Patent Application
20190125753
Provided herein are compounds, compositions and methods for increasing hematopoiesis in vivo and expanding hematopoietic stem cells (HSC) in vitro and in vivo.
A subject having reduced levels of hematopoiesis may not generate sufficient quantities of new blood cells to remain or become healthy. Compositions and methods are needed for increasing hematopoiesis in patients who suffer from, or are susceptible to, depressed hematopoiesis activity or reduced blood cell levels. Such decreased or depressed hematopoiesis activity or reduced blood cell levels may be caused, for example, by chemotherapy, radiation therapy, radiation exposure, accidental radiation exposure, bone marrow transplantation therapy or congenital anemia. Bone marrow transplantation (BMT) or HSC transplantation is a method widely used in clinical practice. By expanding HSC in vitro before transplantation will accelerate the hematopoiesis and increase the chance of success.
A CBP/-catenin antagonist is an agent that disrupts or inhibits, in vitro or in vivo, the interaction between the CBP and 3-catenin proteins. Examples of such CBP/-catenin antagonists can be found, for example, in U.S. Pat. Nos. 6,762,185; 7,531,320; 7,232,822; 7,563,825, and 7,598,253; and U.S. Patent Application Publication Nos. 20120296067; 20120088770; and 20110263607.
More effective compounds and methods for increasing hematopoiesis are also needed for the expansion of HSCs prior to differentiation. In some instances, the CBP/-catenin antagonists have been shown to promote hematopoietic stem cell (HSC) differentiation but not expansion. Without being bound by theory, it is believed that such compounds and methods, alone or in combination with existing CBP/-catenin antagonists, would improve therapies for increasing hematopoiesis in patients.
Thus, in one aspect, there is provided a compound of Formula I
or a pharmaceutically acceptable salt thereof, wherein
In another aspect, a method is provided for increasing and/or supporting hematopoiesis in a subject in need thereof, comprising administering to the subject an effective amount of a compound of Formula I or a pharmaceutically acceptable salt thereof and/or a composition containing one or more compounds as described herein.
In yet another aspect, a method is provided for enhancing expansion of a hematopoietic stem cell or population (HSC), comprising contacting the HSC or population with an effective amount of a compound of Formula I or a pharmaceutically acceptable salt thereof and/or a composition containing one or more compounds as described herein. The contacting can be performed in vitro (e.g., including as part of an ex vivo method) or in vivo.
In still another aspect, a method is provided for inhibiting an interaction between a β-, and/or γ-catenin protein and a p-300 protein in a subject, cell or tissue having a β-, and/or γ-catenin protein and a p-300 protein, by contacting the cell, tissue or administering to the subject an effective amount of a compound of Formula I or a pharmaceutically acceptable salt thereof and/or a composition containing one or more compounds as described herein. In some aspects, the contacting is in vitro. In other aspects, the contacting is in vivo. In some aspects of the methods provided for inhibiting an interaction between a β-, and/or γ-catenin protein and a p-300 protein in a cell, a method for maintaining pluripotency of ES cells and iPS cells is provided.
In some embodiments of any of the methods described herein, the method further comprises administering to the subject or contacting the HSC cell or tissue with an effective amount of a CBP/β-catenin and/or γ-catenin antagonist that promotes hematopoietic stem cell (HSC) differentiation, wherein the antagonist is not a compound of Formula I or a pharmaceutically acceptable salt thereof. In one embodiment of the method, the antagonist is PRI-724 or ICG-001. In some embodiments, the method further comprises protection against fibrosis, which is a common chronic complication associated with radiation damage.
Table 3 and Table 4 are summary of FACS analysis to cells cultured in different growth factor and YH250 concentrations.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described. All technical and patent publications cited herein are incorporated herein by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
As used in the specification and claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise.
The term “about” when used before a numerical value indicates that the value may vary within a reasonable range, such as ±5%, ±1%, and ±0.2%.
As used herein, the term “comprising” is intended to mean that the compositions and methods include the recited elements, but not excluding others. “Consisting essentially of” when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. For example, a composition consisting essentially of the elements as defined herein would not exclude other elements that do not materially affect the basic and novel characteristic(s) of the claimed invention such as the biological activity of the claimed composition or method. “Consisting of” shall mean excluding more than trace amount of other ingredients and substantial method steps recited. Embodiments defined by each of these transition terms are within the scope of this invention.
As used herein, Cm-Cn, such as C1-C12, C1-C8, or C1-C6 when used before a group refers to that group containing m to n carbon atoms.
The term “alkoxy” refers to —O-alkyl.
The term “alkyl” refers to monovalent saturated aliphatic hydrocarbyl groups having from 1 to 12 carbon atoms (i.e., C1-C12 alkyl) or 1 to 6 carbon atoms (i.e., C1-C6 alkyl). This term includes, by way of example, linear and branched hydrocarbyl groups such as methyl (CH3—), ethyl (CH3CH2—), n-propyl (CH3CH2CH2—), isopropyl ((CH3)2CH—), n-butyl (CH3CH2CH2CH2—), isobutyl ((CH3)2CHCH2—), sec-butyl ((CH3)(CH3CH2)CH—), t-butyl ((CH3)3C—), n-pentyl (CH3CH2CH2CH2CH2—), and neopentyl ((CH3)3CCH2—).
The term “aryl” refers to a monovalent, aromatic mono- or bicyclic ring having 6-10 ring carbon atoms. Examples of aryl include phenyl and naphthyl. The condensed ring may or may not be aromatic provided that the point of attachment is at an aromatic carbon atom.
For example, and without limitation, the following is an aryl group:
The term “cycloalkyl” refers to a monovalent, preferably saturated, hydrocarbyl mono-, bi-, or tricyclic ring having 3-12 ring carbon atoms. While cycloalkyl, refers preferably to saturated hydrocarbyl rings, as used herein, it also includes rings containing 1-2 carbon-carbon double bonds. Nonlimiting examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamentyl, and the like. The condensed rings may or may not be non-aromatic hydrocarbyl rings provided that the point of attachment is at a cycloalkyl carbon atom. For example, and without limitation, the following is a cycloalkyl group:
The term “halo” refers to F, Cl, Br, and/or I.
The term “heteroaryl” refers to a monovalent, aromatic mono-, bi-, or tricyclic ring having 2-16 ring carbon atoms and 1-8 ring heteroatoms selected preferably from N, O, S, and P and oxidized forms of N, S, and P, provided that the ring contains at least 5 ring atoms. Nonlimiting examples of heteroaryl include furan, imidazole, oxadiazole, oxazole, pyridine, quinoline, and the like. The condensed rings may or may not be a heteroatom containing aromatic ring provided that the point of attachment is a heteroaryl atom. For example, and without limitation, the following is a heteroaryl group:
The term “heterocyclyl” or heterocycle refers to a non-aromatic, mono-, bi-, or tricyclic ring containing 2-12 ring carbon atoms and 1-8 ring heteroatoms selected preferably from N, O, S, and P and oxidized forms of N, S, and P, provided that the ring contains at least 3 ring atoms. While heterocyclyl preferably refers to saturated ring systems, it also includes ring systems containing 1-3 double bonds, provided that the ring is non-aromatic. Non-limiting examples of heterocyclyl include, azalactones, oxazoline, piperidinyl, piperazinyl, pyrrolidinyl, tetrahydrofuranyl, and tetrahydropyranyl. The condensed rings may or may not contain a non-aromatic heteroatom containing ring provided that the point of attachment is a heterocyclyl group. For example, and without limitation, the following is a heterocyclyl group:
The term “oxo” refers to a C═O group, and to a substitution of 2 geminal hydrogen atoms with a C═O group.
The term “optionally substituted,” unless defined otherwise, refers to a substituted or unsubstituted group. The group may be substituted with one or more substituents, such as e.g., 1, 2, 3, 4 or 5 substituents. Preferably, the substituents are selected from the group consisting of oxo, halo, —CN, NO2, —N2+, —CO2R10, —OR10, —SR10, —SOR10, —SO2R10, —NR11R12, —CONR11R12, —SO2NR11R12, C1-C6 alkyl, C1-C6 alkoxy, —CR10═C(R10)2, —CCR10, C3-C10 cycloalkyl, C3-C10 heterocyclyl, C6-C12 aryl and C2-C12 heteroaryl, wherein each R10 independently is hydrogen or C1-C8 alkyl; C3-C12 cycloalkyl; C3-C10 heterocyclyl; C6-C12 aryl; or C2-C12 heteroaryl; wherein each alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is optionally substituted with 1-3 halo, 1-3 C1-C6 alkyl, 1-3 C1-C6 haloalkyl or 1-3 C1-C6 alkoxy groups. Preferably, the substituents are selected from the group consisting of chloro, fluoro, —OCH3, methyl, ethyl, iso-propyl, cyclopropyl, vinyl, ethynyl, —CO2H, —CO2CH3, —OCF3, —CF3 and —OCHF2.
R11 and R12 independently is hydrogen; C1-C8 alkyl, optionally substituted with —CO2H or an ester thereof, C1-C6 alkoxy, oxo, —CR13═C(R13)2, —CCR, C3-C10 cycloalkyl, C3-C10 heterocyclyl, C6-C12 aryl, or C2-C12 heteroaryl, wherein each R13 independently is hydrogen or C1-C8 alkyl; C3-C12 cycloalkyl; C3-C10 heterocyclyl; C6-C12 aryl; or C2-C12 heteroaryl; wherein each cycloalkyl, heterocyclyl, aryl, or heteroaryl is optionally substituted with 1-3 alkyl groups or 1-3 halo groups, or R11 and R12 together with the nitrogen atom they are attached to form a 5-7 membered heterocycle.
The term “treatment” or “treating” means any treatment of a disease or disorder in a subject, such as a mammal, including:
As used herein, the term “preventing” refers to the prophylactic treatment of a patient in need thereof. The prophylactic treatment can be accomplished by providing an appropriate dose of a therapeutic agent to a subject at risk of suffering from an ailment, thereby substantially averting onset of the ailment.
As used herein, the term “condition” refers to a disease state for which the compounds, salts, compositions and methods provided herein are being used.
As used herein, the term “comprising” or “comprises” is intended to mean that the compositions and methods include the recited elements, but not excluding others. “Consisting essentially of” when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives and the like. “Consisting of” shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention or process steps to produce a composition or achieve an intended result. Embodiments defined by each of these transition terms are within the scope of this invention.
The term “isolated” as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, respectively that are present in the natural source of the macromolecule. The term “isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. The term “isolated” is also used herein to refer to polypeptides, proteins and/or cells that are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides. In other embodiments, the term “isolated” means separated from constituents, cellular and otherwise, in which the cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, which are normally associated in nature. For example, an isolated cell is a cell that is separated form tissue or cells of dissimilar phenotype or genotype. As is apparent to those of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, does not require “isolation” to distinguish it from its naturally occurring counterpart.
As used herein, “stem cell” defines a cell with the ability to divide for indefinite periods in culture and give rise to specialized cells. At this time and for convenience, stem cells are categorized as somatic (adult) or embryonic. A somatic stem cell is an undifferentiated cell found in a differentiated tissue that can renew itself (clonal) and (with certain limitations) differentiate to yield all the specialized cell types of the tissue from which it originated. An embryonic stem cell is a primitive (undifferentiated) cell from the embryo that has the potential to become a wide variety of specialized cell types. An embryonic stem cell is one that has been cultured under in vitro conditions that allow proliferation without differentiation for months to years. A clone is a line of cells that is genetically identical to the originating cell; in this case, a stem cell.
The term “hematopoiesis” intends the formation and development of blood cells. In the embryo and fetus it takes place in a variety of sites including the liver, spleen, thymus, lymph nodes, and bone marrow; from birth throughout the rest of life it is mainly in the bone marrow with a small amount occurring in lymph nodes
As used herein, the term “hematopoietic stem cell” intends a pluripotent cell having the ability to differentiate into a cell of the hematopoietic lineage.
Bone marrow cells contain hematopoietic stem cells that give rise to all lineages, such as lymphoid, myeloid and erythroid lineages. Bone marrow includes stem cells as well as progenitor cells of the lymphoid (T and B cells), myeloid (e.g., granulocytes, macrophages, NK cell, etc.) and erythroid (red blood cells) and platelet lineages. Human hematopoietic stem and progenitor cells express CD34 on their surface while differentiated cells do not. Accordingly, the detection of CD34 can be used to distinguish differentiated from undifferentiated cells.
Hematopoietic precursor cells can be derived either from the patient (autologous transplant) or from a histocompatible donor (allogeneic donor). These cells can be isolated from bone marrow, peripheral blood or from umbilical cord blood. Bone marrow typically is aspirated from the iliac crest. Bone marrow is rich in CD34+ cells; typically 1 to 2% of marrow cells are precursor cells. Peripheral blood typically contains less than 1% CD34+ cells. Umbilical cord blood is very rich in early progenitor cells and may be used as a source of cells for hematopoietic cell transplant.
Separation of CD34+ cells (undifferentiated cells) from differentiated cells can be achieved by a number of different methods. The most widely used is a positive immunological selection based on binding of these cells to anti-CD34-antibodies immobilized on a solid support. Other selection methods include negative selection where all cells not expressing CD34 are isolated away from the CD34+ cells based on their expression of lineage specific cell surface antigens.
The number of progenitor cells that can be harvested at one time from either source is small and, in many cases, is not sufficient for a successful transplant. Several methods have been developed to expand bone marrow cells or progenitor cells obtained from blood apheresis or from umbilical cord blood in in vitro cultures. In vitro expansion of hematopoietic stem cells requires the addition of appropriate growth factors as well as certain growth conditions provided by so called stromal cells. Stromal cells provide physical support to hematopoietic progenitor cells as well as certain growth factors required for the increase of stem cell numbers.
A population of cells intends a collection of more than one cell that is identical (clonal) or non-identical in phenotype and/or genotype.
A “pharmaceutical composition” is intended to include the combination of an active polypeptide, polynucleotide or antibody with a carrier, inert or active such as a solid support, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
As used herein, the term “pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Martin (1975) Remington's Pharm. Sci., 15th Ed. (Mack Publ. Co., Easton).
A “subject,” “individual” or “patient” is used interchangeably herein, and refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, rats, rabbit, simians, bovines, ovine, porcine, canines, feline, farm animals, sport animals, pets, equine, and primate, particularly human. Besides being useful for human treatment, the present invention is also useful for veterinary treatment of companion mammals, exotic animals and domesticated animals, including mammals, rodents, and the like which is susceptible to disease. In one embodiment, the mammals include horses, dogs, and cats. In another embodiment of the present invention, the human is an adolescent or infant under the age of eighteen years of age.
“Host cell” refers not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
The term “suffering” as it related to the term “treatment” refers to a patient or individual who has been diagnosed with or is predisposed to infection or a disease incident to infection. A patient may also be referred to being “at risk of suffering” from a disease because of active or latent infection. This patient has not yet developed characteristic disease pathology.
An “effective amount” is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time period for which the individual dosage unit is to be used, the bioavailability of the therapeutic agent, the route of administration, etc. It is understood, however, that specific dose levels of the therapeutic agents of the present invention for any particular subject depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, and diet of the subject, the time of administration, the rate of excretion, the drug combination, and the severity of the particular disorder being treated and form of administration. Treatment dosages generally may be titrated to optimize safety and efficacy. Typically, dosage-effect relationships from in vitro and/or in vivo tests initially can provide useful guidance on the proper doses for patient administration. In general, one will desire to administer an amount of the compound that is effective to achieve a serum level commensurate with the concentrations found to be effective in vitro. Determination of these parameters is well within the skill of the art. These considerations, as well as effective formulations and administration procedures are well known in the art and are described in standard textbooks. Consistent with this definition, as used herein, the term “therapeutically effective amount” is an amount sufficient to achieve treatment dosages.
The term administration shall include without limitation, administration by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracisternal injection or infusion, subcutaneous injection, or implant), by inhalation spray nasal, vaginal, rectal, sublingual, urethral (e.g., urethral suppository) or topical routes of administration (e.g., gel, ointment, cream, aerosol, etc.) and can be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, excipients, and vehicles appropriate for each route of administration. The invention is not limited by the route of administration, the formulation or dosing schedule.
As used herein, the “lineage” of a cell defines the heredity of the cell, i.e. its predecessors and progeny. The lineage of a cell places the cell within a hereditary scheme of development and differentiation.
As used herein, the term “differentiates or differentiated” defines a cell that takes on a more committed (“differentiated”) position within the lineage of a cell. “Dedifferentiated” defines a cell that reverts to a less committed position within the lineage of a cell.
As used herein, the term “p300/-catenin antagonist” is an agent that disrupts or inhibits, in vitro or in vivo, the interaction between the p300 and β-catenin proteins. In some embodiments, the p300/β-catenin antagonist promotes hematopoietic stem cell (HSC) expansion (i.e., proliferation, as the term is used herein). As used herein, the term CBP/β-catenin antagonist include the compounds of Formula I described herein. In some embodiments, the p300/β-catenin antagonist is selected from a compound of Table 1. In other embodiments, the p300/β-catenin antagonist is YH249 or YH250, having the structures:
As used herein, the term “CBP/β-catenin antagonist” is an agent that disrupts or inhibits, in vitro or in vivo, the interaction between the CBP and β-catenin proteins. In some embodiments, the CBP/β-catenin antagonist promotes hematopoietic stem cell (HSC) differentiation. As used herein, the term CBP/β-catenin antagonist excludes the compounds of Formula I described herein. Exemplary CBP/β-catenin antagonists include those described in U.S. Pat. Nos. 6,762,185; 7,531,320; 7,232,822; 7,563,825, and 7,598,253; and U.S. Patent Application Publication Nos. 20120296067; 20120088770; and 20110263607. The compounds from these patents and patent applications, which are outside the scope of the compounds of Formula I described herein, are incorporated herein by reference as CBP/β-catenin antagonists. Exemplary CBP/β-catenin antagonists include PRI-724 under clinical development by Prism BioLab Co., Ltd (Japan) and ICG-001 having the structure:
The term “pharmaceutically acceptable” refers to safe and non-toxic use for in vivo, preferably, human administration.
The term “pharmaceutically acceptable salt” refers to a salt that is pharmaceutically acceptable.
The term “salt” refers to an ionic compound formed between an acid and a base. When the compound provided herein contains an acidic functionality, such salts include, without limitation, alkali metal, alkaline earth metal, and ammonium salts. As used herein, ammonium salts include, salts containing protonated nitrogen bases and alkylated nitrogen bases. Exemplary, and non-limiting cations useful in pharmaceutically acceptable salts include Na, K, Rb, Cs, NH4, Ca, Ba, imidazolium, and ammonium cations based on naturally occurring amino acids. When the compounds utilized herein contain basic functionality, such salts include, without limitation, salts of organic acids, such as caroboxylic acids and sulfonic acids, and mineral acids, such as hydrogen halides, sulfuric acid, phosphoric acid, and the likes. Exemplary and non-limiting anions useful in pharmaceutically acceptable salts include oxalate, maleate, acetate, propionate, succinate, tartrate, chloride, sulfate, bisulfate, mono-, di-, and tribasic phosphate, mesylate, tosylate, and the likes.
The term “carrier” as used herein, refers to relatively nontoxic chemical compounds or agents that facilitate the incorporation of a compound into cells, e.g., red blood cells, or tissues.
As used herein, a “prodrug” is a compound that, after administration, is metabolized or otherwise converted to an active or more active form with respect to at least one property.
To produce a prodrug, a pharmaceutically active compound can be modified chemically to render it less active or inactive, but the chemical modification is such that an active form of the compound is generated by metabolic or other biological processes. A prodrug may have, relative to the drug, altered metabolic stability or transport characteristics, fewer side effects or lower toxicity. For example, see the reference Nogrady, 1985, Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392. Prodrugs can also be prepared using compounds that are not drugs.
In one aspect, there is provided a compound of Formula I
or a pharmaceutically acceptable salt thereof,
In some embodiments, ring A is cyclohexyl or phenyl substituted with 1-4 groups selected from halo; C1-C6 alkyl, optionally substituted with 1-6 halo; C1-C6 alkoxy, optionally substituted with 1-6 halo; wherein two adjacent alkyl or alkoxy groups can join to form a 5- or 6-membered carbocyclic or heterocyclic ring. In other embodiments, ring A is
In some embodiments, ring B is phenyl, naphthyl, pyridyl, imidazolyl or quinolyl; wherein each is optionally substituted with 1-4 groups selected from halo; C1-C6 alkyl, optionally substituted with 1-6 halo; C1-C6 alkoxy, optionally substituted with 1-6 halo; NR3R4, wherein R3 and R4 are independently hydrogen or C1-C6 alkyl; or heterocyclyl, optionally substituted with C1-C6 alkyl; and two adjacent alkyl, alkoxy or NR3R4 groups can join to form a 5- or 6-membered carbocyclic or heterocyclic ring. In other embodiments, ring B is
R5 is halo and n is 1, 2 or 3. In another embodiment, ring B is
and R6 and R7 are independently halo.
In some embodiments, ring C is
In some embodiments, L1 is
In other embodiments, L1 is
In some embodiments, L2 and L3 are —CH2—.
In some embodiments, the compound is
or a pharmaceutically acceptable salt thereof. In other embodiments, the compound is selected from the non-limiting group of representative compounds provided herein in Table 1.
In another aspect, provided herein is pharmaceutical composition comprising any of the compounds of Formula I as described herein and a pharmaceutically acceptable carrier and/or excipient. Pharmaceutically acceptable carriers or excipients are known in the art, for example, as described in Remington's Pharmaceutical Sciences, Mack Publishing Co., (A. R. Gennaro Ed. 1985). In some embodiments, a compound of Formula I or a pharmaceutically acceptable salt, hydrate, and/or polymorph thereof may be administered in compositions, such as solutions, suspensions, tablets, capsules, lozenges or elixirs for oral administration, suppositories, sterile solutions or suspensions or injectable administration, and the like, or incorporated into shaped articles.
The dose and method of administration of the compound of Formula I will vary from subject to subject and be dependent upon such factors as the type of mammal being treated, its sex, weight, diet, concurrent medication, overall clinical condition, the specific use for which A compound of Formula I is employed, and other factors which those skilled in the medical arts will recognize. In some embodiments, about 0.5 to 500 mg of A compound of Formula I or a pharmaceutically acceptable salt, hydrate, and/or polymorph thereof is combined with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, dye, and/or flavor etc., as called for by accepted pharmaceutical practice.
In some embodiments, the compound of Formula I or a pharmaceutically acceptable salt, hydrate, and/or polymorph thereof, is administered orally in a delayed release enteric coated (EC) tablet. In some embodiments, the compound of Formula I or a pharmaceutically acceptable salt, hydrate, and/or polymorph thereof, is administered orally in an immediate release (IR) capsule. In some embodiments, the compound of Formula I or a pharmaceutically acceptable salt, hydrate, and/or polymorph thereof, is administered orally in a composition comprising dextrose monohydrate, croscarmellose sodium and magnesium stearate. In some embodiments, the composition is granulated and filled into a hard gelatin capsule.
In some embodiments, the compound of Formula I or a pharmaceutically acceptable salt, hydrate, and/or polymorph thereof is administered from about 0.001 mg/kg to about 1000 mg/kg, preferably from about 0.01 mg/kg to about 100 mg/kg, and more preferably from about 0.10 mg/kg to about 20 mg/kg.
In some embodiments, the compound of Formula I or a pharmaceutically acceptable salt, hydrate, and/or polymorph thereof is administered to a patient in a daily dosage of between about 10 mg and about 20 mg, between about 25 mg and 35 mg, or between about 40 mg and about 120 mg. In some embodiments, the compound of Formula I or a pharmaceutically acceptable salt, hydrate, and/or polymorph thereof, is administered in a daily dosage of about 40, 50, 60, 70, 80, 90, 100, 110 or 120 mg. In some embodiments, the compound of Formula I or a pharmaceutically acceptable salt, hydrate, and/or polymorph thereof is administered in a daily dosage of about 10, 15, 20, 25, 30, 40, 60, 80 or 90 mg. In some embodiments, the compound of Formula I or a pharmaceutically acceptable salt, hydrate, and/or polymorph thereof, is administered in a daily dosage of about 40, 60 or 80 mg. In some embodiments, the compound of Formula I or a pharmaceutically acceptable salt, hydrate, and/or polymorph thereof, is administered once, twice or three times a day, preferably once or twice daily.
The compound of Formula I may also be administered in a composition comprising one or more pharmaceutically acceptable carriers or excipients, and/or an effective amount of another therapeutic agent such as a granulocyte-colony stimulating factor (G-CSF) and/or erythropoietin (EPO) or an equivalent thereof.
Without being bound by theory, it is contemplated that compounds, such as those of Formula I described herein, can block or inhibit the interaction between a β-, and/or γ-catenin and p300 and/or promote stem/progenitor cell proliferation. As used herein, the term “block or inhibit the interaction” intends a diminution or reduction in the binding between a β-, and/or γ-catenin and p300 in a cell or tissue having such proteins. The methods also will enhance the interaction between CBP with a β-, and/or γ-catenin. Methods of determining the binding efficiency or interaction between a β-, and/or γ-catenin and p300 or CBP are known in the art and described herein. This method can be performed in vitro in a cell culture or tissue system or in vivo, by administering an effective amount of the compound to a subject. When performed in vitro, they provide a convenient in vitro screen to determine efficacy or dose of the compound or composition containing same use alone or in combination with another active agent. When performed in vivo, they can have therapeutic benefit or be used in an appropriate animal model for pre-clinical or investigational use. As noted herein, the p300/P3-, and/or γ-catenin antagonists described herein promote stem/progenitor cell division in symmetrical non-differentiated patterns and thereby expand stem/progenitor cells.
Bone marrow, stem cell and cord blood stem cell transplantations are performed for many clinical indications such as during the treatment of different types of cancers and aplastic anemia. The dose of the transplanted stem cell is often times an important element of such procedures. Cord blood banking can only be expected to provide modest doses for future stem cell transplantations. In vitro stem cell expansions of the rare stem cells or populations would be far more flexible and convenient and ensure greater chance of clinical success. The methods of this disclosure can be used on any populations known or believed to contain hematopoietic stem cells. The cells or tissue can be isolated from a subject or retrieved from a bank. Thus, the cells or tissue can be primary or cultured cells or tissue.
As shown in the examples below, YH250 and YH249 were found to block the interaction between a β-, and/or γ-catenin and p300 and enhance the interaction between a β-, and/or γ-catenin and CBP. These compounds also resulted in the expansion of hematopoietic stem cells. Thus, it is shown herein that these β-, and/or γ-catenin/p300 antagonists and similar such compounds of Formula I can be used in methods for the expansion of HSCs. One of skill in the art can determine if stem cell expansion has occurred by screening the treated cells or tissue for the number of cells having the appropriate cell markers, e.g., markers that indicate a more immature phenotype or lineage cell markers
Non-limiting exemplary human hematopoietic stem cell markers include cells that are lineage negative, CD34 positive and CD38 negative. Lineage cell markers include T cell markers such as CD3, CD4 and CD8; B cell markers such as CD19 and CD20; and myeloid cell markers such as CD14, CD16 and CD11b.
Non-limiting exemplary mouse hematopoietic stem cell markers include cells that are lineage negative (e.g., T cell, B cell and myeloid markers), stem cell antigen-1 (Sca-1) positive and c-kit positive. These cells are LSK cells (i.e., lineage negative, Sca-1 positive and kit positive). LSK cells contain both long term repopulating and short term repopulating cells. Further markers may be used to identify long term repopulating hematopoietic stem cells. For example a CD34 marker can be used to identify LSK34neg cells as long term repopulating cells. CD135 and CD34 markers can be used to identify LSK135neg34neg cells as long term repopulating cells. CD48 and CD150 markers can be used to identify LSK48neg150posit cells as long term repopulating cells. CD48, CD41 and CD150 markers can be used to identify LSK48neg41neg150posit cells as long term repopulating cells. The above-described markers can also be used to identify LSK135neg34neg48neg150posit cells as the most quiescent long term repopulating cells. Subsequent to expansion, known differentiation factors can be contacted or administered as required by the investigator, researcher or treating physician. The methods of this disclosure can provide an increase of about 5%, or alternatively about 10%, or alternatively about 15%, or alternatively about 20%, or alternatively about 25%, or alternatively about 30%, increase in the number of HSCs obtained after treatment as compared to the number of HSC obtained use of an alternative expansion factor or no factor at all.
In one aspect a method is provided for increasing hematopoiesis in a subject in need thereof, comprising administering to the subject an effective amount of a compound of Formula I:
or a pharmaceutically acceptable salt thereof,
wherein
a composition comprising, or alternatively consisting essentially of, or yet further consisting of one or more of the above compounds and a carrier, such as a pharmaceutically acceptable carrier.
In one embodiment of the method, the subject is a human or an animal, e.g., a mammal, a feline, canine, an equine, a bovine, an ovine, a murine or a rat. In one embodiment of the method, the subject is suffering from, or is susceptible to, decreased or depressed hematopoiesis or blood cell levels. In one embodiment of the method, the decreased or depressed hematopoiesis or blood cell levels are caused by chemotherapy, radiation therapy, bone marrow transplantation therapy or congenital anemia. In further embodiments of the method, the decreased or depressed hematopoiesis or blood cell levels are caused by an unintended exposure to radiation due an accidental release of radiation at, for example, a power plant. Thus, in one aspect, the methods are used to treat conditions caused by chemotherapy, radiation therapy or exposure, bone marrow transplantation or anemia, e.g., congenital anemia by administering to a subject in need thereof an effective amount of a compound of Formula I or a salt thereof, alone or in combination with another therapy as described.
In one embodiment, the method further comprises administering to the subject an effective amount of a CBP/β-, and/or a γ-catenin antagonist that promotes hematopoietic stem cell (HSC) differentiation, wherein the CBP/β-, and/or a γ-catenin antagonist is not a compound of Formula I or a pharmaceutically acceptable salt thereof, and/or a composition comprising one or more compounds as described herein. In one embodiment of the method, the CBP/β-, and/or a γ-catenin antagonist is PRI-724 or ICG-001.
In one embodiment, the method further comprises administering to the subject an effective amount of a supportive growth factor such as granulocyte-colony stimulating factor (G-CSF) and/or erythropoietin (EPO). The administration can be concurrent, prior to or subsequent to the administration of a compound of Formula I and/or a CBP/β-, and/or a γ-catenin antagonist, as determined by the treating physician or veterinarian.
In another aspect, a method is provided for enhancing expansion of a hematopoietic stem cell (HSC), comprising contacting the HSC with a compound, wherein the compound is of Formula I or a pharmaceutically acceptable salt thereof, and/or a composition comprising one or more compounds as described herein. In yet a further aspect, a method is provided for maintaining pluripotency of mouse and Human ES cells and Human iPS cells.
In one embodiment of the method, the cell is autologous or allogeneic and the contacting is in vitro or in vivo. When the contacting is done in vitro, the duration of the contacting step may last for about 8 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, or a duration between any two of these values. When contacting in vivo, the administration amounts and dosages will be determined by the treating physician.
In one embodiment, the method further comprises contacting the HSC with a CBP/β-, and/or a γ-catenin antagonist that promotes hematopoietic stem cell (HSC) differentiation, wherein the CBP/β-catenin antagonist is not a compound of Formula I or a pharmaceutically acceptable salt thereof. In one embodiment of the method, the CBP/β-, and/or a γ-catenin antagonist is PRI-724 or ICG-001. When the contacting is done in vitro, the duration of the contacting step may last for about 8 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, or a duration between any two of these values. When contacting in vivo, the administration amounts and dosages will be determined by the treating physician.
In another aspect, a method is provided for inhibiting an interaction between a β-, and/or a γ-catenin protein and a p-300 protein in a subject, the method comprising administering to the subject an effective amount of a compound of Formula I or a pharmaceutically acceptable salt thereof.
In one embodiment, the method further comprises administering to the subject a CBP/β, and/or a γ-catenin antagonist that promotes hematopoietic stem cell (HSC) differentiation, wherein the CBP/β-, and/or a γ-catenin antagonist inhibits the interaction between a β-, and/or γ-catenin and a CBP protein in the subject and is not a compound of Formula I or a pharmaceutically acceptable salt thereof. In one embodiment of the method, the CBP/β-, and/or a γ-catenin antagonist is PRI-724 or ICG-001.
Unless stated otherwise, the abbreviations used throughout the specification have the following meanings:
Many of the compounds described herein were prepared according to modified versions of known synthetic procedures such as those described in U.S. Pat. Nos. 6,762,185; 7,531,320; 7,232,822; 7,563,825, and 7,598,253; and U.S. Patent Application Publication Nos. 20120296067; 20120088770; and 20110263607. The synthetic procedures from these patents and patent applications are incorporated herein by reference.
For example, a bromoacetal resin (Advanced ChemTech) was added to a solution of each amine (5 equiv, YH-249; 2-(aminomethyl)-6-chloro-N,N-dimethylaniline, YH-250; 2-(aminomethyl)-4,6-dichloro-N,N-dimethylaniline) and DIEA (5 equiv.) in DMSO. The reaction mixture was shaken at 60° C. for 36 hours. The resin was washed with DMF, MeOH, and DCM.
Fmoc-Tyr(tBu)-OH (4 equiv.), HATU (4 equiv.) and DIEA (12 equiv.) in DMF were added to the resin. After the reaction mixture was shaken for 8 hours at room temperature, the resin was washed with DMF, MeOH, and DCM. The resin was added to 25% piperidine in DMF and the reaction mixture was shaken for 1 hour at room temperature and washed with DMF, MeOH, and DCM.
2-(2-(((9H-Fluoren-9-yl)methoxy)carbonyl)-1-methylhydrazinyl)acetic acid (4 equiv.), HATU (4 equiv.), and DIEA (12 equiv.) in DMF were added to the resin. After the reaction mixture was shaken for 8 hours at room temperature, the resin was washed with DMF, MeOH, and then DCM.
The resin was added to 25% piperidine in DMF and the reaction mixture was shaken for 1 hour at room temperature and washed with DMF, MeOH and DCM.
CDI (4 equiv.) in DCM was added to the resin. The reaction mixture was shaken for 8 hours at room temperature. Soon after the resin was washed with DCM, (2,3-dimethoxyphenyl) methanamine (8 equiv.) in DCM was quickly added to the resin. The reaction mixture was shaken for 8 hours at room temperature. The resin was washed with DMF, MeOH, and DCM.
The resin was treated with formic acid for 2 days at room temperature. After the resin was removed by filtration, the filtrate was condensed under a reduced pressure. The crude product was purified by preparative TLC (ANALTECH, thinlayer chromatography plate, AcOEt as a developing solvent) to yield the product.
The binding between β-catenin (or γ-catenin,) with CBP or with p300 plays a pivotal role in the determination of stem/progenitor cell fate: binding of β-, and/or a γ-catenin with CBP promotes expansion (i.e., clonal proliferation), while binding of β-, and/or a γ-catenin with p300 promotes cell differentiation.
Compound ICG-001, by binding to the N-terminus of CBP, blocks the interaction between β-catenin and CBP and thereby increases the binding of β-catenin with p300. Thus, ICG-001 promotes cell differentiation. Also, ICG-001 has been described to significantly inhibit survivin gene expression. To discover compounds that can block the interaction between β-catenin with p300, a two-step screening system was developed. This screening system helped identify compounds that inhibit Wnt/β-catenin pathway but have no significant effect on survivin expression. See the flowchart in
A. Wnt/β-Catenin Activity: The Initial SuperTOPFLASH Screen.
Hek293 cells were stably transfected with plasmid that contains multiple TCF/LEF fragments in front of Luciferase gene. Wnt/β-catenin activity was determined from luciferase expression levels. For example, when ICG-001 was added in this system, luciferase activity was decreased, indicating ICG-001 inhibits Wnt/β-catenin pathway. See
B. Survivin Expression Level: The Secondary Screen.
An endogenous human survivin 1 kb promoter was inserted before a luciferase gene. For example, when ICG-001, a known agent that promotes hematopoietic stem cell (HSC) differentiation, was added to this survivin-reporter system, survivin expression level was significantly decreased in a dose dependent manner. See
YH249 and YH250 were selected from the compounds described herein for further testing. Co-immunoprecipitation (CO-IP) was performed with mouse myoblast C2Cl2 cells. Cells were treated with DMSO (control), or YH249, or YH250 for 24 hours. Cell lyses were pulled down with anti-p300 or anti-CBP antibodies, run on gel and probed with anti-β-catenin antibody. As shown in
YH250 was used in this study. Female C57/Black mice were administrated with either DMSO (solvent control) or YH250 (2 mg/kg) subcutaneously. 24 hrs later, bone marrow Sca-1posit cells were isolated with Anti-Sca-1 microbeads (Miltenyi Biotec, Cat#130-092-529) for co-immunoprecipitation (
Axin2 is a classical Wnt/catenin target genes and plays important regulatory roles in Wnt signaling. The inhibitor of apoptosis (IAP) family member survivin (Birc5) is another Wnt/catenin target gene. We have previously demonstrated that ICG-001, by antagonizing the CBP/catenin interaction, down-regulates survivin expression in a variety of cell types both in vitro and in vivo. To study YH250 effects in bone marrow stem cell population gene expression, female C57/Black mice were treated with DMSO or YH250 (2 mg/kg) overnight and bone marrow Sca-1posit cells were isolated. Then, total RNA were extracted for cDNA synthesis and qPCR assay (
DMSO or YH250 (2 mg/kg) were administrated to female C57/Black mice. 16 hours later, long-term repopulating HSC (Lin-negt/Sca-1-posit/c-kit-posit/CD41negt/CD48negt/CD150posit cells were purified with fluorescent flow cytometry sorting (FACS) for RNA isolation and RNA sequencing (
Other genes that were significantly upregulated in the LTR-HSC and not in the progenitor population included CD52 (3.09 fold), a membrane protein that is found on the surface of HSPCs, and the tetraspanin protein CD53 (2.79 fold), which is associated with the CD34+CD133+ cord blood HSC population. Based upon these results, we investigated the ability of YH250 to promote HSC non-differentiative proliferative divisions in vitro and in vivo.
Table 2 below shows RNA sequence analysis of LSK34135-48CD150+ cells from YH250 vs DMSO treated animals:
DMSO or YH250 (2 mg/kg) were administrated to female C57/BL mice. (Mice were purchased from JAX stock#000664 for CD45.2 or Stock#002014 for CD45.1). 24 hours later, long-term repopulating HSC (LTR-HSC, Lin-negt/Sca-1-posit/c-kit-posit/CD34negt/CD135negt/CD48negt/CD150posit cells were purified with fluorescent flow cytometry sorting (FACS). Then, the purified cells were co-cultured with whole bone marrow cells isolated from GFP-transgenic mice (C57BL/6Tg-UBC-GFP, JAX stock#004353), which serves as supporting system in stem cell medium (Stemspan, Stemcell Technologies, Cat#9650) supplemented with SCF (stem cell factor 10 ng/ml) and TPO (thrombopoietin, 10 ng/ml). 2.5-3 days after co-culture, cells were harvested and analyzed for surface markers of CD48 and CD202b (Tie-2) (
Brief (less than 24 hours) treatment of HSC with YH250 or DMSO was conducted before the CFC or CFU-S12 assay. See the experimental design shown in
In CFU-S12 assay, cells treated with DMSO or YH250 were recovered from plate and injected to lethally irradiated mice (
Isolated bone marrow nucleus cell or HSC from bone marrow or peripheral blood (after HSC mobilization procedure as commonly practiced in the clinic) were cultured in medium containing p300/β-catenin antagonist (such as YH250 or YH249) at proper concentration. The medium can be commercially available serum free HSC culture medium, such as (but not limited) QBSF-60 (Quality Bio), StemPro-34 SFM (Gibco), StemSpan (Stemcell technologies). The medium may contain one or more hematopoietic stem/progenitor cell growth factor(s), such as, but not limited to stem cell factor (SCF), Thrompoietin (TPO), Flk3 ligand, IL-6, GCSF and IL-3. The medium will be totally or partially replaced periodically during cell culture. Cultured cells can be analyzed for HSC phenotype with FACS. Cells can be harvested at proper time for clinical application.
It is contemplated that a relatively brief incubation of human bone marrow (or HSC) with the p300/-catenin antagonists described herein, such as YH250 or YH249, will enhance cell proliferation. It is further contemplated that patients will have quicker and better hematopoiesis in bone marrow/stem cell transplantation by using such treated bone marrow or HSC.
Thus, an exemplary protocol comprises isolating whole bone marrow or HSC from bone marrow or peripheral blood (after HSC mobilization procedure as commonly practiced in the clinic). Red blood cells are removed using conventional methods, such as lysis buffer or centrifugation. To the centrifuged cells, add medium with YH250 or YH249 at 1 pM to 1 mM. The medium can be commercially available serum free HSC culture medium, such as (but not limited) QBSF-60 (Quality Bio), StemPro-34 SFM (Gibco), StemSpan (Stemcell technologies). The cells cultured under conventional methods known in the art and then are harvested after short time (less than 24 hours) incubation, the compounds are removed (by centrifugation or other methods) and used for bone marrow transplantation or other use.
Bone marrow cells were treated with DMSO or YH250 (0.5 μM) for 4 hours, then the compound was removed and the cells cultured in QBSF-58 medium for 14 days with 10 ng/ml of SCF, TPO, Flt3 ligand, and IL-6. At the end of culture, cells were recovered and analyzed for LSK cells with FACS. As shown in
Mouse bone marrow cells were isolated and cultured in QBSF-58 medium with DMSO or YH250 0.5 μM (medium contains 10 ng/ml of SCF, TPO, Flt3 ligand, and IL-6) for 3 days and cells are recovered and analyzed for HSC surface markers with FACS.
Cord blood cells are enriched for HSPC. It has been considered as a source of hematopoietic and mesenchyme stem cell for stem cell therapy. However, the limited number of stem cells in one dose of cord blood cell restricted its clinical efficacy in stem cell transplantation. Since p300/-catenin antagonists have the potential to stimulate stem cell proliferation and expand stem cell pool, we used YH250 in cord blood cell study.
It is further contemplated that longer treatments of HSC with a p300/-catenin antagonist at a proper concentration and time could expand cord blood HSC in vitro. The expanded cord blood cell HSC can be used in clinical for bone marrow transplantation (BMT). Using CD34+ cord blood cells (enriched with hematopoietic stem cells), different combination of growth factors in the presence or absence of p300/-catenin antagonist were used to expand hematopoietic stem cells in different serum free stem cell expansion mediums.
Cord blood CD34+ cells were purchased from Lonza, product code 2C-101B, Lot number 2F3441 and 2F3380. Cord blood cells for BMT were purchased from Allcells (Cat#003F, ID# CB 0809250).
Serum free stem cell mediums were purchased from Stemcell technologies (Stemspan II Cat#09605, and BIT 9500 Serum Substitute, Cat#9500) or Lonza (X-VIVO 20 with Gentamicin L-Gln, Phenol Red, Cat#04-448Q). Growth factors were purchased from R&D systems: recombinant human SCF (Cat#255-SC-010), recombinant human Flt2 ligand (Cat#308-FK-025), recombinant human thombopoietin (Cat#288-TP-005), recombinant human IL-6 (Cat#206-IL-010), recombinant human GCSF (Cat#214-CS-005/CF), recombinant IL-3 (Cat#203-IL-010).
Antibodies were purchased from eBioscience, anti-human CD34 (clone 563, Cat#561290), anti-human CD45 (clone 2D1, Cat#11-9459-42), anti-human CD38 (clone HB7, Cat#25-0388-42), anti-human CD45R (clone H100, Cat#45-0458-73), anti-human CD33 (clone WM-53, Cat#56-0338-42).
Cord blood CD34+ cells were cultured in serum free medium with different growth factor combination and different concentrations of YH250. We tested three serum free mediums with either 4 growth factors (4GF, or SF6T: SCF 100 ng/ml; Flt3 100 ng/ml, IL-6 20 ng/ml, and TPO 20 ng/ml) or 6 growth factors (6GF, or SF6TG3: SCF 100 ng/ml; Flt3 100 ng/ml, IL-6 20 ng/ml, TPO 20 ng/ml, GCSF 10 ng/ml, and IL-3 10 ng/ml). YH250 was at 0.3 μM or 1 μM concentration, DMSO, the solvent for YH250, was used as control. Frozen cord blood CD34+ cells were plated into 96 wells with different medium and different growth factors and different compound concentrations. At day 8 in culture, pictures were taken to record the cell growth and colony morphology (
A method to analyze expanded cord blood CD34+ cell phenotype using FACS is established. In this study, cord blood cells were cultured in vitro with 4GF and different concentrations of p300/-catenin antagonist such as YH250. Because the limited CD34+ cells in culture system, GFP transgenic mouse bone marrow cells were isolated and added to the cells as carrier cells in FACS analysis. The bright GFP positive carrier cells were gated out and the targeting cord blood cells were further analyzed for their surface marker.
Cord blood CD34+ cells were cultured in medium containing either 4GF or 6GF with different concentration of p300/-catenin antagonist YH250. At 7th day in culture, cells were recovered and mixed with carrier cells from GFP mice bone marrow. Antibodies to human HSC surface protein are added to the cells and human CD45posit cells were gated for FACS analysis. As shown in Table 4, in 4GF medium and 10 μM of YH250, there are more human CD45+CD34+ cells compared with DMSO (89.1% vs 79.1%). In 6GF medium, 10 μM of YH250 also gives more human CD45+CD34+ cells compared with DMSO (73.3% vs 66.2%, Table 5).
To examine p300/β-catenin antagonist YH250 effects on cord blood HSC in BMT, cord blood cells were incubated with 4GF or 6GF medium and 10 μM of YH250 for 24 hrs, then cells were recovered and transplanted to irradiated NOD.Cg-PrkdcscidIL1rgtmlwjlTg(CMV-IL2, CSF2, KITLG)1Eva, MloySzJ; JAX stock#013062) mice, these are transgenic NSG mice expressing human interleukin-3, human GM-CSF and human stem cell factor. Blood were collected at day 28th post BMT to determine engraftment. As depicted in
P300/-catenin antagonists stimulate HSPC proliferation both in vitro and in vivo. In this study, 24 or 48 hrs post either DMSO or YH250 administration, BrdU was given to animals and bone marrow cells were isolated and analyzed to determine HSPC proliferation (
The golden standard to determine HSPC number and activity is a competitive repopulation assay. The testing cells and competitor cells are mixed and transplanted to recipients to create an environment the testing cells compete with competitor cells to repopulate the recipients' hematopoietic system. The quantitative engraftment level reflects the testing cell stem/progenitor cell number.
In this study, donor mice (CD45.2) were treated with YH250 or DMSO every 48 hs for 4 round, then, bone marrow cells from the treated mice were recovered and mixed with competitive cells from non-treated normal mice for BMT to lethally irradiated recipients (
YH250 was also tested for stimulation of HSPC (hematopoietic stem/progenitor population) proliferation in irradiated animals. In this study, mice received 7Gy whole body radiation, DMSO or YH250 (s.c., 2 mg/Kg) were administrated 24 hours post radiation, followed with BrdU injection.
In the hematopoietic system, the LTR-HSC or pluripotent HSC sits atop the hematopoietic hierarchy. LTR-HSCs subsequently generate “Short Term Repopulating” hematopoietic stem cells (STR-HSC), and then progenitor cells. After radiation induced myeloid-ablation, there is a significant loss of progenitor cells. To investigate the target cell population in irradiated mice, YH250 or DMSO were administrated to 7Gy irradiated animals 24 hours post radiation. Then, 7 or 14 days post radiation (6 or 13 days after YH250 or DMSO administration), bone marrow cells were recovered from animals and competitive repopulation assay was performed to study YH250 function in stimulating HSPC proliferation. 1×106 testing bone marrow cells were mixed with 2×105 competitor cells (from non-irradiated mice) and injected into lethally irradiated mice (9Gy) (
Notably, in YH250 treated mice, bone marrow cells recovered from either day 7 or day 14 post radiation give similar long-term engraftment (4.1±1.6 vs 3.4±0.57, p=0.70), but higher short term engraftment from cells recovered at day 14 post radiation (day 7 vs day 14, 5.8±2.4 vs 12.3±2.0, p=0.06). This result suggests that after one dose administration of YH250, the LRT-HSC expansion completed by day 6. The expanded LTR-HSC at day 6 post YH250 stimulation then starts to develop and give more STR-HSC by day 14 post radiation. There is no further LTR-HSC expanding from day 6 to day 13 post YH250. Thus, one dose of YH250 administrated 24 hours post 7Gy radiation stimulated symmetric non-differentiation LTR-HSC expansion and followed with STR-HSC expansion.
However, the situation is different with cells that received DMSO treatment. For both short term and long term repopulation, the cells recovered at day 7 post radiation show significantly better engraftment than cells recovered at day 14. In summary, YH250 treatment 24 hours post radiation stimulated HSC expansion.
According to particular aspects of the present invention, if YH250 can stimulate 7Gy irradiated animal HSPC proliferation/expansion, the compound may accelerate the recovery of the hematopoiesis from radiation damage. We first tested if YH250 treatment accelerates bone marrow cell CFC activities, the more differentiated cells. After 7Gy radiation and either DMSO or YH250 administration 24 hours post radiation, bone marrow cells were recovered at different time points (day 6, 11, 13, 15 post treatment) and CFC assays were performed (
YH250 was then tested for stimulating blood count recovery in irradiated animals. In this experiment, animals received 7Gy whole body radiation and then, either YH250 or DMSO was administrated 24 hours post radiation. Animal body weight and peripheral blood counts were measured to monitor hematopoietic recovery (
We next tested if YH250 can rescue animals from lethal dose radiation. Animals received either 9Gy (LD100/60, 100% animals died in 60 days post radiation) or 8.5Gy (LD70/60, 70% animals died in 60 days post radiation) whole body radiation, 24 hours later, DMSO or YH250 (2 mg/Kg) were administrated. As shown in
With 8.5Gy radiation, all the animals (100%) survived in YH250 treatment group while there are only 30% animals survived in DMSO treatment group (p=0.0016,
To test if YH250 treatment caused HSPC exhaustion, bone marrow cells were isolated from animals in DMSO or YH250 treatment group in 8.5Gy irradiated experiment (
Since p300/-catenin antagonists such as YH250 up-regulate survivin gene expression, YH250 was tested for the ability to attenuate radiation induced cell apoptosis by detecting cleaved PARP (Poly ADP ribose polymerase-1) using anti-PARP antibody and FACS analysis (BD Bioscience, Cat#562253, anti-PARP clone F21-852). DMSO or YH250 were administrated 18 hours before 5Gy radiation. 6 hours after radiation, bone marrow cells were isolated and stained for cell surface markers (lineage markers, Sca-1, c-kit) and anti-PARP (
p300/-catenin antagonists, such as YH250, were also tested for the ability to stimulate bone marrow stem/progenitor cell proliferation in chemotherapeutic drug-treated (e.g., 5-FU) animals. As depicted in
To test if p300/-catenin antagonists can also promote multi-dosing chemotherapeutic drug induced bone marrow inhibition, 4 rounds of 5-FU (two doses in each round) administration were given to CD45.2 female mice. 150 mg/kg of 5-FU was given to mice for the first three rounds and then 200 mg/kg for the last round (4th round). The second dose of 5-FU was 6 days apart from the first dose of 5-FU in each round. Either DMSO or YH250 (2 mg/kg) were given to animals 24 hours after the first dose of 5-FU in each round of 5-FU administration (
At the last round of 5-FU treatment from
YH250 and YH249 are structurally similar as shown in Table 1. In fact, they also show similar biological functions with respect to HSPC. When mice bone marrow cells were incubated with DMSO, or YH249 or YH250 for 4 hours and then subject to CFC assay, YH249 treated bone marrow cells give similar colony number in plate as DMSO but larger colonies that resulted in higher cell number in each plate (Table 6).
In a further study, female CD45.1 mice bone marrow cells were incubated with DMSO, or YH249 (0.5 μM) or YH250 (0.5 μM) for 4 hrs in QBSF-58 media, then 2×104 cells were plated in QBSF-58 medium containing SCF, TPO, Flt3 and IL-6 (10 ng/ml for each) for 14 days. Cells were then recovered from plate for FACS analysis. As shown in
We have previously reported that the small molecule IQ-1 could maintain long term mESC pluripotency in a Wnt dependent manner {Miyabayashi, T., et al., Proc. Natl. Acad. Sci., 104, 5668-5673, 2007). We also determined that IQ-1 indirectly, via binding to the PR72/130 subunit of PP2A, inhibited the p300/β-catenin interaction (Id). Therefore, we anticipated that YH249/250 should also maintain mESC pluripotency in a Wnt dependent manner. To test his, R1 mESCs were cultured under feeder free conditions in the presence or absence of YH249/250 for 5 days. The mESCs grown in the presence of YH249/250 and Wnt3a maintained undifferentiated colony morphology (
Specifically,
Although the foregoing has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference.
The disclosure illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including,” containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure claimed.
Thus, it should be understood that although the present disclosure has been specifically disclosed by preferred embodiments and optional features, modification, improvement and variation of the disclosure embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this disclosure. The materials, methods, and examples provided here are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the disclosure.
The disclosure has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the disclosure with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.
In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.
All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.
This application claims the benefit of U.S. Provisional Patent Application No. 62/325,445, filed Apr. 20, 2016, the disclosure of which is incorporated herein in its entirety.
Support from USC Norris Comprehensive Cancer Center Support Grant P30 CA014089 (MK), NIH 1R01CA166161-01A1, 1R21NS074392-01, 1R21AI105057-01 and NIH 1R01 HL112638-01 (MK) is gratefully acknowledged.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US17/28508 | 4/20/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62325445 | Apr 2016 | US |
