Patent Application
20200282009
The present invention, in some embodiments thereof, relates methods of treating diseases associated with vascular smooth muscle cell proliferation.
c-myc is a master regulatory factor of cell proliferation. Pathologic activation of c-myc plays a central role in neoplastic disease pathogenesis by the coordinated upregulation of a transcriptional program influencing cell division, metabolic adaptation and survival. Amplification of c-myc is among the most common genetic alterations observed in cancer genomes. Validation of c-myc as a therapeutic target in cancer is supported by numerous lines of experimental evidence.
A growing body of evidence indicates that c-Myc plays a pivotal role also in cardiovascular diseases. Indeed, alterations of the basal machinery of the cell and perturbations of c-Myc-dependent signaling network are involved in the pathogenesis of certain cardiovascular disorders. Down-regulation of c-Myc induced by intervention with antioxidants or by antisense technology may protect the integrity of the arterial wall as well as neoplastic tissues (reviewed by Napoli et al. 2002 Neoplasia 4(3):185-190).
Nevertheless, a therapeutic approach to target c-Myc has remained elusive. The absence of a clear ligand-binding domain establishes a formidable obstacle toward direct inhibition, which is a challenging feature shared among many compelling transcriptional targets. C-Myc functions as a DNA-binding transcriptional activator upon heterodimerization with another basic-helic-turn-helix leucine zipper, transcription factor, Max. High resolution structures of the complex fail to identify a hydrophobic involution compatible with the positioning of an organic small molecule Reviewed in Delmore et al. Cell 146 904-917, 2011).
4F-benzoyl-TN14003 (also known as BKT140, hereinafter BL-8040), is a 14-residue bio stable synthetic peptide developed as a specific CXCR4 antagonist. It has been shown that BL-8040 binds the CXCR4 receptor with high affinity and long receptor occupancy (Peled A, Wald O, Burger J. Development of novel CXCR4-based therapeutics. Expert Opin Investig Drugs. 2012 March; 21(3):341-53). The peptide exhibits toxic effects on CXCR4-expressing malignancies including: multiple myeloma, acute myeloid leukemia (AML), chronic myeloid leukemia, non-small cell lung cancer and metastatic breast cancer (8,16-18). Currently, BL-8040 is in phase 2 clinical trials for AML and pancreatic cancer. The present inventor has previously shown that over-expression of CXCR4 on tumor cells and simulation of cells with CXCL12 leads to up-regulation of miR-15a/16-1 and consequently down regulation of their target genes BCL-2, MCL-1, and cyclin D1. Furthermore, overexpression of CXCR4 in these cells increases tumorogenesis and shift their oncogenic dependency from BCL2 to CXCR4. The present inventor further showed that that CXCR4 binding agent BL8040 has a similar effect on miR-15a/16-1. Thus, BL8040 induced apoptosis of tumor cell by inhibiting survival signals while keeping apoptotic gene expression low both in vitro and in vivo.
According to an aspect of some embodiments of the present invention there is provided a method of treating a disease associated with vascular smooth muscle cell proliferation in a subject in need thereof, with the proviso that the disease is not cancer, the method comprising administering to the subject a therapeutically effective amount of a CXCR4-antagonistic peptide comprising SEQ ID NO: 1, thereby treating the disease.
According to an aspect of some embodiments of the present invention there is provided a composition comprising a CXCR4-antagonistic peptide comprising SEQ ID NO: 1 for the treatment of a disease associated with vascular smooth muscle cell proliferation in a subject in need thereof with the proviso that the disease is not cancer.
According to an aspect of some embodiments of the present invention there is provided a device for cardiac delivery comprising a CXCR4-antagonistic peptide comprising SEQ ID NO. 1.
According to some embodiments of the invention, the disease is a cardiac disease.
According to some embodiments of the invention, the cardiac disease is selected from the group consisting of cardiac hypertrophy, transplant arteriosclerosis, atherosclerosis, angioplasty restenosis, ventricular stenosis or cardiac vein bypass stenosis.
According to some embodiments of the invention, the disease is selected from the group consisting of essential hypertension, thrombosis, stenosis, ventricular stenosis, diabetic macroangiopathy, myocardial infarction, stroke, vascular dementia, intimal hyperplasia, restenosis, angioplasty restenosis, cardiac vein bypass stenosis, transplant arteriosclerosis and atherosclerosis.
According to some embodiments of the invention, the administering comprises local administering.
According to some embodiments of the invention, the composition is formulated for local administration.
According to some embodiments of the invention, the device is a stent or a catheter.
Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.
Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.
In the drawings:
The present invention, in some embodiments thereof, relates to methods of treating diseases associated with vascular smooth muscle cell proliferation.
Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.
A growing body of evidence indicates that c-Myc can play a pivotal role both in neoplasia and cardiovascular diseases. Indeed, alterations of the basal machinery of the cell and perturbations of c-Myc-dependent signaling network are involved in the pathogenesis of certain cardiovascular disorders. However, c-Myc inhibition remains elusive and to-date there is no clinically advanced therapy that targets c-Myc.
Whist reducing embodiments of the invention to practice, the present inventor has uncovered that inhibition of CXCR4 using the peptide inhibitor BL8040 can block survival signals (by upregulating miR-15a/16-1 and consequently down regulation of their target genes BCL-2, MCL-1, and cyclin D1) and in parallel also represses c-Myc signaling pathway leading to cell death (as schematically illustrated in
Hence, it is now contemplated that BL-8040 can be effectively used in the treatment of c-Myc-related diseases because of its dual effect on cell survival and cell death.
Thus, according to an aspect of the invention there is provided a method of treating a disease associated with vascular smooth muscle cell proliferation in a subject in need thereof, with the proviso that the disease is not cancer, the method comprising administering to the subject a therapeutically effective amount of a CXCR4-antagonistic peptide comprising SEQ ID NO: 1, thereby treating the disease.
Alternatively, there is provided a composition comprising a CXCR4-antagonistic peptide comprising SEQ ID NO: 1 for the treatment of a disease associated with vascular smooth muscle cell proliferation in a subject in need thereof with the proviso that the disease is not cancer.
As used, herein “a disease associated with vascular smooth muscle cell proliferation” refers to a disease which onset or progression depends on vascular smooth muscle cell proliferation, which is governed by c-Myc signaling.
Methods of analyzing c-Myc activity are well known in the art (e.g., using c-Myc reporter kit available from Qiagen).
According to a specific embodiment, the disease is a cardiovascular or coronary heart disease such as cardiac hypertrophy, transplant arteriosclerosis, atherosclerosis, angioplasty restenosis, ventricular stenosis or cardiac vein bypass stenosis.
According to a specific embodiment, the disease is selected from the group consisting of essential hypertension, thrombosis, stenosis, ventricular stenosis, diabetic macroangiopathy, myocardial infarction, stroke, vascular dementia, intimal hyperplasia, restenosis, angioplasty restenosis, cardiac vein bypass stenosis, transplant arteriosclerosis and/or atherosclerosis.
According to a specific embodiment, the disease is a cardiovascular disease selected from the group consisting of atherosclerosis, thrombosis, myocardial infarction, stroke, congestive heart failure, dilated cardiomyopathy, vascular stenosis associated with atherosclerosis, angioplasty treatment, surgical incisions and mechanical trauma.
As used herein “subject in need thereof” refers to a mammal that is suspected of having, or at a risk of having, a disease characterized by vascular smooth muscle cell (“VSMC”) proliferation. Such a mammal can, for example, be a mouse, rat, dog, cat, cow, pig, horse, goat, sheep, rabbit, guinea pig, hamster or primate, such as a human of any gender or age. A mammal is at a risk of having the condition when it, for example, is at an age of being susceptible to the condition, or is genetically predisposed to or has a family history of the condition, or is about to undergo or has recently undergone vascular surgery.
As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
According to specific embodiments, the CXCR4-antagonistic peptide of the present invention comprises 4F-benzoyl-TN14003 (SEQ ID NO: 1, also termed herein as BL-8040) or an analog or derivative that is structurally and functionally related to the peptides disclosed in patent applications WO 2002/020561 and WO 2004/020462, also known as “T-140 analogs”, as detailed hereinbelow.
In various particular embodiments, the T-140 analog or derivative has an amino acid sequence as set forth in the following formula (I) or a salt thereof:
wherein:
A1 is an arginine, lysine, ornithine, citrulline, alanine or glutamic acid residue or a N-a-substituted derivative of these amino acids or A1 is absent;
A2 represents an arginine or glutamic acid residue if A1 is present, or A2 represents an arginine or glutamic acid residue or a N-α-substituted derivative of these amino acids if A1 is absent;
A3 represents an aromatic amino acid residue;
A4, A5 and A9 each independently represents an arginine, lysine, ornithine, citrulline, alanine or glutamic acid residue;
A6 represents a proline, glycine, ornithine, lysine, alanine, citrulline, arginine or glutamic acid residue;
A7 represents a proline, glycine, ornithine, lysine, alanine, citrulline or arginine residue;
A8 represents a tyrosine, phenylalanine, alanine, naphthylalanine, citrulline or glutamic acid residue;
A9 represents a citrulline, glutamic acid, arginine or lysine residue;
A10 represents an arginine, glutamic acid, lysine or citrulline residue wherein the C-terminal carboxyl may be derivatized;
and the cysteine residue of the 4-position or the 13-position can form a disulfide bond, and the amino acids can be of either L or D form.
Exemplary peptides according to formula (I) are peptides having an amino acid sequence as set forth in any one of SEQ ID NOS:1-72, as presented in Table 1 hereinbelow.
According to a specific embodiment, in each one of SEQ ID NOS:1-72, two cysteine residues are coupled in a disulfide bond.
In another embodiment, the analog or derivative has an amino acid sequence as set forth in SEQ ID NO:65 (H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH; TC14003).
In another embodiment, the peptide used in the compositions and methods of the invention consists essentially of an amino acid sequence as set forth in SEQ ID NO:1. In another embodiment, the peptide used in the compositions and methods of the invention comprises an amino acid sequence as set forth in SEQ ID NO:1. In another embodiment, the peptide is at least 60%, at least 70% or at least 80% homologous to SEQ ID NO:1. In another embodiment, the peptide is at least 90% homologous to SEQ ID NO:1. In another embodiment, the peptide is at least about 95% homologous to SEQ ID NO:1. Each possibility represents a separate embodiment of the present invention.
In various other embodiments, the peptide is selected from SEQ ID NOS:1-72, wherein each possibility represents a separate embodiment of the present invention.
In another embodiment, the peptide has an amino acid sequence as set forth in any one of SEQ ID NOS: 1-4, 10, 46, 47, 51-56, 65, 66, 68, 70 and 71. In another embodiment, the peptide has an amino acid sequence as set forth in any one of SEQ ID NOS: 4, 10, 46, 47, 68 and 70. In another embodiment, the peptide has an amino acid sequence as set forth in any one of SEQ ID NOS:1, 2, 51, 65 and 66. In another embodiment, the peptide has an amino acid sequence as set forth in any one of SEQ ID NOS:53-56.
In an embodiment, the peptide has an amino acid sequence as set forth in SEQ ID NO:1. In another embodiment, the peptide has an amino acid sequence as set forth in SEQ ID NO:2. In another embodiment, the peptide has an amino acid sequence as set forth in SEQ ID NO:51. In another embodiment, the peptide has an amino acid sequence as set forth in SEQ ID NO:66.
According to a preferred embodiment, the CXCR4 antagonist is as set forth in SEQ ID NO: 1.
The CXCR4 antagonist and possibly other active ingredients (e.g., for the treatment of vascular smooth muscle cell proliferation diseases as described herein) can each be administered to the subject as active ingredients per se, or in a pharmaceutical composition(s) where (each of) the active ingredients is mixed with suitable carriers or excipients.
Thus, for example, in the case of restenosis, the antagonistic peptide can be administered in conjunction with other therapeutics found effective to limit or eliminate restenosis, such as, for example, anti-platelet, anti-coagulation, anti-inflammatory, and vasodilation therapeutics.
As used, herein a “pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
Herein the term “active ingredient” refers to the peptides accountable for the biological effect. Optionally, a plurality of active ingredient may be included in the formulation, as further described hereinbelow.
Hereinafter, the phrases “physiologically acceptable carrier” and “pharmaceutically acceptable carrier”, which may be used interchangeably, refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
Herein, the term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.
Techniques for formulation and administration of drugs may be found in the latest edition of “Remington's Pharmaceutical Sciences”, Mack Publishing Co., Easton, Pa., which is herein fully incorporated by reference (Remington: The Science and Practice of Pharmacy, Gennaro, A., Lippincott, Williams & Wilkins, Philadelphia, Pa., 20th ed, 2000).
Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations that can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
The pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be accomplished by methods known to those skilled in the art such as intravenously or by use of a catheter or a stent to direct treatment to an affected area. Intravascular devices and their use are known to those skilled in the art.
For example, local administration to the relevant traumatized vessel by way of a catheter is one form of delivery. The peptide (and possibly other active agents, such as described hereinabove) is administered in the vicinity of the lesion via a catheter from inside the lumen, e.g., a porous balloon as described by Wolinsky and Thung, JACC 15: 475-481 (1990), or through the adventitia (i.e., the most outer layer of the vessel wall) with materials aiding slow release of the active ingredient. Other slow release techniques for local delivery include coating stents with the active ingredient compound, e.g., using a binder or gel described in Wilensky et al., Trends in Cardiovascular Med. 3:163-170 (1993). According to a specific embodiment, intracoronary administration using a catheter is contemplated.
Systemic administration is contemplated according to some embodiments of the invention.
The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
According to a specific embodiment the therapeutically effective amount comprises a single administration.
In one embodiment, the CXCR4 antagonist of the invention or the pharmaceutical composition comprising same is administered subcutaneously.
In one embodiment, the CXCR4 antagonist of the invention or the pharmaceutical composition comprising same is administered orally.
For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions (e.g., WFI), preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
Pharmaceutical compositions for potential administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water-based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the active ingredients, to allow for the preparation of highly concentrated solutions.
Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., a sterile, pyrogen-free, water-based solution, before use.
Alternative embodiments include depots providing sustained release or prolonged duration of activity of the active ingredient in the subject, as are well known in the art.
Pharmaceutical compositions suitable for use in the context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals (see the Examples section which follows, and Sekido et al. 2002 Cancer Genet Cytogenet 137(1):33-42). The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1 p. 1).
According to a specific embodiment, treatment with the peptide is accompanied by other Gold standard treatments contemplated for the contemplated diseases. The selection of the specific treatment depends on the specific type of the disease.
Exemplary treatments for cardiac diseases include, but are not limited to, Ace inhibitors, anticoagulation drugs, Beta blockers, Statins.
A more detailed list can be found at:
Specific examples include, but are not limited to:
Commonly prescribed include:
(Also known as ARBs or Angiotensin-2 Receptor Antagonists)
Commonly prescribed include:
ARNIs are a new drug combination of a neprilysin inhibitor and an ARB.
This drug class reduces the heart rate, similar to another class of drugs called beta blockers.
(Also known as Beta-Adrenergic Blocking Agents)
Commonly prescribed include:
Metoprolol succinate (Toprol XL)
Commonly prescribed include:
Hydralazine and Isosorbide Dinitrate (Specifically Benefits African Americans with Heart Failure)
Commonly prescribed:
Hydralazine and isosorbide dinitrate (combination drug)—(Bidil)
(Also known as Water Pills)
Commonly prescribed include:
As used herein the term “about” refers to ±10%
The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.
The term “consisting of” means “including and limited to”.
The term “consisting essentially of” means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof.
Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
The antagonistic peptide as described herein can be combined with other treatment modalities. These other treatments include medication (e.g., blood pressure medication, calcium channel blockers, digitalis, anti-arrhythmics, ACE inhibitors, anti-coagulants, immunosuppressants, pain relievers, vasodilators, etc.), angioplasty, stent placement, coronary artery bypass graft, cardiac assist device (e.g., left ventricular assist device, balloon pump), pacemaker placement, heart transplantation, etc.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.
Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.
Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non-limiting fashion.
Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., Ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al. (Eds.) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, J. E., Ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique” by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocols in Immunology” Volumes I-III Coligan J. E., Ed. (1994); Stites et al. (Eds.), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi (Eds.), “Selected Methods in Cellular Immunology”, W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., Ed. (1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J., Eds. (1985); “Transcription and Translation” Hames, B. D., and Higgins S. J., Eds. (1984); “Animal Cell Culture” Freshney, R. I., Ed. (1986); “Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide to Molecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317, Academic Press; “PCR Protocols: A Guide To Methods And Applications”, Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategies for Protein Purification and Characterization—A Laboratory Course Manual” CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.
Cell Lines
Human NB cell lines SK-N-BE(2), Shy-SY5Y and MHH-NB-11; prostate cancer cell line PC3; multiple myeloma cell line RPMI-8266. The SK-N-BE(2) cell line was kindly provided by Prof. Elizabeth Beierle (Birmingham Ala., USA). The human SHY-SY5Y and MHH-NB-11 cell lines were kindly provided by Prof. Isaac Witz (Tel Aviv University, Israel). All the other cell lines were purchased from ATCC. Cells were maintained at log growth in RPMI medium (Biological Industries) supplemented with 10% fetal calf serum (FCS), 1 mM L-glutamine, 100 U/ml penicillin, and 0.01 mg/ml streptomycin (Biological Industries) in a humidified atmosphere of 5% CO2 at 37° C.
Tumor Tissue Array
Tissue analysis of CXCR4 expression in neuroblastoma (NB) was analyzed in a tissue microarray (US BioMax MC809). Analysis was determined by scoring the staining intensity by two independent investigators.
Flow Cytometry Analysis
Cells were stained with human specific directly-labeled antibodies and analyzed by FACScalibur (Becton Dickinson Immunocytometry Systems), using the CellQuest software. CXCR4 monoclonal antibody, clone 12G5 (R&D Systems). BCL-2 monoclonal antibody, clone BCL-2/100 (eBioscience). For internal staining, cells were first fixed with 4% paraformaldehyde, permeabilized with 0.1% saponin.
Cell Proliferation Assay
Cells were seeded at 2×104 cells/1 ml per well into a 24-well plate in medium supplemented with 0.1% FCS with or without various concentrations of CXCL12 (PeproTech EC). On days 2, 4 and 7, cells were harvested, stained with propidium iodide (PI, Sigma), and the number of viable cells was determined using FACS analysis.
Soft Agar Colony Assay
An agar base layer was prepared as follows: 45 mL RPMI plus 12% FCS was mixed with 15 mL RPMI 32 plus 12% FCS and 15 mL 2.5% agar in double-distilled water. The tumor cells were suspended in RPMI plus 10% FCS. Cell suspension was mixed in a ratio of 1:3 with the agar base solution. This mixture was then plated on top of a preformed solid agar base. CXCL12 (100 ng/mL) or BL-8040 (20 μM) or anti-CXCR4 (1 μg/ml) were added to the mixture. Fourteen days later, the number of colonies was counted in 10 different fields.
Cell Survival Assay
A total of 1×105 cells/mL were cultured in 24-well plates with 1000 mL RPMI 1640 medium supplemented with 10% FCS. 24 hours later, the medium was changed to RPMI 1640 supplemented with either 1% FCS, with BL-8040 or AMD3100 or ABT199. Then 24 hours later, the cells were harvested, washed, and stained with PI (Sigma). The number of live cells was counted and analyzed using FACS analysis.
For SK-N-BE(2)-CXCR4 overexpression, 1×104 cells were seeded onto a 96-well microtiter xCELLigence assay plate (E-Plate) (ACEA Biosciences Inc.) and placed on the Real-Time xCELLigence Cell Analyzer (Roche Applied Science) platform at 37° C. to measure the cell index every 5 min.
Protein Extraction
SK-N-BE(2) cells were seeded into a 12-well plate at 2×105/1 ml of medium per well. The cells were incubated for 48 hours and supernatants were collected. For whole cells: tumor samples and cells were lysed by the addition of lysis buffer containing 50 mM Tris-HCl PH 7.6, 150 mM NaCl, 5 mM EDTA pH 8, 0.5% NP40 and protease inhibitor cocktail (Roche Diagnostics). Lysate was incubated with buffer for 15-20 minutes and then centrifuges at 14,000 rpm, 15 min, at 4° C. Protein amounts were determined by Bradford assay (Bio-Rad).
ELISA Assay
CXCL12 protein levels of cell supernatants or tumor lysates were determined using sandwich-type ELISA commercially available kit according to the manufacture's protocol (R&D Systems). The absorbance was read at 450 nm.
RNA Isolation
Total RNA from various cell lines and mouse tissues was isolated using Trizol reagent (Invitrogen) according to the manufacture's protocol, followed by DNaseI treatment using the DNaseI Kit where needed (Ambion). The concentrations were measured by Nanodrop (ND spectrophotometer) and the integrity was analyzed by gel electrophoreses on 1% agarose gel.
Quantitative Real Time RT-PCR
cDNA was synthesized from 0.5-1 μg total RNA using the Quanta Biosciences qScript™ cDNA Synthesis Kit (95047-100) for mRNA analysis, and using the qScript™ microRNA cDNA Synthesis Kit (95107-100) for miRNAs analysis. Quantiative PCR (qPCR) of miRNAs and mRNA was performed using the CFX384, C1000 touch thermal cycler (Bio-Rad) and a SYBR Green PCR Kit: Quanta Cat. #84018 and #84071, respectively. The fold expression and statistical significance were calculated using the 2-ΔΔ Ct method. All experiments were performed in triplicate.
Western Blot Analysis
Protein extracts were equally loaded onto 10% SDS-polyacrylamide gel, electrophorized and transferred onto a polyvinylidenedifluoride (PVDF) membrane (Bio-Rad Laboratories). Then, membranes were blocked and incubated with primary specific antibody O.N. at 4° C. For BCL-2 mouse monoclonal (Abcam). For cycD1 rabbit monoclonal (Thermo). For pERK1/2 rabbit monoclonal (Cell signaling). For β-Actin mouse monoclonal (MP biomedicals). After the washing procedure (3 times, 10 min in washing solution), the membrane was incubated with a secondary immunopure HRP-conjugated antibody, anti-mouse or anti rabbit (Envision; Dako) (1/10,000), washed (5 times, 6 min) and detected with the EZ-ECL kit (Biological Industries). Photon emission was identified by ChemiDoc MP imaging system (Bio-Rad). Intensities of protein bands were quantified by computerized densitometry using Image lab software (Bio-Rad).
Immunohistochemistry (IHC)
For histological analysis, tumor tissue was cut into 5-mm sections, deparaffinized with xylene, and hydrated through graded ethanol. Endogenous peroxidase was blocked by incubation for 5 minutes in 3% H2O2. A 25-mM citrate buffer (pH 6.0) was used for antigen retrieval, cooked in a pressure cooker for 20 minutes, and left to cool for 30 minutes at room temperature. Slides were washed in Optimax (Pharmatrade) and incubated with primary Ab diluted in CAS-Block (Zymed Laboratories). For Ki67 1:100 (Thermo Scientific), for CXCR4 anti-human CXCR4 monoclonal antibody clone 12G5 1:100 (R&D), for CXCL12 monoclonal CXCL12 antibody 79018 (R&D), for BCL-2 monoclonal BCL-2 antibody clone 124 (cell marque) and for cyclinD1 monoclonal BCL-1 antibody clone sp4 (spring). Staining overnight at 4° C. For all stainings, a conjugated horseradish peroxidase secondary Ab anti mouse or anti rabbit (Envision; Dako) was used for 30 minutes and developing was done with diaminobenzidine for 5 minutes followed by counterstaining with hematoxylin. For ki67, BCL-2 and Cyclin D1, staining levels were quantified using the Image Pro Analyzer image analysis software.
Transduction of Cell Lines
In order to stably overexpress CXCR4, SK-N-BE(2) cells were transduced with the lentiviral bicistronic vector harboring a CXCR4 expression cassette. This was done using a three-plasmid system: pHR′-CMVCXCR4-IRES-GFP-WPRE; envelope coding plasmid VSV-G and a packaging construct CMVDR8.91 according to previously published protocol (23). For transduction of SK-N-BE(2) cells, 1 ml of viral supernatant was used, which is equivalent to an m.o.i. of ˜9.
Flow cytometric analysis (FACS) analyzed the percentage CXCR4+ cells. PC3-CXCR4 and RPMI-CXCR4 cells were previously generated (6,24).
Transfections
Transfections were performed using Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer's protocol. Plasmids were purchased from Dharmacon. MiR-15a: miRIDIAN microRNA hsa-miR-15a-5p mimic. MiR-16-1: miRIDIAN microRNA hsa-miR-16-5p mimic. Negative control miR: miRIDIAN microRNA inhibitor negative control. AntagomiR-15a: miRIDIAN microRNA hsa-miR-15a-5p hairpin inhibitor. AntagomiR-16-1: miRIDIAN microRNA hsa-miR-16-5p hairpin inhibitor. AntagomiR negative control: miRIDIAN microRNA hairpin inhibitor negative control 1. All miRNA-mimics and antagomiRs were transfected at 50 nM or 100 nM. To determine the efficiency of transfection, at 48 hours post-transfection, the cells were analyzed for RNA.
Gene Expression Array
RNA was isolated from tumors and subjected to gene expression profiling using GeneChip human Gene 1.0 ST Array (Affymetrix). The gene expression values were extracted using the Partek Genomics Suite 6.6 software and, after thresholding and filtering procedures, were submitted to fold change and cluster analyses (Gene Expression Omnibus [GEO] GSE94426).
In-Vivo Orthotopic NB Model
Human SK-N-BE(2) cells were orthotopically injected into the left adrenal gland of 6-8 weeks old NSG mice. A 27-gauge needle was introduced through the left adrenal fat pad and 5×10∧5 cells/20 μl PBS were inoculated to the adrenal gland. NSG mice were maintained under defined flora conditions at the Hebrew University Pathogen-Free Animal Facility. Tumor growth and volume were monitored bi-weekly by using T2-weighted MRI until tumors reached the ethical limit volume and mice were randomized to drug-treated or control mice. Mice in the treated group were subjected to 2 treatment protocols. In the first treatment protocol BL-8040 (400 μg) was injected daily, starting three days following inoculation, and was continued for 35 days. In the second treatment protocol mice were monitored for 21 days. As of day 7, mice were treated with a daily injection of BL-8040 (400 μg) for 14 consecutive days.
Study Approval
All animal experiments were approved by the Animal Care Committee of the Hebrew University.
MR Imaging
Magnetic resonance imaging (MRI) scans were performed on a horizontal 4.7T Biospec spectrometer (Bruker Medical) with a 3.5-cm birdcage coil. Mice were anesthetized with Isoflurane (Nicholas Piramal, India; 2% in a mixture of 30:70 O2:N2O). Tumor volume was assessed bi-weekly using T2-weighted (T2W) fast spin echo images (repetition time=2,000 ms; echo time=37 ms; in plane resolution=117 μm; slice thickness-1 mm). Tumor volume was manually assessed using Analyze-7.0 (BIR). For each subject, an exponential growth curve was fitted to the tumor volume data points (Matlab software).
CXCR4 and BCL-2 Inhibitors
BL-8040 (BKT-140, 4F-benzoyl-TN14003) was kindly provided by BioLineRx Ltd. AMD3100 was purchased from Sigma-Aldrich. ABT199 was purchased from Selleck Chemicals.
Statistics
Data are presented as means±SD or ±SE. Statistical comparison of means was performed by a two-tailed unpaired Student's t test. Differences with a P<0.05 were determined as statistically significant. Statistical analysis of NB tumor mice survival following early treatment was performed using the Mantel-cox test.
To appreciate the significance of CXCR4 to neuroblastoma (NB) development, CXCR4 expression was measured in a tissue array composed of 13 NB samples and several cell lines derived from NB tumors. All sections examined stained strongly for CXCR4 (
The effect of CXCR4 on the growth of NB cells was determined. To examine whether CXCR4 has a proliferative effect on the Shy-SY5Y and SK-N-BE(2) cell lines, cells were incubated with different doses of the ligand CXCL12. Upon incubation with CXCL12 (500 ng/ml), the CXCR4 high expressing cell line Shy-SY5Y displayed increased proliferation (
Next, the present inventor investigated whether blocking CXCR4 using the antagonists BL-8040 or AMD3100 would influence NB cell viability. Both cell lines tested displayed decreased viability while exposed to increasing concentrations of BL-8040 (
To further investigate the influence of CXCR4 on the proliferation of NB cells, SK-N-BE(2)-CXCR4 cells were generated. The level of CXCR4 in the overexpressing cells was considerably higher than in control cells as determined by qPCR (
To investigate the role of CXCR4 on NB tumors in-vivo, an orthotopic xenograft tumor model was established. The SK-N-BE(2) cells were chosen for this model since they displayed a high inoculation rate. SK-N-BE(2) cells were injected to the left adrenal gland and mice were monitored bi-weekly for 65 days using MRI, presenting with an exponential growth pattern (
To further test the influence of CXCR4 presence on tumor growth in-vivo SK-N-BE(2) cells overexpressing CXCR4 were implanted to the left adrenal of mice. Tumors arising from SK-N-BE(2) cells overexpressing CXCR4 were 2-folds larger than the tumors from the native cell line 21 days post inoculation (
Following the establishment of the xenograft model the influence of BL-8040 on tumor development in-vivo was assessed. As long as BL-8040 was administered, tumor growth attenuation was exhibited. Discontinuation of BL-8040 treatment, resulted in tumors surge in an exponential growth pattern similar to controls (
Next, it was determined whether administering BL-8040 post tumor formation would also influence tumor development in mice. Tumors extracted from the BL-8040 treated mice were comparably smaller as measured by a 3-fold reduction in tumor weight (
These results indicate that SK-N-BE(2) cells in-vivo express CXCR4 as well as CXCL12. Moreover, as reported previously, CXCR4 overexpression enhances tumor growth. Most significantly, it was demonstrated that treatment with the CXCR4 inhibitor BL-8040, is capable of impairing NB tumor formation and growth resulting in increased survival of animals.
In order to identify genes that are important for NB development and are regulated by CXCR4, the expression patterns of genes between BL-8040 treated and un-treated NB tumors was compared. Gene expression was assessed by screening a human DNA chip array, using the Affymatrix gene chip expression analysis system. Among the differentially expressed genes, increased expression of miR-15a and its parent gene DLEU2 was found (
In an effort to extend our previous results, the influence of the CXCR4 inhibitor BL-8040 on the miR-15a/16-1 axis in-vitro was assessed. SK-N-BE(2) and Shy-SY5Y cells were incubated with BL-8040 (20 μM) and RNA was extracted for microRNA evaluation. QPCR revealed a significant increase in miR-15a, but not miR-16-1 in both cells treated with BL-8040 (
Cell death can be achieved by introducing miR-15a and/or miR-16-1 exogenously into tumor cells (25,26). To test whether miR-15a/16-1 could regulate cell death in NB cells, miR-15a and/or miR-16-1 were transfected into SK-N-BE(2) and Shy-SY5Y cells. Results are shown in
To further explore the involvement of miR-15a/16-1 in BL-8040 induced killing of NB cells, miR-15a/16-1 endogenous cellular levels were reduced by transfection with antagomiRs. Transfection efficacy was evaluated by quantifying miR-15a/16-1 RNA levels by qPCR (
To further study the role of CXCR4 in miR-15a/16-1 regulation their expression pattern in SK-N-BE(2)-CXCR4 cells was tested. Surprisingly, miR-15a/16-1, as measured by qPCR, were significantly upregulated in the SK-N-BE(2)-CXCR4 cells (
To better understand the mechanism by which CXCR4 upregulates miR-15a and miR-16-1 in NB cells. The expression pattern of PAX5, e2f1, e2f7 and c-myc all transcription factors known to target the DLEU2 promoter ((27-29) were examined in the NB cells treated with BL-8040 and in the SK-N-BE(2)-CXCR4 cells. c-myc, a negative regulator of the DLEU2 gene, was reduced significantly both in the NB cells treated with BL-8040 and in the SK-N-BE(2)-CXCR4 cells (
To elaborate on the connection between CXCR4 activation and miR-15a/16-1 expression, this expression pattern was measured in other tumor types. Two additional cell lines were examined: the multiple myeloma RPMI8266 cell line and the prostate cancer PC3 cell line. The levels of miR-15a/16-1 in RPMI-CXCR4 and PC3-CXCR4 cells were significantly upregulated compared to the native controls (
It has previously been demonstrated that both PC3-CXCR4 (6) and RPMI-CXCR4 (24) cells exhibit an increase in ERK1/2 phosphorylation induced by CXCR4 overexpression. Respectively, an increase in ERK1/2 phosphorylation was detected in the SK-N-BE(2) cells overexpressing CXCR4 (
Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.
All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IL2018/051023 | 9/12/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62557197 | Sep 2017 | US |
