Pharmaceutical composition for reducing anticancer drug resistance and enhancing anticancer drug sensitivity and use thereof

Abstract

To provide pharmaceutical compositions for reducing anticancer drug resistance and for enhancing sensitivity to anticancer drugs.SOLUTION: Disclosed is a pharmaceutical composition comprising: a chemokine analogue peptide selected from RISE P-8; and a medicament comprising a targeted drug and a pharmaceutically acceptable buffer, diluent, carrier, adjuvant or excipient, where the targeted drug comprises an antibody for a cell surface receptor (or antigen), a small molecule inhibitor of signaling pathway, a mTOR signaling pathway inhibitor, an anti-angiogenic agent or a proteasome inhibitor.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical composition, and in particular to a pharmaceutical composition for reducing anticancer drug resistance and enhancing anticancer drug sensitivity.

[0002] More specifically, the present invention further relates to pharmaceutical compositions comprising targeted drugs in combination with chemokine analog peptides for overcoming anti-cancer drug resistance, treating drug-resistant cancers and patients with drug-resistant cancers, and inhibiting tumor growth or / and metastasis. [Background technology]

[0003] The increasing prevalence of cancer as a leading cause of death in many countries is partly reflected by rapid global population growth and aging: cancer incidence and mortality rates are rising rapidly worldwide.

[0004] Anticancer therapy is essential for cancer treatment. However, cancer often develops resistance to anticancer therapy. When cancer cells acquire resistance to an anticancer drug, the cells often exhibit resistance to other anticancer drugs that are not used in treatment. In other words, drug resistance is a major problem for cancer treatment.

[0005] For example, tyrosine kinase inhibitors (TKIs) are the standard treatment in clinics for patients with advanced EGFR-mutated non-small cell lung cancer (Sharma SV, 2007). The first-generation EGFR-TKI gefitinib (trade name Iressa®) significantly improved the outcomes of patients with NSCLC harboring activating EGFR mutations. However, after a median response period of 12 months, tumor resistance occurred in all patients, and this resistance was due to the emergence of the EGFRT790M resistance mutation in more than half of these patients (Wheeler DL, 2010).

[0006] Furthermore, ELR-CXC chemokines are associated with angiogenesis associated with tumor development, and their induction mechanism is activation by binding of this type of chemokine, particularly CXCL8, to CXCR1 and CXCR2 on endothelial cells (ECs). It has now been demonstrated that many different types of tumors can secrete ELR-CXC chemokines. For example, one or more ELR-CXC chemokines have been shown to confer resistance to EGFR inhibitors by inducing stem cell properties within cancers (Liu YN, 2015).

[0007] Unfortunately, the therapeutic effect is not always sustainable, as tumors develop resistance mutations and become insensitive to existing drugs. In other words, although there are many different antitumor preparations used in clinical practice, in most cases their efficiency is insufficient and the range of diseases that are sensitive to such treatments is limited.

[0008] Therefore, the development of new, more active preparations and such compositions that are effective in treating and preventing primary and acquired resistance tumors remains an ongoing interest of the present invention.

[0009] Therefore, the present invention attempts to address the above situation encountered in the prior art. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Sharma SV, 2007 [Non-patent document 2] Wheeler DL, 2010 [Non-patent document 3] Liu YN, 2015 Summary of the Invention [Problem to be solved by the invention]

[0011] An object of the present invention is to provide an agent / medicine / pharmaceutical composition for overcoming drug resistance, or an agent / medicine / pharmaceutical composition for overcoming anticancer drug resistance. [Means for solving the problem]

[0012] Another aspect of the 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.

[0013] Another aspect of the invention provides a method for treating cancer in an individual comprising administering to the individual a combination therapy comprising one or more chemokine analog peptides and one or more pharmaceutical agents.

[0014] In other embodiments, there is provided the use of a chemokine analog peptide in the manufacture of a medicament for treating cancer in an individual when administered in combination with a targeted drug, a pharmaceutically acceptable buffer, diluent, carrier, adjuvant or excipient, and the use of a targeted drug, a pharmaceutically acceptable buffer, diluent, carrier, adjuvant or excipient in the manufacture of a medicament for treating cancer in an individual when administered in combination with a chemokine analog peptide.

[0015] A detailed description of the present invention is given in the following embodiments with reference to the accompanying drawings. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 shows the full-length amino acid sequence and structure of RISE P-8. [Figure 2A] FIG. 1 shows the expression of CXCL8, CXCR1, and CXCR2 mRNA in parental and gefitinib-resistant cells. [Figure 2B] FIG. 1 shows the expression of CXCL8, CXCR1, and CXCR2 mRNA in parental and gefitinib-resistant cells. [Figure 2C] FIG. 1 shows the expression of CXCL8, CXCR1, and CXCR2 mRNA in parental and gefitinib-resistant cells. [Figure 2D] FIG. 1 shows the expression of CXCL8, CXCR1, and CXCR2 mRNA in parental and gefitinib-resistant cells. [Figure 3A] FIG. 1 shows IL-8 mRNA expression in parental cells and gefitinib-resistant cells mediated by administered gefitinib. [Figure 3B] FIG. 1 shows IL-8 mRNA expression in parental cells and gefitinib-resistant cells mediated by administered gefitinib. [Figure 4A] FIG. 1 shows the expression of CXCR1 and CXCR2 mRNA in parental cells and gefitinib-resistant cells via administered gefitinib. [Figure 4B] FIG. 1 shows the expression of CXCR1 and CXCR2 mRNA in parental cells and gefitinib-resistant cells via administered gefitinib. [Figure 4C] FIG. 1 shows the expression of CXCR1 and CXCR2 mRNA in parental cells and gefitinib-resistant cells via administered gefitinib. [Figure 4D] FIG. 1 shows the expression of CXCR1 and CXCR2 mRNA in parental cells and gefitinib-resistant cells via administered gefitinib. [Figure 5] FIG. 1 is a graphical representation of RISE P-8 antagonizing human neutrophil responses via CXCL8. [Figure 6A] FIG. 1 shows that gene expression of IL-8, CXCR1, and CXCR2 increases in 3D (suspension) conditions of NSCLC cells. [Figure 6B] FIG. 1 shows that gene expression of IL-8, CXCR1, and CXCR2 increases in 3D (suspension) conditions of NSCLC cells. [Figure 7A] FIG. 1 shows that RISE P-8 significantly reduced IL-8 protein expression in NSCLC cells in a 3D (suspension) state. [Figure 7B] FIG. 1 shows that RISE P-8 significantly reduced IL-8 protein expression in NSCLC cells in a 3D (suspension) state. [Figure 8A]FIG. 1 shows the expression levels of CXCR1, CXCR2, and CXCL8 genes in gefitinib-resistant cancer cells treated with gefitinib compared to treatment with gefitinib in combination with RISE P-8. [Figure 8B] FIG. 1 shows the expression levels of CXCR1, CXCR2, and CXCL8 genes in gefitinib-resistant cancer cells treated with gefitinib compared to treatment with gefitinib in combination with RISE P-8. [Figure 8C] FIG. 1 shows the expression levels of CXCR1, CXCR2, and CXCL8 genes in gefitinib-resistant cancer cells treated with gefitinib compared to treatment with gefitinib in combination with RISE P-8. [Figure 8D] FIG. 1 shows the expression levels of CXCR1, CXCR2, and CXCL8 genes in gefitinib-resistant cancer cells treated with gefitinib compared to treatment with gefitinib in combination with RISE P-8. [Figure 9A] FIG. 1 shows that RISE P-8 causes arrest in the G0 / G1 phase. [Figure 9B] FIG. 1 shows that RISE P-8 arrests the cell cycle of gefitinib-resistant NSCLC cells at the G0 / G1 phase. [Figure 10A] FIG. 1 shows that RISE P-8 can inhibit the anchorage-independent growth rate of gefitinib-resistant NSCLC cell lines when treated simultaneously with or without IL-8. [Figure 10B] FIG. 1 shows that RISE P-8 can inhibit the anchorage-independent growth rate of gefitinib-resistant NSCLC cell lines when treated simultaneously with or without IL-8. [Figure 11A] FIG. 1 shows that RISE P-8 can inhibit the long-term growth of gefitinib-resistant NSCLC cells. [Figure 11B] FIG. 1 shows that RISE P-8 can inhibit the long-term growth of gefitinib-resistant NSCLC cells. [Figure 12A]FIG. 1 shows that RISE P-8 can inhibit the invasive ability of gefitinib-resistant NSCLC cells. [Figure 12B] FIG. 1 shows that RISE P-8 can inhibit the invasive ability of gefitinib-resistant NSCLC cells. [Figure 13A] FIG. 1 shows that RISE P-8 can suppress the invasive ability of Lewis lung carcinoma cells. [Figure 13B] FIG. 1 shows that RISE P-8 can suppress the invasive ability of Lewis lung carcinoma cells. [Figure 14A] FIG. 1 shows that the combined use of RISE P-8 and gefitinib inhibits tumor growth and extends lifespan in vivo. [Figure 14B] FIG. 1 shows that the combined use of RISE P-8 and gefitinib inhibits tumor growth and extends lifespan in vivo. [Figure 14C] FIG. 1 shows that the combined use of RISE P-8 and gefitinib inhibits tumor growth and extends lifespan in vivo. [Figure 14D] FIG. 1 shows that the combined use of RISE P-8 and gefitinib inhibits tumor growth and extends lifespan in vivo. [Figure 15A] FIG. 1 shows how the combined use of RISE P-8 and gefitinib attenuates the expression of IL-8, CXCR1, and CXCR2 mRNA in vivo. [Figure 15B] FIG. 1 shows how the combined use of RISE P-8 and gefitinib attenuates the expression of IL-8, CXCR1, and CXCR2 mRNA in vivo. [Figure 15C] FIG. 1 shows how the combined use of RISE P-8 and gefitinib attenuates the expression of IL-8, CXCR1, and CXCR2 mRNA in vivo. DETAILED DESCRIPTION OF THE INVENTION

[0017] Before describing the present invention in further detail, it should be understood that the detailed description and specific examples, while illustrating embodiments of the present invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art. Those skilled in the art can utilize the present invention to its fullest extent based on the description herein. Unless otherwise defined, all technical and specific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0018] The following definitions are intended to clarify, but not limit, the terms defined. If a particular term used herein is not specifically defined, such term shall not be considered undefined. Rather, these terms shall be used within the meaning accepted by those skilled in the art.

[0019] The term "drug combination" or "combination" as used herein refers to the combined administration of therapeutic agents, which may be chemokine analog peptides or may be combined with targeted drugs. In the context of the present invention, therapeutic agents include chemokine analog peptides or those combined with targeted drugs, which may be administered independently, simultaneously or separately, within a time interval that allows the combination partners to exhibit a synergistic effect.

[0020] As used herein, the term "synergistic" or "synergistic effect" refers to a therapeutic effect achieved by the combinations of the present invention and / or by the methods of treating cancer of the present invention, which effect is greater than the sum of the effects produced by the chemokine analog peptide and the targeted drug used alone or separately. Advantageously, such synergistic effect between therapeutic agents may allow for the use of lower doses of one or both therapeutic agents, provide greater efficacy at the same dose, and / or prevent or delay the development of drug resistance. Synergistic effects can be achieved either by co-formulating the therapeutic agents included in the pharmaceutical combinations or compositions described herein, or by administering them simultaneously in a unit dosage form or as separate formulations administered simultaneously or sequentially.

[0021] As used herein, the term "therapeutically effective amount" refers to an amount of a chemokine analog peptide or a combination of a targeted drug 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, when administered, achieves a desired therapeutic effect. In the context of the present invention, the desired therapeutic effect includes partial or complete inhibition, delay, or prevention of cancer progression, such as cancer metastasis; inhibition, delay, or prevention of cancer recurrence, such as cancer metastasis; and / or prevention of the onset or development of cancer in a subject. With regard to the therapeutic amount of a therapeutic agent, i.e., in combination with a chemokine analog peptide or a targeted drug, it is also considered, within the bounds of sound medical judgment, that the amount of each therapeutic agent used to treat a subject be sufficiently low to avoid undesirable or serious side effects. The therapeutically effective amount when used in combination will vary depending on the age and physical condition 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 used in the pharmaceutical composition containing the therapeutic agent, and other relevant factors.

[0022] The term "subject" as used herein refers to animals, particularly mammals, more particularly humans. As used herein, the term "mammal" refers to warm-blooded vertebrates of the "mammal" class, such as 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 interchangeably with the term patient. In the context of the present invention, the phrase "subject in need thereof" refers to a subject in need of treatment for cancer. Alternatively, the phrase "subject in need thereof" refers to a subject (patient) diagnosed with cancer.

[0023] As used herein, "treating" or "treatment" refers to the treatment of or care of a disease or condition (such as cancer, tumor, neoplastic condition, etc.) in a subject / patient, such as a mammal (particularly a human or companion animal), including ameliorating the disease or condition, i.e., eliminating or causing regression of the disease or condition in the subject / patient, inhibiting the disease or condition, i.e., delaying or preventing the onset of the disease or condition in the subject / patient; or alleviating the symptoms of the disease or condition in the subject / patient.

[0024] As used herein, the term "pharmaceutically acceptable" means that the carriers, diluents, excipients, and / or salts used in the compositions must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. "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 undue toxicity, irritation, allergic response, or other problem or complication, or commensurate with a reasonable benefit / risk ratio.

[0025] "Clinical benefit" refers to a phrase used by doctors and / or clinicians treating cancer. This term encompasses any assessed or recognized benefit experienced by a subject / patient during treatment. As used herein, this term includes one or more of clinical benefits, such as, but not limited to, one or more of: a reduction in tumor size, inhibition or reduction in tumor growth, a delay in time to progression, no new tumors or lesions, a reduction in new tumor formation, an increase in survival or progression-free survival, and no metastasis.

[0026] According to one aspect, the present invention provides a pharmaceutical composition for reducing anticancer drug resistance and enhancing anticancer drug sensitivity, comprising at least a chemokine analog peptide, and further comprising a targeting drug.

[0027] Optionally, in an exemplary embodiment of the present invention, chemokine analog peptides of the present invention include, but are not limited to, RISE P-8 (SEQ ID NO: 1).

[0028] The sequence of RISE P-8 (SEQ ID NO: 1) which can be used in accordance with the present invention is shown below: SEQ ID NO:1: GSKELRCQCIRSYSKPFHPKFIKELRVIPASQFCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAENS

[0029] Furthermore, RISE P-8 was designed as a CXCL8 (IL-8) analogue and was found to be an antagonist of CXCR1 and CXCR2. The complete amino acid sequence and protein structure of RISE P-8 are shown in Figure 1.

[0030] In one embodiment, RISE P-8 directly binds to IL-8 (CXCL8), thereby inhibiting the binding of IL-8 to its receptors CXCR1 and CXCR2 in the present invention.

[0031] In another embodiment, the pharmaceutical composition further comprises a pharmaceutical agent, which comprises a target drug, a pharmaceutically acceptable buffer, diluent, carrier, adjuvant, or excipient.

[0032] The aforementioned targeted drugs of the present invention include antibodies against cell surface receptors (or antigens), small molecule inhibitors of signal transduction pathways, mTOR signal transduction pathway inhibitors, anti-angiogenic agents, and proteasome inhibitors. Preferably, the targeted drug is a small molecule inhibitor of a signal transduction pathway.

[0033] Antibodies against cell surface receptors (or antigens) include, but are not limited to, the anti-CD20 monoclonal antibody rituximab (MabThera), the anti-HER2 / neu antibody trastuzumab (Herceptin), and the anti-HER1 / EGFR antibody cetuximab (Erbitux).

[0034] Small molecule inhibitors of signal transduction pathways include, but are not limited to, EGFR-TK inhibitors (e.g., gefitinib, dasatinib, erlotinib (Tarceva), imatinib, nilotinib (Tasigna), lapatinib, sorafenib, sunitinib, afatinib, osimertinib (Tagrisso) and / or derivatives thereof), c-kit tyrosine kinase inhibitors and / or BCR-ABL tyrosine kinase inhibitors (e.g., imatinib, nilotinib, dasatinib, Votrient (pazopanib)).

[0035] Anti-angiogenic agents include, but are not limited to, anti-VEGF antibodies such as Avastin (bevacizumab), vascular endothelial growth factor receptor (VEGFR) inhibitors (eg, sorafenib, sunitinib, vandetanib).

[0036] mTOR signaling pathway inhibitors include, but are not limited to, temsirolimus and everolimus.

[0037] Proteasome inhibitors include, but are not limited to, bortezomib, carfilzomib, marizomib, ixazomib, oprozomib, and delanzomib.

[0038] There are no particular limitations on the weight, volume, or concentration ratio of the chemokine analog peptide to the targeting drug. Those skilled in the art can select an appropriate ratio between the chemokine analog peptide and the targeting drug depending on the disease, and in particular, the chemokine analog peptide in combination with the targeting drug exhibits a synergistic effect. In one embodiment, the chemokine analog peptide is administered at a dose of about 0.01 mg / kg body weight (of the subject (patient)) to about 500 mg / kg body weight (of the subject (patient)) once to three times a week.

[0039] Furthermore, the present invention provides a pharmaceutical composition for reducing anticancer drug resistance and enhancing anticancer drug sensitivity, and the use of the pharmaceutical composition for treating cancer, inhibiting cancer cell growth, and / or inhibiting cancer cell metastasis.

[0040] The pharmaceutical compositions of the present invention can inhibit 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 cancer, male reproductive system neoplasms, gynecological cancer, blood cancer, skin cancer, and sarcoma.

[0041] The thoracic cancer is selected from lung cancer, such as 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 combined small cell lung carcinoma.

[0042] 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.

[0043] Gastrointestinal cancers include, but are not limited to, esophageal cancer, stomach cancer (gastric cancer), liver cancer (hepatocellular carcinoma), bile duct and biliary tract cancer, pancreatic cancer, colorectal cancer, small intestine cancer, and anal cancer.

[0044] 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 oropharynx cancer, and salivary gland cancer.

[0045] Types of brain tumors include primary or secondary brain tumors, including, but not limited to, astrocytoma, glioblastoma, medulloblastoma, oligodendroglioma, glioma, and brain metastases.

[0046] Endocrine cancers include, but are not limited to, adrenal gland tumors, neuroendocrine tumors, parathyroid tumors, pituitary tumors, and thyroid disorders.

[0047] Urological cancers include, but are not limited to, bladder cancer and urethral cancer.

[0048] Male reproductive system neoplasms include, but are not limited to, prostate cancer, penile cancer, testicular seminoma, and testicular embryonal carcinoma.

[0049] Gynecological cancers include, but are not limited to, cervical cancer, ovarian cancer, uterine cancer (endometrial cancer), vaginal cancer, and vulvar cancer.

[0050] Hematologic cancers include, but are not limited to, leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and multiple myeloma.

[0051] Skin cancers include, but are not limited to, basal cell skin cancer, squamous cell skin cancer, melanoma skin cancer, and Merkel cell skin cancer.

[0052] Sarcomas include, but are not limited to soft tissue sarcoma, osteoblastoma (bone sarcoma), and rhabdomyosarcoma.

[0053] Neoplasias or / and cancers further include, but are not limited to, breast cancer, and neuroblastoma.

[0054] In one embodiment, the chemokine analog peptide or combination with a targeted drug 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.

[0055] In one embodiment, the chemokine analog peptide and / or one or more target drugs 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.

[0056] In another embodiment, the chemokine analog peptide and / or one or more targeting drugs can be administered parenterally, such as by intramuscular, intrathecal, subcutaneous, intraperitoneal, intravenous bolus injection or intravenous infusion. Parenteral administration can be achieved by incorporating the chemokine analog peptide and / or targeting drug(s) into a solution or suspension.

[0057] In one embodiment, one or more of the chemokine analog peptides and / or targeted drugs may be administered in the form of a pharmaceutical composition comprising the chemokine analog peptides and / or one or more targeted drugs and at least one pharmaceutically acceptable diluent, excipient, or carrier.

[0058] Pharmaceutical compositions contain a chemokine analog peptide and / or at least one target drug and one or more pharmaceutically acceptable diluents, excipients, or carriers. For the preparation of pills, tablets, coated tablets, and hard gelatin capsules, pharmaceutically active excipients that can be used include, but are not limited to, lactose, corn starch or derivatives thereof, gum arabic, magnesia, or glucose. For soft gelatin capsules and suppositories, carriers that can be used include, but are not limited to, fats, waxes, natural or hardened oils. Suitable carriers for the preparation of solutions, for example, injection solutions, or emulsions or syrups, include, for example, water, physiological sodium chloride solution, or alcohols, such as ethanol, propanol, or glycerol, sugar solutions such as glucose solutions, or mannitol solutions, or mixtures of the various solvents mentioned. The pharmaceutically acceptable diluents, excipients, or carriers used in the pharmaceutical compositions may be conventionally known pharmaceutically acceptable diluents, excipients, or carriers, and may be selected depending on the dosage form and administration route of the chemokine analog peptide and / or target drug(s).

[0059] In general, compositions intended for pharmaceutical use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions.

[0060] The compositions described herein may be in a form suitable for oral administration, e.g., solid dosage forms such as tablets, capsules, lozenges, or granules; liquid dosage forms such as emulsions, solutions, suspensions; parenteral injection (such as intravenous, subcutaneous, intramuscular, intravascular, or infusion), e.g., sterile solutions, suspensions, or emulsions; or topical administration, e.g., as an ointment, cream, gel, or lotion.

[0061] The composition for oral administration can be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, cachets, emulsions, capsules, syrups, or elixirs. The composition suitable for oral administration can include standard vehicles. Such vehicles are preferably of pharmaceutical grade.

[0062] For ointments and creams, the active ingredients (chemokine analog peptide and / or targeted drug(s)) can be formulated in an oil-in-water or water-in-oil base.

[0063] For intramuscular, intraperitoneal, subcutaneous and intravenous use, sterile solutions of the active ingredients (chemokine analog peptides and / or targeted drug(s)) will typically be used, and the pH of the solutions will be suitably adjusted and buffered.

[0064] Furthermore, the anti-cancer effect of the chemokine analog peptide and / or targeted drug(s) contained in the pharmaceutical composition can be delayed or prolonged by appropriate formulation.

[0065] The effective dose of the chemokine analog peptide and / or target drug(s) used for administration varies depending on the severity of the disease (cancer), the severity of symptoms, age, sex, weight, and sensitivity of the subject (patient), the mode, time, interval, and duration of administration, the nature and type of formulation, etc. In certain embodiments, the chemokine analog peptide and / or one or more target drugs can be administered within a time frame in which both agents are still active. One skilled in the art would be able to determine such a time frame by determining the half-life of the administered therapeutic agents. 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 analog peptide and one or more target drugs can be administered simultaneously or sequentially, when administered sequentially in any order. In another embodiment, the chemokine analog peptide and target drug(s) can be administered so that the peak pharmacokinetic effect of one agent coincides with the peak pharmacokinetic effect of the other.

[0066] However, the chemokine analog peptides and / or targeted drug(s) of the present invention may alternatively be for use in combination with one or more additional cancer therapies, for example, the chemokine analog peptides and / or targeted drug(s) may be used in combination with one, two, three, four, five, or more additional cancer therapies.

[0067] "In combination" in the present invention includes that the pharmaceutical composition is administered to a subject undergoing one or more additional cancer treatments in the same course of treatment.Therefore, this term covers not only the simultaneous administration of the pharmaceutical composition with one or more additional cancer treatments (for example, as a bolus dose or infusion), but also the time-separated administration of these cancer treatments.For example, the pharmaceutical composition can be administered within a treatment schedule / cycle defined by the patient's oncologist, including one or more additional cancer treatments administered before, at the same time, or after the pharmaceutical composition, depending on any of various factors (for example, the severity of symptoms, etc.).

[0068] In another embodiment, the therapeutically effective amount of chemokine analog peptide or / and targeted drug(s) for the treatment of a particular cancer will depend on the type and nature of the cancer, its size, progression, and metastatic status, and will be determined in consultation with your physician.

[0069] For example, in some embodiments, the pharmaceutical compositions of the present disclosure are administered once per month, twice per month, three times per month, every other week, once per week, twice per week, three times per week, four times per week, five times per week, six times per week, every other day, once per day, twice per day, or three times per day.

[0070] The dosage of the pharmaceutical composition used will vary depending on the activity of the particular chemokine analog peptide and the condition being treated, but as a guideline, a dosage selected in the range of 0.01 mg / kg to 500 mg / kg body weight, particularly in the range of 0.02 mg / kg body weight / dose to 1 mg / kg body weight / dose, may be mentioned. Conversely, this dosage regimen may be continued for as many days as is appropriate for the patient in question, dividing the daily dose into several separate administrations, if necessary.

[0071] A typical, non-limiting acceptable dosage for administration of chemokine analog peptides is about 0.01 mg / kg to about 500 mg / kg body weight of subject (patient) once to three times per week.

[0072] A typical, non-limiting, tolerated dose of gefitinib is about 250 mg / kg body weight (subject (patient)) once daily.

[0073] A representative, non-limiting, tolerated dose of osimertinib (Tagrisso) is approximately 80 mg / kg body weight (subject (patient)) once daily.

[0074] A typical, non-limiting tolerated dose of afatinib is about 40 mg / kg body weight (subject (patient)) once daily.

[0075] A representative, non-limiting, tolerated dose of erlotinib (Tarceva) is about 150 mg / kg body weight (subject (patient)) once daily.

[0076] A typical, non-limiting tolerated dose for MabTera (rituximab) administration is approximately 90 mg / m per dose. 2 ~about 120mg / m 2 Body surface area.

[0077] A typical, non-limiting, tolerated dose of Herceptin (trastuzumab) is about 2 mg / kg body weight (subject (patient)) per week.

[0078] A representative, non-limiting, tolerated dose of Votrient (pazopanib) for oral administration is about 800 mg / kg body weight (subject (patient)) once daily.

[0079] A typical, non-limiting, acceptable dose for administration of imatinib is about 400 mg / kg to about 800 mg / kg body weight (subject (patient)) per day.

[0080] A typical, non-limiting, tolerated dose of nilotinib (Tasigna) is about 400 mg / kg to about 800 mg / kg body weight (subject (patient)) per day.

[0081] In one embodiment, the combinations provided by the present invention have been evaluated in specific assay systems and under several different administration schedules in vitro. Experimental details are provided below. The data presented herein clearly demonstrate that chemokine analog peptides, particularly RISE P-8, exhibit synergistic effects when combined with targeted drugs. Furthermore, in one embodiment, subjects achieve clinical benefit.

[0082] Additional specific embodiments of the present invention include, but are not limited to, the following. [Example]

[0083] cell line Lewis lung carcinoma (LL / 2) is a cell line established from the lungs of C57BL mice bearing tumors resulting from the transplantation of primary Lewis lung carcinoma. This lung cancer cell line is grown in Dulbecco's modified Eagle's medium containing 10% FBS, 1% penicillin-streptomycin, and 1% L-glutamine at 37°C in a humidified cell culture incubator with 5% CO2.

[0084] Human NSCLC cell lines, PC9, gefitinib-resistant PC9 (PC9GR), HCC827, and gefitinib-resistant HCC827 (HCC827GR), were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 1% L-glutamine at 37°C in a humidified cell culture incubator at -5% CO. After thawing the frozen GR cell lines for 1 week, gefitinib resistance was confirmed by adding 3 μM gefitinib to the growth medium for 48 hours, and then replacing the medium with fresh growth medium. [Example]

[0085] Quantification of the expression levels 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.

[0086] All real-time PCR reactions were performed on a StepOnePlus™ real-time PCR system (Applied Biosystems™) using 2x qPCR BIO Probe Mix Hi-ROX Reagents (PCR Biosystems, UK) and the UPL Probe System (IL-8, CXCR1, CXCR2). 18s was used as an internal loading control. The sequences of the primers included were: 5'-gagcactccataaggcacaaa-3' (SEQ ID NO: 2) (forward primer for CXCL8) and 5'-atggttccttccggtggt-3' (SEQ ID NO: 3) (reverse primer for CXCL8); 5'-gaccaacatcgcagacacat-3' (SEQ ID NO: 4) (forward primer for CXCR1) and 5'-tgcttgtctcgttccacttg-3' (SEQ ID NO: 5) (reverse primer for CXCR1); 5'-ggctaagcaaaatgtgatatgtacc-3' (SEQ ID NO: 6) (forward primer for CXCR2) and 5'-caaggttcgtccgtgttgta-3' (SEQ ID NO: 7) (reverse primer for CXCR2). Gene expression was calculated using the following formula: gene expression = 2 -ΔΔCt . [Example]

[0087] Verification of gefitinib-resistant cancer cell lines To verify the establishment of gefitinib-resistant NSCLC cell lines, MTT with a gefitinib gradient was performed on parental and gefitinib-resistant (GR) cells. The drug resistance of the GR cell lines was 100-1000 orders of magnitude higher than that of the parental cells (Table 1). The IC50 value of gefitinib in PC9 cells was 0.6009 µM compared to 92.43 µM in PC9GR cells (154-fold higher resistance). The IC50 value of gefitinib in HCC827 cells was 0.056 µM compared to 125.5 µM in HCC827GR cells (1517-fold higher resistance).

[0088] [Table 1] [Example]

[0089] Expression levels of CXCL8, CXCR1, and CXCR2 genes in gefitinib-resistant cancer cells compared with parental cancer cells with or without gefitinib induction Figures 2A-2D show that IL-8 mRNA expression is almost the same or not significantly different between parental cells (PC9 (Figure 2A) and HCC827 (Figure 2B)) and GR cells (PC9GR (Figure 2A) and HCC827GR (Figure 2B)). However, CXCR1 and CXCR2 mRNA expression in GR cells (PC9GR (Figure 2C) and HCC827GR (Figure 2D)) is significantly enhanced by approximately 10- to 50-fold, respectively, compared to parental cells. *P<0.05; **P<0.01; ***P<0.001, Student's t-test.

[0090] Furthermore, the present invention also demonstrated the stimulation of IL-8 mRNA expression in HCC827 and HCC827GR through gefitinib. The data showed that IL-8 mRNA expression was not significantly induced in HCC827 after gefitinib treatment (Figure 3A). In contrast, IL-8 mRNA expression in HCC827GR was increased 24 hours after treatment with gefitinib (5 μM) (Figure 3B).

[0091] Similarly, CXCR1 and CXCR2 mRNA expression data of HCC827 and HCC827GR stimulated by gefitinib showed that CXCR1 and CXCR2 mRNA expression was not obviously induced in HCC827 after gefitinib treatment (Figures 4A and 4B). In contrast, CXCR1 and CXCR2 mRNA expression in HCC827GR increased 24 hours after treatment with gefitinib (5 μM) (Figures 4C and 4D). [Example]

[0092] P-8 affects the chemotaxis of human neutrophils via CXCL8 Referring to Figure 5, neutrophil chemotaxis was assessed using a modified Boyden chamber microchemotaxis assay. CXCL8 alone or in combination with RISE P-8 was placed in the lower compartment of a Boyden chamber well, and purified neutrophils were placed in the upper compartment. Migrated neutrophils were detected by lysis. The neutrophil migration rate was assessed and expressed as a chemotaxis index (CI) value, i.e., CI = (intensity RISE P-8 -Strength HBSS ) / (strength CXCL8 -Strength HBSS ) x 100%. The results demonstrate that RISE P-8 effectively antagonizes the neutrophil response to CXCL8 and competes with CXCL8 for CXCR1 and CXCR2. [Example]

[0093] Expression levels of CXCR1, CXCR2, and CXCL8 genes in gefitinib-resistant cancer cells compared with parental cancer cells survive in an adhesion-independent state PC9 (Figure 6A) and PC9GR (Figure 6B) showed enhanced expression of IL-8 (CXCL8) / CXCR1 / CXCR2 mRNA in 3D (suspension) culture compared to 2D, indicating that the expression of CXCL8, CXCR1, and CXCR2 mRNA differs between adherent and suspension cultures for cancer cell survival and growth. [Example]

[0094] RISE P-8 significantly reduces IL-8 protein expression in NSCLC cancer cells in 3D (suspension) conditions Cells were treated with serum-free medium for 16–18 h and incubated for 10 min in growth medium. 6 Suspended at 2x10 cells / mL 6Cells / well were seeded into coated or uncoated 6-well plates and treated with or without RISE P-8 (200 ng / ml) for 24 hours. In PC9 (Figure 7A) and PC9GR (Figure 7B), RISE P-8 appears to significantly attenuate IL-8 autocrine secretion only in suspension culture. Data are shown as mean ± SD. *P<0.05; ***P<0.001, Student's t-test. Thus, RISE P-8 treatment significantly reduces IL-8 secretion from cancer cells surviving in an adhesion-independent manner. [Example]

[0095] Expression levels of CXCR1, CXCR2, and CXCL8 genes in gefitinib-resistant cancer cells treated with gefitinib compared to treatment with gefitinib in combination with RISE P-8 Treatment of gefitinib-resistant cancer cells with 5 μM gefitinib for 24 hours significantly increased the mRNA expression of IL-8, CXCR1, and CXCR2 in PC9GR (Figure 8A) and HCC827GR (Figure 8B). However, treatment of gefitinib-resistant cancer cells with gefitinib in combination with RISE P-8 (200 ng / mL) reduced the mRNA expression of IL-8, CXCR1, and CXCR2 in PC9GR (Figure 8C) and HCC827GR (Figure 8D). Data are shown as mean ± SD. *<0.05; **P<0.01; ***P<0.001, Student's t-test. Thus, RISE P-8 can compete with IL-8 and attenuate its feedback to regulate the gene expression of IL-8, CXCR1, and CXCR2. [Example]

[0096] Gefitinib-resistant cancer cells are arrested in the G0 / G1 phase by treatment with RISE P-8 Cancer cells were treated with 5 μM gefitinib or / and RISE P-8 (200 ng / mL) or / and CXCL8 (100 ng / mL) for 24 hours, then harvested and fixed in 75% ice-cold ethanol overnight at 20°C. After fixation and washing, the cancer cells were stained with propidium iodide (PI) and the cell cycle of the cancer cells was analyzed using a flow cytometer and FlowJo 7.6.1 to acquire and analyze data. We demonstrated that the cell cycle of HCC827GR (Figure 9A) and PC9GR (Figure 9B) was arrested at G0 / G1 phase. Furthermore, co-treatment of cancer cells with gefitinib and RISE P-8 significantly arrested the proportion of all cells in G0 / G1 phase compared with those treated with gefitinib alone (Figures 9A and 9B). Data are shown as mean ± SD. *P<0.05; **P<0.01; ***P<0.001, Student's t-test. In other words, the present invention infers that co-treatment of RISE P-8 with gefitinib can induce cell apoptosis and reduce cell proliferation. [Example]

[0097] Anchorage-independent growth of gefitinib-resistant cancer cells can be inhibited by RISE P-8 Cell proliferation assays were performed using a Cell Counting Kit-8 (CCK-8) (Dojindo, Kumamoto, Japan). Gefitinib-resistant cancer cells were treated with gefitinib (5 μM) and RISE P-8 (200 ng / mL) or gefitinib, RISE P-8, and IL-8 (100 ng / mL) for 24 hours. The proliferation rate of gefitinib-resistant cancer cells was significantly attenuated in PC9GR (Figure 10A) and HCC827GR (Figure 10B) compared with the control and IL-8 groups (Figures 10A and 10B). Data are shown as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001, Student's t-test). These data further confirm that co-treatment of cancer cells with gefitinib and RISE P-8 resulted in downregulation of CXCL8, CXCR1, and CXCR2. [Example]

[0098] RISE P-8 can inhibit long-term proliferation in gefitinib-resistant cancer cells Meanwhile, gefitinib-resistant cancer cells were treated with gefitinib (5 μM) and RISE P-8 (200 ng / mL), or with gefitinib and RISE P-8 and IL-8 (100 ng / mL), or with gefitinib and IL-8, respectively, for colony formation assays. Colony formation assays were also performed to evaluate cell proliferation. These results revealed that the colony formation rate of RISE P-8-treated cells was significantly slower than that of the control and IL-8 groups in the long term in PC9GR (Figure 11A) and HCC827GR (Figure 11B). Data are shown as mean ± SD. *P<0.05; **P<0.01; ***P<0.001, Student's t-test. [Example]

[0099] RISE P-8 inhibits the invasive potential of gefitinib-resistant lung adenocarcinoma cells Cell invasion was measured 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 in a 5% CO atmosphere. 2.5x10 4 Cells were seeded in 0.2 mL of serum-free growth medium onto the Matrigel-coated upper chamber and treated with the drug IL-8 (100 ng / ml) with or without RISE P-8 (200 ng / ml). After 24 h of incubation at 37°C and 5% CO2, the upper chamber membrane was fixed with methanol and stained with propidium iodide (PI). Invaded cells at the bottom of the membrane were imaged and counted using an inverted fluorescence microscope (Observer.Z1, Zeiss) in five random fields (magnification, 100x) for each specimen.

[0100] Gefitinib-resistant cancer cells were treated with gefitinib (5 μM) and RISE P-8 (200 ng / mL), or with gefitinib and RISE P-8 and IL-8 (100 ng / mL), or with gefitinib and IL-8, respectively, for the invasion assay.

[0101] The results of the transwell invasion assay showed that RISE P-8 reduced the invasion rate in PC9GR (Figure 12A) and HCC827GR (Figure 12B) compared with the control group. Meanwhile, the invasion rate of the "IL-8 + RISE P-8 group" in PC9GR (Figure 12A) and HCC827GR (Figure 12B) was significantly prevented compared with the "IL-8 group" in PC9GR (Figure 12A) and HCC827GR (Figure 12B). Data are shown as mean ± SD. *P<0.05; **P<0.01, Student's t-test. [Example]

[0102] Inhibitory effect of RISE P-8 on the invasive potential of Lewis lung carcinoma cells Referring to Figures 13A and 13B, the present invention also demonstrated the invasion of LL / 2 cells (Lewis lung carcinoma) treated with CXCL8, MIP-2, or RISE P-8. LL / 2 cancer cells are characterized by their rapid growth and a strong tendency to invade the lung. LL / 2 cells were seeded on Matrigel-coated polycarbonate filters to analyze their invasive ability. The cells were then incubated in a chamber for 24 hours, analyzed by PI staining, and counted under a microscope. Representative fields (100x magnification) of invasive cells on a transwell membrane and the invasion rates are shown. LL / 2 cells were treated with CXCL8 (100 ng / ml), RISE P-8 (200 ng / ml), or MIP-2 (50 ng / ml), respectively. Figures 13A-13B show quantitative representations of the invasion rates. Data are presented as mean ± SD. *P<0.05; **P<0.01, Student's t-test. The same results were observed in the data on MIP-2-induced cell infiltration (Figure 13B). MIP-2 is a mouse homolog of CXCL8. RISE P-8 also significantly reduced the number of infiltrating cells. Thus, the present invention further demonstrated that CXCL8 can stimulate LL / 2 invasive activity, whereas RISE P-8 significantly suppressed CXCL8-stimulated cell infiltration. [Example]

[0103] The combination of RISE P-8 and gefitinib inhibits tumor growth and extends lifespan in vivo Male BALB / c nude mice (5 weeks old) were purchased from the National Laboratory Animal Center. PC9GR was injected subcutaneously into the backs of BALB / c nude mice. RISE P-8 (500 μg / kg) was injected intraperitoneally three times, and gefitinib (80 mg / kg) was administered orally twice weekly. Tumor size in tumor-bearing mice was recorded up to 72 days after treatment. Tumor size was significantly suppressed in the "gefitinib + RISE P-8 group" (Figures 14A-14B). The results show that combining RISE P-8 with gefitinib significantly reduced tumor size compared to the "gefitinib group" and effectively extended the lifespan of tumor-bearing mice by at least 14 days compared to the other groups (Figure 14C). [Example]

[0104] The combined use of RISE P-8 and gefitinib attenuates the expression of IL-8, CXCR1, and CXCR2 mRNA in vivo After sacrificing PC9GR tumor-bearing BALB / c nude mice on day 72, IL-8, CXCR1, and CXCR2 gene expression in tumor tissues was examined in the "gefitinib" group and the "gefitinib + RISE P-8" group (Figures 15A-C). Data are shown as mean ± SEM (n = 8). *P < 0.05; **P < 0.01; ***P < 0.001, Student's t-test).

[0105] In summary, the pharmaceutical composition synergistically attenuates the expression of IL-8, CXCR1, and CXCR2 in the tumor microenvironment, inhibits tumor growth by downregulating IL-8, blocks cancer cell migration and invasion by downregulating IL-8, and treats or reduces metastasis. Furthermore, the above drug-resistant cells overexpress IL-8.

[0106] Each feature disclosed in this specification (including any accompanying claims, abstract, and drawings), unless otherwise stated, may be replaced by alternative features serving the same, equivalent, or similar purpose. Thus, unless otherwise stated, each disclosed feature is only an example of a generic series of equivalent or similar features.

[0107] As indicated, these modifications can be made to the present invention in light of the foregoing description of illustrated embodiments thereof and are 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 latitude of modifications, variations, and substitutions is contemplated in the foregoing disclosure. It will be understood that, in some cases, some features of embodiments of the present invention may be applied without the corresponding use of other features without departing from the scope and spirit of the invention as described. Accordingly, many modifications may be made to adapt a particular situation or material to the essential scope and spirit of the present invention.

Claims

1. A pharmaceutical composition for reducing anticancer drug resistance and enhancing anticancer drug sensitivity, comprising: a chemokine analog peptide, wherein the chemokine analog peptide is RISE P-8 consisting of the sequence set forth in SEQ ID NO: 1; 1. A pharmaceutical composition comprising gefitinib and a pharmaceutically acceptable buffer, diluent, carrier, adjuvant or excipient, wherein the pharmaceutical composition is administered in a therapeutically effective amount to a subject in need thereof, wherein the subject has been diagnosed with non-small cell lung cancer, and the non-small cell lung cancer is resistant to gefitinib.

2. 10. Use of the chemokine analog peptide and medicament of claim 1 in the manufacture of a medicament for treating cancer, wherein the cancer is resistant to targeted drugs.

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