NZ711682B2 - Kits and methods for the treatment of cancer using gliadin peptides - Google Patents

Abstract

Kits and methods for treating cancer comprising administration of a gliadin peptide to a patient are disclosed herein. A kit according to the invention comprises a pharmaceutical composition comprising a gliadin peptide and instructions for administering the peptide to a patient. The kit may further comprise a pharmaceutical composition comprising at least one chemotherapeutic agent such as a receptor tyrosine kinase inhibitor and instructions for co¬ administering the compounds. A method of treating cancer according to the invention comprises administering a gliadin peptide to a patient and may further comprise co-administering at least one chemotherapeutic agent such as a receptor tyrosine kinase inhibitor. Co-administration of a gliadin peptide and receptor tyrosine kinase inhibitor to a patient with cancer is effective to decrease or prevent resistance of the cancer to the receptor tyrosine kinase inhibitor. The gliadin peptide is an alpha-gliadin peptide p31-43 with the sequence LGQQQPFPPQQPY and is to be used for treating non-small cell lung cancer (NSCLC) and pancreatic cancer.

Description

KITS AND METHODS FOR THE TREATMENT OF CANCER USING GLIADIN PEPTIDES This application contains, as a te part of the disclosure, a sequence listing in computer-readable form (Filename: 47321A_SeqListing.txt; Size: 795 bytes; Created: March 4, 2014) which is incorporated by nce in its ty.

REFERENCE TO RELATED APPLICATION This ation claims the benefit of priority under 35 U.S.C. §ll9(e) of US.

Provisional Patent Application Serial No. 61/774,285 filed March 7, 2013, which is incorporated herein by reference.

FIELD OF THE INVENTION The invention is directed to kits for treating cancer comprising a gliadin peptide and methods for treating cancer comprising administration of a gliadin peptide to a patient. The gliadin peptide may be co—administered with at least one chemotherapeutic agent such as a receptor tyrosine kinase inhibitor to increase the ncer effect of the chemotherapeutic agent(s).

BACKGROUND OF THE INVENTION With millions of people world—wide dying from cancer each year, there is an ever present need for improved therapeutic options. A host of chemotherapeutic agents have been ped in an effort to combat the various forms of the disease. Examples of classes of herapeutic agents include alkylating agents, antibiotics, antimetabolites, entiating agents, mitotic inhibitors, steroids, topoisomerase inhibitors, and tyrosine kinase inhibitors (TKIs). TKIs block the phosphorylation of proteins to inhibit activation of signal transduction pathways that t tumor development and progression. Receptor tyrosine kinase inhibitors (RTKIs) are TKIs that ically target the activity of receptor tyrosine kinase (RTK) proteins such as epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), platelet—derived growth factor receptor (PDGFR), and vascular endothelial growth factor receptor (VEGFR). However, the effectiveness of RTKIs and other herapy drugs is often hindered by the intrinsic or acquired resistance of cancer cells to anticancer agents.

One contributing factor to the resistance of tumors to chemotherapy is the presence of cancer stem cells (CSCs) within cancer cell populations. CSCs are undifferentiated cells that constitute a small subset (typically less than 10%) of cancer cells. CSCs are so named because they possess some of the characteristics of embryonic stem cells and can differentiate into a variety of cancer cell types. CSCs are therefore tumorigenic and can lead to cancer relapse and metastasis. Many chemotherapeutic drugs kill differentiated cancer cells, but fail to ively ate CSCs, allowing those cells to proliferate and the cancer to persist, resulting in the overall resistance of the tumor to eradication.

Two RTKIs approved for use in treating cancer are erlotinib (Tarceva®, OSI Pharmaceuticals) and gefitinib a®, AstraZeneca). Both drugs target and inhibit EGFR.

Activation of EGFR following the g of epidermal growth factor (EGF) or another ligand to the receptor results in the ATP—driven phosphorylation of tyrosine residues located in the intracellular domain of the receptor. The phosphorylated tyrosines then interact with other intracellular proteins and activate signal uction pathways to promote cell survival and proliferation. Increased activation of EGFR is associated with a y of cancer types, especially tumors derived from epithelial cells. The increase in receptor activity can result from mutations in the kinase domain of EGFR, amplification of EGFR gene expression, or pression of the EGFR n (Yauch et al., Clinical Cancer Research. 2005;11(24):8686— 98). Erlotinib and nib, as well as other RTKIs, interfere with the ATP—binding domain of RTKs to suppress receptor activation and block downstream signal transduction.

Erlotinib and nib were the first RTKIs approved for use in treating non—small cell lung cancer (NSCLC). Lung cancer is the g cause of cancer deaths worldwide, and about 85—90% of lung cancer patients have NSCLC (Gottschling et al. Lung Cancer. 2012; 77(1):183— 91). The effectiveness of erlotinib and gefitinib in treating NSCLC has been limited, with most patients continuing to eXhibit disease progression ing tion of therapy (Witta et al., Cancer Research. 2006; 66(2):944—950). Patients with certain EGFR mutations have been found to respond better to treatment with RTKIs than those with wild—type EGFR. Approximately 70— 80% of NSCLC patients with EGFR mutations are sensitive to RTKI therapy, however, virtually all patients eventually acquire resistance (Suda et al. Journal of Thoracic Oncology. 2011; 6(7):1152—61). Additionally, the prevalence of the mutations is relatively rare, occurring in less than 20% of patients (Yauch 2005, supra). Overall, only about 10% of ian NSCLC patients exhibit significant changes in e progression following therapy with erlotinib or gefitinib (Gottschling 2012, , coring the need for improved therapeutic methods.

Gliadin is a n found in wheat and d grains and is one of the main components of gluten. The four main types of gliadin are alpha, beta, gamma, and omega.

Gliadin can be digested into a number of active peptides, including some that trigger T—cell immunity or cytotoxicity. Gliadin has been extensively studied for its role in celiac disease, a chronic inflammatory ion related to dietary gluten, but has not been disclosed or suggested for use as an anticancer agent, either alone or in combination with conventional chemotherapeutic agents. In fact, treatment of various cell types, including cancer cells, with gliadin peptides has been trated to activate the EGFR pathway and induce cell proliferation e et al. Gut. 2007;56(4):480—488), which strongly suggests that gliadin administration is contraindicated for the treatment of cancer.

SUMMARY OF THE INVENTION The ion provides a kit comprising a ceutical composition comprising a gliadin peptide and instructions for administration of a therapeutically effective amount of the peptide to a patient with cancer. A kit according to the invention may further comprise a pharmaceutical ition comprising at least one chemotherapeutic agent and instructions for co—administering therapeutically effective amounts of the gliadin peptide and chemotherapeutic agent(s) to a patient with cancer.

The invention also provides a method of treating cancer comprising administering a therapeutically effective amount of a gliadin peptide to a patient with cancer. A method according to the invention may further comprise co—administering a therapeutically ive amount of at least one chemotherapeutic agent to a patient with cancer.

The gliadin peptide according to the invention may be an alpha—gliadin peptide.

Examples of suitable alpha—gliadin peptides include at least alpha—gliadin peptide p31—43, e.g., alpha—gliadin peptide p31—43, gliadin peptide p31—49 and gliadin peptide p31—55. In one aspect, at least one chemotherapeutic agent according to the invention is a RTKI. A RTKI according the invention may be an EGFR inhibitor. Examples of suitable EGFR inhibitors include gefitinib and erlotinib. In one embodiment, co—administration of a gliadin peptide and RTKI to a patient with cancer is effective to decrease or prevent resistance of the cancer to the RTKI.

DETAILED DESCRIPTION OF THE ION The invention provides kits and methods for treating cancer comprising administering a gliadin peptide to a patient. A kit according to the invention comprises a pharmaceutical composition comprising a gliadin peptide and may further se instructions for stration of a therapeutically effective amount of the peptide to a patient with cancer. The kit may further comprise a pharmaceutical composition comprising at least one herapeutic agent such as a RTKI and instructions for co—administering therapeutically effective amounts of the gliadin e and chemotherapeutic agent(s) to a t with cancer.

A method of ng cancer ing to the invention ses administering a therapeutically effective amount of a gliadin peptide to a patient with cancer. The method may further comprise co—administering a therapeutically effective amount of at least one chemotherapeutic agent such as a RTKI to a patient with cancer.

The gliadin peptide may be an alpha, beta, gamma, or omega n peptide. In one aspect, the gliadin peptide according to the invention is an alpha—gliadin peptide or a tive or fragment thereof. Examples of suitable alpha—gliadin peptides include at least alpha—gliadin peptide p3l—43, e.g., alpha—gliadin peptide p3l—55, alpha—gliadin peptide p3l—49, and alpha— gliadin peptide p3l—43. In a preferred embodiment, the alpha—gliadin peptide is alpha—gliadin peptide p3l—43 or a derivative or fragment thereof. gliadin peptide p3l—43 has the amino acid sequence LGQQQPFPPQQPY (SEQ ID NO: 1) and is often referred to as the "toxic" n peptide because it induces an innate inflammatory immune response and results in intestinal . Treatment of cancer cells with alpha—gliadin peptide p3l—43 induces cell proliferation (Barone et al. PLOS ONE. 2010; 5(8):e12246), so the peptide has not been reported for use in treating , much less included in pharmaceutical compositions or kits for treating tumors.

A gliadin peptide according to the invention may be obtained following enzymatic digestion of a gliadin protein or can be chemically synthesized using conventional s known in the art.

A pharmaceutical composition comprising a gliadin peptide comprises the peptide in combination with a pharmaceutically able carrier, diluent, and/or excipient(s). Routes of administration suitable for administering a pharmaceutical composition comprising gliadin to a patient include, but are not limited to, oral, intramuscular, intravenous, respiratory/inhalation, and subcutaneous.

According to the invention, a gliadin peptide can be co—administered with at least one chemotherapeutic agent to a patient with cancer. es of herapeutic agents that may be co—administered with a n peptide include, but are not limited to, alkylating agents, antibiotics, antimetabolites, differentiating , mitotic inhibitors, steroids, topoisomerase inhibitors, TKIs (such as RTKIs), and combinations thereof. In one , a gliadin peptide can be co—administered with at least one RTKI. Examples of RTKIs include, but are not limited to, afatinib, axitinib, canertinib, cediranib, erlotinib, gefitinib, grandinin, imatinib, lapatinib, leflunomide, lestaurtinib, neratinib, pazopanib, quizartinib, regorafenib, semaxanib, sorafenib, sunitib, sutent, tivozanib, tocerabib, vandetanib, vatalanib, monoclonal antibodies that bind specific RTKs, and combinations f. A preferred RTKI according to the invention is an inhibitor of EGFR. Examples of EGFR inhibitors include, but are not d to, gefitinib and erlotinib. In one embodiment, co—administration of a gliadin peptide and RTKI to a patient with cancer is ive to decrease or prevent resistance of the cancer to the RTKI.

The kits and s disclosed herein can be used to treat a human patient with cancer or any other mammal. The invention is useful for treating many types of cancer including bladder cancer, breast , colon cancer, endometrial , kidney cancer, leukemia, lung cancer, lymphoma, pancreatic cancer, prostate cancer, skin cancer, and thyroid cancer. The invention is particularly ive for treating cancers Whose progression is dependent on changes in ellular adhesion. In one , the invention is used to treat lung , including NSCLC.

As used herein, the following definitions may be useful in aiding the skilled practitioner in understanding the invention: The term "therapeutically effective" depends on a patient’s ion and the specific compound administered. The term refers to an amount effective to achieve a desired clinical effect. In some embodiments, a therapeutically effective amount is an amount effective to inhibit growth of cancer cells, prevent metastasis, or result in cell death. Therapeutically effective WO 38556 s of known herapeutic agents are known in the art. For example, a therapeutically effective amount of a RTKI is generally about 5 mg/kg/day to about 150 mg/kg/day, about 10 mg/kg/day to about 100 mg/kg/day, and/or about 25 mg/kg/day to about 75 mg/kg/day, depending on the drug. For a gliadin peptide, a therapeutically effective amount is generally a dosage necessary to achieve a plasma concentration of about 5 ug/mL to about 200 ug/mL, about ug/mL to about 100 ug/mL, and/or about 15 ug/mL to about 50 ug/mL, for example, about ug/mL. Dosages and the ncy of administration for use according to the present disclosure may vary according to such factors as the route of administration, the nature and ty of the disease to be treated, and the size and general condition of the patient.

Appropriate dosages can be determined by procedures known in the pertinent art, e.g., clinical trials that may involve dose escalation studies and protocols described herein. Generally, a clinician titers the dosage and modifies the route of administration to obtain the optimal therapeutic effect. Purely by way of illustration, the dosage of a gliadin peptide needed to achieve a therapeutically effective amount ranges from about 100 ug/kg/day to about 100 mg/kg/day, about 200 ug/kg/day to about 75 mg/kg/day, about 500 ug/kg/day to about 50 mg/kg/day, about 750 ug /kg/day to about 25 mg/kg/day, and/or about 1 mg/kg/day to about 15 mg/kg/day, depending on the factors mentioned above. Some conditions require prolonged treatment, which may or may not entail administering lower doses over multiple administrations.

If desired, a dose is administered as two, three, four, five, six or more sub—doses administered separately at riate als throughout the day. The treatment period will depend on the particular condition and may last one day to l days, weeks, months, or years.

The term "monotherapy" means that a n peptide is stered in a manner such that its pharmacological effects on cancer cells and tumors do not overlap with the pharmacological effects of a chemotherapeutic agent. During monotherapy, a gliadin peptide is necessarily administered alone. Gliadin peptide monotherapy may occur before, after, or both before and after, treatment using a chemotherapeutic agent, so long as the chemotherapeutic agent is no longer therapeutically effective at the time the gliadin e is administered.

The terms "co—administering" and "combination y" mean that a gliadin peptide and at least one herapeutic agent are administered in a manner that permits all the compounds to exert pharmacological effects during an overlapping period of time. The gliadin peptide and chemotherapeutic agent(s) may be administered in the same pharmaceutical composition or in separate compositions, and via the same or different routes of administration.

The gliadin peptide and chemotherapeutic agent(s) may be co—administered at the same time or at different times as long as both compounds exert pharmacological effects during an overlapping period of time. For example, the compounds may both be stered to a patient within a time period of about 2, 4, 6, 8, 12, 24, or 48 hours. Either the gliadin peptide or the chemotherapeutic agent(s) may be administered first. As long as subsequent compounds are administered while a therapeutically effective concentration of the first compound is present, the gliadin peptide and the chemotherapeutic agent(s) are considered to be inistered in ance with the teachings of the invention.

The term "chemotherapeutic agent" means any compound that is toxic with respect to cancer cells. A herapeutic agent can be a small molecule, n, polypeptide, peptide, c acid, and combinations thereof. Exemplary classes of chemotherapeutic agents are provided above. Specific examples of chemotherapeutic agents include, but are not d to, azacitidine, axathioprine, bevacizumab, bleomycin, capecitabine, carboplatin, chlorabucil, cisplatin, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, etoposide, fluorouracil, gemcitabine, herceptin, idarubicin, mechlorethamine, lan, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, tafluposide, teniposide, tioguanine, retinoic acid, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, the specific exemplary RTKIs listed above, and combinations thereof. Additional examples of chemotherapeutic agents are known in the art.

The term "receptor tyrosine kinase inhibitor" or "RTKI" means any nd capable of inhibiting the activity of a member of the receptor tyrosine kinase (RTK) family of proteins.

A RTKI can be a small molecule, protein, polypeptide, peptide, nucleic acid, and combinations thereof. Examples of protein targets for RTKIs include, but are not limited to, members of the following RTK families: ephrin or, epidermal growth factor receptor, fibroblast growth factor receptor, insulin receptor, insulin—like growth factor receptor, phin ors, platelet—derived growth factor or, and vascular elial growth factor receptor. Specific exemplary RTKIs are listed above.

The term "derivative or fragment" means a peptide having a structure and biological ty similar to a gliadin peptide. A derivative or fragment shares at least 70%, 80%, or 90% amino acid sequence homology with a gliadin peptide. In various embodiments, a derivative or fragment shares at least 70%, 80%, or 90% amino acid ce homology with alpha—gliadin peptide p3l—55, alpha—gliadin peptide p3l—49, or alpha—gliadin peptide . A derivative or fragment may be a chemically modified gliadin peptide. For example, a tive or fragment can be a gliadin peptide chemically modified to e the stability, membrane penetration, or immunogenicity of the peptide. Examples of chemical modifications that can be used to form derivatives and fragments of n peptides include, but are not limited to, polymer conjugation, lipidization, use of amino acid analogs, ylation, and cationization. In one aspect, a suitable derivative or fragment of a gliadin peptide contains at least the amino acid sequence PPQQPY (SEQ ID NO:2).

Advantageously, co—administering a gliadin peptide and at least one herapeutic agent such as a RTKI to a patient with cancer is effective to se or prevent resistance of the cancer cells to the chemotherapeutic agent(s). The co—administration of a gliadin peptide and at least one herapeutic agent, therefore, increases and prolongs the efficacy of the chemotherapeutic agent(s). While not intending to be bound by a single theory, it is believed that the anticancer effect achieved from administering a gliadin peptide alone or in combination with at least one chemotherapeutic agent according to the invention can be attributed to the impact of the gliadin peptide on the intracellular transport of cargo.

Proteins and other molecules are trafficked within the cytosolic compartment of cells in es known as endosomes as part of the endocytic pathway. Early endosomes are vesicles that receive les internalized from the plasma membrane. Early endosomes mature into late endosomes, also known as esicular bodies (MVBs). The late endosomes/MVBs eventually fuse with lysosomes, resulting in the enzymatic degradation of the internalized cargo.

As an alternative to lysosomal ation, some les are sorted into recycling endosomes and trafficked back to the plasma membrane or to other intracellular sites. n peptides, particularly alpha—gliadin peptide p3l—43, have been shown to interfere with the endocytic pathway by delaying maturation of early endosomes to late endosomes (Barone 2010, supra).

Thus, gliadin peptides interfere with the degradation of ns, chemotherapeutic agents, and other molecules cked within endosomes in the cytosolic tment. By preventing the degradation of ellular molecules, gliadin peptides can enhance the anticancer activity of chemotherapeutic agents.

The endosomal sorting complex required for transport (ESCRT) protein complexes (ESCRT—0, l, ESCRT—2, and 3) te the sorting of cargo into MVBs for eventual degradation within lysosomes. Hepatocyte growth factor—regulated tyrosine kinase substrate (HRS), a component of ESCRT—0, regulates the trafficking of molecules in early and late endosomes (Henne et al. Developmental Cell. 2011; 77—91; Barone 2010, supra).

Amino acids 719—731 of HRS, located at the carboxyl terminus, contain the binding domains necessary for the localization of HRS to endosomal membranes (Barone 2010, supra). The sequence of HRS amino acids 719—731 (PSQDASLPPQQPY; SEQ ID NO:3) is very similar to the alpha—gliadin peptide p31—43 peptide. Out of 13 es, seven are identical, including six contiguous amino acids (PPQQPY; SEQ ID NO:2), and two are similar between the sequences, with the only significant difference being an N—terminal leucine in alpha—gliadin e p31—43 compared to the e in HRS 719—731 (Barone 2010, supra). Alpha—gliadin peptides comprising at least alpha—gliadin p31—43, e.g., alpha—gliadin peptide , alpha—gliadin peptide p31—49 and alpha—gliadin peptide p31—55, can therefore compete with HRS binding, thereby interfering with HRS localization within endosomal membranes (Barone 2010, supra).

As a result, after cells are treated with alpha—gliadin peptide p31—43, the amount of HRS in the cytosol is increased while ne—associated HRS is decreased (Barone 2010, supra). The decreased presence of HRS in the endosomal membranes disrupts the normal trafficking of cargo within the cell, leading to impaired degradation of intracellular molecules. By promoting the retention of cargo, including toxic molecules, that would otherwise ordinarily be d from the cell, administration of gliadin peptides to a patient is able to decrease cell viability, which supports the use of the peptides for the treatment of cancer.

In addition to their potential for use in treating cancer without additional therapeutic agents, gliadin peptides also show promise for use in combination with one or more chemotherapeutic agents because the impact of the peptides on the endocytic pathway can affect the cking of chemotherapeutic agents and their targets. After out—competing HRS for g sites associated with endosomal membranes, alpha—gliadin peptides comprising at least alpha—gliadin p31—43, e.g., alpha—gliadin peptide p31—43, alpha—gliadin e p31—49 and alpha— gliadin peptide p31—55, localize to endosomes. Vesicles carrying alpha—gliadin peptides comprising at least alpha—gliadin peptide p31—43 move more slowly than normal vesicles (Barone 2010, supra). The delay of ellular transport d by gliadin peptide p31—43 is not influenced by the cargo within the vesicles, so all molecules trafficked intracellularly, including chemotherapeutic agents, are potentially affected (Barone 2010, supra). The prolonged lic transit of cargo can extend the time in which a chemotherapeutic agent is able to accumulate, leading to higher intracellular concentrations of drug and increased cytotoxicity. The extended presence of the drug within the cell also allows the drug to exert its cological effect within a cell for a longer period of time, enhancing the drug’s efficacy. Co—administration of a gliadin peptide with at least one chemotherapeutic agent can therefore potentiate the activity of a wide range of anticancer drugs having various mechanisms of action.

The effect of gliadin peptides on the endocytic pathway can also exert anticancer therapeutic effects by influencing cellular phenotype. Epithelial and mesenchymal are two main classes of cellular phenotypes. Epithelial cells are highly zed, with us cell junctions maintaining adherence between neighboring cells. In contrast, hymal cells are disorganized and lack strong intercellular junctions, which increases their migratory potential.

During a s known as the hymal transition (MT), epithelial cells and non—epithelial cells differentiate into mesenchymal cells. The transition results in the loss of cell—cell adhesion and increased cell motility, as well as increased resistance to apoptosis, thereby promoting the invasiveness, i.e., metastasis, of tumors. During MT, expression of cell junction proteins such as e—cadherin is decreased, and expression of hymal markers such as vimentin and ectin increases.

Low expression of e—cadherin has been associated with the progression of a number of cancer types (Rao et al. Cell Biol. Int. 2011; 35(9):945—51; Yilmaz et al. Molecular Cancer Research. 2010; :629—42). Administration of a n peptide can prevent a mesenchymal phenotype by impairing e—cadherin degradation and promoting recycling of the junction protein back to the plasma membrane to maintain cell—to—cell on. The effect of gliadin on e— cadherin retention may explain why the presence of plasma gliadin leads to reduced enterocyte height and villous y in untreated celiac patients (Barone 2010, supra) because changes in cellular adhesion through the loss of e—cadherin are necessary to promote vertical growth of intestinal cells. Additionally, by interfering with HRS and the ESCRT complexes, gliadin peptides can prevent the degradation of focal adhesions that connect cells to the extracellular matrix (Tu et al. Proceedings of the National Academy of Sciences. 2010;107(37):l6107—l2).

The intact focal adhesions also help maintain the non—mesenchymal phenotype and inhibit transition to a mesenchymal state. Because administration of a gliadin peptide ing to the invention can block MT and prevent cell growth and cellular migration (and thus metastasis of cancer cells), the treatment according to the ion can effectively control a spectrum of cancer types.

The effect of gliadin es on MT can also se the efficacy of co—administered chemotherapeutic agents. Tumor metastasis complicates cancer treatment and is a major butor to patient death. A mesenchymal phenotype has been identified as predictive of drug sensitivity, with expression of mesenchymal markers signaling a poor se to chemotherapy (Yauch 2005, supra; Buck et al. Molecular Cancer Therapeutics. 2007;6(2):532—4l; Frederick et al. Molecular Cancer Therapeutics. 2007;6(6):l683—l69l). E—cadherin expression is substantially absent in resistant cancer cell lines, and restoration of e—cadherin sion can increase drug sensitivity, resulting in cell growth inhibition and sis following treatment (Witta 2006, supra). The administration of gliadin peptides to promote ion of e—cadherin and a non—mesenchymal phenotype can therefore improve the se of cancer cells to a co— administered chemotherapeutic agent. For example, a mesenchymal phenotype is associated with lower amounts of e—cadherin and with both intrinsic and acquired resistance to EGFR— ic RTKIs in NSCLC (Suda 2011, supra). Non—mesenchymal cells rely on EGFR—mediated pathways for cell survival and eration, but in the mesenchymal state, EGFR signaling is reduced and cells are believed to rely on EGFR—independent mechanisms for cell survival and proliferation (Thomson et al. Clin. Exp. Metastasis. 2008;25(8):843—54). Use of a gliadin peptide to in e—cadherin and prevent transition to a mesenchymal state will therefore decrease drug resistance and prolong the sensitivity of cancer calls to the cytotoxic effects of an EGFR—specific RTKI such as gefitinib or erlotinib. Co—administration of a gliadin peptide is ed to act synergistically with other classes of chemotherapeutic agents as well, resulting in improved options for ation therapy to treat cancer.

The anticancer effect achieved from administering a gliadin e alone or in ation with at least one chemotherapeutic agent according to the invention may also be attributed to the unexpected advantageous effect of the gliadin peptide on undifferentiated cells.

Gliadin peptides are surprisingly effective at g CSCs that are resistant to other chemotherapeutic agents, ing them an effective anticancer agent when used alone, i.e., as monotherapy. For example, a gliadin peptide may be administered as an initial (first—line) therapy, i.e., before other ncer therapies (e.g., chemotherapeutic agents, ion, and/or surgery) are attempted. As r example, a gliadin peptide may be administered as a subsequent (e.g., second— or third—line) therapy following an anticancer therapy that is no longer eutically effective.

The therapeutic effect of a gliadin peptide administered alone as a erapy compared to gliadin co—administered with a chemotherapeutic agent may be ed by the ism of action of the chemotherapeutic agent. In particular, monotherapy administration of a gliadin peptide following treatment with a chemotherapeutic agent whose main site of action is in the s, e.g., alkylating agents, otics, topoisomerase inhibitors, and other agents that damage DNA, has been found to be surprisingly effective at inhibiting resistant cancer cells that survive treatment with the chemotherapeutic agent, particularly relative to co—administration of both compounds. For such herapeutic agents, it is theorized that monotherapy using a gliadin peptide may prevent interference with the localization of the chemotherapeutic agent to the nucleus (whereas it is theorized that co—administration can promote such interference, thereby possibly attenuating the eutic effect of the chemotherapeutic agent). For chemotherapeutic agents whose primary site of action is outside the nucleus, e.g., differentiating agents, mitotic inhibitors, steroids, and TKIs, co—administration of a gliadin peptide and the chemotherapeutic agent is surprisingly effective at inhibiting cancer cell growth and can surprisingly achieve synergistic therapeutic efficacy greater than monotherapy with the gliadin peptide or chemotherapeutic agent alone. The administration of a gliadin peptide before and/or after treatment with at least one chemotherapeutic (e.g., monotherapy when the compounds do not exert pharmacological effects during an overlapping period of time) or during administration of at least one chemotherapeutic (i.e, co—administration when the compounds do exert pharmacological effects during an overlapping period of time) is therefore effective to decrease the number of CSCs and prevent cancer relapse and metastasis.

The ability of gliadin es to increase the therapeutic efficacy of a co—administered chemotherapeutic agent may also be attributable in part to the interaction of the gliadin peptide with proteins important for maintaining an undifferentiated phenotype. In addition to HRS, the alpha gliadin peptide p31—43 shares the six amino acid sequence PPQQPY (SEQ ID NO: 2) with residues found within the kinase domain of —dependent kinase 12 (CDK12). The kinase domain of CDK12 is important for its interaction with cyclin K (Cch) (Dai et al., J. Biol. Chem. 2012; 287(30):25344—52). Both CDK12 and Cch are highly expressed in embryonic stem cells, but their expression decreases upon differentiation (Dai 2012, supra). The proteins are important for enewal of embryonic stem cells, and inhibition of either leads to cell differentiation (Dai 2012, supra). Based on the foregoing and the results shown in the Examples below, it is believed that alpha—gliadin peptide p31—43 may interfere with the interaction between CDK12 and Cch and inhibit the activity of the two proteins, thereby promoting cellular entiation and potentially increasing the sensitivity of the tumor to the herapeutic agent. The abilities of gliadin peptides to kill CSCs and/or promote cellular differentiation unexpectedly provide ages not ed from other chemotherapeutic . The therapeutic efficacy achievable using a combination of a gliadin peptide and at least one herapeutic agent to treat cancer is surprising and unexpected considering the characterization of the activity of the compounds as being contrary. For example, gliadin es are known to drive cells into S— phase of the cell cycle, thereby promoting cell proliferation (Barone 2007, supra), while chemotherapeutic agents generally are cytotoxic, particularly to rapidly dividing cells. RTKIs such as erlotinib and gefitinib generally act to arrest cells in se to inhibit cell growth (Arora et al., JPET. 2005;315(3):971—79). Thus, the activity of a gliadin peptide and chemotherapeutic agent would be expected to at least counteract each other. Similarly, the ty of an EGFR activator such as a gliadin peptide and an EGFR inhibitor such as a RTKI would be expected to be contrary to each other. However, because gliadin peptides cause EGFR and other ors to be ed back to the cell membrane instead of degraded within lysosomes, the time during which EGFR remains phosphorylated is extended (Barone 2007, supra; Barone 2010, supra). Such a prolonged activation of EGFR and other RTKs may increase the sensitivity of the cells to RTKIs. The co—administration of a gliadin peptide and an EGFR—specific RTKI is therefore effective for treating ts with wild—type EGFR and those expressing mutant receptor proteins.

The co—administration of a gliadin peptide and at least one chemotherapeutic agent such as a RTKI provides an unexpected and singly effective anticancer therapy. The gliadin peptide acts in concert with the chemotherapeutic agent(s) to achieve enhanced therapeutic cy. When a n e and RTKI are co—administered to a patient with , resistance of the cancer to the RTKI is decreased or prevented. A patient suffering from both untreated celiac disease and cancer could be expected to respond better to anti—cancer therapy using RTKIs or other chemotherapeutic agents. Celiac disease is a chronic inflammatory disease of the small intestine that es an immunogenic response to wheat gluten and similar proteins. Adopting a gluten—free diet mitigates the symptoms of celiac e. In patients suffering from celiac disease, gliadin peptides are ant to degradation and transported intact into serum in significantly higher amounts compared to healthy subjects and ts with treated celiac disease (Matysiak—Budnik et al. Gastroenterology. 2003;125(3):696—707), creating a condition known as "leaky gut syndrome. 77 The increased permeability of gliadin through the lining of the digestive track and into systemic ation would allow gliadin peptides to reach tumor sites and increase the sensitivity of the cancer cells to chemotherapy. Thus, according to one aspect, the t to be treated is not suffering from celiac disease.

The invention is further explained by the following Examples which should not be construed as limiting its scope.

Example 1 Cancer cells of various types including NSCLC are obtained from American Type Culture Collection (ATCC; Manassas, VA) or biopsies from cancer patients and maintained in growth medium. Cells are plated in multi—well cell e plates and divided into the ing experimental groups: (1) cells incubated with growth medium only; (2) cells incubated with growth medium mented with multiple concentrations of alpha—gliadin peptide p3l—43, alpha—gliadin peptide p3l—49, or alpha—gliadin peptide p3l—55 from 5 ug/mL to 200 ug/mL; (3) cells incubated with growth medium supplemented with multiple concentrations of erlotinib from 0.1 uM to 10 uM; (4) cells ted with growth medium supplemented with 5 ug/mL to 200 ug/mL alpha—gliadin peptide p3l—43, alpha—gliadin peptide p3l—49, or gliadin peptide p3l—55 and 0.1 uM to 10 uM erlotinib; (5) cells incubated with growth medium supplemented with multiple concentrations of gefitinib from 0.1 uM to 10 uM; and (6) cells incubated with growth medium supplemented with 5 ug/mL to 200 ug/mL alpha—gliadin peptide p3l—43, alpha— gliadin peptide p3l—49, or alpha—gliadin peptide p3l—55 and 0.1 uM to 10 uM gefitinib.

Cell Viability is measured after 3 to 5 days and the half—maximal inhibitor concentration (IC50) is determined from the dose—response curve. Co—treatment with a gliadin e and erlotinib significantly decreases the IC50 of erlotinib compared to the IC50 for erlotinib administered alone. Similarly, co—treatment with a gliadin peptide and gefitinib significantly decreases the IC50 of gefitinib compared to the IC50 of gefitinib administered alone.

Mice are injected subcutaneously with NSCLC cells in the flank region. Tumors are allowed to grow to about 100 cubic eters to 200 cubic millimeters. The animals are diVided into the following experimental groups and treated for 14 days: (1) animals receiVing a once daily saline injection into the tumor site; (2) animals receiVing a once daily injection of 5 ug/mL to 200 ug/mL of alpha—gliadin peptide p3l—43, alpha—gliadin e , or alpha— n peptide p3l—55 into the tumor site; (3) animals receiVing a once daily oral dose of up to 100 mg/kg erlotinib; (4) animals receiVing a once daily oral dose of up to 100 mg/kg gefitinib; (5) s receiVing a once daily injection of 5 ug/mL to 200 ug/mL of alpha—gliadin peptide p3l—43, alpha—gliadin peptide p3l—49, or alpha—gliadin peptide p3l—55 into the tumor site and once daily oral dose of up to 100 mg/kg erlotinib; (6) animals receiVing a once daily injection of ug/mL to 200 ug/mL alpha—gliadin e p3l—43, gliadin peptide p3l—49, or alpha— gliadin peptide p3l—55 into the tumor site and once daily oral dose of up to 100 mg/kg gefitinib.

Tumor volumes are evaluated using calipers over the course of treatment to determine growth inhibition. Co—administration of a gliadin peptide and nib or a gliadin peptide and gefitinib results in a significant inhibition of tumor growth compared to administration of erlotinib or gefitinib alone.

Example 3 An efficacy study in humans is conducted to evaluate the effect of co—administration of (1) alpha—gliadin peptide p31—43, gliadin e p31—49, or alpha—gliadin peptide p31—55 and nib or (2) alpha—gliadin peptide p31—43, alpha—gliadin peptide p31—49, or alpha—gliadin peptide p31—55 and erlotinib in patients with NSCLC. Control group patients are dosed with up to 250 mg daily of gefitinib or up to 150 mg daily of erlotinib. In the experimental group, patients are stered gliadin peptide p31—43, alpha—gliadin peptide p31—49, or alpha— gliadin peptide p31—55 daily in addition to gefitinib or erlotinib. The alpha—gliadin peptide p31— 43, alpha—gliadin peptide p31—49, or alpha—gliadin peptide p31—55 is administered to achieve a plasma concentration of about 5 ug/mL to about 200 ug/mL. Tumor mass and metastasis are evaluated after one, two, and three months of therapy. Patients receiving a gliadin e in on to a RTKI t a significantly reduced primary tumor mass in the lungs compared to patients receiving a RTKI alone. Patients receiving the combination therapy also exhibit significantly fewer metastatic tumors compared to the control group patients. The clinical trial demonstrates the value of including administration of a n peptide in combination therapy to treat cancer.

For Examples 4 to 8, human cancer cell lines A549, NCI—H1975, and PANC—l were obtained from ATCC and maintained in RPMI 1640 media (Life logies, Inc., Grand Island, NY) containing 10% fetal bovine serum, 2 mM amine and 1% antibiotic— antimycotic solution (10 units/uL penicillin, 10 ug/uL streptomycin and 25 ug/mL amphotericin B). Cells were kept at 37 °C in a fied atmosphere of 5% C02 and grown until they reached a confluency of 90%. Cells were then washed, trypsinized, and counted using a Coulter counter. (Beckman, Brea, CA).

A549 NSCLC cells were maintained and cultured as bed above. Alpha—gliadin peptide p31—43 (Anaspec Inc., Fremont, CA), gefitinib (LC Laboratories, Woburn, MA), and erlotinib (LC Laboratories) were used to treat the cells. Cells were plated at a density of 10,000 cells/well in 24—well cell culture plates and allowed to adhere for 24 hours. The cells were then incubated for 72 hours with the following: (1) vehicle (DMSO/water); (2) 5 ug/mL, 20 ug/mL, or 70 ug/mL alpha—gliadin peptide p31—43; (3) 1 uM gefitinib in DMSO/water; (4) 1 uM erlotinib; (5) 5 ug/mL, 20 ug/mL, or 70 ug/mL alpha—gliadin peptide p31—43 and 1 uM gefitinib 2014/021660 in DMSO/water; or (6) 5 ug/mL, 20 ug/mL, or 70 ug/mL gliadin peptide p31—43 and 1 uM erlotinib in DMSO/water. All experiments were conducted in sextuplicate. For combination therapy using alpha—gliadin peptide p3l—43 and gefitinib/erlotinib, the two compounds were administered to the cells simultaneously. Following 72 hours of treatment, growth inhibition was ted by the 3—(4,5—dimethylthiazol—2—yl)—2,5—diphenyltetrazolium bromide (MTT) assay (Roche stics Corporation, Indianapolis, IN) according to the manufacturer’s instructions.

The absorbance at 570 nm was measured using a plate reader (BioTek, Winooski, VT). Table 1 shows the mean absorbance at 570 nm and the percent growth inhibition for gliadin— and RTKI— treated A549 cells compared to vehicle—treated cells.

Table 1: Effects of alpha-gliadin e p31-43 alone or in combination with gefitinib or erlotinib on A549 cell proliferation following 72-hour treatment Mean absorbance . . % Inhibition p-Value (570 nm) Conuol -- (DMSO/water) ug/mL gliadin p > 0.05 ug/mL gliadin 0.15 3.4 p > 0.05 70 ug/mL gliadin 0.18 9.4 p > 0.05 1 ug/mL gefitinib 0.19 9.4 p > 0.05 1 ug/mLerlotinib 0.12 3.4 p > 0.05 ug/mL gliadin + 1.50 0.12 0 p > 0.05 1 ug/mL erlotinib ug/mL gliadin 1.19 0.08 20.1 p > 0.05 + 1 ug/mL erlotinib 70 ug/mL gliadin 0.86 0.04 42.3 p < 0.05 + 1 ug/mL erlotinib ug/mL n + 1.24 0.04 16.8 p > 0.05 1 ug/mL gefitinib ug/mL gliadin 1.14 0.02 23.5 p > 0.05 + 1 ug/mL gefitinib 70 ug/mL n 1.30 0.16 12.8 p > 0.05 + 1 ug/mL gefitinib A549 cells treated with alpha—gliadin peptide p31—43 alone at a dose of 20 ug/mL or 70 ug/mL exhibited comparable growth inhibition ed to cells treated with gefitinib or erlotinib alone. Treatment with a combination of alpha—gliadin peptide p31—43 and gefitinib or erlotinib ed in increased growth inhibition compared to alpha—gliadin peptide p3l—43 or each RTKI alone. Co—administration of alpha—gliadin peptide p3l—43 and gefitinib or erlotinib surprisingly had a synergistic effect on growth inhibition, with the combination therapy resulting in greater growth inhibition than the sum of the individual growth inhibitory effects of the alpha— gliadin peptide p3 l—43 and the RTKI.

Overall, the results demonstrated that a gliadin peptide administered alone was an effective anticancer treatment and inhibited cancer cell growth as well as a benchmark herapeutic agent. Combination y using a gliadin peptide and a RTKI advantageously resulted in increased tion of cancer cell growth compared to either the gliadin peptide or RTKI alone and also produced a surprising and unexpected synergistic antitumor .

Example 5 NCI—Hl975 NSCLC cells were maintained and cultured as described in Example 4.

NCI—Hl975 cells harbor an activating mutation in EGFR (L858R) and an additional mutation (T790M), which confers resistance to EGFR TKIs including erlotinib and gefitinib. Alpha— gliadin peptide p3l—43, gefitinib, and erlotinib were used to treat the cells. Cells were plated at a density of 10,000 cells/well in 24—well cell e plates and allowed to adhere for 24 hours.

The cells were then incubated for 72 hours with the following: (1) vehicle (DMSO/water); (2) 5 ug/mL, 20 ug/mL, or 70 ug/mL alpha—gliadin peptide p3l—43 in DMSO/water; (3) 1 uM gefitinib in DMSO/water; (4) 1 uM erlotinib in DMSO/water; (5) 5 ug/mL, 20 ug/mL, or 70 ug/mL alpha—gliadin peptide p3l—43 and 1 uM nib in DMSO/water; or (6) 5 ug/mL, 20 ug/mL, or 70 ug/mL alpha—gliadin peptide p3l—43 and 1 uM nib in ater. All experiments were conducted in sextuplicate. For ation therapy using alpha—gliadin peptide p3l—43 and gefitinib/erlotinib, the two compounds were administered to the cells simultaneously. Following 72 hours of treatment, growth inhibition was evaluated by the MTT assay according to the manufacturer’s instructions. The absorbance at 570 nm was ed using a plate reader. Table 2 shows the mean ance at 570 nm and the percent growth inhibition for gliadin— and RTKI—treated NCI—Hl975 cells compared to vehicle—treated cells.

Table 2: Effects of alpha-gliadin peptide p31-43 alone or in ation with gefitinib or erlotinib on NCI-H1975 cell proliferation following 72-hour treatment Dose Mean ance % Inhibition p-value (570 nm) Conuol 2.04 (DMSO/water) ug/mL gliadin ug/mL gliadin 70 ug/mL gliadin 1 ug/mL gefitinib 1 ug/mL erlotinib ug/mL gliadin + 1 ug/mL erlotinib ug/mL gliadin + 1 ug/mL erlotinib 70 ug/mL gliadin + 1 tinib ug/mL gliadin + 1.33 0.05 34.8 p < 0.001 1 ug/mL gefitinib ug/mL gliadin 1.11 0.06 45.6 p < 0.001 + 1 ug/mL gefitinib 70 ug/mL gliadin 0.79 0.06 61.3 p < 0.001 + 1 ug/mL gefitinib NCI—H1975 cells d with alpha—gliadin peptide p31—43 alone exhibited significant growth inhibition ed to control cells. The growth inhibition in cells treated with alpha— gliadin peptide p31—43 alone was greater than in cells treated with gefitinib or erlotinib alone.

Treatment with a combination of alpha—gliadin e p31—43 and gefitinib or erlotinib achieved significant growth inhibition of the cancer cells at all concentrations tested. Additionally, the combination therapy resulted in increased growth tion compared to alpha—gliadin peptide p31—43 or each RTKI alone. As shown in Table 2, co—administration of gliadin peptide p31—43 and gefitinib or erlotinib surprisingly was able to have a synergistic effect on growth inhibition, with the combination therapy resulting in greater growth inhibition than the sum of the individual growth inhibitory effects of the alpha—gliadin peptide p31—43 and the RTKI.

Overall, the results trated that a gliadin peptide stered alone was effective at significantly inhibiting the growth of RTKI—resistant cancer cells and achieved 2014/021660 greater therapeutic efficacy than a ark RTKI. Combination therapy using a gliadin peptide and a RTKI advantageously resulted in significantly increased inhibition of cancer cell growth compared to either the gliadin peptide or RTKI alone and also produced a surprising and unexpected synergistic antitumor effect.

Example 6 PANC—l pancreatic carcinoma cells were maintained and cultured as described in Example 4. Alpha—gliadin peptide p31—43, gefitinib, and erlotinib were used to treat the cells.

Cells were plated at a density of 10,000 cells/well in 24—well cell culture plates and allowed to adhere for 24 hours. The cells were then incubated for 72 hours with the following: (1) vehicle water); (2) 5 ug/mL, 20 ug/mL, or 70 ug/mL alpha—gliadin peptide p31—43 in DMSO/water; (3) 1 uM gefitinib in DMSO/water; (4) 1 uM erlotinib in DMSO/water; (5) 5 ug/mL, 20 ug/mL in DMSO/water, or 70 ug/mL alpha—gliadin peptide p31—43 and 1 uM gefitinib in DMSO/water; or (6) 5 ug/mL, 20 ug/mL, or 70 ug/mL alpha—gliadin peptide p31—43 and 1 uM erlotinib in DMSO/water. All experiments were conducted in sextuplicate. For combination therapy using alpha—gliadin peptide p31—43 and gefitinib/erlotinib, the two compounds were administered to the cells simultaneously. Following 72 hours of ent, growth inhibition was evaluated by the MTT assay according to the cturer’s instructions.

The absorbance at 570 nm was ed using a plate reader. Table 3 shows the mean absorbance at 570 nm and the t growth inhibition for gliadin— and RTKI—treated PANC—l compared to vehicle—treated cells.

Table 3: Effects of alpha-gliadin e p31-43 alone or in combination with gefitinib or erlotinib on PANC-l cell proliferation following 72-hour treatment Dose Mean absorbance S.E. p-value (570 nm) (DMSO/water) ug/mL n 1.59 0.18 15.0 p > 0.05 ug/mL gliadin 1.39 0.15 25.7 p > 0.05 70 ug/mL gliadin 26.2 1 ug/mL gefitinib 0 1 ug/mL nib 0 ug/mL gliadin + 1.35 0.11 27.7 p > 0.05 1 ug/mL erlotinib ug/mL gliadin + 1 ug/mL erlotinib 70 ug/mL gliadin + 1 ug/mL erlotinib ug/mL gliadin + 1.33 0.06 28.9 p > 0.05 1 ug/mL gefitinib ug/mL gliadin 1.15 0.05 38.5 p < 0.001 + 1 ug/mL gefitinib 70 ug/mL gliadin 1.14 0.07 39.0 p < 0.001 + 1 ug/mL gefitinib PANC—l cells treated with alpha—gliadin peptide p31—43 alone exhibited greater growth inhibition than cells treated with gefitinib or erlotinib alone. Treatment with a combination of alpha—gliadin peptide p31—43 and gefitinib or erlotinib achieved significant growth inhibition of the cancer cells. Additionally, the combination therapy resulted in significantly increased growth inhibition compared to alpha—gliadin peptide p31—43 or each RTKI alone. As shown in Table 3, inistration of alpha—gliadin peptide p31—43 and gefitinib or erlotinib surprisingly could have a synergistic effect on growth inhibition, with the combination therapy resulting in greater growth inhibition than the sum of the dual growth tory effects of the alpha—gliadin peptide p31—43 and the RTKI.

Overall, the results demonstrated that a gliadin peptide stered alone was effective at inhibiting the growth of RTKI—resistant cancer cells and achieved greater therapeutic efficacy than a ark RTKI. Combination therapy using a gliadin peptide and a RTKI advantageously resulted in significantly increased inhibition of cancer cell growth ed to either the gliadin peptide or RTKI alone and also produced a surprising and unexpected synergistic antitumor . Examples 4 to 6 demonstrated that co—administration of a gliadin peptide and a chemotherapeutic agent provided an advantageous therapeutic effect.

Example 7 PANC—l human atic carcinoma cells (ATCC) were maintained and cultured as described in Example 4. Alpha—gliadin e p31—43 and S—Fluorouracil (S—FU) (Fisher Scientific, Pittsburgh, PA) were used to treat the cells. Cells (passage 27) were plated at a density of 1 x 104 cells/well in 24—well cell culture plates and allowed to adhere for 24 hours.

Cells were then incubated with vehicle (water) or 5—FU at increasing concentrations for 72 hours.

All experiments were ted in triplicate. After incubation with 5—FU at trations ranging from 0 uM to 400 uM, cells were detached with trypsin and counted. Table 4 shows the mean cell number and percent growth inhibition following treatment with 5—FU.

Table 4: Anti-proliferative effects of S-FU on PANC-l cells following 72-hour treatment S-FU Dose (uM) Mean Cell Number/mL S.E. % Inhibition p-value 0 (vehicle) 317,600 7,738 —— —— 6.25 0 2,342 63.3 p < 0.001 12.5 117,500 4,474 63.0 p < 0.001 .0 118,200 4,288 62.8 p < 0.001 50.0 100,200 1,791 63.0 p < 0.001 100.0 117,600 2,006 68.5 p < 0.001 200.0 83,900 1,895 73.6 p < 0.001 400.0 66,200 1,935 79.2 p < 0.001 Cells (passage 30) were then plated at a density of 5 X 103 cells/well in 24—well cell culture plates and allowed to adhere for 24 hours. Cells were incubated with the following: (1) vehicle (DMSO/water); (2) 6.25 uM 5—FU; (3) 70 ug/mL alpha—gliadin peptide p3l—43; (4) 6.25 uM 5—FU and 70 ug/mL alpha—gliadin peptide p3l—43 (high combination); or (5) 3.1 uM 5—FU and ug/mL alpha—gliadin peptide p3 l—43 (low combination). All ents were conducted in triplicate. For combination therapy using alpha—gliadin peptide p3l—43 and 5—FU, the two nds were administered to the cells simultaneously. Media was refreshed with the respective treatments every 72 hours. After 14 days of treatment, cells were trypsinized and counted. Table 5 shows the mean cell number and percent growth inhibition following treatment.

Table 5: Effects of S-FU alone or in combination with gliadin e p31-43 on PANC-l cell proliferation following a 14-day treatment regimen Dose Mean Cell . . % Inhibition Number/mL 0 (vehicle) 810,467 6.25 uM 5—FU 33,233 255 70 ug/mL n 833,800 9500 6.25 uM 5—FU + 70 ug/mL gliadin 49,367 575 3.1 uM 5—FU + 35 ug/mL gliadin 42,033 135 95 p < 0.001 In order to examine the effects of alpha—gliadin peptide p31—43 on PANC—l cells exposed to 5—FU for an extended period of time (14 days), an experiment was conducted on the surviving, i.e., resistant, cell tion (4%) from the previous experiment. Briefly, after ng viable cells on day 14 of treatment, surviving cells were replated at a density of 5,000 cells per well. The following day, cells were treated with e or 100 ug/mL or 200 ug/mL of alpha—gliadin peptide p31—43. Media containing the tive treatments was refreshed on day 3 and 6. On day 7, cells were trypsinized and counted. Table 6 shows the mean cell number and percent growth inhibition following treatment with alpha—gliadin peptide p31—43.

Table 6: Effects of alpha-gliadin peptide p31-43 0n surviving cell population of S-FU- ant PANC-l cells Dose Cell Number/mL % Inhibition Control 8200 -- 100 ug/ml gliadin 3700 55% 200 ug/ml gliadin 1900 77% The herapeutic agent 5—FU, which damages DNA, suppressed eration of PANC—l pancreatic cancer cells. The drug inhibited growth of PANC—l cells by 96% following treatment with 6.25 uM for 14 days. Co—administration of 5—FU and a gliadin peptide achieved significant growth inhibition compared to control cells. The results shown in Tables 3 and 5 suggest that the therapeutic efficacy of co—administering a gliadin peptide and a herapeutic agent compared to either alone could be affected by the site of action (nucleus or cytoplasm) of the herapeutic agent.

Surprisingly, monotherapy administration of a gliadin peptide alone was effective in killing the cancer cells that prior ent with 5—FU did not eliminate. Following 5—FU treatment, a surviving population of 5—FU—resistant cells ing to 4% of the initial population remained viable. When the surviving cell population from 5—FU—treated PANC—l cells were exposed to alpha—gliadin peptide p31—43 (100 ug/mL or 200 ug/mL) for 7 additional days, cell proliferation was significantly suppressed. The ability of the gliadin peptide to effectively kill cells resistant to a potent chemotherapeutic agent such as S—FU was surprising and unexpected.

Example 8 A549 cells were maintained and cultured as bed in Example 4. Alpha—gliadin e p3l—43 and cisplatin sion, as, CA) were used to treat the cells. Cells (passage 32) were plated at a density of l x 104 cells/well in 24—well cell culture plates and allowed to adhere for 24 hours. Cells were then incubated with vehicle (0.9% sodium chloride) or cisplatin at increasing concentrations for 72 hours. All experiments were conducted in triplicate. After tion with cisplatin at concentrations ranging from 0 uM to 6.6 uM, cells were detached with trypsin and counted. Table 7 shows the mean cell number and percent growth inhibition following treatment with cisplatin.

Table 7: Anti-proliferative effects of cisplatin on A549 cells following 72-hour treatment Cis latin Dose M Mean Cell % tion -value 0 vehicle 193 017 —— —— 0.103 166,883 13.5 p < 0.001 0.207 150,917 21.8 p < 0.001 0.413 111,917 42.0 p < 0.001 0.825 48,400 74.9 . < 0.001 1.650 16117 91.6 <0.001 3.300 10,033 452 94.8 p < 0.001 3.600 3,222 136 98.3 p < 0.001 Cells (passage 34) were then plated at a density of 5 x 103 cells/well in 24—well cell e plates and allowed to adhere for 24 hours. Cells were incubated with the following: (1) vehicle (DMSO/water); (2) 3.3 uM cisplatin; (3) 70 ug/mL alpha—gliadin peptide p3l—43; (4) 3.3 uM cisplatin and 70 ug/mL alpha—gliadin peptide p3l—43 (high combination); or (5) 1.65 uM cisplatin and 35 ug/mL alpha—gliadin peptide p3l—43 (low combination). All treatments were conducted in triplicate. For combination therapy using alpha—gliadin peptide p3l—43 and cisplatin, the two compounds were administered to the cells aneously. Media was refreshed with the respective treatments every 72 hours. After 14 days of treatment, cells were trypsinized and counted. Table 8 shows the mean cell number and t growth inhibition following treatment.

Table 8: Effects of cisplatin alone or in combination with alpha-gliadin peptide p31-43 on A549 cell eration following a 14-day treatment regimen Dose Mean Cell S.E. % p-Value Number/mL tion 0 (vehicle) 364,683 5334 " '- 3.3 uM Cisplatin 5,780 950 98 p < 0.001 70 ug/mL gliadin 325,217 1761 11 p > 0.05 3.3 uM Cisplatin + 70 ug/mL n 91,100 2350 75 p < 0.001 1.65 uM Cisplatin + 35 ug/mL gliadin 132,783 1961 64 p < 0.001 In order to examine the effects of gliadin peptide p31—43 on A549 cells exposed to Cisplatin for an extended period of time (14 days), a second experiment was conducted on the surviving, i.e., resistant cell population (2%) from the previous experiment. Briefly, after counting viable cells on day 14 of treatment, the surviving cells were replated at a density of ,000 cells per well. The following day, cells were treated with vehicle or 100 ug/mL or 200 ug/mL of alpha—gliadin peptide . Media containing the respective treatments was refreshed on day 3 and 6. On day 7, cells were trypsinized and counted. Table 9 shows the mean cell number and growth inhibition following treatment with alpha—gliadin peptide p31—43.

Table 9: Effects of alpha-gliadin peptide p31-43 on surviving cell population of tin- resistant A549 cells —_CeiiNumber/mL % Inhibition 100 ug/mL gliadin 1,300 200 ug/mL gliadin 65% The chemotherapeutic agent cisplatin, which damages DNA and is characterized as an alkylating agent, suppressed proliferation of A549 lung cancer cells. tin inhibited eration of A549 cells by 98% following treatment with 3.3 uM for 14 days. Co—administration of Cisplatin and a gliadin peptide ed significant growth inhibition compared to control cells.

The results shown in Tables 3 and 8 suggested that the therapeutic efficacy of co—administering a gliadin peptide and a herapeutic agent compared to either alone could be affected by the site of action (nucleus or cytoplasm) of the chemotherapeutic agent.

Surprisingly, administration of a n peptide alone was effective in killing the cancer cells that prior treatment with cisplatin did not eliminate. Following cisplatin treatment, a surviving population of tin—resistant cells amounting to 2% of the initial population remained viable. When the surviving cell population from cisplatin—treated A549 cells were exposed to alpha—gliadin peptide p3 l—43 (100 ug/mL or 200 ug/mL) for 7 additional days, cell proliferation was significantly suppressed. The ability of the gliadin peptide to effectively kill cells resistant to a potent chemotherapeutic agent such as cisplatin was surprising and unexpected.

Overall, the results in Examples 7 and 8 demonstrated that co—administering a gliadin peptide and a chemotherapeutic agent was effective in significantly inhibiting the growth of cancer cells. Surprisingly, administering a n peptide alone achieved significant suppression of cancer cells that ed prolonged ent with a potent chemotherapeutic agent. The surprising and cted ability of a gliadin peptide to advantageously and effectively kill the most resistant cancer cells, e.g., CSCs, ted the gliadin peptide could be used in monotherapy or combination therapy to inhibit tumor growth and prevent cancer relapse.

Administration of a n peptide was therefore effective to decrease or prevent resistance of the cancer to the chemotherapeutic agent.

Example 9 The toxicity of alpha—gliadin peptide p3l—43 in animals was evaluated. Five— to six— week old female BALB/c mice were dosed with vehicle (100 uL DMSO/saline) administered subcutaneously once a day for five days (Group 1) or 200 ug of alpha—gliadin peptide p3l—43 in e administered subcutaneously once a day for five days (Group 2). To prepare the n solution, 70 uL of sterile DMSO was added to 1 mg of alpha—gliadin peptide p3l—43 and vortexed to dissolve the peptide. After the peptide was dissolved, 430 uL of sterile 0.9% sodium chloride was added and ed to create a 1000 ug/500 uL solution of alpha—gliadin peptide p3 l—43.

Toxicity was evaluated using daily weight measurements and behavior assessments.

The alpha—gliadin e p31—43 was associated with no treatment—related deaths. At the end of the study, the mean body weights (i SE) were 19.8 i 0.4 grams for Group 1 (n=5) and 20.0 i 0.3 grams for Group 2 (n=5). No behavior changes were observed in alpha—gliadin peptide p31—43 treated animals as compared to control mice. gliadin peptide p31—43 was therefore tolerated at a dosing level of 10 mg/kg/day without apparent toxicity. e 10 The ability of alpha—gliadin p31—43 to induce apoptosis in cancer cells was assessed.

A549 cells were maintained and cultured in RPMI 1640 media containing 10% fetal bovine serum, 2 mM L—glutamine and 1% otic—antimycotic solution. Cells were grown in the presence of 5% C02 at 37 °C in an incubator. ion of apoptosis following ent with alpha—gliadin peptide p31—43 alone or in combination with gefitinib was determined using the terminal deoxynucleotidyl transferase dUTP nick—end labelling (TUNEL) assay.

Briefly, A549 cells (1 x 105) were plated in chamber slides and allowed to adhere overnight. The cells were incubated for 72 hours with the following: (1) vehicle control; (2) 1 uM gefitinib; (3) 100 ug/mL, 200 ug/mL, or 500 ug/mL alpha—gliadin peptide p31—43; (4) 100 ug/mL, 200 ug/mL, or 500 ug/mL alpha—gliadin peptide p31—43 and 1 uM gefitinib. For combination therapy using alpha—gliadin peptide p31—43 and gefitinib, the two compounds were administered to the cells simultaneously. After the 72—hour treatment, the cells were fixed with 4% formaldehyde in PBS (pH 7.4) for 25 minutes at room temperature, then washed twice for 5 s in PBS, permeabilized in 0.2% Triton X—100 solution in PBS for 5 minutes at room temperature, and finally washed twice for 5 minutes in PBS. Apoptosis was measured using the dTM Colorimetric TUNEL System (Promega, n, WI) according to the manufacturer's instructions. At the end of the assay, the cells were mounted and ed under the microscope. Staining of apoptotic cells was observed for cells treated with alpha—gliadin and/or gefitinib, and the percentage of cells that were tic was ined by counting the number of stained cells within a representative sample. Table 10 shows the percent of cells that were apoptotic following each treatment and the p—value determined using y ANOVA analysis of the data for the gliadin— and gefitinib—treated cells compared to the control cells.

Table 10: Effect of gefitinib alone or in combination with alpha-gliadin peptide p31-43 on the induction of apoptosis Dose Mean % tic S.E. p-value Control 3 2 _ nib 19 p > 0.05 100 ug/mL gliadin 37 p < 0.01 200 ug/mL gliadin 36 p < 0.01 500 ug/mL n 38.5 100 ug/mL gliadin + 28.5 200 ug/mL gliadin + 37 gefitinib 500 ug/mL gliadin + 55 gefitinib A significantly higher percentage of cells d with alpha—gliadin peptide p3l—43 alone were tic, compared to cells treated with vehicle or gefitinib alone. onally, significantly more cells were apoptotic following combination therapy using alpha—gliadin peptide p3l—43 and gefitinib, compared to cells treated with vehicle or gefitinib alone. The most effective treatment for inducing apoptosis was the combination of 500 ug/mL gliadin peptide p3l—43 and gefitinib. Overall, the results demonstrated that alpha—gliadin peptide alone or in combination with gefitinib was a potent inducer of apoptosis in human lung cancer cells.

Example 11 The activity of gefitinib alone or in combination with alpha—gliadin peptide p3 l—43 was evaluated using an A549 human lung cancer xenograft model. Six—week old female nude mice (Harlan Laboratories, Indianapolis, IN) were quarantined for 3 days and housed 5 mice per cage, with a 12—hour light—dark cycle, and a relative ty of 50%. Drinking water and diet were supplied to the animals ad libitum. All animals were housed under pathogen—free conditions. On day 4, 5 X 106 A549 cells in 100 uL of RPMI 1640 media were ed subcutaneously into the right flank of the mice. Starting 24 hours post—inoculation, animals were dosed once daily for 14 days as follows: Group 1 — vehicle (2% DMSO in saline) administered intravenously; Group 2 — 150 mg/kg gefitinib administered by gavage; and Group 3 — 150 mg/kg gefitinib administered by gavage WO 38556 and 200 ug alpha—gliadin p31—43 administered intravenously. For combination therapy using alpha— gliadin peptide p31—43 and gefitinib, the two compounds were administered to the cells simultaneously. The animals were monitored for two weeks following the 14—day treatment period. Tumor measurements were initiated as soon as the tumor formed a palpable mass and measured twice weekly. Table 11 shows the mean body weights for the treatment groups over the course of the study.

Table 11: Effect of nib alone or in combination with alpha-gliadin peptide p31-43 on body weight Mean Body Weight (grams) Dosing Period Recover Period Study 21 25 Day: Group 1 21.26 $ 22.34 $ ol) 0.89 0.94 Group 2 21.12 $ 22.00 $ (gefitinib) 0.60 0.52 Group 3 (gefitinib All treatments were well tolerated and associated with no drug—related deaths. No significant body weight loss was noted for any of the treatment groups. The mean body weights in grams ($ SE.) at ation were: Group 1 = 22.80 $ 0.88, Group 2 = 22.50 $ 0.44, and Group 3 = 22.92 $ 0.44. Table 12 shows the mean tumor volumes for the treatment groups over the course of the study.

Table 12: Effect of gefitinib alone or in combination with alpha-gliadin peptide p31-43 on tumor volume Mean Tumor Volume (mms) Dosing Period Recovery Period Day: Groupl 44.78$ 60.44$ 86.58$ 118.60 169.34 227.86 326.78 402.86 (control) 6.48 13.00 15.44 $29.24 $40.87 $67.54 $91.72 $95.41 Group2 47.74i 51.67i 49.30i 60.43i 75.40i 95.22i 157.69 239.25 (gefitinib) 2.87 5.69 3.77 5.35 9.05 11.15 i14.76 $32.78 Group3 (gefitinib 27.24i 31.06i 40.89i 44.87i 50.12i 82.16i 112.79 145.46 and 7.12 8.13 1.11 3.13 4.01 8.72 i 13.65 i 19.74 gliadin) At study termination day (Day 28), mean tumor volumes in cubic millimeters (i S.E.) were: Group 1 = 402.86 i 95.4, Group 2 = 239.25 i 32.78, and Group 3 = 145.46 i 19374.

Table 13 shows the mean tumor volumes for the treatment groups over the course of the study.

Table 13: Effect of gefitinib alone or in combination with alpha-gliadin peptide p31-43 on tumor volume Mean Percent Tumor Growth Inhibition Dosing Period Recovery Period Day: Group2 —6.61% 14.51% 43.06% 49.05% 55.48% 58.21% 51.74% 40.61% (gef1t1n1b) Group3 96$me 39.18% 48.61% 52.77% 62.16% 70.40% 63.94% 65.48% 63.89% gliadin) The percent mean tumor growth inhibition values were 40.6% for Group 2 and 63.9% for Group 3. The tumor doubling times were 17.21 days for Group 1, 24.48 days for Group 2, and 22.89 days for Group 3. The tumor growth tion T/C ratio was 57.16 for Group 2 and 44.06 for Group 3.

Overall, the results demonstrated that combination therapy using gefitinib and a gliadin peptide produced a or anticancer effect compared to gefitinib alone. The combination therapy was olerated and not toxic to the animals, but was still effective at reducing the tumor . The mean tumor volume following combination therapy was reduced by more than 60% compared to control animals. The mean tumor volume was also significantly smaller (about 40%) following combination therapy compared to the mean tumor volume following treatment with nib alone. Additionally, the combination therapy exhibited therapeutic efficacy significantly more rapidly than gefitinib alone, ing close to 50% tumor growth inhibition within one week and maintaining greater than 60% growth inhibition for two weeks following cessation of treatment.

The foregoing Examples are provided to further rate the invention without being limiting. The data demonstrated that a gliadin peptide was an effective anticancer agent when used alone and was able to inhibit growth and induce apoptosis in drug—resistant cancer cells derived from a variety of different cancer types. A gliadin peptide administered alone was unexpectedly effective in killing even the most resistant cancer cells that survived prolonged treatment with a potent herapeutic agent. Co—administration of a gliadin peptide and a chemotherapeutic agent was shown to significantly inhibit the growth of cancer cells, for a number of chemotherapeutic agents. inistration of a gliadin peptide with a RTKI resulted in greater therapeutic efficacy than the gliadin peptide or RTKI alone, and synergistic effects were surprisingly achieved. Despite exhibiting potent antitumor effects in vivo and the y to dramatically reduce tumor volume and growth, administration of a gliadin peptide did not cause overall toxicity.

In view of the evidence described in the above es, the use of a gliadin peptide, alone or in ation with a herapeutic agent, provides unexpected and surprisingly ive anticancer therapy.

While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the ion. It is therefore intended to cover in the claims all such changes and modifications that are within the scope of this invention.

Claims (13)

1. Use of a therapeutically effective amount of a gliadin peptide in the manufacture of a medicament for treating cancer in a patient, wherein the gliadin peptide ses SEQ ID NO:1 and wherein the cancer is selected from the group consisting of non-small cell lung cancer and pancreatic cancer.

2. The use of claim 1, wherein the gliadin peptide is formulated for co-administration with at least one chemotherapeutic agent.

3. The use of claim 2, wherein the chemotherapeutic agent is selected from the group ting of idine, axathioprine, bevacizumab, bleomycin, capecitabine, carboplatin, bucil, tin, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, etoposide, fluorouracil, gemcitabine, herceptin, idarubicin, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, tafluposide, teniposide, tioguanine, retinoic acid, valrubicin, vinblastine, vincristine, ine, vinorelbine, receptor tyrosine kinase inhibitors, and combinations thereof.

4. The use of claim 2, wherein the at least one chemotherapeutic agent is a or tyrosine kinase inhibitor.

5. The use of claim 4, wherein the receptor ne kinase inhibitor is an epidermal growth factor receptor (EGFR) inhibitor.

6. The use of claim 5, wherein the EGFR inhibitor is gefitinib.

7. The use of claim 5, wherein the EGFR inhibitor is erlotinib.

8. The use of any one of claims 1-7, wherein the gliadin peptide is an alpha-gliadin peptide or derivative or fragment thereof.

9. The use of any one of claims 1-8, wherein the gliadin peptide is an gliadin peptide selected from the group consisting of alpha-gliadin peptide p31-55, alpha-gliadin e , and derivatives and fragments thereof.

10. The use of any one of claims 1-9, wherein the medicament and at least one chemotherapeutic agent are formulated for co-administration in amounts such that coadministration is effective to decrease or prevent resistance of the cancer to the chemotherapeutic agent.

11. The use of any one of claims 1-10, wherein the ment and at least one chemotherapeutic agent are formulated for co-administration in amounts such that coadministration is effective to increase the efficacy of the chemotherapeutic agent.

12. The use of any one of claims 1-11, wherein the patient does not have mutations in the EGFR gene known to increase sensitivity to EGFR inhibitors.

13. The use of any one of claims 1-12, wherein the patient does not have celiac disease.