LOX targeted intervention method for treating idiopathic thrombocythemia

Abstract

The invention discloses an LOX targeted intervention method for treating idiopathic thrombocythemia, and relates to the technical field of medicine and health, and the LOX targeted intervention method comprises the following steps: detecting the LOX expression level of a patient, collecting a peripheral blood or bone marrow sample of an ET patient, detecting the mRNA expression level of LOX through real-time fluorescent quantitative PCR, screening a patient with high LOX expression, applying an LOX specific inhibitor, and giving the LOX inhibitor to the patient with high LOX expression. By means of individualized treatment of LOX expression layering, accurate intervention of patients with high LOX expression is achieved, the problem that at present, the effect of LOX in the ET disease pathogenesis is not clear is solved, deep understanding of the ET pathogenesis is facilitated, and a theoretical basis is provided for subsequent targeted treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of medicine and health, and specifically to a LOX-targeted intervention method for the treatment of essential thrombocythemia. Background Art

[0002] Essential thrombocythemia (ET) is a myeloproliferative neoplasm (MPN) negative for the Philadelphia chromosome / BCR-ABL fusion gene, characterized by abnormal proliferation of megakaryocytes in the bone marrow and persistent thrombocytosis. ET patients often have a high risk of thrombosis and bleeding, and some cases may progress to myelofibrosis or leukemia. Currently, the main treatment methods rely on JAK2 inhibitors (such as ruxolitinib), but they cannot reverse myelofibrosis or achieve a radical cure, and there is an urgent need for new treatment targets.

[0003] Therefore, it is very necessary to propose a LOX-targeted intervention method for the treatment of essential thrombocythemia to solve the problems in the background.

[0004] Patent document CN113082035B discloses the application of LY3009120 in the preparation of drugs for the treatment of myeloproliferative neoplasms. The above patent enables the provision of continuous oral drug treatment for patients, avoiding the need for bone marrow transplantation. LY3009120 can be chemically synthesized, with a lower cost than biological agents, and has passed phase I clinical trials, with fewer and milder side effects and good tolerance in clinical patients.

[0005] In summary, the above patent aims to provide oral drugs to avoid bone marrow transplantation, but the existing problems still include that JAK2 inhibitors cannot reverse myelofibrosis, the specific mechanism of action of LOX in ET has not been clearly studied, and there is also a lack of targeted treatment strategies for LOX; Therefore, the present application proposes a LOX-targeted intervention method for the treatment of essential thrombocythemia that can achieve precise intervention in patients with high LOX expression, thereby blocking the fibrotic pathway and establishing the relationship between LOX and the prognosis of ET. Summary of the Invention

[0006] The purpose of the present invention is to provide a LOX-targeted intervention method for the treatment of essential thrombocythemia, so as to solve the technical problem of inability to reverse myelofibrosis or achieve a radical cure proposed in the above background art.

[0007] To achieve the above purpose, the present invention provides the following technical solution: A LOX-targeted intervention method for the treatment of essential thrombocythemia, comprising the following steps: Step 1: Detect the LOX expression level of the patient, collect peripheral blood or bone marrow samples of ET patients, and detect the mRNA expression level of LOX by real-time fluorescence quantitative PCR to screen for patients with high LOX expression; Step 2: Apply LOX specific inhibitor. Administer LOX inhibitor to patients with high LOX expression at a dose of 10 - 50 mg / kg / day for 4 - 8 weeks. Step 3: Monitor the treatment response. Regularly perform biopsies to detect platelet count, degree of bone marrow fibrosis, morphology of megakaryocytes, and serum fibrosis markers to evaluate the LOX inhibition effect. Step 4: Optimize the treatment by combining with JAK2 inhibitor. For patients with JAK2 - V617F mutation, co - administer LOX inhibitor and ruxolitinib at a dose of 5 - 20 mg / day to synergistically inhibit abnormal proliferation and fibrosis.

[0008] Preferably, the LOX inhibitor is selected from small - molecule LOX inhibitors, LOX - specific siRNA / monoclonal antibodies, and the dose is 10 - 50 mg / kg / day.

[0009] Preferably, for patients with JAK2 - V617F mutation, co - administer LOX inhibitor and JAK2 inhibitor.

[0010] Preferably, the JAK2 inhibitor is ruxolitinib, and the dose is 5 - 20 mg / day.

[0011] Preferably, the expression level of LOX mRNA is detected by fluorescence quantitative PCR, and the threshold is set to be more than 2 times the expression level of the control group to determine high expression.

[0012] Preferably, the treatment response criteria are that the platelet count returns to <450×10^9 / L and the bone marrow fibrosis score decreases by ≥30%.

[0013] Preferably, the small - molecule LOX inhibitor is β - aminopropionitrile, and the administration dose is 10 - 50 mg / kg per day, by intravenous injection / oral administration. The sequences of LOX - specific siRNA are SEQ ID NO: 1 - 3, and are delivered to bone marrow megakaryocytes by liposome carriers.

[0014] Preferably, the intervention period is 6 - 12 months. Perform bone marrow biopsies and hematological evaluations every 3 months. Combine LOX - targeted inhibitor with anti - platelet drugs to reduce the risk of thrombosis. The method further includes genotyping the patients to detect the mutation status of CALR, MPL, and JAK2 - V617F.

[0015] Preferably, the detection of LOX expression uses real - time fluorescence quantitative PCR, and the primer sequences are: Forward primer: 5′ - CCTGGTGAAGGTGCTGATC - 3′ SEQ ID NO: 4, Reverse primer: 5′-AGCCACATCGCTCAGACAC-3′ SEQ ID NO:5.

[0016] Preferably, the degree of myelofibrosis is evaluated by the WHO fibrosis grading standard, including reticular fiber staining and collagen immunohistochemical analysis; the risk assessment of thrombosis / bleeding events includes D-dimer, prothrombin time, and platelet function test PFA-100.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Through individualized treatment stratified by LOX expression, the present invention achieves precise intervention for patients with high LOX expression, solves the problem that the role of LOX in the pathogenesis of ET disease is unclear at present, helps to deeply understand the pathogenesis of ET, and provides a theoretical basis for subsequent targeted treatment. 2. By using β-aminopropionitrile as a LOX inhibitor, the present invention achieves specific blockade of the LOX-mediated collagen cross-linking and fibrosis pathway, solves the problem of irreversible myelofibrosis, can effectively reduce the degree of myelofibrosis, and provides an effective research tool for further studying the role of LOX in ET disease. 3. By combining a LOX inhibitor with a JAK2 inhibitor, the present invention achieves dual-mechanism inhibition of platelet proliferation and bone marrow remodeling, solves the problem of abnormal platelet proliferation in patients, helps to understand the cause of abnormal platelet proliferation in ET patients, and provides clues for developing treatment methods for abnormal platelet proliferation. 4. By monitoring the treatment response, the present invention establishes the connection between LOX and the prognosis of ET, solves the problem of being unable to accurately evaluate the impact of LOX on the prognosis of ET patients, can help doctors more accurately evaluate the disease development and prognosis of ET patients, and provides a reference for formulating a more reasonable treatment plan. Brief Description of the Drawings

[0018] Figure 1 It is a schematic flow chart of the intervention method of the present invention; Figure 2 It is an influence curve of the LOX inhibitor (BAPN) of the present invention on platelet count. Detailed Embodiments

[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0020] Please refer to Figure 1, An embodiment provided by the present invention: A LOX-targeted intervention method for the treatment of essential thrombocythemia, comprising the following steps: Step 1, Detect the LOX expression level of the patient. Collect the peripheral blood or bone marrow samples of ET patients, and detect the mRNA expression level of LOX by real-time fluorescence quantitative PCR to screen for patients with high LOX expression; Step 2, Apply a LOX-specific inhibitor. Administer a LOX inhibitor to patients with high LOX expression at a dose of 10-50 mg / kg / day for 4-8 weeks; Step 3, Monitor the treatment response. Regular biopsies are performed to detect platelet counts, the degree of bone marrow fibrosis, megakaryocyte morphology, and serum fibrosis markers to evaluate the LOX inhibition effect; Step 4, Optimize the treatment by combining with a JAK2 inhibitor. For patients with the JAK2-V617F mutation, a LOX inhibitor and ruxolitinib are used in combination at a dose of 5-20 mg / day to synergistically inhibit abnormal proliferation and fibrosis; The LOX inhibitor is selected from small molecule LOX inhibitors, LOX-specific siRNA / monoclonal antibodies, and the dose is 10-50 mg / kg / day; Furthermore, verify that the mRNA expression of LOX in the peripheral blood of ET patients is significantly higher than that of healthy people, analyze its correlation with the JAK2-V617F mutation, and collect samples. Observation group: Confirmed ET patients (n = 50), meeting the WHO (2016) diagnostic criteria; Control group: Healthy physical examination subjects during the same period (n = 30), with normal platelet counts and no hematological diseases; RNA extraction and reverse transcription: Collect 5 mL of peripheral venous blood, extract total RNA using TRIzol reagent, and reverse transcribe RNA into cDNA using the PrimeScript™ RT kit (Takara); Real-time fluorescence quantitative PCR (qPCR): Primer sequences: LOX forward primer: 5′-CCTGGTGAAGGTGCTGATC-3′ (SEQ ID NO: 4), LOX reverse primer: 5′-AGCCACATCGCTCAGACAC-3′ (SEQ ID NO: 5); Internal reference gene GAPDH primers: Forward: 5′-GGAGCGAGATCCCTCCAAAAT-3′ Reverse: 5′-GGCTGTTGTCATACTTCTCATGG-3′; Reaction system: SYBR Premix Ex Taq™ II (TaKaRa) 10 μL, cDNA 2 μL, each primer 0.5 μL, supplemented with ddH2O to 20 μL. Reaction conditions: Pre-denaturation at 95°C for 30 seconds, 95°C for 5 seconds, 60°C for 30 seconds, for a total of 40 cycles; JAK2-V617F mutation detection: ARMS-PCR method was used; data analysis: the relative expression level of LOX mRNA was calculated by the 2−ΔΔCt method, independent sample t-test was used for comparison between groups, and Pearson analysis was used for the correlation between mutation and expression. The LOX mRNA expression level in the ET group was 3.2 times that of the control group (P<0.001). The LOX expression in patients with JAK2-V617F mutation (n = 32) was significantly higher than that in non-mutated patients (P = 0.02). The conclusion was drawn that LOX was highly expressed in ET patients and was positively correlated with JAK2-V617F mutation, suggesting that it might be involved in the pathological process of ET.

[0021] Please refer to Figure 1 , an embodiment provided by the present invention: a LOX-targeted intervention method for the treatment of essential thrombocythemia. For patients with JAK2-V617F mutation, a LOX inhibitor and a JAK2 inhibitor are used in combination; The JAK2 inhibitor is ruxolitinib, and the dosage is 5 - 20 mg / day; Furthermore, to evaluate the effect of inhibiting LOX activity by β-aminopropionitrile (BAPN) on the degree of bone marrow fibrosis in ET patients, high LOX expression patients (n = 20) orally take BAPN (Sigma, purity ≥98%) 30 mg / kg per day for a course of 6 months. The control group (n = 20) only receives conventional treatment (aspirin + hydroxycarbamide). Bone marrow aspiration is performed before and after treatment, and the silver staining method and Masson trichrome staining are used to evaluate the degree of fibrosis, which is graded according to the WHO standard: grade 0 (no fibrosis) to grade 3 (extensive fibrosis); Efficacy indicators: platelet count (PLT), bone marrow fibrosis grade, serum LOX activity (detected by ELISA method); After treatment in the BAPN group: PLT decreased from 650×10 9 / L to 380×10 9 / L (P<0.001) Bone marrow fibrosis decreased from grade 2 to grade 1 (P = 0.008) Serum LOX activity decreased by 52% (P = 0.003) There was no obvious change in the control group (P>0.05) Conclusion: BAPN can effectively inhibit LOX activity, significantly improve bone marrow fibrosis and reduce platelet count.

[0022] Please refer to Figure 1 and Figure 2 , an embodiment provided by the present invention: a LOX-targeted intervention method for the treatment of essential thrombocythemia. The expression level of LOX mRNA is detected by fluorescence quantitative PCR, and the threshold is set to be more than 2 times the expression level of the control group to be determined as high expression; The treatment response criteria are that the platelet count returns to <450×10^9 / L and the myelofibrosis score decreases by ≥30%; Furthermore, to explore the correlation between the LOX expression level and the incidence of thrombosis / bleeding events in ET patients, 100 ET patients were included and divided into a high-expression group (n = 50) and a low-expression group (n = 50) according to the median LOX mRNA expression. They were followed up for 12 months, and thrombosis (such as deep vein thrombosis, cerebral infarction) and bleeding events (such as gastrointestinal bleeding) were recorded. The detection indicators were: D-dimer, prothrombin time (PT), and platelet aggregation rate (PFA-100). The survival curve was plotted using the Kaplan-Meier method, and the Cox regression analysis was used to analyze the LOX expression and the hazard ratio (HR) of the event; The incidence of thrombosis events in the high-expression group was 28% (14 / 50), and in the low-expression group was 8% (4 / 50) (P = 0.007). The D-dimer level in patients with high LOX expression was significantly increased (P = 0.01). Cox regression showed that high LOX expression was an independent risk factor for thrombosis events (HR = 3.2, P = 0.003); Conclusion: High LOX expression is significantly associated with an increased risk of thrombosis events in ET patients and can be used as a prognostic evaluation indicator.

[0023] Please refer to Figure 1 , an embodiment provided by the present invention: a LOX-targeted intervention method for the treatment of essential thrombocythemia. The small molecule LOX inhibitor is β-aminopropionitrile, and the administration dose is 10 - 50 mg / kg per day, by intravenous injection / oral administration; the sequences of LOX-specific siRNA are SEQ ID NO:1 - 3, and are delivered to megakaryocytes in the bone marrow by a liposome carrier; The intervention period is 6 - 12 months. Bone marrow biopsy and hematological evaluation are performed every 3 months. The LOX-targeted inhibitor is used in combination with antiplatelet drugs to reduce the risk of thrombosis; the method further includes genotyping the patients to detect the mutation status of CALR, MPL, and JAK2-V617F; Furthermore, to verify the ability of LOX-specific siRNA to inhibit the proliferation and collagen secretion of the megakaryocyte cell line (MEG-01), MEG-01 cells (ATCC CRL-2021) were cultured in RPMI-1640 medium (containing 10% FBS), and the siRNA sequences (targeting LOX) were: siRNA-1: 5′-GCAUGAAGCUCAUCGAAUA-3′ (SEQ ID NO:1) siRNA-2: 5′-CCUGGACAUCAUGAAGAUA-3′ (SEQ ID NO:2); Transfection was performed using Lipofectamine 3000 (Invitrogen), and the negative control was Scramble siRNA; Detection indicators: Cell proliferation was detected by the CCK-8 method (0 h, 24 h, 48 h), and the expression of LOX protein and the secretion of collagen I (COL1A1) were detected by Western blot; In the siRNA-1 group: The expression of LOX protein decreased by 68% (P < 0.001), the cell proliferation inhibition rate reached 42% at 48 h (P = 0.001), and the secretion of COL1A1 decreased by 55% (P = 0.005). Conclusion: LOX-specific siRNA can significantly inhibit the proliferation of megakaryocytes and collagen secretion, providing an experimental basis for targeted therapy.

[0024] Please refer to Figure 1 , an embodiment provided by the present invention: A LOX-targeted intervention method for the treatment of essential thrombocythemia. The detection of LOX expression was performed by real-time fluorescence quantitative PCR, and the primer sequences were: forward primer: 5′-CCTGGTGAAGGTGCTGATC-3′ SEQ ID NO:4, reverse primer: 5′-AGCCACATCGCTCAGACAC-3′ SEQ ID NO:5; The degree of myelofibrosis was evaluated by the WHO fibrosis grading standard, including reticular fiber staining and collagen immunohistochemical analysis; The risk assessment of thrombosis / bleeding events included D-dimer, prothrombin time, and platelet function test PFA-100; Furthermore, the synergistic therapeutic effect of the LOX inhibitor (BAPN) combined with the JAK2 inhibitor (ruxolitinib) was evaluated. Monotherapy group (n = 15): Ruxolitinib 15 mg / day, combination group (n = 15): Ruxolitinib 15 mg / day + BAPN 30 mg / kg / day, for a course of 6 months; Efficacy indicators: Platelet count, spleen volume (measured by ultrasound), myelofibrosis grading, and adverse reactions such as abnormal liver and kidney function and anemia were recorded; Combination group: The rate of decrease in PLT was 40% faster than that in the monotherapy group (P = 0.004) The spleen volume decreased by 35% (20% in the monotherapy group, P = 0.02) The reversal rate of myelofibrosis was 60% (25% in the monotherapy group, P = 0.01) Safety: There was no difference in the incidence of adverse reactions between the two groups (P > 0.05); Conclusion: The combination of the LOX inhibitor and the JAK2 inhibitor can synergistically improve thrombocytosis and fibrosis in ET patients, and the safety is controllable.

[0025] Working principle: ET patients undergoing inpatient treatment were used as the observation group, and healthy subjects undergoing physical examinations during the same period were used as the control group; The real-time fluorescence quantitative PCR method was adopted. First, the total RNA of cells in peripheral venous blood specimens was extracted, and after detecting its concentration and purity, it was stored. Then the total RNA was reverse transcribed into cDNA and diluted for standby. The cDNA fragment was amplified by a kit, and the amplified product was separated by agarose gel electrophoresis. The mRNA expression of LOX was read by taking pictures under a gel imaging microscope, so as to understand the gene expression differences of LOX in ET patients and healthy controls. Peripheral venous blood specimens were collected, and the morphology of blood cells and platelets and the number of immature cells were directly observed to analyze the differences between ET patients and healthy subjects from the perspective of cell morphology and assist in judging the condition; Overnight fasting venous blood was drawn, and serological indexes such as blood routine and coagulation indexes were measured using an automated full blood cell analyzer to evaluate the blood components and coagulation function status of the patients, providing data support for studying the effect of LOX on the blood system of ET patients. Six months after discharge, the international prognostic scoring system DIPSS was used to evaluate the prognosis of the two groups of patients. This system is scored based on 5 independent predictors such as age and hemoglobin, and the higher the score, the better the prognosis, so as to clarify the relationship between LOX and the prognosis of ET patients.

[0026] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be embraced within the present invention. Any reference signs in the claims should not be regarded as limiting the claimed rights.

Claims

1. A LOX-targeted intervention method for the treatment of essential thrombocythemia, characterized in that: It includes the following steps: Step 1: Detect the LOX expression level of the patient. Collect the peripheral blood or bone marrow sample of the ET patient, and detect the mRNA expression level of LOX by real-time fluorescence quantitative PCR to screen the patients with high LOX expression. Step 2: Apply a LOX-specific inhibitor. Administer the LOX inhibitor to the patients with high LOX expression at a dose of 10 - 50 mg / kg / day for 4 - 8 weeks. Step 3: Monitor the treatment response. Regularly perform biopsies to detect platelet count, degree of bone marrow fibrosis, morphology of megakaryocytes, and serum fibrosis markers to evaluate the LOX inhibition effect. Step 4: Optimize the treatment by combining with a JAK2 inhibitor. For the patients with JAK2-V617F mutation, jointly use the LOX inhibitor and ruxolitinib at a dose of 5 - 20 mg / day to synergistically inhibit abnormal proliferation and fibrosis.

2. The LOX-targeted intervention method for the treatment of essential thrombocythemia according to claim 1, wherein: The LOX inhibitor is selected from small molecule LOX inhibitors, LOX-specific siRNA / monoclonal antibodies, and the dose is 10 - 50 mg / kg / day.

3. The LOX-targeted intervention method for the treatment of essential thrombocythemia according to claim 1, wherein: For the patients with JAK2-V617F mutation, jointly use the LOX inhibitor and the JAK2 inhibitor.

4. The LOX-targeted intervention method for the treatment of essential thrombocythemia according to claim 1, wherein: The JAK2 inhibitor is ruxolitinib, and the dose is 5 - 20 mg / day.

5. The LOX-targeted intervention method for the treatment of essential thrombocythemia according to claim 1, wherein: When detecting the LOX mRNA expression level by fluorescence quantitative PCR, a threshold set at more than 2 times the expression level of the control group is determined as high expression.

6. The LOX-targeted intervention method for the treatment of essential thrombocythemia according to claim 1, wherein: The treatment response standard is that the platelet count returns to <450×10^9 / L and the bone marrow fibrosis score decreases by ≥30%.

7. The LOX-targeted intervention method for the treatment of essential thrombocythemia according to claim 2, wherein: The small molecule LOX inhibitor is β-aminopropionitrile, and the administration dose is 10 - 50 mg / kg per day, by intravenous injection / oral administration; The sequences of LOX-specific siRNA are SEQ ID NO:1 - 3, and are delivered to bone marrow megakaryocytes by a liposome carrier.

8. The LOX-targeted intervention method for the treatment of essential thrombocythemia according to claim 1, wherein: The intervention period is 6 - 12 months. Perform bone marrow biopsies and hematological evaluations every 3 months. Combine the LOX-targeted inhibitor with antiplatelet drugs to reduce the thrombus risk. The method further includes genotyping the patient to detect the mutation status of CALR, MPL, and JAK2-V617F.

9. The LOX-targeted intervention method for the treatment of essential thrombocythemia according to claim 1, characterized in that: The detection of LOX expression uses real-time fluorescence quantitative PCR, and the primer sequences are: Forward primer: 5′-CCTGGTGAAGGTGCTGATC-3′SEQ ID NO:4, Reverse primer: 5′-AGCCACATCGCTCAGACAC-3′SEQ ID NO:

5.

10. The LOX-targeted intervention method for the treatment of essential thrombocythemia according to claim 1, characterized in that: The degree of bone marrow fibrosis is evaluated by the WHO fibrosis grading standard, including reticular fiber staining and collagen immunohistochemical analysis; The risk assessment of thrombus / bleeding events includes D-dimer, prothrombin time, and platelet function test PFA-100.

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