WO2013026373A1

WO2013026373A1 - Use of berbamine for treating chronic myeloid leukemia

Info

Publication number: WO2013026373A1
Application number: PCT/CN2012/080297
Authority: WO
Inventors: 徐荣臻; 谢福文; 赖洪喜; 荣风光
Original assignee: 杭州本生药业有限公司
Priority date: 2011-08-19
Filing date: 2012-08-17
Publication date: 2013-02-28

Abstract

The present invention relates to uses of berbamine or a pharmaceutically acceptable salt thereof as a pharmaceutical composition to treat chronic myeloid leukemia, in particular to the Imatinib tolerant chronic myeloid leukemia.

Description

Use of berbamine in the treatment of chronic myeloid leukemia

The present invention relates to the treatment of chronic myeloid leukemia, and in particular to the use of berberine or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of chronic myeloid leukemia.

Background technique

About 20% of chronic myeloid leukemia (CML), which accounts for all adult leukemia, is characterized by the presence of the Abelson TK gene (Abl) on chromosome 9 and the break point cluster region on chromosome 22. (Bcr) The Philadelphia (Ph) chromosome produced by juxtaposition. For this disease, the usual treatment is bone marrow transplantation, but this method has serious side effects and often leads to the loss of treatment opportunities due to the lack of a suitable bone marrow donor. Traditional drug therapy is often treated with alpha interferon, but it also has many serious adverse effects.

It was later discovered that the oncogenic Bcr-Abl tyrosine kinase produced in Ph ⁺ CML patients is critical for the growth of CML tumor cells, and thus Bcr-AW kinase becomes! A compelling target for 3⁄4+ CML case treatment. Imatinib (IM) is a Bcr-Abl tyrosine kinase inhibitor (TKI). In addition to being effective in accelerated and dangerous phases, imatinib can also be used in chronic phase patients with ineffective interferon alpha therapy. . Imatinib can interfere with both the signaling pathways of tumor cell growth and specific abnormal chromosomes, such as the characteristic Bcr-AbL in CML.

After 2000, the first-generation tyrosine kinase inhibitor (TKI) represented by imatinib brought a breakthrough in the treatment of CML. A large-scale prospective, randomized clinical trial (IRIS study) confirmed that imatinib 400 mg/d has excellent efficacy as a first-line option for the initial CML chronic phase (CP): an 8-year overall survival (OS) rate of 85 is expected %, If only CML-related deaths are calculated, the 8-year OS rate can reach 93%. The 8-year event-free survival (EFS) rate and the progression-free survival (PFS) rate for the accelerated (AP)/quick period (BP) are expected to be 81% and 92%. Survival is much higher than interferon ± ^ ₁ A sugar glycosides historical controls. At 8 years, 83% of 553 patients achieved complete cytogenetic response (CCR), and 86% of the 98 consecutive patients who underwent molecular monitoring received primary molecular response (MMR). Obtaining CCR or MMR at 1 year of treatment indicates long-term disease stability. Moreover, there were few cases of late recurrence or termination of medication due to adverse reactions in the first 3 years. However, there are still a small number of patients with unsatisfactory efficacy, loss of response, progression to AP/BP, or discontinuation of imatinib due to adverse effects (Deininger M, O'Brien SG, Guilhot F, et al. 2009) .

In addition, inhibition of CML in the chronic phase with Abl tyrosine kinase inhibitor (TKI) such as imatinib (IM) is highly effective but not effective in curing the disease. This is mainly due to the inability of these kinase inhibitors to kill leukemia stem cells that cause CML pathogenesis, drug resistance and relapse.

'J, guanamine (BBM) is a structurally unique bisbenzylisoquinoline isolated from the traditional Chinese medicine Bigber 檗 (Berberis is is). The literature reports that berbamine has anti-inflammatory, anti-tumor, elevated white blood cells, anti-tuberculosis, blood pressure lowering, anti-myocardial hypoxia-ischemia, anti-arrhythmia and so on.

However, the use of small guanamine drugs approved for clinical use is mainly used to raise white blood cells, as an auxiliary drug for anti-tumor therapy, rather than a direct anti-tumor drug. As a drug for raising leukocyte drugs, the clinical dose is 120 mg/time, 3 times a day, orally (2 mg/kg based on 60 kg body weight).

There is still a need in the art for efficient and safe anti-leukemia drugs, particularly for the treatment of chronic myeloid leukemia.

Summary of the invention

The inventors have found that high doses of berbamine are capable of killing leukemia stem cells and killing imatinib-resistant leukemia stem cells.

Accordingly, in a first aspect, the present invention relates to the use of berbamine or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of leukemia at a dose of from 5 mg/kg/day to 200 mg/kg/day. In one embodiment of the use of the invention, the leukemia is chronic myeloid leukemia. In one embodiment of the use of the invention, the dosage is from 20 mg/kg/day to 80 mg/kg/day.

In a further embodiment of the use of the invention, the dosage is from 30 mg/kg/day to 60 mg/kg/day.

In a further embodiment of the use of the invention, the dosage is from 40 mg/kg/day to 50 mg/kg/day.

In one embodiment of the use of the invention, the medicament is formulated for oral administration.

In a second aspect, the invention relates to the use of Berberine or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of imatinib-resistant chronic myeloid leukemia.

In one embodiment of the use of the invention, the medicament is administered at a dose of from 30 mg/kg/day to 100 mg/kg/day.

In a further embodiment of the use of the invention, the medicament is administered at a dose of from 36 mg/kg/day to 90 mg/kg/day.

In a further embodiment of the use of the invention, the medicament is administered at a dose of from 45 mg/kg/day to 60 mg/kg/day.

In one embodiment of the use of the invention, the medicament may be formulated for oral administration.

In a third aspect, the present invention relates to a daily dosage independent unit for treating leukemia, comprising a small guanamine or a pharmaceutically acceptable salt thereof in the form of a pharmaceutical composition in an amount sufficient to provide a body weight of 5 per kg patient A daily dose of 200 mg.

In one embodiment of the daily dose independent unit of the invention, the amount of the small guanamine or a pharmaceutically acceptable salt thereof is sufficient to provide a daily dose of from 20 to 80 mg per kg of patient body weight.

In a further embodiment of the daily dose independent unit of the present invention, the berbamine or its The amount of the pharmaceutically acceptable salt is sufficient to provide a daily dose of 30 - 60 mg per kg of patient body weight. In a further embodiment of the daily dosage unit of the invention, the amount of the small amide or a pharmaceutically acceptable salt thereof is sufficient to provide a daily dose of 40 to 50 mg per kg of patient body weight.

In one embodiment of the daily dose independent unit of the invention, the daily dose independent unit is for the treatment of imatinib-resistant chronic myeloid leukemia.

In a further embodiment of the daily dose-independent unit of the invention, the amount of the small amide or a pharmaceutically acceptable salt thereof is sufficient to provide a daily dose of from 30 to 100 mg per kg of patient body weight.

In a further embodiment of the daily dosage unit of the invention, the amount of the small amide or a pharmaceutically acceptable salt thereof is sufficient to provide a daily dose of from 36 to 90 mg per kg of patient body weight.

In a further embodiment of the daily dosage unit of the invention, the amount of the small amide or a pharmaceutically acceptable salt thereof is sufficient to provide a daily dose of 45 to 60 mg per kg of patient body weight.

In a further embodiment of the daily dose-independent unit of the invention, the daily dose-independent unit contains one or more pharmaceutical tablets of berbamine or a pharmaceutically acceptable salt thereof.

In a further embodiment of the daily dose independent unit of the invention, the individual units are separate packages or separate sub-packages.

In a fourth aspect, the invention relates to a method of treating leukemia in a patient comprising administering to said patient d, guanamine or a pharmaceutically acceptable salt thereof at a dose of from 5 mg/kg/day to 200 mg/kg/day.

In one embodiment of the method of the invention, the leukemia is chronic myeloid leukemia. In one embodiment of the method of the invention, the dosage is from 20 mg/kg/day to 80 mg/kg/day.

In a further embodiment of the method of the invention, the dosage is from 30 mg/kg/day to 60 mg/kg/day.

In a further embodiment of the method of the invention, the dosage is from 40 mg/kg/day to 50 mg/kg/day. In one embodiment of the method of the invention, the administration is oral.

In a fifth aspect, the present invention relates to a method of treating imatinib-resistant chronic myeloid leukemia in a patient comprising administering to the patient berberine or a pharmaceutically acceptable salt thereof.

In one embodiment of the method of the invention, the dose administered is from 30 mg/kg/day to 100 mg/kg/day.

In a further embodiment of the method of the invention, the dose administered is from 36 mg/kg/day to 90 mg/kg/day.

In a further embodiment of the method of the invention, the dose administered is from 45 mg/kg/day to 60 mg/kg/day.

In one embodiment of the method of the invention, the administration is oral.

In a sixth aspect, the present invention relates to berbamine or a pharmaceutically acceptable salt thereof for use in a method of treating leukemia in a patient, the method comprising administering a dose of from 5 mg/kg/day to 200 mg/kg/day The patient is a guanamine or a pharmaceutically acceptable salt thereof.

In one embodiment of the berbamine or a pharmaceutically acceptable salt thereof of the present invention, the leukemia is 'ft myeloid leukemia.

In one embodiment of the berbamine or a pharmaceutically acceptable salt thereof of the present invention, the dosage is from 20 mg/kg/day to 80 mg/kg/day.

In a further embodiment of the berbamine or a pharmaceutically acceptable salt thereof of the present invention, the dosage is from 30 mg/kg/day to 60 mg/kg/day.

In a further embodiment of the berbamine or a pharmaceutically acceptable salt thereof of the present invention, the dosage is from 40 mg/kg/day to 50 mg/kg/day.

In one embodiment of the berbamine or a pharmaceutically acceptable salt thereof of the present invention, the administration is oral.

In a seventh aspect, the present invention relates to a small indamine or a pharmaceutically acceptable salt thereof for use in a method of treating imatinib-resistant chronic myeloid leukemia in a patient, the method comprising administering The patient is a guanamine or a pharmaceutically acceptable salt thereof. In one embodiment of the berbamine or a pharmaceutically acceptable salt thereof of the present invention, the dose is from 30 mg/kg/day to 100 mg/kg/day. In a further embodiment of the berbamine or a pharmaceutically acceptable salt thereof of the present invention, the dose is from 36 mg/kg/day to 90 mg/kg/day. In a further embodiment of the berbamine or a pharmaceutically acceptable salt thereof of the present invention, the dosage is from 45 mg/kg/day to 60 mg/kg/day. In one embodiment of the berbamine or a pharmaceutically acceptable salt thereof of the present invention, the administration is oral.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the in vivo anti-leukemia activity of a single dose of berbamine. (A) Effect of BBM on established CML xenograft growth. (B) Effect of BBM on the body weight of tumor-bearing mice. Figure 2 shows the pathological observation of CML xenografts. (A) Excised typical CML xenografts of tumor-bearing mice and control untreated mice 45 days after oral BBM (150 mg/kg; 10 days per day). (B) Effect of BBM on tumor structure and cell proliferation, tumors were excised and sectioned from BBM-treated and untreated mice. Tissues were stained with H&E to detect histomorphology or immunostained with anti-Ki-67 antibody to detect cell proliferation. Sections were counterstained with eosin to detect nuclei. Arrows indicate Ki-67 positive cells. Scale bar, 15 μηι. Figure 3 shows the in vivo antitumor activity of the third dose regimen. (A) Effect of the tri-dosing regimen on established CML xenograft growth. (Β) The effect of ΒΒΜ on the body weight of tumor-bearing mice. Figure 4 shows the in vivo antitumor activity of the sputum-dose regimen against sputum-resistant CML. (Α) The effect of ΒΒΜ on the growth of established ΙΜ-resistant CML xenografts. (Β) The effect of ΒΒΜ on the body weight of tumor-bearing mice. Figure 5 shows the in vivo antitumor activity of the third dose regimen. (A) ΙΜ confrontation-resistant CML The effect of xenograft growth. (B) Effect of IM on the body weight of tumor-bearing mice. detailed description

We used a rat-type leukemia xenograft model to study the efficacy of berbamine in the treatment of chronic myeloid leukemia. As a result, it has been unexpectedly found that administration of a dose of 5 times or more of the dose of the conventional berberine can be used as a monotherapy for leukemia, especially chronic myeloid leukemia. More surprisingly, the treatment of leukemia with berbamine achieved even better therapeutic effects than imatinib and was effective against imatinib-resistant chronic myeloid leukemia.

In the rat-derived leukemia xenograft model, the 100 mg/kg sputum dose of the melamine treatment regimen achieved a therapeutic effect of 70% (14/20) of the remission of chronic myeloid leukemia xenografts within 10 days. The 300 mg/kg berberine treatment regimen achieved a therapeutic effect of 83% (5/6) of imatinib-resistant chronic myeloid leukemia xenograft regression within 10 days.

Refer to "Pharmacological Experimental Methodology" (Third Edition, Xu Shuyun, etc., People's Health Publishing House), Chapter 6 Pharmacological Experimental Design and Statistical Analysis, page 203, Table 6-9, Dose Conversion Coefficients for Different Animals, Mice and Humans The amount used is approximately 10:1.

Berylamine can be purchased on the market.

As used herein, the term "pharmaceutically acceptable salts of the compounds of formula (I)" is exemplified by the formation of pharmaceutically acceptable anions (eg, sulfonate, mesylate, malate, acetate, citrate). , malonate, tartarate, succinate, benzoate, resistance An organic acid addition salt formed from an organic acid of blood acidate, α-ketoglutarate, and α-glycerophosphate. Suitable inorganic salts can also be formed, including but not limited to hydrochlorides, sulfates, nitrates, bicarbonates and carbonates, phosphates, hydrobromides, hydroxamates, and the like.

Pharmaceutically acceptable salts can be obtained using standard procedures well known in the art, for example, by reacting a sufficient amount of a basic compound with a suitable acid that provides a pharmaceutically acceptable anion.

As used herein, the term "polymorph" refers to the solid crystalline form of a compound of the invention or a complex thereof. Different polymorphs of the same compound may exhibit different physical, chemical and/or spectral properties. Different physical properties include, but are not limited to, stability (e.g., to heat or light), compressibility and density (important for formulation and product production), and dissolution rate (which can affect bioavailability). Differences in stability can result in chemical reactivity (eg, differential oxidation, such that when formulated from one polymorph, fades faster than when formed from another polymorph) or mechanical properties (eg, as storage) Changes in the kinetically favorable polymorphic tablet granules converted to thermodynamically more stable polymorphs) or both (for example, tablets of one polymorph are more susceptible to breakage at high humidity) . The different physical properties of polymorphs can affect their processing. For example, one polymorph may be more likely to form a solvate than the other or may be more difficult to filter or wash away from the shield due to, for example, differences in the shape or size distribution of its particles.

As used herein, the term "hydrate" refers to a compound of the invention or a salt thereof, which further comprises a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces.

The guanamine of the compound of the present invention has the stereochemistry shown by the structural formula of Formula I. The definitions and conventions of stereochemistry used herein generally follow MCGRAW-HILL DICTIONARY OF CHEMICAL TERMS (SP Parker, Ed,, McGraw-Hill Book Company, New York, 1984); and ELIEL, E. and WILEN, S., STEREOCHEMISTRY OF ORGANIC COMPOUNDS (John Wiley & Sons, Inc., New York, 1994).

The invention also provides a pharmaceutical composition comprising a compound of formula I of the invention. The invention provides a pharmaceutical composition comprising at least one of the compounds of formula I of the invention as described above, and optionally a pharmaceutically acceptable excipient.

Methods of preparing various pharmaceutical compositions containing a certain amount of active ingredient are known,

REMINGTON'S PHARMACEUTICAL SCIENCES, Martin, E. W., ed" Mack Publishing Company, 19th ed. (1995), a method of preparing the pharmaceutical composition comprising incorporating a suitable pharmaceutical excipient, carrier, diluent, and the like.

The pharmaceutical preparation of the present invention is produced by a known method, including a conventional mixing, dissolving or lyophilizing method.

The compounds of the invention may be formulated into pharmaceutical compositions and administered to the patient in a variety of ways suitable for the chosen mode of administration, e.g., orally or parenterally (by intravenous, intramuscular, topical or subcutaneous routes).

Thus, the compounds of the invention may be administered systemically, e.g., sputum, in combination with a pharmaceutically acceptable carrier such as an inert diluent or an assimilable edible carrier. They can be enclosed in hard or soft shell gelatin capsules and can be compressed into tablets. For oral therapeutic administration, the active compound may be combined with one or more excipients and in the form of swallowable tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. Form use. Such compositions and preparations should contain at least 1% of active compound. The ratio of such compositions and formulations may of course vary and may range from about 1% to about 99% by weight of a given unit dosage form. In such therapeutically useful compositions, the amount of active compound is such that an effective dosage level can be obtained.

Tablets, lozenges, pills, capsules, and the like may also contain: a binder such as tragacanth, acacia, corn starch or gelatin; an excipient such as dicalcium phosphate; a disintegrant such as corn starch , potato starch, alginic acid, etc.; a lubricant such as stearic acid; and a sweetener such as sucrose, fructose, lactose or aspartame; or a flavoring agent such as mint, wintergreen or cherry. When the unit dosage form is a capsule, it may contain a liquid carrier such as vegetable oil or polyethylene glycol in addition to the above types of materials. Various other materials may be present, as a coating, or otherwise modified The physical form of the solid unit dosage form. For example, tablets, pills or capsules may be coated with gelatin, wax, shellac or sugar. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methylparaben or propylparaben as a preservative, a dye and a flavoring such as cherry or orange flavor. Of course, any material used to prepare any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. Furthermore, the active compound can be incorporated into sustained release formulations and sustained release devices.

The active compound can also be administered intravenously or intraperitoneally by infusion or injection. An aqueous solution of the active compound or a salt thereof may be prepared, optionally mixed with a non-toxic surfactant. Dispersants in glycerin, liquid polyethylene glycols, triacetin and mixtures thereof, and oils can also be prepared. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent microbial growth.

The pharmaceutical dosage form suitable for injection or infusion may comprise a sterile aqueous solution or dispersion of the active ingredient (optionally encapsulated in a liposome) comprising a ready-to-use preparation suitable for sterile injectable or infusible solutions or dispersions. Or sterile powder. In all cases, the final dosage form must be sterile, liquid, and stable under the conditions of manufacture and storage. The liquid carrier can be a solvent or liquid dispersion medium including, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), vegetable oil, non-toxic glycerides, and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by liposome formation, by maintaining the desired particle size in the case of dispersing agents, or by the use of surfactants. Microbial action can be prevented by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.). In many cases, it will be preferable to include isotonic agents, such as sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use of compositions which delay the absorbent (e.g., aluminum monostearate and gelatin).

Sterile injectable solutions are prepared by combining the required active compound in a suitable solvent with the various other ingredients listed above, followed by filter sterilization. In the case of a sterile powder for the preparation of a sterile injectable solution, the preferred method of preparation is vacuum drying and Freeze-drying techniques which produce a powder of the active ingredient plus any additional ingredients present in the previously sterile filtration solution. Useful solid carriers include powdered solids (e.g., talc, clay, microcrystalline cellulose, silica, alumina, etc.). Useful liquid carriers include water, ethanol or ethylene glycol or a water-ethanol/ethylene glycol mixture, and the compounds of the present invention may be dissolved or dispersed in an effective amount, optionally with the aid of a non-toxic surfactant. Adjuvants (such as fragrances) and additional antimicrobial agents can be added to optimize the properties for a given use.

Thickeners (such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified inorganic materials) can also be used with liquid carriers to form coatable pastes, gels, ointments. , soap, etc., used directly on the user's skin.

The above preparations may be presented in unit dosage form, which is a unit dispersion unit containing a unit dosage unit suitable for administration to humans and other mammalian bodies. The unit dosage form can be a capsule or tablet, or a lot of capsules or tablets. The pharmaceutical preparation of the present invention may also be prepared in the form of a daily dosage unit, which may be in the form of a separate package of individual daily dosage units, or may be a single package comprising a plurality of daily dosage units, for example, A plurality of daily dose independent units for one session or a plurality of daily dose independent units for multiple courses are included. For example, a single package may include 1 to 10 daily dose independent units, 1 20 daily dose independent units, 1, 30 daily dose independent units, 1 - 40 daily dose independent units, 1 - 50 daily doses Independent unit or more daily dose independent units, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 , 30, 35, 40, 45, 50 or more daily dose independent units. The daily dosage unit may be formulated as a single dose, such as a tablet for single oral administration, a buccal tablet, a tablet, a capsule, an elixir, a suspension, a syrup, a wafer, etc. Form, or in the form of an injection solution or suspension for a single injection or infusion. The daily dose independent unit may also be formulated in a multi-dose form, such as being divided into corresponding multi-dose according to the number of times of daily administration. For example, it may be in the form of 2-6 doses, 2-5 dose forms, 2-4 dose forms, 2-3 dose forms, 3-5 dose forms or 3-4 dose forms. Each of the multiple dose forms of a single daily dose of discrete units may be equal or unequal, for example, one or more of the multiple doses may be higher than the other dose or doses, or multiple doses of a single daily dose of separate units Each dose in the form may be incrementally increasing or decreasing.

Recently, we have demonstrated that berbamine and its T-bio can kill IM-resistant CML cells in vitro, but do not kill normal hematopoietic cells, suggesting that berbamine may have the potential to target and differentiate CML LSC. And our results indicate that berbamine can directly target and eliminate CML SC in vivo.

To identify protein targets that regulate the basic functions of LSC, we used BBM to link to the agarose affinity matrix and used as a molecular probe. SDS-PAGE analysis revealed that two proteins of approximately 62 and 54 kDa were captured by the berbamine probe. These two proteins were identified by mass spectrometry (MS) as CaMKII gamma isoforms 1 (62 kDa) and 3 (54 kDa), respectively, and independently confirmed by Western blotting. GST pull-out experiments demonstrated that berbamine binds directly to CaMKII gamma protein.

To further investigate whether berbamine specifically binds to CaMKII gamma protein in cells, we transiently overexpress EGFP-tagged CaMKII γΐ in 293 Τ cells. Confocal fluorescence microscopy analysis showed that berbamine was colocalized with EGFP-CaM II γΐ in a specific manner. To assess the effect of berbamine on kinase activity in tumor cells, we examined the protein levels of phosphorylated CaMKII y and total CaMKIl Y in CML cells treated with berbamine and found that berbamine inhibited CaMKII in a dose-dependent manner. Phosphorylation of γΐ. In addition, analysis of CaMKII y kinase activity indicated that berbamine inhibited CaM II-γ activity in a dose-dependent manner with an IC50 value of about 1.0 μM. Collectively, these results show that berbamine interacts directly with CaMKII gamma protein and inhibits phosphorylation of this kinase in CML cells.

To gain insight into the mechanism by which CaMKIIy activity was inhibited, we constructed a model of CaMKIIy complexed with calmodulin by using Modeller based on X-ray crystals of the CaMKII delta/oping protein complex. BBM molecule is located in ATP of CaMKIIy kinase Combined inside the bag. Consistent with this finding, we observed that the inhibitory effect of BBM on CaMKIIy activity was overcome by increasing ATP concentration. These results indicate that BBM may be an ATP competitive inhibitor of CaMKIIy kinase.

Since our findings have demonstrated that CaMKIiy is a target for berbamine, we hypothesized that CaMKIIy may play an important role in LSC. We therefore analyzed the activation status of CaMKIIy in CD34+ CML stem cells sorted by FACS from CML patients and sorted normal hematopoietic stem/progenitor cells from cord blood samples. Western blot analysis indicated that both total CaMKIIyl protein and phosphorylated CaMKIIyl protein were highly expressed in CD34 ⁺ CML LSC, but were weakly expressed or undetectable in CD34-CML cells, CD34+ HSC and normal hematopoietic cells. Furthermore, RT-PCR analysis revealed a significant increase in CaMKIIy transcripts in CML tumor cells at the time of primitive cell crisis compared to normal hematopoietic cells. Taken together, these findings indicate that CAMKIIyl is highly activated in CML stem cells but not highly activated in normal hematopoietic stem cells, which means that the kinase may be a CML LSC-specific marker. These results guide us to test whether CaMKIIyl is required for survival and self-renewal of CML LSC. We next evaluated the effect of CaMKIIyl expression on the growth of leukemic clonogenic cells. The results show that down-regulation of CaMKIIyl expression significantly inhibits the growth of leukemic clonal cells in vitro, suggesting that CaMKIIy may affect the carcinogenic potential of CML cells. To test this concept, we established a stable CML cell line with high CaMKIIyl expression using human CMK K562 cells transfected with EGFP-tagged CaMKIIyl expression shield. We observed that overexpression of CaMKIIyl resulted in an increase in the number of colonies compared to the control (139 ± 3.61 / well versus 67 ± 2.65 / well). Notably, colonies induced by CaMKIIyl overexpression were larger in size than the control colonies. Consistent with this, overexpression of CaMKIIyl also significantly amplifies the number of mitotic cells in leukemia colonies. These results indicate that CaMKIIyl can enhance the colony forming ability and proliferative capacity of leukemia cells by promoting the survival and self-renewal of leukemia stem cells. In fact, the total number and percentage of CD34+ leukemia stem cells in the CML cell population with CaMKIIyl overexpression was significantly increased compared to the control cell population.

To further confirm these observed phenomena, an equal amount of CaMKIIyl overexpressed white Hematological cells and control leukemia cells were transplanted into mice to determine if overexpression of CaMKIIyl affects LSC survival and self-renewal in vivo. As expected, the size of leukemia xenografts with CaMKIIyl overexpression was much greater at 30 days than the control leukemia xenografts, suggesting that overexpression of CaMKIIyl can drive tumor growth in nude mice. In addition, the number of morphologically undifferentiated stem cell-like mother cells was significantly increased in tumors with CaM IIyl overexpression compared to controls (33% vs. 1.0%). In summary, these results demonstrate that overexpression of CaMKIIyl does promote CML LSC. Survive and self-renewal.

To elucidate the underlying mechanisms that contribute to the CaMKIIy-mediated LSC survival and self-renewal, we investigated whether CaMKIIy affects the protein levels of β-catenin, which is a major regulator of CML LSC survival and self-renewal, in CML cells. . We observed that the protein shield level of β-catenin was significantly reduced after treatment of CML cells with the CaMKIIy inhibitor BBM, while high levels of β-catenin were detected in CML CD34 ⁺ LSCs with high expression of CaMKIIyl. Similarly, overexpression of CaMKIIyl dramatically increased the protein level of β-catenin with an increase in phosphorylated GSIG-β protein. These results indicate that CaMKIIy can act as an upstream regulator of the GSK3p/p_catenin signaling axis of CML LSC. To obtain biochemical evidence supporting this theory, an immunoprecipitation experiment was performed. Leukemia cell lysates were incubated with CaMKIIy antibody or GSK3P antibody, and then the immune complexes were purified, separated by SDS-PAGE, and subjected to Western blot analysis using GSK3P and β-catenin antibodies. We found that both GSK3P and β-catenin proteins are present in protein complexes immunoprecipitated by CaMKIIy antibodies. As expected > CaMKIIyl protein is also present in the cross-immunoprecipitated complex using the GSK3p antibody. Most importantly, we found that the substrate phosphorylation motif of CaMKII gamma and the sequence of Ser21/9 comprising GSK-3α/β and the substrate of CaMKII β that have been shown to phosphorylate GSK-3ct/p in Ser21/9 The acidification motif is identical to the sequence of Ser21/9 comprising GSK-3a/p. These results indicate that the new CaM IlY/GSK3p/p-catenin signaling axis regulates LSC survival and self-renewal in CML cells.

Recently, CaM I y has been shown to be directly phosphorylated and activated in myeloid leukemia cells. STAT3. Furthermore, STAT3 is critical for the survival and self-renewal of cancer stem cells from several tumors. Consistent with previous reports, we confirmed that overexpression of CaMKIIyl increased the level of phosphorylated STAT3 protein in CML cells. Similarly, we also observed that phospho-STAT3 protein levels were significantly reduced after treatment of K562 leukemia cells with the CaMKIIy inhibitor BBM. To reveal whether STAT3 is involved in this new CaMKIlY/GSK3p/p-catenin axis, we performed co-immunoprecipitation with STAT3 antibody and then used Western blot analysis using CaMKII _y , GSK3 and β-catenin antibodies. We observed a STAT3-precipitate complex containing CaMKIIy, but no STAT3-precipitate complex containing GS 3p and β-catenin was observed, suggesting that CaMKIIy and STAT3 can form independent signal axes, which are associated with this CaMKIIy/GSK3p/p - The catenin signal axis synergistically promotes survival and self-renewal of CML LSCs.

In our findings, we present a working model of CaMKEyl-mediated promotion of leukemia stem cell survival and self-renewal: constitutive activation of leukemia stem cells recruits GSK3-p/p-linked proteins for inhibition Sexual phosphorylation and subsequent proteasomal degradation inactivate GSK. When the molecule shuts down GSK3P in a leukemia cell, it does not block β-linked protein. This in turn causes β-catenin to act as a promoter of STAT3 to drive LSC survival and self-renewal.

Overall, we have confirmed that CaMKIIy is a target for the anti-leukemia activity of the natural Chinese medicine product BBM. Our findings suggest that CaMKiry may be a novel leukemia stem cell-specific marker and a CaMKII _y /GSK-3/p-catenin that regulates CML LSC survival and self-renewal signaling pathways and CaMK]^/STAT3 signaling pathways The important molecular switch. Confirmation of CaMKIIy kinase as a target for BBM illustrates the reason for the strong anti-LSC activity of berbamine. These data also show that targeting CaMKII gamma kinase may be a new strategy for the treatment of chronic myeloid leukemia.

We conducted a further study on the effect of berbamine on myeloid leukemia using a rat-type leukemia xenograft model. It was found that high dose d and guanamine can be used as a monotherapy for white. Blood diseases, especially chronic myelogenous leukemia, and the treatment of leukemia with berbamine achieve even better therapeutic effects than imatinib, and are effective against imatinib-resistant chronic myeloid leukemia. In the following examples, the invention will be explained more specifically. However, it is to be understood that the following examples are intended to illustrate the invention and not to limit the scope of the invention.

Example 1:

(1) Experimental materials

Experimental animals: The rat was purchased from the Shanghai Animal Center of the Chinese Academy of Sciences.

Reagents: Berberine hydrochloride was purchased from Chengdu Xiaolong Natural Pharmaceutical Life Technology Co., Ltd.

Instrument: C02 Cell Incubator (Model.' BB16UY/BB5060UV)

K562 cells: From the Institute of Cancer Research, Zhejiang University.

(2) Experimental method

Seven-week-old rats were subcutaneously inoculated with human chronic myeloid leukemia cell K562 at a dose of 5 X 10 ⁷ cells per mouse. After 24 hours, they were randomly divided into groups of 21 each. Medication group: The berbamine group was administered orally, at a dose of 100 mg per kilogram of body weight, once a day (administered). Control group: Replace with saline for 10 days. A total of 30 days were observed, during which the tumor weight, body weight, activity and diet were measured. At the end of the experiment, the mice were sacrificed and tumor tissues were taken and the actual weight was measured.

(3) Experimental results

The experimental results are shown in Table 1.

Table 1 Berylamine once a day, 100mg/kg each time, combined with 10 days of treatment regimen anti-leukemia effect

(Results on the 30th day)

Tumor weight (: gram) ■ J, mouse weight (g) Mouse number control group, berbamine group, control group, berbamine group

1 0.208 0 17.68 17.395

2 0.249 0 19.02 16.52

3 0.723 0 15.85 18.32

4 0.444 0.574 14.99 18

5 1.535 0 17.95 19.29

6 0.954 0 16.553 21.75

7 1.342 0 15.1 21.31

8 1.023 0.194 16.59 18.62

9 0.522 0 17.68 19.44

10 0.14 0 19.28 17.32

11 1.591 0.27 14.934 14.76

12 0.427 0.164 18.82 18.03

13 2.1 0 18.171 17.454

14 0.649 0.822 16.028 11.793

15 0.283 0 16.406 12.365

16 0.718 0.787 16.871 11.46

17 0.887 0 15.288 12.033

18 0.827 0 15.974 18.482

19 L674 0 13.405 15.115

20 1.445 0 14.192 19.609

21 2.354 14.164 ―

X 0.9569 0.14055 16.426 16.9533

SD 0.63422 0.26822 1.642476 3.086031 The experimental results show that a single dose of 100 mg/kg (equivalent to 10 mg/kg per day) for 10 consecutive days The protocol administered BBM orally to rats carrying CML xenografts. This regimen caused significant tumor regression, with 70% of CML xenografts remaining for up to 30 days of regression. The tumor weights of the control group and the BBM-treated group were 0.96 ± 0.64 g and 0.14 ± 0.27 g, respectively (Fig. 1A), and the body weight of the tumor-bearing mice increased after treatment with BBM (Fig. 1B). Histological analysis indicated that tumors from BBM-treated mice exhibited necrosis, while tumor tissues from control mice consisted mainly of proliferating tumor cells (Fig. 2A, B and C:). Example 2

(1) Experimental materials

Imatinib purchased from Swiss Novartis Pharmaceutical Co., Ltd.

Instrument: Cell culture incubator (model BB1 ⁶ UV/BB50 ⁶ 0UV)

Imatinib resistance and resistance K562 cells: from the Institute of Cancer Research, Zhejiang University (both chronic myeloid leukemia cells)

(2) Experimental method

Seven-week-old rats were subcutaneously inoculated with human chronic myeloid leukemia cell K562 at a dose of 5 X 10 ⁷ cells per mouse. After 24 hours, randomized groups, 7 in the control group, 6 in the berbamine group, and 7 in the imatinib group. Medication group: The citrate group and the imatinib group were administered orally, at a dose of lOOmg per kilogram of body weight, 3 times a day (glycated λ control group: replaced with normal saline, used for 10 days. A total of 45 days of observation) During the experiment, the weight, body weight, activity and diet of the rats were measured. At the end of the experiment, the mice were sacrificed, the tumor tissues were taken, and the actual weight was measured.

(3) Experimental results The experimental results are shown in Table 2.

Table 2 Comparison of the anti-leukemia effects of imatinib 3 times per day with 100 mg/kg of berbamine in combination with the same treatment regimen

The results of the experiment showed that when the BBM regimen was a 10-dose regimen for 10 consecutive days, we observed complete regression of CML xenograft tumors (Fig. 3A). The weights of the control and BBM-treated groups were 16.69±2.92 g and 17.61 ± 1.70 g, respectively. (Fig. 3B). Most importantly, this three-dose regimen of berbamine resulted in complete regression of IM-resistant CML xenograft tumors (Fig. 4A), and the body weights of the control and BBM treated groups are shown in Figure 4B. The survival rate of the control group was 28.6% (2/7), the tumor-free rate was 0% (0/7); the survival rate of the berbamine group was 100% (6/6), and the tumor-free rate was 83.3%. (5/6); The survival rate of the imatinib group was 100% (7/7), and the tumor-free rate was 42.9% (3/7). The same three-dose regimen of imatinib did not cause regression. The tumor weights of the control and IM treatment groups were 2.44±0.25 g and 1.02±1.23 g, respectively (Fig. 5A). The body weights of the IM and treated groups were 13.0 ± 0.25 g and 15.00 ± 3.78 g, respectively (Fig. 5B). These results indicate that berbamine can directly target and eliminate CML LSC in vivo. Example 3

(1) Experimental materials

Berbamine citrate was prepared by mixing citric acid and berbamine at a weight of 1:2.

Instrument: Cell culture incubator (model BB16UV/BB ⁵ 060UV)

K562 cells: (from Zhejiang University Cancer Institute)

(2) Experimental method

Seven-week-old rats were subcutaneously inoculated with human chronic myeloid leukemia cells K562 5 X 107 per mouse. After 24 hours, randomize. The drug group: The berbamine hydrochloride and the citrate citrate group were administered orally, at a dose of lOOmg per kilogram of body weight, 3 times a day (stomach). Control group: Replace with saline for 10 days. A total of 30 days were observed, during which the tumor weight, body weight, activity and diet were measured. At the end of the experiment, the mice were sacrificed and tumor tissue was taken and the actual weight was measured.

(3) Experimental results

The experimental results are shown in Table 3. Table 3 different small guanamine salts 3 times a day, 100mg/kg each time, combined with 10 days of treatment program anti-leukemia effect comparison weight (g) tumor weight (: g)

The experimental results show that both berbamine hydrochloride and berbamine citrate can eliminate chronic myeloid leukemia xenografts in mice.

Claims

Claim

A use of berberine or a pharmaceutically acceptable salt thereof for the preparation of a medicament for treating leukemia at a dose of 5 mg/kg/day to 200 mg/kg/day.

2. Use according to claim 1 wherein the leukemia is chronic myeloid leukemia.

3. Use according to claim 1 or 2, wherein the dose is from 10 mg/kg/day to 100 mg/kg/day.

4. Use according to claim 1 or 2, wherein the dosage is from 20 mg/kg/day to 80 mg/kg/day.

5. Use according to claim 1 or 2, wherein the dose is from 30 mg/kg/day to 60 mg/kg/day.

6. Use according to claim 1 or 2, wherein the dosage is from 40 mg/kg/day to 50 mg/kg/day.

7. Use according to claim 1 wherein the medicament is formulated for oral administration.

8. Use of berbamine or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of imatinib-resistant chronic myeloid leukemia.

9. The use according to claim 8, wherein the medicament is administered at a dose of from 30 mg/kg/day to 100 mg/kg/day.

10. Use according to claim 8, wherein the medicament is administered at a dose of from 36 mg/kg/day to 90 mg/kg/day.

11. The use according to claim 8, wherein the medicament is administered at a dose of 45 mg kg/day to 60 mg/kg/day.

12. Use according to claim 8, wherein the medicament can be formulated for oral administration.

13. A daily dosage unit for the treatment of leukemia, comprising a small guanamine or a pharmaceutically acceptable salt thereof in the form of a pharmaceutical composition in an amount sufficient to provide a daily dose of from 5 to 200 mg per kg of patient body weight.

14. A daily dose independent unit according to claim 13 wherein the amount of the small amide or a pharmaceutically acceptable salt thereof is sufficient to provide a daily dose of from 10 to 100 mg per kg of patient body weight.

15. A daily dose independent unit according to claim 13 wherein the amount of the small amide or a pharmaceutically acceptable salt thereof is sufficient to provide a daily dose of from 20 to 80 mg per kg of patient body weight.

16. The daily dosage unit according to claim 13 wherein said berberine or a pharmaceutically acceptable salt thereof is present in an amount sufficient to provide a daily dose of from 30 to 60 mg per kg of patient body weight.

17. A daily dosage unit according to claim 13 wherein the amount of the small amide or a pharmaceutically acceptable salt thereof is sufficient to provide a daily dose of from 40 to 50 mg per kg of patient body weight.

18. A daily dose independent unit according to claim 13 for use in the treatment of imatinib resistant chronic myeloid leukemia.

19. A daily dosage unit according to claim 18, wherein the amount of the small amide or a pharmaceutically acceptable salt thereof is sufficient to provide a daily dose of from 30 to 100 mg per kg of patient body weight.

20. A daily dose-independent unit according to claim 18, wherein the amount of berbamine or a pharmaceutically acceptable salt thereof is sufficient to provide a daily dose of from 36 to 90 mg per kg of patient body weight.

21. A daily dosage unit according to claim 18, wherein the amount of the small amide or a pharmaceutically acceptable salt thereof is sufficient to provide a daily dose of 45 to 60 mg per kg of patient body weight.

22. A daily dosage unit according to any one of claims 13 to 18, which comprises a pharmaceutical tablet comprising one or more 'J, guanamine or a pharmaceutically acceptable salt thereof.

23. A daily dose individual unit according to claim 22, wherein said individual units are separate packages or separate sub-packages.