Pharmaceutical composition for preventing or treating cancer comprising propafenone and mTOR inhibitor or hypoglycemic agent

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of cancer using propafenone in combination with an mTOR inhibitor, or propafenone and a hypoglycemic agent. By including propafenone and an mTOR inhibitor, or propafenone and a hypoglycemic agent, as active ingredients, the pharmaceutical composition according to the present invention can be expected to have a significantly superior cancer prevention or treatment effect compared to the use of the drug alone.

Description

Technology Field

[0001] The present invention relates to a pharmaceutical composition for the prevention or treatment of cancer using propafenone in combination with an mTOR inhibitor, or propafenone in combination with a hypoglycemic agent. Background Technology

[0002] Cancer is one of the major causes of death worldwide, along with infectious and cardiovascular diseases, and the mortality rate due to cancer is on the rise every year. Surgical therapy, radiation therapy, and chemotherapy are used to treat cancer. While chemotherapy is primarily used to inhibit the proliferation of cancer cells, it is also used before and after surgery to increase the success rate of surgical procedures, as well as to suppress cancer metastasis, prevent recurrence, or alleviate symptoms. Currently, about 60 different types of anticancer drugs are in use, and active research is underway to develop new drugs or to repurpose existing treatments for other diseases, whose safety has been confirmed, as anticancer agents.

[0003] Meanwhile, propafenone is an antiarrhythmic drug that treats abnormal heartbeats by targeting sodium channels. It is effective in suppressing ventricular ectopic activity by non-selectively blocking beta receptors of the sympathetic nervous system and blocking the intracellular influx of sodium ions. Propafenone is primarily prescribed to patients with ventricular arrhythmia or supraventricular arrhythmia. Recently, the possibility of combining propafenone with colchicine to treat inflammatory diseases such as chronic gout and familial Mediterranean fever has been suggested, and propafenone has been proposed as a type of ADRB2 inhibitor to be used in combination with CXCR4 inhibitors to inhibit the formation of CXCR4-ADRB2 heterodimers that are overexpressed in cancer tissues. However, there are no instances of applying propafenone to cell or disease animal models to verify its effects on alleviating inflammation or treating cancer, and to date, propafenone has not been recognized as a treatment for other diseases or as a combination drug. Therefore, significant research is required to confirm the new efficacies of propafenone and repurpose it as a novel therapeutic agent. Prior art literature

[0004] U.S. Patent No. 9,555,029 U.S. Patent No. 10,709,763 The problem to be solved

[0005] The present invention aims to provide a pharmaceutical composition for the prevention or treatment of cancer using propafenone in combination with a conventional drug. means of solving the problem

[0006] One aspect of the present invention aims to provide a pharmaceutical composition for the prevention or treatment of cancer comprising propafenone and an mTOR inhibitor as active ingredients.

[0007] The “propafenone” used in the present invention is a drug used to treat heart diseases characterized by rapid heart rate, such as atrial arrhythmia and ventricular arrhythmia, by non-selectively blocking beta-1 receptors, which primarily increase heart rate and cardiac contractility in the sympathetic nervous system, and beta-2 receptors, which are involved in bronchodilation, peripheral vascular and uterine smooth muscle relaxation. Although there have been attempts to use propafenone as a treatment for other diseases, the mechanism other than the drug mechanism targeting sodium channels has not been clearly elucidated, and there have been no studies on the treatment of cancer using propafenone alone or in combination with conventional treatments or anticancer drugs.

[0008] The “mTOR (mammalian target of rapamycin)” used in the present invention is a type of serine / threonine protein kinase, a protein involved in signal transduction within mammalian cells, and is known as the target protein of rapamycin. It transmits signals through mTOR complex 1 and mTOR complex 2 to regulate various biological activities such as cell growth, cell proliferation, cell motility, cell survival, protein synthesis, autophagy, and transcription. The “mTOR inhibitor” used in the present invention is a substance that inhibits the activity of mTOR. First-generation drugs of the rapamycin family, which have binding sites for mTOR and FKBP12; second-generation drugs known as mTOR kinase inhibitors, which inhibit the ATP binding site within mTOR kinase; and third-generation drugs, which are a combination of the first and second generations, have been developed. These mTOR inhibitors were initially developed as antifungal agents, but due to their excellent ability to suppress immunity and cell proliferation by inhibiting abnormally activated mTOR, they are used to treat immune rejection caused by organ transplantation, lymphangioleiomyomatosis, and kidney cancer.

[0009] According to one embodiment of the present invention, the mTOR inhibitor may be any compound known in the art that inhibits mTOR activity without limitation, and may be, for example, a compound composed of rapamycin or an analog thereof, an mTOR kinase inhibitor, and a combination thereof.

[0010] More specifically, the rapamycin analog may be one or more selected from the group consisting of Sirolimus, Temsirolimus, Everolimus, Ridaforolimus, Umirolimus, and Zotarolimus, but is not limited thereto.

[0011] The above mTOR kinase inhibitors may be one or more selected from the group consisting of Torin-1, Torin-2, Vistusertib, Dactolisib, Voxtalisib, BGT226, SF1126, PKI-587, NVPBE235, Sapanisertib, AZD8055, and AZD2014, but are not limited thereto.

[0012] In addition, the above mTOR inhibitor may be RapaLink-1, a compound in which sapanicertip is linked to rapamycin.

[0013] The combination of propafenone and mTOR inhibitors can inhibit the growth of cancer cells and cause cancer cell death, and may exhibit a synergistic effect on cancer cell death compared to the use of propafenone or mTOR inhibitors alone.

[0015] In addition, another aspect of the present invention aims to provide a pharmaceutical composition for the prevention or treatment of cancer comprising propafenone and a hypoglycemic agent as active ingredients.

[0016] The above propafenone is the same as previously described, so it is omitted to avoid redundancy.

[0017] The “hypoglycemic agent” used in the present invention is a substance that activates the secretion of insulin from the pancreas to lower blood glucose levels and is mainly used to treat diabetes.

[0018] According to one embodiment of the present invention, the hypoglycemic agent may be used without limitation as long as it is an insulin secretion-promoting substance known in the art, and may be, for example, one or more selected from the group consisting of glucose uptake inhibitors, glucose transporter (GLUT) inhibitors, glycolysis inhibitors, insulin secretion promoters, and insulin sensitivity improvers.

[0019] More specifically, the glucose absorption inhibitor may be an SGLT2 (sodium-glucose cotransporter 2) inhibitor or a σ-glucosidase inhibitor, but is not limited thereto.

[0020] Herein, the SGLT2 inhibitor may be one or more selected from the group consisting of dapagliflozin, ipragliflozin, empagliflozin, ertugliflozin, and pharmaceutically acceptable salts thereof, but is not limited thereto. The σ-glucosidase inhibitor may be one or more selected from the group consisting of acarbose, voglibose, and pharmaceutically acceptable salts thereof, but is not limited thereto.

[0021] The above glucose transporter inhibitor may be one or more selected from the group consisting of BAY-876, STF-31, WZB-117 and Fasentin, but is not limited thereto.

[0022] The above-mentioned inhibitor may be 2-deoxyglucose, but is not limited thereto.

[0023] The above insulin secretion enhancer may be one or more selected from the group consisting of GLP-1 receptor agonists, DPP-4 (dipeptidyl peptidase-4) inhibitors, repaglinide, miitiglinide, nateglinide, gliclazide, glimepiride, and glipizide, but is not limited thereto.

[0024] Here, the GLP-1 receptor agonist may be one or more selected from the group consisting of lixisenatide, liraglutide, and dulaglutide, but is not limited thereto. The DPP-4 inhibitor may be one or more selected from the group consisting of sitagliptin, vildagliptin, saxagliptin, linagliptin, gemigliptin, teneligliptin, alogiptin, evogliptin, and anagliptin, but is not limited thereto.

[0025] The above insulin sensitivity improving agent may be one or more selected from the group consisting of pioglitazone, lobeglitazone, and biguanide compounds, but is not limited thereto.

[0026] Here, biguanide compounds refer to compounds that have an imidodicarbonimidic diamide as a backbone, such as metformin, phenformin, and buformin.

[0027] The combination of propafenone and hypoglycemic agents can inhibit the growth of cancer cells and kill cancer cells by inducing AMPK activity and inhibiting mTOR, and can show a synergistic effect on cancer cell death compared to the use of propafenone or hypoglycemic agents alone.

[0028] As used in this invention, "cancer" (or "tumor") refers collectively to diseases caused by cells that possess aggressiveness—dividing and growing beyond normal growth limits—invasiveness—infiltrating surrounding tissues, and metastaticness—spreading to other parts of the body. Here, "cancer" is also used synonymously with "malignant tumor."

[0029] According to one embodiment of the present invention, the cancer may be one or more selected from the group consisting of brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, brain lymphoma, oligodendroglioma, intracranial tumor, ependymoma, brainstem tumor, head and neck tumor, laryngeal cancer, oropharyngeal cancer, nasal cavity cancer, nasopharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, thyroid cancer, oral cancer, thoracic tumor, small cell lung cancer, non-small cell lung cancer, thymic cancer, mediastinal tumor, esophageal cancer, breast cancer, abdominal tumor, stomach cancer, liver cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, small intestine cancer, colorectal cancer, anal cancer, bladder cancer, urethral cancer, kidney cancer, cervical cancer, endometrial cancer, uterine sarcoma, ovarian cancer, vaginal cancer, prostate cancer, testicular cancer, penile cancer, and skin cancer.

[0030] As used in the present invention, "cancer prevention or treatment" includes acts that inhibit the growth or proliferation of cancer cells or delay the progression of cancer, and means improving or alleviating the symptoms of cancer.

[0031] The term "pharmaceutical composition for the prevention or treatment of cancer" as used in the present invention refers to a substance used for the purpose of treating, alleviating, managing, or preventing cancer in animals, including humans.

[0032] The pharmaceutical composition according to the present invention can be formulated into various forms according to conventional methods and used. For example, it can be formulated into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, and syrups, and can be formulated into external preparations, suppositories, and sterile injectable solutions. In addition, the pharmaceutical composition may further include a pharmaceutically acceptable carrier. The carrier is one that is commonly used in formulations and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. The pharmaceutical composition of the present invention may additionally include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc., in addition to the above components.

[0033] The dosage of the above pharmaceutical composition may vary depending on the formulation method, administration method, administration time and / or administration route of the above pharmaceutical composition, and may vary depending on various factors including the type and degree of response to be achieved by administering the above pharmaceutical composition, the type of individual to be administered, age, body weight, general health condition, symptoms or severity of disease, gender, diet, excretion, and components of other compositions used simultaneously or at other times with the individual, as well as similar factors well known in the pharmaceutical field, and a person with ordinary knowledge in the art can easily determine and prescribe a dosage effective for the intended treatment. For example, it may be 0.001 to 1000 mg / kg, 0.01 mg / kg to 100 mg / kg, or 0.1 mg / kg to 10 mg / kg per day, but is not limited thereto.

[0034] In addition, the above pharmaceutical composition may be administered to mammals, such as rats, mice, livestock, and humans, via various routes. The route and method of administration of the above pharmaceutical composition may be independent and are not particularly limited thereto; any route and method of administration may be used as long as the pharmaceutical composition can reach the intended site. The above pharmaceutical composition may be administered orally or parenterally. Examples of parenteral administration methods include intravenous administration, intraperitoneal administration, intramuscular administration, transdermal administration, or subcutaneous administration. Methods of applying, spraying, or inhaling the above pharmaceutical composition to the diseased site may also be used, but are not limited thereto.

[0035] A pharmaceutical composition for cancer prevention or treatment containing such propafenone may contain an mTOR inhibitor or a hypoglycemic agent in a ratio of 1:0.001 to 1000.

[0036] More specifically, propafenone and an mTOR inhibitor or hypoglycemic agent may be included in a weight ratio of 1:0.001 to 1000, 1:0.01 to 1000, 1:0.1 to 1000, 1:1 to 1000, 1:0.001 to 100, 1:0.1 to 100, 1:1 to 100, 1:0.001 to 10, 1:0.01 to 10, 1:0.1 to 10, or 1:1 to 10, but are not limited thereto. Effects of the invention

[0037] The pharmaceutical composition according to the present invention includes propafenone and an mTOR inhibitor, or propafenone and a hypoglycemic agent, as active ingredients, thereby allowing for the expectation of significantly superior cancer prevention or treatment effects compared to the use of the drug alone. Brief explanation of the drawing

[0038] Figure 1 confirms the anticancer effect of the combination of propafenone and an mTOR inhibitor, and is a cell image when (a) Rapalink-1 or (b) Temsirolimus was treated as an mTOR inhibitor. Figure 2 is a graph showing the (a) tumor weight and (b) body weight of each group, in which PBS was administered once daily (Mon-Fri) for 5 weeks to confirm the anticancer effect of the combination of propafenone and an mTOR inhibitor using an animal cancer model. PBS was administered to the control group (Ctrl), propafenone to the propafenone administration group (Pro), temsirolimus to the mTOR inhibitor administration group (Tem), and propafenone and temsirolimus to the combination administration group (Pro+Tem). Figure 3 confirms the anticancer effect of the combination of propafenone and a hypoglycemic agent, and shows cell images of (a) 786-O, H1975, and BT549 cells as cancer cell lines and (b) HREC, BEAS-2B, and MCF-10A cells as normal cell lines, when treated with propafenone while cultured in high-concentration glucose medium (High Glu) or low-concentration glucose medium (Low Glu) or in high-concentration glucose medium (GLUTi) containing a glucose transporter inhibitor. Figure 4 confirms the anticancer effect of the combination of propafenone and a hypoglycemic agent, showing the results of changes in the activity of signaling molecules (p-S6K, p-AMPK, and p-ERK) when propafenone (0, 10, 20 μM) was treated under high or low glucose conditions using (a) 786-O, H1975, and BT549 cells as cancer cell lines and (b) HREC, BEAS-2B, and MCF-10A cells as normal cell lines. Figure 5 is a graph showing the (a) tumor weight and (b) body weight of each group, in which PBS was administered once a day (Mon to Fri) for 5 weeks to confirm the anticancer effect of the combination of propafenone and a hypoglycemic agent using an animal cancer model, PBS was administered to the control group (Ctrl), propafenone to the propafenone administration group (Pro), BAY-876 to the hypoglycemic agent administration group (Bay), and propafenone and BAY-876 to the combination administration group (Pro+Bay). Specific details for implementing the invention

[0039] The present invention will be described in more detail below. However, this description is provided merely as an example to aid in understanding the invention, and the scope of the invention is not limited by this exemplary description.

[0041] Example 1. Anticancer effect by combination of propafenone and mTOR inhibitor

[0042] To confirm the cancer cell death effect when propafenone and mTOR inhibitors are used in combination, cell and animal experiments were conducted using propafenone and Rapalink-1 and Temsirolimus as mTOR inhibitors.

[0044] 1-1. Experiment using cancer cell lines

[0045] 786-O cells, a renal carcinoma cell line, were cultured in RPMI (Hyclone, SH30027.01) medium containing 1% penicillin / streptomycin (Invitrogen, 15140122) and 10% FBS (fetal bovine serum) (Hyclone, SV30207.02) and maintained in a humidified incubator at 5% CO2 and 37°C.

[0046] 1 x 10 cells in a 12-well plate 5After seeding the wells, propafenone (0, 10, 30, 40 μM), laparlink-1 (0, 3, 10 nM), and / or temsirolimus (0, 30, 100 nM) were treated for 48 hours. After treatment, cell morphology was observed under a light microscope.

[0047] As a result, as shown in Figure 1, when propafenone, Lapalink-1, or temsirolimus were treated alone, there was no change in the number of cells regardless of the drug concentration, but when propafenone was treated in combination with mTOR inhibitors such as Lapalink-1 and temsirolimus, it was confirmed that the number of cells decreased as the concentration of the mTOR inhibitor increased.

[0049] 1-2. Animal Experiments

[0050] Animal experiments were conducted in compliance with animal ethics regulations after obtaining approval from the Yonsei University Committee on Laboratory Animal Ethics. Six-week-old BALB / C nude mice (Zabio Co., Ltd.) were used for the animal experiments. For the experiment, 1 x 10⁶ 786-O cells were used. 7 The solution was dissolved in 100 μl of PBS and injected subcutaneously into mice. One week later, the cells-transplanted mice were randomly divided into four groups. PBS was administered to the control group (Ctrl), propafenone 20 mg / kg to the propafenone group (Pro), temsirolimus 20 mg / kg to the mTOR inhibitor group (Tem), and propafenone 20 mg / kg and temsirolimus 20 mg / kg to the combination group (Pro+Tem) via intraperitoneal injection once daily (Monday to Friday) for five weeks. Body weight was measured after each weekly injection, and after the administration was completed, the animals were euthanized in compliance with animal ethics regulations to excise the tumors, and the tumor weight was measured.

[0051] As a result, as shown in Figure 2, in the combination therapy group administered both propafenone and temsirolimus, there was almost no difference in body weight compared to the control group, but it was confirmed that the tumor size was significantly reduced compared to the propafenone or mTOR inhibitor administration group.

[0053] These results suggest that the combination of propafenone and mTOR inhibitors selectively exhibits a cancer cell death effect.

[0055] Example 2. Anticancer effect by combined use of propafenone and a hypoglycemic agent

[0056] 2-1. Anticancer effects of propafenone according to glucose conditions

[0057] To determine the effect of glucose concentration on the cancer cell death effect induced by propafenone, cellular changes in various cancer cells or normal cells were observed using high-concentration glucose medium, low-concentration glucose medium, and a glucose transporter inhibitor (GLUT inhibitor).

[0058] 786-O (kidney cancer), H1975 (lung cancer), and BT549 (breast cancer) cells were used as cancer cell lines, and HREC (kidney), BEAS-2B (lung), and MCF-10A (breast) cells were used as normal cell lines. 786-O, H1975, BT549, and BEAS-2B cells were cultured in RPMI (Hyclone, SH30027.01) medium containing 1% penicillin / streptomycin (Invitrogen, 15140122) and 10% FBS (Hyclone, SV30207.02). HREC cells were cultured in Renal Epithelial Cell Basal Medium (ATCC, PCS-400-030) containing 1% penicillin / streptomycin and 10% FBS, and MCF-10A cells were cultured in F12 (Hyclone, SH30023.01) medium containing 1% penicillin / streptomycin and 10% FBS. All cells were maintained in a humidified incubator at 5% CO2 and 37°C.

[0059] 4 x 10 cells per well in a 12-well plate4 The cells were cultured by dispensing them into / wells. After replacing the medium with high-concentration glucose medium (DMEM (Hyclone, SH30022.01), containing 1% penicillin / streptomycin and 10% FBS) or low-concentration glucose medium (DMEM (Gibco, 11966-025), containing 1% penicillin / streptomycin and 10% FBS) or high-concentration glucose medium containing 5 μM of the GLUT inhibitor BAY-876 (Sigma, SML1774), propafenone (0, 10, 30 μM) was administered for 24 hours. Cell morphology was observed under a light microscope after treatment.

[0060] As a result, as shown in Figure 3, in the case of cancer cell lines, the number of cells decreased in a concentration-dependent manner with propafenone when cultured in a low-concentration glucose medium or under low-concentration glucose conditions where the intracellular uptake of glucose was inhibited, whereas no change in the number of cells according to the concentration of propafenone was observed under high-concentration glucose conditions. Meanwhile, in the case of normal cells, it was confirmed that there was no change in the number of cells regardless of glucose concentration or propafenone concentration.

[0062] And, to confirm changes in intracellular signaling pathways, a Western blot was performed.

[0063] First, 4 x 10 cells per well in a 12-well plate 4The cells were inoculated into / wells and cultured. After replacing the medium with high-concentration or low-concentration glucose medium, propafenone (0, 10, 20 μM) was added for 24 hours. Subsequently, sample buffer (containing Tris (pH 6.8) 50 mM, 2% SDS, 0.002% bromophenol blue, 10% glycerol, and 5% β-mercaptoethanol) was added to the cells after removing the medium, and the mixture was incubated for 5 minutes. The supernatant obtained was used as the protein sample. Protein in each protein sample was quantified using BSA (bovine serum albumin), and then equal amounts of protein were separated by 8% SDS-PAGE (sodium dodecylsulfate-polyacrylamide gel electrophoresis), transferred to a PVDF (polyvinylidene fluoride) membrane, and reacted with the primary antibody. After washing the membrane, it was reacted with a secondary antibody, and the protein band was confirmed by developing it with an enhanced chemiluminescence solution (ECL). Here, p-S6K (Cell Signaling Technology, 9234), p-AMPK (Cell Signaling Technology, 2535), p-ERK (Cell Signaling Technology, 4377), and Vinculin (Santa Cruz, sc-73614) were used as primary antibodies, and Goat Anti-Rabbit IgG antibody (GeneTex, GTX213110-01) and Goat Anti-Mouse IgG antibody (GeneTex, GTX213111-01) were used as secondary antibodies.

[0064] As a result, as shown in Figure 4, under high glucose conditions, there was no change in the activity of S6K, AMPK, and ERK according to propafenone concentration regardless of cell type; however, in cancer cell lines under low glucose conditions, it was confirmed that S6K activity was inhibited and AMPK activity increased in a concentration-dependent manner with propafenone. Therefore, it was found that the anticancer effect of propafenone occurs when the glucose concentration is maintained at a low level.

[0066] 2-2. Animal Experiments

[0067] To confirm the cancer cell death effect induced by propafenone under hypoglycemic stimulation, animal experiments were conducted using propafenone in combination with BAY-876 as a hypoglycemic agent.

[0068] Animal experiments were conducted in compliance with animal ethics regulations after obtaining approval from the Yonsei University Committee on Laboratory Animal Ethics. Six-week-old BALB / C nude mice (Zabio Co., Ltd.) were used for the animal experiments. For the experiment, 1 x 10⁶ 786-O cells were used. 7 The solution was dissolved in 100 μl of PBS and injected subcutaneously into mice. One week later, the cells-transplanted mice were randomly divided into four groups. PBS was administered to the control group (Ctrl), 20 mg / kg of propafenone to the propafenone group (Pro), 5 mg / kg of BAY-876 to the hypoglycemic agent group (Bay), and 20 mg / kg of propafenone and 5 mg / kg of BAY-876 to the combination group (Pro+Bay) via intraperitoneal injection once daily (Monday to Friday) for five weeks. Body weight was measured after each weekly injection, and after the administration was completed, the animals were euthanized in compliance with animal ethics regulations to excise the tumors, and the tumor weight was measured.

[0069] As a result, as shown in Figure 5, there was almost no difference in body weight between the treatment groups, but it was confirmed that the tumor size was significantly reduced in the combination treatment group administered both propafenone and BAY-876 compared to the control group, the propafenone treatment group, or the BAY-876 treatment group.

[0071] These results suggest that the combination of propafenone and hypoglycemic agents selectively exhibits a cancer cell killing effect.

[0073] The present invention has been described above with reference to its preferred embodiments. Those skilled in the art will understand that the present invention may be embodied in modified forms without departing from the essential characteristics of the invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the invention is defined by the claims, not by the foregoing description, and all variations within the scope of the claims should be interpreted as being included in the invention.

Claims

Claim 1 A pharmaceutical composition for the prevention or treatment of cancer comprising propafenone and an mTOR activity inhibitor as active ingredients; wherein the cancer is one or more cancers selected from the group consisting of renal cancer, lung cancer, breast cancer, and skin cancer in which intracellular mTOR (mammalian target of rapamycin) is inhibited by the mTOR activity inhibitor that inhibits mTOR activity, and the mTOR activity inhibitor is an mTOR inhibitor or a hypoglycemic agent, wherein the mTOR inhibitor is rapamycin, Temsirolimus, RapaLink-1, an mTOR kinase inhibitor, or a combination thereof, wherein the mTOR kinase inhibitor is sapanisertib, and the hypoglycemic agent is BAY-876 (CAS No. 1799753-84-6). Claim 2 delete Claim 3 delete Claim 4 delete Claim 5 delete Claim 6 delete Claim 7 delete Claim 8 delete Claim 9 delete Claim 10 delete Claim 11 delete Claim 12 delete Claim 13 delete Claim 14 delete

Images