Patent Application
20190240176
The instant application contains a sequence listing which has been submitted with the instant application via EFS-Web. The sequence listing file is named 222102-2780_ST25.txt and is incorporated herein by reference in its entirety.
Reprogrammed cellular metabolism is a common characteristic observed in various cancers. Yet it remains poorly understood whether metabolic changes directly regulate cancer development and progression. Cells sense intrinsic and extrinsic nutrient status and respond by modulating metabolic processes to control their proliferation and differentiation. It is evident that deregulated cell energetics is one of the hallmarks of cancer and that cancer cells alter their metabolic processes to meet the biosynthetic demands of rapid growth and to enhance their fitness in the tumor microenvironment. The discovery of aerobic glycolysis, known as the Warburg effect, provided the initial clue to help the understanding of how the metabolism of tumor cells differs from non-tumor cells. Cancer metabolism research in the past decade has revealed that aerobic glycolysis is just one example of the metabolic alterations in cancer and that such alterations can serve as therapeutic targets. Importantly, many of the metabolic changes in cancer are not passive, but rather active; in fact, oncogenic events can directly reprogram cellular processes in glucose, fatty acid and amino acid metabolisms. Ras mutations are associated with enhanced glucose usage via activation of the glucose transporter GLUT1. Another example is the oncogenic transcription factor c-Myc and its role in glutamine (Gln) metabolism. Although Gln is not an essential amino acid, the growth of Myc-expressing tumor cells is dependent upon this amino acid. Gln not only affects proliferation but also modulates cell differentiation in cancer. It has long been known that glutaminase is an effective treatment for several malignancies by lowering the plasma Gln level, and increased serum amino acid levels are often correlated with poor prognosis in multiple cancers. Recent studies have identified indispensable roles of the serine biosynthetic pathway in lung and breast cancers. Despite the accumulating evidence, it remains uncertain whether these metabolic alterations directly regulate cancer development and progression in vivo.
Embodiments of the present disclosure provide for compositions and methods for treating chronic myeloid leukemia, compositions and methods for modulating cancer progression, and the like.
An embodiment of the present disclosure includes a method of treating blast crisis condition in chronic myeloid leukemia in a subject, including administering to the subject a therapeutically effective amount of a composition including a tyrosine kinase inhibitor, gabapentin (or derivatives thereof), and rapamycin (or derivatives thereof), or a pharmaceutically acceptable salt or a prodrug thereof.
An embodiment of the present disclosure includes a method of modulating cancer progression and development, including administering to the subject a therapeutically effective amount of a composition including a tyrosine kinase inhibitor, gabapentin (or derivatives thereof), and rapamycin (or derivatives thereof), or a pharmaceutically acceptable salt or a prodrug thereof, wherein the composition interrupts the branched-chain amino acid transamination pathway.
An embodiment of the present disclosure includes a pharmaceutical composition including a therapeutically effective amount of a composition which contains a tyrosine kinase inhibitor, gabapentin (or derivatives thereof), and rapamycin (or derivatives thereof), or a pharmaceutically acceptable salt of the composition or a prodrug of the composition, and a pharmaceutically acceptable carrier.
An embodiment of the present disclosure includes a method of treating blast crisis condition in chronic myeloid leukemia in a subject, including: administering to the subject a therapeutically effective amount of each of a tyrosine kinase inhibitor, gabapentin (or derivatives thereof), and rapamycin (or derivatives thereof), or a pharmaceutically acceptable salt or a prodrug thereof.
An embodiment of the present disclosure includes a method of modulating cancer progression and development, comprising administering to the subject a therapeutically effective amount of each of a tyrosine kinase
Other compositions, methods, features, and advantages will be or become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional compositions, methods, features and advantages be included within this description, be within the scope of the present disclosure, and be protected by the accompanying claims.
Further aspects of the present disclosure will be more readily appreciated upon review of the detailed description of its various embodiments, described below, when taken in conjunction with the accompanying drawings.
Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.
Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C., and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20° C. and 1 atmosphere.
Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible.
As used in the specification and claims, the singular forms “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof.
The terms “subject”, “individual”, or “patient” as used herein are used interchangeably and refer to an animal preferably a warm-blooded animal such as a mammal. Mammal includes without limitation any members of the Mammalia. A mammal, as a subject or patient in the present disclosure, can be from the family of Primates, Carnivora, Proboscidea, Perissodactyla, Artiodactyla, Rodentia, and Lagomorpha. In a particular embodiment, the mammal is a human. In other embodiments, animals can be treated; the animals can be vertebrates, including both birds and mammals. In aspects of the disclosure, the terms include domestic animals bred for food or as pets, including equines, bovines, sheep, poultry, fish, porcines, canines, felines, and zoo animals, goats, apes (e.g., gorilla or chimpanzee), and rodents such as rats and mice.
In the context of certain aspects of the disclosure, the term “subject” generally refers to an individual who will receive or who has received treatment (e.g., administration of a composition of the disclosure, and optionally one or more other agents) for a condition characterized by a cancer (e.g., leukemia). In certain aspects, a subject may be a healthy subject. Typical subjects for treatment include persons afflicted with or suspected of having or being pre-disposed to a disease disclosed herein, or persons susceptible to, suffering from or that have suffered a disease disclosed herein. A subject may or may not have a genetic predisposition for a disease disclosed herein.
When referring to a subject or patient, the term “administering” refers to an administration that is oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation or via an implanted reservoir. The term “parenteral” includes subcutaneous, intravenous, intramuscular, intra-articular, intra-peritoneal, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injections or infusion techniques. In some embodiments, the administration is intracaviteal.
The term “diagnosed” as used herein, refers to the recognition of a disease by its signs and symptoms (e.g., resistance to conventional therapies), or genetic analysis, pathological analysis, histological analysis, and the like.
The terms “administering” and “administration” as used herein refer to a process by which a therapeutically effective amount of a composition of the disclosure are delivered to a subject for prevention and/or treatment purposes. Compositions are administered in accordance with good medical practices taking into account the subject's clinical condition, the site and method of administration, dosage, patient age, sex, body weight, and other factors known to physicians.
The terms “administration of” and “administering” a compound or composition as used herein refers to providing a compound of the disclosure or a prodrug of a compound of the disclosure to the individual in need of treatment. The compounds of the present disclosure may be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracisternal injection or infusion, subcutaneous injection, or implant), by inhalation spray, nasal, vaginal, rectal, sublingual, or topical routes of administration and may be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles appropriate for each route of administration.
The terms “treat” or “treatment” as used herein refer to both therapeutic treatment and prophylactic or preventative measures, where the object is to prevent or slow down (lessen) an undesired physiological change or disorder resulting from the disease. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of the extent of disease, stabilized (i.e., not worsening) state of disease, and delay or slowing of progression of the symptoms recognized as originating from a stroke. The term “treatment” can also refer to prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented or onset delayed.
As used herein, the terms “prophylactically treat” and “prophylactically treating” refer completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
The term “modulate” refers to the activity of a composition to affect (e.g., to promote or retard) an aspect of cellular function, including, but not limited to, cell growth, proliferation, apoptosis, and the like.
The term “unit dosage form,” as used herein, refers to physically discrete units suitable as unitary dosages for human and/or animal subjects, each unit containing a predetermined quantity of a compound or composition calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for unit dosage forms depend on the particular compound employed, the route and frequency of administration, and the effect to be achieved, and the pharmacodynamics associated with each compound or composition in the subject.
As used herein, a “pharmaceutical composition” and a “pharmaceutical formulation” are meant to encompass a composition suitable for administration to a subject, such as a mammal, especially a human. In general, a “pharmaceutical composition” or “pharmaceutical formulation” is sterile, and preferably free of contaminants that are capable of eliciting an undesirable response within the subject (e.g., the compound(s) in the pharmaceutical composition is pharmaceutical grade). Pharmaceutical compositions can be designed for administration to subjects or patients in need thereof via a number of different routes of administration including oral, intravenous, buccal, rectal, parenteral, intraperitoneal, intradermal, intracheal, intramuscular, subcutaneous, inhalational and the like.
A “pharmaceutically acceptable excipient”, “pharmaceutically acceptable diluent”, “pharmaceutically acceptable carrier”, and “pharmaceutically acceptable adjuvant” means an excipient, diluent, carrier, and/or adjuvant that are useful in preparing a pharmaceutical composition that are generally safe, non-toxic and neither biologically nor otherwise undesirable, and include an excipient, diluent, carrier, and adjuvant that are acceptable for veterinary use and/or human pharmaceutical use. “A pharmaceutically acceptable excipient, diluent, carrier and/or adjuvant” as used in the specification and claims includes one or more such excipients, diluents, carriers, and adjuvants.
The terms “therapeutically effective amount” and “an effective amount” are used interchangeably herein and refer to that amount of the composition being administered that is sufficient to effect the intended application including but not limited to disease treatment. For example, an effective amount of the composition will relieve to some extent one or more of the symptoms of the disease being treated, and/or that amount that will prevent, to some extent, one or more of the symptoms of the disease that the host being treated has or is at risk of developing. The therapeutically effective amount may vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated, e.g., the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The term also applies to a dose that will induce a particular response in target cells. The specific dose will vary depending on the particular composition chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried.
“Pharmaceutically acceptable salt” refers to those salts (organic or inorganic) that retain the biological effectiveness and optionally other properties of the free bases. Pharmaceutically acceptable salts can be obtained by reaction with inorganic or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, malic acid, maleic acid, succinic acid, tartaric acid, citric acid, and the like.
In the event that embodiments of the disclosed compositions form salts, these salts are within the scope of the present disclosure. Reference to a composition of any of the formulas herein is understood to include reference to salts thereof, unless otherwise indicated. The term “salt(s)”, as employed herein, denotes acidic and/or basic salts formed with inorganic and/or organic acids and bases. In addition, when composition contains both a basic moiety and an acidic moiety, zwitterions (“inner salts”) may be formed and are included within the term “salt(s)” as used herein. Pharmaceutically acceptable (e.g., non-toxic, physiologically acceptable) salts are preferred, although other salts are also useful, e.g., in isolation or purification steps which may be employed during preparation. Salts of the compounds of the composition may be formed, for example, by reacting the composition or compounds of the composition with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization.
Embodiments of the composition that contain a basic moiety may form salts with a variety of organic and inorganic acids. Exemplary acid addition salts include acetates (such as those formed with acetic acid or trihaloacetic acid, for example, trifluoroacetic acid), adipates, alginates, ascorbates, aspartates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates, hemisulfates, heptanoates, hexanoates, hydrochlorides (formed with hydrochloric acid), hydrobromides (formed with hydrogen bromide), hydroiodides, 2-hydroxyethanesulfonates, lactates, malates (salts formed with malic acid), maleates (formed with maleic acid), ethanesulfonates (formed with ethanesulfonic acid), methanesulfonates (formed with methanesulfonic acid), 2-naphthalenesulfonates, nicotinates, nitrates, oxalates, pectinates, persulfates, 3-phenylpropionates, phosphates (formed with phosphoric acid), picrates, pivalates, propionates, salicylates, succinates, sulfates (such as those formed with sulfuric acid), sulfonates (such as those mentioned herein including those formed with p-toluenesulfonic acid), tartrates, thiocyanates, toluenesulfonates such as tosylates, undecanoates, and the like.
Embodiments of the composition that contain an acidic moiety may form salts with a variety of organic and inorganic bases. Exemplary basic salts include ammonium salts, alkali metal salts such as sodium, lithium, and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases (for example, organic amines) such as benzathines, dicyclohexylamines, hydrabamines (formed with N,N-bis(dehydroabietyl)ethylenediamine), N-methyl-D-glucamines, N-methyl-D-glucamides, t-butyl amines, and salts with amino acids such as arginine, lysine, and the like.
Basic nitrogen-containing groups may be quaternized with compounds such as lower alkyl halides (e.g., methyl, ethyl, propyl, and butyl chlorides, bromides and iodides), dialkyl sulfates (e.g., dimethyl, diethyl, dibutyl, and diamyl sulfates), long chain halides (e.g., decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides), aralkyl halides (e.g., benzyl and phenethyl bromides), and others. Solvates of the compounds of the disclosure are also contemplated herein.
The term “prodrug” refers to an inactive precursor of a composition that is converted into a biologically active form in vivo. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent compound. They may, for instance, be bioavailable by oral administration whereas the parent compound is not. The prodrug may also have improved solubility in pharmaceutical compositions over the parent drug. A prodrug may be converted into the parent drug by various mechanisms, including enzymatic processes and metabolic hydrolysis. Harper, N.J. (1962). Drug Latentiation in Jucker, ed. Progress in Drug Research, 4:221-294; Morozowich et al. (1977). Application of Physical Organic Principles to Prodrug Design in E. B. Roche ed. Design of Biopharmaceutical Properties through Prodrugs and Analogs, APhA; Acad. Pharm. Sci.; E. B. Roche, ed. (1977). Bioreversible Carriers in Drug in Drug Design, Theory and Application, APhA; H. Bundgaard, ed. (1985) Design of Prodrugs, Elsevier; Wang et al. (1999) Prodrug approaches to the improved delivery of peptide drug, Curr. Pharm. Design. 5(4):265-287; Pauletti et al. (1997). Improvement in peptide bioavailability: Peptidomimetics and Prodrug Strategies, Adv. Drug. Delivery Rev. 27:235-256; Mizen et al. (1998). The Use of Esters as Prodrugs for Oral Delivery of β-Lactam antibiotics, Pharm. Biotech. 11:345-365; Gaignault et al. (1996). Designing Prodrugs and Bioprecursors I. Carrier Prodrugs, Pract. Med. Chem. 671-696; M. Asgharnejad (2000). Improving Oral Drug Transport Via Prodrugs, in G. L. Amidon, P. I. Lee and E. M. Topp, Eds., Transport Processes in Pharmaceutical Systems, Marcell Dekker, p. 185-218; Balant et al. (1990) Prodrugs for the improvement of drug absorption via different routes of administration, Eur. J. Drug Metab. Pharmacokinet, 15(2): 143-53; Balimane and Sinko (1999). Involvement of multiple transporters in the oral absorption of nucleoside analogues, Adv. Drug Delivery Rev., 39(1-3):183-209; Browne (1997). Fosphenytoin (Cerebyx), Clin. Neuropharmacol. 20(1): 1-12; Bundgaard (1979). Bioreversible derivatization of drugs—principle and applicability to improve the therapeutic effects of drugs, Arch. Pharm. Chemi. 86(1): 1-39; H. Bundgaard, ed. (1985) Design of Prodrugs, New York: Elsevier; Fleisher et al. (1996). Improved oral drug delivery: solubility limitations overcome by the use of prodrugs, Adv. Drug Delivery Rev. 19(2): 115-130; Fleisher et al. (1985). Design of prodrugs for improved gastrointestinal absorption by intestinal enzyme targeting, Methods Enzymol. 112: 360-81; Farquhar D, et al. (1983). Biologically Reversible Phosphate-Protective Groups, J. Pharm. Sci., 72(3): 324-325; Han, H. K. et al. (2000). Targeted prodrug design to optimize drug delivery, AAPS PharmSci., 2(1): E6; Sadzuka Y. (2000). Effective prodrug liposome and conversion to active metabolite, Curr. Drug Metab, 1(1):31-48; D. M. Lambert (2000) Rationale and applications of lipids as prodrug carriers, Eur. J. Pharm. Sci., 11 Suppl 2:S15-27; Wang, W. et al. (1999) Prodrug approaches to the improved delivery of peptide drugs. Curr. Pharm. Des., 5(4):265-87.
Gbp, gabapentin; Pgb, pregabalin; Gaba, gamma-aminobutyric acid; TI-1, 2-(1-Aminocyclohexyl)acetic acid hydrochloride; TI-3, 4-Amino-3-phenylbutanoic acid; JK-1, Tranexamic acid.
Embodiments of the present disclosure provide for methods of treating cancer (e.g. leukemia), pharmaceutical compositions for treating cancer, methods of modulating cancer progression and development, and the like. Embodiments of the present disclosure can be used to treat blast crisis phase chronic myelogenous leukemia (BC-CML), which is fundamentally different from chronic phase chronic myelogenous leukemia (CP-CML) and is characterized by differentiation arrest and propagation of immature progenitor cells, resistance to current treatments because of secondary mutations with a poor prognosis and shorter median survival. Embodiments of the present disclosure include a combination or “cocktail” approach to treating BC-CML using a mix of different compounds.
In an embodiment, a method of treating blast crisis condition in chronic myeloid leukemia in a subject includes administering to a subject of need of treatment a therapeutically effective amount of a composition. In an embodiment, the composition includes a tyrosine inhibitor, gabapentin or derivative thereof, and rapamycin or derivative thereof, or a pharmaceutically acceptable salt or a prodrug of the composition and/or one or more components of the composition. In an embodiment, the amount of each of the tyrosine inhibitor, gabapentin, and rapamycin (inclusive of derivatives of one or more of these) can be about 1 to 50 weight percent or about 1 to 35 weight percent.
In one embodiment, the composition components are administered as individual components by the same route of administration or by different routes of administration, with administration of each component or components at substantially the same time or at times frames that achieve the desired outcome. In one embodiment, the composition components are formulated into a “cocktail composition”, using methods known by one skilled in the art.
A “tyrosine kinase inhibitor” is meant a molecule that inhibits the function or the production of one or more tyrosine kinases. Tyrosine kinase inhibitors include small molecule inhibitors of tyrosine kinases, antibodies to tyrosine kinases, and antisense oligomers and RNAi inhibitors that reduce the expression of tyrosine kinases.
In an embodiment, the tyrosine kinase inhibitors can include tyrosine kinase inhibitors used to treat CP-CML. In an embodiment, the tyrosine kinase inhibitors can include one or more of the following: curcumin, difluorinated curcumin (DFC), [3-{5-[4-cyclopentyloxy)-2-hydroxybenzoyl]-2-[(3-hydroxy-1,2-benzisoxazol-6-yl) methoxy]phenyl}propionic acid] (T5224, Roche), nordihydroguaiaretic acid (NDGA), dihydroguaiaretic acid (DHGA), [(E,E,Z,E)-3-methyl-7-(4-methylphenyl)-9-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2,4,6,8-nonatetraenoic acid (SR 1302, Tocris Biosciences), (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI), TPI-2, TPI-3, triptolide, genistein, imatinib mesylate (Gleevec®), nilotinib (Tasigna®), dasatinib (Sprycel®), ponatinib (Iclusig®), leflunomide (SU101), ZD1839 (Iressa®), OSI-774 (Tarceva®), CI-1033, SU5416, SU6668, ZD4190, ZD6474, PTK787, PKI166, GW2016, EKB-509, EKB-569, CEP-701, CEP-751, PKC412, SU11248, MLN518, trastuzumab (Herceptin®), C225 (Fibitux®), rhu-Mab VEGF (Avastin®), MDX-H210, 2C4, MDX-447, IMC-ICI 1, EMD 72000, RH3, and ABX-EGF. For each of the tyrosine kinase inhibitors, pharmaceutically acceptable salts or a prodrugs can be used as well.
In an embodiment, gabapentin or derivatives of gabapentin can be used in the composition, where the derivatives of gabapentin, when combined with the two other components, can obtain similar or the same results. For each of the gabapentin or derivatives of gabapentin, pharmaceutically acceptable salts or prodrugs can be used as well.
In an embodiment, gabapentin refers to 1-(aminomethyl)cyclohexane acetic acid and derivatives of gabapentin as well as pharmaceutically acceptable salts, esters, solvates, hydrates, and polymorphs thereof, can also be used in the composition. (See also U.S. Pat. Nos. 4,024,175, 4,087,544, 4,894,476, 4,960,931 and 6,683,112 for various gabapentin derivatives, which are included herein by reference) 1-(aminomethyl)cyclohexane acetic acid is a γ-aminobutyric acid (GABA) analogue with a molecular formula of C9H17NO2 and a molecular weight of 171.24. 1-(aminomethyl)cyclohexane acetic acid is freely soluble in water and in both basic and acidic aqueous solutions. I-(aminomethyl)cyclohexane acetic acid has a structure of:
Gabapentin may be obtained from a variety of commercial sources, such as Shanghai Zhongxi International Trading Co., Shanghai, China; Hikal Limited, Bangalore, Karnaraka, India; Erregierre S.p.A., San Paolo d'Argon (BG), Italy; MediChem, SA, Sant Joan Despi (Barcelona), Spain; Ranbaxy Laboratories, New Delhi, India; Procos S.p.A., Gamed, Italy; Zambon Group, Milan, Italy; Hangzhuo Chiral Medicine Chemicals Co., Hangzhuo, China; InterChem Corporation USA, Paramus, N.J.; SST Corporation, Clifton, N.J.; Teva Pharmaceuticals USA, North Whales, Pa.; Plantex USA, Hakensack, N.J.; and Sigma-Aldrich, St. Louis, Mo., or an appropriate distributor.
In an embodiment, rapamycin or derivatives of rapamycin can be used in the composition, where the derivatives of rapamycin, when combined with the two other components, can obtain similar or the same results. For each of the rapamycin or derivatives of rapamycin, pharmaceutically acceptable salts or a prodrugs can be used as well.
Rapamycin, in addition to naturally occurring forms of rapamycin, includes rapamycin analogs and derivatives. Many such analogs and derivatives are known in the art. Examples include those compounds described in U.S. Pat. Nos. 6,329,386; 6,200,985; 6,117,863; 6,015,815; 6,015,809; 6,004,973; 5,985,890 5,955,457; 5,922,730; 5,912,253; 5,780,462; 5,665,772; 5,637,590; 5,567,709; 5,563,145; 5,559,122; 5,559,120; 5,559,119; 5,559,112; 5,550,133; 5,541,192; 5,541,191; 5,532,355; 5,530,121; 5,530,007; 5,525,610; 5,521,194; 5,519,031; 5,516,780; 5,508,399; 5,508,290; 5,508,286; 5,508,285; 5,504,291; 5,504,204; 5,491,231; 5,489,680; 5,489.595; 5,488,054; 5,486,524; 5,486,523; 5,486,522; 5,484,791; 5,484,790; 5,480,989; 5,480,988; 5,463,048; 5,446,048; 5,434,260; 5,411,967; 5,391,730; 5,389,639; 5,385,910; 5,385,909; 5,385,908; 5,378,836; 5,378,696; 5,373,014; 5,362,718; 5,358,944; 5,346,893; 5,344,833; 5,302,584; 5,262,424; 5,262,423; 5,260,300; 5,260,299; 5,233,036; 5,221,740; 5,221,670; 5,202,332; 5,194,447; 5,177,203; 5,169,851; 5,164,399; 5,162,333; 5,151,413; 5,138,051; 5,130,307; 5,120,842; 5,120,727; 5,120,726; 5,120,725; 5,118,678; 5,118,677; 5,100,883; 5,023,264; 5,023,263; and 5,023,262; all of which are incorporated herein by reference. In particular, rapamycin can include, CCI-779, Everolimus (also known as RADOOI), and ABT-578. CCI-779 is an ester of rapamycin (42-ester with 3-hydroxy-2-hydroxymethyl-2-methylpropionic acid), disclosed in U.S. Pat. No. 5,362,718. Everolimus is an alkylated rapamycin (40-O-(2-hydroxyethyl)-rapamycin, disclosed in U.S. Pat. No. 5,665,772.
In an embodiment, a method of modulating cancer progression and development a subject includes administering to a subject of need of treatment a therapeutically effective amount of the composition. In an embodiment, the composition includes a tyrosine inhibitor, gabapentin or derivative thereof, and rapamycin or derivative thereof, or a pharmaceutically acceptable salt or a prodrug of the composition and/or one or more components of the composition. In an embodiment, the cancer can include BC-CML. Additional details regarding the specific way in which the composition can modulate cancer progression is described in detail in Example 1.
In an embodiment, a pharmaceutical composition comprising a therapeutically effective amount of a composition and a pharmaceutically acceptable carrier. In an embodiment, the composition includes a tyrosine inhibitor, gabapentin or derivative thereof, and rapamycin or derivative thereof, or a pharmaceutically acceptable salt or a prodrug of the composition and/or one or more components of the composition. In an embodiment, the pharmaceutical composition can be used to treat a disease such as cancer (e.g., BC-CML and Acute Myeloid Leukemia).
In various embodiments, the ratios of tyrosine inhibitor, gabapentin or derivative thereof, and rapamycin or derivative thereof can be about 50:200:5 to about 50:67:5, about 50:200:5, or about 50:67:5. In a particular aspect, an illustrative ratios are as follows: Imatinib 50 mg/kg, Gabapentin 200 mg/kg and rapamycin 5 mg/kg) and Imatinib 50 mg/kg, Gabapentin 67 mg/kg and rapamycin 5 mg/kg
Embodiments of the present disclosure include a compound (e.g., tyrosine inhibitor, gabapentin or derivative thereof, and rapamycin or derivative thereof) as identified herein and formulated with one or more pharmaceutically acceptable excipients, diluents, carriers and/or adjuvants. In addition, embodiments of the present disclosure include a compound formulated with one or more pharmaceutically acceptable auxiliary substances. In particular the compound can be formulated with one or more pharmaceutically acceptable excipients, diluents, carriers, and/or adjuvants to provide an embodiment of a composition of the present disclosure.
A wide variety of pharmaceutically acceptable excipients are known in the art. Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy,” 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds., 7th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3rd ed. Amer. Pharmaceutical Assoc.
The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.
In an embodiment of the present disclosure, the compound can be administered to the subject using any means capable of resulting in the desired effect. Thus, the compound can be incorporated into a variety of formulations for therapeutic administration. For example, the compound can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols.
In pharmaceutical dosage forms, the compound may be administered in the form of its pharmaceutically acceptable salts, or a subject active composition may be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. The following methods and excipients are merely exemplary and are in no way limiting.
For oral preparations, the compound can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
Embodiments of the compound can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
Embodiments of the compound can be utilized in aerosol formulation to be administered via inhalation. Embodiments of the compound can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.
Furthermore, embodiments of the compound can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. Embodiments of the compound can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.
Unit dosage forms for oral or rectal administration, such as syrups, elixirs, and suspensions, may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more compositions. Similarly, unit dosage forms for injection or intravenous administration may comprise the compound in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.
Embodiments of the compound can be formulated in an injectable composition in accordance with the disclosure. Typically, injectable compositions are prepared as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation may also be emulsified or the active ingredient encapsulated in liposome vehicles in accordance with the present disclosure.
In an embodiment, the compound can be formulated for delivery by a continuous delivery system. The term “continuous delivery system” is used interchangeably herein with “controlled delivery system” and encompasses continuous (e.g., controlled) delivery devices (e.g., pumps) in combination with catheters, injection devices, and the like, a wide variety of which are known in the art.
Mechanical or electromechanical infusion pumps can also be suitable for use with the present disclosure. Examples of such devices include those described in, for example, U.S. Pat. Nos. 4,692,147; 4,360,019; 4,487,603; 4,360,019; 4,725,852; 5,820,589; 5,643,207; 6,198,966; and the like. In general, delivery of the compound can be accomplished using any of a variety of refillable, pump systems. Pumps provide consistent, controlled release over time. In some embodiments, the compound can be in a liquid formulation in a drug-impermeable reservoir, and is delivered in a continuous fashion to the individual.
In one embodiment, the drug delivery system is an at least partially implantable device. The implantable device can be implanted at any suitable implantation site using methods and devices well known in the art. An implantation site is a site within the body of a subject at which a drug delivery device is introduced and positioned. Implantation sites include, but are not necessarily limited to, a subdermal, subcutaneous, intramuscular, or other suitable site within a subject's body. Subcutaneous implantation sites are used in some embodiments because of convenience in implantation and removal of the drug delivery device.
Drug release devices suitable for use in the disclosure may be based on any of a variety of modes of operation. For example, the drug release device can be based upon a diffusive system, a convective system, or an erodible system (e.g., an erosion-based system). For example, the drug release device can be an electrochemical pump, osmotic pump, an electro-osmotic pump, a vapor pressure pump, or osmotic bursting matrix, e.g., where the drug is incorporated into a polymer and the polymer provides for release of drug formulation concomitant with degradation of a drug-impregnated polymeric material (e.g., a biodegradable, drug-impregnated polymeric material). In other embodiments, the drug release device is based upon an electrodiffusion system, an electrolytic pump, an effervescent pump, a piezoelectric pump, a hydrolytic system, etc.
Drug release devices based upon a mechanical or electromechanical infusion pump can also be suitable for use with the present disclosure. Examples of such devices include those described in, for example, U.S. Pat. Nos. 4,692,147; 4,360,019; 4,487,603; 4,360,019; 4,725,852, and the like. In general, a subject treatment method can be accomplished using any of a variety of refillable, non-exchangeable pump systems. Pumps and other convective systems are generally preferred due to their generally more consistent, controlled release over time. Osmotic pumps are used in some embodiments due to their combined advantages of more consistent controlled release and relatively small size (see, e.g., PCT published application no. WO 97/27840 and U.S. Pat. Nos. 5,985,305 and 5,728,396). Exemplary osmotically-driven devices suitable for use in the disclosure include, but are not necessarily limited to, those described in U.S. Pat. Nos. 3,760,984; 3,845,770; 3,916,899; 3,923,426; 3,987,790; 3,995,631; 3,916,899; 4,016,880; 4,036,228; 4,111,202; 4,111,203; 4,203,440; 4,203,442; 4,210,139; 4,327,725; 4,627,850; 4,865,845; 5,057,318; 5,059,423; 5,112,614; 5,137,727; 5,234,692; 5,234,693; 5,728,396; and the like.
In some embodiments, the drug delivery device is an implantable device. The drug delivery device can be implanted at any suitable implantation site using methods and devices well known in the art. As noted herein, an implantation site is a site within the body of a subject at which a drug delivery device is introduced and positioned. Implantation sites include, but are not necessarily limited to a subdermal, subcutaneous, intramuscular, or other suitable site within a subject's body.
In some embodiments, the composition can be delivered using an implantable drug delivery system, e.g., a system that is programmable to provide for administration of the composition. Exemplary programmable, implantable systems include implantable infusion pumps. Exemplary implantable infusion pumps, or devices useful in connection with such pumps, are described in, for example, U.S. Pat. Nos. 4,350,155; 5,443,450; 5,814,019; 5,976,109; 6,017,328; 6,171,276; 6,241,704; 6,464,687; 6,475,180; and 6,512,954. A further exemplary device that can be adapted for the present disclosure is the Synchromed infusion pump (Medtronic).
Suitable excipient vehicles for the compound are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents or pH buffering agents. Methods of preparing such dosage forms are known, or will be apparent upon consideration of this disclosure, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 17th edition, 1985. The composition or formulation to be administered will, in any event, contain a quantity of the compound adequate to achieve the desired state in the subject being treated.
Compositions of the present disclosure can include those that comprise a sustained-release or controlled release matrix. In addition, embodiments of the present disclosure can be used in conjunction with other treatments that use sustained-release formulations. As used herein, a sustained-release matrix is a matrix made of materials, usually polymers, which are degradable by enzymatic or acid-based hydrolysis or by dissolution. Once inserted into the body, the matrix is acted upon by enzymes and body fluids. A sustained-release matrix desirably is chosen from biocompatible materials such as liposomes, polylactides (polylactic acid), polyglycolide (polymer of glycolic acid), polylactide co-glycolide (copolymers of lactic acid and glycolic acid), polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxcylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone. Illustrative biodegradable matrices include a polylactide matrix, a polyglycolide matrix, and a polylactide co-glycolide (co-polymers of lactic acid and glycolic acid) matrix.
In another embodiment, the pharmaceutical composition of the present disclosure (as well as combination compositions) can be delivered in a controlled release system. For example, the compound may be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump may be used (Sefton (1987). CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al. (1980). Surgery 88:507; Saudek et al. (1989). N. Engl. J. Med. 321:574). In another embodiment, polymeric materials are used. In yet another embodiment a controlled release system is placed in proximity of the therapeutic target thus requiring only a fraction of the systemic dose. In yet another embodiment, a controlled release system is placed in proximity of the therapeutic target, thus requiring only a fraction of the systemic. Other controlled release systems are discussed in the review by Langer (1990). Science 249:1527-1533.
In another embodiment, the compositions of the present disclosure (as well as combination compositions separately or together) include those formed by impregnation of the compound described herein into absorptive materials, such as sutures, bandages, and gauze, or coated onto the surface of solid phase materials, such as surgical staples, zippers and catheters to deliver the compositions. Other delivery systems of this type will be readily apparent to those skilled in the art in view of the instant disclosure.
Embodiments of the composition (e.g., tyrosine inhibitor, gabapentin or derivative thereof, and rapamycin or derivative thereof) can be administered to a subject in one or more doses. Those of skill will readily appreciate that dose levels can vary as a function of the specific the composition administered, the severity of the symptoms and the susceptibility of the subject to side effects. Preferred dosages for a given composition are readily determinable by those of skill in the art by a variety of means and are well above those amounts that might be found in some food products.
In an embodiment, multiple doses of the composition are administered. The frequency of administration of the composition can vary depending on any of a variety of factors, e.g., severity of the symptoms, and the like. For example, in an embodiment, the composition can be administered once per month, twice per month, three times per month, every other week (qow), once per week (qw), twice per week (biw), three times per week (tiw), four times per week, five times per week, six times per week, every other day (god), daily (qd), twice a day (qid), or three times a day (tid). As discussed above, in an embodiment, the composition is administered continuously.
The duration of administration of the composition analogue, e.g., the period of time over which the composition is administered, can vary, depending on any of a variety of factors, e.g., patient response, etc. For example, the composition in combination or separately, can be administered over a period of time of about one day to one week, about two weeks to four weeks, about one month to two months, about two months to four months, about four months to six months, about six months to eight months, about eight months to 1 year, about 1 year to 2 years, or about 2 years to 4 years, or more.
Embodiments of the present disclosure provide methods and compositions (e.g., tyrosine inhibitor, gabapentin or derivative thereof, and rapamycin or derivative thereof) for the administration of the composition to a subject (e.g., a human) using any available method and route suitable for drug delivery, including in vivo and ex vivo methods, as well as systemic and localized routes of administration.
Routes of administration include intranasal, intramuscular, intratracheal, subcutaneous, intradermal, topical application, intravenous, rectal, nasal, oral, and other enteral and parenteral routes of administration. Routes of administration can be combined, if desired, or adjusted depending upon the composition and/or the desired effect. The composition can be administered in a single dose or in multiple doses.
Embodiments of the composition can be administered to a subject using available conventional methods and routes suitable for delivery of conventional drugs, including systemic or localized routes. In general, routes of administration contemplated by the disclosure include, but are not limited to, enteral, parenteral, or inhalational routes.
Parenteral routes of administration other than inhalation administration include, but are not limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, and intravenous routes, i.e., any route of administration other than through the alimentary canal. Parenteral administration can be conducted to effect systemic or local delivery of the composition. Where systemic delivery is desired, administration typically involves invasive or systemically absorbed topical or mucosal administration of pharmaceutical preparations.
In an embodiment, the composition can also be delivered to the subject by enteral administration. Enteral routes of administration include, but are not limited to, oral and rectal (e.g., using a suppository) delivery.
Methods of administration of the composition through the skin or mucosa include, but are not limited to, topical application of a suitable pharmaceutical preparation, transdermal transmission, injection and epidermal administration. For transdermal transmission, absorption promoters or iontophoresis are suitable methods. Iontophoretic transmission may be accomplished using commercially available “patches” that deliver their product continuously via electric pulses through unbroken skin for periods of several days or more.
While embodiments of the present disclosure are described in connection with the Examples and the corresponding text and figures, there is no intent to limit the disclosure to the embodiments in these descriptions. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of embodiments of the present disclosure.
Reprogrammed cellular metabolism is a common characteristic observed in various cancers1,2. However, whether metabolic changes directly regulate cancer development and progression remains poorly understood. Embodiments of the present disclosure show that BCAT1, a cytosolic aminotransferase for the branched-chain amino acids (BCAAs), is aberrantly activated and functionally required for chronic myeloid leukemia (CML). BCAT1 is up-regulated during CML progression and promotes BCAA production in leukemia cells by aminating the branched-chain keto acids. Blocking BCAT1 expression or enzymatic activity induces cellular differentiation and impairs the propagation of blast crisis CML (BC-CML) both in vitro and in vivo. Stable isotope tracer experiments combined with NMR-based metabolic analysis demonstrate the intracellular production of BCAAs by BCAT1. Direct supplementation with BCAAs ameliorates the defects caused by BCAT1 knockdown, indicating that BCAT1 exerts its oncogenic function via BCAA production in BC-CML cells. Importantly, BCAT1 expression not only is activated in human BC-CML and de novo acute myeloid leukemia but also predicts disease outcome in patients. As an upstream regulator of BCAT1 expression, Musashi2 (MSI2) was identified as an oncogenic RNA binding protein that is required for BC-CML. MSI2 is physically associated with the BCAT1 transcript and positively regulates its protein expression in leukemia. Taken together, the present disclosure reveals that altered BCAA metabolism activated through the MSI2-BCAT1 axis drives cancer progression in myeloid leukemia.
To understand the contribution of α-amino acid (AA) metabolism to the cancer progression of CML, blood AA levels were analyzed in murine models that recapitulate the chronic and blast crisis phases of human CML3,4. Using amine-specific fluorescent labeling coupled with high-performance liquid chromatography, sixteen AAs were quantified in the blood plasma from leukemic mice (
Although BCAT1 catalyzes transamination in both directions, the breakdown of BCAAs is the predominant reaction in most cell types6. In order for BCAT1 to generate BCAAs via the reverse reaction, the corresponding branched-chain keto acids (BCKAs), as well as glutamate, must be present as substrates. All three BCKAs, keto-isovalerate (KIV), keto-isocaproate (KIC) and keto-methylvalerate (KMV), were found present in both the blood plasma and leukemia cells (
Given that Bcat1 is highly expressed and augments intracellular BCAAs in BC-CML, Bcat1 may functionally contribute to the acute properties of BC-CML. To test this possibility, Bcat1 expression was inhibited using a short hairpin RNA (shRNA)-mediated gene knockdown approach. The immature lineage-negative (Lin−) cells were sorted from primary BC-CML samples, a population that contains the leukemia-initiating cells of this cancer, and introduced two independent retroviral shRNA constructs (
To examine whether Bcat1 loss affects the propagation of BC-CML in vivo, Lin− cells expressing shBcat1 were transplanted into conditioned recipient mice. Whereas 75% of the recipients transplanted with control cells succumbed to the disease within 30 days, only 47% (shBcat1-a) and 31% (shBcat1-b) of the mice transplanted with Bcat1-knockdown cells developed the disease, and more than half of these mice survived even when followed out to 60 days (
It was next examined whether the enforced expression of Bcat1 could drive blastic transformation in hematopoietic cells. Although a significant increase in Bcat1 expression was observed compared with the vector control, Bcat1 expression alone did not enhance the colony-forming ability of either LSK or Lin− c-Kit+ hematopoietic cells isolated from normal bone marrow (
These results demonstrate that Bcat1 is essential for the development of BC-CML in mice, while normal bone marrow HSPCs show a very limited dependence on this metabolic enzyme. To investigate the contribution of BCAT1 to human leukemia, a panel of 13 peripheral blood samples from healthy and leukemic subjects was examined and it was found that human BCAT1 expression was higher in BC-CML than in either normal or CP-CML cells (
To understand how the BCAT1-driven change in metabolism promotes leukemia growth, intracellular AA concentrations were analyzed upon BCAT1 inhibition and it was found that all three BCAAs were significantly reduced by shBCAT1 or Gbp treatment compared with the controls (
To further investigate the BCAT1-mediated regulation of CML progression, we performed gene correlation analyses using tumor gene expression datasets available in the GEO and TCGA databases. It was found that BCAT1 and MSI2 are often co-expressed in several types of cancer, including leukemias, colorectal and breast cancers (
BCAT1 inhibition by Gabapentin is shown in
The up-regulation and functional requirements of BCAT1 have been reported in glioblastoma and in colorectal and breast tumors22,23. Interestingly, Musashi proteins also regulate the same spectrum of cancers including myeloid leukemia18-21,24,25, suggesting a highly conserved role for the MSI-BCAT1 pathway in multiple cancer types. Despite the conservation of this pathway, the metabolic role of BCAT1 seems distinct and dependent on the tissue of origin; in the brain, BCAT1 catalyzes BCAA breakdown and glutamate production to enhance tumor growth in glioblastoma23, whereas BCAT1 promotes BCAA production in leukemia. Mayers et al. recently showed that two different types of tumors, specifically pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung carcinoma (NSCLC)26, exhibit different usages of BCAAs. Despite the same initiating events of Kras activation and p53 deletion, NSCLC cells actively utilize BCAAs by enhancing their uptake and oxidative breakdown to BCKAs, whereas PDAC cells display decreased uptake and thus little dependency on BCAAs. Consistently, BCAT1 and BCAT2 are required for tumor formation in NSCLC but not in PDAC. Although BCAT1 is functionally required for tumor growth in a broad range of malignancies, these reports and the present disclosure highlight the context-dependent role of the BCAT1 metabolic pathway in cancer.
C57BL6/J mice were from the Jackson Laboratory. Mice were bred and maintained in the facility of the University Research Animal Resources at University of Georgia. All mice were 8-16 weeks old, age- and sex-matched and randomly chosen for experimental use. No statistical methods were used for sample size estimates. All animal experiments were performed according to protocols approved by the University of Georgia Institutional Animal Care and Use Committee.
Cells were suspended for cell sorting in Hanks' balanced salt solution (HBSS) containing 5% (vol/vol) fetal bovine serum (FBS) and 2 mM EDTA as previously described27. The following antibodies were used to define lineage positive cells: 145-2C11 (CD3ε), GK1.5 (CD4), 53-6.7 (CD8), RB6-8C5 (Ly-6G/Gr1), M1/70 (CD11b/Mac-1), TER119 (Ly-76/TER119), 6B2 (CD45R/B220), and eBio1D3 (CD19). Red blood cells were lysed with RBC Lysis Buffer (eBioscience) before staining for lineage markers. For the Lin− Sca-1+ cKit+ (LSK) bone marrow cell sorting, the antibodies 2B8 (cKit/CD117) and D7 (Sca-1/Ly-6A/E) antibodies were also used. To determine donor-derived chimerism in the transplantation-based assays, peripheral blood from the recipients was obtained by the submandibular bleeding method and prepared for analysis as previously described20. All antibodies were purchased from eBioscience. Apoptosis assays were performed by staining cells with Annexin V and 7-AAD (BioLegend). Cell cycle status was analyzed by staining cells with 2.5 μg/ml PI containing 0.1% BSA and 2 μg/ml RNase after fixation with 70% ethanol. Flow cytometric analysis and cell sorting were carried out on the Moflo XDP, Cyan ADP (Beckman Coulter) or S3 (Bio-Rad), and the data were analyzed with FlowJo software (Tree Star Inc.).
Retroviral BCR-ABL1 and NUP98-HOXA9 vectors and lentiviral FG12-UbiC-GFP vector were obtained from Addgene. Mouse Bcat1 cDNA (IMAGE clone ID 30063465) was cloned into MSCV-IRES-GFP and Human BCAT1 cDNA (NITE clone ID AK056255) was cloned into FG12-Ubc-hCD2. The short hairpin RNA constructs against Bcat1 (shBcat1) were designed and cloned in MSCV-LTRmiR30-PIG (LMP) vector from Open Biosystems or TtRMPVIR from Addgene according to their instructions. The target sequences are (SEQ ID 1) 5′-CCCAGTCTCTGATATTCTGTAC-3′ for shBcat1-a, (SEQ ID 2) 5′-TCCGCGCCGTTTGCTGGAGAAA-3′ for shBcat1-b and (SEQ ID 3) 5′-CTGTGCCAGAGTCCTTCGATAG-3′ for luciferase as a negative control (shCtrl). Lentiviral short hairpin RNA (shRNA) constructs were cloned in FG12 essentially as described previously28. The target sequences are (SEQ ID 4) 5′-CGCAGAGTGTACCGGAGA-3′ for shBCAT1-c, (SEQ ID 5) 5′-TGCCCAATGTGAAGCAGT-3′ for shBcat1-d and (SEQ ID 6) 5′-TGCGCTGCTGGTGCCAAC-3′ for luciferase as a negative control. Virus was produced in 293FT cells transfected using polyethylenimine with viral constructs along with VSV-G and gag-pol. For lentivirus production Rev was also co-transfected. Viral supernatants were collected for two days followed by ultracentrifugal concentration at 50,000×g for 2 h.
The human BC-CML cell line K562, the human acute leukemia cell lines MV4-11 and U937 were maintained in Roswell Park Memorial Institute 1640 medium (RPMI-1640) with 10% FBS, 100 IU/ml penicillin and 100 μg/ml streptomycin. The human acute promyelocytic leukemia cell line HL60 was maintained in RPMI supplemented with 20% FBS. All human cell lines were obtained from ATCC, and cell line authentication testing was performed by ATCC-standardized STR analysis to verify their identity in July 2016. For the colony forming assays, the cells were transduced with lentiviral shRNA and plated in triplicate in 1.2% methylcellulose medium (R&D systems) supplemented with 100 IU/ml penicillin and 100 μg/ml streptomycin, 10% FBS. Where indicated, either BCAAs (L-Leucine, L-Valine, L-Isoleucine, 4 mM each, Sigma-Aldrich), L-alanyl-L-glutamine (4 mM, GlutaMax™, Life Technologies), rapamycin (50 nM, Tocris) or gabapentin (Gbp; Tokyo Chemical Industry Co.) was added to the medium. Gbp was freshly dissolved in PBS before use. Colonies were scored on days 9 to 14. For liquid culture of murine cells, freshly isolated adult LSK cells or Lin− BC-CML cells were plated into a 96-well U bottom plate in X-Vivo15 (with Gentamicin and Phenol Red; Lonza) supplemented with 50 μM 2-mercaptoethanol, 10% FBS, 100 ng/ml stem cell factor (SCF, eBioscience) and 20 ng/ml thrombopoietin (TPO, Peprotech). For the BC-CML and LSK colony formation assays, BCR-ABL+ NUP98-HOXA9+ or infected construct-positive cells were sorted and plated in triplicate in Iscove's modified medium (IMDM)-based methylcellulose medium (Methocult M3434, StemCell Technologies). Colonies were scored on days 7 to 10.
Mice bearing CP- and BC-CML were generated essentially as previously described3,4,29-31. In brief, CP-CML was modeled by transducing the oncogene BCR-ABL1 into hematopoietic stem/progenitor cells (HSPCs) defined by the LSK surface marker phenotype from normal bone marrow, which were transplanted into conditioned recipient mice. BC-CML was modeled by transplanting LSK cells infected with two oncogenes, BCR-ABL1 and NUP98-HOXA9, which are associated with myeloid BC-CML in humans. LSK cells were sorted from healthy C57BL6/J bone marrow and cultured in X-Vivo15 media supplemented with 50 μM 2-mercaptoethanol, 10% FBS, 100 ng/ml SCF and 20 ng/ml TPO. After incubation overnight, cells were infected with retroviruses carrying the oncogenes. Viruses used were as follows: MSCV-BCR-ABL-IRES-YFP to generate CP-CML, or MSCV-BCR-ABL-IRES-YFP and MSCV-NUP98-HOXA9-IRES-tNGFR to generate BC-CML. Cells were harvested 48 h after infection and transplanted retro-orbitally into groups of C57BL6/J mice. Recipients were lethally irradiated (9.5 Gy) for CP-CML and sublethally (6 Gy) for BC-CML. For Bcat1 overexpression, LSK cells were infected with MSCV-BCR-ABL-IRES-YFP and MSCV-Bcat1-IRES-GFP, and doubly infected cells were FACS-purified and transplanted into recipients that were sublethally irradiated. For Bcat1 knockdown by retroviral shRNA transduction, the Lin− population from BC-CML cells was sorted and infected with either control shCtrl (against luciferase) or shBcat1-a/b (against Bcat1) retrovirus for 48 h. Infected cells were sorted based on GFP expression, and 1,000 or 2,000 cells were transplanted in sublethally irradiated C57BL6/J recipients. For conditional Bcat1 knockdown by a Dox-inducible shRNA system, animals were analyzed for donor chimerism at day 10 post-transplantation, and then Dox treatment was initiated by feeding Dox-containing rodent chow (0.2 mg/g diet; S3888, BioServ). After transplantation, recipient mice were maintained on antibiotic water (sulfamethoxazole/trimethoprim) and evaluated daily for signs of morbidity, weight loss, failure to groom, and splenomegaly. Premorbid animals were sacrificed, and relevant tissues were harvested and analyzed by flow cytometry and histopathology. For secondary BC-CML transplantations, cells recovered from terminally ill primary recipients were sorted for Lin− donor cells and transplanted into secondary recipients. Where indicated, sorted live BC-CML cells from the spleen were cytospun and stained with Wright's stain solution (Harleco) for cytopathologic evaluation by a board-certified veterinary pathologist.
Patient blood samples were obtained at the Institute of Medical Science, the University of Tokyo (IMSUT) Hospital with written informed consent according to the procedures approved by the Institutional Review Board. Mononuclear cells from the subjects were viably frozen and stored in liquid nitrogen. For in vitro colony formation with BCAT1 knockdown, primary hCD34+ cells sorted from patient bone marrow samples were cultured in IMDM supplemented with 10% FBS, 100 IU/ml penicillin and 100 μg/ml streptomycin, 55 μM 2-mercaptoethanol, SCF, IL-3, IL-6, FLT3L and TPO. After 24 h of culture, the cells were transduced with lentiviral shRNA (cloned in FG12-UbiC-GFP), and the GFP-positive infected cells were sorted at 48 h, and 5,000-50,000 cells were plated in complete methylcellulose medium (Methocult H4435, StemCell Technologies). For the colony forming assays with Gbp, sorted hCD34+ cells from the primary patient specimens were cultured in complete methylcellulose medium with the indicated concentrations of Gbp. Colonies were scored on days 9 to 14.
For the focused gene expression analysis of BCAT1, BCAT2 and MSI2 in human CML progression, the GEO dataset GSE4170 was retrieved and analyzed using Python v2.7 and the SciPy statistical toolkit. Pearson correlation coefficients were used to find patterns of co-expression. For co-expression analysis of BCAT1 and MSI2 across multiple cancer types, the GEO datasets GSE14671 (CML), GSE10327 (medulloblastoma), GSE20916 (colorectal), GSE14548 (breast) and TCGA datasets LAML (AML) and LUAD (lung adenocarcinoma) were collected and analyzed in a similar fashion.
Total cellular RNAs were isolated using RNAqueous-Micro kit (Ambion) and cDNAs were prepared from equal amounts of RNAs using Superscript III reverse transcriptase (Life Technologies). For standard PCRs, the reactions were performed with DreamTaq PCR Master Mix (Life Technologies), cDNA and 0.5 μM of each primer. PCR conditions were as follows: 1 min at 94° C., followed by 35 cycles at 94° C. for 30 s, 58° C. for 30 s, and 72° C. for 30 s. PCR primer sequences are as follows:
(SEQ ID 9) Bcat1-F1, 5′-TGTGGCTGTACGGCAAGGACAAC-3′; (SEQ ID 10) Bcat1-R2, 5′-GTAGCTCGATTGTCCAGTCACT-3′. Quantitative real-time PCRs were performed using EvaGreen® qPCR Master Mix (Bio-Rad) on an iQ5 (Bio-Rad), or using TaqMan Gene Expression Assays on an Applied Biosystems® 7500 Real-Time PCR Systems (Life Technologies). Results were normalized to the level of β-2-microglobulin. PCR primer sequences are as follows:
(SEQ ID 11) mB2m-F, 5′-ACCGGCCTGTATGCTATCCAGAA-3′; (SEQ ID 12) mB2m-R, 5′-AATGTGAGGCGGGTGGAACTGT-3′;
(SEQ ID 13) hB2M-F, 5′-ATGAGTATGCCTGCCGTGTGA-3′; (SEQ ID 14) hB2M-R, 5′-GGCATCTTCAAACCTCCATG-3′;
(SEQ ID 15) hBCAT1-F, 5′-TGGAGAATGGTCCTAAGCTG-3′; (SEQ ID 16) hBCAT1-R, 5′-GCACAATTGTCCAGTCGCTC-3′;
(SEQ ID 17) hMYC-F, 5′-GAGCAAGGACGCGACTCTCC-3′; (SEQ ID 18) hMYC-R, 5′-GCACCGAGTCGTAGTCGAGG-3′. The following genes were analyzed with TaqMan Gene Expression Assays: Bcat1 (Mm00500289_m1), Bcat2 (Mm00802192_m1), Gpt1 (Mm00805379_g1), Gpt2 (Mm00558028_m1), Got1 (Mm00494698_m1), Got2 (Mm00494703_m1).
Leukemia cells or peripheral blood samples drawn from mice bearing myeloid leukemia were used for amino acid and keto acid analysis by high-performance liquid chromatography (HPLC)-fluorescence detection, as described32-34. In brief, two hundred thousand live leukemia cells per sample were sorted and washed twice with ice-cold PBS to remove media components prior to amino acid extraction. The blood plasma was prepared by centrifugation of the peripheral blood samples at 2,000×g at 4° C. for 10 min. Plasma fractions were then treated with 45% methanol/45% acetonitrile containing 6-aminocaproic acid (internal standard for amino acid analysis) or α-ketovalerate (internal standard for keto acid analysis) on ice for 10 min. Cell samples were treated with 80% methanol instead of 45% methanol/acetonitrile mixture. After removing the insoluble particles by centrifugation, the supernatants were collected and dried using a SpeedVac at 30-45° C. For amino acid quantification, the dried samples were treated with the amine-reactive 4-fluoro-7-nitro-2,1,3-benzoxadizole (NBD-F) to derivatize the amino acids. HPLC separation of NBD-amino acids was carried out on an Inertsil ODS-4 column (3.0×250 mm, 5 μm, GL Sciences, Tokyo, Japan) at a flow rate of 0.6 ml min−1. We used two types of mobile phase conditions for the separation of 16 amino acids. The mobile phases included (A) 25 mM citrate buffer containing 25 mM sodium perchlorate (pH 6.2) and (B) water/acetonitrile (50/50, v/v). The gradient conditions were as follows: t=0 min, 10% B; t=20 min, 50% B; and t=30 min, 100% B. For NBD-Asn, Ser, Thr, Gln and Phe analysis, 25 mM citrate buffer containing 25 mM sodium perchlorate (pH 4.4) was used as the mobile phase A. NBD-amino acids were detected with excitation and emission wavelengths of 470 and 530 nm, respectively. For keto acid quantification, dried samples were treated with o-phenylenediamine (OPD) to derivatize alpha-keto acids, followed by liquid-liquid extraction with ethyl acetate. HPLC separation of OPD-keto acids was carried out on an Inertsil ODS-4 column (3.0×250 mm, 5 μm) at a flow rate of 0.6 ml min−1. Mobile phase was water/methanol (55/45, v/v). The fluorescence detection was carried out at the emission wavelength of 410 nm with excitation of 350 nm.
Primary mouse leukemia cells from the spleens of the mice bearing myeloid leukemia were used for the analysis of leucine uptake essentially as described previously35,36. In brief, live leukemia cells were sorted and washed with HBSS to remove media components. The cells were incubated at 37° C. for 1-3 min with pre-warmed HBSS containing 10 μM [(U)-14C]-L-leucine (Moravek Inc., specific activity, 328 mCi/mmol). The cells were subsequently washed twice with cold HBSS and lysed using 100 mM NaOH. The solubilized cell lysates were mixed with the EcoLume liquid scintillation cocktail (MP Biomedicals), and radioactivity was measured using an L56500 liquid scintillation counter (Beckman Coulter). Leucine uptake was quantified using a calibration curve of [14C]-L-leucine reference standard samples.
Cells were cultured and labeled in media supplemented with either 170 μM [(U)-13C]-L-valine, 30 or 170 μM [(U)-13C]-ketoisovalerate (KIV) sodium salt (for 13C tracer experiments; Cambridge Isotope Laboratories) or 2 mM [amine-15N]-L-glutamine (for 15N tracer experiments; Cambridge Isotope Laboratories). The concentrations are based on the standard RPMI-1640 media formulation. At the time of collection, the cells were washed twice with ice-cold PBS and extracted with 80% methanol on ice for 10 min. After removing the insoluble particles by centrifugation, the supernatants were collected and dried using a SpeedVac at 30° C. The cell extracts were dissolved in a total volume of 90 μL 99.96% D2O containing 0.1 mM DSS-d6 and transferred to 3-mm NMR tubes (Shigemi Inc.). Calibration samples (150-250 mM) were prepared from 98% 15N-enriched glutamine, glutamic acid, valine, leucine, isoleucine and alanine (Isotec Inc.) and 13C-enriched KIV and 13C,15N-enriched valine (Cambridge Isotope Laboratories) in D2O containing 0.1 mM DSS. All signals were identified either with authentic samples or by reference to literature values. Two-dimensional proton correlated spectra (COSY and TOCSY) were also collected in some cases to confirm assignments. The data were collected at 25° C. on Agilent DD2 spectrometers at 600 or 900 MHz equipped with cryogenically cooled probes. The 1H data were collected with a 20-sec relaxation delay for accurate integration. The 15N data were acquired with a two-dimensional heteronuclear multiple bond correlation experiment (gNhmbc) derived from the Agilent pulse program library with the transfer delay set for a 15N-1H coupling value of 4 Hz. Typically, data sets were 2000×64 complex points with the 15N dimension set between 30 and 46 ppm, and 64 scans per point. The 13C data were acquired with a two-dimensional heteronuclear single bond correlation experiment (HSQCAD) from the Agilent pulse program library, and the datasets were 1202×64 complex points with the 13C dimension set between 10 and 80 ppm with 16 scans per point. One-dimensional spectra were also collected using the same heteronuclear correlation experiments for 15N and 13C. The data were processed using MestReNova software (Mestrelab Research S.L.). One-dimensional proton data were processed with 0.3 Hz line broadening and polynomial baseline correction. The gNhmbc and HSQC data were processed with linear prediction and zero-filling in the 15N and 13C dimensions. Integration was achieved by summing over peak areas with the contribution of noise subtracted in the 15N spectra. To calculate the concentrations in the 15N tracer experiments, the 1H and gNhmbc spectra of the calibration samples were integrated, and a scaling factor was derived from the ratio of the known concentration of each 98% enriched 15N-amino acid and the integral values from the gNhmbc data. These factors are a function of the 3-bond coupling between the 15N-amine and β-protons as well as the number of those protons. Therefore, the concentrations of each amino acid in cell extracts can be estimated from their integral values by applying the respective scaling factor. For quantification of 13C-labeled compounds, the methyl groups in the 1H and HSQC spectra of the calibration references were integrated, and a scaling factor was derived essentially as described above and used to calculate concentrations from the HSQC data of each sample.
Anti-FLAG monoclonal antibody M2 (Sigma-Aldrich), anti-MSI2 monoclonal antibody EP1305Y (Abcam) and normal Rabbit IgG PP64B (Millipore) were used for immunoprecipitation. For Western blotting the following antibodies were used: mouse monoclonal BCAT1 (clone ECA39, BD Transduction Laboratories) and Bcat1 OTI3F5 (OriGene), rabbit monoclonal S6K (#9202 and #2708), pS6K (#9234), AKT (#4691), pAKT, T308 (#13038) and pAKT, S473 (#4060) from Cell Signaling, rabbit monoclonal MSI2 EP1305Y, mouse monoclonal HSP90 F-8 (Santa Cruz Biotech) and mouse monoclonal β-tubulin BT7R (Thermo Fisher Scientific).
K562 cells were lysed in 50 mM Tris/HCl (pH 7.5) containing 150 mM NaCl, 5 mM EDTA, 1% NP-40, and the Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). We performed immunoprecipitations with anti-FLAG, anti-MSI2 or rabbit normal IgG and protein G magnetic beads (Life Technologies) for 1 h at 4° C. The immunoprecipitated protein-RNA complexes were washed three times with low- and high-salt wash buffers (300 mM or 550 mM NaCl, respectively), followed by three washes in PBS. Total RNAs were purified from the washed beads using the RNAqueous-Micro kit (Ambion) and subjected to RT-qPCR analysis for quantification. The fold enrichment of the transcript amount in the RIP fraction over the amount present in the input sample before RIP (RIP/input) was calculated for each sample.
Statistical analyses were carried out using GraphPad Prism software version 6.0f (GraphPad Software Inc.). Data are shown as the mean±the s.e.m. Two-tailed unpaired Student's t-tests or Mann-Whitney U tests were used to determine statistical significance. For Kaplan Meier survival analysis, log-rank tests were used for statistical significance (*p<0.05, **p<0.01, ***p<0.001).
It should be noted that ratios, concentrations, amounts, and other numerical data may be expressed herein in a range format. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a concentration range of “about 0.1% to about 5%” should be interpreted to include not only the explicitly recited concentration of about 0.1 wt % to about 5 wt %, but also include individual concentrations (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within the indicated range. In an embodiment, “about 0” can refer to 0, 0.001, 0.01, or 0.1. In an embodiment, the term “about” can include traditional rounding according to significant figures of the numerical value. In addition, the phrase “about ‘x’ to ‘y’” includes “about ‘x’ to about ‘y’”.
It should be emphasized that the above-described embodiments of the present disclosure are merely possible examples of implementations, and are set forth only for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the above-described embodiments of the disclosure without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure.
This application claims the benefit of and priority to U.S. Provisional Application Ser. No. 62/413,028, having the title “METHODS OF TREATMENT FOR MYELOID LEUKEMIA”, filed on Oct. 26, 2016, the disclosure of which is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2017/058457 | 10/26/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62413028 | Oct 2016 | US |
