTek plates (MatTek Corporation). Post-acquisition snapshots were taken from time-lapse movies at time points indicated in the figures. For EGFP-CT20 Bax fusion proteins, time-lapse movies were initiated two hours after transfection and images acquired through 12 hours of expression using a Plan-Apochromat 10× objective. For DD-CT20 Bax fusion proteins, cells were incubated with 1 nM MitoTracker Red 580 for 30 minutes prior to imaging. Time-lapse movies were recorded for up to 12 hours using a Plan-Apochromat 63× Oil DIC objective.
Visualization of the uptake and effects of the fluorescent dye (DiI)-loaded nanoparticles in HCT-116, MCF-7, and MDA-MD-231 cells was observed using a 10× air objective with a numerical aperture of 0.3, using ex/em of 514/587. All cells were loaded with MitoTracker. Visualization of the HCT-116 cell lines was observed using Plan-Apochromat 63× Oil objective. MCF-7 and MDA-MB-231 images were captured using Plan-Apochromat 40× Oil objective. All time-lapse images were generated in 2D by capturing 6 time points per hour for 24 hours of the same field.
h. Treatments and Detection of Apoptotic Cells by Flow Cytometry
HCT-116, MCF-7, or MDA-MB-231 cells were collected at a final concentration of 1×106 cells/mL and assayed using the SYTOX® AADvanced™ dead cell stain solution (Invitrogen). Cells were analyzed using the BD FACSCanto flow cytometer. SYTOX® AADvanced™ was visualized at 488 nm and emissions collected at 695 nm. Analysis of data was done using FSC Express software (DeNovo). Membrane asymmetry was assessed using the Violet Ratiometric Membrane Asymmetry Probe/Dead Cell Apoptosis Kit (Invitrogen) according to the manufacturer's protocol.
To evaluate apoptosis, cells were pre-treated with 100 μM of the pan-caspase inhibitor Z-VAD-fmk (EMD Biosciences) or transiently transfected with Bcl-2, then treated with either CT20/nanoparticles or cisplatin (CDDP) alone or in combination as indicated in figure legends. Following treatment, cells were analyzed for cell death and membrane asymmetry.
i. Synthesis of Polymeric Nanoparticles Encapsulating the Bax CT20 Bax Peptide
The Bax CT20p peptide was encapsulated into hyperbranched polymeric (HBPE) nanoparticles following a previously reported method (Santra et al., 2010). A fluorescent dye (DiI) was co-encapsulated with the peptide. In brief, 1.0 μL, of DiI dye (10 μg/μL) and 36 μL, of CT20p peptide (0.05 μg/μL) solution in 250 μL, of DMSO were mixed in 250 μL of a DMSO solution containing the HBPE polymer (12 mg) for a ratio of ˜0.15 μg peptide:1 mg nanoparticles. The resulting polymer-DiI/Bax mixture in DMSO was added to deionized water (2.5 mL) to form the HBPE (Bax CT20/DiI) nanoparticles. The resulting nanoparticles were purified using a PD-10 column and dialyzed (MWCO 6-8K) against PBS (pH=7.4). Dynamic light scattering and zeta potential analysis of the nanoparticle reveals a size diameter of 88±2 nm and zeta potential of −54.5 mV.
j. Synthesis of Aminated Polymeric Nanoparticles Encapsulating the CT20 Bax Peptide
The HBPE nanoparticles contain functional carboxylic groups on their surface that resulted in a negative charge. To introduce a positively charged surface, the nanoparticles were aminated using water-soluble carbodiimide chemistry [EDC: 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride and NHS: N-hydroxysuccinimide chemistry], following a previously reported method (Santra et al., 2010). Briefly, to a solution of HBPE (Bax CT20/DiI) nanoparticles (1.0 mmol) in PBS (pH=7.4), a solution of EDC (10 mmol) and NHS (10 mmol) in MES buffer (pH=6.0) was added. Afterwards, ethylenediamine (10 mmol) in DMSO was added to obtain aminated DiI/Bax co-encapsulation polymeric nanoparticles (PNPs), which were purified and dialyzed. Dynamic light scattering and zeta potential analysis of the nanoparticle reveals a nanoparticle size diameter or 91±3 nm and zeta potential of +10.3 mV. All nanoparticles were stored at 4° C. A final working concentration of 350 pM was determined by testing the toxicity of 7 nM, 1.4 nM, 0.7 nM, and 0.35 nM using HCT-116+/+ cells.
k. Determination of Calcein Release
Calcein release from artificial membranes was measured on a JASCO 810 spectropolarimeter (Jasco Inc.) with a Peltier water cooled thermostat and a photomultiplier tube mounted at 90 degrees for fluorescence measurements. Large unilamellar vesicles (LUVs) were prepared with the following modifications: Lipids (Avanti Polar Lipids) in chloroform were mixed in the following molar ratios: 52.5% 1-palmitoyl-2-oleyl-sn-glycero-3-phosphatidylcholine (POPC), 21% 1-palmitoyl-2-oleyl-sn-glycero-3-phosphatidylethanolamine (POPE), 13% bovine liver L-α phosphatidylinositol (PI), 10% cholesterol and 3.5% 1-palmitoyl-2-oleyl-sn-glycero-3-phosphatidylglycerol (POPG). These rations mimicked the outer mitochondrial membrane. After removing chloroform and desiccating, the dried lipid film was resuspended in 50 mM HEPES, pH 7, supplemented with 110 mM NaCl and 80 mM calcein and extruded with Avanti's mini extruder (Alabaster, Ala.). External calcein was removed by gel filtration through a 1.5×50 cm Econo-Column (Bio-Rad) freshly packed with Sephadex 50 (GE Healthcare). CT20 Bax peptides were added at concentrations equivalent to those used with cells in culture. Calcein fluorescence was excited at 495 nm and emission spectra recorded between 510 and 550 nm (excitation/emission slits: 10/3 nm). Samples were maintained at 37° C. and a final measurement taken at 24 hours. Maximum calcein release was obtained by the addition of Triton x 100 (0.1% final concentration) to calcein loaded LUVs without nanoparticles that had had been incubated at 37° C. for 24 hours after the addition of 10 μL DMSO.
1. In Vivo Experiments
One to two million MBA-MD-231 cells were harvested from culture and injected subcutaneously into the right and left flanks of 16 female nude mice (nu/nu, Charles River). After 2-3 weeks, tumor volume and growth was assessed by ultrasound (VisualSonics Vevo 2100). Mice with tumors were injected intratumorally with PBS, unloaded polymeric nanoparticles, or CT20 Bax peptide-loaded polymeric nanoparticles at 4× the concentrations described above. Injections were performed twice over a 4-5 day period. Mice were observed 0-5 days post-treatments and tumor volume assessed by ultrasound.
2. C-Terminal α9 Helix of Bax Enables Membrane Binding and Permeabilization
To develop a novel anti-cancer agent that could be used to sensitize cancer cells to treatment, the death-inducing activity of Bax was examined. The protein's C-terminal domain was a point of focus because of its putative membrane-binding properties (Ausili et al., 2008; Martinez-Senac et al., 2001; Nechushtan et al., 1999; Schinzel et al., 2004). Mutagenesis of the C-terminus of full-length Bax was performed (Table 2). As the constitutive expression of N-terminal-tagged Bax induced spontaneous cell death. To avoid this, full-length HA-tagged Bax was expressed in stably transfected Flp-In T-REx 293 cells. Bax constructs were integrated into the genome at a single FRT (recombination) site and the levels of Bax expression in these isogenic cell lines did not cause apoptosis. These experiments determined the role of the C-terminus and amino acids K189/K190 in the localization of Bax under normal cellular conditions.
Localization of full-length, wild-type (WT) Bax (Bax-KK) was distributed among cytosolic and mitochondrial lysates (
Substitution of K189/K190 with negatively charged residues, aspartic acid (D) or glutamic acid (E), resulted in the cytosolic retention of Bax (Bax-DD, Bax-EE) (
Analysis of the effects of mutations of K189/K190 in full-length Bax indicated that the C-terminal domain was a preferred region from which to fashion a membrane-binding and possibly a membrane-permeabilizing peptide. Using a commercially synthesized peptide composed of the last twenty amino acids (172-192) of the C-terminus of Bax (CT20), the ability of CT20p peptide to insert and permeabilize lipid membranes was examined. Liposomes contained phospholipids that composed the mitochondrial outer membrane and were loaded with calcein.
3. Expression of the CT20p Peptide Induces Cell Death
Having established the importance of the C-terminal domain of Bax in the intracellular localization of the protein and demonstrated the membrane-binding and permeabilization properties of the CT20 Bax peptide, the capacity of the CT20p peptide to induce cell death was next evaluated. Specifically, whether the CT20p peptide had cytotoxic properties based on its capacity to associate with lipid membranes of organelles like mitochondria was assessed. The CT20p peptide domain was fused to a short destabilization domain (DD) for detection and inducible expression. The CT20p peptide fused to DD (DD-CT20) was inducibly expressed in HCT-116 cells and peptide localization and induction of cell death was examined. For a control, the action of the DD-CT20 (KK and EE, LL and RR mutants) peptides to DD-tagged full-length (FL) Bax was compared. Induced expression of DD-FL Bax did not cause cell death in the absence of apoptotic stimuli.
Gene expression was induced for four hours after previous transfection of cells with DD-FL Bax or DD-CT20 peptide constructs. Constitutive GFP expression on the bi-cistronic plasmid was controlled by an IRES element and used to detect transfected cells. The mitochondrial translocation of DD-tagged Bax full-length (FL-Bax) and DD-tagged CT20 peptides, wild-type and EE, LL and RR mutants, was examined in Bax+/+ HCT-116 cells by immunoblot. As shown in
Expression of DD-CT20 Bax also caused the mitochondrial translocation of a small amount endogenous Bax (
These results were confirmed by immunofluorescence (
Experiments attaching the CT20 peptides to EGFP confirmed the findings that fusion of the CT20p peptide could confer membrane binding properties (
In
The toxicity of DD-CT20 KK peptide was shown when expression was induced in HCT-116 cells (
4. Delivery of the CT20 Peptide Using Polymeric Nanoparticles Kills Cancer Cells
To determine whether the CT20p peptide could directly kill cancer cells, the CT20 wild type peptide was encapsulated in polymeric nanoparticles. As the CT20p peptide is amphipathic, it can be encapsulated within the hydrophobic pockets of aliphatic polymeric nanoparticles.
In
The uptake of DiI-loaded nanoparticles and effect upon the viability of HCT-116 cells was evaluated. HCT-116 cells were treated with nanoparticles loaded with DiI or DiI+CT20 peptide (0.07 nM) for 24 hours.
To demonstrate that nanoparticles alone (i.e., without the CT20 peptide) did not cause death, a DNA-binding dye (Sytox) was utilized to detect membrane rupture of dead cells.
To visualize the effect of CT20 Bax peptide-nanoparticles upon mitochondria, Bax-containing or Bax-deficient HCT-116 cells were stained with Mitotracker and imaged live cells. Bax+/+ (
Similarly, HCT-116 cells were treated with nanoparticles loaded with CT20 peptide (350 pM). Cell death was measured and the loss of membrane integrity was detected within three hours of treatment with CT20 Bax peptide-nanoparticles (
Whether the CT20 peptide was able to kill the breast cancer cells, MCF-7 and MDA-MB-231, was examined. As shown in
5. The Death-Inducing Activity of the CT20p Peptide is Independent of Caspases and Resistant to Bcl-2.
To investigate the mechanism by which the CT20p peptide was inducing cell death, MDA-MB-231 cells were treated with CT20 Bax peptide/nanoparticles (350 pM). A caspase inhibitor (e.g., ZVAD-Fmk and/or CDDP) was added or Bcl-2 was overexpressed. As shown in
In
While Sytox is good indicator of cell death, it does not distinguish between apoptotic and necrotic cell death. Therefore, to determine whether the CT20p peptide induced apoptotic cell death, the amount of membrane asymmetry caused by flipping of phospholipids in the plasma membrane using a violet ratiometric probe was determined. In
To demonstrate that the CT20 peptide could kill cancer cells in vivo, a small scale murine tumor experiment was performed. MDA-MB-231 cells were implanted in the flanks of nude mice. Initial growth of tumors was detected after 2 weeks. At this time tumors were measured, and over a 4-5 day period, the mice were treated with intratumoral injections of PBS, unloaded or empty nanoparticles (NP), or CT20 peptide/nanoparticles. The CT20 peptide prevented tumor growth as compared to empty nanoparticles or a PBS-treated tumor. (
Moreover, the ultrasound data in
Collectively, these data demonstrated that the C-terminal domain of Bax has membrane-binding capacity. The CT20p peptide permeabilized membranes and caused cell death. The CT20 peptide, unlike the full-length Bax protein, caused a lethal cascade that resulted in membrane rupture that is not characteristic of conventional apoptosis. The amphipathic features of the CT20 peptide made it amenable to encapsulation in nanoparticles and delivery to cancer cells. The CT20 peptide caused the death of colon and breast cancer cells, even in the absence of endogenous Bax or expression of Bcl-2, and led to reduced tumor volume in a murine model. The CT20 peptide caused cell death in a manner that was different from the apoptotic mechanism activated by CDDP.
Central to the lethal function of Bax is the membrane binding capacity of domains like the C-terminal alpha-9 helix. Loss of the C-terminus, or mutagenesis of K189/K190, modulated the ability of Bax to associate with mitochondria. The CT20 peptide, derived from the C-terminal a9 helix of Bax, penetrated and permeabilized lipid vesicles. The CT20 Bax peptide, when encapsulated in nanoparticles, was toxic at picomolar concentrations. The CT20 Bax peptide/nanoparticle mixture induced morphological features of cell death such loss of membrane integrity and cell shrinkage, but did not cause the changes in membrane symmetry that characterizes apoptosis. Use of the Bax CT20 peptide/nanoparticles as an anti-cancer agent was demonstrated when breast cancer cell lines succumbed to treatment as indicated by increased loss of membrane integrity that was not impaired by caspase inhibition or Bcl-2 expression. The CT20 peptide caused a form of cell death that was mechanistically different from typical anti-cancer drugs like CDDP.
The data indicate that the CT20p peptide kills cells caused the formation of pores in mitochondrial membranes. The CT20p peptide was efficient at forming pores in zwitterionic and anionic lipid membranes, leading to the release of calcein from loaded lipid vesicles.
Pore-forming proteins or peptides that can spontaneously insert into lipid membranes and form stable pores have significant biological interest and clinical application. As demonstrated herein, the CT20 peptide, a shorter version of the α9 helix of Bax (amino acids 172-192), caused cell death indicated by membrane rupture. These findings indicate that the CT20 peptide may cause necrotic-like cell death, rather than conventional apoptosis, and therefore has the potential for additive effects in combinatorial therapies with agents like CDDP that induce apoptosis. The data presented herein show that a small amount of the CT20 peptide was lethal under conditions when the apoptotic machinery is intact (such as Bax-containing HCT-116 cells) or when the apoptosis machinery is defective (such as Bax deficient HCT-116 cells). The CT20 peptide was also effective under conditions of effector caspase inhibition or Bcl-2 over expression. These data indicate that the CT20 peptide may be a potent killing tool in cancers with abnormal levels of anti-apoptotic proteins, as well as cancers in which other survival signaling mechanism may be irregular.
An analysis of formation of relatively large membrane pores by Bax C-terminal 20-reside peptide (CT20p) and two mutants where the two native lysines are replaced either with glutamates (charge reversal mutation) or leucines (charge neutralization mutation) were examined. The three peptides demonstrate distinct potencies to form pores in both zwitterionic and anionic membranes that transport calcein (Mr˜623). The most efficient pores are formed by the wild-type peptide in anionic membranes. The kinetics of calcein release at various concentrations of the three peptides allowed identification of the second-order rate constants of pore formation within the membrane, the affinity constants of peptide units composing the pore, and the oligomeric pore structure. Nucleation of the pore is shown to be relatively slow and involve 2-3 peptide molecules, followed by a faster process of assembly of the pore that includes up to eight peptide molecules. Structural studies led to a model of an octameric transmembrane pore with an inner diameter of 20-22 A. Analysis of the kinetics of calcein release from lipid vesicles allows determination of rate constants of pore formation, peptide-peptide affinities within the membrane, the oligomeric state of transmembrane pores, and the role of the lysine residues.
In summary, the C-terminal 20-residue stretch of Bax has the capability of forming relatively large membrane pores with a radius of at least 13 Å. Replacement of the two lysine residues close to the C-terminus with anionic glutamate or nonpolar leucine residues reduce the pore forming activity of the peptide, but the mutant peptides are still quite capable pore formers. In general, the pore formation is a two-stage process, nucleation and assembly of the final pore structure that includes up to eight peptide molecules, and is stabilized by intermolecular interaction energies of −10 to −13 kcal/mol, which is quite significant for 20-mer peptides.
Additionally, the pore structure was analyzed by polarized Fourier transform infrared, circular dichroism, and fluorescence experiments on the peptides reconstituted in phospholipid membranes. The peptides assumed an α/β-type secondary structure within membranes. Both β-strands and α-helices are significantly tilted relative to the membrane normal, by 30-60 degrees. The tryptophan residue embed into zwitterionic membranes at 8-9 A from membrane center. Membrane anionic charge causes a deeper insertion of tryptophan for BaxC-KK and BaxC-LL but not BaxC-EE. Combined with pore stoichiometry, these data suggest a pore model where eight peptide molecules form an “α/β-ring” structure with pore inner diameter of 20-22 Å. These results identified a strong membranotropic activity of Bax C-terminus and proposed a new mechanism by which peptides can efficiently perforate cell membranes and thus be used as cytotoxic agents.
Additionally, membrane insertion of the peptide and subsequent pore formation was mediated mainly by hydrophobic rather than electrostatic interactions. In the presence of bulk water, the peptides assume an α/β-type structure, with the β-strands and α-helices significantly tilted relative to the membrane normal. Altogether, the data are consistent with an octameric “α/β-ring” structure with an internal pore diameter of at least 13 Å to 20-22 Å that can effectively transfer calcein and larger molecules. The data establish a foundation for characterization of the molecular structure of the α/β ring transmembrane pore formed by Bax-derived peptides.
Example of Killing Microbial Cells
All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention.
More specifically, certain agents which are both chemically and physiologically related can be substituted for the agents described herein while the same or similar results can be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.
This application is a National Phase Application of International Application No. PCT/US2012/052354 filed Aug. 24, 2012, which claims priority to U.S. Provisional Application No. 61/527,524 filed Aug. 25, 2011 and to U.S. Provisional Application No. 61/645,891 filed May 11, 2012, each of which is incorporated herein fully by reference.
This invention was made with government support under GM083324 awarded by the National Institutes of Heath/NIGMS. The United States government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2012/052354 | 8/24/2012 | WO | 00 | 2/25/2014 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2013/029011 | 2/28/2013 | WO | A |
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| 20040191843 | Wright et al. | Sep 2004 | A1 |
| 20100099742 | Stassi et al. | Apr 2010 | A1 |
| 20140255299 | Khaled et al. | Sep 2014 | A1 |
| Number | Date | Country |
|---|---|---|
| 2846629 | Feb 2013 | CA |
| 2747773 | Jul 2014 | EP |
| WO-2013029011 | Feb 2013 | WO |
| WO-2013086430 | Jun 2013 | WO |
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| International Preliminary Report on Patentability issued Jun. 10, 2014 for International Patent Application No. PCT/US2012/068590, which was filed on Dec. 7, 2012 and published as WO 2013/086430 on Jun. 13, 2013 (Inventor—Khaled // Applicant—University of Central Florida Research Foundation, Inc.) (pp. 1-9). |
| Number | Date | Country | |
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| 20140255299 A1 | Sep 2014 | US |
| Number | Date | Country | |
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| 61527524 | Aug 2011 | US | |
| 61645891 | May 2012 | US |
