Pharmaceutical composition for eliminating resistance caused by cancer chemotherapy and enhancing the effect of cancer chemotherapy and use thereof

Abstract

To provide novel, safe therapies directed to preventing, treating, and inhibiting cancer, including inhibiting cancer cell metastasis, cancer cell proliferation, tumor growth, and / or angiogenesis.SOLUTION: A pharmaceutical composition for relieving anticancer drug resistance and enhancing sensitivity of anticancer drug, comprises: a chemokine receptor antagonistic modified peptide, selected from ONCO P-8 (SEQ ID NO:1: GSKELRCQCIRSYSKPFHPKFIKELRVIPASQFCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAENS); and a medicament comprising a chemotherapeutic drug, a pharmaceutically acceptable buffer, diluent, carrier, adjuvant or excipient.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to a pharmaceutical composition, and in particular to a pharmaceutical composition for removing anticancer drug resistance and increasing anticancer drug sensitivity.

[0002] More specifically, the present invention further relates to pharmaceutical compositions comprising chemotherapeutic agents in combination with chemokine receptor antagonist-modified peptides for overcoming anticancer drug resistance, treating drug-resistant cancers and patients with drug-resistant cancers, and inhibiting tumor growth and / or metastasis. [Background technology]

[0003] Cancer poses a significant burden to human health, accounting for an estimated 13% of all deaths each year. Rapid population growth and aging worldwide has resulted in a significant increase in cancer as a leading cause of death in many countries.

[0004] Cancer treatment remains one of the greatest challenges in medicine today. Treatment of late-stage and metastatic cancer remains a major challenge. For example, pancreatic cancer is a devastating malignancy, and developing effective treatments with reduced side effects has proven challenging. Pancreatic cancer is a fatal human malignancy, with a 5-year survival rate of less than 14% for stage I and less than 1% for stage IV. Due to the fact that most cases are diagnosed at an advanced stage, treatment options for pancreatic cancer are extremely limited and primarily palliative. Although the molecular and genetic basis of pancreatic cancer has been extensively studied, treatment options remain limited.

[0005] Currently, cancer treatment requires classical chemotoxic chemotherapy, surgery, and / or radiation therapy to eradicate cancer cells in patients, all of which pose significant disadvantages to the patient.

[0006] Chemotherapy is considered the standard treatment for many cancers. Chemotherapy often reduces tumor size, allowing for subsequent surgery, followed by radiation therapy and further adjuvant chemotherapy. However, given that most current anticancer drugs do not significantly differentiate between cancerous cells and normal cells, the systemic toxicity and adverse effects associated with these chemotherapeutic agents limit their therapeutic efficacy. Furthermore, there are a variety of chemotherapeutic agents available for the treatment of tumor diseases.

[0007] On the other hand, surgery may be contraindicated due to the patient's health or may be unacceptable to the patient. Furthermore, surgery may not completely remove tumor tissue. Radiation therapy is only effective if the irradiated cancer tissue exhibits higher sensitivity than normal tissue, and radiation therapy may also induce serious side effects.

[0008] Many tumor treatments are currently in use, including any chemotherapeutic and biological agents as well as radiation therapy.

[0009] Gemcitabine is a chemotherapy drug approved for a wide range of tumors, including, but not limited to, pancreatic and colorectal cancer. However, the efficacy of gemcitabine is modest, and life expectancy is rarely extended, especially in patients with pancreatic cancer. The side effects of gemcitabine administration are relatively mild compared to other chemotherapy drugs, and consist of bone marrow suppression, which increases the risk of infection; decreased platelet count, which increases the risk of bleeding, nausea, and vomiting; increased liver function blood test values, and fatigue. However, in general, gemcitabine has replaced other treatments due to its less toxic effects on patients and the resulting better quality of life.

[0010] On the other hand, a new model of tumorigenesis has recently gained widespread acceptance, which assumes that only a small fraction of the total tumor mass is responsible for tumorigenic activity within the tumor. This small fraction of tumorigenic cells in the new model are transformed cells with stem cell-like properties, termed "cancer stem cells" (CSCs).

[0011] Because surviving cancer stem cells can repopulate tumors and lead to relapse, it is essential that anti-cancer therapies include strategies against CSCs. Selective targeting of cancer stem cells can treat patients with aggressive, unresectable tumors and refractory or recurrent cancers, and can also prevent tumor metastasis and recurrence. The development of specific therapies targeting cancer stem cells offers hope for improving the survival and quality of life of cancer patients, especially those with metastatic cancer.

[0012] However, the most serious problem facing oncologists in treating cancer is the development of drug resistance in tumors, which reduces the cytotoxicity of chemotherapeutic agents. Some cancers are drug resistant before treatment, while others develop drug resistance during treatment.

[0013] Furthermore, solid tumor growth is usually angiogenesis (neovascularization) dependent. Endothelial cells (ECs) in the vasculature play a key role in angiogenesis, and therefore there is a need for therapeutic agents that target this activity. The proliferation, migration, and differentiation of vascular endothelial cells during angiogenesis are understood to be regulated in both normal and disease states by the complex interplay of various chemokines and chemokine receptors. It has now been demonstrated that many different types of tumors can secrete, in particular, an ELR-CXC chemokine called CXCL8, in conjunction with CXCR1 and CXCR2, on endothelial cells (ECs). Summary of the Invention [Problem to be solved by the invention]

[0014] In view of these problems, there is a need in the art for new and safe therapeutic approaches aimed at preventing, treating, and inhibiting cancer, including inhibiting cancer cell metastasis, cancer cell proliferation, tumor growth, and / or angiogenesis. [Means for solving the problem]

[0015] An object of the present invention is to provide a drug / medicine / pharmaceutical composition for overcoming drug resistance or a drug / medicine / pharmaceutical composition for overcoming anti-cancer drug resistance.

[0016] Another aspect of the present invention provides methods of treating cancer, methods of treating drug-resistant cancer, and pharmaceutical compositions for use in treating cancer in a subject, such as a subject with drug-resistant cancer.

[0017] Another aspect of the present invention provides a method of treating cancer in an individual, the method comprising administering to the individual a combination therapy comprising one or more chemokine receptor antagonist-modified peptides and one or more drugs.

[0018] Other embodiments provide for the use of a chemokine receptor antagonist-modified peptide in the manufacture of a medicament for treating cancer in an individual when administered in combination with a chemotherapeutic agent, a pharmaceutically acceptable buffer, diluent, carrier, adjuvant or excipient; and the use of a chemotherapeutic agent, a pharmaceutically acceptable buffer, diluent, carrier, adjuvant or excipient for treating cancer in an individual when administered in combination with a chemokine receptor antagonist-modified peptide.

[0019] Collectively, these and other objectives have been achieved by the present disclosure, which demonstrates enhanced efficacy and survival rates and reduced side effects and toxicity commonly associated with chemotherapeutic agents.

[0020] In the following embodiments, the present invention will be described in detail with reference to the accompanying drawings. [Brief explanation of the drawings]

[0021] [Figure 1] This shows that ONCO P-8 can neutralize the effect of CXCL8 and reduce the invasive rate of human pancreatic cancer (PDAC) cells. Data are shown as mean ± SD. *P<0.05; ***P<0.001, Student's t-test. [Figure 2]This shows that ONCO P-8 can neutralize the effect of CXCL8 and reduce the colony formation efficiency of BxPC-3. Data are shown as mean ± SD. **P<0.01; ***P<0.001, Student's t-test. [Figure 3A] Establishment of a gemcitabine-resistant pancreatic ductal adenocarcinoma (PDAC) cell line. This figure shows the establishment of BxPC-3GR. [Figure 3B] Establishment of a gemcitabine-resistant pancreatic ductal adenocarcinoma (PDAC) cell line. This figure shows the establishment of MIAPaCa2. [Figure 4A] IL-8, CXCR1, and CXCR2 mRNA expression in parental cells and gemcitabine-resistant cells treated with gemcitabine. This figure shows mRNA expression in BxPC-3GR compared to BxPC-3. [Figure 4B] Figure 1 shows IL-8, CXCR1, and CXCR2 mRNA expression in parental cells and gemcitabine-resistant cells treated with gemcitabine. This figure shows mRNA expression of MIAPaCa2GR compared to MIAPaCa2. Data are shown as mean ± SD. *P<0.05; **P<0.01; ***P<0.001, Student's t-test. [Figure 5A] IL-8, CXCR1, and CXCR2 mRNA expression in gemcitabine-resistant cells following gemcitabine administration or gemcitabine administration in combination with ONCO P-8. This figure shows mRNA expression in BxPC-3GR compared to BxPC-3. [Figure 5B] Figure 1 shows IL-8, CXCR1, and CXCR2 mRNA expression in gemcitabine-resistant cells treated with gemcitabine or gemcitabine in combination with ONCO P-8. This figure shows mRNA expression of MIAPaCa2GR compared to MIAPaCa2. Data are shown as mean ± SD. *P<0.05; **P<0.01; ***P<0.001, Student's t-test. [Figure 6A]ONCO P-8 can suppress the invasive ability of gemcitabine-resistant PDAC cells. This figure shows the invasive rate of BxPC-3GR treated with gemcitabine in combination with ONCO P-8, or gemcitabine in combination with ONCO P-8 and IL-8, or gemcitabine in combination with IL-8, respectively. [Figure 6B] ONCO P-8 can suppress the invasive ability of gemcitabine-resistant PDAC cells. This figure shows the invasive rate of MIAPaCa2GR treated with gemcitabine in combination with ONCO P-8, gemcitabine in combination with ONCO P-8 and IL-8, or gemcitabine in combination with IL-8. [Figure 7] We show that the growth rate of gemcitabine-resistant cancer cells can be enhanced by gemcitabine and inhibited by ONCO P-8. [Figure 8A] We show that ONCO P-8 can reduce the migration rate of gemcitabine-resistant cancer cells. [Figure 8B] We show that ONCO P-8 can reduce the invasive rate of gemcitabine-resistant cancer cells. [Figure 9A] 1 shows a comparison of cell morphology and CXCR1 / CXCR2 gene expression between BxPC-3GR parental cells and BxPC-3GR-derived spheroids. This figure shows the cell morphology of BxPC-3GR parental cells. [Figure 9B] Figure 1 shows a comparison of cell morphology and CXCR1 / CXCR2 gene expression between BxPC-3GR parent cells and BxPC-3GR-derived spheroids. This figure shows a comparison of cell morphology between BxPC-3GR parent cells and BxPC-3GR-derived spheroids. This figure shows the cell morphology of BxPC-3GR-derived spheroids. [Figure 9C] Figure 1 shows a comparison of cell morphology and CXCR1 / CXCR2 gene expression between BxPC-3GR parental cells and BxPC-3GR-derived spheroids. This figure shows that CXCR1 and CXCR2 RNA expression on BxPC-3GR-derived spheroids is higher than that of parental BxPC-3GR cells. [Figure 10A] RT-PCR analysis of RNA expression of the key transcription factor NANOG in BxPC-3GR cells and BxPC-3GR-derived spheroids. Drug resistance-related CSC markers: NANOG, OCT4, SOX2. Cell invasion-related CSC markers: ALDHA1, BMI-1. Tumor proliferation-related CSC markers: KLF4, nestin. *P<0.05; **P<0.01; ***P<0.001, Student's t-test. [Figure 10B] RNA expression analysis of the key transcription factor OCT4 by RT-PCR in BxPC-3GR cells and BxPC-3GR-derived spheroids. Drug resistance-related CSC markers: NANOG, OCT4, SOX2. Cell invasion-related CSC markers: ALDHA1, BMI-1. Tumor proliferation-related CSC markers: KLF4, nestin. *P<0.05; **P<0.01; ***P<0.001, Student's t-test. [Figure 10C] RNA expression analysis of the key transcription factor SOX2 by RT-PCR in BxPC-3GR cells and BxPC-3GR-derived spheroids. Drug resistance-related CSC markers: NANOG, OCT4, SOX2. Cell invasion-related CSC markers: ALDHA1, BMI-1. Tumor proliferation-related CSC markers: KLF4, nestin. *P<0.05; **P<0.01; ***P<0.001, Student's t-test. [Figure 10D] RT-PCR analysis of RNA expression of the key transcription factor ALDHA1 in BxPC-3GR cells and BxPC-3GR-derived spheroids. Drug resistance-related CSC markers: NANOG, OCT4, SOX2. Cell invasion-related CSC markers: ALDHA1, BMI-1. Tumor proliferation-related CSC markers: KLF4, nestin. *P<0.05; **P<0.01; ***P<0.001, Student's t-test. [Figure 10E]RT-PCR analysis of RNA expression of the key transcription factor BMI-1 in BxPC-3GR cells and BxPC-3GR-derived spheroids. Drug resistance-related CSC markers: NANOG, OCT4, SOX2. Cell invasion-related CSC markers: ALDHA1, BMI-1. Tumor proliferation-related CSC markers: KLF4, nestin. *P<0.05; **P<0.01; ***P<0.001, Student's t-test. [Figure 10F] RNA expression analysis of the key transcription factor KLF4 by RT-PCR in BxPC-3GR cells and BxPC-3GR-derived spheroids. Drug resistance-related CSC markers: NANOG, OCT4, SOX2. Cell invasion-related CSC markers: ALDHA1, BMI-1. Tumor proliferation-related CSC markers: KLF4, nestin. *P<0.05; **P<0.01; ***P<0.001, Student's t-test. [Figure 10G] RT-PCR analysis of RNA expression of the key transcription factor nestin in BxPC-3GR cells and BxPC-3GR-derived spheroids. Drug resistance-related CSC markers: NANOG, OCT4, SOX2. Cell invasion-related CSC markers: ALDHA1, BMI-1. Tumor proliferation-related CSC markers: KLF4, nestin. *P<0.05; **P<0.01; ***P<0.001, Student's t-test. [Figure 11] CXCR1 / CXCR2 mRNA expression in BxPC-3GR-derived spheroids after treatment with 100 nM gemcitabine (GEM) is shown. *P<0.05; **P<0.01; ***P<0.001, Student's t-test. [Figure 12A] RT-PCR analysis was performed to measure the effect of ONCO P-8 in combination with gemcitabine on the expression level of NANOG in BxPC-3GR-derived spheroids. Drug resistance-related CSC markers: NANOG, OCT4, SOX2. Cell invasion-related CSC markers: ALDHA1, BMI-1. Tumor growth-related CSC markers: KLF4, nestin. Drug concentrations: ONCO P-8 (400 ng / ml; i.e., 47 nM), gemcitabine (100 nM). *P<0.05; **P<0.01; ***P<0.001, Student's t-test. [Figure 12B] RT-PCR analysis was performed to measure the effect of ONCO P-8 in combination with gemcitabine on the expression level of OCT4 in BxPC-3GR-derived spheroids. Drug resistance-related CSC markers: NANOG, OCT4, SOX2. Cell invasion-related CSC markers: ALDHA1, BMI-1. Tumor growth-related CSC markers: KLF4, nestin. Drug concentrations: ONCO P-8 (400 ng / ml; i.e., 47 nM), gemcitabine (100 nM). *P<0.05; **P<0.01; ***P<0.001, Student's t-test. [Figure 12C] RT-PCR analysis was performed to measure the effect of ONCO P-8 in combination with gemcitabine on the expression level of SOX2 in BxPC-3GR-derived spheroids. Drug resistance-related CSC markers: NANOG, OCT4, SOX2. Cell invasion-related CSC markers: ALDHA1, BMI-1. Tumor growth-related CSC markers: KLF4, nestin. Drug concentrations: ONCO P-8 (400 ng / ml; i.e., 47 nM), gemcitabine (100 nM). *P<0.05; **P<0.01; ***P<0.001, Student's t-test. [Figure 12D] RT-PCR analysis was performed to measure the effect of ONCO P-8 in combination with gemcitabine on the expression level of ALDHA1 in BxPC-3GR-derived spheroids. Drug resistance-related CSC markers: NANOG, OCT4, SOX2. Cell invasion-related CSC markers: ALDHA1, BMI-1. Tumor growth-related CSC markers: KLF4, nestin. Drug concentrations: ONCO P-8 (400 ng / ml; i.e., 47 nM), gemcitabine (100 nM). *P<0.05; **P<0.01; ***P<0.001, Student's t-test. [Figure 12E]RT-PCR analysis was performed to measure the effect of ONCO P-8 in combination with gemcitabine on the expression level of BMI-1 in BxPC-3GR-derived spheroids. Drug resistance-related CSC markers: NANOG, OCT4, SOX2. Cell invasion-related CSC markers: ALDHA1, BMI-1. Tumor proliferation-related CSC markers: KLF4, nestin. Drug concentrations: ONCO P-8 (400 ng / ml; i.e., 47 nM), gemcitabine (100 nM). *P<0.05; **P<0.01; ***P<0.001, Student's t-test. [Figure 12F] RT-PCR analysis was performed to measure the effect of ONCO P-8 in combination with gemcitabine on the expression level of KLF4 in BxPC-3GR-derived spheroids. Drug resistance-related CSC markers: NANOG, OCT4, SOX2. Cell invasion-related CSC markers: ALDHA1, BMI-1. Tumor growth-related CSC markers: KLF4, nestin. Drug concentrations: ONCO P-8 (400 ng / ml; i.e., 47 nM), gemcitabine (100 nM). *P<0.05; **P<0.01; ***P<0.001, Student's t-test. [Figure 12G] RT-PCR analysis was performed to measure the effect of ONCO P-8 in combination with gemcitabine on the expression level of nestin in BxPC-3GR-derived spheroids. Drug resistance-related CSC markers: NANOG, OCT4, SOX2. Cell invasion-related CSC markers: ALDHA1, BMI-1. Tumor growth-related CSC markers: KLF4, nestin. Drug concentrations: ONCO P-8 (400 ng / ml; i.e., 47 nM), gemcitabine (100 nM). *P<0.05; **P<0.01; ***P<0.001, Student's t-test. [Figure 13A]This figure shows that the combined use of ONCO P-8 and gemcitabine inhibits tumor growth and extends the lifespan of PDAC xenograft tumor models. PDAC xenograft tumor models were treated with ONCO P-8, gemcitabine combined with ONCO P-8, gemcitabine alone, or PBS. Gemcitabine was administered at approximately 100 mg / kg of subject weight twice weekly. ONCO P-8 was administered at approximately 500 μg / kg of subject weight three times weekly. This figure shows the tumor size (mm3) of the xenograft models. [Figure 13B] This shows that the combined use of ONCO P-8 and gemcitabine inhibits tumor growth and extends the lifespan of PDAC xenograft tumor models. PDAC xenograft tumor models were treated with ONCO P-8, gemcitabine combined with ONCO P-8, gemcitabine alone, or PBS. Gemcitabine was administered at approximately 100 mg / kg of subject weight twice weekly. ONCO P-8 was administered at approximately 500 μg / kg of subject weight three times weekly. The figure shows the weight (g) of the xenograft models. [Figure 13C] This figure shows that the combined use of ONCO P-8 and gemcitabine inhibits tumor growth and extends the lifespan of PDAC xenograft tumor models. PDAC xenograft tumor models were treated with ONCO P-8, gemcitabine in combination with ONCO P-8, gemcitabine alone, or PBS. Gemcitabine was administered at approximately 100 mg / kg of subject weight twice weekly. ONCO P-8 was administered at approximately 500 μg / kg of subject weight three times weekly. This figure shows the survival rate (%) of the xenograft models. The survival rate of xenograft models treated with ONCO P-8 or gemcitabine in combination with ONCO P-8 was approximately 100%. [Figure 14A]This figure shows that the combined use of ONCO P-8 and gemcitabine suppresses gemcitabine-resistant tumor growth and extends the lifespan of a gemcitabine-resistant PDAC xenograft tumor model. The gemcitabine-resistant PDAC xenograft tumor model was treated with gemcitabine in combination with ONCO P-8 or gemcitabine alone. Gemcitabine was administered at approximately 100 mg / kg of subject weight via the intraperitoneal (IP) route twice weekly. ONCO P-8 was administered at approximately 500 μg / kg of subject weight via the intraperitoneal (IP) route three times weekly. This figure is a schematic diagram showing the experimental procedure for the gemcitabine-resistant PDAC xenograft tumor model. [Figure 14B] This figure shows that the combined use of ONCO P-8 and gemcitabine suppresses gemcitabine-resistant tumor growth and extends the lifespan of gemcitabine-resistant PDAC xenograft tumor models. Gemcitabine-resistant PDAC xenograft tumor models were treated with gemcitabine in combination with ONCO P-8 or gemcitabine alone. Gemcitabine was administered at approximately 100 mg / kg of subject weight via the intraperitoneal (IP) route twice weekly. ONCO P-8 was administered at approximately 500 μg / kg of subject weight via the intraperitoneal (IP) route three times weekly. This figure shows tumor size (mm3) in gemcitabine-resistant xenograft tumor models. Data are presented as mean ± SEM (n=8). *P<0.05; **P<0.01; ***P<0.001, Student's t-test. [Figure 14C] This shows that the combined use of ONCO P-8 and gemcitabine suppresses gemcitabine-resistant tumor growth and extends the lifespan of a gemcitabine-resistant PDAC xenograft tumor model. The gemcitabine-resistant PDAC xenograft tumor model was treated with gemcitabine in combination with ONCO P-8 or gemcitabine alone. Gemcitabine was administered intraperitoneally (IP) at approximately 100 mg / kg of subject weight twice weekly. ONCO P-8 was administered intraperitoneally (IP) at approximately 500 μg / kg of subject weight three times weekly. This figure shows the appearance of tumors obtained after sacrifice on day 72 (scale bar = 10 mm). [Figure 14D]This figure shows that the combined use of ONCO P-8 and gemcitabine suppresses gemcitabine-resistant tumor growth and extends the lifespan of a gemcitabine-resistant PDAC xenograft tumor model. Gemcitabine-resistant PDAC xenograft tumor models were treated with gemcitabine in combination with ONCO P-8 or gemcitabine alone. Gemcitabine was administered at approximately 100 mg / kg of subject weight via the intraperitoneal (IP) route twice weekly. ONCO P-8 was administered at approximately 500 μg / kg of subject weight via the intraperitoneal (IP) route three times weekly. This figure shows the survival rate of the "ONCO P-8 + gemcitabine group" compared to the "gemcitabine group" up to day 72. Data are presented as mean ± SEM (n=8). *P<0.05; **P<0.01; ***P<0.001, Student's t-test). [Figure 15A] Figure 1 shows that the combined use of ONCO P-8 and gemcitabine reduces IL-8, CXCR1 and CXCR2 mRNA expression in gemcitabine-resistant PDAC xenograft tumor model.This figure shows the IL-8 mRNA expression comparison between "gemcitabine + ONCO P-8 group" and "gemcitabine group". [Figure 15B] Figure 1 shows that the combined use of ONCO P-8 and gemcitabine reduces IL-8, CXCR1 and CXCR2 mRNA expression in gemcitabine-resistant PDAC xenograft tumor model.This figure shows the CXCR1 mRNA expression comparison between " gemcitabine + ONCO P-8 group " and " gemcitabine group ". [Figure 15C] Figure 1 shows that the combined use of ONCO P-8 and gemcitabine reduces IL-8, CXCR1 and CXCR2 mRNA expression in gemcitabine-resistant PDAC xenograft tumor model.This figure shows the CXCR2 mRNA expression comparison between " gemcitabine + ONCO P-8 group " and " gemcitabine group ". [Figure 16A] Binding capacity of ONCO P8-CXCR2 complex formation compared to IL-8-CXCR2 complex formation. This figure shows the binding capacity of ONCO P8-CXCR2 complex formation. [Figure 16B]Binding capacity of ONCO P8-CXCR2 complex formation compared to IL-8-CXCR2 complex formation. This figure shows the binding capacity of IL-8-CXCR2 complex formation. [Figure 17A] Binding capacity of ONCO P8-CXCR1 complex formation compared to IL-8-CXCR1 complex formation. This figure shows the binding capacity of ONCO P8-CXCR1 complex formation. [Figure 17B] Binding capacity of ONCO P8-CXCR1 complex formation compared to IL-8-CXCR1 complex formation. This figure shows the binding capacity of IL-8-CXCR1 complex formation. DETAILED DESCRIPTION OF THE INVENTION

[0022] The following definitions are intended to clarify, not limit, the terms defined. If a particular term used herein is not specifically defined, such term should not be considered indefinite. Rather, the term is used within the meaning accepted by those skilled in the art.

[0023] As used herein, the term "pharmaceutical agent" means a compound that has pharmacological activity or effect on a patient. The terms "pharmaceutical agent," "active ingredient," "compound," and "drug" are used interchangeably herein.

[0024] The term "cytokine" refers to a small functional peptide that, under physiological conditions, regulates intercellular communication within various body tissues. Cytokines are also called interleukins, monokines, lymphokines, chemokines, and growth factors. Local tissue or circulating cytokine levels are altered in many cancers, which can affect development / progression, treatment, and prognosis. For example, elevated cytokine levels have been associated with reduced anticancer activity of various treatments. Cytokines have also been demonstrated to exacerbate the toxic effects of chemotherapeutic agents and affect drug metabolism. Inflammatory cytokines, such as interferons and interleukins, produced in the tumor microenvironment are involved in stimulating or suppressing disease progression.

[0025] As used herein, the term "pharmaceutical combination" or "combination" refers to the combined administration of a therapeutic agent, which may be a chemokine receptor antagonist-modified peptide and / or may be combined with a chemotherapeutic agent. In the present invention, the therapeutic agent includes a chemokine receptor antagonist-modified peptide and / or is combined with a chemotherapeutic agent, which may be administered independently, simultaneously or separately within a time interval that allows the combination partners to exhibit a synergistic effect.

[0026] As used herein, the term "synergistic" or "synergistic effect" refers to a therapeutic effect achieved by the combination of the present invention and / or the method of treating cancer of the present invention, which effect is greater than the sum of the effects achieved by using the chemokine receptor antagonist-modified peptide and the chemotherapeutic agent alone separately. Advantageously, such synergy between therapeutic agents allows for the use of lower dosages of one or both therapeutic agents, resulting in greater efficacy at the same dosage, and / or prevents or delays the development of drug resistance. A synergistic effect can be achieved by co-formulation of the therapeutic agents contained in the pharmaceutical combinations or compositions described herein, or by simultaneous administration of the therapeutic agents using a unit dosage form or by simultaneous or sequential administration as separate formulations.

[0027] As used herein, the term "therapeutically effective amount" refers to a certain amount of a chemokine receptor antagonist-modified peptide or / in combination with a chemotherapeutic agent that is effective to produce a desired therapeutic response in a particular patient (subject) suffering from cancer. In particular, the term "therapeutically effective amount" includes an amount of a therapeutic agent that achieves a desired therapeutic effect upon administration. In the present invention, the desired therapeutic effect includes partial or complete inhibition, delay, or prevention of cancer progression, including cancer metastasis, in a subject; inhibition, delay, or prevention of cancer recurrence, including cancer metastasis; and / or prevention of cancer onset or development. With regard to therapeutic agents, i.e., therapeutic amounts of a chemokine receptor antagonist-modified peptide or / in combination with a chemotherapeutic agent, it is also considered, within the scope of sound medical judgment, that the amount of each therapeutic agent used to treat a subject be low enough to avoid undesirable or severe side effects. When used in combination, the therapeutically effective amount will vary with the age and health of the end user, the severity of the cancer, the duration of treatment, the nature of any other concomitant therapy, the specific type of therapeutic agent used in the treatment, the particular pharmaceutically acceptable carrier utilized in the pharmaceutical composition containing the therapeutic agent, and other relevant factors.

[0028] As used herein, the term "subject" refers to an animal, particularly a mammal, more particularly a human. As used herein, the term "mammal" refers to a warm-blooded vertebrate of the class Mammalia, including humans. The term mammal includes animals such as cats, dogs, rabbits, cows, horses, sheep, goats, monkeys, mice, rats, gerbils, guinea pigs, pigs, and humans. The term "subject" can be used synonymously with the term patient. In the present invention, the expression "subject in need thereof" refers to a subject in need of cancer treatment. Alternatively, the expression "subject in need thereof" refers to a subject (patient) diagnosed with cancer.

[0029] The term "administration" or "administering" includes the route by which the compound of the present invention is introduced into a subject to perform its function. Examples of administration routes that can be used include injection (subcutaneous, intravenous, parenteral, intraperitoneal, intrathecal), oral, inhalation, rectal, and transdermal. Pharmaceutical preparations can be administered in a form suitable for each administration route.

[0030] As used herein, "treating" or "treatment" means to cure or treat a disease or condition (e.g., cancer, tumor, neoplastic condition) in a subject / patient, such as a mammal (especially a human or companion animal), including ameliorating the disease or condition, i.e., eliminating or regressing the disease or condition in the subject / patient; inhibiting the disease or condition, i.e., delaying or arresting the onset of the disease or condition in the subject / patient; or alleviating the symptoms of the disease or condition in the subject / patient.

[0031] As used herein, the term "pharmaceutically acceptable" means that the carriers, diluents, excipients, and / or salts used in the composition must be compatible with other formulation ingredients and not deleterious to the recipient. "Pharmaceutically acceptable" also means that a composition or dosage form is, within the scope of sound medical judgment, suitable for use in subjects, such as animals or humans, without excessive toxicity, irritation, allergic response, or other problem or complication, and is commensurate with a reasonable benefit / risk ratio.

[0032] As used herein, the term "neoplasm" means abnormal cell or tissue growth and is understood to include benign, i.e., non-cancerous, and malignant, i.e., cancerous, growths. The term "neoplastic" means of or relating to a neoplasm. The term "antineoplastic agent" is understood to mean a substance that produces an antineoplastic effect in a tissue, system, animal, mammal, human, or other subject.

[0033] Diseases that can be treated using the compounds of the present invention include, but are not limited to, cancer, e.g., cancerous tumors. "Cancer" is intended to mean any disease caused by or resulting in inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both.

[0034] As used herein, the term "pancreatic cancer" refers to any cancer that originates in pancreatic cells, including metastatic and localized forms of pancreatic cancer. In certain embodiments, certain subpopulations of pancreatic cancer patients can be treated with the combination therapy of the present invention. The drug combination of the present invention can be used to promote increased efficacy and reduced dosages of both drugs required to achieve efficacy.

[0035] The term "cancer stem cells" or CSCs refers to cells with tumor-initiating and tumor-maintaining capabilities, including the ability to proliferate extensively, form new tumors, and sustain cancer growth, i.e., cells with limitless proliferative potential that promote tumor formation and growth. CSCs are biologically distinct from bulk tumor cells and possess stem cell-associated properties, particularly the ability to self-renew and proliferate and give rise to all cell types found in a particular cancer specimen. The term "cancer stem cells" or CSCs encompasses both genetic changes in stem cells (SCs) and genetic changes in cells that result in CSCs.

[0036] CSCs are also called tumor-initiating cells, cancer stem-like cells, stem-like cancer cells, highly tumorigenic cells, tumor stem cells, solid tumor stem cells, or hyper-malignant cells. On the other hand, CSCs have been demonstrated to be essentially involved in tumorigenesis, cancer metastasis, and cancer recurrence.

[0037] As used herein, the term "differentiation" of cancer stem cells refers to both the conversion of cancer stem cells into multipotent tumor progenitor cells and the conversion of multipotent tumor progenitor cells into unipotent tumor progenitor cells and / or well-differentiated tumor cells.

[0038] The term "expression" refers to the biosynthesis of a gene product. For example, in the case of a coding sequence, expression includes transcription of the coding sequence into mRNA and translation of the mRNA into one or more polypeptides. Conversely, expression of a non-coding sequence includes only transcription of the non-coding sequence into a transcript.

[0039] As used herein, the term "antiproliferative effect" refers to the ability of an agent or agents of the invention to inhibit cancer cell growth and division or reduce the incidence of cancer cell growth and division in an individual. In certain embodiments, an agent or agents of the invention exert an antiproliferative effect by affecting cytokine production. In certain embodiments, an agent or agents of the invention reduce the incidence of cancer cell growth and division in an individual by about 1% to about 99.0% compared to existing drugs known to have antiproliferative effects.

[0040] As used herein, the term "anti-invasive effect" refers to the ability of an agent or agents of the present invention to reduce the incidence of cancer or tumor invasion in an individual. In one embodiment, an agent or agents of the present invention reduces the incidence of tumor invasion in an individual by about 1% to about 99.0% compared to an existing drug known to have an anti-invasive effect.

[0041] As used herein, the term "anti-migration effect" refers to the ability of an agent or agents of the present invention to prevent cancer cell migration or reduce the incidence of tumor cell migration in an individual. The process of invasion is triggered when these cells cross the basement membrane and extracellular matrix and progress to intravasation as they invade the lymphatic or vascular circulation. Metastatic cells then migrate through the circulatory system and invade the vascular basement membrane and extracellular matrix in a process known as extravasation. Ultimately, these cells attach to a new location, proliferate, and generate a secondary tumor. In certain embodiments, an agent or agents of the present invention reduces the incidence of tumor cell migration in an individual by about 1% to about 99.0% compared to existing drugs known to have anti-migration effects.

[0042] As used herein, the term "anti-metastatic effect" refers to the ability to prevent or reduce the occurrence of at least one of the following steps in the metastatic process: (1) detachment of cancer cells from the primary site, (2) introduction and invasion into new blood vessels, (3) egress from the blood circulation, and (4) establishment of new colonies at distant locations. In certain embodiments, the agent or agents of the invention reduce the incidence of a step in the metastatic process in an individual by about 1% to about 99.0% compared to existing drugs known to have anti-metastatic effects.

[0043] "Clinical benefit" refers to a term used by doctors and / or clinicians treating cancer. This term encompasses any benefit understood or perceived by a subject / patient during treatment. As used herein, this term includes, but is not limited to, one or more clinical benefits being one or more of: reduction in tumor size; inhibition or reduction in tumor growth; delay in progression, time to no new tumors or lesions, reduction in the formation of new tumors, increased survival or progression-free survival, and freedom from metastasis.

[0044] In one aspect, the present invention provides a pharmaceutical composition for removing anticancer drug resistance and increasing anticancer drug sensitivity, the composition comprising at least a chemokine receptor antagonist-modified peptide, and further comprising a chemotherapeutic agent.

[0045] The terms "amino acid sequence," "protein," "polypeptide," and "peptide" are used interchangeably herein to refer to two or more amino acids, or "residues," covalently linked by an amide bond or equivalent. The amino acid sequence may be joined by non-natural and non-amide chemical bonds.

[0046] Optionally, in an exemplary embodiment of the invention, chemokine receptor antagonist-modified peptides of the invention include, but are not limited to, ONCO P-8 (SEQ ID NO: 1).

[0047] The sequence of ONCO P-8 (SEQ ID NO: 1) which can be used in accordance with the present invention is shown below: SEQ ID NO:1: GSKELRCQCIRSYSKPFHPKFIKELRVIPASQFCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAENS

[0048] Furthermore, ONCO P-8 was designed as a similar protein to CXCL8 (IL-8) and is an antagonist of CXCR1 and CXCR2.

[0049] In one embodiment, in the present invention, ONCO P-8 directly binds to IL-8 (CXCL8), thereby inhibiting the binding of IL-8 to its receptors CXCR1 and CXCR2.

[0050] In another embodiment, the pharmaceutical composition further comprises a drug, which comprises a chemotherapeutic agent, a pharmaceutically acceptable buffer, diluent, carrier, adjuvant, or excipient.

[0051] Some different types of chemotherapeutic agents include, for example, antimetabolites, antibiotics, alkylating agents, plant alkaloids, hormones (e.g., corticosteroids, sex hormones), immunomodulators, anticoagulants, antithrombotic agents, nitrosoureas, mitotic inhibitors, L-asparaginase, tretinoin, and other natural products. Specific, non-limiting examples of these classes of drugs, and cancers that can be treated by their use, are provided below.

[0052] Antimetabolites are believed to interfere with normal metabolic pathways, including those requiring the creation of new DNA. Common antimetabolites include, but are not limited to, folate antagonists (e.g., methotrexate, pemetrexed (Alimta®)), purine antagonists (e.g., 6-mercaptopurine, 6-thioguanine, fludarabine, dacarbazine, cladribine, and pentostatin), and pyrimidine antagonists (e.g., gemcitabine (Gemzar®), capecitabine, 5-fluorouracil (5-FU (e.g., capecitabine)), cytarabine (cytosine arabinoside or Ara C), and azacitidine (Vidaza®)).

[0053] Gemcitabine is a pyrimidine analogue known as antimetabolite, which belongs to a general group of chemotherapy agents and also acts as a radiosensitizer. Gemcitabine exhibits cell division phase specificity, primarily killing cells undergoing DNA synthesis, i.e., S-phase cells, and blocking cellular progression through the G1 / S-phase boundary.

[0054] Other antimetabolites that can be used together include, but are not limited to, tegafur, raltitrexed, hydroxyurea, and floxuridine.Gallium nitrate is another antimetabolite that inhibits ribonucleotide reductase.The antimetabolites described above can be used in combination with one or more other antimetabolites and / or one or more different classes of chemotherapeutic agents.

[0055] As noted above, another class of chemotherapeutic agents that can be used includes anticancer antibiotics, including, for example, anthracyclines (e.g., doxorubicin (adriamycin), epirubicin, daunorubicin, and idarubicin), dactinomycin, idarubincin, plicamycin, mitomycin, and bleomycin.

[0056] More specifically, the antitumor antibiotic is selected from the group consisting of, but not limited to, actinomycin D, mitoxantrone, aclarubicin, and neocarzinostatin.

[0057] Alkylating agents are chemotherapeutic agents that attack negatively charged sites on DNA (e.g., oxygen, nitrogen, phosphorus, and sulfur atoms), bind to DNA, and thus alter replication, transcription, and even base pairing. DNA alkylation also leads to DNA strand breaks and cross-linking. By altering DNA in this way, cellular activity is effectively stopped and cancer cells are killed. Common alkylating agents include, but are not limited to, procarbazine, isophosphamide (IFO), cyclophosphamide, melphalan, chlorambucil, dacarbazine, busulfan, thiotepa, altretamine, cisplatin, carboplatin, nitrosoureas, carmustine, alkyl sulfonates, ethyleneimines, altretamine (hexamethylmelamine), nitrogen mustard (mechlorethamine), temozolomide, imidazoterazine, streptozocin, triazenes, temozolomide, mechlorethamine, mustine, uramustine, uracil mustard, ifosfamide, oxaliplatin, lomustine (CCNU), etc. Alkylating agents such as those described above can be used in combination with one or more other alkylating agents and / or with one or more different classes of chemotherapeutic agents.

[0058] Additional types of chemotherapeutic agents that can be administered according to the present invention are plant alkaloids, such as vinblastine, vincristine, etoposide, teniposide, topotecan, irinotecan, paclitaxel, and docetaxel.

[0059] Still further, in some embodiments, the plant alkaloid is selected from the group consisting of berberine, oxyberberine, berbamine, palmatine, magnoflorine, phellodendrine, jateoridine, candicine, menisperine, coptisine, wollenine, columbamine, epiberberine, hydrastine, canadine, hydrastidine, oxycyanthine, berberrubine, and isotetridine.

[0060] Further types of anti-cancer agents that can be used in accordance with the present invention are anticoagulants and antithrombotic agents, for example, heparin (e.g., low molecular weight heparin or heparin sulfate) or warfarin.

[0061] In further embodiments, the antimitotic agent is selected from the group consisting of, but not limited to, paclitaxel, docetaxel, vinblastine, vincristine, and etoposide.

[0062] Hormonal therapy agents block cell receptors, suppress the in vivo production of hormones, and / or remove or alter hormone receptors on cells, the net result of all of this is slowing or stopping tumor growth. Conventional hormone therapy agents include, but are not limited to, antiestrogens (e.g., tamoxifen, toremifene, fulvestrant, raloxifene, droloxifene, idoxifene, etc.), progestogens (e.g., megestrol acetate, etc.), aromatase inhibitors (e.g., anastrozole, letrozole, exemestane, vorozole, exemestane, fadrozole, aminoglutethimide, exemestane, 1-methyl-1,4-androstadiene-3,17-dione, etc.), antiandrogens (e.g., bicalutamide, nilutamide, flutamide, cyproterone acetate, etc.), luteinizing hormone-releasing hormone agonists (LHRH agonists) (e.g., goserelin, leuprolide, buserelin, etc.); 5-α-reductase inhibitors, such as finasteride, etc.

[0063] The "immunomodulator" can be any suitable immunomodulator, such as, for example, a cytokine or adjuvant, obtained from any suitable source, such as a mammal, e.g., a human. Desirably, the immunomodulator induces or stimulates an immune response to a viral antigen expressed by the cell line. Cell targeting means can also be considered immunomodulators. Similarly, antibodies (or fragments thereof that react with antigens), antisense molecules, dsRNAi, and the like, can also be considered immunomodulators to the extent that they inhibit or block the ability of viral gene products to block the action of interferon, as desired.

[0064] Examples of suitable immunomodulatory cytokines include interferons (e.g., IFNα, IFNβ, and IFNγ), interleukins, tumor necrosis factors (e.g., TNFα and TNFβ), erythropoietin (EPO), FLT-3 ligand, gIp10, TCA-3, macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF), as well as functional fragments of any of the foregoing. The most preferred immunomodulatory cytokine is GM-CSF, e.g., human GM-CSF, including functional fragments thereof. Examples of adjuvants include, but are not limited to, heat shock proteins, CpG, Listeria monocytogenes, aluminum hydroxide (for use with soluble antigens), aluminum phosphate (alum; for use with soluble antigens), muramyl dipeptide, muramyl tripeptide, Mycobacterium tuberculosis, QuilA (a purified saponin from the plant Quillaja saponaria) alone or in further combination with glycosides, cholesterol, and / or phospholipids, empty adenovirus capsids, and the like.

[0065] There are no particular restrictions on the weight, volume, or concentration ratio between the chemokine receptor antagonist-modified peptide and the chemotherapeutic agent. Those skilled in the art can select an appropriate ratio between the chemokine receptor antagonist-modified peptide and the chemotherapeutic agent depending on the disease, particularly when the chemokine receptor antagonist-modified peptide exhibits a synergistic effect in combination with the chemotherapeutic agent. In one embodiment, the chemokine receptor antagonist-modified peptide is administered at a dose of about 0.01 mg / kg to about 500 mg / kg of subject (patient) body weight once to three times a week.

[0066] Additionally, the present invention provides use of the pharmaceutical composition for removing anticancer drug resistance and increasing sensitivity to anticancer drugs, and the pharmaceutical composition for treating cancer, inhibiting cancer cell proliferation and / or inhibiting cancer metastasis.

[0067] The pharmaceutical composition of the present invention can suppress angiogenesis-related or angiogenesis-dependent diseases in a subject. Angiogenesis-related or angiogenesis-dependent diseases include, but are not limited to, vascular invasion and abnormal cell proliferation, such as tumors or cancers. Cancers in the present invention include, but are not limited to, thoracic cancer, abdominal cancer, gastrointestinal cancer, head and neck cancer, brain cancer, endocrine cancer, urinary tract cancer, male reproductive system neoplasms, gynecological cancer, blood cancer, skin cancer, and sarcoma.

[0068] The thoracic cancer is selected from lung cancer, including small cell lung cancer (SCLC) and / or non-small cell lung cancer (NSCLC). NSCLC can be selected from lung adenocarcinoma, squamous cell carcinoma, and / or large cell carcinoma. SCLC can be selected from small cell carcinoma and mixed small cell / large cell carcinoma or mixed small cell lung carcinoma.

[0069] Abdominal cancers include, but are not limited to, liver cancer, colorectal cancer, pancreatic cancer, kidney cancer (renal cell carcinoma), stomach cancer (gastric cancer), adrenocortical carcinoma, primary peritoneal carcinoma, and peritoneal mesothelioma.

[0070] Gastrointestinal cancers include, but are not limited to, esophageal cancer, stomach cancer (gastric cancer), liver cancer (hepatocellular carcinoma), gallbladder and biliary tract cancer, pancreatic cancer, colorectal cancer, small intestine cancer, and anal cancer.

[0071] Head and neck cancers include, but are not limited to laryngeal and hypopharyngeal cancer, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, oral cavity and oropharyngeal cancer, and salivary gland cancer.

[0072] Types of brain tumors include primary or secondary brain tumors, including, but not limited to, astrocytoma, glioblastoma, medulloblastoma, oligodendroglioma, glioma, and brain metastases.

[0073] Endocrine cancers include, but are not limited to, adrenal gland tumors, neuroendocrine tumors, parathyroid tumors, pituitary tumors, and thyroid disorders.

[0074] Urological cancers include, but are not limited to, bladder cancer and urethral cancer.

[0075] Male reproductive system neoplasms include, but are not limited to, prostate cancer, penile cancer, testicular seminoma, and testicular embryonal carcinoma.

[0076] Gynecological cancers include, but are not limited to, cervical cancer, ovarian cancer, uterine cancer (endometrial cancer), vaginal cancer, and vulvar cancer.

[0077] Hematologic cancers include, but are not limited to, leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and multiple myeloma.

[0078] Skin cancers include, but are not limited to, basal cell skin cancer, squamous cell skin cancer, melanoma skin cancer, and Merkel cell skin cancer.

[0079] Sarcomas include, but are not limited to soft tissue sarcoma, osteosarcoma (bone sarcoma), and rhabdomyosarcoma.

[0080] Neoplasias or / and cancers further include, but are not limited to, breast cancer, and neuroblastoma.

[0081] In certain embodiments, the chemokine receptor antagonist modified peptide or / and combination with a chemotherapeutic agent can be administered by conventional routes of administration, including but not limited to oral, intravascular, intradermal, transdermal, intramuscular, intraperitoneal, intratumoral, parenteral, nasal, rectal, sublingual, topical, aerosol, or intratracheal.

[0082] In certain embodiments, the chemokine receptor antagonist-modified peptide and / or one or more chemotherapeutic agents may be administered in a form suitable for oral administration, such as tablets, lozenges, aqueous or oily suspensions, granules, powders, cachets, emulsions, capsules, syrups, elixirs, etc.

[0083] In another embodiment, the chemokine receptor antagonist-modified peptide and / or one or more chemotherapeutic agents can be administered parenterally, such as intramuscularly, intrathecally, subcutaneously, intraperitoneally, by intravenous bolus injection or intravenous infusion. Parenteral administration can be achieved by incorporating the chemokine receptor antagonist-modified peptide and / or chemotherapeutic agent into a solution or suspension.

[0084] In one embodiment, the chemokine receptor antagonist-modified peptide and / or one or more chemotherapeutic agents may be administered in the form of a pharmaceutical composition comprising said chemokine receptor antagonist-modified peptide and / or one or more chemotherapeutic agents and at least one pharmaceutically acceptable diluent, excipient or carrier.

[0085] The pharmaceutical composition comprises a chemokine receptor antagonist-modified peptide and / or at least one chemotherapeutic agent and one or more pharmaceutically acceptable diluents, excipients, or carriers. Pharmaceutically active excipients that can be used for making pills, tablets, coated tablets, and hard gelatin capsules include, but are not limited to, lactose, corn starch or its derivatives, gum arabic, magnesia, or glucose. Carriers that can be used for soft gelatin capsules and suppositories include, but are not limited to, fats and oils, waxes, natural or hardened oils, etc. Suitable carriers for making solutions include, for example, water, physiological sodium chloride solution, phosphate-buffered saline (PBS), or alcohols, such as ethanol, propanol, or glycerol, sugar solutions such as glucose solutions or mannitol solutions, or mixtures of the various solvents mentioned above, for example, injection solutions, or emulsions or syrups. The pharmaceutically acceptable diluents, excipients, or carriers used in the pharmaceutical composition may be conventionally known pharmaceutically acceptable diluents, excipients, or carriers, and these can be selected depending on the dosage form and administration route of the chemokine receptor antagonist-modified peptide and / or chemotherapeutic agent.

[0086] In general, compositions for pharmaceutical use may be prepared by any method known in the art for the manufacture of pharmaceutical compositions. The compositions described herein may be in a form suitable for oral administration, such as, for example, a solid dosage form, such as a tablet, capsule, lozenge, or granule; a liquid dosage form, such as an emulsion, solution, or suspension; a form suitable for parenteral injection (including intravenous, subcutaneous, intramuscular, intravascular, or infusion), such as, for example, a sterile solution, suspension, or emulsion; or a form suitable for topical administration, for example, as an ointment, cream, gel, or lotion.

[0087] Compositions for oral administration may be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, cachets, emulsions, capsules, syrups, or elixirs. Compositions suitable for oral administration may contain standard vehicles, which are preferably of pharmaceutical grade.

[0088] In the case of ointments and creams, the active ingredients (chemokine receptor antagonist-modified peptide and / or chemotherapeutic agent) may be formulated in an oil-in-water or water-in-oil base.

[0089] For intramuscular, intraperitoneal, subcutaneous, and intravenous use, sterile solutions of the active ingredients (chemokine receptor antagonist-modified peptide and / or chemotherapeutic agent) are usually used, and the pH of the solutions should be adjusted and buffered appropriately.

[0090] Furthermore, the anti-cancer efficacy of the chemokine receptor antagonist-modified peptide and / or chemotherapeutic agent contained in the pharmaceutical composition can be delayed or prolonged by appropriate formulation.

[0091] However, the effective amount of the chemokine receptor antagonist-modified peptide and / or chemotherapeutic agent used for administration will vary depending on the severity of the disease (cancer), the severity of symptoms, age, sex, weight, and sensitivity of the subject (patient), the method, time, interval, and duration of administration, and the nature and type of formulation. In certain embodiments, the chemokine receptor antagonist-modified peptide and / or one or more chemotherapeutic agents can be administered during a time frame in which both agents are still active. Those skilled in the art should be able to determine such a time frame by measuring the half-life of the administered therapeutic agent. As previously indicated herein, in the pharmaceutical combinations and / or methods for treating cancer and / or uses for treating cancer according to the present invention, the chemokine receptor antagonist-modified peptide and one or more chemotherapeutic agents can be administered simultaneously or sequentially, and if sequential, can be administered in any order. In another embodiment, the chemokine receptor antagonist-modified peptide and chemotherapeutic agent can be administered so that the peak pharmacokinetic effect of one agent coincides with the peak pharmacokinetic effect of the other agent.

[0092] Alternatively, however, the chemokine receptor antagonist-modified peptides and / or chemotherapeutic agents of the present invention may be used in combination with one or more additional cancer therapeutic agents, for example, the chemokine receptor antagonist-modified peptides and / or chemotherapeutic agents may be used in combination with one, two, three, four, five or more cancer therapeutic agents.

[0093] By the term "in combination," the present invention includes administering the pharmaceutical composition to a subject receiving one or more cancer therapeutic agents in the same course of therapy. Thus, the term encompasses not only the concomitant administration of the pharmaceutical composition with one or more additional cancer therapeutic agents (e.g., as a bolus or infusion), but also the temporally separated administration of these cancer therapeutic agents. For example, the pharmaceutical composition can be administered within a treatment schedule / cycle determined by the patient's oncologist to include one or more additional cancer therapeutic agents administered before, simultaneously with, or after the pharmaceutical composition, depending on any of a variety of factors, such as the severity of symptoms.

[0094] In another embodiment, the therapeutically effective amount of chemokine receptor antagonist-modified peptide or / and chemotherapeutic agent for the treatment of a particular cancer will depend on the type and nature of the cancer, its size, progression, and metastatic status, and should be determined in consultation with a treating physician.

[0095] For example, in some embodiments, the pharmaceutical compositions of the present disclosure are administered monthly, twice monthly, three times monthly, every other week, once weekly, twice weekly, three times weekly, four times weekly, five times weekly, six times weekly, every other day, daily, twice daily, or three times daily.

[0096] The dosage of the pharmaceutical composition used will vary depending on the activity of the specific chemokine receptor antagonist-modified peptide and the condition to be treated, but may be selected from the range of 0.01 to 500 mg / kg of body weight per single dose, particularly from the range of 0.02 to 1 mg / kg of body weight per single dose. While this administration regimen is appropriate for the patient, the dosage may be divided into several separate administrations if desired.

[0097] A representative, non-limiting acceptable dosage for administration of the chemokine receptor antagonist-modified peptide is about 0.01 mg / kg to about 500 mg / kg of subject (patient) body weight, one to three times per week.

[0098] A representative, non-limiting acceptable dosage for gemcitabine administration is about 10 mg / kg to about 500 mg / kg of subject (patient) body weight, once to three times per week.

[0099] In one embodiment, the combination provided by the present invention was evaluated in a specific assay system and under several different in vitro administration schedules. Experimental details are provided herein. The data presented herein clearly demonstrate that chemokine receptor antagonist-modified peptides, particularly ONCO P-8, exhibit synergistic effects when combined with targeted drugs. Furthermore, in one embodiment, subjects can achieve clinical benefits.

[0100] Additional specific embodiments of the present invention include, but are not limited to, the following. [Example]

[0101] cell line Human pancreatic cancer (PDAC) cell lines, BxPC3 and MIAPaCa2, were used in this study. The gemcitabine-resistant BxPC3 cell line (BxPC3GR) was generated as a gemcitabine-resistant cell line by culturing BxPC3 cells in medium containing increasing concentrations of gemcitabine and then maintaining them in 0.5 μM gemcitabine. The gemcitabine-resistant MIAPaCa2 cell line (MIAPaCa2GR) was generated by culturing MIAPaCa2 cells in medium containing increasing concentrations of gemcitabine and then maintaining them in 0.12 μM gemcitabine. BxPC3 and BxPC3GR are maintained in RPMI 1640 medium containing 10% FBS, 1% penicillin-streptomycin, 100 mM L-glutamine, 100 nM HEPES, 10 nM sodium pyruvate, and 25 nM glucose in a humidified cell culture incubator at 37°C with 5% CO. MIAPaCa2 and MIAPaCa2GR are maintained in DMEM containing 10% FBS, 1% penicillin-streptomycin, and 1% L-glutamine. [Example]

[0102] Quantification of CXCR1, CXCR2 and CXCL8 genes Total RNA from cells was isolated using RNA Extraction Reagent (REzol® C & T, Protech Technology Enterprise Co., Taiwan) and quantified using a spectrophotometer (Nanodrop 1000, Thermo Scientific). First-stranded cDNA was synthesized using the PrimeScript RT Reagent Kit (Perfect Real Time) (RR037A, Takara Bio, Japan) according to the user's manual.

[0103] All real-time PCR reactions were performed on a StepOne® Real-Time PCR System (Applied Biosystems®) using specific primers and Taqman probes or a universal probe library (Roche Applied Science). 18 s was used as an internal loading control. Reactions were performed as follows: denaturation at 50°C for 2 min and 95°C for 10 min, 50 cycles of 95°C for 15 s and 60°C for 1 min. Gene expression was calculated by the following formula: Gene Expression = 2 - ΔΔCt. The sequences of all primers used are listed below (F: forward primer; R: reverse primer):

[0104] [Table 1] [Example]

[0105] ONCO P-8 can suppress the invasive ability of human pancreatic cancer (PDAC) cells Cell invasive ability measurements were performed using 8 μm pore size transwells (Corning FluoroBlok®). Transwell inserts were coated with 60 μl of Matrigel (300 μg / ml in serum-free medium (BD Bioscience)) overnight at 37°C under a 5% CO2 atmosphere. 2.5 × 104 cells in 0.2 mL of serum-free growth medium were seeded into the Matrigel-coated upper chamber and then treated with IL-8 (200 ng / ml) with or without ONCO P-8 (400 ng / ml). After 24 hours of incubation at 37°C under 5% CO2, the membranes of the upper chambers were fixed with methanol and stained with propidium iodide (PI). Invaded cells at the bottom of the membrane were imaged and counted in five random fields (magnification ×100) for each specimen using an inverted fluorescence microscope (Observer.Z1, Zeiss).

[0106] To investigate whether cell invasion is mediated by CXCL8 (IL-8) or its signal, the present invention uses ONCO P-8 to conduct invasion experiments. The present results show that ONCO P-8 can reduce the invasive rate in BxPC-3 cell lines. In addition, the present data also show that CXCL8 (IL-8) can enhance cell invasion. However, ONCO P-8 can neutralize the effect of CXCL8 and reduce the invasive rate of BxPC-3 cells (Figure 1). [Example]

[0107] ONCO P-8 can suppress colony formation of human pancreatic cancer (PDAC) cells Cells were seeded into 24-well plates at a concentration of 1,000 cells per well, and treatment was performed 24 hours after seeding. Treatments included treating cells with IL-8 (200 ng / ml), IL-8 (200 ng / ml) + ONCO P-8 (400 ng / ml), or ONCO P-8 (400 ng / ml). After 2 weeks, the medium was removed, the cells were washed with PBS, and stained with 0.1% w / v crystal violet (dissolved in methanol; 32675, Fluka® Analytical, USA). The colony area was identified by camera and analyzed using ImageJ analysis software. The results were calculated as follows: Colony formation rate (%) = (treated area of ​​colonies / control area of ​​colonies) x 100%

[0108] Similarly, PDAC colony formation data indicate that CXCL8 (IL-8) can enhance cell invasion, whereas ONCO P-8 neutralizes the effect of CXCL8 and reduces the colony formation rate of BxPC-3 (Figure 2). [Example]

[0109] Verification of gemcitabine-resistant cancer cell lines To verify the establishment of gemcitabine-resistant pancreatic ductal adenocarcinoma (PDAC) cell lines, MTT of gemcitabine gradient was performed using parental and gemcitabine-resistant (GR) cells.

[0110] BxPC-3 and BxPC-3GR were treated with 0-100 nM gemcitabine and measured by MTT (Figure 3a). Similarly, MIAPaCa2 and MIAPaCa2GR were treated with 0-100 nM gemcitabine and measured by MTT (Figure 3b).

[0111] Drug resistance in GR cell lines is 34-39 times higher than in parental cells (Table 1). In comparison, the IC of gemcitabine in BxPC-3 cells was 50 The IC value for gemcitabine in MIAPaCa2 cells was 6.128 nM, compared with 236.3 nM in BxPC-3GR cells (38.6-fold increased resistance). 50 The value was 250.7 nM in MIAPaCa2GR cells and 8620 nM in MIAPaCa2GR cells (34.4-fold resistance).

[0112] [Table 2] [Example]

[0113] The expression levels of CXCL8, CXCR1 and CXCR2 genes in gemcitabine-resistant cancer cells with and without gemcitabine induction were compared with those in parental cancer cells.

[0114] Parental and gemcitabine-resistant BxPC3 and MIAPaCa2 cells were harvested and examined for IL8, CXCR1, and CXCR2 mRNA expression by Q-PCR. See Figures 4A-4B. GR cell lines exhibit significantly higher expression than parental cells. In other words, CXCL8 (IL-8) mRNA, CXCR1 mRNA, and CXCR2 mRNA expression in GR cells (BxPC-3GR (Figure 4A) and MIAPaCa2GR (Figure 4B)) are significantly elevated compared to parental cells (BxPC-3 (Figure 4A) and MIAPaCa2 (Figure 4B)), respectively. Data are presented as mean ± SD. *P<0.05; **P<0.01; ***P<0.001, Student's t-test. [Example]

[0115] ONCO P-8 attenuates high IL-8 / CXCR1 / CXCR2 gene expression in gemcitabine-resistant PDAC cell lines Furthermore, the present invention also demonstrated that IL-8, CXCR1, and CXCR2 mRNA expression in BxPC-3GR (Figure 5A) and MIAPaCa2GR (Figure 5B) gemcitabine-resistant cancer cells was reduced after treatment with 0.1 μM gemcitabine in combination with ONCO P-8 (400 ng / mL) for one month. Data are shown as mean ± SD. *P<0.05; **P<0.01; ***P<0.001, Student's t-test. Thus, ONCO P-8 competes with IL-8, weakens its feedback, and regulates the downregulation of IL-8, CXCR1, and CXCR2 expression. [Example]

[0116] ONCO P-8 can suppress the invasive ability of gemcitabine-resistant PDAC cells For the invasive ability assay, gemcitabine-resistant cancer cells were treated with gemcitabine (100 nM) combined with ONCO P-8 (400 ng / mL), or gemcitabine combined with ONCO P-8 and IL-8 (200 ng / mL), or gemcitabine combined with IL-8, respectively.

[0117] The results of the transwell invasion assay showed that ONCO P-8 reduced the invasive rate of BxPC-3GR (Figure 6A) and MIAPaCa2GR (Figure 6B) compared with the control group. Meanwhile, ONCO P-8 significantly reduced the invasive rate of the "IL-8 + ONCO P-8 group" in BxPC-3GR (Figure 6A) and MIAPaCa2GR (Figure 6B) compared with the "IL-8 group" in BxPC-3GR (Figure 6A) and MIAPaCa2GR (Figure 6B). Data are shown as mean ± SD. *P < 0.05; **P < 0.01, Student's t-test. [Example]

[0118] ONCO P-8 inhibits the growth rate of gemcitabine-resistant cancer cells Cell proliferation assays were performed using a Cell Counting Kit-8 (CCK-8) (Dojindo, Kumamoto, Japan). Gemcitabine-resistant cancer cells were treated for 24 hours with gemcitabine (100 nM) combined with ONCO P-8 (400 ng / mL, 47 nM) or gemcitabine combined with ONCO P-8 and IL-8 (200 ng / mL, 23.8 nM). The proliferation rate of gemcitabine-resistant cancer cells (BxPC-3GR) was significantly attenuated compared with the IL-8 group (Figure 7). Data are shown as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001, Student's t-test). These data further confirm that cancer cells co-treated with gemcitabine and ONCO P-8 downregulated CXCL8, CXCR1, and CXCR2 (Figures 5A-5B). [Example]

[0119] ONCO P-8 can attenuate the migration and invasion abilities of gemcitabine-resistant cancer cells. Furthermore, the present invention also demonstrated that ONCO P-8 can reduce the migration rate (Figure 8A) and invasive rate (Figure 8B) of gemcitabine-resistant cells. Additionally, the present data also demonstrate that CXCL8 can enhance cell migration and invasion. However, ONCO P-8 can neutralize the effect of CXCL8 and reduce the invasive rate of BxPC-3GR. Drug concentrations: ONCO P-8 (400 ng / ml), reparixin (200 nM), IL-8 (200 ng / ml). Statistical significance: *P<0.05; **P<0.01; ***P<0.001. [Example]

[0120] Comparison of cell morphology and gene expression between gemcitabine-resistant cancer cells and gemcitabine-resistant cancer cell-derived spheroids Chemotherapy kills most cells in a tumor but is thought to leave tumor stem cells behind, which may be an important mechanism of resistance.

[0121] To investigate whether drug resistance is mediated by CXCR1 and CXCR2, several characteristics of cancer stem cells (CSCs) against BxPC-3GR were analyzed (Figures 9A-C, 10A-G, 11, 12A-G).

[0122] Cell morphology of BxPC-3GR parent cells (Figure 9A) and BxPC-3GR-derived spheroids (Figure 9B). Gray arrows indicate BxPC-3GR-derived spheroids. CXCR1 and CXCR2 RNA expression in BxPC-3GR-derived spheroids was higher than in parental BxPC-3GR cells (Figure 9C).

[0123] Furthermore, the data show that CSC-specific markers are more abundant in BxPC-3GR-derived spheroids than in parental BxPC-3GR cells (Figures 10A-G). These results indicate that CSC propensity aids cell resistance to chemotherapy. Furthermore, suppression of CXCR1 / CXCR2 by ONCO P-8 can reduce the expression of these CSC-specific markers. These results also suggest that ONCO P-8 may prevent cancer cells from becoming chemotherapeutic-resistant by suppressing CSC transformation.

[0124] In other words, the present invention demonstrated that ONCO P-8 can suppress cell proliferation, survival, invasion, migration, and CSC markers of pancreatic cancer cells, and revealed the functional properties of ONCO P-8 through IL-8R (CXCR1 / CXCR2) overexpression in cancer cells. [Example]

[0125] The combined use of ONCO P-8 and gemcitabine inhibits tumor growth and extends in vivo survival To evaluate the in vivo efficacy of ONCO P-8, we used the BxPC-3 model in 4-week-old male BALB / c nude mice. BxPC-3 cells were subcutaneously inoculated into the mice. Seven days later, the mice were treated with PBS or ONCO P-8 (500 μg / kg) via IP injection three times a week. The initial tumor size after the first treatment was 128.5 ± 47.8 mm3. ONCO P-8 significantly suppressed in vivo tumor growth (Figure 13A). Furthermore, ONCO P-8 treatment did not result in a decrease in mouse body weight (Figure 13B), indicating that ONCO P-8 may not have toxic effects in vivo. These results indicated that ONCO P-8 significantly suppressed tumor growth and significantly improved the survival rate of BALB / c nude mice (Figure 13C). [Example]

[0126] The combined use of ONCO P-8 and gemcitabine suppresses gemcitabine-resistant tumor growth and extends in vivo survival Furthermore, we established a nude mouse xenograft model with the BxPC-3GR cell line (Figure 14A). Male BALB / c mice aged 4–6 weeks were purchased from BioLASCO Taiwan Co., Ltd. 2.5 × 10 BxPC-3GR cells in 0.2 ml of PBS mixed with an equal volume of Matrigel (suspended in cold PBS at a final concentration of 1.2 × 10 cells / ml) were injected subcutaneously into the back of each mouse (n = 8 per group). After 1 week of inoculation, gemcitabine (gemcitabine hydrochloride >99%, G4177, LC Laboratories) was injected i.p. twice weekly in the "gemcitabine group." ONCO P-8 (500 μg / kg) was administered intraperitoneally (i.p.) three times weekly in the "ONCO P-8 + gemcitabine group." After 72 hours of drug treatment, all mice were sacrificed.

[0127] The tumor size of the "ONCO P-8 combined with gemcitabine group" was significantly reduced compared to the "gemcitabine group" (Figure 14B, Figure 14D). All tumor volumes were calculated using the following formula: tumor size (mm3) = 0.52 × (width × length). The results are shown as tumor size ± SEM.

[0128] Meanwhile, the survival rate up to day 72 in the "ONCO P-8 + gemcitabine group" was effectively increased by 30% compared to the "gemcitabine group" (Figure 14D). After sacrifice, mRNA expression in tumor tissue was examined. [Example]

[0129] The combined use of ONCO P-8 and gemcitabine attenuates IL-8, CXCR1, and CXCR2 mRNA expression in gemcitabine-resistant tumor models BxPC-3GR tumor-bearing BALB / c nude mice were sacrificed on day 72, and IL-8, CXCR1, and CXCR2 mRNA expression in tumor tissues from the gemcitabine and gemcitabine + ONCO P-8 groups was examined (Figures 15A-15C). The results show a significant decrease in gene expression of IL-8 (Figure 15A), CXCR1 (Figure 15B), and CXCR2 (Figure 15C) in the gemcitabine + ONCO P-8 group compared with the gemcitabine group. Data are shown as mean ± SEM (n = 8). *P < 0.05; **P < 0.01; ***P < 0.001, Student's t-test. [Example]

[0130] Comparison of antagonism between ONCO P-8 and IL-8 for binding to CXCR1 and CXCR2 In the present invention, we demonstrated this by measuring ONCO P-8-CXCR2 complex formation and IL-8-CXCR2 complex formation using indirect ELISA. ELISA binding assays showed that ONCO P-8 (Fig. 16A) could bind to CXCR2 3-4 times more potently than IL-8 (Fig. 16B). The K d The K value of ONCO IL-8 was 139 nM.d The value was 471 nM.

[0131] Furthermore, the present invention was further demonstrated by measuring ONCO P-8-CXCR1 complex formation and IL-8-CXCR1 complex formation using indirect ELISA. ELISA binding assays showed that ONCO P-8 (Figure 17A) could bind to CXCR1 3-4 times more potently than IL-8 (Figure 17B). The K d The K value of IL-8 was 286 nM. d The value was 853 nM. d The formula includes: (1) i = A 450nm / A max 450nm , i = ligand / ligand max ;(2)1 / (1-i)=(Lig tot / i) / K d -b, b=fqS rec / K d .

[0132] In summary, the pharmaceutical composition synergistically reduces the expression of IL-8, CXCR1, and CXCR2 in the tumor microenvironment, suppresses tumor growth by downregulating IL-8, inhibits cancer cell migration and invasion, and treats or reduces metastasis. Furthermore, the above drug-resistant cells overexpress IL-8.

[0133] Each feature disclosed in this specification (including any accompanying claims, abstract, and drawings), unless expressly stated otherwise, may be replaced by alternative features serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0134] As indicated, these modifications may be made to the present invention in light of the foregoing description of illustrated embodiments of the invention, and are intended to be included within the spirit and scope of the present invention. Thus, while the present invention has been described herein with reference to specific embodiments thereof, a range of modifications, variations, and substitutions is contemplated in the foregoing disclosure. In some instances, some features of the embodiments of the invention may be employed without the corresponding use of other features without departing from the spirit and scope of the invention as described. Accordingly, many modifications may be made to adapt a particular situation or material to the basic scope and spirit of the present invention.

Claims

1. A pharmaceutical composition for removing anticancer drug resistance and increasing anticancer drug sensitivity, comprising: a chemokine receptor antagonist modified peptide, which is ONCO P-8 as set forth in SEQ ID NO: 1; a drug comprising gemcitabine and a pharmaceutically acceptable buffer, diluent, carrier, adjuvant or excipient; The pharmaceutical composition is administered in a therapeutically effective amount to a subject in need thereof, wherein the subject has been diagnosed with cancer, the cancer is resistant to gemcitabine, the cancer is pancreatic cancer or pancreatic ductal adenocarcinoma, and the ONCO P-8 is capable of binding to CXCR2 and the ONCO P-8 is capable of binding to CXCR1.

2. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition attenuates the expression of IL-8, CXCR1, and CXCR2 in the tumor microenvironment through a synergistic effect, suppresses tumor growth by downregulating IL-8, blocks cancer cell migration and invasion by downregulating IL-8, treats metastasis or reduces metastatic spread, and overcomes resistance to anticancer drugs, wherein drug-resistant cells overexpress IL-8, and the drug-resistant cells are selected from cancer cells.

3. 2. The pharmaceutical composition of claim 1, wherein the cancer cells further comprise cancer stem-like cells, stem-like cancer cells, tumor stem cells, tumor-initiating cells, tumor progenitor cells, multipotent tumor progenitor cells, or unipotent tumor progenitor cells.

4. 2. The pharmaceutical composition of claim 1, wherein the subject with drug-resistant cancer is selected from mammals, and the mammals are selected from cats, dogs, rabbits, cows, horses, sheep, goats, monkeys, mice, rats, gerbils, guinea pigs, pigs, and humans.

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