FIELD OF THE INVENTION

[0001] This invention relates to the field of the treatment of cancer cells and more particularly to the use of PNC -27, PNC-28 and related cell membrane-pore forming peptides for killing chemotherapy-resistant cancer cells, immunotherapy-resistant cancer cells and radiation therapy-resistant cancer cells, but not killing normal cells.

BACKGROUND OF THE INVENTION

[0002] The American Cancer Society estimates that in 2017 an additional 1.7 million people will be diagnosed as having cancer. If the cancer is a di screte tumor, then a surgical procedure may be sufficient to remove all of a patient's cancer cells. In many cancer patients, however, the cancer has spread from a discrete tumor and surgery can only remove the discrete tumor, thus leaving the patient still having cancer. In these patients, among the residual cancer cells that surgery missed, are some cancer cells which are resistant to treatments such as radiation therapy, chemotherapy, and/or immunotherapy. Residual cancer cells multiply over time to spread and kill the cancer patient in many cases as is clear from Table 1 ACS 2014 data (American Cancer Society). Table 1 shows that 56 to 94 percent of cancer patients die within five years if they have been diagnosed with a pancreas, liver, bile duct, lung, bronchus, esophagus, stomach, gallbladder, colon, brain, nervous system, myeloid cells, plasma cells, leukocytes, lymphatic system, or ovary. Five-year mortality from a cancer of the cervix, oral cavity, pharynx, colon, rectum, small intestine, bones & joints, heart, renal pelvis, soft tissues, kidney, eye, orbit, uterine corpus, breast, and bladder is also significant. [0003] Table 1 - 2014 ACS Statistics for Five Year Cancer Patient Survival

[0004] Cancer patients are treated with radiation therapy after or before cancer surgery. Radiation has limited effectiveness for killing cancer cells for several reasons. Firstly, only a low radiation dose of about 60 Gy (Gray units; 1 Gy = 1 joule of radiation energy absorbed per kilogram tissue) can be used, otherwise too many normal cells are killed. However, a 60 Gy radiation dose fails 30-50% of the time to stop cancer tumor growth, even if there is also cancer chemotherapy (Willers, 2013). Secondly, some cancer cells are radiation-resistant and are classified as being cancer stem cells or as clonogenic cancer cells which can produce an expanding family of daughter cells which can give rise to a recurrent tumor (Willers, 2013). Thirdly, radiation needs oxygen to effectively kill cancer cells but tumors often have hypoxic areas. [0005] Treatment of cancer patients almost al ways invol ves some use of chemotherapy drugs and when one drug does not work then another drug can be tried, but ultimately chemotherapy does not eliminate cancer in many patients. There can be one or several reasons for the presence of chemotherapy resistant cancer cells in a cancer patient. Firstly, it is believed that about 50% of all cancer tumors have some cancer cells that are initially resistant to chemotherapy (Lippert, 2010). Secondly, after chemotherapy treatment is initiated, new cancer cells due to natural selection and random genetic mutations may prevail which are

chemotherapy resistant cancer cells. Chemotherapy resistant cancer cells have many ways to resist chemotherapy. For example, a chemotherapy resistant cancer cell may have a mutation preventing formation of a metabolic enzyme needed to change a chemotherapy prodrug into the chemotherapy drug inside the cancer cell (Housman, 2014). Another problem can be that the chemotherapy resistant cancer cell has a mutation which results in a reduced binding of the chemotherapy drug to its cancer cell target or which results in a reduced protein synthesis of the chemotherapy drug target in the cancer cell. Another problem can be that the chemotherapy resistant cancer cell has a mutation that alters the signal transduction process following the binding of the chemotherapy drug to its cellular target (Housman, 2014). Another problem can be that the chemotherapy resi stant cancer cell has a mutati on that increases tran sport of the chemotherapy drug out of the cancer cell and thereby reduces the cancer drug concentration inside the cancer cells. Chemotherapy resistant cancer cells have enhanced mechanisms for chemotherapy drug removal by ATP-binding cassette (ABC) transporter family proteins:

MDR1, MRP1, and BCRP (Housman, 2014). Chemotherapy resistant cancer cells are also known to have an increased ability to reverse chemotherapy drug-induced DNA damage by having enhanced mechanisms for DNA damage, detection, and repair (DDR) (Willers, 2013).

[0006] Targeted cancer therapy concerns the administering to cancer patients specific drugs or other substances intended to block the growth and metastases of cancer by interfering with specific molecular targets involved in the growth, progression, and spread of cancer. Targeted cancer therapy includes molecular targeted drugs, molecularly targeted therapy, precision medicines, and specific cancer pathway inhibitors. Targeted therapies differ from

chemotherapeutic therapy, in that the former act on specific molecular targets that are associated with cancer whereas most chemotherapies act on both rapidly-dividing normal cells and cancerous cells. Targeted therapies are selected or designed based on a biological target inside a cancer cell. Targeted therapies are often cytostatic and block tumor cell proliferation, whereas chemotherapy agents can kill some tumor cells. A limitation of targeted cancer therapy is that known targets for targeted cancer therapy can change as cancer cell DNA mutates which expresses the protein target and/or because the target's function is continuously regulated in a cancer cell. For example Ras, a cytoplasmic signaling protein, is mutated in as many as 25 percent of all cancers and in the majority of pancreatic cancers studied to date. This has made it impossible to develop a targeted inhibitor of the Ras signaling pathway. Thus, targeted cancer therapy fails to kill many of the cancers which are resistant to other cancer treatments due to cancer cell mutations.

[0007] Cancers are often treatment resistant to immunotherapy due to downregulation or immune suppression by cancer cells. The immune system recognizes and is poised to eliminate cancer and the immune system has immune checkpoints which are various inhibitory pathways that regulate the duration and intensity of immune responses in peripheral tissues so as to minimize collateral tissue damage. However, these immune checkpoint pathways can be manipulated by cancer cells, thus circumventing immune destruction and this has become one of the hallmarks of cancer. In NSCLC, expression of programmed death ligand-1 (PD-L1 , B7- Hl) reflects an immune-active microenvironment and is a mechanism designed to evade elimination by the immune system. Exhausted T-cells in the microenvironment show overexpression of programmed cell death protein 1 (PD-1), which binds to PD-L1 and decreases effector cytokine production and cytolytic activity, leading to the failure of cancer elimination. While there has been some improvements in immunotherapy with the introduction of the anti-PD-1 or anti-PD-Ll immunotherapies, it has become apparent that some cancers contain cells which mutate to resist immunotherapy and further proliferate. There can be a hyperprogression of tumors, as the phenomenon is known, with shared specific genetic characteristics. In some patients with amplifications in the MDM-2 gene family, also known as the HDM-2 family, and in some patients with alterations in the EGFR gene, the treatment of the cancer with anti-PD-1 or anti-PD-Ll immunotherapies quickly failed, and the patients' cancers progressed rapidly. Many cancer patients fail treatments with immunotherapies such as atezolizumab, avelumab (Bavencio); pembrolizumab (Keytruda); and nivolumab (Opdivo). Another major class of immunotherapies known as anti-CTLA-4 treatments, such as ipilimumab (Y ervoy), which target a different mechanism to unleash immune cells to fight tumors also exhibit failures to treat some cancer patients adequately for their survival to occur. [0008] Cancer cells in a tumor are now appreciated as forming heterogeneous cellular populations and it is becoming more and more clear that any human cancer tumor might harbor a few cancer cells having radiation resistance, chemotherapy resistance, and/or immunotherapy resistance. It is commonly observed under the microscope that the histologic diversity of cells is much more pronounced in a cancer tumor compared to normal tissue. Cancer cells are also found to have many phenotypic variations in their expression of surface antigens and cytoplasmic proteins, and activity of biochemical processes. Also, cancer cells in tumors of various kinds of cancers have a great range of functional variations in for example, their cancer cell proliferation rate, cancer cell invasiveness of normal tissues, forming metastases, and activation of neo-angiogenesis. Research has shown that some of the underlying causes for this are genomic heterogeneity, hierarchical organization of tumor cells, environmental influences, and randomness of cancer cell derangements. Thus, cancer cells are highly de-regulated, facultative, immortal cells with a high potential to become resistant to conventional cancer treatments such as radiation therapy, chemotherapy, targeted cancer drug therapy, and/or immunotherapy.

[0009] The genomic heterogeneity of some cancer cells can be a result of cancer cell genomic instability and the increased proliferation rate of cancer cells. Cancer cells which become more easily mutated and which have an increased rate of proliferation can more rapidly evolve to survive natural selection pressures in their tumor microenvironments. Also, cancer cells live longer than non-cancer cells and give rise to descendant cells. Clones of the hardier cancer cells in a chaotic tumor microenvironment will increase in relative number as a tumor grows. There appears to be a continual replacement within the tumor mass of predominant clonal populations of cancer cells with a simultaneous coexistence of multiple clonal populations. In addition, within each clonal population in a tumor there may be a cellular hierarchical organization based on a cancer stem cell model.

[0010] The cancer stem cell (CSC) model offers several hypotheses as to how some cancer cells in a cancer tumor may have an intrinsic resistance to radiation therapy, chemotherapy, and/or immunotherapy. The CSC model hypothesizes that a tumor will contain at least three basic functional groups of cancer cells: (1) cancer stem cells, (2) progenitor cancer cells, and

(3) mature cancer cells. CSCs will be a self-renewing population with some CSCs dividing to form two new CSCs, and with some CSCs dividing to form a CSC and a progenitor cancer cell

(PCC). The relative size of the CSC population in a tumor would be cancer-type dependent. Progenitor cells proliferation would be the cause for rapid tumor mass growth. Ultimately some PCCs would be expected to differentiate into mature cancer cells (MCC) which cannot divide but could still contribute to tumor mass. Also, it is hypothesized that a CSC may arise from a progenitor cancer cell that mutates to acquire a self-renewal capability. In addition, it is hypothesized that another source of CSCs may be differentiated epithelial cells from the epithelial-mesenchymal transition (EMT) process. EMT is an intermediate step in the spreading of cancer in a cancer patient by metastasis. Thus, some oncologists believe that cells with traits of cancer stem cells should be targeted so that a cancer treatment may kill all cancer cells in a cancer patient.

[0011] The cancer stem cell (CSC) model includes the concept that CSCs are an

infrequently dividing population. This makes CSCs much less vulnerable to radiation therapy and to chemotherapy because these treatments are more potent against rapidly dividing cells than against infrequently dividing cells such as CSCs. Also, compared to PCCs, CSCs may inherit or acquire resistance to radiation or chemotherapy. For example CSCs may have one of more of the following resistance mechanisms: an elevated activity of DNA damage detection and repair mechanisms, an aberration in an apoptosis pathway, a transport of cancer drugs out of the CSCs, a lower production of a reactive oxygen species (ROS) and/or a reactive nitrogen species (RNS) (Liou, 2010), and a higher production of an interleukin (Fulawka, 2014). An important concern raised by the CSC model is whether the CSCs that can resist radiation and chemotherapy treatments can persist and proliferate to develop into an undetectable, residual, cancer disease (URCD) in a cancer patient. Then later, the URCD may foster a population of progenitor cancer cells (PCCs) which can lead to a cancer tumor recurrence. Thus, needed is a more effective cancer treatment for a cancer patient that would be capable of preventing URCD development during or following conventional cancer treatments.

[0012] Another cancer treatment issue is the highly abnormal characteristics of the tumor microenvironment compared to normal tissue microenvironments. Tumor microenvironments have extracellular variations in hypoxia, extracellular acidosis, hydrostatic pressure, oncotic pressure, and signaling molecules. In addition, due to the variations in the tumor

microenvironment, the cancer cells therein have variations in their increases in glycolysis, their increases in glucose dependence, their increases in ion transport across intracellular membranes and plasma membranes, their increases in production of signaling molecules, and in their shifts of the cellular redox towards oxidation (Liou, 2010). The altered biochemistry of cancer cells creates an energy drain on the cancer patient and cancer cells drive mechanisms for suppression there of the patient's immune system pathways. There is also transcriptional noise between cells from variations in the timing of transcription between cells (Fulawka, 2014). The heterogeneity of the ongoing stresses in the tumor microenvironment create a wide range in the variety and degree of Darwinian selection pressures which can drive tumor cell heterogeneity and large adaptive changes making cancer cells more resistant to conventional cancer treatment.

[0013] The p53 protein is a vital regulator of the cell cycle. In several ways, the p53 protein blocks oncogenic effects of numerous oncogene proteins that induces mitosis. The p53 protein can block protein transcription that induces mitosis. Also, the p53 protein can induce transcription of proteins that can block mitosis, and promote apoptosis (cell-initiated

programmed cell death). Absence of the p53 protein is associated with cell transformation and malignant disease. The MDM-2 (mouse double minute 2) protein is a negative regulator of p53, for example modulates the cellular response of p53 to cytotoxic/DNA damaging treatments. While in the prior art the MDM-2 protein is named HDM-2 in human settings by some researchers, the present patent application specification and claims uses MDM-2 and HDM-2 as simply two names for same protein.

[0014] In many cell lines and several tumor samples, overexpression of HDM-2 typically correlates with a decreased response to both chemotherapy and radiation therapy. HDM-2 is overexpressed in more than forty different types of malignancies, including solid tumors, sarcomas and leukemia. Because of its prevalent expression and its interactions with p53 and other signaling molecules, HDM-2 is hypothesized to play an important role in cancer development and progression.

[0015] Despite many publications exploring the expression level, functions and interactions of MDM-2 in animal cancer models, there is still not a clear-cut picture of what role HDM-2 (the human analog of MDM-2) may have for cancer patients with respect to using HDM-2 in cancer patient diagnosis and prognosis. It is known that different cancers behave differently, and the effects of HDM-2 are variable. Due to the ubiquitous nature of HDM-2 and its involvement in various signaling pathways, some of the results obtained about the effects of MDM-2 expression are paradoxical. One example of this paradox is with the soft tissue sarcomas (STS) where studies find the sarcomas overexpress MDM-2, however the effect of MDM-2 expression on cancer patient prognosis is conflicting. For example, one study has found that MDM-2 was associated with heightened proliferation in the tumor whereas another study reported MDM-2 to not be correlated with proliferation in the tumor (Rayburn, 2005).

[0016] A genetic mutations analysis of metastatic and locally advanced recurrent breast cancer in 43 breast cancer patients by Meric-Berstam (2014) found high concordance between the genetic mutations in primary tumors and recurrent/metastatic tumors. In addition, some genes were amplified: MCL1, CCND1, FGFR1, MYC, IGF1R, MDM-2, MDM-4, A T3, CDK4, AKT2. MDM-2 and 9 other genes were focally amplified more than six-fold. CDK4 and MDM-2 amplifications were greater in tumor recurrences. The p53-pathway alteration was common in triple-negative tumor samples. However, correlations in the Meric-Berstam study are based on few matched sets of primary and recurrent tumor data, and questionable given the variability in time points of biopsies and adjuvant therapy in the study.

[0017] Conventional cancer treatment methods kill cancer cells by acting upon one or more biochemical processes inside the cancer cell that may activate an intracellular apoptosis pathway (programmed cell death pathway). A change due to a gene mutation in the biochemical processes preceding apoptosis or in the apoptosis pathway processes may block the killing of the cancer cells by conventional cancer treatments including radiation therapy, chemotherapy, and/or immunotherapy. Some cancer cells are inherently resistant to such cancer treatments or become resistant for reasons mentioned.

[0018] Another problem with prior art cancer treatments which oncologists find unavoidable as collateral damage is the killing of some normal cells when killing cancer cells or during surgery having to remove normal tissues near the cancer tumor. Another serious problem is that cancer patient may suffer severe long-lasting side effects from conventional cancer treatments. In addition, as shown in Table 1, the current cancer treatments fail to save the lives of many cancer patients from many of the cancers. Oncologists believe that these cancer treatment failures are due to the failure of best clini cal treatments to kill the chemotherapy-resistant cancer cells, radiation therapy resistant cancer cells, and/or immunotherapy-resistant cancer cells in many cancer patients. Clearly there is an urgent need to overcome these problems in treating cancers. Many cancers continue to mutate to form clones of cancer treatment cancer cell tumors in cancer patients which are treatment resistant to current clinical treatments with radiation therapy, chemotherapy, targeted cancer therapy, and immunotherapy. SUMMARY OF THE INVENTION

[0019] In general, the present invention relates to methods of killing cancer cells resistant to current cancer treatments including chemotherapy, radiation therapy, targeted cancer therapy, and immunotherapy. More specifically, embodiments of the present invention comprise methods of using cancer cell membrane pore-forming peptides for killing treatment resistant cancer cells while not killing normal cells. The methods of using cancer cell membrane pore- forming peptides for killing treatment resistant cancer cells while not killing normal cells concerns using cancer cell membrane pore-forming peptides including PNC-27, PNC-28 and related cancer cell membrane pore-forming peptides.

[0020] Some embodiments of the present invention are a method of killing a cancer treatment resistant cancer cell in a mammal using a cell membrane pore-forming peptide, the method comprising the steps of: (1) administering to the mammal a pharmaceutical formulation by a route of administration, the pharmaceutical formulation comprising the cell membrane pore-forming peptide which comprises a first amino acid sequence attached at its carboxyl terminal end to an amino terminal end of a second amino acid sequence, wherein the first amino acid sequence can bind to an HDM-2 protein, and wherein the second amino acid sequence can function as a membrane resident peptide; (2) binding the first amino acid sequence of the cell membrane pore-forming peptide to a part of the HDM-2 protein on an outer face of the cell membrane of the cancer treatment resistant cancer cell in the mammal; (3) residing the second amino acid sequence of the cell membrane pore-forming peptide in the cell membrane of the cancer treatment resistant cancer cell in the mammal; (4) forming a pore with the cell membrane pore-forming peptide in the cell membrane of the cancer treatment resistant cancer cell as a result of the first amino acid sequence of the cell membrane pore-forming peptide binding the HDM-2 protein on the outer face of the cell membrane of the cancer treatment resistant cancer cell and as the result of the second amino acid sequence of the cell membrane pore-forming peptide residing in the cell membrane of the cancer treatment resistant cancer cell; (5) using the pore formed by the cell membrane pore-forming peptide in the cell membrane of the cancer treatment resistant cancer cell as a pathway for diffusing a substance between extracellular and intracellular spaces of the cancer treatment resistant cancer cell; and

(6) using the substance diffusing between the extracellular and intracellular spaces of the cancer treatment resistant cancer cell for killing the cancer treatment resistant cancer cell in the mammal . In some embodiments of the present in vention, the treatment resi stant cancer cells are killed in a mammal which is a human. Generally it is expected that normal cells in the human cancer patient are not killed by the cell membrane pore forming peptide embodiments of the present invention.

[0021] In some preferred embodiments of the present invention, the first amino acid sequence of the cell membrane pore-forming peptide comprises between 6 to 15 con tiguous amino acids of amino acid sequence PPLSQETFSDLWKLL,

wherein optionally an L of the amino acid sequence PPLSQETFSDLWKLL may be substituted by an He or a Val,

wherein optionally an S of the amino acid sequence PPLSQETFSDLWKLL may be substituted by a Thr,

wherein optionally a Q of the amino acid sequence PPLSQETFSDLWKLL may be substituted by an Asn,

wherein optionally an E of the amino acid sequence PPLSQETFSDLWKLL may be substituted by an Asp,

wherein optionally a T of the amino acid sequence PPLSQETFSDLWKLL may be substituted by a Ser,

wherein optionally an F of the amino acid sequence PPLSQETFSDLWKLL may be substituted by a Met, a Leu, or a Tyr,

wherein optionally a D of the amino acid sequence PPLSQETFSDLWKLL may be substituted by a Glu,

wherein optionally a W of the amino acid sequence PPLSQETFSD LWKLL may be substituted by a Tyr, and

wherein optionally a K of the amino acid sequence PPLSQETFSDLWKLL may be substituted by an Arg, a Gin, or a Glu.

[0022] In some preferred embodiments of the present invention, the second amino acid sequence of the cell membrane pore-forming peptide is selected from the group consisting of:

amino acid sequence KKWKM RNQFWVKVQRG,

and wherein optionally an alpha helical stabilizing amino acid may be added at the amino terminal of the HDM2 binding component, with the alpha helical stabilizing amino acid selected from the group consisting of Leu, Glu, Met, Phe, and a combination thereof.

[0023] In some preferred embodiments of the present invention, the second amino acid sequence of the cell membrane pore forming peptide may contain at least one d-amino acid and/or may have leupeptin or another peptidase-inhibiting peptide attached to the carboxyl terminal end of the cell membrane pore forming peptide so as to reduce the action of peptidases in the mammal to damage the cell membrane pore forming peptide of the invention.

[0024] In some particularly preferred embodiments of the present invention, the invention is a method of using a cell membrane pore forming peptide for killing a cancer treatment resistant cancer cell which is a chemotherapy resistant cancer cell. In some particularly preferred embodiments of the present invention, the invention is a method of using a cell membrane pore- forming peptide for killing a cancer treatment resistant cancer cell which is a radiation therapy resistant cancer cell. In some particularly preferred embodiments of the present invention, the invention is a method of using a cell membrane pore-forming peptide for killing a cancer treatment resistant cancer cell which is a targeted cancer therapy resistant cancer cell. In some particularly preferred embodiments of the present invention, the invention is a method of using a cell membrane pore forming peptide for killing a cancer treatment resistant cancer cell which is an immunotherapy resistant cancer cell.

[0025] The method of practicing some embodiments of the present invention is

advantageous for killing cancer treatment resistant cancer cell in a mammal wherein the cancer treatment resistant cancer cell may be for example a cancer which is selected from the group consisting of a pancreatic cancer, a lung cancer, a breast cancer, a liver cancer, an intrahepatic bile duct cancer, an acute myeloid leukemia cancer, a bronchus cancer, an esophagus cancer, a gallbladder cancer, a stomach cancer, a brain cancer, a nervous system cancer, a myeloma cancer, an ovary cancer, a uterine cancer, a cervix cancer, a chronic myeloid cancer, and oral cavity cancer, a pharynx cancer, a colon cancer, a rectum cancer, a small intestine cancer, a bone cancer, a joint cancer, a soft tissue cancer, a heart cancer, an acute lymphocytic leukemia cancer, a non-Hodgkin lymphoma cancer, a kidney cancer, a renal pelvis cancer, a chronic lymphocytic leukemia cancer, a urinary bladder cancer, an eye cancer, and eye orbit cancer, a uterine corpus cancer, a Hodgkin lymphoma cancer, a melanoma cancer, a skin cancer, a lymph node cancer, a penile cancer, an adrenal cancer, a thymus cancer, a thyroid cancer, a prostrate cancer, a metastatic cancer, a cancer stem cell cancer, a cancer progenitor cell cancer, and a combination thereof.

[0026] For some embodiments of the present invention, the method of the invention involves measuring a level of the substance in the mammal which may be released from cancer cells in the mammal. The substance in the mammal which may be released can be an enzyme released from the cancer cell cytoplasm, and various cytoplasmic enzymes are known, and any of these enzymes could be released, and include cytoplasmic enzyme lactate dehydrogenase.

[0027] A particularly preferred embodiment of the present invention involves methods of the invention the wherein the cell membrane pore-forming peptide is PNC-27. Another particularly preferred embodiment of the present invention involves methods of the invention the wherein the cell membrane pore-forming peptide is PNC-28.

[0028] In practicing some method embodiments of the present invention, the route of administration is selected from the group consisting of an oral route, a parenteral route, an intravenous route , an intramuscular route, a subcutaneous route, a pump implanted in the mammal, a route of administration to the mammal which uses a continuous administration by a pump implanted subcutaneously, a route of administration to the mammal which uses a continuous administration by a pump implanted intraperitoneally, an injection into a tumor, an injection into an arterial circulation of a tumor, an injection into a hepatic artery, an injection into a hepatic vein, an injection into CNS spinal fluid, an intraocular injection, a nasal route, a rectal route, an administration route into a bladder, an administration route into a cervix, an administration route into a brain, an administration route into an artery, an intraperitoneal route, an administration route into a lymphatic circulation, an administration route into a lymph node, an injection into a lymph node, an inhalation route, a lung ventilator apparatus route, and a combination thereof.

[0029] In practicing some method embodiments of the present invention, the

pharmaceutical formulation comprises a mixture of the membrane pore forming peptide in an excipient selected from the group consisting of with an isotonic saline, a pH buffered aqueous solutions, ethanol, glycerol, propylene glycol, a polyol, a polyethylene glycol, a surfactants, a fatty acid, a micro-emulsion, a liposome, a microsphere, a peptide nanoparticle, an emulsion, a gelatin, a vegetable oil, a saccharide, a polysaccharide, an excipient for an oral table

formulation, an excipient for an oral capsule formulation, a hard gelatin capsule, a soft shell gelatin capsule, an elixir, a fruit juice, a sugar cube, a candy, a suspension, a syrup, an excipient for an oral formulation, an excipient for a suppository, an excipient for an intravenous solution, an excipient for a syringe injection formulation, an excipient for a catheter injectable formulation, an excipient for a transdermal patch, an excipient for a parenteral injection, an excipient for an eye drop formulation, and excipient for an injection into the CNS, an excipient for an inhalant, and a combination thereof, and wherein the amount of the membrane pore forming peptide per 100 mg of the excipient in the pharmaceutical formulation is selected from the group consisting of about 0.01 microgram to about 1 microgram, about 1 microgram to about 100 micrograms, about 100 micrograms to about 10 milligrams, about 10 milligrams to about 50 milligrams, about 50 milligrams to about 5 grams, about 5 grams to about 20 grams and any combination thereof.

[0030] In practicing some method embodiments of the present invention, the frequency of administration of a dose of the pharmaceutical formulation is selected from the group consisting of a single dose, a single dose every hour, a single dose every three hours, a single dose every six hours, a single dose every twelve hours, a single dose every twenty-four hours, a single dose every two days, a single dose every three days, a single dose every fourth day, a single dose every fifth day, a single dose per week, a single dose per two weeks, a single dose per three weeks, a single dose per month, a single dose per two months, a single dose per three months,a single dose per four months, a single dose per five months, a single dose per year, a single dose per two years, a single dose per three years, a single dose per four years, a single dose per five years, a dose which is an intravenous infusion for between about one minute to about fifteen minutes, a dose which is an intravenous infusion for as long as thirty minutes, a dose which is an intravenous infusion for as long as one hour, a dose which is an intravenous infusion for as long as two hours, a dose which is an intravenous infusion for as long as three hours, a dose which is an intravenous infusion for as long as four hours, a dose which is an intravenous infusion for as long as eight hours, a dose which is an intravenous infusion for as long as twelve hours, a dose which is an intravenous infusion for as long as twenty-four, a dose which is an intravenous infusion for as long as two days, a dose which is an intravenous infusion for as long as three days, a dose which is an intravenous infusion for as long as four days, a dose which is an intravenous infusion for as long as five days, a dose which is an intravenous infusion for as long as a week, a dose which is an intravenous infusion for as long as two weeks, a dose which is an intravenous infusion for as long as three weeks, a dose which is an intravenous infusion for as long as a month, a dose which is an intravenous infusion for as long as two months, a dose which is an intravenous infusion for as long as three months, a dose which is an intravenous infusion for as long as four months, a dose which is an intravenous infusion for as long as five months, a dose which is an intravenous infusion for as long as a year, a continuous administration by pumps implanted subcutaneous! y, a continuous administration by pumps implanted intraperitoneally, and a combination thereof.

[0031] In practicing some method embodiments of the present invention, the dose of the membrane pore forming peptide per kilogram of body weight of the mammal is selected from the group consisting of about 0.1 mg to about 20 mg, about 0.001 mg to about 0.1 mg, about 0.1 mg to about 1 mg, about 1 mg to about 10 mg, about 10 mg to about 50 mg, about 20 mg to about 100 mg, and a combination thereof. In practicing some method embodiments of the present invention, note that the pharmaceutical formulation of the membrane pore-forming peptide and excipient may be for example, a mixture wherein the amount of the membrane pore forming peptide per 100 mg of the excipient in the pharmaceutical formulation is selected from the group consisting of about 0.01 microgram to about 1 microgram, about 1 microgram to about 100 micrograms, about 100 micrograms to about 10 milligrams, about 10 milligrams to about 50 milligrams, about 50 milligrams to about 5 grams, about 5 grams to about 20 grams and any combination thereof.

In practicing some method embodiments of the present invention, a mammal is administered the therapeutic dose of the pharmaceutical formulation comprising the cancer cell membrane pore forming peptide as a cancer treatment plan selected from the group consisting of the cancer treatment plan for killing a treatment-resistant cancer stem cell in the mammal at any time in life of the mammal, the cancer treatment plan for killing a treatment-resistant cancer progenitor cancer cell in the mammal at any time in the life of the mammal, the cancer treatment plan for killing the treatment-resistant cancer stem cell in the mammal, the cancer treatment plan for killing the treatment-resistant cancer progenitor cancer cell in the mammal, the cancer treatment plan for killing the treatment resistant cancer cell in the mammal which has metastasized from the primary tumor, the cancer treatment plan for killing the treatment- resistant cancer cell in the mammal as a prophylactic treatment, the cancer treatment plan for killing the treatment-resistant cancer cell in a tumor of the mammal which is inoperable, the cancer treatment plan for killing the treatment-resistant cancer cell in a blood circulation of the mammal without causing an immune system suppression, the cancer treatment plan for killing the treatment-resistant cancer cell in the mammal without killing a normal cell in the mammal, the cancer treatment plan for killing the treatment-resistant cancer stem cell in the mammal without killing the normal cell in the human, the cancer treatment plan for killing the treatment- resistant cancer cell in the mammal as a treatment preceding chemotherapy to kill

chemotherapy sensitive cancer cells, the cancer treatment plan for killing the treatment- resistant cancer cell in the mammal as an adjunct to the chemotherapy to kill the chemotherapy sensitive cancer cells, the cancer treatment plan for killing the treatment-resistant cancer cell in the mammal after the chemotherapy to kill the chemotherapy sensitive cancer cells, the cancer treatment plan for killing the treatment-resistant cancer cell in the mammal as the adjunct to a radiation therapy to kill radiation therapy sensitive cancer cells, the cancer treatment plan for killing the treatment-resistant cancer cell in the mammal preceding the radiation therapy to kill the radiation therapy sensitive cancer cells, the cancer treatment plan for killing the treatment-resistant cancer cell in the mammal after the radiation therapy to kill the radiation therapy sensitive cancer cells, the cancer treatment plan for killing the treatment-resistant cancer cell in the mammal as the adjunct to an immunotherapy to kill immunotherapy sensitive cancer cells, the cancer treatment plan for killing the treatment-resistant cancer cell in the mammal preceding the immunotherapy to kill the immunotherapy sensitive cancer cells, the cancer treatment plan for killing the treatment-resistant cancer cell in the mammal after the immunotherapy to kill the immunotherapy sensitive cancer cells, the cancer treatment plan for killing the treatment resistant cancer cell in the mammal as an adjunct to a targeted cancer pathway chemotherapy, the cancer treatment plan for killing the treatment resistant cancer cell in the mammal preceding the targeted cancer pathway chemotherapy, the cancer treatment plan for killing the treatment resistant cancer cell in the mammal after the targeted cancer pathway chemotherapy, the cancer treatment plan for killing the treatment resistant cancer cell in the mammal as an adjunct to a cancer surgery, the cancer treatment plan for killing the treatment resistant cancer cell in the mammal preceding the cancer surgery, the cancer treatment plan for killing the treatment resistant cancer cell in the mammal after the cancer surgery, the cancer treatment plan for killing the treatment resistant cancer cell in the mammal as an adjunct to another cancer treatment, the cancer treatment plan for killing the treatment resistant cancer cell in the mammal preceding the other cancer treatment, the cancer treatment plan for killing the treatment resistant cancer cell in the mammal after the other cancer treatment, the cancer treatment plan for killing treatment resistant cancer cell to prevent a cancer reappearance in the mammal after the mammal appears to have become cancer free, and a combination thereof.

Some preferred embodiments comprise a method of killing a cancer treatment resistant cancer cell in a human cancer patient using a cell membrane pore-forming peptide, the method comprising the steps of:

(a) administering to the human cancer patient a therapeutic dose of a pharmaceutical

formulation by a route of administration, the pharmaceutical formulation comprising the cell membrane pore-forming peptide which comprises a first amino acid sequence attached at its carboxyl terminal end to an amino terminal end of a second amino acid sequence, wherein the first amino acid sequence can bind to an HDM-2 protein, and

wherein the second amino acid sequence can function as a membrane resident peptide;

(b) binding the first amino acid sequence of the cell membrane pore-forming peptide to a part of the HDM-2 protein on an outer face of the cell membrane of the cancer treatment resistant cancer cell in the mammal;

(c) residing the second amino acid sequence of the cell membrane pore-forming peptide in the cell membrane of the cancer treatment resistant cancer cell in the mammal;

(d) forming a pore with the cell membrane pore-forming peptide in the cell membrane of the cancer treatment resistant cancer cell as a result of the first amino acid sequence of the cell membrane pore forming peptide binding the HDM-2 protein on the outer face of the cell membrane of the cancer treatment resistant cancer cell and as the result of the second amino acid sequence of the cell membrane pore forming peptide residing in the plasma cell membrane of the cancer treatment resistant cancer cell;

(e) using the pore formed by the cell membrane pore-forming peptide in the cell membrane of the cancer treatment resistant cancer cell as a pathway for diffusing a substance between extracellular and intracellular spaces of the cancer treatment resistant cancer cell; and

(f) using the substance diffusing between the extracellular and intracellular spaces of the cancer treatment resistant cancer cell for killing the cancer treatment resistant cancer cell in the mammal. For example, in this regard the substance in the mammal which may be released can be a protein or an enzyme released from the cancer cell cytoplasm. Many cytoplasmic enzymes and proteins are known, and any of these could be released and measured in practicing the present invention, such as lactate dehydrogenase enzyme.

[0032] It is a particularly important and preferred series of method embodiments of the present invention, that at the time in the mammal or human when the cell membrane pore forming peptide is killing a treatment resistant cancer cell, that an insignificant number, and preferably none of the normal cells in the mammal or in the human are killed by a cell membrane pore forming peptide used in an embodiment of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 depicts a line graph plotting experimental results from treating an in vitro chemotherapy-resistant A875 human melanoma cells culture (n = 40,000 cells per test well) to PNC-27 concentrations: 0 uM, 10 uM, 20 uM, 40 uM, 80 uM and 160 uM. Note uM means micromolar concentration. After 24 hours of treatment with PNC-27, no viable cancer cells remained when the cells were treated with PNC-27 concentration of about 40 uM. The killing of the treatment-resistant A875 human melanoma cells by PNC-27 was quantified by the MTT cell viability assay. Graphed is the viability of chemotherapy-resistant A875 human melanoma cells (n = 40,000 cells per test well) as a function of PNC-27 concentration in the experiment. The viability of the A875 cells is measured using OD575 nm data (optical density

measurements at 575 nanometers wavelength) from a 24 hour MTT cell proliferation test.

[0034] Figure 2 depicts a dose-response bar graph based on a 24 hour cell LDH release response tested on chemotherapy-resistant SKOV-3 Human ovarian carcinoma cells at PNC-27 and PNC-29 concentrations of 0, 20, 40, 60, 80, and 120 micromolar (uM). PNC-29 is an inactive peptide which serves as a control. It can be seen that PNC-29 had no concentration dependent effect on SKOV-3 cells LDH release. The absorbance level of 3.5 was the same as for total cell lysis induced by treatment with detergent.

[0035] Figure 3 is a schematic depiction of a cluster of three SW 1222 cancer stem cells in black & white to present the outcome of a fluorescent microscopy experiment, which used PNC-27, membrane-damaged SW 1222 cancer stem cells, a green fluorescent antibody to PNC- 27 and a red fluorescent antibody to HDM-2 to study and locate where PNC-27 and HDM-2 co- localize.

DETAILED DESCRIPTION OF THE INVENTION

[0036] In general, the present invention relates to peptides with novel methods for killing chemotherapy-resistant cancer cells, radiation therapy-resistant cancer cells, targeted cancer therapy, and cancer cells which are resistant to immunotherapy, and that do not kill normal cells or non-cancerous cells. In some peptide embodiments of the present invention, the peptide comprises a portion of the amino acid sequence present in the p53 protein which can bind to the HDM-2 protein. Also in these peptide embodiments of the present invention, the peptide further comprises a portion of an amino acid sequence of a membrane residency peptide (MRP). Thus, in some peptide embodiments of the present invention, the peptide comprises a first sequence of amino acids which is an HDM-2 binding domain and comprises a second amino acid sequence which is an MRP.

[0037] For example, a preferred peptide embodiment of the present invention is PNC-27 that is a 32 amino acid peptide. Beginning at the amino end of the peptide, the PNC-27 amino acids sequence is presented immediately below. PNC-27 amino acids nos. 1-15 correspond to amino acids nos. 12-26 of the p53 protein that can bind to the HDM-2 protein. The PNC-27 amino acid nos. 16-32 below are an amino acids sequence of an MRP.

[0038] PNC-27 Amino Acid Sequence:

[0039] Another preferred embodiment of the present invention is PNC-28. In relation to the PNC-27 amino acid sequence depicted above, PNC-28 has amino acids nos. 6-15, which correspond to amino acids nos. 17-26 of the p53 protein that can bind to the HDM-2 protein. PNC-28 amino acid nos. 16-32 are same MRP amino acid sequence present in PNC-27.

[0040] PNC-28 Amino Acid Sequence (using sequence numbering for PNC-27):

[0041] Another preferred embodiment of the present in vention i s PNC-26. In relation to the PNC-27 amino acid sequence depicted above, PNC-26 has amino acids nos. 1-9, which correspond to amino acids nos. 12-20 of the p53 protein that can bind to the HDM-2 protein. PNC-26 amino acid nos. 16-32 are the same MRP amino acid sequence present in PNC-27.

[0042] PNC-26 Amino Acid Sequence (using sequence numbering for PNC-27)

[0043] Below Table 2 has in vitro dose-response data from US patent No. 7,539,327 (the '327 patent) for PNC-27 and PNC-28 killing of cancer cells of commercial culture cell lines which are known to be sensitive to chemotherapy and/or to radiation therapy. Table 2 provides name of the cancer cell culture line, the type of peptide, the peptide concentration tested in the cancer cell culture, and the time needed for the peptide to kill the cancer cells.

Table 2 - In Vitro PNC-27 and PNC-28 Dose-Response Data for Killing Chemotherapy Sensiti ve Cancer Commercial Cell Culture Lines (taken from US Patent No. 7,539,327)

[0044] While U.S. patent No. 9,539,327 teaches that PNC-27 and PNC-28 peptides can be used for killing chemotherapy sensitive and radiation sensitive cancer cells, the prior art does not teach that PNC-27 or another peptide of the present invention can be used in vitro for killing cancer cells in a commercial culture cell line where the cancer cells are chemotherapy resistant cancer cells, radiation resistant cancer cells, or immunotherapy resistant cancer cells.

In addition, whereas conventional cancer treatments are well known in general to be toxic to normal cells, it is highly noteworthy that PNC-27, PNC-28, and other peptides of the present invention do not kill normal cells. Table 3 below presents some test data using commercial established cancer cell lines in vitro. The Table 3 test data shows that normal cells survive prolonged exposure to PNC-27 and PNC-28 peptides at concentrations of these peptides, which will kill cancer cells.

[0045] Table 3 - PNC-27 and PNC-28 Peptides Do Not Kill Normal Cell Culture Lines

[0046] Table 4 below presents some test data using primary cell culture in vitro. The Table 4 test data shows that normal cells survive prolonged exposure to PNC-27 and PNC-28 peptides at concentrations of these peptides, which will kill cancer cells.

[0047] Table 4 - PNC-27 and PNC-28 Do Not Kill Normal Cells in Primary Cell Cultures

[0048] It is also very clinically important that PNC-27 is not toxic to normal macrophages given the important role of macrophages in the immune system. PNC-27 will however kill cultured K562 human chronic myelogenous leukemia cells. Kanovsky et al. (2001) has also reported that PNC-28 does not inhibit differentiation of hematopoietic stem cells in vitro. These stem cells used by Kanovsky were obtained from human female umbilical cord blood. The Kanovsky study indicates that PNC-28 does not suppress bone marrow function and this is an important safety feature of PNC-28 and other peptides of the present invention. In contrast, it is well known that most chemotherapeutic agents as well as radiation therapy are capable of causing a fatal anemia, leukopenia and thrombocytopenia because they suppress bone marrow function.

In Vivo Anti-Cancer Effect of PNC Peptides

[0049] Michl et al. (2006) tested the in vivo anti-cancer effectiveness of PNC-28. Nude mice were given a peritoneal implant of one million TUC-3 rat pancreatic cancer cells. The TUC-3 cancer cells have a high potential to metastasize. PNC-28 was then administered for two weeks to the peritoneum of each mouse using an exALzet pump. The PNC-28 administration eradicated the implanted pancreatic cancer cells during the two weeks and then over the following two weeks there was no evidence for the presence of tumor cells in the mice, at which time the experiment was ended. Similar experimental results were obtained in nude mice when the PNC-28 was administered by exAlzet pump to a subcutaneous space at a considerable distance from the peritoneal implant of cancer cells. When an inactive anti-cancer peptide known as PNC-29 was administered instead of the PNC-28, then the cancer cells were not killed and there was extensive tumor growth with peritoneal carcinomatosis and metastasis.

[0050] In another in vivo Michl study, the anti-cancer effectiveness of PNC-28 was further tested when TUC-3 rat pancreatic cancer cells were implanted in the right shoulder region of nude mice. Some of the mice were administered PNC-28 subcutaneously in the left hind region by exALzet pump. In control experiments, other mice were implanted with these TUC-3 tumor cells, but were administered the inactive peptide PNC-29 by the exAlzet pump. The anti-cancer peptide PNC-28 prevented tumor growth, whereas the inactive peptide PNC-29 permitted rapid tumor growth. It was also noted that the PNC-28 treated mice had small residual masses but no tumor cells were found in these residual masses during microscopy examination of the masses. [0051] In a third in vivo Michl study, further tested the anti-cancer effectiveness of PNC-28 in nude mice after the implanted the TUC-3 rat pancreatic cancer cells had been allowed to proliferate to a critical-size tumors. After the tumor cells had proliferated, then exAlzet pumps were implanted in the nude mice to administer PNC-28. The PNC-28 administration was able to kill the cancer tumors. Michl concluded that PNC-28 could work in vivo to kill cancer cells which have a high metastatic potential. Michl studies also reported that there was no evidence for any morbidity in the nude mice treated with the PNC-28, and that these mice maintained their normal behavior and weights. In contrast, the nude mice that had been treated with the inactive peptide, PNC-29 harbored rapid-growth tumors, suffered significant weight loss and had sluggish behavior.

Mechanism of Cancer Cell Killing by PNC-27 and PNC-28

[0052] High-resolution transmission electron microscopy (TEM) studies by Do et al. (2003) on a breast cancer cell and by Bowne et al. (2008) on a pancreatic cancer cell line reported that PNC-27 and PNC-28 peptides can induce significant formation of sizable pores (holes) in the cancer cell membranes. No such pores formed in cell membranes of normal primary human fibroblasts treated with PNC-27 or PNC-28. Rosal et al. (2004) inferred from studies using two- dimensional NMR that the three-dimensional structure of PNC-27 in a biological milieu may consist of two alpha-helical segments. The first PNC-27 alpha helical segment corresponds to the region of PNC-27, which is a HDM-2-binding domain. This first PNC-27 alpha helix conformation was determined to result from amino acid residues nos. 3-15 of the PNC-27 peptide. The second PNC-27 alpha helical segment corresponds to amino acid residues nos. 19- 32 of the MRP sequence in PNC-27. Between the first and second PNC-27 alpha helices is a three amino acid loop formed by amino acid residues nos.16- 18. In these alpha-helices, some of the amino acids have a hydrophobic amino acid side group and become oriented on one structural face of the alpha helix while the amino acids with a hydrophilic (polar) amino acid side chains form the opposite structural face of the alpha helix. Such structurally opposite faces in a molecule are known as an amphipathic structure. Alpha-helix amphipathic molecules have also been discovered in membrane-active peptides such as melittin and magainin. However, unlike some of the peptide embodiments of present invention, melittin and magainin lack a selective toxicity for killing only cancer cells. Melittin and magainin are selective for red blood cells and bacterial cell membranes, respectively. [0053] The specificity of PNC-27 and some other peptide embodiments of the present invention to induce trans-membrane pore formations only in cancer cell membranes may be related to the ability of PNC-27 and some other peptide embodiments of the present invention to bind to HDM-2. Cancer cells express HDM-2 whereas normal cells and untransformed cells either do not express HDM-2 or express HDM-2 in very low amounts per cell. For the present invention, the terms trans-membrane pore formation and cell membrane pore formation shall have the same meaning.

[0054] Sarafraz-Yazdi et al., 2010 reported that PNC-27 co-localizes with HDM-2 in cancer cells but PNC-27 does not co-localize with HDM-2 on normal cells or untransformed cells since they do not express this protein on their cell surfaces. However, when Sarafraz-Yazdi transfected normal MCF-10-2A human breast epithelial cells with a plasmid encoding the gene for full-length HDM-2 and a membrane localization signal CAAX box on its carboxyl terminal end, then these normal breast epithelial cells formed HDM-2. Sarafraz-Yazdi also transfected some normal breast epithelial cells with a truncated HDM-2 gene designed to cause expression of a HDM-2 protein lacking a binding site for PNC-27. In this case, when the cells were treated with PNC-27, the cells were not killed. This result showed that PNC-27 needs to bind to HDM- 2 in order for the PNC-27 to kill a cell. Note that the bioassay used to quantify the killing of cells used in the PNC-27 experiments measured the level of LDH. LDH is a cytoplasmic enzyme known as lactate dehydrogenase. LDH rapidly leaks from cells that develop transmembrane pores. Cell killing was also quantified by the well known MTT assay. Sarafraz- Yazdi et al. proposed that cells can be killed by trans-membrane pore formation if the peptide PNC-27 is able to bind to cancer cell membrane HDM-2.

Mechanism of PNC-27 Induced Trans-Membrane Pore Structure

[0055] The above results strongly suggest that , to exert its cytotoxic effects on cancer cells, PNC-27 must bind to HDM-2 in the cancer cell membrane. This binding then induces formation of trans-membrane pores that result in tumor cell necrosis. These events occur exclusively in the cancer cell membrane and not within the intracellular environment. This conclusion is further supported by the work of Sookraj et al (2008) wherein PNC-27 was labeled with a green (amino terminal) fluorescent probe and a red (carboxyl terminal) fluorescent probe. These probes were located sufficiently close to one another such that the two colors overlapped to give a yellow fluorescence that could be seen only if the PNC-27 molecule remained intact. This peptide killed MCF-7 breast cancer cells over a 24 hour period during which time there was a strong yellow fluorescent signal exclusively on the cancer cells' membranes. This result suggests that PNC-27 exerts its cytotoxic effect exclusively in the cancer cell membrane,

[0056] Thus these results show that the pores formed through the cancer cell membrane are composed of complexes of PNC-27 with HDM-2 and that these complexes form near the cell surface. Therefore, PNC-27 does not enter into the cell but remains on the cell surface at the mouth of the pore. This finding explains why PNC-27 i s not susceptible to cancer cell-mediated resistance, which requires the presence of the anti-cancer drug in the cell that can then either be extruded from the cell or metabolized to an inactive compound.

[0057] As a result of the novel observation by the inventor, that PNC-27 co-localizes with HDM-2 that is present on an exterior face of a cancer cell membrane and there is a formation of cell membrane pores which can kill the cancer cell, the inventor conceived of a new invention method for killing a treatment-resistant cancer in a cancer patient using a pharmaceutical formulation of a peptide embodiment of the present invention. The method for killing a treatment-resistant cancer in a cancer patient comprises the steps of: administering a

pharmaceutical formulation of a cell membrane-pore forming peptide to the cancer patient; co- localizing the cell membrane-pore forming peptide in the cancer patient to an HDM-2 protein on a exterior surface of a treatment-resistant cancer cell membrane, wherein the cell membrane- pore forming peptide comprises an HDM-2 binding amino acid sequence, and wherein the cell membrane-pore forming peptide further comprises a membrane resident amino acid sequence; forming a cell membrane pore with the cell membrane-pore forming peptide co-localized to the HDM-2 protein on the exterior face of the treatment-resistant cell membrane; and killing the treatment-resistant cancer cell with the plasma membrane pore formed by the cell membrane- pore forming peptide co-localized to the HDM-2 protein on the exterior face of the treatment- resistant cell membrane in the cancer patient, wherein the cell membrane pore allows a movement of molecules across the treatment-resistant cell membrane to kill the treatment- resistant cancer cell.

Testing of PNC-27 Against Chemotherapy-Resistant Cancer Cell Lines

[0058] The inventor has had PNC-27 tested against a variety of chemotherapy-resistant cancer cell lines and the test results are summarized in Table 5. Table 5 indicates the cancer cell lines tested: (1) the PNC-27 IC ₅₀ to kill 50% of the cancer cells in each cell line test sample, (2) the minimum lethal concentration (MLC) of PNC-27 that kills all of the cancer cells in a test sample, and (3) the total time taken to kill all of the cancer cells in the test sample. As can be seen from this table, each line is resistant to a variety of chemotherapeutic agents, and some of these, especially A875 melanoma cells, are resistant to a wide variety of agents. Importantly, PNC-27 kills SW1222 colon cancer cells that contain a high percentage of tumor stem cells.

[0059] Table 5 - Chemotherapy Drug Resistant Cancer Cell Lines Killed by PNC-27

[0060] Figure 1 depicts an example of a PNC-27 test against a chemotherapy-resistant melanoma cancer cell line based on using an MTT assay to quantify the cancer cell killing by the PNC-27. In Figure 1, a line graph plots experimental results from treating an in vitro chemotherapy-resistant A875 human melanoma cells culture (n = 40,000 cells per test well) to

PNC-27 concentrations: 0 uM, 10 uM, 20 uM, 40 uM, 80 uM and 160 uM. Note uM means micromolar concentration. After 24 hours of treatment with PNC-27, no viable cancer cells remained when the cells were treated with PNC-27 concentration of about 40 uM. The killing of the treatment-resistant A875 human melanoma cells by PNC-27 was quantified by the MTT cell viability assay. The MTT assay is a colorimetric assay for assessing cell metabolic activity. NAD(P)H-dependent cellular oxidoreductase enzymes may, under defined conditions, reflect the number of viable cells present. These enzymes are capable of reducing the tetrazolium dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to its insoluble formazan, which has a purple color. MTT, a yellow tetrazole, is reduced to purple formazan in living cells. A solubilization solution (usually either dimethyl sulfoxide, an acidified ethanol solution, or a solution of the detergent sodium dodecyl sulfate in diluted hydrochloric acid) is added to dissolve the insoluble purple formazan product into a colored solution. The absorbance of this colored solution can be quantified by measuring at a certain wavelength (usually between 500 and 600 nm) by a spectrophotometer. In this case, the viability of the A875 cells was measured using optical density measurements at 575 nanometers wavelength from a 24 hour MTT cell proliferation test.

[0061] Figure 2 depicts another example of a PNC-27 test against a chemotherapy-resistant ovarian carcinoma cancer cell line based on using an LDH (lactate dehydrogenase) assay to quantify the cancer cell killing due to the PNC-27. The large number of large pores that PNC- 27 creates in a cancer cell plasma membrane leads to a rapid LDH release from the cell's cytoplasm into the cell culture medium. Figure 2 is a dose-response bar graph based on a 24 hour cell LDH enzyme release response tested on chemotherapy-resistant SKOV-3 Human ovarian carcinoma cells at PNC-27 and PNC-29 concentrations of 0, 20, 40, 60, 80, and 120 micromolar (uM). PNC-29 is an inactive peptide which serves as a control and it can be seen that PNC-29 has no concentration dependent effect on LDH release by SKOV-3 cells.

[0062] The method of practicing the invention may further comprise measuring a level of the substance that has a cell cytoplasmic origin in a mammal. The inventor found that LDH

(lactate dehydrogenase enzyme) is released from the cytoplasm cancer cells that are made porous by exposure to the cell membrane pore forming peptide of the present invention. A substance leaking from the cancer cell pores can become elevated in a blood sample or urine sample or in another type of biological sample from the mammal provided that: (1) the substance can freely diffuse without significant binding or degradation of the substance; and (2) the substance has a higher concentration inside the cell cytoplasm than outside the cell. The level of a substance known as LDH has been conveniently quantified the method of practicing the invention, a mammal may be for example an intracellular enzyme. The level of the substance such as LDH may be measured for example in a blood, or urine sample taken from the mammal or in a cancer biopsy sample or other tissue sample. The change in the level of the substance such as LDH can be used to infer when the cell membrane pore forming peptide has formed pores in cancer cells. Conceivably, the lack of an increase in the level of the substance such as LDH might be used to infer that the mammal no longer harbors treatment resistant cancer cells.

[0063] A mammal which may be treated by the present invention in some embodiments by a method which comprises killing cancer treatment resistant cancer cells, wherein the mammal may have a cancer treatment resistant cancer cells and be for example, a human child, a human adolescent, a young human adult, or a human adult, or be a human suspected of having a cancer. Other mammals might be for example a horse, a dog, a cat, a farm animal, or another mammal such as an endangered species which needs a method treatment of a cancer by an embodiment of the present invention.

[0064] Fluorescent microscopy experiments were conducted by applicant using PNC-27, SW 1222 cancer stem cells, a green fluorescent antibody system to PNC-27 and a red fluorescent antibody system to HDM-2 in order to study and locate where PNC-27 and HDM-2 co-localize in SW 1222 cancer stem cells. The experiments included an intracellular marker which binds to nuclear DNA, known as DAPI, which is 4,6-diamidino-2-phenylindole and which binds strongly to A-T (Adenine-Thymidine) nucleotide regions of DNA. Cell plasma membrane damage to the SW 1222 cells was confirmed when the DAPI blue-stained the intracellular space of the SW 1222 cancer stem cells. In addition, the DAPI bound to DNA region could be seen as blue fluorescent nuclear bodies inside the SW 1222 cancer stem cells. In the fluorescent microscopy experiments, the SW 1222 cancer stem cells were first treated with PNC-27 and then incubated with a green-fluorescence-labeled anti-PNC-27 antibody system and then with anti-HDM-2 red-fluorescence-labeled antibody system. The result was a co-localization of the green fluorescence of the PNC-27 antibody and the red fluorescence of the HDM-2 antibody in the extracellular space surrounding clusters of SW 1222 cancer stem cells. This co-localization of the red and green fluorescence in the cell membrane is also revealed by the presence of the combined yellow fluorescence in some parts of the fluorescent micrographs. Note that the words cancer and tumor have the same meaning. [0065] Figure 3 represents a black & white schematic depiction of the cluster of three SW 1222 cancer stem cells from the above fluorescent microscopy experiment. The damaged cell membranes of these three SW 1222 cancer stem cells are depicted by dotted lines 301 in FIG. 3. In FIG 3, the DAPI-stained nuclear bodies are simply depicted as small thin black ellipses 302. In FIG 3, the extracellular space 303 between the dashed perimeter line and the cell membrane surfaces the three SW 1222 cancer stem cells is where the PNC-27 antibody green fluorescence co-localized with the HDM-2 antibody red fluorescence.

[0066] Applicant's discovery that PNC-27 strongly co- localizes with HDM-2 present in the membranes of SW-1222 tumor stem cells implies to applicant that PNC-27 can destroy tumors by killing their progenitor cells (the so-called tumor stem cells). These progenitor cancer cells are also refractory to treatment by currently available methods and therefore continue to "generate" tumor cells. It is contemplated by the inventor that the killing of the tumor stem cells in a cancer patient may cut off a significant source of cancer cells with chemotherapy- resistance, radiation therapy resistance, immunotherapy-resistance and targeted cancer therapy resistance. Finding that PNC-27 can kill cancer cells that are resistant to chemotherapy drugs, immunotherapy drugs, targeted cancer therapies, and radiation therapies is new information. In addition, it is also particularly advantageous that PNC-27 and its related peptides can be used to kills cancer cells that are resistant to chemotherapy drugs, targeted cancer therapies, immunotherapy drugs, and radiation therapies without inducing potentially fatal harmful side- effects such as bone-marrow suppression, immune-compromised states, bleeding, anemias and pancytopenias. Furthermore, finding that PNC-27 kills cancer cells that are resistant to chemotherapy, immunotherapy, targeted cancer therapies, and radiation therapies makes PNC- 27 and related peptides promising candidates for treating refractory and advanced tumors in patients. Refractory and advanced tumors are considered unbeatable and are the reason why many patients with "untreatable" cancers are referred to hospice centers from hospital medical centers.

[0067] Some embodiments of the presen t invention comprise methods of using the pore formed by the cell membrane pore-forming peptide in the cell membrane of the cancer treatment resistant cancer cell as a pathway for diffusing a substance between the extracellular and the intracellular spaces of the cancer treatment resistant cancer cell. Also these method embodiments of the present invention further comprise using the substance diffusing between the extracellular and intracellular spaces of the cancer treatment resistant cancer cell for killing the cancer treatment resistant cancer cell in the mammal. Regarding these method embodiments of the present invention, it is contemplated that free calcium ions are one example of a substance capable of diffusing between the extracellular and intracellular spaces of the cancer treatment resistant cancer cell. Calcium ions are also a substance capable of killing cancer treatment resistant cancer cells. Mammalian cells live with a relatively high extracellular free calcium ion concentration of between 1-3 millimolar, and yet at the same time must have a low intracellular free calcium ion concentration of between 10-100 nanomolar. Thus, the cell membrane of mammalian cells works to maintain an extracellular [Ca++] : intracellular [Ca++] chemical ion gradient of about 10,000-fold. The direction of this free calcium ion chemical gradient favors a net calcium ion diffusion from the extracellular space to the intracellular space. The present invention comprises methods using cell membrane pore-forming peptides for creating a plurality of large membrane pores as shown in Do et al, 2003 and Bowne et al, 2008. Applicant contemplates that using cell membrane pore-forming peptides for creating a plurality of large membrane pores is a method for enabling a significant net flux of a substance such as calcium ions by diffusion from the extracellular space to the intracellular space.

Calcium ions in a sufficient concentration bean function as a substance for killing the treatment resistant cancer cells. A micromolar free calcium ion concentration in the cell cytoplasm is known to alter or to inhibit various normal functions of many biochemical pathways in the cytoplasm as well as in organelles such as the mitochondria. Higher calcium ion concentrations of about 10 micromolar are known to activate lethal cytoplasmic proteases. In addition the integrity of the membrane cytoskeleton filaments is calcium ion sensitive. Thus, applicant envisions that treatment resistant cancer cells can be killed by a method of using a membrane pore-forming peptide for forming a pore in a cancer cell membrane and permitting a diffusion of a substance which comprises free calcium ions from the extracellular space to the intracellular space of the cancer cell. These calcium ions can activate cytoplasmic proteases when the cancer cell intracellular free calcium ion concentration becomes elevated to 10 micromolar. The calcium ion activated cytoplasmic proteases then kill the cancer cell. Calcium ions are but one example of one substance which can be used for killing the treatment resistant cancer cells in accordance with some embodiment methods of the present invention.

[0068] Applicant contemplates that many other intracellular substances including potassium ions, ATP, ADP, creatine phosphate, AMP, inorganic phosphate, and numerous intracellular metabolites can diffuse across the cell membrane pore formed by the cell membrane pore forming peptide. The pores are large size as shown by the loss of large size enzymes such as LDH (see FIG 2). Applicant contemplates that the loss of essential intracellular enzymes and many other essential intracellular substances can be a means for killing a treatment resistant cancer cell. In addition, Applicant contemplates many other extracellular substances including sodium ions, and protons, and tumor cell microenvironment toxins including various free radicals can diffuse across the cell membrane pore formed by the cell membrane pore forming peptide. Applicant contemplates that the influx of sodium ions, and protons, and tumor cell microenvironment toxins including various free radicals into a cancer cell can be a means for killing a treatment resistant cancer cell. Furthermore, when a treatment resistant cancer cell has substantially lost the integrity of its cell membrane due to a plurality of cell membrane pore forming peptides, there can no longer be energy conservation by the cell as major ATP- consuming membrane enzymes such as the Na-K ATPase consume ATP in an unregulated manner.

[0069] The following prior art publications are incorporated by reference in their entirety.

1. Kanovsky, M., Raffo, A., Drew, L., Rosal, R., Do, T., Friedman, F.K., Rubinstein, P., Visser, I., Robinson, R., Brandt-Rauf, P.W., Michl, J., Fine, R.L. and Pincus, M.R. (2001) entitled "Peptides from the Amino Terminal mdm-2 Binding Domain of p53, Designed from Conformational Analysis, Are Selectively Cytotoxic to Transformed Cells." Proc. Natl. Acad. Sci. USA 98, 12438-12443.

2. Michl, J., Scharf, B., Schmidt, A., Hannan, R., von Gizycki, H., Friedman, F.K., Brandt- Rauf, P.W., Fine, R.L. and Pincus, M.R. (2006) entitled "PNC-28, a p53 Peptide that Is Cytotoxic To Cancer Cells, Blocks Pancreatic Cancer Cell Growth in vivo." Int. J. Cancer, 119, 1577-1585.

3. Do, T.N., Rosal, R.V., Drew, L., Raffo, A.J., Michl, J., Pincus, M.R., Friedman, F.K., Petrylak, D.P., Cassai, N., Szmulewicz, J., Sidhu, G., Fine, R.L. and Brandt-Rauf, P.W. (2003) entitled "Preferential Induction of Necrosis in Human Breast Cancer Cells by a p53 Peptide Derived from the mdm-2 Binding Site." Oncogene 22, 1431-1444.

4. Bowne, W.B., Sookraj, K.A., Vishnevetsky, M., Adler, V., Yadzi, E., Lou, S., Koenke, J., Shteyler, V., Ikram, K., Harding, M., Bluth, M.H., Ng, M., Brandt-Rauf, P.W., Hannan, R., Bradhu. S., Zenilman, M., Michl, J. and Pincus, M.R. (2008) entitled "The Penetratin Sequence in the Anti-Cancer PNC-28 Peptide Causes Tumor Necrosis Rather than Apoptosis of Human Pancreatic Cancer Cells." Ann. Surg. Oncol.15, 3588-3600. 5. Rosal, R. Pincus, M.R., Brandt-Rauf, P.W., Fine, R.L., Michl, J. and Wang, H. (2004) entitled "NMR Solution Structure of a Peptide from the mdm-2 Binding Domain of the p53 Protein that is Selectively Cytotoxic to Cancer Cells." Biochemistry, 43, 1754-1861.

6. Sarafraz-Yazdi, E., Bowne, W.B., Adler, V., Sookra, K.A., Wud, V., Shteyler, V.,Patel,H., Oxbury, W. Brandt-Rauf, P.W., Zenilman, M.E.,Michl, J. and Pincus,M.R. (2010) entitled "Anticancer Peptide PNC-27 Adopts an HDM-2-Binding Conformation and Kills Cancer Cells by Binding to HDM-2 in their Membranes." Proc.Natl Acad. Sci. USA 107, 1918-1923.

7. Beaufort CM et al (2014) entitled "Ovarian Cancer Cell Line Panel (OCCP): Clinical importance of in vitro morphological subtypes." PLOS 19, 1-16 el03988.

8. Sookraj KA, Bowne WB, Adler V, Sarafraz-Yazdi E, Michl J and Pincus MR (2009) The anti-cancer peptide, PNC-27, induces tumor cell lysis as the intact peptide. Cancer Chemother Pharmacol on-line, February 25, 1166-1 177 DO! 10.1007/s00280-009-l 166-7.

[0070] Table 6: Amino Acid Names, Three and One-Letter Abbreviations

[0071] In a first aspect of the invention, there is provided a peptide comprising at least about six contiguous amino acids of the following amino acid sequence: PPLSQETFSDLWKLL (SEQ ID NO:1), wherein the peptide comprising at least about six contiguous amino acids is fused to a leader sequence. Preferably, the peptide comprises from at least about eight (8) to at least about fifteen (15) amino acid residues. In a preferred embodiment, a peptide comprising from at least about eight (8) to at least about 15 (fifteen) amino acids of the sequence set forth in SEQ ID NO:1 has the following amino acid sequence: PPLSQETFSDLWKLL (SEQ ID NO:1). In another preferred embodiment, a peptide comprising from at least about eight (8) to at least about 15 (fifteen) amino acids of the sequence set forth in SEQ ID NO:1 has the following amino acid sequence: PPLSQETFS (SEQ ID NO:2). In still another preferred embodiment, a peptide comprising from at least about eight (8) to at least about fifteen (15) amino acids of the sequence set forth in SEQ ID NO:1 has the following amino acid sequence: ETFSDLWKLL (SEQ ID NO:3).

[0072] Leader sequences (also known as MRP) which function to import the peptides of the invention into a cell may be derived from a variety of sources. An Example of an MRP is the MRP used in PNC-27, PNC-28, and PNC-26 which is KKWKMRRNQFWVKVQRG which is the transmembrane-penetrating peptide sequence from antennapedia, also known as penetratin. Control peptide PNC-29 contains the peptide sequence MPFSTGKRIMLGE (known as X13) which is an unrelated peptide from cytochrome P450) with its carboxyl terminal end attached to penetratin. Preferably, the leader sequence comprises predominantly positively charged amino acid residues since a positively charged leader sequence stabilizes the alpha helix of a subject peptide. Examples of leader sequences which may be linked to the peptides of the present invention are described in Futaki, S. et al (2001) Arginine-Rich Peptides, J. Biol. Chem.

276,:5836-5840, and include but are not limited to the following membrane-penetrating leader sequences (numbering of the amino acid residues making up the leader sequence of the protein is indicated parenthetically immediately after the name of the protein in many cases):

[0073] Other membrane penetrating leader sequences may also be used.

[0074] Preferably, the positively charged leader sequence of the penetratin leader sequence of antennapedia protein is used. This leader sequence has the following amino acid sequence: KKWKMRRNQFWVKVQRG (SEQ ID NO:4). Preferably, the leader sequence is attached to the carboxyl terminal end of the p53 peptide to enable the synthetic peptide to kill transformed and malignant cells.

[0075] Structurally related amino acid sequences may be substituted for the disclosed sequences set forth in S EQ ID NOs: 1, 2, 3, or 4 in practicing the present invention. Any of the sequences set forth in SEQ ID NOs: 1, 2 or 3, including analogues or derivatives thereof, when joined with a leader sequence, including, but not limited to the sequence set forth in SEQ ID

NO: 4, will be referred to herein as either a "synthetic peptide" or "synthetic peptides." Rigid molecules that mimic the three-dimensional structure of these synthetic peptides are called peptidomimetics and are also included within the scope of this invention. Alpha helix stabilizing amino acid residues at either or both the amino or carboxyl terminal ends of the p53 peptide may be added to stabilize the alpha helical conformation which is known to be the conformation of this region of the p53 protein when it binds to the MDM-2 protein. Examples of alpha helical stabilizing amino acids include Leu, Glu (especially on the amino terminal of the helix), Met and Phe.

[0076] Amino acid insertional derivatives of the peptides of the present invention include amino and/or carboxyl terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in a subject peptide although random insertion is also possible with suitable screening of the resulting product. Deletional variants may be made by removing one or more amino acids from the sequence of a subject peptide. Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. For the present invention, amino acid substitutions can be made in accordance with the following table.

[0077] TABLE 7 = Substitutional Amino Acid Variants For Peptides of the Invention

[0078] When the synthetic peptide is derivatised by amino acid substitution, the amino acids are generally replaced by other amino acids having like properties such as Hydrophobicity, hydrophilicity, electronegativety, bulky side chains and the like. As used herein, the terms "derivative", "analogue", "f agment", "portion" and "like molecule" refer to a subject peptide having the amino acid sequence as set forth in SEQ ID NOs:l, 2, 3, or 4, having an amino acid substitution, insertion, addition, or deletion, as long as said derivative, analogue, fragment, portion, or like molecule retains the ability to enter and selectively kill transformed or neoplastic cells.

[0079] Peptide embodiments of the present invention may become inactivated over time perhaps by peptide degradation, extrusion, or inactivating binding to other molecules. Pincus US Patent No. 9,539,327 teaches that peptide-based compounds, including PNC-27 and PNC- 28, may be altered to include a D-amino acid on the amino terminal end in order to slow peptidase activity of the molecule. Similarly, leupeptin, a known peptidase activity inhibitor, may be attached to the carboxyl terminal end of PNC-27 and PNC-28 in order to slow peptidase activity and lengthen the half-life of the molecules. Since it is conceived that some peptide embodiments of the present invention following their administration to a cancer patient may have a half-life in the cancer patient as brief as a few minutes, then in some peptide

embodiments of the present invention, the peptide is optionally modified by a substitution of some 1-amino acids with d-amino acids, or by adding a leupeptin substituent, or adding on another known-in-the-art modification of peptides known to inhibit a protease. Leupeptin is also known as N-acetyl-L-leucyl-L-leucyl-L-argininal. Leupeptin is a protease inhibitor that can inhibit cysteine, serine and threonine peptidases.

[0080] The synthetic peptides of the present invention may be synthesized by a number of known techniques. For example, the peptides may be prepared using the solid-phase technique initially described by Merrifield (1963) in J. Am. Chem. Soc. 85:2149-2154. Other peptide synthesis techniques may be found in M. Bodanszky et al. Peptide Synthesis, John Wiley and Sons, 2d Ed., (1976) and other references readily available to those skilled in the art. A summary of polypeptide synthesis techniques may be found in J. Sturart and J. S. Young, Solid Phase Peptide Synthesis, Pierce Chemical Company, Rockford, 111., (1984). Peptides may also be synthesized by solution methods as described in The Proteins, Vol. Π, 3d Ed., Neurath, H. et al., Eds., pp. 105-237, Academic Press, New York, N.Y. (1976). Appropriate protective groups for use in different peptide syntheses are described in the texts listed above as well as in J. F. W. McOmie, Protective Groups in Organic Chemistry, Plenum Press, New York, N.Y. (1973). The peptides of the present invention may also be prepared by chemical or enzymatic cleavage from larger portions of the p53 protein or from the full length p53 protein. Likewise, leader sequences for use in the synthetic peptides of the present invention may be prepared by chemical or enzymatic cleavage from larger portions or the full length proteins from which such leader sequences are derived, the disclosure of which is incorporated by reference herein as if fully set forth.

[0081] Additionally, the peptides of the present in vention may also be prepared by recombinant DNA techniques. For most amino acids used to build proteins, more than one coding nucleotide triplet (codon) can code for a particular amino acid residue. This property of the genetic code is known as redundancy. Therefore, a number of different nucleotide sequences may code for a particular subject peptide selectively lethal to malignant and transformed mammalian cells. The present invention also contemplates a deoxyribonucleic acid (DNA) molecule that defines a gene coding for, i.e., capable of expressing a subject peptide or a chimeric peptide from which a peptide of the present invention may be enzymatically or chemically cleaved.

[0082] When applied to cells grown in culture, synthetic peptides are selectively lethal to malignant or transformed cells, resulting in dose dependent reduction in cell number. The effect is observable generally within two to three and at most 48 hours. A line of rat pancreatic acinar cells (BMRPA.430) grown in culture was transformed with K-ras. The normal cell line displays the architecture typical of pancreatic acinar cells; the transformed cells (TUC-3) lack the differentiated morphology of acinar cells, appearing as typical pancreatic cancer cells. When

BMRPA.430 cells were treated with a synthetic peptide with the primary structure of SEQ ID

NO: 1 coupled to leader sequence SEQ ID NO:4, at a dosage of 100 ug/mL, the cells were not affected. However, when TUC-3 cells were treated with a peptide with the primary structure of SEQ ID NO:1 coupled to leader sequence SEQ ID NO:4, at a dosage of 100 ug/ml, they died within three to four days. Similar results were obtained when the same experiment was performed but SEQ ID NO:1 was substituted with either SEQ ID NO:2, or SEQ ID NO:3. Additionally, transformed and malignant cell death was observed in human breast carcinoma cell lines and Melanoma and HeLa cells treated with a synthetic peptide with the primary structure of SEQ ID NO:1 coupled to leader sequence SEQ ID NO:4, at a dosage of 100 ug/ml. In contrast, the same synthetic peptide at the same dosage had no effect on non-malignant and non-transformed human breast or fibroblast cell lines.

[0083] When the leader sequence set forth in SEQ ID NO:4 was positioned at the carboxyl terminal end of PNC29, a control protein having the following amino acid sequence:

MPFSTGKRIMLGE (SEQ ID NO: 25), there was no effect on malignant or normal cells.

[0084] Additionally, the peptide having the amino acid sequence as set forth in SEQ ID NO:3 fused at the carboxyl terminal end to the leader peptide set forth in SEQ ID NO:4, has no effect on the ability of human stem cells to differentiate into hematopoietic cell lines in the presence of growth factors. This indicates that this peptide will not be injurious to bone marrow cells when administered as a chemotherapeutic agent. See Kanovsky et al., (Oct. 23, 2001) Proc. Nat. Acad. Sci. USA 98(22); 12438-12443, the disclosure of which is incorporated by reference herein as if fully set forth.

[0085] For additional details on HDM-2 binding component, an HDM-2 binding peptide sequence, a MDM-2 binding component, an MDM-2 binding peptide sequences, a membrane resident sequence, a membrane resident component, a pentratin, a leader sequence, and using a D-amino acid on the amino terminal end to slow peptidase activity to prolong therapeutic half- life, see the disclosures of US Patent Nos. 7,531,515; 7,745,405; 7,883,888; 8,822,419; and 9,539,327 which are incorporated by reference herein as if fully set forth.

[0086] When cultured cancer cells were treated with a peptide with the primary structure of SEQ ID NO:1 without a leader sequence attached, at a dosage of 100 ug ml., the cells were unaffected. Similarly, when cultured cancer cells were treated with leader sequence SEQ ID NO:4, the presently preferred leader sequence, at the same dosage, the cell were also unaffected.

[0087] The synthetic peptides of the present invention may be administered preferably to a human patient as a pharmaceutical composition containing a therapeutically effective dose of at least one synthetic peptide according to the present invention together with a pharmaceutical acceptable carrier. The term "therapeutically effective amount" or "pharmaceutically effective amount" means the dose needed to produce in an indi vidual, suppressed growth including selective killing of neoplastic or malignant cells, i.e., cancer cells.

[0088] Preferably, compositions containing one or more of the synthetic peptides of the present invention are administered intravenously for the purpose of selectively killing neoplastic cells, and therefore, treating neoplastic or malignant disease such as cancer.

Examples of different cancers which may be effectively treated using one or more the peptides of the present invention include but are not limited to: breast cancer, prostate cancer, lung cancer, cervical cancer, colon cancer, melanoma, pancreatic cancer and all solid tissue tumors (epithelial cell tumors) and cancers of the blood including but not limited to lymphomas and leukemias.

[0089] Administration of the synthetic peptides of the present invention may be by oral, intravenous, intranasal, suppository, intra-peritoneal, intramuscular, intradermal or

subcutaneous administration or by infusion or implantation. When administered in such manner, the synthetic peptides of the present invention may be combined with other

ingredients, such as carriers and/or adjuvants. There are no limitations on the nature of the other ingredients, except that they must be pharmaceutically acceptable, efficacious for their intended administration, cannot degrade the activity of the active ingredients of the compositions, and cannot impede importation of a subject peptide into a cell. The peptide compositions may also be impregnated into transdermal patches, or contained in subcutaneous inserts, preferably in a liquid or semi-liquid form which patch or insert time-releases therapeutically effective amounts of one or more of the subject synthetic peptides.

[0090] The pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The ultimate solution form in all cases must be sterile and fluid.

Typical carriers include a solvent or dispersion medium containing, e.g., water buffered aqueous solutions, i.e., biocompatible buffers, ethanol, polyols such as glycerol, propylene glycol, polyethylene glycol, suitable mixtures thereof, surfactants or vegetable oils. Sterilization may be accomplished utilizing any art-recognized technique, including but not limited to filtration or addition of antibacterial or antifungal agents. Examples of such agents include paraben, chlorbutanol, phenol, sorbic acid or thimerosal. Isotonic agents such as sugars or sodium chloride may also be incorporated into the subject compositions.

[0091] As used herein, a "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and the like. The use of such media and agents are well-known in the art.

[0092] Production of sterile injectable solutions containing the subject synthetic peptides is accomplished by incorporating one or more of the subject synthetic peptides described hereinabove in the required amount in the appropriate solvent with one or more of the various ingredients enumerated above, as required, followed by sterilization, preferably filter sterilization. In order to obtain a sterile powder, the above solutions are vacuum-dried or freeze- dried as necessary.

[0093] Inert diluents and/or edible carriers and the like may be part of the pharmaceutical compositions when the peptides are administered orally. The pharmaceutical compositions may be in hard or soft shell gelatin capsules, be compressed into tablets, or may be in an elixir, suspension, syrup or the like.

[0094] The subject synthetic peptides are thus compounded for convenient and effective administration in pharmaceutically effective amounts with a suitable pharmaceutically acceptable carrier in a therapeutically effective dosage. Examples of a pharmaceutically effective amount include peptide concentrations in the range from about at least about 25 ug/ml to at least about 300 ug ml.

[0095] A precise therapeutically effective amount of synthetic peptide to be used in the methods of the invention applied to humans cannot be stated due to variations in stage of neoplastic disease, tumor size and aggressiveness, the presence or extent of metastasis, etc. In addition, an individual's weight, gender, and overall health must be considered and will effect dosage. It can be generally stated, however, that the synthetic peptides of the present invention be administered in an amount of at least about 10 mg per dose, more preferably in an amount up to about 1000 mg per dose. Since the peptide compositions of the present in vention will eventually be cleared from the bloodstream, re-administration of the pharmaceutical compositions is indicated and preferred.

[0096] The synthetic peptides of the present invention may be administered in a manner compatible with the dosage formulation and in such an amount as will be therapeutically effective. Systemic dosages depend on the age, weight, and condition of the patient and the administration route. An exemplary suitable dose for the administration to adult humans ranges from about 0.1 to about 20 mg per kilogram of body weight. Preferably, the dose is from about 0.1 to about 10 mg per kilogram of body weight.

[0097] In accordance with the present invention, there i s also provided a method of treating neoplastic disease. The method comprises administering to a subject in need of such treatment, a therapeutically effective amount of a synthetic peptide hereinbefore described, including analogs and derivatives thereof. Thus for example, in one embodiment, an effective amount of a peptide comprising at least about six contiguous amino acids as set forth in SEQ ID NO:1 or an analog or derivative thereof fused on its carboxyl terminal end to a leader sequence may be administered to a subject. In another embodiment, an effective amount of a peptide comprising at least from about eight (8) to at least about ten (10) contiguous amino acids as set forth in SEQ ID NO:1 or an analog or derivative thereof, fused on its carboxyl terminal end to a leader sequence, may be administered to a subject. For example, an effective amount of a peptide having the amino acid sequence as set forth in SEQ ID NO:1 or an analog or derivative thereof, fused on its carboxyl terminal end to a leader sequence may be administered to a subject. An effective amount of a peptide having the amino acid sequence as set forth in SEQ ID NO:2 or an analog or derivative thereof, fused on its carboxyl terminal end to a leader sequence may also be administered to a subject. In still another embodiment, an effective amount of a peptide having the amino acid sequence set forth in SEQ ID NO: 3 or an analog or derivative thereof, fused on its carboxyl terminal end to a leader sequence may be administered to a subject. In accordance with a method of treatment, a mixture of synthetic peptides may be administered. Thus, for example, in addition to administering one of the peptides, or analogs or derivatives thereof hereinbefore described in an effective amount, mixtures of two or more peptides or analogs or derivatives hereinbefore described may be administered to a subject.

[0098] The foregoing specification, and the experimental results reported therein are illustrative and are not limitations of the scope of applicant's invention. Those skilled in the art will appreciate that various modifications can be made without departing from applicant's invention. Where a term is provided in the singular, the inventors also contemplate aspects of the invention described by the plural of that term. As used in this specification and in the appended claims, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise, e.g., "a tip" includes a plurality of tips. Thus, for example, a reference to "a method" includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, constructs and materials are now described. All publications mentioned herein are incorporated herein by reference in their entirety. Where there are discrepancies in terms and definitions used in references that are incorporated by reference, the terms used in this application shall have the definitions given herein.

[0099] It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the appended claims.