Methods and compositions for the use of bisbenzylisoquinoline for the treatment of malignant tumors

Abstract

The present invention provides pharmaceutical compositions and methods of use of bisbenzylisoquinoline, e.g., 6,6',7,12-tetramethoxy-2,2'-dimethyl-berbaman, and analogs, derivatives, isomers, and modified forms of the compound, e.g., in crystalline, salt, or salt form, in combination with pharmaceutically acceptable acids or other agents, for the treatment of acute, chronic, and preleukemic conditions, as well as lymphoma and solid tumors. These include, but are not limited to, preneoplastic and neoplastic diseases and solid tumors, such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), atypical chronic myeloid leukemia (aCML), and acute myeloid leukemia (AML), polycythemia vera (PV), chronic lymphoblastic leukemia (CLL), myeloproliferative syndrome (MPS), myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), myelofibrosis (MF), and polycythemia vera (PV).

Description

Technical Field

[0001] Cross - reference to Related Applications This application is a U.S. utility patent application claiming the benefit of priority to both U.S. Patent Provisional Application No. 63 / 453,063, filed Mar. 17, 2023, titled "Methods of Use and Compositions of Bisbenzylisoquinolines such as 6,6’,7,12 - tetramethoxy - 2,2’ - dimethyl - berbaman or Analogs for the Treatment of Leukemic Stem Cells, Cancer Stem Cells and Precancerous Stem Cells", and U.S. Patent Provisional Application No. 63 / �53,068, filed Mar. 18, 2023, titled "Leukemic Stem Cell Treatment", each of which is incorporated herein by reference in its entirety.

Background Art

[0002] While the effectiveness of cancer chemotherapy drugs may be limited by both systemic and local toxicity, treatment failure and cancer recurrence are also a result of drug resistance and regenerative properties of small populations of tumor cells identified as cancer stem cells (CSCs). Several properties of CSCs, such as cell surface markers (CD44, CD24, and CD133), have been identified, which may enable the development of more suitable therapies for eradicating these cell populations. CSCs survive by relying on specific signaling pathways (e.g., Wnt / β-catenin, Notch, NF-κB, NLRP3, and Hedgehog, which regulate CSC properties), unlike normal stem cells. In CSCs, this reduces apoptotic signaling and increases proliferation signaling, which allows for the overexpression of drug efflux pumps, thereby enabling avoidance of toxic chemotherapeutic drug concentrations. Furthermore, the interaction between the bone marrow microenvironment and leukemia stem cells via the Wnt / β-catenin, NF-κB, and NLRP3 inflammatory pathways provides opportunities for therapeutic intervention. Understanding the survival characteristics of CSCs makes it possible to identify and develop therapeutic drugs that are particularly suitable for their elimination. Cancers identified in populations rich in CSCs include brain cancer, head and neck cancer, breast cancer, pancreatic cancer, lung cancer, liver cancer, stomach cancer, colorectal cancer, lymphoma, and leukemia.

[0003] Tetrandrin (6,6',7,12-tetramethoxy-2,2'-dimethylberbam) (also known as d-tetrandrin, TET, NSC#77037, and ES-3000) is a bis-benzylisoquinoline alkaloid extracted from the roots of the plant Stephania tetrandra S.moore. ES-3000 (tetrandrin) is under development by Escend Pharmaceuticals, Inc. (Escend) for the treatment of leukemia and related hematological malignancies, including relapsed or refractory acute myeloid leukemia (AML), chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), and polycythemia vera (PV).

[0004] Bis-benzylisoquinoline (BBI) represents a large class of alkaloids having two benzylisoquinoline units linked by ether crosslinking, including methylene oxy bonds or direct carbon-carbon bonds. BBI has a variety of biological activities. The inventions of this disclosure provide bis-benzylisoquinoline, e.g., 6,6',7,12-tetramethoxy-2,2'-dimethyl-berbaman (also known as tetrandrin, d-tetrandrin, TET, NSC#77037, and ES-3000), in combination with pharmaceutically acceptable acids or other agents for the treatment of acute, chronic, and preleukemic conditions, as well as lymphoma and solid tumors, as well as analogs, derivatives, isomers, and modified forms of this compound, e.g., crystalline, salt form, or salt, as well as pharmaceutical compositions and methods of use of the compound. This includes, but is not limited to, preneoplastic and neoplastic diseases and solid tumors, such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), atypical chronic myeloid leukemia (aCML), and acute myeloid leukemia (AML), polycythemia vera (PV), chronic lymphoblastic leukemia (CLL), myeloproliferative syndrome (MPS), myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), myelofibrosis (MF), and polycythemia vera (PV). The present invention also relates to the use of bisbenzylisoquinoline, e.g., 6,6',7,12-tetramethoxy-2,2'-dimethyl-berbaman (tetrandrin), for the treatment and / or conditioning of patients undergoing bone marrow transplantation and hematopoietic stem cell transplantation (HSCT). These drugs can be used as single agents or in combination with other therapeutic agents, such as alkylating agents, antimetabolites, antitumor antibiotics, topoisomerase inhibitors, mitotic inhibitors, and corticosteroids, or targeted therapies, such as TKIs and antibody-targeted chemotherapy, to treat cancers and leukemias in which stem cells may be the cause of treatment resistance.

[0005] Indications for MDS, MPN, CML, aCML, AML, and PV are bone marrow disorders that affect hematopoietic stem cells. MDS is a precursor to leukemia and is not a single disease but a group of disorders that affect blood cell formation. In all forms of MDS, bone marrow abnormalities result in a decrease in the concentration of blood cells circulating in the bloodstream. In healthy individuals, stem cells in the bone marrow produce daughter cells. These daughter cells go through several developmental stages within the bone marrow. Eventually, they mature into red blood cells (RBCs), white blood cells (WBCs), or platelets. They are released from the bone marrow and circulate in the bloodstream, performing important functions such as carrying oxygen throughout the body, fighting infections, and protecting the body from bleeding by aiding blood clotting.

[0006] In MDS patients, bone marrow stem cells do not function properly. Instead of producing healthy, mature RBCs, WBCs, and platelets, the bone marrow produces cells that remain immature and tend to die prematurely. As a result, MDS patients have a decrease in the concentration of one or more types of blood cells in their bloodstream (cytopenia), such as anemia (decreased RBC concentration), leukopenia (decreased WBC concentration), and thrombocytopenia (decreased platelet concentration). Decreased blood cell concentration or low blood cell count causes the symptoms of MDS. The disease progresses over time in two ways. In the majority of MDS patients, the number of healthy blood cells produced or that survive decreases. This can lead to severe anemia (decreased RBCs), an increased risk of infection (due to decreased WBCs), or a severe risk of bleeding (due to decreased platelets). In approximately 30% of MDS patients (depending on the subtype), the number of extremely immature abnormal cells (blast cells or blasts) increases in the bone marrow, and MDS progresses to acute leukemia. This risk of developing leukemia is referred to as the "low, intermediate, or high-risk" subtype of MDS.

[0007] The following is the MDS classification system for MDS subtypes as defined by the World Health Organization (WHO):

[0008] Refractory cytopenia with monosystemic dysplasia (RCUD). This involves too few red blood cells (RBCs) in the blood (anemia), too few white blood cells (neutropenia), or too few platelets (thrombocytopenia).

[0009] Refractory anemia with ring sideroblasts (RARS). Ring sideroblasts are an early stage of red blood cell counts (RBCs) that have accumulated an abnormal amount of iron. WBC and platelet counts may be normal, but the percentage of blasts in the bone marrow and blood is low.

[0010] Refractory cytopenia with multiple systemic dysplasia (RCMD). The bone marrow shows dysplastic changes in cells that produce at least two types of blood cells (RBCs, WBCs, or platelets). The percentage of blasts in the bone marrow and blood is low. In cases of multiple systemic dysplasia and ring sideroblasts, this is classified as RCMD-RS.

[0011] Refractory anemia with supernumerary blasts-1 (RAEB-1) and refractory anemia with supernumerary blasts-2 (RAEB-2). Blasts accounting for at least 5% (RAEB-1) or at least 10% (RAEB-2), but less than 20%, in the bone marrow.

[0012] MDS is associated with an isolated abnormality of chromosome 5 [del(5q)]. A portion of chromosome 5 is deleted. Typically, this means too few red blood cells (RBCs) and a low percentage of blast cells in the bone marrow and blood.

[0013] MDS, unclassifiable (MDS-U). This subtype means that the bone marrow shows dysplastic changes in cells that produce WBCs or platelets (but not in cells that produce RBCs). The patient has normal percentages of blasts in the bone marrow and blood, and MDS does not fit any of the other subtypes.

[0014] Some problems related to hematopoietic formation are not limited to myelodysplasticity (related to the abnormal production of cells in the bone marrow) or myeloproliferativeity (related to the overproduction of cells in the bone marrow). Instead, these problems exhibit characteristics of both MDS and MPN, and include the following:

[0015] Chronic myelomonocytic leukemia (CMML). The main characteristic of CMML is an excess of both myelocytes and monocytes, which are types of white blood cells, in the blood.

[0016] Juvenile myelomonocytic leukemia (JMML). Similar to CMML, this occurs in young children. It results in high concentrations of myelocytes and monocytes.

[0017] Atypical chronic myeloid leukemia (aCML), BCR-ABL1 negative. In aCML, there is an excessive amount of granulocytes, a type of white blood cell, and aCML closely resembles chronic myeloid leukemia (CML). However, CML patients have a chromosomal alteration called the Philadelphia chromosome, while aCML patients do not.

[0018] MDS / MPN, unclassifiable. This disease exhibits characteristics of both MDS and MPN and does not fit any other subtype. This subtype is refractory anemia with ring sideroblasts and thrombocytosis (RARS-T).

[0019] In CML, BCR-ABL tyrosine kinase inhibitors (TKIs), such as imatinib mesylate (IM), nilotinib, and dasatinib, have revolutionized the treatment of Philadelphia-positive (Ph+) leukemia in both CML and B-cell acute lymphoblastic leukemia (B-ALL) by targeting and inactivating proliferation signals derived from BCR-ABL. However, clinical resistance to these TKIs negates their potential therapeutic effects in Ph+ leukemia.

[0020] Resistance to TKIs is a problem in some populations of CML patients. Resistance is particularly important in patients who develop the T315I BCR-ABL kinase domain (KD) mutation, which accounts for approximately 15% of all mutations detected after TKI failure. The T315I mutation leads to resistance to imatinib mesylate (IM) and second-generation TKIs, including dasatinib (D), nilotinib (N), bosutinib (B), ponatinib, and bafetinib.

[0021] In the case of AML, the outcomes for adults with AML are poor, with long-term overall survival (OS) being only 40-50% in younger patients and a median OS of less than one year in older patients. Adding cytarabine infusion with intermittent anthracycline (7+3) to the standard remission induction regimen has not yielded significant additional benefits. Recently, it has been recognized that leukemia stem cells (LSCs), which can produce identical daughter cells and differentiated cells, can persist and maintain AML. Because LSCs have different characteristics from the bulk AML population, they are difficult to eliminate with standard chemotherapy and are therefore a cause of disease resistance and relapse. [Overview of the project]

[0022] A method for treating patients with hematological malignancies is disclosed, comprising treating the patient with bisbenzylisoquinoline before and / or after treatment with standard therapeutic agents, including alkylating agents, antimetabolites, antitumor antibiotics, topoisomerase inhibitors, mitotic inhibitors, and corticosteroids or targeted therapy agents (e.g., TKIs) for treating cancer and leukemia stem cells. Examples of conventional chemotherapeutic agents include, but are not limited to, daunorubicin, doxorubicin, cytarabine, cisplatin, gemcitabine, vinblastine, etoposide, decitabine, azacitidine, and venetoclax. Examples of targeted therapy agents include, but are not limited to, tyrosine kinase inhibitors, such as imatinib, dasatinib, nilotinib, bosutinib, and ponatinib. Bisbenzylisoquinoline treatment is preferably continued until the patient shows a hematological or cytological response to leukemia. If a patient's leukemia cells develop resistance to a TKI or other chemotherapy agent, bisbenzylisoquinoline therapy is repeated along with repeated treatment with a TKI or other chemotherapy regimen. Bisbenzylisoquinoline therapy reduces the leukemia stem cell population, inhibits the development of additional leukemia stem cells, and reduces or eliminates leukemia stem cells, including clonal populations that are resistant to TKI therapy or other chemotherapy regimens, which would otherwise expand during treatment without bisbenzylisoquinoline. Clonal populations containing the bcr-abl genotype with the T315I mutation are an example of such populations. If necessary, bisbenzylisoquinoline therapy is repeated until the patient shows a hematological or cytological response to leukemia. Thereafter, the patient may be treated with the same or a different TKI or chemotherapy regimen, with or without bisbenzylisoquinoline therapy, to further improve the patient's outcome. These drugs utilize different mechanisms of action, and the response to treatment varies among them, but serious adverse reactions may exist that could limit the effectiveness of treatment.

[0023] The present invention describes the use of bisbenzylisoquinoline, e.g., 6,6',7,12-tetramethoxy-2,2'-dimethyl-berbaman (tetrandrin), as well as derivatives, isomers, and analogues, prodrugs, modified forms, e.g., salt forms, or salts thereof, with pharmaceutically acceptable acids in the inhibition of molecular signaling pathways involved in the survival, self-renewal, and differentiation of cancer stem cells and leukemia stem cells, including but not limited to Alox5, Stat3, Wnt / beta-catenin, Msr2, Blk, Myc, Survivin, Cyclin D, Osteopontin, Tenascin C, L1CAM, NF-κB, NLRP3, and CaMKII.

[0024] The present invention describes the use of bisbenzylisoquinoline, e.g., 6,6',7,12-tetramethoxy-2,2'-dimethyl-berbaman (tetrandrin), as well as derivatives, isomers, and analogues, modified forms, e.g., salt forms, or salts thereof, with pharmaceutically acceptable acids in the inhibition of molecular protein targets involved in the survival of cancer and leukemia stem cells, including but not limited to lipoxygenase, Mcl-1, cyclin-D1, beta-catenin, Bcl-2, Bcl-xL NF-κB, CamKIIγ, pCaMKIIγ, NLRP3, and VEGF.

[0025] The invention of the present disclosure relates to the use of bisbenzylisoquinolines, such as 6,6’,7,12-tetramethoxy-2,2’-dimethyl-berbaman (tetrandrine), and derivatives, isomers, and analogs, modified forms, such as salt forms, or salts thereof, with pharmaceutically acceptable acids only, or in combination with chemotherapeutic agents or immunosuppressive agents including epoetin alfa + GCSF, azacitidine (Vildaza), decitibine (Dacogen), cedazuridine, Inqovi® (decitabine and cedazuridine), cytarabine, fludarabine, lenalidomide (Revlimid), for immunosuppressive, immunomodulatory, and anti-inflammatory activities enabling the treatment of MDS, MPN, CML, aCML, AML, PV, and related diseases.

[0026] The invention of the present disclosure relates to the use of bisbenzylisoquinolines, such as 6,6’,7,12-tetramethoxy-2,2’-dimethyl-berbaman (tetrandrine), and derivatives, isomers, and analogs, modified forms, such as salt forms, or salts thereof, with pharmaceutically acceptable acids only, or in combination with tyrosine kinase inhibitors including imatinib, dasatinib, nilotinib, bosutinib, ponatinib, AZD0530, NPB-001-05, AT9283, BAY43-9006, bafetinib, AP24534, lestaurtinib, tozasertib, danusertib, XL228, KW-2449, AT-9283, VE-465, DCC-2036, PKC412, PF-03814735, and batatinib, for the treatment of solid tumor cancers, lymphomas, MPN, CML, aCML, AML, PV, and related myelodysplastic diseases.

[0027] Tetrandrine is the preferred bisbenzylisoquinoline, although other bisbenzylisoquinoline analogs can also be used. Initial treatment with tetrandrine is preferably about 2 - 15 mg / kg of tetrandrine per day, more preferably 5 - 10 mg / kg of tetrandrine per day. In the case of oral delivery, the preferred initial treatment with tetrandrine can be expressed as a fixed dose of 360 mg per day, more preferably 540 mg per day. Tetrandrine treatment can be for 7 days or more. However, the treatment can also be for 14 - 21 consecutive days in a 28 - day cycle, or can be long - term with once - daily dosing continuing for 6 - 8 weeks or up to 3 months. In some cases, the amount and / or duration can be less than 5 mg / kg or less than 350 mg of tetrandrine per day and less than 7 days. The therapy can also be administered over a long period. Further, tetrandrine derivatives may be provided for parenteral use such as IV as a bolus or infusion.

[0028] The methods described above can also be modified such that treatment with a TKI or chemotherapy regimen is complemented by combination treatment with a bisbenzylisoquinoline. In such cases, (1) the amount of bisbenzylisoquinoline may be less than the amount used when administered alone, (2) the time for bisbenzylisoquinoline treatment may be reduced (e.g., 2 - 7 days in the case of tetrandrine), or (3) both the amount and time of bisbenzylisoquinoline treatment may be reduced. Further, the amount of the TKI or other chemotherapy regimen may also be less than when administered alone, or a different dosing schedule may be used.

[0029] The disclosed invention also includes a method for treating leukemia patients who have developed resistance to imatinib-containing TKIs or chemotherapy regimens such as 7+3 (7 days of cytarabine plus the first 3 days of daunorubicin) or 5 days of high-dose cytarabine. Treatment with bisbenzylisoquinoline is intended to reduce the leukemia cell population and inhibit the development or proliferation of leukemia stem cells that have acquired treatment resistance. Such patients may be treated concurrently with TKIs or other chemotherapy regimens, or subsequently with TKIs or other chemotherapy regimens after the patient has demonstrated a hematological or cytological response to leukemia. Furthermore, other anti-leukemia agents may be administered to the patient before, during, or after administration of bisbenzylisoquinoline or TKIs or other chemotherapy regimens. These additional treatments include the use of SRC kinase inhibitors, as well as protein synthesis inhibitors such as omasetaxin, conventional standard chemotherapy agents such as interferon alpha and alkylating agents, antimetabolites, antitumor antibiotics, topoisomerase inhibitors, mitotic inhibitors, and corticosteroids or targeted therapies. Conventional chemotherapy agents include, but are not limited to, daunorubicin, doxorubicin, cytarabine, cisplatin, gemcitabine, vinblastine, etoposide, decitabine, azacitidine, and venetoclax. Targeted therapies include, but are not limited to, tyrosine kinase inhibitors such as imatinib, dasatinib, nilotinib, bosutinib, and ponatinib. Solid tumors and lymphomas that may benefit from the treatment of bisbenzylisoquinoline, e.g., 6,6',7,12-tetramethoxy-2,2'-dimethyl-berbaman (tetrandrin), either alone or in combination with chemotherapeutic agents or targeted therapies, as well as derivatives, isomers, and analogs, prodrugs, modified forms, e.g., salt forms, or salts of this compound include triple-negative breast cancer (TNBC), other breast cancers, pancreatic cancer, brain cancer, head and neck cancer, lung cancer, liver cancer, gastric cancer, and colorectal cancer, all of which have cancer stem cell enrichment populations with known surface antigens for identification as biomarkers.

[0030] Furthermore, the disclosed inventions describe novel improvements, pharmaceutical ingredients, dosage forms, excipients, solvents, diluents, drug delivery systems, preservatives, toxicity monitoring and improvement, and techniques or agents for avoiding or reducing toxicity. The disclosed inventions also relate to the use of drug delivery systems, prodrugs, polymer conjugates, routes of administration, antibody conjugates, and other agents for enhancing the activity of compounds, inhibiting suboptimal cellular effects or repair of sublethal damage, or “transitioning” cells to a more destructive cellular phase such as apoptosis. In some cases, the use of bisbenzylisoquinoline, e.g., 6,6',7,12-tetramethoxy-2,2'-dimethyl-berbaman (tetrandrin), and derivatives, isomers, and analogues, modified forms, e.g., salt forms, or salts thereof, in combination with pharmaceutically acceptable acids, such as, but not limited to, antibodies, vaccines, cytokines, lymphokines, gene and antisense therapies, and CAR-TCR cell therapies, offers novel approaches and significant improvements to suboptimal therapeutic agents.

[0031] Bisbenzylisoquinoline, e.g., 6,6',7,12-tetramethoxy-2,2'-dimethyl-berbaman (tetrandrin), along with derivatives, isomers, and analogues, prodrugs, modified forms, e.g., salt forms, or salts thereof, with the help of a pharmaceutically acceptable acid, may be prepared synthetically, semi-synthetically, biochemically, or by extraction processes from plant materials containing drugs, precursors, or intermediates, including but not limited to Stephania tetrandra and Stephania abuta.

[0032] Pharmaceutically acceptable salts include salts with inorganic acids such as hydrochloride, sulfate, nitrate, and phosphate, or salts of organic acids such as acetate, propionate, succinate, oxalate, benzoate, fumarate, maleate, methanesulfonate, isethionate, theophylline acetate, salicylate, phenolphthalate, and methylene-bis-β-hydroxynaphthoate, or substituted derivatives of these. These may also include citrate and malate forms.

[0033] The disclosed invention also relates to bisbenzylisoquinoline, e.g., 6,6',7,12-tetramethoxy-2,2'-dimethyl-berbaman (tetrandrin), in its pure state or in the form of a composition in combination with any other pharmaceutically compatible product that may be inactive or physiologically active, accompanied by a pharmaceutically acceptable acid, as well as to pharmaceutical compositions comprising derivatives, isomers, and analogs, prodrugs, modified forms, e.g., crystals, salts, or salts of this compound. The agents according to the inventions of the present disclosure may be used orally, parenterally, rectally, topically, or as a patch.

[0034] For oral administration, solid compositions may be used in the form of tablets, pills, powders (solutions, gelatin capsules, wafer capsules), medical foods, or granules. In these compositions, the active ingredient according to the disclosed invention is mixed with one or more inert excipients or diluents, such as starch, cellulose, sucrose, lactose, or silica, whether or not under argon flow. These compositions may also contain one or more substances other than diluents, such as one or more lubricants, colorants, coatings, or varnishes, such as magnesium stearate or talc.

[0035] Liquid compositions for oral administration may include pharmaceutically acceptable solutions, suspensions, emulsions, syrups, or elixirs containing water, ethanol, glycerol, vegetable oil, or an inert diluent such as liquid paraffin. These compositions may also contain substances other than diluents, such as humectants, sweeteners, thickeners, flavorings, or stabilizing products.

[0036] Parenterally administered sterile compositions may preferably be aqueous or non-aqueous solutions, suspensions, or emulsions. Water, propylene glycol, polyethylene glycol, vegetable oil, olive oil, injectable organic esters, such as ethyl oleate, or other suitable organic solvents may be used as solvents or vehicles (e.g., DMSO, DMA, and ethanol). These compositions may also contain auxiliary agents, particularly wetting agents, isotonic agents, emulsifiers, dispersants, and stabilizers. Sterilization may be carried out in several ways, for example, by sterile filtration, by incorporating a sterilizing agent into the composition, by irradiation, or by heating. They may also be prepared in the form of sterile solid compositions that can be dissolved at the time of use in a co-solvent, sterile water, or any other sterile injection medium.

[0037] The composition for rectal administration is a suppository or rectal capsule containing, in addition to the active product, an excipient such as cocoa butter, semi-synthetic glyceride, or polyethylene glycol.

[0038] Compositions for topical administration may be, for example, creams, lotions, mouthwashes, nasal sprays, aerosols, or patches.

[0039] The dosage depends on the desired effect, duration of treatment, and route of administration used, whether used as monotherapy or in combination with other therapies. Generally, the dosage for adults is 5–1000 mg orally per day, with single doses ranging from 20–450 mg of the active substance each time, which may be three times per day. Generally speaking, physicians and / or regulatory health authorities determine the appropriate dosage based on age and weight, as well as all other factors specific to the subject being treated, including the patient's medical condition or approved regulatory guidelines.

[0040] Tetrandrine (6,6',7,12-tetramethoxy-2,2'-dimethylverbam), or ES-3000, is currently under development by Escend Pharmaceuticals, Inc. (Escend). It may be useful in the treatment of relapsed or refractory AML, MDS, and PV when combined with ASTX727, a fixed-dose oral combination of decitabine (DEC) and sedazulidine, which was recently approved by the FDA for use in myelodysplasia. The combination of two oral agents, with an established safety profile and potential combinatorial effects, represents a novel and much-needed approach to the treatment of low, intermediate, and high-risk MDS or AML.

[0041] ASTX727 is the reference tablet and the investigational drug name for Iqovi [decitabine (35 mg) and sedazulidine (100 mg)] tablets. Various dosage combinations of ES-3000 can be administered with ASTX727. The administration schedule for ES-3000 can be once, twice, or three times daily, and can vary from approximately 1 to 14 days to approximately 1 to 7 days, which can be combined with standard doses of ASTX727 (Inqovi®) for approximately 1 to 5 days, approximately 1 to 4 days, approximately 1 to 3 days, and approximately 1 to 2 days. The dosage of ES-3000 may be 20-100 mg, 25-90 mg, 30-80 mg, 40-60 mg, and approximately 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, and 100 mg, as well as 105 mg, 110 mg, 115 mg, and 120 mg, once, twice, or three times daily. It can also be administered long-term for several weeks to several months.

[0042] Tetrandrin has several known mechanisms of action, including inhibition of calcium channels and activated large potassium channels, which are generally overexpressed in malignant tumors and have been shown to play a certain role in cancer and leukemia (Arcangeli 2010, Arcangeli 2012, Wang 1995, Huber 2013, Lang 2014, Dohner 2015). Tetrandrin has also been reported to reverse P-glycoprotein (P-gp)-mediated drug resistance (Zhu 2005, Jin 2005), and although tetrandrin itself is not a substrate of P-gp, it directly inhibits P-gp function in a dose-dependent manner without altering P-gp protein expression levels (Susa 2010). More recently, tetrandrin has been shown to inhibit the Wnt / β-catenin pathway, which has been identified as a novel target for AML (Simon 2005, He 2011, Xu 2012). Wnt / β-catenin is essential for the survival and self-renewal of leukemia stem cells in AML (Griffiths 2010, Wang 2010). Furthermore, tetrandrin has been demonstrated to competitively bind to calmodulin (CaM) and may be a novel CaM antagonist (Ma 2013). Calmodulin protein-dependent kinase (CaMKIIγ) is overexpressed in leukemia stem cells and blast cells and modulates the Wnt / β-catenin and STAT3 pathways (Si 2008).

[0043] The anticancer activity of TET has been demonstrated in vitro against human breast, colon, and hepatocellular carcinoma cell lines (IC50 range 1.5–10.4 μM), inhibiting drug efflux and increasing intracellular drug accumulation in MDR-overexpressing cancer cells. TET has also been shown to inhibit wnt / β-catenin signaling and tumor growth in human colorectal (HCT116) cells. In the same study, TET showed synergistic anticancer activity with 5-FU, reducing the migratory and invasive capabilities of HCT116 cells (He 2011). Screening of NCI60 cell lines demonstrated 50% growth inhibition (Log GI50) in the range of -5.0 to -6.6 (0.25–10 μM) in leukemia cell lines (Developmental Therapeutics Program, NIH / NCI). TET has been reported to have potent and specific activity in reversing P-glycoprotein-mediated drug resistance. In colorectal mouse xenografts, TET reduced tumor growth by inducing apoptosis (Wu 2010).

[0044] The published clinical data support the safety and efficacy of tetrandrine in cancer patients and provide another compelling rationale for the clinical evaluation of tetrandrine in leukemia. A Phase I dose-escalation trial was conducted by the National Cancer Institute (NCI) under IND 9613 (also known as 089613). Thirty-two patients with advanced solid tumors received IV doses of tetrandrine ranging from 50 to 875 mg / m2. The recommended dose for Phase II trials was 300 mg / m2 IV (repeated single dose once weekly); or 200 mg / m2 IV (repeated once daily for 5 days, every two weeks) (IND 9613 Tetrandrine Investigators Reports). Further supporting public reports include clinical trials in the United States using the trademarked CBT-1® TET (Oldham 1998, Oldham 2000, Kelly 2012) and two trials in China (Xu 2006, Liu 2012). The drug known as CBT-1 is the same formulation ingredient (NSC77037) used in the US Phase I trial (IND#089613). CBT-1 has been published as NSC-77037 (tetrandrine) by Fanelli (2016).

[0045] The chemical structure of tetrandrin is shown in Figure 1. Tetrandrin is the common name for NSC 77037 (d-tetrandrin, which has the chemical structure described as 6',7,12-tetramethoxy-2,2'-dimethyl-berbaman). Its IUPAC name is 9,20,21,25-tetramethoxy-15,30-dimethyl-7,23-dioxa-15,30-diazaheptacyclo[22.6.2.23,6.18,12.114,18.027,31.022,33]hexatriaconta-3,5,8(34),9,11,18,20,22(33),24(32),25,27(31),35-dodecaene, and its chemical formula C38H42N2O6 has a molecular weight of 622.76 and its CAS registry number is 518-34-3.

[0046] The inventions of this disclosure are, in part, based on the discovery that bisbenzylisoquinoline, tetrandrine, reduces the leukemia stem cell population, including those that have developed resistance to TKI treatment or other chemotherapy regimens. This discovery provides an anti-stem cell therapy protocol in which bisbenzylisoquinoline is administered to leukemia patients to reduce the leukemia stem cell population, followed by treatment with a TKI or chemotherapy regimen. If resistance to a TKI or chemotherapy regimen develops, treatment with the same or a different bisbenzylisoquinoline reduces the leukemia stem cell population, including leukemia stem cells resistant to the TKI and leukemia stem cells resistant to other chemotherapy regimens. Treatment with the same or a different TKI or other chemotherapy regimen can then be resumed. This cycle can be repeated as needed to improve hematological and cytogenetic responses.

[0047] The inventions of this disclosure are also, in part, based on the discovery that it is possible to treat leukemia patients who have developed resistance to TKIs or other chemotherapy regimens with bisbenzylisoquinoline to reduce the leukemia cell population and inhibit the development of such resistant leukemia stem cells. Such patients may be treated concurrently with TKIs or other chemotherapy regimens, or subsequently with TKIs or other chemotherapy regimens after the patient has shown a hematological or cytological response to leukemia. Repeated cycles of such treatments may be used to induce a persistent, progression-free response.

[0048] Other anti-leukemia agents may be administered to patients before, during, or after the administration of bisbenzylisoquinoline, TKIs, or other chemotherapy regimens. Such additional therapies include the use of immunomodulators such as SRC kinase inhibitors, aurora kinase inhibitors, interferon-alpha, conventional chemotherapy agents such as hydroxyurea, cytarabine (ara-C), daunorubicin, doxorubicin, hypomethylating agents, decitabine, 5-azacitidine, and venetoclax, and liposomal formulations of anthracyclines and fraudulent nucleosides such as CPX-351 (Vyxeos®).

[0049] Bisbenzylisoquinolines, such as tetrandrine, may be used in bone marrow decompression conditioning to reduce toxicity and improve outcomes of hematopoietic stem cell transplantation (HSCT) procedures aimed at curative treatment of leukemia patients. The use of bisbenzylisoquinolines in bone marrow decompression conditioning before HSCT can be performed before, after, and in addition to bone marrow decompression agents and procedures, including but not limited to cyclophosphamide, busulfan, fludarabine, melphalan, clofarabine, amsacrine, cytarabine, decitabine, sedazuridine, and radiation.

[0050] The present invention describes the use of bisbenzylisoquinoline, e.g., 6,6',7,12-tetramethoxy-2,2'-dimethyl-berbaman (tetrandrin), and derivatives, isomers, and analogs, modified forms, e.g., salt forms, or salts thereof, with pharmaceutically acceptable acids in the inhibition of molecular signaling pathways involved in the survival, self-renewal, and differentiation of cancer stem cells and leukemia stem cells, including but not limited to Alox5, Stat3, Wnt / beta-catenin, Msr2, Blk, Myc, Survivin, Cyclin D, Osteopontin, Tenascin C, L1CAM, and CaMKII.

[0051] The present invention describes the use of bisbenzylisoquinoline, e.g., 6,6',7,12-tetramethoxy-2,2'-dimethyl-berbaman (tetrandrin), as well as derivatives, isomers, and analogues, modified forms, e.g., salt forms, or salts thereof, with pharmaceutically acceptable acids in the inhibition of molecular protein targets involved in the survival of cancer and leukemia stem cells, including but not limited to lipoxygenase, Mcl-1, cyclin-D1, beta-catenin, Bcl-2, Bcl-xL NF-kappa B, CamKIIγ, pCaMKIIγ, and VEGF.

[0052] The inventions of this disclosure describe the use of bisbenzylisoquinoline, e.g., 6,6',7,12-tetramethoxy-2,2'-dimethyl-berbaman (tetrandrin), and derivatives, isomers, and analogues, modified forms, e.g., salt forms, or salts, of this compound, either alone with pharmaceutically acceptable acids or in combination with immunosuppressants including epoetin alfa + GCSF, azacitidine (Vildaza), decitibin (Dacogen), and lenalidomide, for immunosuppressive and anti-inflammatory activity that enables the treatment of MDS, MPN, CML, aCML, AML, PV, and related diseases. These agents utilize different mechanisms of action, and the clinical response to treatment varies among them, although serious adverse side effects may limit the therapeutic effect.

[0053] The inventions of this disclosure relate to the treatment of solid tumor cancer, MPN, CML, aCML, AML, PV, and related diseases with only pharmaceutically acceptable acids, or imatinib, dasatinib, nilotinib, bosutinib, ponatinib, AZD0530, NPB-001-05, AT9283, BAY43-9006, bafetinib, AP24534, restaurtinib, tozacertib, danucertib, XL228, KW-2449, AT This document describes the use of bisbenzylisoquinoline, e.g., 6,6',7,12-tetramethoxy-2,2'-dimethyl-berbaman (tetrandrin), as well as derivatives, isomers, and analogues, modified forms, e.g., salt forms, or salts of this compound, in combination with pharmaceutically acceptable acids, including tyrosine kinase inhibitors such as -9283, VE-465, DCC-2036, PKC412, PF-03814735, and batalanib. These drugs utilize different mechanisms of action, and the response to treatment varies among them, although serious adverse reactions exist that limit the therapeutic effect.

[0054] Solid tumors that may benefit from the treatment of bisbenzylisoquinoline, e.g., 6,6',7,12-tetramethoxy-2,2'-dimethyl-berbaman (tetrandrin), either alone or in combination with chemotherapeutic agents or targeted therapies, with pharmaceutically acceptable acids, as well as derivatives, isomers, and analogues, modified forms, e.g., salt forms, or salts of this compound include TNBC, other breast cancers, Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, pancreatic cancer, brain cancer, head and neck cancer, lung cancer, liver cancer, gastric cancer, and colorectal cancer, all of which have cancer stem cell enrichment populations with known surface antigens for identification.

[0055] As used herein, leukemia refers to CML, AML, ALL, CLL, and APL. Leukemia also includes preleukemic syndromes such as MDS, MPN, and PV.

[0056] As used herein, TKI refers to any thymidine kinase inhibitor. Examples of TKIs include, but are not limited to, imatinib mesylate (IM) and second-generation TKIs such as dasatinib (D), nilotinib (N), bosutinib (B), and befatinib.

[0057] Other chemotherapy regimens used herein include, but are not limited to, the 7+3 regimen (7 days of cytarabine plus the first 3 days of daunorubicin), liposomal formulations of cytarabine with daunorubicin, high-dose cytarabine administered for 5 days, and hypomethylating agents such as 5-azacitidine, sedazulidine, and decitabine.

[0058] As used herein, leukemia stem cells refer to pluripotent stem cells characterized by genetic transformation resulting in uncontrolled cell division. For example, in CML, the BCR-ABL fusion gene (Philadelphia chromosome). Leukemia stem cells may be defined by a surface antigen profile including CD34+CD38-CD47+CCL-1+CD96+TIM3+CD32+CD25+.

[0059] When used herein, a cytological response to treatment with bisbenzylisoquinoline and / or TKIs or other chemotherapy regimens is a response occurring in both peripheral blood and bone marrow. There are at least three cytological responses: (1) a cytogenetic response (CR), (2) a major cytogenetic response (MCR), and a complete cytogenetic response (CcyR). Determining such a response is based on measuring the number of peripheral blood cells and / or bone marrow cells that have markers associated with a particular leukemia. Such markers include cell surface antigens, abnormal proteins, and gene modifications. A cytogenetic response occurs when the number or percentage of cells with these markers decreases during treatment. In the case of CML, a major cytogenetic response occurs when the number or percentage of these cells decreases to less than 35%. A complete cytogenetic response occurs when no cells containing the markers are detected at all. For example, CML is characterized by Ph+ chromosomes. A cytogenetic response occurs if the number of Ph+ chromosomes decreases even slightly during treatment. A decrease in the percentage of Ph+ to 35% or less is considered a major cytogenetic response, while 0% Ph+ is a complete cytogenetic response.

[0060] As used herein, a hematological response occurs when there is a change in the patient's white blood cell count after treatment with bisbenzylisoquinoline and / or a TKI or other chemotherapy regimen. In CML, the change in white blood cell count may occur in both peripheral blood and bone marrow, although the change may be observed only in the peripheral blood. The response may be a partial decrease in white blood cell count or a complete decrease to normal levels (e.g., 10,000–12,000 cells / ml).

[0061] When used herein, the molecular response in CML occurs when BCR-ABL1 transcript levels decrease after treatment with bisbenzylisoquinoline and / or TKIs. Following quantitative real-time PCR (qRT-PCR) analysis, the BCR-ABL1 / control gene transcript ratio is determined using an international scale (IS) standardized baseline. The major molecular response (MMR) occurs when there is a decrease of 3log10 (≤0.10%IS) or greater in BCR-ABL1 transcript. Currently, there is no universal definition of the complete molecular response (CMR).

[0062] As used herein, minimal residual disease (MRD) is the name given to the small number of leukemia cells (bone marrow-derived cancer cells) that remain in a patient during treatment, or after treatment if the patient is in remission (no symptoms or signs of the disease). This is the main cause of relapse in cancer and leukemia. MRD is known to be enriched in leukemia stem cells. In CML, the presence of MRD is determined by the detection of t(9;22), and the detection of t(9;22) is considered the standard of care for all CML patients.

[0063] As used herein, the term bisbenzylisoquinoline includes all members of its chemical family, including alkaloid derivatives of the Menispermaceae family, Stephania tetrandra S.moore, and other related species.

[0064] The agents according to the present invention consist of bisbenzylisoquinoline, e.g., 6,6',7,12-tetramethoxy-2,2'-dimethyl-berbaman (tetrandrin), in its pure state or in the form of a composition with any other pharmaceutically compatible product that may be inactive or physiologically active, accompanied by a pharmaceutically acceptable acid, as well as derivatives, isomers, prodrugs, and analogs, modified forms, e.g., salt forms, or salts of this compound. The agents of the present invention may be used orally, parenterally, rectally, topically, or as a patch.

[0065] Bisbenzylisoquinoline formulations are suitable for oral, topical, or parenteral (including subcutaneous, intramuscular, and intravenous) administration. These formulations can conveniently be provided in unit dosage forms and can be prepared by conventional pharmaceutical techniques. Such techniques involve associating the active ingredient with a pharmaceutical carrier(s) or excipient(s). Generally, formulations are prepared by homogeneously and closely associating the active ingredient with a liquid carrier, a micronized solid carrier, an emulsion, or both, and then, if necessary, shaping the product.

[0066] Bisbenzylisoquinoline formulations suitable for oral administration may be presented as isolated units such as capsules, cachets, or tablets, each containing a predetermined amount of the active ingredient; as powders or granules; as solutions or suspensions in aqueous or non-aqueous liquids; as oil-in-water or water-in-oil emulsions; and as boluses. These can be formulated for immediate release, sustained release, or controlled retention.

[0067] Tablets may be prepared by compression or molding, along with one or more auxiliary components as desired. Compressed tablets may be prepared by compressing the active ingredient in a free-flowing form, such as a powder or granules, mixed with optionally a binder, lubricant, inert diluent, preservative, surfactant, or dispersant, in a suitable machine. Molded tablets may be prepared by molding a mixture of powder compounds moistened with an inert liquid diluent in a suitable machine. Tablets may optionally be coated or split, and may be formulated to provide sustained or controlled release of the active ingredient within them over a period of several minutes, several hours, or several days.

[0068] When an oral formulation is used, the oral bisbenzylisoquinoline dosage form is preferably administered to the subject in the range of 0.05 to 15.0 mg / kg. In a preferred embodiment, bisbenzylisoquinoline is administered to the subject in the range of 2 to 10.0 mg / kg. In a more preferred embodiment, bisbenzylisoquinoline is administered to the subject in the range of 6 to 10 mg / kg, or as 420 mg to 700 mg, either once daily or divided into multiple doses (increments).

[0069] Examples of bisbenzylisoquinoline preparations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions that may contain antioxidants, buffers, bacteriostatic agents, and solutes that make the preparation isotonic with the blood of the recipient, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickeners. The preparations may be presented in unit-dose or multi-dose containers, such as sealed ampoules and vials, and may be stored in a freeze-dried state requiring only the addition of a sterile liquid carrier such as sterile water for injection immediately before use. Immediate injection solutions and suspensions can be prepared from the aforementioned types of sterile powders, granules, and tablets. A unit parenteral dose may contain 280 to 1,050 mg, more preferably 420 to 700 mg, of bisbenzylisoquinoline per unit dose.

[0070] A preferred unit dose formulation of bisbenzylisoquinoline comprises the daily dose or unit, divided daily dose, or multidose use dose of the administered component as described herein, or a suitable fraction thereof.

[0071] In particular, in addition to the components mentioned above, the formulation may include other agents conventional in the art, taking into account the type of formulation in question. For example, those suitable for oral administration may include flavoring agents, sustained-release and controlled-release excipients, and coating and formulation techniques.

[0072] Furthermore, the disclosed inventions describe novel improvements, pharmaceutical ingredients, dosage forms, excipients, solvents, diluents, drug delivery systems, preservatives, toxicity monitoring and improvement, and techniques or agents for avoiding or reducing toxicity. The disclosed inventions also relate to the use of drug delivery systems, novel prodrugs, polymer conjugates, novel routes of administration, antibody conjugates, and other agents for enhancing the activity of a compound, inhibiting suboptimal cellular effects or repair of sublethal damage, or “transitioning” cells to more destructive cellular responses such as apoptosis. In some cases, the use of bisbenzylisoquinoline, e.g., 6,6',7,12-tetramethoxy-2,2'-dimethyl-berbaman (tetrandrin), and derivatives, isomers, and analogues, modified forms, e.g., salt forms, or salts thereof, in combination with pharmaceutically acceptable acids, such as antibodies, vaccines, cytokines, lymphokines, gene and antisense therapies, CAR-T, or other conventional chemotherapeutic agents or biological therapies, provides novel approaches and significant improvements to suboptimal therapeutic agents.

[0073] Bisbenzylisoquinoline, e.g., 6,6',7,12-tetramethoxy-2,2'-dimethyl-berbaman (tetrandrin), along with derivatives, isomers, and analogues, modified forms, e.g., salt forms, or salts thereof, with pharmaceutically acceptable acids, can be prepared synthetically, semi-synthetically, biochemically, or by extraction processes from plant materials containing the agent, including but not limited to Stephania tetrandra and Stephania abuta.

[0074] Pharmaceutically acceptable salts include salts with inorganic acids such as hydrochloride, sulfate, nitrate, and phosphate, or salts of organic acids such as acetate, propionate, succinate, oxalate, benzoate, fumarate, maleate, methanesulfonate, isethionate, sodium theophylline acetate, salicylate, phenolphthalate, and methylene-bis-β-hydroxynaphthoate, or substituted derivatives of these. These may also include citrate and malate forms.

[0075] The disclosed invention also relates to bisbenzylisoquinoline, e.g., 6,6',7,12-tetramethoxy-2,2'-dimethyl-berbaman (tetrandrin), in its pure state or in the form of a composition in combination with any other pharmaceutically compatible product that may be inactive or physiologically active, accompanied by a pharmaceutically acceptable acid, as well as to pharmaceutical compositions comprising derivatives, isomers, and analogs, prodrugs, modified forms, e.g., crystals, salts, or salts of this compound. The agents according to the inventions of the present disclosure may be used orally, parenterally, rectally, topically, or as a patch.

[0076] For oral administration, solid compositions may be used in the form of tablets, pills, powders (gelatin capsules, wafer capsules), medical foods, or granules. In these compositions, the active ingredient according to the disclosed invention is mixed with one or more inert diluents or excipients, such as starch, cellulose, sucrose, lactose, or silica, whether or not under an argon or nitrogen stream. These compositions may also contain substances other than diluents or excipients. For example, one or more lubricants, colorants, coatings, or varnishes, such as magnesium stearate or talc, may be used.

[0077] Liquid compositions for oral administration may include pharmaceutically acceptable solutions, suspensions, emulsions, syrups, or elixirs containing water, ethanol, glycerol, vegetable oil, or an inert diluent such as liquid paraffin. These compositions may also contain substances other than diluents, such as humectants, sweeteners, thickeners, flavorings, or stabilizing additives.

[0078] Parenterally administered sterile compositions may preferably be aqueous or non-aqueous solutions, suspensions, or emulsions. Water, propylene glycol, polyethylene glycol, vegetable oils, particularly olive oil, injectable organic esters, such as ethyl oleate, or other suitable organic solvents may be used as solvents or vehicles. These compositions may also contain auxiliary agents, particularly wetting agents, isotonic agents, emulsifiers, dispersants, and stabilizers.

[0079] Sterilization can be carried out in several ways, for example, by sterile filtration, by incorporating a sterilizing agent into the composition, by irradiation, or by heating. They may also be prepared in the form of sterile solid compositions that can be dissolved at the time of use in a cosolvent, sterile water, or any other sterile injection medium.

[0080] The composition for rectal administration is a suppository or rectal capsule containing, in addition to the active product, an excipient such as cocoa butter, semi-synthetic glyceride, or polyethylene glycol.

[0081] Compositions for topical administration may be, for example, creams, lotions, mouthwashes, nasal sprays, aerosols, or patches.

[0082] The dosage depends on the duration of treatment and the desired effect based on the route of administration used. These are generally 5 to 1000 mg per day orally for adults, with single or multiple daily doses ranging from 5 to 450 mg of the active substance per dose. The physician will determine the appropriate dosage based on all factors specific to the patient being treated, including age and weight, as well as indications, toxicity concerns, and guidance from regulatory authorities such as the FDA and EMA. [Examples]

[0083] Tetrandrin has inhibitory activity against CML stem cells and is highly effective in treating BCR-ABL-induced CML in mice. Figure 1 shows the structure of tetrandrin.

[0084] Example 1 This example demonstrates that tetrandrin reduces the development of circulating leukemia cells, decreases spleen weight, and reduces the development of LSCs in a mouse model of CML. The methods are as follows: Twenty C57BL / 6 mice were used as donors for bone marrow transduction. Four days before cell collection, they were primed with intravenous injection of 5-fluorouracil. Bone marrow cells were collected from the femur and tibia. Bone marrow cells were infected twice with retroviruses including MSCV-BCR-ABL-IRES-GFP. Sixty recipient mice were irradiated with two doses of lethal radiation at 550 cGy, followed by bone marrow transplantation by intravenous injection of 500,000 cells / mouse. Mice were treated one week after bone marrow transplantation. Fifteen mice in each group: +Placebo: 3 times a day, 2 times with water and 1 time with 0.1N HCl, 6 hours apart each time. + Imatinib: 100 mg / kg in water, twice daily, every 12 hours. +Tetranandrine: 150 mg / kg in 0.1N HCl, once daily +Imatinib +ES3000 combination: Three times a day - imatinib twice and tetrandrine once, 6 hours apart. Figures 7A and 7B show the number of leukemia cells in peripheral blood and leukemia stem cells, respectively, as well as the number of days after bone marrow transplantation in C57BL / 6 mice.

[0085] Example 2 This example demonstrates that tetrandrin reduces β-catenin expression in K562 (human myeloid leukemia) cells. Western blotting of β-catenin and β-actin protein levels in untreated and tetrandrin-treated K562 cell lines.

[0086] Antibodies for Western blotting analysis of β-catenin and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Protein lysates were prepared by lysing cells in RIPA buffer and immunoprecipitation.

[0087] Figure 3 shows the results of Example 2.

[0088] Example 3 This example demonstrates that tetrandrin removes BCR-ABL1+ secondary colonies in a colony-forming cell (CFC) assay of human CML myeloid cells.

[0089] Cloning progenitor cells were set up using human CML bone marrow-derived cells in the methylcellulose-based culture medium described above. The test substance was added to the medium to the final concentration described above. Standard cultures containing IMDM + 10% FBS and solvent control cultures containing DMSO were also started. After adding the test compound and cells, the tubes were vortexed to ensure that the compound and cells were evenly distributed throughout the matrix. The cultures were prepared at 0.1 × 10⁶ per culture for each control and each compound concentration. 6The cells were set up in a triple configuration (lot number BBM1000-E1110025705111110G4). The cultures were placed in a humidified incubator (37°C, 5% CO2). After 14-16 days of incubation, trained personnel evaluated and scored the colonies.

[0090] a. Replating: After counting erythrocytes and bone marrow progenitor cells, individual CFU-GM colonies were picked from the matrix and diluted in 20 μL of IMDM + 10% FBS. The colonies were dispersed into this medium by gentle pipetting. This cell mixture was then added to the wells of a 96-well plate containing 180 μL of ColonyGel medium as described above. Colonies were picked from the solvent control culture and treated with 10 μM tetrandrin in the original culture (both ICs). 50 Samples were also taken from 24 wells with the concentration closest to the value. The 96-well plate was then placed in a humidified incubator for 14 days, and each well was evaluated for secondary colony growth. Both the number of wells supporting clonal growth and the number of colonies per well were recorded.

[0091] Colony collection, RNA isolation, and cDNA conversion: After 14–16 days in the culture, individual colonies were picked from tetrandrin under solvent control and 10 μM test conditions and placed in 75 μL of buffer RLT (Qiagen). Secondary replated colonies were collected, washed with 2 mL of 1× PBS, frozen at -80°C, and then lysed in 75 μL of buffer RLT (Qiagen). RNA was isolated using the Rneasy Micro kit according to the manufacturer's instructions. Due to the low cell count in each colony, 20 ng of carrier RNA was included in each reaction to promote better RNA yield. After elution, the entire isolate was subjected to cDNA conversion using a high-volume cDNA conversion kit (Life Technologies).

[0092] Analysis of BRC-ABL transcripts using qPCR: Using cDNA derived from both primary and secondary replated colonies, qPCR assay was used to determine whether the colonies were positive for the bcr-abl transcript. The primers and probes were designed based on a publication from Luthra, et al., and were designed to anneal to bcr-abl (target) and abl (endogenous control) cDNA. Each target was assayed in a double-run cycle in a 20 μL reaction volume. The samples were denatured at 95°C for 10 minutes, then amplified for 40 rounds at 95°C for 15 seconds and 60°C for 60 seconds. Figure 5 shows the results of Example 3.

[0093] Example 4 This example demonstrates that tetrandrin improves the survival of mice with BCR-ABL-WT-induced CML.

[0094] The results of the experiments described in Examples 1-4 demonstrate the following: (a) Tetrandrin reduces the development of circulating leukemia cells in a mouse model of CML, (b) reduces spleen weight, and (c) reduces the development of leukemia stem cells; (d) Tetrandrin reduces β-catenin expression in K562 cells; and (e) Tetrandrin eliminates BCR-ABL1+ secondary colonies in a colony-forming cell (CFC) assay of human CML myeloid cells. [Brief explanation of the drawing]

[0095] [Figure 1] The chemical structure of ES-3000, also known as tetrandrin (TET), is shown. [Figure 2] The effect of TET treatment on K562 cells is shown graphically as cell viability. [Figure 3] PAGE shows that β-catenin protein is present as a bicot in all K562 cell extracts, including untreated cells. [Figure 4] PAGE is shown, revealing clear beta-actin bands observed in all extracts of K562 cells treated with 0-40 μM tetrandrin. [Figure 5] We present a plot showing that beta-catenin levels were significantly lower in extracts of K562 cells treated with >10 μM tetrandrin. [Figure 6] We present a plot showing that beta-actin levels in extracts of K562 cells treated with up to 40 μM tetrandrin were not significantly affected by drug treatment. [Figure 7A] The bar graph shows samples of circulating leukemia cells from recipient mice treated with tetrandrin or imatinib + tetrandrin. [Figure 7B] This bar graph shows the results of leukemia stem cell samples from recipient mice treated with tetrandrine or imatinib + tetrandrine on day 8 after bone marrow transplantation. [Figure 8] The graph shows electrophysiological measurements of CHO cells stably transfected with hERG to investigate the possibility that the test substance inhibits hERG. The post-compound current, expressed as a percentage of the pre-compound current, is plotted against the concentration for each compound.

[0096] Experimental evidence of the efficacy of ES-3000 in human leukemia Experiment I: Effect of tetrandrin on β-catenin in human leukemia (K562) cells

[0097] The ability of tetrandrin (TET) to reduce β-catenin expression in an in vitro assay was tested in human erythroleukemia (K562) cells. The first part of the study consisted of cell exposure to tetrandrin and preparation of cell lysates. The study was conducted by Applied Immunology, Menlo Park, CA.

[0098] In short, the method for the first part of the study was as follows: K562 cells were grown and maintained in culture medium (RPMI, 10% FBS, Pen Strep, and glutamic acid). The cells were then exposed to 0, 0.1, 1, 5, 10, 20, and 40 μM TET at 37°C for 24 hours. After drug exposure, cell viability was checked using trypan blue, followed by cell lysis. The supernatant was collected and protein concentration was tested using the Pierce BCA kit. Protein assay tubes were read at 562 nm using Spectramax, and Softmax(c) software was used for protein concentration analysis. 8 × 10⁶ per drug concentration. 6 Using individual K562 cells, the protein concentration in the cell lysate was determined to be approximately 1 mg / ml by BCA protein assay, as shown in Table 1. TIFF2026509524000001.tif78160

[0099] Experiment II: Effects of tetrandrin on beta-catenin and beta-actin levels in human myeloid leukemia K562 cell line

[0100] The second part involved quantifying β-catenin protein levels by Western blotting. The ability of TET to reduce β-catenin expression in an in vitro assay was tested in human erythroleukemia (K562) cells. The test was performed by Alamo Laboratories, San Antonia, TX.

[0101] In short, the method for quantifying β-catenin protein by Western blotting was as follows: Extracts from K562 cells (untreated and treated with various tetrandrin concentrations) were concentrated to 0.4 ml using a Centricon-3 column to obtain a protein concentration of approximately 2 mg / ml. The samples were mixed with 5 × Laemmli buffer and loaded onto an 8–16% Tris-glycine gradient gel. Polyacrylamide gel electrophoresis (PAGE) was then performed at 6°C for 100 minutes at 85 volts to transfer the protein onto a PVDF membrane at 8°C for 85 minutes at 70 volts. The membrane was blocked with 5% milk in Tris-buffered saline containing 0.1% TW-20 (TBST). The membrane was incubated with primary antibody at 4°C for 18 hours, followed by three washes with TBST for 5 minutes each, and incubated with secondary antibody in TBST at 25°C for 2 hours. The membrane was washed again with TBST as described above, incubated with ECL-2 substrate for 5 minutes, and scanned with Kodak Image Station. After scanning, the primary and secondary antibodies were removed from the blot and reprobed with anti-beta-actin antibody as described above. Protein bands in the WB images were quantified using the Molecular Imaging software pack MIS-4.0 software and compiled into an Excel spreadsheet.

[0102] The results, as shown in Table 2, demonstrated that tetrandrin dose-dependently reduced β-catenin levels in K562 cells. It is also important to note that β-catenin protein was present as a doublet in all K562 cell extracts, including untreated cells. This doublet may be due to proteolysis, post-translational modification, or mRNA splicing. A clear β-catenin band was observed in extracts of K562 cells treated with 0–10 μM tetrandrin (Figure 3). However, a conclusive β-catenin band was not present in K562 cells treated with 20 μM and 40 μM tetrandrin. Furthermore, a clear beta-actin band was observed in all extracts of K562 cells treated with 0–40 μM tetrandrin (Figure 4). Quantification of the protein bands in Figures 3 and 4 revealed that beta-catenin levels were significantly lower in K562 cell extracts treated with >10 μM tetrandrin (Figure 5), while beta-actin levels in K562 cell extracts treated with up to 40 μM tetrandrin were not significantly affected by drug treatment (Figure 6). The inventors conclude that TET reduces β-catenin expression in K562 cells.

[0103] The effect of TET treatment on the cell viability of K562 is shown in Table 2 and graphically in Figure 2. TIFF2026509524000002.tif67165

[0104] Experiment III: Effects of tetrandrin in cells derived from leukemia patients (in vitro) Study Title: In vitro evaluation of one test compound for human chronic myeloid leukemia using a colony-forming cell assay Test Report: END 01 Testing organization: Reach Bio, Seattle, WA

[0105] An in vitro study was conducted to determine whether TET is effective against human chronic myeloid leukemia (CML) stem-like cells. Colony-forming cell (CFC) assays were performed using bone marrow cells derived from newly diagnosed CML patients.

[0106] Human bone marrow low-density cells (Conversant Bio, Alabama) derived from the bone marrow of CML patients were stored in the gas phase of liquid nitrogen until needed. On the day of the experiment, the cells were rapidly thawed, and the contents of the vial were diluted in 10 mL of Iscove's modified Dulbecco medium containing 10% fetal bovine serum (IMDM + 10% FBS), and washed by centrifugation (approximately 1200 rpm, 10 minutes, room temperature). The supernatant was discarded, and the cell pellet was resuspended in a known volume of IMDM + 10% FBS. Cell count (3% glacial acetic acid) and viability (trypan blue exclusion test) were assessed by treatment with tetrandrin (10 μM) for 14 days. After treatment, primary and secondary colonies were grown and analyzed by qPCR to determine whether the cells were BCR-ABL or ABL only.

[0107] Human erythrocyte (CFU-E and BFU-E) and bone marrow (CFU-GM) colonies were counted by trained personnel. Furthermore, the distribution of colony types and general colony and cell morphology were analyzed. Differences in the number of colonies detected in isotyped cultures represented historical coefficients of variation for colony counting using these types of assays. The number and distribution of colonies detected in the solvent control (0.2% DMSO) were statistically no different from the standard control (without tetrandrin or imatinib compounds or DMSO). For statistical analysis, the number of colonies in compound-treated cultures was compared to the solvent control cultures. The potential effects of tetrandrin and CML progenitor cells are shown in Table 3. TIFF2026509524000003.tif125165

[0108] Interestingly, since the culture media used for culturing the progenitor cells did not contain erythropoietin, the erythrocyte colonies detected in these cultures exhibited Epo-independent clonal growth. ES-3000 had a significant effect on CFU-GM colonies at 30 μM and 10 μM concentrations, and simultaneously had the same effect on the erythrocyte lineage. Imatinib, an experimentally positive control, also had a significant effect on CFU-GM progenitor cells at 30 μM and 10 μM concentrations, but was more toxic to the erythrocyte lineage compared to ES-3000 (30-1 μM). IC 50 Based on the determined values, imatinib was found to be more toxic than ES-3000 (Table 4). TIFF2026509524000004.tif34165

[0109] Experiment IV: Replating assay These CML samples supported secondary clonal growth determined by replating assay (Table 3). In solvent-control cultures, 66.7% of wells supported secondary growth with one or more colonies and an average of 3 CFCs per well. In cultures started with colonies derived from the original culture containing 3 μM imatinib, 16.7% of wells supported secondary growth with one or more colonies and an average of 2 CFCs per well. In cultures started with colonies derived from the original culture containing 10 μM ES-3000, 54.2% of wells supported secondary growth with one or more colonies and an average of 3 CFCs per well.

[0110] Experiment V: qPCR assay of bcr-abl TaqMan®-based qPCR testing was used to determine whether colonies were normal (bcr-abl negative and abl positive) or diseased (bcr-abl and abl positive). The following scoring guidelines were followed to score colonies as positive or negative: All colonies were assayed in double series for each target. If a cytogram (Ct) was obtained in a single reaction, that reaction was scored 1. If no Ct was recorded, a score of 0 was given. From there, a logic test was used to score colonies into four different categories: bcr-abl and abl, abl only, bcr-abl only, or both negative. To be included in a category, a colony had to score 1 in both reactions. However, if a colony scored 1 in only one of the two reactions, that colony was excluded from the analysis. If a colony scored 1 for each reaction (both bcr-abl and abl), that colony was placed in the bcr-abl and abl category and indicated as a diseased colony. Alternatively, if a colony scored 1 for only both the BCR-ABL reaction, or 1 for only both ABL reactions, the colony was placed in the BCR-ABL only or ABL only category, respectively. If a colony scored 0 for both targets (BCR-ABL and ABL), the colony was placed in the negative category for both.

[0111] A total of 48 primary colonies and 31 secondary replated colonies were evaluated for bcr-abl and abl using qPCR. Three colonies were excluded from the analysis using the scoring logic described above. Thus, 20 out of 21 colonies (95.2%) were scored as positive for both bcr-abl and abl. After excluding 7 colonies treated with 10 μM tetrandrin from the analysis, 11 out of 17 colonies were positive for both bcr-abl and abl (64.7%), 1 out of 17 was positive only for abl (5.8%), 1 out of 17 was positive only for bcr-abl (5.8%), and 4 out of 17 colonies were negative for both targets (23.5%). Most secondary colonies treated under solvent control conditions were scored positive for both BCR-ABL and ABL (10 out of 14, 71.4%), one out of 14 (7.1%) was ABL-positive only, and one out of 14 (7.1%) was BCR-ABL-positive only. Two out of 14 colonies were scored negative for both targets. Thirteen secondary colonies treated with 10 μM tetrandrin were evaluated. However, most colonies were scored negative for both BCR-ABL and ABL (9 out of 10, 90%), suggesting that the transcript levels of the colonies were outside the detection level of the assay. Only one out of 10 colonies (10%) was scored positive for ABL only. No colonies were scored positive for both BCR-ABL and ABL. In addition, several secondary colonies treated with 3 μM imatinib were evaluated for BCR-ABL and ABL. Of the four colonies, only one was positive for both targets, while the remaining three colonies were negative for both. The results are shown in Tables 5 and 6. Table 5 shows that in the solvent control culture, 66.7% of the wells supported secondary growth with one or more colonies and an average of 3 CFCs per well. In the culture started with colonies derived from the original culture containing 3 μM imatinib, 16.7% of the wells supported secondary growth with one or more colonies and an average of 2 CFCs per well.In cultures started with colonies derived from the original culture containing 10 μM ES-3000, 54.2% of the wells supported secondary growth with one or more colonies and an average of three CFCs per well. TIFF2026509524000005.tif53165TIFF2026509524000006.tif64165

[0112] In conclusion, the test compound ES-3000, a semi-solid culture system supporting the proliferation of chronic myeloid progenitor cells, was evaluated and compared with imatinib for primary and secondary clonal proliferation. This patient sample supported Epo-independent proliferation (i.e., erythrocyte colonies were detected in the culture despite no Epo being added to the medium). Both ES-3000 and imatinib inhibited erythrocyte and myeloid colonies, but the IC50 value of imatinib was lower than that of ES-3000. Secondary cultures were evaluated by picking individual colonies from solvent control cultures and cultures supplemented with 3 μM imatinib and 10 μM ES-3000 (representing their respective IC50 values) and replating them in fresh medium. In solvent-controlled cultures, 66.7% of colonies supported secondary proliferation, compared to 16.7% in imatinib-treated cultures and 54.2% in ES-3000-treated cultures. Table 6 shows the results of qPCR assays used to detect bcr-abl transcripts in the picked colonies. 95% of colonies from the primary plating solvent-controlled culture were bcr-abl positive, compared to 65% of colonies treated with ES-3000. Analysis of colonies obtained from secondary replating revealed that most colonies treated with ES-3000 were below the detection limit, while the majority of solvent-controlled colonies remained bcr-abl positive.

[0113] Experiment VI: Effects of tetrandrin in a mouse model of leukemia Study Title: Inhibition of CML stem cells by alkaloids that reduce β-catenin Test Report: In Vivo Report UMass Examination administered by: University of Massachusetts Medical School (Dr. Li's Lab), Worchester, MA

[0114] The efficacy of tetrandrin was tested on CML stem cells in an in vivo model. The objective of this study was to determine the survival rates of leukemia cells and leukemia stem cells in response to tetrandrin treatment. Donor C57BL / 6(B6) mice were primed with intravenous 5-fluorouracil for 4 days, and then bone marrow cells were collected from the femur and tibia and subsequently transfected twice with a retrovirus containing MSCV-BCR-ABL-IRES-GFP. Recipient mice were irradiated with two doses of lethal radiation at 550 cGy, followed by 5x10⁻¹⁴ 5 Bone marrow transplantation was performed by intravenous injection of cells / mice. One week after transduction, blood from recipient mice was tested for disease induction by GFP FACS analysis (Figure 7A). All mice tested positive. Treatment was initiated on day 8 post-bone marrow transplantation (Figure 7B). Mice were randomized into four groups and treated orally with either vehicle [3 times / day, 2 times], imatinib [100 mg / kg, 2 times / day], tetrandrine [150 mg / kg, 1 time / day], or imatinib + tetrandrine [3 times / day: 2 times with imatinib, 1 time with tetrandrine].

[0115] The results of this trial showed that tetrandrine administered orally once daily was superior to imatinib administered twice daily in inhibiting the development of both circulating leukemia cells and leukemia stem cells, while the combination of tetrandrine and imatinib further improved the efficacy of tetrandrine.

[0116] Experiment VII: Effect of tetrandrin on CamKIIγ activity Study Title: ES-3000 Inhibition of CAMKII Gamma Test Report: CamKII Activity Report ES-3000 Study conducted by: Institute for Rare and Neglected Diseases Drug Discovery, Mountain View, CA

[0117] Enzyme assays were performed to evaluate the inhibition of CAMKII gamma kinase by ES-3000 (tetrandrin). CAMKII gamma kinase activity was measured according to the Promega protocol using ATP GLO purchased from Promega. CAMKII enzyme system (V3531) purchased from Promega. ADP Glo was used for detection. The assay was optimized for linearity between protein and time. Conditions for inhibitor screening: 25 ng of CAMKII, 20 minutes incubation, inhibitor or DMSO (5%), room temperature, total volume 20 μL. The inhibitor, ATP (10 or 50 μM), substrate and buffer were added to the tube and the reaction was initiated with the enzyme. Staurosporine was used as a positive control. The reaction was terminated at 20 minutes by adding the ADP Glo reactant according to the manufacturer's instructions. All reactions were performed in double series. The inhibition percentage (%) was calculated by dividing the average activity under each condition by the average value of the DMSO control and multiplying by 100.

[0118] The results of individual experiments using 10 μM or 50 μM ATP are shown in Tables 7 and 8, respectively. TIFF2026509524000007.tif86165TIFF2026509524000008.tif55163

[0119] The test results showed good signal-to-noise ratio. Staurosporine was effective as a positive control, and ES-3000 dose-dependently inhibited CAMKII gamma kinase activity.

[0120] Table 9 shows the inhibition of CamKIIγ activity in the presence of various concentrations of ES-3000 (tetrandrine), staurosporine, and vervamin, which demonstrates dose-dependent inhibition. TIFF2026509524000009.tif68165

[0121] Experiment VIII: Effects of TET on hERG Test Title: hERG Inhibitory Ability of Test Substance ES-3000 Test Report: CYP1328_R1 Testing organization: Cyprotex US, LLC, Watertown, MA

[0122] The objective of this study was to evaluate the ability of the test substance to inhibit hERG. The experiment was performed on an IonWorks® HT instrument (Molecular Devices Corporation) that automatically and simultaneously performs electrophysiological measurements of 48 single cells in a dedicated 384-well plate (PatchPlate). All cell suspensions, buffers, and test compound solutions were kept at room temperature during the experiment. The cells used were Chinese hamster ovary (CHO) cells (Cytomyx, cell line obtained from the UK) stably transfected with hERG. Single-cell suspensions were prepared in extracellular solution (Dulbeccio phosphate-buffered saline containing calcium and magnesium, pH 7-7.2), and aliquots were automatically added to each well of the PatchPlate®. The cells were then positioned over small holes at the bottom of each well by applying a vacuum directly beneath the plate to form an electroseal. Reduced pressure was applied through a single common compartment to all wells filled with intracellular solution (buffered to pH 7.2 with HEPES). The resistance of each seal is measured via a common ground electrode in the intracellular compartment and individual electrodes placed in each of the upper wells. Next, electrical access to the cells is achieved by circulating the perforating agent amphotericin under the PatchPlate and then measuring the pre-compound hERG current. The electrodes are placed in the extracellular compartment and a holding potential of -80mV is applied for 15 seconds. Next, the hERG channel is activated by adding a depolarization step to +40mV for 5 seconds, then fixing at -50mV for 4 seconds to draw out the hERG tail current, and then returning to -80mV over 0.3 seconds. Next, the test compounds are automatically added to the upper wells of the PatchPlate® from a 96-well microtiter plate containing each compound within a certain concentration range. Solutions are prepared by diluting the DMSO solution of the test compounds extracellularly (final DMSO concentration 0.25%). After the test compounds are in contact with the cells for 300 seconds, the current is recorded using the same voltage-step protocol as in the pre-compound scan. Quinidine, an established hERG inhibitor, is included as a positive control, and a buffer containing 0.25% DMSO is included as a negative control.If the results for all compounds on the plate are rejected and the IC50 values ​​for quinidine or the negative control are outside the quality control limits, the experiment is repeated. Each concentration is tested in four replicate wells on PatchPlate®. However, only cells with a seal resistance greater than 50 mOhm and a compound pre-current of at least 0.1 nA are used to evaluate hERG blockade. Subsequently, the compound post-current is expressed as a percentage of the compound pre-current and plotted against the concentration for each compound (Figure 8). The tests were performed by Cyprotex US, LLC, Watertown, MA. The experimental conditions (Table 10) and results (Table 11) are shown below. TIFF2026509524000010.tif21165TIFF2026509524000011.tif25165

[0123] Observations and conclusions from experimental tests The present invention relates to novel compositions and methods for improving the usefulness of biotherapeutic agents, including bisbenzylisoquinoline, e.g., 6,6',7,12-tetramethoxy-2,2'-dimethyl-berbaman (tetrandrin) and derivatives, for patients with cancer, infectious diseases, and immune disorders, with suboptimal performance. The present invention relates to improved pharmaceutical ingredients, dosage forms, excipients, solvents, diluents, drug delivery systems, preservatives, more accurate drug administration, improved dose determination and scheduling, toxicity monitoring and improvement, techniques or agents for avoiding or reducing toxicity, techniques and tools for identifying / predicting patients who may benefit from improved outcomes with therapeutic agents by using diagnostic kits or phenotypic or genotypic identification via the use of pharmacokinetic or metabolic monitoring approaches, and novel developments of drug delivery systems, novel prodrugs, polymer conjugates, novel routes of administration, and other drug uses to enhance the activity of compounds, inhibit suboptimal cellular effects or repair of sublethal damage, or "transition" cells into a more destructive cellular phase such as apoptosis. In some cases, embodiments of the present invention include the use of these suboptimal therapeutic agents in combination with radiotherapy or other conventional chemotherapeutic agents or biological therapeutic agents, such as antibodies, vaccines, cytokines, lymphokines, gene and antisense therapies.

[0124] As used herein, “suboptimal” means “not meeting expectations,” and the terms “suboptimal therapy” and “suboptimal therapeutic agent” include agents whose Phase I toxicity excluded further human clinical evaluation, agents from Phase II trials in which limited or non-significant tumor responses were identified, agents from Phase III clinical trials whose outcomes were not medically or statistically significant enough to justify submission to or approval by regulatory authorities for commercialization, or marketed agents with a response rate of less than 25% as monotherapy or whose side effects are so severe as to limit their broad benefit. Furthermore, this includes allergic reactions, adverse or fatal side effects, and organ damage from suboptimal therapeutic agents. Suboptimal active agents include, but are not limited to, bisbenzylisoquinoline, e.g., 6,6',7,12-tetramethoxy-2,2'-dimethyl-berbaman (tetrandrin), and its derivatives, Avastin® (bevacizumab), Rituxan® (rituximab), Neavar® (sorafenib), dasatinib, imatinib, nilotinib, Provenge® (ciplucel-T), Tarceva® (erlotinib), and Iressa® (gefitinib). More specifically, the methods and compositions of the present invention also focus on improving bisbenzylisoquinoline, e.g., 6,6',7,12-tetramethoxy-2,2'-dimethyl-berbaman (tetrandrin) and its derivatives.

[0125] Tetrandrin, or TET, has been shown to possess multiple pharmacological activities associated with anticancer function. These include inhibition of Wnt / β-catenin, reduction of MDR1 expression and inhibition of P-gp function, activation of apoptosis, and suppression of proliferation via various activation and survival signaling pathways. The inventors further investigated some of the pharmacological properties most relevant to the use of TET for leukemia. The inventors' tests demonstrated that TET reduces β-catenin in human (K562) leukemia cells, reduces bcr-able-positive primary colonies, and eliminates bcr-able-positive secondary colonies in a colony-forming cell assay. In a mouse model of leukemia, TET reduced the development of leukemia cells in the peripheral blood of mice and leukemia stem cells in the bone marrow. In an in vitro enzyme assay, TET dose-dependently inhibited the activation of CaMKIIγ.

[0126] In an in vitro evaluation of TET's ability to inhibit hERG, 5.2 μM IC5 was observed. 50 This became clear, and this is based on previous human clinical experience. max Regarding cardiac conduction-related toxicity using this method, it is 13 times safer (Oldham 1998, Oldham 2000). QT changes are related to IC 50 Although it may be observed at concentrations below 249 ng / mL, a reasonable interpretation is that because tetrandrin has a high protein-binding potential, QT prolongation in the target population can be expected at plasma levels above 249 ng / mL.

[0127] Furthermore, the Wnt / β-catenin pathway has been identified as a novel target in AML (Wang 2010), and ES-3000 has been shown to reduce β-catenin. More recent studies have shown that TET can competitively bind to calmodulin (CaM) and may be a novel CaM antagonist (Ma 2013). Calmodulin protein-dependent kinase (CaMKIIγ) is overexpressed in leukemia stem cells and blast cells and modulates the Wnt / β-catenin and STAT3 pathways (Si 2008). Assessing patient β-catenin and CaMKIIγ levels to determine the correlation between response and expression levels may provide valuable information in selecting patients who would most benefit from ES-3000 treatment.

[0128] The diverse mechanisms and safety profiles of tetrandrin offer opportunities for its development as a treatment for relapsed or refractory myeloid leukemia and related hematological disorders.

[0129] TIFF2026509524000012.tif140165TIFF2026509524000013.tif184165

[0130] References Arcangeli A, Becchetti A, New trends in cancer therapy: targeting ion channels and transporters.Pharmaceuticals,2010.3:1202-1224. Arcangeli A, Pillozzi S, Becchetti A, Targeting ion channels in leukemias: a new challenge for treatment. Curr Med Chem, 2012.19(5):683-96. Dohner H,Weisdorf DJ,Bloomfield CD,Acute Myeloid Leukemia.The New England Journal of Medicine,2015.373(12):1136-1152. Fanelli M,Hattinger CM,Vella S,Tayanti E,Michelacci F,Gudeman B,Barnett D,Picci P,Serra M,Targeting ABCB1 and ABCC1 with their specific inhibitor CBT-1 can overcome drug resistance in osteosarcoma.2016,Curr Canc Drug Targets16:261-274. Gralla EJ,Coleman GL,Jonas AM,Toxicology studies with d-Tetrandrine(NSC-77037),a plant alkaloid with vascular and lymphotoxic effects in dogs and monkeys.Cancer Chemotherapy Report Part 3,1974.5:79-84. Griffiths EA,Gore SD,Hooker CM,et al.,Acute myeloid leukemia is characterized by Wnt pathway inhibitor promoter hypermethylation.Leuk&Lymph,2010.51(9):1711-1719. He BC,Gao J-L,Zhang B-Q,et al.Tetrandrine inhibits Wnt / β-catenin signalling and suppresses tumor growth of human colorectal cancer.Mol Pharmacol,2011.79(2):211-9. Huang Y-T,Hong C-Y,Tetrandrine.Cardiovascular Drug Reviews,1998.16(1):1-15. Huber SM,Oncochannels.Cell Calcium,2013.53(4):241-55. Jin J,Wang FP,Wei H and Liu G.Reversal of multidrug resistance of cancer through inhibition of P-glycoprotein by 5-bromoTetrandrine.Cancer Chemotherapy and Pharmacology.2005.55:179-188. Ju A,Kang Y,et al.Pharmacokinetics,tissue distribution and excretion study of tetrandrine in rats.Journal of Chinese Pharmaceutical Sciences.2015;24(8). Kelly RJ,Robey RW,Chen CC,Draper D,Luchenko V,Barnett D,Oldham RK,Caluag Z,Frye AR,Steinberg SM,Fojo T,Bates SE,A pharmacodynamic study of the P-glycoprotein antagonist CBT-1 in combination with paclitaxel in solid tumors.2012,The Oncologist.17:512. Lang F,Stoumaras C,Ion channels in cancer:future perspectives and clinical potential.Phil Trans R Soc B,2014.369:20130108. Liu W,Zhang J,Ying C,et al.Tetrandrine combined with gemcitabine and cisplatin for patients with advanced non-small cell lung cancer improve efficacy.Int J Biomed Sci,2012.8(1):28-35. Ma L,Wang Z,Liu S,et al.Screening calmodulin-binding ligands using intensity-fading matrix-assisted laser desorption / ionization mass spectrometry.Rapid Commun Mass Spectrom,2013.27:p.1527-1534. National Cancer Institute,Annual Progress Report for IND#089613,1976. Ng LT,Chiang L-C,Lin Y-T,et al.Antiproliferative and apoptotic effects of Tetrandrine on different human hepatoma cell lines.Am J Chin Med,2006.34(1):125-35. Oldham RK,Reid WK,Preisler HD,Barnett D.A phase I and pharmacokinetic study of CBT-1 as a multidrug resistance modulator in the treatment of patients with advanced cancer.Cancer Biother Radiopharm.1998;13:71-80. Oldham RK,Reid WK,Barnett D,Phase I study of CBT-1 and Taxol in patients with Taxol resistant cancers.2000,Cancer Biother Radiopharm.15(2):153-159. Si J,Collins SJ,Activated Ca2+ / Calmodulin-dependent protein kinase IIγ is a critical regulator of myeloid leukemia cell proliferation.Cancer Res,2008.68(10):3733-3742. Simon M,Grandage VL,Linch DC and Khwaja A.Constitutive activation of the Wnt / β-catenin signalling pathway in acute myeloid leukaemia.Oncogene 2005.24:2410-2420. Susa M,Choy E,Yang C,Schwab J,Mankin H,Hornicek F,Duan Z.2010.Multidrug resistance reversal agent,NSC77037,identified with a cell-based screening assay.J Biomol Screen.15,287-296. Wang G,Lemos JR,Tetrandrine:a new ligand to block voltage-dependent Ca2+ and Ca2+-activated K+ channels.Life Sci,1995.56(5):295-306. Wang Y,Krivtsov A,Sinha A, The wnt / β-catenin pathway is required for the development of leukemia stem cells in AML.Science,2010.Mar 26;327(5973):1650-1653. Wu J-M,Chen Y,Chen J-C,Lin T-Y,Tseng S-H,Tetrandrine induces apoptosis and growth suppression of colon cancer cells in mice.2010,Cancer Letters.287:187-195. Wu K,Zhou M,Wu Q-X,Yuan S-X,Wang D-X,Jin J-L,Huang J,Yang J-Q,Sun W-J,Wan L-H,He B-C,The role of IGFBP-5 in mediating the antiproliferation effects of Tetrandrine in human colon cancer cells.2015,Int.J Oncology.46:1203-1213. Xu W-L,Shen H-L,Ao Z-F,Combination of Tetrandrine as a potential-reversing agent with daunorubicin,etoposide and cytarabine for the treatment of refractory and relapsed acute myelogenous leukemia.Leuk Res,2006.30:407-413. Xu XH,Gan YC,Xu GB,Chen T,Zhou H,Tang JF,Gu Y,Xu F,Xie,YY,Zhao XY,Xu RZ,Tetrandrine citrate eliminates imatinib-resistant chronic myeloid leukemia cells in vitro and in vivo by inhibiting Bcr-Abl / β-catenin axis.J Zhejiang Univ-Sci B(Biomed & Biotechnol),2012 13(11):867-874. Zheijiang Jinhua Conba Bio-Pharma,Co.Ltd.,(Jinhua,China),Tetrandrine Tablet Package Insert 2014. Zhu X,Sui M,Fan W,In vitro and in vivo characterizations of Tetrandrine on the reversal of p-glycoprotein-mediated drug resistance to paclitaxel.2005,Anticancer Research.25:1953-1962

Claims

1. A method for treating preneoplastic and neoplastic diseases and solid tumors in subjects requiring such treatment, comprising administering to the subject a compound of formula I (tetrandrine) having the following structural formula: a. In the formula, R １ and R １ ’ are the same or different short-chain carbon-based ligands including, but not limited to, CH ３ , CO ２ CH ３ , or H; R ２ is CH ３ or C ２ H ５ , and R ３ is CH ３ or hydrogen, and has an "S" isomer configuration at the C-1' chiral carbon position. b. The disease is selected from the group consisting of myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), leukemia, and solid tumors. c. The effective dose is 0.01 to 1000 mg / m². ２ The method, including the method described above.

2. The method according to claim 1, wherein the disease is selected from the group consisting of myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML), atypical chronic myeloid leukemia (aCML), acute myeloid leukemia (AML), and polycythemia vera (PV).

3. The aforementioned patient, a. Before treating with tyrosine kinase inhibitors, other chemotherapy regimens, or immunotherapy drugs, b. After treatment with the tyrosine kinase inhibitor, or other chemotherapy regimens, or immunotherapy agents, c. Tetrandolin is administered concurrently with the tyrosine kinase inhibitor, other chemotherapy regimens, or immunotherapy. d. The method according to claim 2, wherein the leukemia is intrinsically or exogenously resistant and has minimal residual disease after treatment with a tyrosine kinase inhibitor or other chemotherapy regimen, or is refractory to or resistant to a tyrosine kinase inhibitor or other chemotherapy regimen.

4. The aforementioned disease, a. Treatment by inhibiting or killing cancer stem cells or leukemia stem cells. b. Treatment by inhibiting molecular pathways selected from the group consisting of Alox5, Stat3, Wnt / beta-catenin, Msr2, Blk, Myc, Survivin, Cyclin D, Osteopontin, Tenascin C, L1CAM, and CaMKII, and c. The method according to claim 1, wherein the patient is affected by at least one of the treatments involving inhibition of a molecular protein target involved in the survival of cancer and leukemia stem cells, selected from the group consisting of lipoxygenase, Mcl-1, cyclin-D1, beta-catenin, Bcl-2, Bcl-xL NF-kappa B, CamKIIγ, pCaMKIIγ, and VEGF.

5. The method according to claim 3, further comprising administering an additional anti-leukemic agent(s), the additional anti-leukemic agent(s)(s) being selected from existing approved treatments for leukemia, MDS, or MPN, the additional anti-leukemic agent(s) being selected from one or more of the following: tyrosine kinase inhibitors, SRC kinase inhibitors, JAK2 kinase inhibitors, aurora kinase inhibitors, interferon alpha, hydroxyurea, anthracycline, cytarabine, ara-C, daunorubicin, or doxorubicin, and combinations of two or more anti-leukemic agents.

6. The above method of tetrandrin is a. Oral administration, b. Parenteral administration (infusion IV), and / or c. The method according to claim 1, wherein the drug is administered by at least one of the following methods: subcutaneous administration.

7. The frequency of tetrandrin administration is, a. After injection, it lasts for several hours to several days. b. Once every two weeks, c. Dose escalation based on patient tolerance. d. Selected and intermittent boost dose administration; e. Dosage by bolus dose, single dose, multiple daily doses, and multiple daily doses, f. The method according to claim 1, wherein the release of the dose is at least one of immediate release, sustained release, and controlled release.

8. The method according to claim 1, wherein the bisbenzylisoquinoline is administered once daily.

9. The method according to claim 1, wherein the bisbenzylisoquinoline is administered for 7 to 14 days or more.

10. The bisbenzylisoquinoline was administered to the patient at a dose of approximately 0.01 mg / m². ２ ~1000 mg / m² ２ The method according to claim 1, administered in an amount.

11. The method according to claim 1, wherein the administration is twice a day for seven days or more.

12. The dose of bisbenzylisoquinoline is approximately 0.01 mg / m². ２ ~1000 mg / m² ２ The method according to claim 10.

13. The method according to claim 1, wherein the bisbenzylisoquinoline is administered for 7 to 14 days or more.

14. The dose of bisbenzylisoquinoline is approximately 0.01 mg / m². ２ ~1000 mg / m² ２ The method according to claim 11.

15. A method of administering to patients who require bone marrow depletion conditioning before hematopoietic stem cell transplantation (HSCT), a. One or more bone marrow depletion conditioning agents, i. Cyclophosphamide, ii. Busulfan, iii. radiation, iv. Fludarabine, v. Melphalan, vi. Clopharabine, vii. Amsaklin, viiii. Cytarabine, ix. Decitabine, x. Sedazuridine The method comprising administering bisbenzylisoquinoline to the patient before administering a treatment including the above.

16. A method for treating subjects having cancer stem cell-associated preneoplastic and neoplastic diseases such as myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), chronic myeloid leukemia (CML), atypical chronic myeloid leukemia (aCML), acute myeloid leukemia (AML), polycythemia vera (PV), and lymphoma, non-leukemic solid tumors, the method comprising the step of administering to the subject in need of such treatment an effective amount of bisbenzylisoquinoline, for example 6,6',7,12-tetramethoxy-2,2'-dimethyl-berbaman (tetrandrin) and derivatives, isomers, and analogs of the compound, as well as prodrugs, esters, modified forms, such as crystals, salts, or salts, together with a pharmaceutically acceptable acid or in combination with other agents.

17. The method according to claim 15, wherein tetrandrin, or derivatives, isomers, and / or analogs, modified forms, such as crystals, salts, or salt forms of the compound, in combination with a pharmaceutically acceptable acid or a first other agent, is administered together with a pharmaceutically acceptable acid, a carrier, and / or in a pharmaceutically compatible product, or in combination with a second other agent.

18. Other drugs mentioned above include alemtuzumab, amvochlorin (chlorambucil), amvochlorin (chlorambucil), arzera (ofatumumab), bendamustine hydrochloride, campus (alemtuzumab), chlorambucil, fludarabine (fludarabine phosphate), fludarabine phosphate, gazyva (obinutuzumab), ibrutinib, imbruvica (ibrutinib), leukeran (chlorambusin), lymphoridine (chlorambucil) Sil), obinutuzumab, azacitidine, decitabine, Incobi (sedazulidine and decitabine), sedazulidine, lenalidomide, doxorubicin hydrochloride, arsenic trioxide, daunorubicin hydrochloride, cyclophosphamide, cytarabine, Cytosal-U (cytarabine), Cytoxan (cyclophosphamide), JAK2 inhibitors (e.g., fedratinib, duclabacitinib, ritrecitinib), Keytruda (pembrolizumab) ), Neosal (cyclophosphamide), Opdivo (nivolumab), Rubidomycin (daunorubicin hydrochloride), Tarabine PFS (cytarabine), Trisenox (arsenic trioxide), Vincasal PFS (vincristine sulfate), vincristine sulfate, Bosulif (bosutinib), bosutinib, busulfan, busulfex (busulfan), Clafen (cyclophosphamide), dasatinib, Gleevec (imatinib mesylate) The method according to claim 17, comprising: ), Iclusig (ponatinib hydrochloride), imatinib mesylate, Myrelan (busulfan), nilotinib, omacetaxin mepesuccinate, ponatinib hydrochloride, Sprycel (dasatinib), Synribo (omacetaxin mepesuccinate), tarabine PFS (cytarabine), tasigna (nilotinib), bortezomib (Velcade), panobinostat, CAR-TCR therapy, and related agents.

19. The method and composition according to claim 1, wherein the compound exists as a powder, tablet, capsule, liquid, patch, or medical food(s).

20. The method according to claim 1, wherein the administration schedule of bisbenzylisoquinoline, e.g., 6,6',7,12-tetramethoxy-2,2'-dimethyl-berbaman (tetrandrin) and derivatives, isomers, and analogs, modified forms, e.g., salt forms, or salts of the same compound, in combination with a pharmaceutically acceptable acid or other agent, comprises: once daily; once weekly; once weekly for three weeks; once every two weeks; once every two weeks for three weeks (with a rest period of one to two weeks); intermittent boost dose administration; once daily for several weeks; and / or immediate-release, sustained-release, or controlled-release oral administration.

21. A method for treating a subject having chronic BCR-ABL-induced myeloid leukemia characterized by oncogene-mediated beta-catenin elevation, comprising administering a therapeutically effective dose of a beta-catenin inhibitor to the subject and administering a therapeutically effective dose of a BCR-ABL tyrosine kinase inhibitor to the subject, wherein leukemia stem cells are inhibited.

22. The method according to claim 21, wherein the beta-catenin inhibitor is bisbenzylisoquinoline, for example 6,6',7,12-tetramethoxy-2,2'-dimethyl-berbaman (tetrandrin), and derivatives, isomers, and analogs, modified forms, for example, salt forms, or salts thereof, administered in combination with a pharmaceutically acceptable acid or other agent.

23. The method according to claim 21, wherein the BCR-ABL kinase inhibitor is selected from the group consisting of imatinib, dasatinib, nilotinib, bosutinib, ponatinib, restaurtinib, tozacertib, danucertib, batalanib, nexavar (sorafenib tosylate), midostaurin, AZD0530 (salacatinib), NPB-001-05 (imatinib-resistant Bcr-Abl inhibitor), AT9283 (aurora kinase inhibitor), bafetinib, XL228 (multitarget protein kinase inhibitor), KW-2449 (multi-kinase inhibitor), AT-9283 (multitarget protein kinase inhibitor), VE-465 (aurora kinase inhibitor), DCC-2036 (levacitinib), and PF-03814735 (aurora kinase inhibitor).

24. The method according to claim 21, further comprising administering a therapeutically effective dose of a beta-catenin inhibitor.

25. The method according to claim 21, further comprising administering a therapeutically effective dose of an Hsp90 inhibitor.

26. The method according to claim 21, further comprising administering a therapeutically effective dose of an anticancer agent selected from the group consisting of protein synthesis inhibitors, metabolites, alkylating agents, steroids, interferon alpha 2b, and any combination of two or more of these.

27. The method according to claim 21, wherein the subject is resistant to the anticancer effects of a therapeutic agent selected from the group consisting of imatinib, dasatinib, and nilotinib.

28. The method according to claim 21, further comprising administering a therapeutically effective dose of JAK2.

29. The method according to claim 28, which is not limited to, but includes, fedratinib, duclabacitinib, and ritrecitinib as the JAK2 inhibitor.

30. The method according to claim 21, further comprising administering a therapeutically effective dose of an FLT-3 inhibitor.

31. The method according to claim 30, which includes, but is not limited to, sorafenib, midostaurin, and restauritinib, gilteritinib, xartinib, and clenolanib as FLT-3 inhibitors.

32. R1 and R' are CH ３ CO ２ CH ３ Examples include, but are not limited to, H, and R, which are the same or different short-chain carbon ligands. ２ CH ３ or C ２ H ５ And R ３ CH ３ Alternatively, it is hydrogen, having an "S" isomer configuration at the C-1' chiral carbon position, and the disease is selected from the group consisting of myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), leukemia, and solid tumors, and the effective dose is 0.01 mg / m² ２ ~1000 mg / m² ２ The method according to claim 1, including the method described in claim 1.

33. R １ and R １ ', or R ２ The method according to claim 1, wherein is an aliphatic, aromatic, heterocyclic, halogenated amino, sulfhydryl, carboxylic acid, ketone, ester, carboxamide, ester salt, glycopeptide protein, nucleic acid, or antisense antibody substituent.

34. R １ and R １ 'or R ２ The method according to claim 1, wherein the drug is in the form of a prodrug, ester, or salt.

35. The method according to claim 5, wherein the additional anti-leukemia agent is combined in a single dosage form.

36. The method according to claim 16, wherein the aforementioned drugs are combined in a single dosage form.

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