Patent Application
20230263825
This disclosure relates to the field of anti-cancer therapy. In particular, the disclosure relates to perborate salts, compositions comprising the same, and uses thereof as anti-cancer agents.
Cancer is the general name of various diseases that result in the continuous growth and division of cells in the body due to the disruption of control mechanisms. Ten properties that distinguish cancer cells from normal cells include: the ability to synthesize the growth factors they need, immunity to signals that restrict their growth, the ability to escape controlled cell death pathways, the ability to divide unlimitedly, the ability to form a blood vessel network that can feed themselves, the ability to metastasize and invade other tissues and organs through blood vessels, immunity to the ability to escape the system, increase tumor inflammation, irregular metabolic pathways, and an unstable genome that is open to mutations (Hanahan & Weinberg, 2011).
Cancer is the second most fatal disease after cardiovascular diseases, with lung cancer causing the most deaths amongst all cancer types. Surgical, radiotherapeutic and/or chemotherapeutic methods are commonly used as cancer treatments, each having variable levels of efficacy depending on the type of cancer.
For treatments that use chemotherapy, even the most effective chemotherapy drugs are not able to eliminate cancer completely, and merely serve to extend the patient's life. Furthermore, chemotherapy drugs do not solely affect cancer cells. Chemotherapy drugs may be targeted to cancer cells, e.g. by using cancer's interest in sugar, but this does not prevent entry to healthy cells. As a result, chemotherapies have many side effects such as emesis, vertigo, weight loss, fever, decreased blood values and bleeding. These side effects are only some of the 23 known side effects of 250 chemotherapy drugs supported by the NCI (National Cancer Institute). In addition, the side effects of existing chemotherapeutic drugs vary from patient to patient.
Thus, there remains a need for alternative treatments for treating various cancers in order to eliminate the cancer, or extend a patient's life.
In one aspect, the present disclosure provides a method for reducing gene expression of one or more genes in a cancer cell or a tumor cell, the method comprising contacting the cell with an effective amount of a perborate salt, wherein the one or more genes are selected from the group consisting of Paxillin (PXN), Vinculin (VCL), TLN1, TNS2, RHOA, CDC42, KRAS, CCN2, C-Met and BIRC5.
In some embodiments, the one or more genes is PXN. In some embodiments, the one or more genes is VCL. In some embodiments, the one or more genes is TLN1. In some embodiments, the one or more genes is TNS2. In some embodiments, the one or more genes is RHOA. In some embodiments, the one or more genes is CDC42. In some embodiments, the one or more genes is KRAS. In some embodiments, the one or more genes is CCN2. In some embodiments, the one or more genes is C-met. In some embodiments, the one or more genes is BIRC5 (survivin).
In various embodiments, the cell is characterized by upregulation of the EGF/EGFR pathway as compared to normal cells. In some embodiments, the cancer or tumor cell is a bladder cancer cell, a bone cancer cell, a brain tumor cell, a breast cancer cell, a cervical cancer cell, a colorectal cancer cell, an endometrial cancer cell, a gastric cancer (adenocarcinoma) cell, a leukemia cell, a liver cancer cell, a lung cancer cell, a lymphoma cell, an oral cancer cell, an ovarian cancer cell, a pancreatic cancer cell, a prostate cancer cell, a renal cancer cell, a skin cancer cell or a thyroid cancer cell. In some embodiments, the cancer or tumor cell is a lung cancer cell, a pancreatic cancer cell, a chordoma cell, a breast cancer cell or an adenocarcinoma cell. In some embodiments, the cancer or tumor cell is a lung cancer cell. In some embodiments, the cancer or tumor cell is a pancreatic cancer cell. In some embodiments, the cancer or tumor cell is a chordoma cell.
In another aspect, the present disclosure provides a pharmaceutical composition comprising a perborate salt and a pharmaceutically acceptable carrier, diluent or adjuvant. The pharmaceutical composition may comprise an effective amount of a perborate salt. The pharmaceutical composition may further comprise a chemotherapy agent, such as Cisplatin and/or 5-fluorouracil.
In another aspect, the present disclosure provides a perborate salt for use in the treatment of a cancer. For example, an effective amount of a perborate salt for use in the treatment of cancer. The present disclosure further provides a combination comprising an effective amount of a perborate salt and a chemotherapy agent, such as Cisplatin and/or 5-fluorouracil.
In another aspect, the present disclosure provides a method for treating a cancer in a subject, the method comprising administering a perborate salt to the subject. For example, the method comprises administering an effective amount of a perborate salt to the subject. The method may further comprise administering a chemotherapy agent, such as Cisplatin and/or 5-fluorouracil, to the subject.
In another aspect, the present disclosure provides a pharmaceutical composition comprising an effective amount of a perborate salt and a pharmaceutically acceptable carrier, diluent or adjuvant.
In another aspect, the present disclosure provides an effective amount of a perborate salt for use in the treatment of a cancer. The cancer may be a lung cancer, a pancreatic cancer, a chordoma, a breast cancer or an adenocarcinoma. For example, the cancer may be small cell lung cancer. Also provide is a combination comprising the effective amount of the perborate salt and a chemotherapy agent.
In another aspect, the present disclosure provides use of a perborate salt for treatment of a cancer in a subject. For example, use of an effective amount of a perborate salt for treatment of a cancer in a subject. The perborate salt may be in combination with a chemotherapy agent, such as Cisplatin and/or 5-fluorouracil.
In another aspect, the present disclosure provides use of a perborate salt in preparation of a medicament for treatment of a cancer in a subject. For example, use of an effective amount of a perborate salt in preparation of a medicament for treatment of a cancer in a subject. The perborate salt may be in combination with a chemotherapy agent, such as Cisplatin and/or 5-fluorouracil.
In some embodiments, the perborate salt may be in anhydrous form or solvent addition form. The perborate salts may be associated with a non-stoichiometric amount of a solvent, water and/or buffer. The solvent may be, for example, a pharmaceutically acceptable solvent. In some embodiments, the perborate salt may have the general formula XBO3·nH2O (n=1 to 6). In some embodiments, the perborate salt is a tetrahydrate perborate salt.
The perborate salt may be a group 1 or group 2 metal salt (e.g. where X is a group 1 or group 2 metal ion). In some embodiments, the perborate salt may be a group 1 metal salt. In some embodiments, the perborate salt may be a sodium perborate salt, a potassium perborate salt or a lithium perborate salt. In some embodiments, the perborate salt is a sodium perborate salt, e.g. having the formula NaBO3·nH2O (n=1 to 6). In some embodiments, the perborate salt is sodium perborate tetrahydrate.
In various embodiments, the cancer is characterized by upregulation of the EGF/EGFR pathway as compared to normal cells. In various embodiments, the cancer is a bladder cancer, a bone cancer, a brain tumor, a breast cancer, a cervical cancer, a colorectal cancer, an endometrial cancer, a gastric cancer (adenocarcinoma), a chordoma, a liver cancer, a lung cancer, a lymphoma, an oral cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a renal cancer, a skin cancer, a thyroid cancer or a leukemia. In some embodiments, the cancer is a lung cancer, a pancreatic cancer, a chordoma, a breast cancer or an adenocarcinoma. In some embodiments, the cancer is a lung cancer, such as small cell lung cancer. In some embodiments, the cancer is a pancreatic cancer. In some embodiments, the cancer is a chordoma.
The perborate salt may be provided at a dosage of at least about 0.05 mg/kg/day. The perborate salt may be provided at a dosage of between about 0.05 mg/kg/day to about 10 mg/kg/day. The perborate salt may be provided at a dosage of up to about 115 mg/kg/day.
In another aspect, the present disclosure provides a method for reducing gene expression of one or more genes in a cancer cell or a tumor cell, the method comprising contacting the cell with an effective amount of a perborate salt, wherein the one or more genes are involved in a cell signalling pathway selected from the group consisting of: cell cycle pathway, DNA replication pathway, EGF/EGFR signal pathway, Focal Adhesion-P13K-AKT pathway, focal adhesion pathway, G protein signaling pathway, Hippo-Merlin Signaling dysregulation pathway, MAPK signaling pathway, actin-cytoskeleton pathway, TGF Beta signaling pathway and WNT signaling pathway.
The one or more genes may be a gene involved in the cell cycle pathway, such as one or more genes selected from the group consisting of: ANAPC5, ANAPC11, CCNA2, CCNB1, CCNB2, CCNB3, CCND3, CCNE1, CCNE2, CDC20, CDC23, CDC25A, CDC25B, CDC25C, CDC45, CDC6, CDC7, CDK1, CDK2, CDK4, CDKN2A, CDKN2C, CUL1, E2F1, E2F2, E2F3, HDAC1, HDAC2, MCM2, MCM3, MCM4, MOMS, MCM6, MCM7, MYC, ORC1, ORC6, PCNA, PLK1, PTTG1, RAD21, RBL1, RBX1, BUB1, BUB3, SKP2, SMC1A, STAG1, TFDP1, TGFB1, TTK, YWHAB, and YWHAG.
The one or more genes may be a gene involved in the DNA replication pathway, such as one or more genes selected from the group consisting of: CDC6, CDC7, CDK2, CDT1, GMNN, MCM10, MCM2, MCM3, MCM4, MOMS, MCM6, MCM7, PCNA, POLA2, POLD2, POLD3, POLE, POLE2, RFC2, RFC3, RFC4, RPA2, RPA3, and PRIM1.
The one or more genes may be a gene involved in the EGF/EGFR pathway, such as one or more genes selected from the group consisting of: AP2S1, ARHGEF1, AURKA, E2F1, INPP5D, MYBL2, NEDD4, PCNA, PEBP1, PIK3R1, PLCE1, PRKCB, RAF1, RALB, RALBP1, RPS6KA1, RPS6KA3, SHC1, and STMN1.
The one or more genes may be a gene involved in the focal adhesion pathway, such as one or more genes selected from the group consisting of: ANGPT2, CDC37, COL3A1, EIF4E, EIF4E2, ELAVL1, EPHA2, EPOR, FGF18, FGF2, FGFR1, GNB2, GNG4, GNG10, GNG12, HSP9OAA1, HSP90AB1, HSP90B1, IKBKG, IRS1, ITGB3, LAMA1, LAMB1, LAMC3, PIK3R1, PPP2CA, PPP2CB, PPP2R1A, PPP2R1B, PPP2R3C, RAB11B, RAF1, SLC2A3, TBC1D1, THBS1, ITGB3, LAMA1, LAMB1, THBS1, LAMC3, SHC1, SHC4, STYK1, PRKCB, ACTN1, ACTN4, FLNA, ZYX, VASP, VCL, PARVB, PARVA, RHOA, PIK3R1, PPP1CA, PPP1CC, MYL9, MYL12B, MYLK, ACTB, ACTG1, PAK4, CCND3, BIRC3, and RAF1.
The one or more genes may be a gene involved in the G protein pathway, such as one or more genes selected from the group consisting of: AKAP5, AKAP8, AKAP9, GNB2, GNG4, GNG10, GNG12, GNAl2, PDE7A, PRKAR1B, RRAS, CALM1, GNA12, ARHGEF1, RHOA, PRKCH, PRKD3, and PRKCQ.
The one or more genes may be selected from a gene involved in the Hippo pathway, such as one or more genes selected from the group consisting of: AJUBA, CD44, EPHA2, FGFR1, FOXM1, ITGB3, LATS2, MYC, NF2, PAK4, PPP1CA, PPP1CC, PRKAR1B, RBX1, and TEAD4.
The one or more genes may be a gene involved in the MAPK/ERK pathway such as one or more genes selected from the group consisting of: ARRB1, CASP3, CDC25B, DUSP6, DUSP9, ECSIT, FGF18, FGF2, FGFR1, FLNA, GNA12, HSPA1A, HSPA1B, HSPA2, HSPA8, IKBKG, MAP2K3, MAP2K6, MAP2K7, MAP3K11, MAPK11, MYC, NFKB1, PLA2G4E, PPP5C, PPP5D1, RAF1, RASGRP1, RPS6KA3, RRAS, STMN1, and TGFB1.
The one or more genes may be a gene involved in the actin cytoskeleton pathway, such as one or more genes selected from the group consisting of: ACTB, ACTG1, ACTN1, ARHGEF1, ARPC5, FGF18, FGF2, FGFR1, GNG12, MSN, MYH10, MYLK, PAK4, PIK3R1, RAF1, RHOA, RRAS, TMSB4X, and VCL.
The one or more genes may be a gene involved in the TGF beta signal pathway, such as one or more genes selected from the group consisting of: CCNB2, CDK1, CITED1, CUL1, EID2, FOXP3, HDAC1, ITGB3, MAP2K6, MMP1, MYC, PARD6A, PIK3R1, RAF1, RBL1, RBX1, RHOA, SHC1, SIK1, TERT, TFDP1, TGFB1, TGFB1I1, THBS1, TRAP1, WWP1, and ZEB2.
The one or more genes may be a gene involved in the WNF signal pathway, such as one or more genes selected from the group consisting of: CAMK2G, CCND3, CTBP1, CTNNB1, GPC4, LRP6, MYC, PLAU, PRICKLE1, PRKCB, RHOA, ROR2, SFRP1, VANGL1, WNT5A, and WNT5B.
The chemotherapy agent may comprise Altretamine; Bendamustine; Busulfan; Carboplatin; Carmustine; Chlorambucil; Cisplatin; Cyclophosphamide; Dacarbazine; Ifosfamide; Lomustine; Mechlorethamine; Chlormethine; Melphalan; Oxaliplatin; Temozolomide; Thiotepa; Trabectedin; Carmustine; Lomustine; Streptozocin; Azacitidine; 5-fluorouracil (5-FU); 6-mercaptopurine (6-MP); Capecitabine (Xeloda); Cladribine; Clofarabine; Cytarabine (Ara-C); Decitabine; Floxuridine; Fludarabine; Gemcitabine (Gemzar); Hydroxyurea; Methotrexate; Nelarabine; Pemetrexed (Alimta); Pentostatin; Pralatrexate; Thioguanine; Trifluridine/tipiracil combination; Daunorubicin; Doxorubicin; Epirubicin; Idarubicin; Valrubicin; Bleomycin; Dactinomycin; Mitomycin-C; Mitoxantrone; Irinotecan; Topotecan; Etoposide (VP-16); Mitoxantrone; Teniposide; Cabazitaxel; Docetaxel; Nab-paclitaxel; Paclitaxel; Vinblastine; Vincristine; Vinorelbine; Prednisone; Methylprednisolone; Dexamethasone; Arsenic trioxide; Asparaginase; Eribulin; Hydroxyurea; Ixabepilone; Mitotane; Omacetaxine; Pegaspargase; Procarbazine; Romidepsin; Vorinostat, Raltitrexed or any combinations thereof. In some embodiments, the chemotherapy agent comprises Cisplatin. In some embodiments, the chemotherapy agent comprises 5-fluorouracil.
Other aspects and features of the present invention will become apparent to those of ordinary skill in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying claims.
In drawings which illustrate embodiments of the invention,
In the context of the present disclosure, various terms are used in accordance with what is understood to be the ordinary meaning of those terms.
This disclosure is based in part on the discovery that perborate salts may be used for reducing or decreasing gene expression of one or more genes in a cancer cell or a tumor cell. The one or more genes may be selected from the group consisting of PXN, VCL, TLN1, TNS2, RHOA, CDC42, KRAS, CCN2, C-Met and BIRC5. The one or more genes may be involved in a cell signalling pathway, the cell signalling pathway being a cell cycle pathway, a DNA replication pathway, an EGF/EGFR signal pathway, a Focal Adhesion-PI3K-AKT pathway, a focal adhesion pathway, a G protein signaling pathway, a Hippo-Merlin Signaling dysregulation pathway, a MAPK signaling pathway, an actin-cytoskeleton pathway, a TGF Beta signaling pathway or a WNT signaling pathway. In various embodiments, the perborate salts as described herein may be used in the preparation of a medicament or a composition for treatment of a cancer.
Further aspects of the disclosure make use of compositions comprising a perborate salt and a pharmaceutically acceptable carrier, diluent or adjuvant. Methods of treating a cancer are also provided. Such methods may include administering a perborate salt or a composition of a perborate salt, or an effective amount of a perborate salt or a composition of a perborate salt, to a subject in need thereof. The compositions, methods and uses comprising perborate salts as described herein may be in combination with one or more chemotherapy agent.
The term “perborate salt” refers to a compound of the general formula XBO3·nH2O, where X is a metal element and n is an integer from 0 to 6. For the hydrated perborate salt, n is an integer from 1 to 6.
In some embodiments, the metal element may be a group 1 metal element, such as lithium, sodium, potassium, rubidium, cesium or francium. For example, the metal element may be lithium, sodium or potassium. In some embodiments, the metal element may be sodium.
In alternative embodiments, the metal element may be a group 2 metal element such as magnesium, calcium, strontium, barium or radium.
The perborate salt may be anhydrous (e.g. XBO3). The perborate salt may be a monohydrate, dihydrate, trihydrate, or tetrahydrate. In some embodiments, the perborate salt is a monohydrate salt. In some embodiments, the perborate salt is a dihydrate salt. In some embodiments, the perborate salt is a trihydrate salt. In some embodiments, the perborate salt is a tetrahydrate salt. In some embodiments, the perborate salt is a solvate.
The perborate salts contain a perborate anion [(B(OH)2OO)2]2− consisting of a —B—O—O—B—O—O— core with two hydroxyl groups attached to each boron atom. The embodiments of perborate salts as described include all possible stereochemical alternatives, including those illustrated or described herein. For example, the ring may adopt a chair conformation—see Formula (Ia):
Sodium perborate may be manufactured by reaction of borax Na2B4O7 and sodium hydroxide (NaOH) to give sodium metaborate NaBO2, which may then be reacted with hydrogen peroxide to give hydrated sodium perborate.
The raw material cost of the perborate salts (tetrahydrate and others) is approximately USD$350 per ton. The raw materials required to synthesise perborate salts are abundant in nature and they are readily synthesized in comparison to other cancer drugs. Perborate salts are stable and have a relatively long shelf life compared to other chemotherapy drugs. They are suitable candidates for incorporating in drug delivery systems which may be designed to target ‘hard to reach’ cancer cells, e.g. the perborate salts can readily be coated with polyethylene glycol or be loaded in liposomes or other types of target delivery vehicles such as nanoparticles.
While the present disclosure exemplifies the use of sodium perborate as an anti-cancer agent, it is known in the art that other perborate salts have similar physical properties and therefore are expected to have similar physiological and pharmokinetic properties.
The perborates have a long history of safe use in bleach-containing cleaning products, e.g. laundry detergents and machine dishwashing detergents, and are known to have low toxicity via oral, inhalation and dermal exposure routes in humans. Furthermore, sodium perborate solutions have been classified as not cytotoxic (Masetti, 2018).
Boron itself is a mineral found in foods such as nuts and is commonly taken as a medical supplement for conditions such as boron deficiency, menstrual cramps and osteoarthritis. Boron regulates calcium, potassium and magnesium metabolism in the body, and is associated with proper bone growth and development (Dessordi, 2017; Meacham, 1995). Boron is also a known food preservative. It has been shown that taking up to 18 mg of boron daily in adults does not cause any side effects (Murray, 1995). It has also been shown that boron homeostasis in mammalian systems is excreted through the urinary tract within 96 hours (Toxicological Review Of Boron And Compounds, CAS No. 7440-42-8).
While perborates are precursors of boric acid in the body, the biological effects and toxicology profiles of boric acid and other boron derivatives are noticeably different for both animals and humans (Biatek, 2019; Reproductive and General Toxicology of some Inorganic Borates and Risk Assessment for Human Beings, 1995; SCCS/1249/09, 2010; SCCS/1345/10, 2010). The difference in biological activity is confirmed by
In the human body, sodium perborate tetrahydrate undergoes multiple reactions. For example, sodium perborate tetrahydrate may dissociate into hydrogen peroxide, sodium metaborate and water (see Scheme I), or hydrogen peroxide and sodium borate (borax). Hydrogen peroxide further degrades to water and oxygen. However, NMR and Raman spectroscopy indicate that in dilute solution, an equilibrium exists that still contains peroxoborate anions and that these peroxoborate species are able to deliver the hydroperoxide anion at a lower pH than when H2O2 is used (Scheme II; A. McKillop, W. R. Sanderson, Tetrahedron, 1995, 51, 6145-6166). Furthermore, some borates are not metabolized at all, and the fact that perborates can be detected in urine indicates that some perborates are not converted to boric acid in the body (Reproductive and General Toxicology of some Inorganic Borates and Risk Assessment for Human Beings, 1995; Large-scale human metabolic phenotyping and molecular epidemiological studies via 1H NMR spectroscopy of urine investigation of borate preservation—PMID: 19453167).
Sodium perborate also reacts with acetic acid in the body (Scheme III; A. McKillop, W. R. Sanderson, Tetrahedron, 1995, 51, 6145-6166).
As shown in
Video analysis of MRC5 cells treated with sodium perborate tetrahydrate also showed that the damage to healthy cells only slowed the division and movement of these cells, and these effects disappeared when the boron moves away from the environment (see Example 3).
Toxicology studies performed in rats also confirmed that sodium perborate tetrahydrate had no significant toxicity to healthy tissues (see Table 2).
The perborate salts were found to have significant toxicity to cancer cell lines, with minimal toxicity to healthy cell lines (see
The following anti-cancer activities were observed, but are not exhaustive:
It was also found that administering a perborate salt in combination with a chemotherapy agent significantly reduced the concentration of the chemotherapy agent required to achieve an anti-cancer effect (see
The term “reducing gene expression”, otherwise known as gene silencing, gene knockdown or gene suppression, will be readily understood by those in the art to mean any effect that renders a gene inactive, or less active. For example, a gene that is suppressed two-fold means that said gene has half of its usual level of activity. Methods for measuring gene activity will be known to those skilled in the art. For example, gene expression measurement may be achieved by quantifying levels of the gene product, which is often a protein (e.g. Western Blot, ELISA), or by measuring levels of mRNA or DNA (e.g. Northern Blot, Microarray). Interestingly, it was also found that the perborate salts increased the expression of survivin in healthy cells (see
While existing cancer drugs generally produce a single effect (e.g. suppressing division), the use of perborate salts may produce multiple anti-cancer effects simultaneously, producing rapid and effective results, with no or minimal side effects compared to current treatments.
The pharmaceutical compositions of the present invention may include a pharmaceutically acceptable carrier, diluent or adjuvant to achieve a composition usable as a dosage form. The pharmaceutical composition may be formulated for administration orally, intravenously, intraperitoneally, rectally, intramuscularly, or subcutaneously. In some embodiments, the pharmaceutical compositions are formulated for oral use.
The pharmaceutical compositions according to the invention can be in different forms, in particular in a form chosen from the group comprising tablets, capsules, pills, syrups, suspensions, solutions, powders, granules, emulsions, microspheres and injectable solutions and solid lipid nanoparticles, in particular solid lipid nanoparticles.
The pharmaceutical compositions of the present invention may be used alone or in combination with other biologically active ingredients. A pharmaceutical composition of the present invention, alone or in combination with other active ingredients, may be administered to a subject in a single dose or multiple doses over a period of time. In various embodiments, the perborate salts may be used as a pre-treatment or a co-treatment of a cancer in a subject. In some embodiments, the perborate salts may be formulated in a pharmaceutical composition in combination with a chemotherapy agent. In some embodiments, the perborate salts may be administered in combination with a chemotherapy agent. In some embodiments, the administration of the perborate salt with the chemotherapy agent is simultaneous. In some embodiments, the administration of the perborate salt with the chemotherapy agent is sequential.
The term “effective amount” of a perborate salt or a pharmaceutical composition comprising a perborate salt includes a therapeutically effect amount or a prophylactically effective amount. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as decreased cancer proliferation, increased life span or increased life expectancy. A therapeutically effective amount may vary according to factors such as the disease state, age, sex and weight of the subject, and the ability of the perborate salt to elicit a desired response in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the formulation are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as the prevention, or the prevention of the progression, of a cancer. Typically, a prophylactic dose is used in subjects prior to or at an earlier state of disease. Methods of determining reduction or elimination of cancer or tumor cells are well known in the art, and need not be repeated herein.
It is to be noted that dosage values may vary with the severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. The amount of composition may vary according to factors such as the disease state, age, sex and weight of the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time or the dosage may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It may be advantageous to formulate compositions in dosage unit form for ease of administration and uniformity of dosage.
In some embodiments, the “effective amount” of perborate salt refers to a dosage of at least about 0.05 mg/kg/day. The dosage may be at least about 0.1 mg/kg/day. The dosage may be at least about 0.125 mg/kg/day. The dosage may be at least about 0.15 mg/kg/day. The dosage may be at least about 0.2 mg/kg/day. The dosage may be at least about 0.25 mg/kg/day. The dosage may be at least about 0.5 mg/kg/day. The dosage may be at least about 1 mg/kg/day. The dosage may be at least about 2 mg/kg/day. The dosage may be at least about 3 mg/kg/day. The dosage may be at least about 4 mg/kg/day. The dosage may be at least about 5 mg/kg/day. The dosage may be at least about 6 mg/kg/day. The dosage may be at least about 7 mg/kg/day. The dosage may be at least about 10 mg/kg/day. The dosage may be at least about 20 mg/kg/day. The dosage may be at least about 30 mg/kg/day. The dosage may be at least about 40 mg/kg/day. The dosage may be at least about 50 mg/kg/day. The dosage may be at least about 60 mg/kg/day. The dosage may be at least about 70 mg/kg/day. The dosage may be at least about 80 mg/kg/day. The dosage may be at least about 90 mg/kg/day. The dosage may be at least about 100 mg/kg/day.
The dosage may be between about 0.05 mg/kg/day and about 15 mg/kg/day. The dosage may be between about 0.1 mg/kg/day and about 10 mg/kg/day. The dosage may be between about 0.125 mg/kg/day and about 8 mg/kg/day. The dosage may be between about 0.05 mg/kg/day and about 7 mg/kg/day. The dosage may be between about 0.05 mg/kg/day and about 5 mg/kg/day. The dosage may be between about 10 mg/kg/day and about 20 mg/kg/day. The dosage may be between about 10 mg/kg/day and about 50 mg/kg/day. The dosage may be between about 20 mg/kg/day and about 50 mg/kg/day. The dosage may be between about 20 mg/kg/day and about 70 mg/kg/day. The dosage may be between about 10 mg/kg/day and about 120 mg/kg/day. The dosage may be between about 50 mg/kg/day and about 120 mg/kg/day. The dosage may be between about 50 mg/kg/day and about 70 mg/kg/day. The dosage may be between about 70 mg/kg/day and about 120 mg/kg/day.
The dosage may be up to about 0.25 mg/kg/day, up to about 0.5 mg/kg/day, up to about 1 mg/kg/day, up to about 2 mg/kg/day, up to about 4 mg/kg/day, up to about 5 mg/kg/day, up to about 6 mg/kg/day, up to about 7 mg/kg/day, up to about 8 mg/kg/day, up to about 10 mg/kg/day, or up to about 15 mg/kg/day. In some embodiments, the dosage of sodium perborate tetrahydrate may be up to about 50 mg/kg/day. In some embodiments, the dosage of sodium perborate tetrahydrate may be up to about 70 mg/kg/day. In some embodiments, the dosage of sodium perborate tetrahydrate may be up to 115 mg/kg/day.
In some embodiments, perborate salts as described herein may be used in combination with other treatment methods. For example, the perborate salts may be used as neoadjuvant (prior), adjunctive (during), and/or adjuvant (after) therapy in combination with other therapies known to one of ordinary skill in the art. As discussed above, the perborate salts may be administered in combination with a chemotherapy agent. In some embodiments, a non-toxic dosage of the perborate salt is used in a combination treatment, for example, up to about 115 mg/kg/day of sodium perborate tetrahydrate, up to about 70 mg/kg/day or up to about 15 mg/kg/day. In some embodiments, the effective dosage of the chemotherapy agent is at least 30% lower than a standard dosage of the chemotherapy agent. In some embodiments, the effective dosage of the chemotherapy agent is at least 40% lower than a standard dosage of the chemotherapy agent. In some embodiments, the effective dosage of the chemotherapy agent is at least 50% lower than a standard dosage of the chemotherapy agent. In some embodiments, the dosage of the chemotherapy agent is the EC50 dose or lower. In some embodiments, the dosage of the perborate salt is a dose that does not kill a cancer cell. Thus, the skilled person will appreciate that such dose of perborate salt that is used in a combination treatment will depend on the cancer being treated.
The perborate salts may be administered to a subject. As used herein, a “subject” may be a human, non-human primate, rat, mouse, cow, horse, pig, sheep, dog, cat, etc. The subject may be suspected of having or at risk for having a cancer. Diagnostic methods for cancers are known to those of ordinary skill in the art.
Lung cancer is divided into two subtypes: small cell lung cancer and non-small cell lung cancer. Approximately 85% of lung cancer cases are non-small cell lung cancers and 15% are small cell lung cancers. Small cell lung cancer cells are much more aggressive than the other type as they have the ability to divide rapidly, and they can easily metastatize to other tissues, especially the brain, bone and lymph, through the blood vessels (Nana-Sinkam & Powell, 2013). Surgical, radiotherapeutic and chemotherapeutic methods are generally used in the treatment of lung cancer. In cases of non-small cell lung cancer with a more mild prognosis than small cell lung cancer, tyrosine kinase inhibitors (such as axitinib and cediranib), small molecule inhibitors (such as c1-994 and cisplatin) and monoclonal antibodies (such as bevacizumab and figitumumab) are used for treatment (Lemjabbar-Alaoui et al., 2015). In small cell lung cancer, small molecule inhibitors such as cisplatin and etoposide are commonly used. However, despite these treatments, 98% of small cell lung cancer patients die within 5 years (Jackman & Johnson, 2005).
The present disclosure shows that perborate salts may be an effective treatment against various lung cancers and have minimal toxicity to healthy lung tissue.
Pancreatic cancer is one of the most common cancer types and the mortality rate within 5 years is 95% (Jackman & Johnson, 2005). Pancreatic cancer, which has 2 subspecies: exocrine and endocrine, causes the death of 500 thousand people every year. Pancreatic cancer is the fastest spreading cancer in the body. It is known to metastasize to bone, brain, liver and lung tissues (Simon et al., 2018). In pancreatic cancer, surgery is performed if possible before chemotherapy treatment. The most commonly used chemotherapy drugs for pancreatic cancer are gemicitabin and 5-fluorouracil (Zhang & Yang, 2020).
The present disclosure shows that perborate salts may be an effective treatment against pancreatic cancer and have minimal toxicity to healthy tissue.
Although a rare type of cancer, chordoma is an aggressive bone tumor that is thought to originate from ectopic notochord residues in the embryonic period encountered on the axial skeleton, with a high risk of recurrence and local metastasis. It is observed on the clivus and axial skeleton (Chugh et al., 2007). For chordomas, radical surgery is preferred as the main treatment method, since chordomas are resistant to chemotherapy and radiotherapy (Carrabba et al., 2008). The 5 year survival rate of chordoma is about 30-50% (Golden & Small, 2019).
The present disclosure shows that perborate salts may be an effective treatment against chordoma and have minimal toxicity to healthy tissue.
The cell cycle pathway refers to the phases of cell division. In order for the cell to divide and multiply, this pathway must work effectively. It has been found that perborate salts can suppress many genes in this pathway and therefore prevent cancer cell division. Genes involved in the cell cycle pathway whose expression was found to decrease two-fold and above following treatment with a perborate salt include: ANAPC5, ANAPC11, CCNA2, CCNB1, CCNB2, CCNB3, CCND3, CCNE1, CCNE2, CDC20, CDC23, CDC25A, CDC25B, CDC25C, CDC45, CDC6, CDC7, CDK1, CDK2, CDK4, CDKN2A, CDKN2C, CUL1, E2F1, E2F2, E2F3, HDAC1, HDAC2, MCM2, MCM3, MCM4, MOMS, MCM6, MCM7, MYC, ORC1, ORC6, PCNA, PLK1, PTTG1, RAD21, RBL1, RBX1, BUB1, BUB3, SKP2, SMC1A, STAG1, TFDP1, TGFB1, TTK, YWHAB and YWHAG (see
Cells must first copy their DNA in order to divide. Blocking the pathway for DNA replication prevents the cancer cell from dividing and multiplying. It has been found that perborate salts can prevent the division of cancer cells by lowering the expression levels of genes involved in the DNA replication pathway. Genes involved in the DNA replication pathway whose expression was found to decrease two-fold and above following treatment with a perborate salt include: CDC6, CDC7, CDK2, CDT1, GMNN, MCM10, MCM2, MCM3, MCM4, MCM5, MCM6, MCM7, PCNA, POLA2, POLD2, POLD3, POLE, POLE2, RFC2, RFC3, RFC4, RPA2, RPA3, and PRIM1 (see
The epidermal growth factor receptor (EGFR) signaling pathway regulates growth, differentiation, migration, attachment and survival of cells. A disorder in this pathway prevents cancer cells from growing, differentiating, migrating, clinging and surviving. It has been found that perborate salts inhibit the function of the EGF pathway by suppressing the expression of genes in this pathway. Genes involved in the EGF/EGFR signaling pathway whose expression was found to decrease two-fold and above following treatment with a perborate salt include: AP2S1, ARHGEF1, AURKA, E2F1, INPP5D, MYBL2, NEDD4, PCNA, PEBP1, PIK3R1, PLCE1, PRKCB, RAF1, RALB, RALBP1, RPS6KA1, RPS6KA3, SHC1, and STMN1 (see
The EGF/EGFR pathway is upregulated in many cancers, including bladder cancers, bone cancers, brain tumors, breast cancers, cervical cancers, colorectal cancers, endometrial cancers, gastric cancers (adenocarcinomas), liver cancers, lung cancers, lymphomas, oral cancers, ovarian cancers, pancreatic cancers, prostate cancers, renal cancers, skin cancers, thyroid cancers and leukemias. Thus, the perborate salts as disclosed herein, which inhibit the function of the EGF/EGFR pathway by suppressing expression of genes in this pathway, may be used to treat these cancers via downregulation of the EGF/EGFR pathway.
This pathway allows cells to move, e.g. allows cancer cells to metastasize by breaking off from one tissue and connecting to another. It has been found that perborate salts prevents cancer cells from moving and metastasizing by suppressing the expression of many genes in this pathway.
This pathway is a signal transmission pathway that enables the pathways inside the cell to work with an external stimulus. A disorder in this pathway will disrupt the signaling of cancer cells and inhibit its functions such as division, movement and growth. It has been found that perborate salts reduce the expression of many genes in this pathway. Genes involved in the G protein signaling pathway whose expression was found to decrease two-fold and above following treatment with a perborate salt include: AKAP5, AKAP8, AKAP9, GNB2, GNG4, GNG10, GNG12, GNA12, PDE7A, PRKAR1B, RRAS, CALM1, GNA12, ARHGEF1, RHOA, PRKCH, PRKD3, and PRKCQ (see
This pathway is related to the proliferation of cells and their apoptosis. A disorder in this pathway will prevent cancer cells from dividing and multiplying. It has been found that perborate salts disrupt the expression level of many genes in this pathway. Genes involved in the G protein signaling pathway whose expression was found to decrease two-fold and above following treatment with a perborate salt include: AJUBA, CD44, EPHA2, FGFR1, FOXM1, ITGB3, LATS2, MYC, NF2, PAK4, PPP1CA, PPP1CC, PRKAR1B, RBX1 and TEAD4 (see
This pathway provides communication between the stimulator that binds to a receptor outside the cell and the cell nucleus. A defect in this pathway negatively affects many functions, primarily cell division and cell cycle. It has been found that perborate salts reduce the expression of many genes in this pathway. Genes involved in the MAPK signaling pathway whose expression was found to decrease two-fold and above following treatment with a perborate salt include: ARRB1, CASP3, CDC25B, DUSP6, DUSP9, ECSIT, FGF18, FGF2, FGFR1, FLNA, GNA12, HSPA1A, HSPA1B, HSPA2, HSPA8, IKBKG, MAP2K3, MAP2K6, MAP2K7, MAP3K11, MAPK11, MYC, NFKB1, PLA2G4E, PPP5C, PPP5D1, RAF1, RASGRP1, RPS6KA3, RRAS, STMN1, and TGFB1 (see
This pathway is another basic pathway that allows cells to move. A disorder in this pathway will prevent a cancer cell from moving and as a result, prevent metastasis and spreading. It has been found that perborate salts reduce the expression of many genes in this pathway. Genes involved in the actin-cytoskeleton pathway whose expression was found to decrease two-fold and above following treatment with a perborate salt include: ACTB, ACTG1, ACTN1, ARHGEF1, ARPC5, FGF18, FGF2, FGFR1, GNG12, MSN, MYH10, MYLK, PAK4, PIK3R1, RAF1, RHOA, RRAS, TMSB4X, and VCL (see
This pathway is one of the main pathways that controls the differentiation and growth of cells. A disorder in this pathway will prevent the growth and differentiation of cancer cells. It has been found that perborate salts reduce the expression level of many genes in this pathway. Genes involved in the TGF Beta signaling pathway whose expression was found to decrease two-fold and above following treatment with a perborate salt include: CCNB2, CDK1, CITED1, CUL1, EID2, FOXP3, HDAC1, ITGB3, MAP2K6, MMP1, MYC, PARD6A, PIK3R1, RAF1, RBL1, RBX1, RHOA, SHC1, SIK1, TERT, TFDP1, TGFB1, TGFB1I1, THBS1, TRAP1, WWP1, and ZEB2 (see
This pathway controls the intracellular signal pathways that start with a stimulus that binds to the cell receptors. It is one of the pathways that control the destiny, movement and migration of the cell. A disorder in this pathway prevents the cell from continuing its life and prevents its division and migration. It has been found that perborate salts reduce the expression level of many genes in this pathway. Genes involved in the WNG signaling pathway whose expression was found to decrease two-fold and above following treatment with a perborate salt include: CAMK2G, CCND3, CTBP1, CTNNB1, GPC4, LRP6, MYC, PLAU, PRICKLE1, PRKCB, RHOA, ROR2, SFRP1, VANGL1, WNT5A, and WNT5B (see
These examples illustrate various aspects of the disclosure, evidencing a variety of methods for reducing gene expression of one or more genes in a cancer or a tumor cell by contacting the cell with an effective amount of a perborate salt. Selected examples are illustrative of advantages that may be obtained compared to alternative methods, and these advantages are accordingly illustrative of particular embodiments and not necessarily indicative of the characteristics of all aspects of the invention.
As used herein, the term “about” refers to an approximately +/−10% variation from a given value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.
Cell death pathways were analysed using the Annexin V method, performed by flow cytometry. In this method, Annexin V dye is used to detect cells entering the apoptosis death pathway (Sigma-Aldrich Cat No: 11858777001), while PI (Propidium Iodide) dye is used to detect cells entering the necrosis death pathway (Sigma-Aldrich Cat No: 11858777001).
Cells were grown in complete RPMI media (completed with 10% Fetal Bovine Serum and 1% penicillin streptomycin amphotericin) in a humidified CO2 incubator (80% humidity, 5% CO2 and 37° C.). At 70-80% confluency, cells were harvested using Trypsin/EDTA solution. After 3 days of sodium perborate tetrahydrate treatment (15 μg/mL), the cells were harvested and divided into 4 equal tubes. The first tube did not contain any dye and was used as a negative control. Only PI dye was placed in the second tube and necrotic cells were detected. Annexin V dye was placed in the third tube and apoptotic cells were detected. In the fourth tube, two dyes were put together. The result were analyzed and are shown in
An MTS reduction (MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay) was conducted to analyze the cytotoxic effect of sodium perborate tetrahydrate on lung, chordoma and pancreatic cells and normal cells. In this method, cell viability is determined via the activity of mitochondrial dehydrogenase enzyme, which reduces the tetrazolium dye MTS to its insoluble formazan (absorbance maximum of 490 nm in PBS).
Cells were counted and seeded in 96-well culture dishes. BxPC3 and PSN-1 cells were contacted with sodium perborate tetrahydrate at concentrations of 125 μg/mL, 62.5 μg/mL, 31.25 μg/mL, 15.625 μg/mL, 7.8125 μg/mL and 3.90615 μg/mL. DMS114 cells were contacted with sodium perborate tetrahydrate at concentrations of 250 μg/mL, 125 μg/mL, 62.5 μg/mL, 31.25 μg/mL, 15.625 μg/mL, 7.8125 μg/mL and 3.90615 μg/mL. ASPC and MRC5 cells were contacted with sodium perborate tetrahydrate at concentrations of 500 μg/mL, 250 μg/mL, 125 μg/mL, 62.5 μg/mL, 31.25 μg/mL, 15.625 μg/mL. CH22, JHC7, UM-Chor1, MUG-Chor1, UCH1 and NPH cells were contacted with sodium perborate tetrahydrate at concentrations of 500 μg/mL, 250 μg/mL and 125 μg/mL. The positive control comprised 20% Dimethyl Sulfoxide and the negative control comprised complete growth media. Seeding densities were as follows: DMS114: 20.000 cells/well; MRC5: 5.000 cells/well; CH22: 2.000 cells/well; JHC7, UM-Chor1, MUG-Chor1, UCH1 and NPH: 5.000 cells/well; PSN1: 10.000 cells/well; BxPC3 and ASPC: 7.500 cells/well. The effect of sodium perborate tetrahydrate on cell viability was measured as follows. The cell culture medium was discarded and 100 μl of MTS solution (10% glucose solution and 1% MTS-PMS solution in PBS) was added into each well. After 1 hour incubation in a humidified incubator, an absorbance reading was taken to determine mitochondrial dehydrogenase activity and cell survival was calculated relative to the negative control.
The results show that sodium perborate tetrahydrate was effective in decreasing cell survival of lung cancer, pancreatic and chordoma cells lines, with minimal impact on the cell survival of normal cells lines (see
The DMS114 and MRC5 cells from Example 2 were analyzed during the 3 day treatment period with sodium perborate tetrahydrate. Roughly 20 photos/hour were taken of the cells over the 72 hour (3 day) period. The resulting 1500 were merged to create a video which was used to observe the effect of sodium perborate on movement and division of DMS114 and MRC5 cells. The video analysis showed that the maximum effect on cell survival of DMS114 cells was achieved after 18 hours and that movement of close to 100% of DMS114 cells was stopped. The video analysis also showed that sodium perborate tetrahydrate had the effect of slowing cell division and movement of healthy cells to some extent, but that the effects diminished once the sodium perborate tetrahydrate moved away from the cellular environment.
Changes in the expression level of the cells treated with sodium perborate tetrahydrate for 3 days were carried out using the polymerase chain reaction method. Primers used were created with Primer BLAST software on NCBI (National Center for Biotechnology). After sodium perborate tetrahydrate treatment, the total RNAs of the cells were isolated (Macherey-Nagel Cat No: 740955250) and cDNA synthesis was made from these RNAs (QIAGEN Cat No: 205313). Synthesized cDNAs were mixed with SYBR Green mixture (Biorad Cat No: 1725122) and required primers to a final volume of 10 μl and expression levels of genes were analyzed using a BIO-RAD™ PCR device. The results are shown in
Cells were treated with sodium perborate tetrahydrate for 3 days and changes in the levels of gene expression were determined by Microarray. Affected genes and related pathways are shown in
Rats were treated orally with varying dosages of sodium perborate tetrahydrate ranging from 0.125 mg/kg/day to 8 mg/kg/day. After 32 days, the rats were sacrificed and their organs were examined under a microscope. The histopathology results showed no significant organ damage (see Tables 1 and 2 below).
The cytoxic effect of boric acid, sodium pentaborate pentahydrate and sodium perborate tetrahydrate on small cell lung cancer cells (DMS114) was tested using the method described in Example 2 and the results are shown in
These examples show that the biological and cytotoxic effects of boric acid and other boron derivatives such as sodium pentaborate pentahydrate are not equivalent to the perborate salts such as sodium perborate tetrahydrate.
The cytoxic effect of sodium perborate tetrahydrate, Cisplatin, 5-Fluorouracil (5-FU), and their combinations on PSN-1 (a pancreatic adenocarcinoma cell line) was tested using the method described in Example 2. According to the cytotoxicity experiments, the EC50 concentration of sodium perborate tetrahydrate is 7.5 μg/mL (
Surprisingly, it was found that when the chemotherapeutic agents Cisplatin and 5-FU were combined with a non-toxic dose of sodium perborate tetrahydrate (4 μg/mL), the EC50 value of Cisplatin decreased from 2 mM to 1 mM (
Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. The word “comprising” is used herein as an open-ended term, substantially equivalent to the phrase “including, but not limited to”, and the word “comprises” has a corresponding meaning. As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a thing” includes more than one such thing. Citation of references herein is not an admission that such references are prior art to the present invention. Any priority document(s) and all publications, including but not limited to patents and patent applications, cited in this specification are incorporated herein by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein and as though fully set forth herein. The invention includes all embodiments and variations substantially as hereinbefore described and with reference to the examples and drawings.
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| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/CA2020/050950 | Jul 2020 | WO | international |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2021/050932 | 7/7/2021 | WO |
