CN110314222B

CN110314222B - Application of bortezomib and panobinostat or vorinostat composition in preparation of drug-resistant MLL leukemia treatment drugs

Info

Publication number: CN110314222B
Application number: CN201910724720.5A
Authority: CN
Inventors: 刘晗; 葛茂林; 李丹; 乔智; 孙燕
Original assignee: Ruinjin Hospital Affiliated to Shanghai Jiaotong University School of Medicine Co Ltd
Current assignee: Ruinjin Hospital Affiliated to Shanghai Jiaotong University School of Medicine Co Ltd
Priority date: 2019-08-07
Filing date: 2019-08-07
Publication date: 2023-05-26
Anticipated expiration: 2039-08-07
Also published as: CN110314222A

Abstract

The invention relates to an application of a bortezomib and panobinostat or vorinostat composition in preparing a medicament for treating drug-resistant MLL leukemia. The invention proves that panobinostat and vorinostat can restore the sensitivity of MLL leukemia drug-resistant cells, reverse bortezomib drug resistance in mice, reduce the generation of drug-resistant cells and prolong the life cycle. The invention is helpful for solving the drug resistance problem of MLL leukemia and further improving the treatment effect of malignant tumors in personalized medical age.

Description

Application of bortezomib and panobinostat or vorinostat composition in preparation of drug-resistant MLL leukemia treatment drugs

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of a bortezomib and panobinostat or vorinostat composition in preparation of medicines for treating drug-resistant MLL leukemia.

Background

Proteasome inhibitors (proteasome inhibitor, PI) are effective in inducing apoptosis in tumor cells and have been tested clinically on a variety of malignancies since the end of the 90 s of the 20 th century. Three proteasome inhibitors (bortezomib, carfilzomib and iferum Sha Zuo meters) have been approved by the U.S. food and drug administration (food and drug administration, FDA) for clinical treatment of Multiple Myeloma (MM) and Mantle Cell Lymphoma (MCL).

Bortezomib (trade name valcade, mo Ke) is the first proteasome inhibitor approved for clinical use. Bortezomib is able to bind to the catalytic site of the 26S proteasome, thereby inhibiting the proteolytic activity of β5, β2 and β1 subunits. The inhibitor was initially approved for first-line treatment of relapsed and refractory multiple myeloma patients, showed impressive clinical effects as a monotherapy drug, and was subsequently successfully used in combination with other drugs to improve clinical outcome. Clinical efficacy of bortezomib was evaluated in a number of different drug combination combinations, including in combination with lenalidomide and dexamethasone (dexamethasone), immunomodulatory drugs (immunomodulatory drugs, IMiDs), the CD38 monoclonal antibody daratumumab (daratumumab), histone deacetylase (histone deacetylase, HDAC) inhibitors, and the like, all resulted in positive therapeutic effects.

Mixed lineage leukemia (mixed lineage leukemia, MLL) caused by 11q23 chromosomal translocation is a common acute leukemia, found in both acute lymphoblastic leukemia (acute lymphoblastic leukemia, ALL) and acute myeloid leukemia (acute myeloid leukemia, AML), accounting for about 10% of the total acute leukemia population, and more particularly more than 80% of secondary leukemia caused by treatment with infant leukemia and topoisomerase inhibitors. MLL leukemia is extremely dangerous and has extremely poor prognosis, and even stem cell transplantation cannot significantly improve the prognosis, and is considered to be the most refractory leukemia, and there is a need to find effective treatment methods. In recent years, with the intensive research on the biological functions of MLL and the pathogenesis of MLL leukemia, a plurality of novel targets of MLL leukemia, including GSK3, BRD4, DOT1L and other molecules, are discovered successively, and corresponding treatment strategies are also developed.

Recently, proteasome inhibitors have been found to specifically kill MLL leukemia cells by activating the inherent tumor inhibitory activity of MLL fusion proteins, resulting in cell cycle arrest and induction of apoptosis, and have been successfully applied clinically, thus providing a good therapeutic effect and laying a good foundation for the treatment of MLL.

Although proteasome inhibitors greatly improve the therapeutic effects of multiple myeloma and lymphoma, patients eventually inevitably develop drug resistance, which ultimately leads to tumor recurrence. The problem of drug resistance of patients is also encountered when the proteasome inhibitor is applied to treat MLL leukemia in the patients, so that treatment failure is caused, and the clinical application of the proteasome inhibitor is seriously influenced.

Patent document CN109789138A, publication date 2019.05.21, discloses a pharmaceutical composition for treating hematological cancer, comprising formula 1

A Histone Deacetylase (HDAC) inhibitor, a proteasome inhibitor, or an immunomodulatory drug, and a steroidal anti-cancer agent. And demonstrate the improved efficacy of the composition according to the invention by measuring therapeutic synergy, it was found that the composition of the invention exhibits a synergistic effect when administered as a first active ingredient in combination with a second active ingredient and a third active ingredient as an HDAC inhibitor, and thus has excellent anticancer activity against hematological cancers, particularly multiple myeloma.

Patent document CN101528037a, publication No. 2009.09.09, discloses a method of treating multiple myeloma using SAHA and bortezomib, comprising administering to a patient: i) SAHA (suberoylanilide hydroxamic acid) or a pharmaceutically acceptable salt or hydrate thereof; and ii) (1R) -3-methyl-1- [ [ (2S) -1-oxo-3-phenyl-2- [ (pyrazinylcarbonyl) amino group]Propyl group]Amino group]Butyl group]Boric acid (bortezomib), or a pharmaceutically acceptable salt or hydrate thereof, wherein 200mg to 800mg of SAHA, or a pharmaceutically acceptable salt or hydrate thereof, is orally administered daily for at least one treatment period of days 4-11 of a 21-day cycle, and 0.7-1.3mg/m is intravenously administered daily for at least one treatment period of

days

1, 4, 8 and 11 of a 21-day cycle ² Bortezomib, or a pharmaceutically acceptable salt or hydrate thereof.

The combination of the signal transduction pathway inhibitor and the acute myeloid leukemia drug resistance and the mechanism research thereof in the 2008-level doctor's academy of university of southern medical science disclose that LBH589 and Bortezomib can obviously down regulate the expression of HL-60/ADM cell MRP1, improve the uptake rate of doxorubicin in cells and reverse drug resistance, and the combination effect of the two drugs is more obvious; LBH589 is combined with Bortezomib to treat HL-60/ADM cells, so that the activity of a PI3K/Akt/NF- κB signal path can be reduced, the expression of P53 protein is up-regulated, the expression of Bcl-2 and XIAP proteins is inhibited, the cleavage and activation of Caspase-3, 8 and PARP are promoted, and the inhibition of the protein is jointly involved in reversing AML drug resistance.

However, no drug with obvious effect on drug-resistant MLL leukemia is currently seen.

Disclosure of Invention

The invention aims to provide a new application of a composition of bortezomib and panobinostat or vorinostat, aiming at the defects in the prior art.

In a first aspect, the invention provides the use of a composition of bortezomib and panobinostat in the manufacture of a medicament for the treatment of drug-resistant MLL leukemia.

In the composition, the molar ratio of bortezomib to panobinostat is 20:1 or 2:1.

In a second aspect, the invention provides a pharmaceutical composition comprising bortezomib and panobinostat in the form of an injection, wherein the bortezomib and panobinostat are dissolved in the same solvent system.

In a third aspect, the invention provides the use of a composition of bortezomib and panobinostat in the preparation of a reagent for reversing the resistance of MLL leukemia resistant cells.

The MLL leukemia drug-resistant cells are B cell leukemia cell strain RS4 subjected to MLL ectopic rearrangement; 11 or SEM.

In a fourth aspect, the invention provides the use of a bortezomib and vorinostat composition in the manufacture of a medicament for the treatment of drug-resistant MLL leukemia.

In the composition, the molar ratio of bortezomib to vorinostat is 1:20.

In a fifth aspect, the present invention provides a pharmaceutical composition comprising bortezomib and panobinostat in a dosage form of an injection, the bortezomib and vorinostat being dissolved in the same solvent system.

In a sixth aspect, the invention provides the use of a bortezomib and vorinostat composition in the preparation of a reagent for reversing the resistance of MLL leukemia resistant cells.

The invention has the advantages that:

MLL leukemia, because of its unique ectopic rearrangement mechanism, results in a high degree of malignancy, is still currently "sharp sword" suspended on the head of the patient, and especially has a very high proportion in infant leukemia, resulting in serious effects, so it is particularly urgent to develop corresponding therapeutic strategies and improve their application. However, the mechanism of the onset and drug resistance of MLL leukemia is not clear at present, and the tumor of the blood system is a group of diseases with strong heterogeneity, so the research of drug resistance MLL leukemia therapeutic drugs is difficult and serious.

The invention provides a combined treatment strategy of a proteasome inhibitor (bortezomib) and an HDAC inhibitor (panobinostat, LBH 589) and vorinostat (SAHA) in MLL leukemia, and compared with continuous single-drug treatment of the proteasome inhibitor, the combined treatment strategy of the proteasome inhibitor and the HDAC inhibitor can eliminate tumors in patients by reducing the generation of drug-resistant cells, prolong the survival time of the patients and treat the MLL leukemia more effectively.

The invention improves the clinical transformation and application effects of the proteasome inhibitor for treating the MLL leukemia, and is important for further improving the effect of the medicines in the personalized medical age.

Based on the invention, the composition of bortezomib and vorinostat can be used as a reagent for treating MLL leukemia drug-resistant cells, researching the occurrence and development mechanism of MLL leukemia drug resistance, screening reversal drugs and the like.

The invention also obtains the optimal ratio of the bortezomib and the HDAC inhibitor (LBH 589 and SAHA) to be used together, thereby being beneficial to the development of clinical medicines.

Drawings

FIG. 1 construction of MLL drug-resistant cell lines and detection of their sensitivity to bortezomib. (A) MTT assay for detection of different MLL cells SEM and RS4;11 sensitivity to bortezomib, and detection was performed 24 hours after the addition of bortezomib at the corresponding concentration. (B) Annexin V method detects SEM and RS4 of different states; percentage of apoptosis of 11 cells under treatment with bortezomib at the corresponding concentration, the drug treatment time was 16 hours.

Fig. 2 hdac inhibitors LBH589 and SAHA were administered in combination with bortezomib. SEM and RS4; the 11 tolerant cells were treated with LBH589 or SAHA at the corresponding concentrations shown in the figures in combination with DMSO or 50nM bortezomib, respectively, for 24 hours and the MTT assay was used to examine the inhibitory effect.

Fig. 3 hdac inhibitors LBH589 and SAHA were able to restore sensitivity to drug resistant cells. (A) SEM-resistant cells were treated with DMSO, LBH589 (5 nM) or SAHA (2. Mu.M) in combination with bortezomib, respectively, and their sensitivity was examined by MTT method. (B) RS4; the sensitivity of 11 tolerant cells was tested by MTT assay by treatment with DMSO, LBH589 (50 nM) or SAHA (2. Mu.M) in combination with bortezomib, respectively, for 24 hours.

Fig. 4 hdac inhibitors reverse proteasome inhibitor resistance in mice. (A) And (3) injecting SEM drug-resistant cells into the irradiated mice through tail veins, respectively adopting different schemes to carry out tail vein injection bortezomib and/or intraperitoneal injection SAHA for treatment, extracting peripheral blood of the mice at corresponding time in the illustration, and detecting the proportion of CD133 positive cells by a flow cytometer. (B) Mice were sacrificed at the end of their observation, and bone marrow cells were extracted for flow-through detection of CD133 positive cell proportion. (C) Survival curves for mice from different treatment groups, wherein control group (vehicle, n=8, average survival 26.4 days), SAHA single drug treatment group (SAHA, n=7, average survival 28.3 days), bortezomib single drug treatment group (bortezomib, n=7, average survival 27.9 days), combination treatment group (bortezomib+saha, n=8, average survival 40.0 days). Data are expressed as mean ± standard error, data statistics using a two-tailed t-test or time series test (log-rank test), n.s. differences are not significant, P <0.05, P <0.01, P <0.001.

Detailed Description

The following detailed description of the invention provides specific embodiments with reference to the accompanying drawings.

Example 1

1 materials and methods

The study applied the following cell lines: b-cell leukemia cell line RS4 with ectopic MLL rearrangement; 11 and SEM were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DMSZ). All suspension cell lines were cultured in RPMI 1640 medium containing 10% fetal bovine serum and placed at 37℃in 5% CO ₂ Culturing in a 95% air humidity incubator. Changing liquid of cells every other day, and adjusting cell concentration to 5×10 ⁵ Individual/ml and 2X 10 ⁶ Between individual/ml. The proteasome inhibitor bortezomib resistant cells (resistance cells) were obtained by adding a gradually increasing concentration of bortezomib to the parent cells (parent cells) for at least four weeks. The specific drug concentration is the half-growth inhibitory concentration of the drug corresponding to the parent cell (half maximal inhibitory concentration, IC ₅₀ ) I.e. 5nM bortezomib was added, and after three days of treatment the solution was changed and new drug was added for continued treatment for four weeks. Most cells undergo apoptosis during drug treatment, and a small number of residual cells survive and continue to proliferate, and the population of cells that can tolerate drug treatment is referred to as tolerizing cells.

Female NOD-SCID immunodeficiency mice of 6-8 weeks old are purchased from Vetolihua laboratory animal technology company, fed into animal laminar flow chamber of barrier facility, fed at 24-26 deg.C and relative humidity 40-60%, fed by artificial lighting, and feed and water are sterilized at high temperature. Intravenous injection to the tail of each mouse was 5X 10 ⁶ SEM drug-resistant cells, leukemia onset and leukemia progression were observed after cell injection. In general, CD133 positive cells can be detected in the bone marrow of mice of 1-2 weeks, and CD133 positive cells can be detected in the peripheral blood of mice of about 2-3 weeks.

The specific method for detecting the peripheral blood cells comprises the following steps: 10 mu L of heparin is added into a 1.5ml centrifuge tube in advance, tail ends of the mice are cut off by using an ophthalmic scissors, peripheral blood of the mice is taken out by using a 1.5ml centrifuge tube, and a red blood cell lysate with the volume of 5 times is added for 5min at room temperature. Centrifuging 500g for 5min to collect cells, dividing the cells into two parts, wherein one part of the cells is not labeled with an antibody as a control, and setting an adjusting voltage for a flow cytometer; another aliquot was resuspended in 50. Mu.L of DPBS containing 1. Mu.L of anti-CD 133 fluorescent antibody and incubated at room temperature for 20-30min. 1mL of DPBS was added for washing, 500g was centrifuged for 5min, and the supernatant was discarded. Each tube was filled with 500. Mu.L of DPBS buffer, examined by flow cytometry, analyzed by FlowJo software, and the positive ratios of the different cells were calculated separately. Mice were treated with random groupings after tumor formation, treated for 4 consecutive weeks, then monitored for disease, and sacrificed at the point of their impending death. Animal care and sacrifice was performed according to the methods approved by the animal care and use committee of the university of Shanghai transportation animal experiment center.

The specific method for bone marrow cell detection comprises the following steps: NOD-SCID immunodeficient mice were sacrificed using cervical dislocation. The tibia of the mice was removed, the muscles removed, and placed in pre-chilled DPBS with 1% bsa. The tibia was cut off at both ends with surgical scissors, and the tibia was rinsed with a 1ml syringe to obtain bone marrow cells. The cell suspension was collected by filtration through a 70 μm sieve. Centrifuge at 400g for 5min at 4℃and discard the supernatant. Cells were lysed with erythrocyte lysate ACK for 5min, and lysis was terminated by adding 3 volumes of PBS. The mixture was centrifuged at 400g for 5min at 4℃to discard the supernatant, thereby obtaining mouse bone marrow cells.

Mice injected with SEM drug-resistant cells were treated with random groupings after tumor formation. Mice were randomly divided into three groups according to experimental design, treatment was started 7 days after transplantation, the first group was a control group, and the same amount of DMSO was injected; the second group is bortezomib single drug treatment group: 1mg/kg bortezomib Mi Wei intravenously, 2 times per week, mice receiving bortezomib single drug treatment also received drug carrier treatment without drug, while performing intraperitoneal injection of the same amount of DMSO five times a week; the third group is SAHA single drug treatment group: 5mg/kg SAHA was injected intraperitoneally 5 times per week; the fourth group was bortezomib and SAHA combination treatment group: 1mg/kg bortezomib Mi Wei was intravenously injected 2 times per week, while 5mg/kg SAHA was intraperitoneally injected five times a week. All mice were treated for 4 weeks and peripheral blood flow assays were taken every 5 days starting on day 10 to record CD133 positive cell fractions. On day 25 3 mice were sacrificed per group and bone marrow cells in the hind leg tibia were removed and the CD133 positive cell fraction was detected with a flow cytometer, respectively.

The MTT assay detects cell sensitivity: at 5X 10 per well ⁴ Individual cells were seeded in 96-well plates and treated with concentration gradients of 0, 1, 2, 5, 10, 20, 50, 100nM, respectively, after 24h 10 μl MTS was added to each well and incubated for 4h at 37 ℃ and absorbance values were measured at 490nM in a spectrophotometer. 3 duplicate wells were made for each drug concentration and 3 experiments were repeated. Calculation of IC from drug to cell dose-response curve ₅₀ . Drug resistant cells were treated simultaneously with the HDAC inhibitors LBH589 and SAHA (vehicle DMSO), and the effect of the combination of the above inhibitors with bortezomib on drug resistant cells was observed, and the combination index (Combination index, CI) was analyzed by calculation using CompuSyn software.

Apoptosis detection was performed using PE Annexin V Apoptosis Detection Kit kit, and cell processing was performed according to the kit-related procedure. Collection of 1X 10 ⁵ Cells were resuspended in 100. Mu.L of 1 Xbinding buffer and transferred to a flow tube by washing 2 times with pre-chilled PBS. Add 2. Mu.L PE Annexin V and 2. Mu.L 7-AAD, mix well and incubate at room temperature in the dark for 15min. 400 μL of 1×binding buffer was added, and flow cytometry was performed over 1h, and analyzed by FlowJo software to calculate the apoptosis ratios of the different cells, respectively.

2 experimental results

The parental MLL leukemia cell lines (SEM and RS4; 11) which are sensitive to the proteasome inhibitor are respectively subjected to external drug addition treatment, and drug-resistant cell lines are obtained by screening by gradually increasing the concentration of the proteasome inhibitor bortezomib in the culture medium. After approximately 4 weeks of drug treatment, these cells developed resistance to bortezomib (a in fig. 1). To further verify the above results, we performed apoptosis assays on these in vitro cell line models. Apoptosis was examined by labeling Annexin V and the results showed that drug resistant cell lines did not undergo significant apoptosis with increasing drug dose compared to the parental cells, indicating that these cells were resistant to bortezomib (B in fig. 1).

To study the therapeutic effect of HDAC inhibitors in resistant cells, we examined the efficacy of HDAC inhibitors in combination with bortezomib by treating resistant cells with HDAC inhibitors LBH589 and SAHA, respectively, in combination with bortezomib. We first determined the optimal concentrations of LBH589 and SAHA for use in combination with bortezomib by MTT experiments. We found that in SEM resistant cells, the killing effect on SEM resistant cells was most pronounced by treatment with 5nM LBH589 or 2. Mu.M SAHA in combination with bortezomib, respectively. And RS4; in 11 resistant cells, RS4 was treated with 50nm lbh589 or 2 μm SAHA in combination with bortezomib, respectively; the killing effect of 11 resistant cells was most pronounced (fig. 2).

Subsequently, we respectively use LBH589 or SAHA with the concentrations and bortezomib to treat drug-resistant cells in combination, and cell sensitivity detection experiments show that compared with the use of bortezomib alone, the combination can obviously reduce the IC of the drug-resistant cells ₅₀ Values resulted in a significant decrease in tolerance cells (a and B in fig. 3). We used the drug combination index (Combination index, CI) to judge the synergy between the two drugs, where a CI of less than 0.9 indicates that the drugs have a synergistic effect and a CI of less than 0.3 is a strong synergistic effect. Our results show that bortezomib in combination with panobinostat or vorinostat acts on MLL resistant cells, both show significant synergy and are seen in SEM and RS4 at concentrations of bortezomib and vorinostat of 0.1 μm and 5 μm, respectively; the CI in 11 cells was less than 0.1, indicating a very strong synergistic effect between the two drugs. These results indicate that the combined use of HDAC inhibitors and proteasome inhibitors has a significant synergistic effect on MLL-tolerant cells (tables 1-4).

TABLE 1 combination index of different concentrations of bortezomib in combination with vorinostat on SEM resistant cells

TABLE 2 combination index of different concentrations of bortezomib in combination with panobinostat on SEM resistant cells

Table 3 bortezomib at different concentrations in combination with vorinostat for RS4; combination index of 11 resistant cells

Table 4 different concentrations of bortezomib in combination with panobinostat on RS4; combination index of 11 resistant cells

Subsequently, we transplanted SEM cells resistant to proteasome inhibitors into NOD/SCID mice and evaluated the in vivo efficacy of bortezomib alone or in combination with SAHA. NOD/SCID mice were previously tumor-resistant by SEM-tail intravenous injection, tail vein or intraperitoneal injection (Vehicle, 1mg/kg bortezomib, 5mg/kg SAHA, 1mg/kg bortezomib in combination with 5mg/kg SAHA) was started after 1 week, while tumor cell proportion in peripheral blood was flow-detected by CD133 antibody labeling, mice were sacrificed at the end of their observation, and the proportion of tumor cells in bone marrow was detected.

Examination of SEM-resistant cell ratios in peripheral blood and bone marrow cells found that bortezomib and SAHA single drug treatment had no significant therapeutic effect on these xenograft mice compared to the control group, whereas bortezomib combined with SAHA significantly reduced the tumor cell ratio, indicating that the combined use of both drugs significantly increased killing of SEM-resistant cells in mice (fig. 4 a and B). By observing the survival of mice we found that bortezomib alone (average number of survival days 27.857) and SAHA (average number of survival days 28.3) did not significantly extend the survival of mice compared to the control group (average number of survival days 26.375), but that bortezomib alone in combination with SAHA significantly extended the survival of mice (average number of survival days 40.0) (fig. 4C). These results show that bortezomib single drug treatment was not effective in these transplanted mice, consistent with the results that these cells had developed tolerance to bortezomib treatment. In contrast, bortezomib-in-SAHA-treated mice exhibited significant reactivity and significantly increased overall survival, these results indicate that proteasome inhibitor-in-HDAC inhibitor treatment may be effective against proteasome inhibitor-induced tolerogenic cells, suggesting that our combination regimen is viable in vivo.

The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and additions may be made to those skilled in the art without departing from the method of the present invention, which modifications and additions are also to be considered as within the scope of the present invention.

Claims

1. The application of a bortezomib and panobinostat composition in preparing a medicament for treating a bortezomib-resistant MLL leukemia is characterized in that the molar ratio of bortezomib to panobinostat in the composition is 20:1 or 2:1.

2. The application of the bortezomib and panobinostat composition in preparing a reagent for reversing the drug resistance of MLL leukemia bortezomib drug-resistant cells is characterized in that the MLL leukemia bortezomib drug-resistant cells are B cell leukemia cell strain RS4, 11 or SEM with MLL ectopic rearrangement.

3. Use of a composition of bortezomib and vorinostat in the manufacture of a medicament for the treatment of MLL leukemia resistant to bortezomib, wherein the molar ratio of bortezomib to vorinostat in the composition is 1:20.

4. The application of the bortezomib and vorinostat composition in preparing a reagent for reversing the drug resistance of MLL leukemia to bortezomib-resistant cells is characterized in that the MLL leukemia bortezomib-resistant cells are B-cell leukemia cell strain RS4, 11 or SEM with MLL ectopic rearrangement.