Application of hD1R protein in the preparation of drugs for the treatment of AML

Abstract

This invention relates to the field of biomedical technology, and in particular to the application of hD1R protein in the preparation of drugs for the treatment of AML. Addressing the current lack of systematic and in-depth research on whether hD1R protein can directly act on AML cells and whether it has a synergistic effect with existing standard chemotherapy drugs (such as anthracyclines), this invention, through a series of in vivo and in vitro experiments and the construction of a NOD / SCID mouse transplantation model, confirms that hD1R protein alone can effectively inhibit AML cell proliferation, induce apoptosis, inhibit AML cell colony formation, and downregulate the expression of the key anti-apoptotic gene Bcl2 in AML cells. It also confirms that hD1R treatment can significantly prolong the survival of model mice. Furthermore, by setting up a Dox+hD1R group (combined experimental group), it is confirmed that when hD1R protein is used in combination with anthracycline chemotherapy drugs, it exhibits significant synergistic effects in inhibiting AML cell proliferation, promoting AML cell apoptosis, downregulating the expression of the anti-apoptotic gene Bcl2 in AML cells, and inhibiting AML cell colony formation.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of hD1R protein in the preparation of drugs for the treatment of AML. Background Technology

[0002] Acute myeloid leukemia (AML) is the most common acute leukemia in adults. It is characterized by the malignant proliferation, impaired differentiation, and apoptosis of myeloid progenitor cells in the bone marrow, leading to the accumulation of a large number of immature cells (primitive cells) and suppression of normal hematopoietic function. Patients often present with symptoms such as anemia, infection, bleeding, and infiltration of tissues and organs, ultimately leading to organ failure. Although the treatment efficacy of AML has significantly improved in recent years with combination chemotherapy (such as the "7+3" regimen, i.e., anthracyclines combined with cytarabine) and allo-HSCT, the long-term survival rate of AML patients remains low. Only 35-40% of adult patients achieve long-term survival, and most eventually develop drug resistance and relapse, resulting in very limited treatment options and a very poor prognosis.

[0003] To further improve the prognosis and overall survival of AML patients, an increasing number of studies are shifting their focus from traditional cytotoxic chemotherapy to precision medicine centered on molecular targets. With the development of technologies such as genomics, transcriptomics, and proteomics, the molecular pathological characteristics of AML are gradually being elucidated, and new oncogenes or signaling pathways are constantly being discovered. Based on this, designing molecularly targeted drugs with higher specificity and fewer toxic side effects by identifying key signaling molecular pathways has become the most promising direction in the comprehensive treatment of AML patients, and it is expected to overcome the current challenges of drug resistance and rapid progression.

[0004] The Notch signaling pathway is a highly conserved intercellular communication pathway that is activated by the binding of Notch receptors to ligands on the surface of neighboring cells. It mediates signal transduction between adjacent cells and regulates cell proliferation, differentiation, and apoptosis. In the normal hematopoietic system, hematopoietic cells and stromal cells of the hematopoietic microenvironment express Notch receptors and ligands, respectively. The binding of Notch receptors and ligands activates the Notch signaling pathway; therefore, Notch signaling plays a crucial regulatory role in the proliferation and differentiation of hematopoietic cells. Studies have shown that abnormal alterations in Notch signaling are associated with the pathogenesis of various hematologic malignancies such as lymphoma and leukemia. In hematologic malignancy cells, abnormal activation of Notch signaling plays different roles in tumor suppression or tumor promotion, exhibiting a significant duality. This contradictory finding suggests that the specific function of Notch signaling in AML is highly dependent on its cellular background, microenvironment, and the downstream target genes it activates.

[0005] hD1R protein is a genetically engineered, soluble Notch ligand that targets endothelial cells. It was first synthesized and reported by the Department of Genetics and Developmental Biology at the Fourth Military Medical University, primarily for research on the expansion of umbilical cord blood hematopoietic stem and progenitor cells and their impact on angiogenesis. However, to date, systematic and in-depth research is lacking regarding whether hD1R protein can directly act on AML cells, and whether it has a synergistic effect with existing standard chemotherapy drugs (such as anthracyclines). Summary of the Invention

[0006] To address the aforementioned problems, this invention aims to provide the application of hD1R protein in the preparation of drugs for the treatment of AML. Currently, there is a lack of systematic and in-depth research on whether hD1R protein can directly act on AML cells and whether it has a synergistic effect with existing standard chemotherapy drugs (such as anthracyclines). This invention, through a series of experimental verifications, confirms that hD1R protein, used alone or in combination with anthracyclines, can effectively inhibit AML cell proliferation, induce apoptosis, and reduce stem cell characteristics, exhibiting significant anti-leukemic activity, thus providing a new drug combination strategy for the clinical treatment of AML.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: On the one hand, the present invention provides the use of hD1R protein in the preparation of medicaments for the treatment of AML.

[0008] Furthermore, the hD1R protein achieves therapeutic effects on AML by inhibiting AML cell proliferation and / or promoting AML cell apoptosis.

[0009] Furthermore, the inhibition of AML cell proliferation and / or promotion of AML cell apoptosis includes downregulating Bcl2 gene expression within AML cells.

[0010] On the other hand, the present invention also provides the use of hD1R protein and anthracycline chemotherapy drugs in the preparation of medicaments for the treatment of AML.

[0011] Furthermore, the anthracycline chemotherapy drugs include doxorubicin, epirubicin, daunorubicin, idarubicin, arubicin, and pharmaceutically acceptable derivatives thereof.

[0012] In another aspect, the present invention also provides a medicament for treating AML, the medicament comprising hD1R protein and pharmaceutically acceptable excipients.

[0013] In another aspect, the present invention also provides a pharmaceutical composition for treating AML, the pharmaceutical composition comprising hD1R protein and anthracycline chemotherapy drugs, and pharmaceutically acceptable excipients.

[0014] The beneficial effects of this invention are: compared with the prior art, the improvement of this invention lies in that... 1. This invention, through a series of in vivo and in vitro experiments, confirms that hD1R protein alone can effectively inhibit the proliferation of AML cells and induce their apoptosis. Furthermore, hD1R protein can significantly inhibit the colony formation of AML cells, indicating that hD1R protein has a targeted inhibitory effect on leukemia stem cells with self-renewal capacity, demonstrating that hD1R protein has a direct and potent anti-leukemia effect. In addition, hD1R protein promotes apoptosis of leukemia cells by downregulating the expression of the key anti-apoptotic gene Bcl2 in AML cells, revealing the specific molecular mechanism by which hD1R protein acts on the apoptosis signaling pathway.

[0015] 2. This invention constructs a NOD / SCID mouse transplantation model to simulate human leukemia symptoms. In mouse simulation experiments, it was demonstrated that hD1R treatment can significantly prolong the survival of model mice and effectively reduce the leukemia burden in their peripheral blood, proving its effectiveness and therapeutic potential in animals.

[0016] 3. This invention, through setting up a Dox+hD1R group (combined experimental group), demonstrated that the combined use of hD1R protein and commonly used anthracycline chemotherapy drugs (such as doxorubicin) exhibits significant synergistic effects in inhibiting AML cell proliferation, promoting AML cell apoptosis, downregulating the expression of the anti-apoptotic gene Bcl2 in AML cells, and inhibiting AML cell colony formation. This lays a solid foundation for developing more efficient and less toxic combination drug regimens. This invention not only provides a basis for the application of hD1R protein as a monotherapy, but more importantly, it pioneers a new strategy for the combined use of hD1R protein and standard chemotherapy drugs, providing new directions and candidate drugs for solving the problems of drug resistance and relapse in the clinical treatment of AML. Attached Figure Description

[0017] Figure 1 This invention relates to the effect of different culture conditions on the viability of AML cells.

[0018] Figure 2 This invention relates to the effect of different culture conditions on apoptosis in AML cells.

[0019] Figure 3 This invention relates to the effect of different culture conditions on the colony-forming ability of AML cells.

[0020] Figure 4 This invention relates to the effect of different culture conditions on Bcl2 mRNA expression in AML cells.

[0021] Figure 5The Kaplan-Meier survival curves of NOD / SCID mice implanted with patient AML cells according to the present invention were obtained after treatment with PBS and hD1R protein, respectively.

[0022] Figure 6 On day 28 after NOD / SCID mice implanted with patient AML cells according to the present invention were treated with PBS and hD1R protein, respectively, the peripheral blood AML cell infiltration (human CD45) was measured. + Cell ratio). Detailed Implementation

[0023] To enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0024] Example 1: Original AML-CD34 + Cells are a population of primitive leukemia cells bearing the CD34 surface marker, isolated directly from the bone marrow or peripheral blood of newly diagnosed, untreated AML patients. They represent the most clinically relevant "living material" for studying the pathogenesis and targeted therapy of AML. Human umbilical vein endothelial cells (HUVECs) are primary endothelial cells isolated from the umbilical vein of newborns and are one of the most classic and widely used in vitro models in vascular biology research. This embodiment uses primary AML-CD34... + To investigate the effect of hD1R protein on primary AML-CD34 cells by co-culturing cells with human umbilical vein endothelial cells (HUVECs). + Inhibition of cell proliferation. Specific research details are as follows: 1. hD1R protein purification test: Based on the pET32a-hD1R vector, *E. coli* BL21 was transformed and induced at 25°C for 24 h to achieve soluble fusion expression with TrxHis. Purification was performed using a nickel chelation column targeting the His tag. After protein lysis, high-purity TrxHis-hD1R was obtained. Trx was removed by Thrombin restriction enzyme digestion, followed by dialysis purification of the hD1R protein. All purification steps were strictly performed according to the ProBond™ purification manual and the Thrombin restriction enzyme digestion kit instructions. After filtration and sterilization, hD1R protein was obtained. The average concentration of protein obtained from different batches ranged from 0.7 to 1 mg / mL. 12% SDS-PAGE analysis detected hD1R expression at a molecular weight of approximately 18 kDa. The purified protein was used for subsequent experiments.

[0025] 2. Isolation and culture of human umbilical vein endothelial cells (HUVECs): Under aseptic conditions, umbilical cords approximately 20 cm long were collected from healthy mothers within 2 hours postpartum. The clamped ends of the cord were cut off. A needle was inserted into one end of the umbilical vein, and the vein was thoroughly rinsed with PBS until no blood remained. The distal end of the umbilical vein was then clamped with hemostatic forceps, and the vein was filled with 10 mL of 0.2% collagenase solution. The vein was incubated at 37°C for 20 min, and the cord was gently massaged. The digestive fluid was collected in a centrifuge tube. The umbilical vein was rinsed twice with 30 mL of PBS, and the rinsing fluid was collected together in a centrifuge tube. The tube was centrifuged at 1500 rpm for 10 min, and the supernatant was discarded. The cells were resuspended in complete umbilical vein endothelial cell culture medium (M199, 20% FBS, 30 μg / mL ECGS, 20 units / mL heparin, 100 U / mL penicillin, and 100 μg / mL streptomycin). After counting, the cells were sputtered at 1×10⁻⁶. 5 / mL of cells were seeded into 24-well plates, and complete culture medium was added to each well. The plates were incubated at 37°C, saturated humidity, and 5% CO2. After 24 hours, the cells that did not adhere were removed. The culture medium was changed every 3 days until the cells reached about 90% confluence. The cells were then passaged, and the 3rd to 5th generation umbilical vein endothelial cells were used as supporting cells for subsequent experiments.

[0026] 3. Original AML-CD34 + Cell isolation and culture: Bone marrow samples were collected from AML patients. Bone marrow blood was diluted 1:1 with PBS and mixed thoroughly. The diluted bone marrow blood was then slowly added 1:1 onto the lymphocyte separation medium (1.077 g / ml). Mononuclear cells on the surface of the lymphocyte separation medium were collected. Sorting was performed according to the Human CD34 MicroBead Kit instructions. CD34 was obtained. + Cells were cryopreserved at -80°C for subsequent experiments.

[0027] Frozen CD34 + Cells were thawed and revived using StemSpan. TM Resuspend the cells in serum-free medium and seed them in 6-well plates containing HUVECs as supporting cells, with a cell concentration of approximately 1 × 10⁶ cells per well. 5 Cells were cultured at 37°C with 5% CO2 for 48 hours. The resulting suspension cells were then collected for further analysis. AML cells were then grouped and observed under the following different culture conditions: ①PBS group (blank group): HUVECs + 5GF + PBS + CD34 + Cell co-culture; ②hD1R group (experimental group): HUVECs + 5GF + hD1R + CD34 + Cell co-culture; ③Dox+hD1R group (combined experimental group): HUVECs+5GF+hD1R+Dox+CD34 + Cell co-culture; ④Dox group (control group): HUVECs+5GF+Dox+CD34 + Cell co-culture (Dox group); During the culture process, the working concentration of hD1R protein was 2.5 μg / ml, the Dox concentration was 200 ng / ml, and the same volume of control liquid PBS was used.

[0028] 4. CCK8 assay for cell proliferation: Cells cultured under different culture conditions as described in Method 3 above were collected in suspension. The cells were washed once with PBS and resuspended in culture medium. 90 μL / well was seeded into a 96-well plate, and 10 μL / well of CCK8 reagent was added. After 4 hours, the OD value was measured using a microplate reader at 450 nm.

[0029] CCK8 test results are referenced. Figure 1 As shown, by Figure 1 It can be seen that, compared with the PBS group (blank group), the hD1R group (experimental group) significantly inhibited AML-CD34. + The role of hD1R protein in cell proliferation, specifically its ability to significantly inhibit AML-CD34. + Cell proliferation; compared with the Dox group (control group), the Dox+hD1R group (combined experimental group) showed greater activity against AML-CD34. + Cell proliferation can be synergistically inhibited, that is, the combined use of hD1R protein and Dox can inhibit AML-CD34. + The inhibition of cell proliferation has a synergistic effect.

[0030] 5. Flow cytometry detection of apoptosis: Cells cultured under different culture conditions in Method 3 above were collected in suspension, washed with flow cytometry solution, and 5 μL of Annexin V-FITC and PI were added respectively. The cells were incubated in the dark for 15 min and then detected by flow cytometry to determine the apoptosis status.

[0031] Flow cytometry results refer to Figure 2 As shown, by Figure 2 It can be seen that, compared with the PBS group (blank group), the hD1R group (experimental group) can significantly induce AML-CD34. + Cell apoptosis occurs, meaning the hD1R protein significantly induces AML-CD34.+ The effect of apoptosis; compared with the Dox group (control group), the Dox+hD1R group (combined experimental group) was able to synergistically promote AML-CD34. + Apoptosis of cells, i.e., the combined use of hD1R protein and Dox, promotes AML-CD34. + Apoptosis has a synergistic effect.

[0032] Example 2: Colony-forming units (CFUs) were measured in cells cultured under different culture conditions in Part 3 of Example 1. The specific research details are as follows: Cells cultured under different culture conditions in Part 3 of Example 1 were collected into suspension, washed twice with PBS, and then subjected to a 2×10⁻⁶ solution. 3 / mL was suspended in 1.1ml of methylcellulose semi-solid medium H4434 and seeded in 35mm diameter Petri dishes, with one replicate for each specimen. During incubation, two identical small Petri dishes and one small Petri dish containing only sterile water and without a lid were placed together in a 10cm diameter Petri dish. After incubation at 37℃ in a 5% CO2 humidified incubator for 14 days, colony morphology was observed under an inverted microscope and CFU were counted.

[0033] Community forming unit (CFU) test results as follows Figure 3 As shown, by Figure 3 It can be seen that, compared with the PBS group (blank group), the hD1R group (experimental group) can significantly inhibit AML-CD34. + Cell colony formation, specifically the hD1R protein, can significantly inhibit AML-CD34. + Cell colony formation; compared with the Dox group (control group), the Dox+hD1R group (combined experimental group) was able to synergistically inhibit AML-CD34. + Cell colony formation, i.e., the combined use of hD1R protein and Dox, inhibits AML-CD34. + Cell colony formation has a synergistic effect. This result further indicates that hD1R protein can reduce the stemness of leukemia cells, has the effect of killing tumor stem cells, and could become a drug targeting leukemia stem cells.

[0034] Example 3: To analyze the reasons why hD1R induces apoptosis in AML cells, this study extracted total RNA from cells cultured under different conditions in Part 3 of Example 1, and detected the expression level of Bcl2 mRNA in the cells using real-time quantitative PCR. The specific research content is as follows: Cells cultured under different culture conditions in Part 3 of Example 1 were collected in suspension, and Trizol was added to lyse the cells. Total RNA was extracted from the cells according to the reagent instructions, and the expression level of Bcl2 mRNA in AML cells cultured under different culture conditions was detected by real-time quantitative PCR.

[0035] Real-time quantitative PCR detection results as follows Figure 4 As shown, by Figure 4 It can be seen that, compared with the PBS group (blank group), the hD1R group (experimental group) showed better response to AML-CD34. + The expression of the intracellular anti-apoptotic gene Bcl2 mRNA was significantly inhibited, meaning that hD1R protein could significantly inhibit AML-CD34. + The expression level of the intracellular anti-apoptotic gene Bcl2 mRNA was reduced, thereby promoting AML cell apoptosis; compared with the Dox group (control group), the Dox+hD1R group (combined experimental group) was able to synergistically downregulate AML-CD34. + Intracellular expression of the anti-apoptotic gene Bcl2 mRNA, i.e., hD1R protein, combined with Dox, downregulates AML-CD34. + The expression level of intracellular anti-apoptotic gene Bcl2 mRNA has a synergistic effect.

[0036] Example 4: This embodiment constructs a NOD / SCID mouse transplantation model to simulate human leukemia in mice, thereby testing the therapeutic effect of hD1R protein on transplanted mice. Specific research details are as follows: 1. Constructing a patient-derived mouse model of leukemia transplantation: NOD / SCID mice, female, 6-8 weeks old, were housed in sterile cages and fed a high-pressure sterile diet. Prior to transplantation, they underwent whole-body irradiation with a sublethal dose of 1 Gy, followed by intravenous injection via the tail vein within 4 hours. The transplanted cells were obtained from bone marrow mononuclear cells from AML patients; the isolation of bone marrow mononuclear cells is described in Part 3 of Example 1. Two × 10⁶ cells were used per mouse. 6 Cells were seeded via the tail vein. After AML cell seeding, blood was drawn from the orbital cavity weekly, and the proportion of human hCD45 in peripheral blood was measured by flow cytometry to monitor the AML burden in the peripheral blood. When the average tumor burden reached a certain level, the human hCD45 concentration in the peripheral blood was increased. +When the cell percentage was 0.5-1%, mice were randomly divided into two groups (n=10 per group) based on AML burden. One group received intraperitoneal injection of hD1R 50ug / time (hD1R group) once daily for 7 days, and the other group received intraperitoneal injection of PBS 0.2ml (PBS group) once daily for 7 days. The day of grouping was designated as day 0. Drug administration began on the second day after grouping (day 1). Mice behavior, weight, and survival status were observed daily.

[0037] Mouse Kaplan-Meier survival curve reference Figure 5 As shown, by Figure 5 It can be seen that hD1R protein has a good anti-leukemia effect on transplanted mice. The average survival of mice injected with hD1R protein was 58 days, which was significantly longer than that of mice injected with PBS (average survival was 36.5 days) (P < 0.001).

[0038] Mouse peripheral blood AML cell infiltration status as reference Figure 6 As shown, flow cytometry analysis of the hCD45 ratio in the peripheral blood of mice on day 28 revealed that the hCD45 ratio in the peripheral blood of mice injected with hD1R was significantly lower than that in the PBS group, suggesting that hD1R protein significantly slows down the growth of leukemia cells and reduces the leukemia burden.

[0039] Based on the above examples, it is demonstrated that the hD1R protein can be used for the prevention and treatment of relapsed / refractory acute myeloid leukemia. The hD1R protein has a synergistic effect with anthracycline chemotherapy drugs (Dox) in anti-leukemia treatment and can be used in combination therapy for acute myeloid leukemia.

[0040] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. Use of the hD1R protein in the preparation of a medicament for treating AML.

2. Use according to claim 1, characterized in that: The hD1R protein achieves the treatment of AML by inhibiting the proliferation of AML cells and / or promoting the apoptosis of AML cells.

3. Use according to claim 2, characterized in that: The inhibition of the proliferation of AML cells and / or the promotion of the apoptosis of AML cells includes down-regulating the expression of the Bcl2 gene in AML cells.

4. Use of the hD1R protein and an anthracycline chemotherapy drug in the preparation of a medicament for treating AML.

5. Use according to claim 4, characterized in that: The anthracycline chemotherapy drug includes doxorubicin, epirubicin, daunorubicin, idarubicin, aclarubicin, and pharmaceutically acceptable derivatives thereof.

6. A medicament for treating AML, characterized by: The medicament includes the hD1R protein and a pharmaceutically acceptable excipient.

7. A pharmaceutical composition for treating AML, characterized by: The pharmaceutical composition includes the hD1R protein and the anthracycline chemotherapy drug, and a pharmaceutically acceptable excipient.

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