Application of cyclic creatine in preparation of diffuse large B-cell lymphoma treatment medicine
By studying the creatine metabolism in DLBCL patients, it was found that GAMT upregulation was related to adverse prognosis. Using ring creatine to interfere with creatine energy metabolism and significantly kill DLBCL cells, solving the problem that existing treatment methods are not effective in patients with relapsed/refractory DLBCL. Problem, effective suppression of DLBCL is achieved.
Patent Information
- Application Number
- CN202411587841.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-08
- Publication Date
- 2025-02-07
AI Technical Summary
The existing treatment of diffuse large B-cell lymphoma is not effective in relapsed/refractory patients, and the mechanism of creatine metabolism in the progression of DLBCL is unknown.
Through studying endogenous creatine synthesis and exogenous creatine carriers, it was found that the levels of creatine and creatine creatine creatine in DLBCL patients were elevated, and GAMT was upregulated in the key rate-limiting enzyme related to the endogenous creatine synthesis pathway. Using creatine analog ring creatine interferes with creatine energy metabolism, limits the energy supply of tumor cells, and significantly kills DLBCL cells.
Cyclic creatine interferes with creatine energy metabolism, significantly kills DLBCL cells, inhibits cell proliferation, induces cell apoptosis, and effectively improves patient prognosis.
Smart Images
Figure CN119386010A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of targeted therapy, and specifically relates to the application of cyclocreatine in preparing a drug for treating diffuse large B-cell lymphoma. Background Art
[0002] Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL). The combination of anti-CD20 monoclonal antibody (moAb) rituximab and chemotherapy has significantly improved the outcome of DLBCL. DLBCL is currently considered curable under the standard first-line chemoimmunotherapy regimen R-CHOP. However, in fact, only some patients can benefit from this treatment, and 30-40% of patients are still refractory or relapse after initial remission. First-line treatment failure, especially primary refractory and early relapsed DLBCL patients have a poor overall prognosis and short survival. The current standard treatment for patients with relapsed / refractory diffuse large B-cell lymphoma (R / RDLBCL) is salvage chemotherapy followed by transplantation, but the efficacy is not ideal. There is still a huge unmet medical need, and it is urgent to explore new treatment strategies to break the dilemma. Revealing the pathogenesis of DLBCL and finding new targeted treatment strategies are of great scientific significance for improving patient prognosis and quality of life.
[0003] Metabolic reprogramming is a core feature of tumors. During the occurrence and development of tumors, the malnutrition microenvironment promotes the metabolic reprogramming of tumor cells to meet their needs for rapid proliferation and survival. Metabolic abnormality is one of the important characteristics of tumors. Oncogene activation, tumor suppressor gene inactivation, tumor cell-specific microenvironment, and abnormal activation of certain signaling pathways will regulate metabolic enzymes or metabolic regulatory molecules at multiple levels such as transcription and post-translational modification, causing metabolic reprogramming by affecting multiple mechanisms. Rapidly proliferating cancer cells require a lot of energy to maintain growth. In this case, energy consumption may be an effective strategy to limit cancer cell proliferation. Many studies in recent years have shown that energy metabolic reprogramming is a key factor driving lymphoma disease progression and malignant evolution. Precisely targeting the metabolic weaknesses of tumor cells has important potential clinical application value in the treatment of PTCL.
[0004] Creatine is a nitrogen-containing organic acid. Creatine can be absorbed directly from the diet or synthesized endogenously. Endogenous creatine synthesis mainly occurs through the kidney-liver axis. Arginine and glycine are catalyzed by glycine amidinotransferase (GATM) in the kidney to generate the creatine precursor guanidinoacetate (GAA). Subsequently, GAA enters the blood circulation, is taken up by hepatocytes, and is converted into creatine under the catalysis of guanidinoacetate N-methyltransferase (GAMT). Exogenous creatine enters the blood circulation from hepatocytes and is taken up by cells expressing creatine transporters (CRT / SLC6A8). Under the action of creatine kinase (CK), creatine in the cells is converted into phosphocreatine to provide energy for the cells. In recent years, more and more studies have focused on the role of creatine in cancer, and it has been found that creatine and its analogs inhibit tumor growth by regulating the energy buffering of tumor cells or CD8+T cells in the tumor microenvironment. Some clinical trials have also begun to try to improve muscle mass in cancer patients by supplementing creatine. However, there is also evidence that creatine can promote the progression and metastasis of cancer. Therefore, targeting creatine metabolism has been a hot topic in tumor research in recent years.
[0005] Currently, there are no reports on the mechanism of action of creatine metabolism in the progression of DLBCL. Summary of the invention
[0006] The present invention proposes for the first time the use of creatine analog cyclocreatine in the preparation of therapeutic drugs for diffuse large B-cell lymphoma. First, in view of the unclear mechanism of creatine metabolism in DLBCL, the present invention studies endogenous creatine synthesis and exogenous creatine transporters through key technical means such as public database transcriptome sequencing, molecular biology technology and animal models. The study found that the elevated levels of creatine and creatine-creatine in DLBCL patients are closely related to the poor prognosis of patients; the key rate-limiting enzymes (GATM, GAMT, CKB) related to the endogenous creatine synthesis pathway in DLBCL are upregulated, with GAMT being the most significantly upregulated, which is related to the poor prognosis of patients. As a potential therapeutic target, GAMT's high expression activates endogenous creatine synthesis, causing tumor energy metabolism remodeling, and intracellular creatine is phosphorylated by creatine kinase to form phosphocreatine. Phosphocreatine participates in ATP metabolism, provides energy for cells, and promotes cell proliferation. Promote disease progression; secondly, it was confirmed that the creatine analog cyclocreatine limits the energy supply of tumor cells by interfering with creatine energy metabolism. Cyclocreatine interferes with creatine energy metabolism and can significantly kill tumor cells.
[0007] Furthermore, creatine was used to interfere with the phosphorylation of creatine kinase to form phosphocreatine in diffuse large B-cell lymphoma cells.
[0008] Furthermore, cyclocreatine was used to interfere with creatine energy metabolism in diffuse large B-cell lymphoma cells.
[0009] Furthermore, cyclocreatine is used to inhibit cell proliferation in diffuse large B-cell lymphoma.
[0010] Furthermore, cyclocreatine was used to induce apoptosis in diffuse large B-cell lymphoma.
[0011] Furthermore, cyclocreatine effectively killed diffuse large B-cell lymphoma cells and showed drug concentration dependence in the concentration range of 0 to 10 mM;
[0012] Beneficial effects: The present invention inhibits creatine metabolism and the conversion of creatine to creatine phosphate through creatine analogs, further inhibits ATP production in tumor cells, interferes with creatine energy metabolism, reduces intracellular energy production, thereby inhibiting tumor growth and exerting anti-tumor efficacy. BRIEF DESCRIPTION OF THE DRAWINGS
[0013] Figure 1 A diagram showing the pathway for upregulating endogenous creatine production in DLBCL based on public databases combined with in vitro experiments;
[0014] Figure 2 The figure is a schematic diagram showing the expression of GAMT, the rate-limiting enzyme of the endogenous creatine pathway, in DLBCL, and the results of cell proliferation and apoptosis after knocking out the GAMT gene;
[0015] Figure 3 This is a graph showing the results of a test on cyclocreatine killing DLBCL cells;
[0016] Figure 4 This is a graph showing the results of a test on cyclocreatine inhibiting DLBCL tumor growth. DETAILED DESCRIPTION
[0017] The technical solution of the present invention is described in detail below through test examples, but the protection scope of the present invention is not limited to the test examples.
[0018] The following test methods are used in the present embodiment of the present invention:
[0019] 1.CCK8 cell proliferation ability detection:
[0020] 100ul of DLBCL cell lines treated under different conditions were added to a 96-well plate, with 5 replicate wells in each group. 10ul CCK8 was added to each well in a light-proof condition, incubated for 2-4 hours, and the absorbance value was read at a wavelength of 450nm.
[0021] 2. Flow Cytometry:
[0022] Take 100,000 DLBCL cells, centrifuge them, wash them with PBS, discard the supernatant, add Annexin V-FITC binding solution to gently resuspend the cells, add 5 μl Annexin V-FITC and 5 μl propidium iodide staining solution, mix gently, incubate at room temperature in the dark for 10 minutes, and perform flow cytometry detection.
[0023] 3.Western Blot:
[0024] The drug-treated cells were collected and lysed with RIPA cell lysis buffer, and the proteins were collected after centrifugation and quantified by BCA method. Then, equal amounts of proteins in each group were separated by electrophoresis and electrotransferred to PVDF membranes. After blocking with 5% skim milk for two hours, the primary antibody was incubated overnight, washed with TBST three times, each time for 15 minutes, and then incubated with the secondary antibody at room temperature for two hours. The protein expression was detected by ECL luminescence liquid.
[0025] 4. Creatine / creatine phosphate level determination:
[0026] Collect 2×10 6 The cells were lysed with lysis buffer and the supernatant was transferred to a 10 kDa MWCO spin column after centrifugation. The sample was then centrifuged again and the filtrate was collected. The metabolites were freeze-dried in vacuum and then detected by LC-MS (ABSCIEXQTRAP 6500+), and the protein concentration was determined using the BCA assay.
[0027] 5. Construction of DLBCL Mouse Model
[0028] In this example, a DLBCL mouse model was established by transplanting DLBCL cell lines into NOD SCID mice. Female mice aged 5 to 6 weeks were selected and subcutaneously inoculated with Farage cells in the axilla of the forelimbs. 7 Farage cells were resuspended in PBS to an appropriate density and then mixed with matrix gel at a volume ratio of 1:1 for inoculation. After the tumor was visible to the naked eye, the tumor diameter was measured regularly to calculate the tumor volume. The tumor size calculation formula was: tumor volume (mm 3 ) = 0.5 × (tumor long diameter × tumor short diameter 2 When the tumor grows to 100-300 mm 3 The mice were randomly divided into 2 groups, 6-10 mice in each group, set as negative control group (ddH2O, 50mg / kg / d), and cyclocreatine (50mg / kg / d) was intraperitoneally injected once a day. The tumor size and body weight were detected every 2-3 days, and the monitoring was continued for 9 days. After the 9th day, the tumor weight was measured and photographed to determine the anti-tumor efficacy of the test substance.
[0029] Example 1: Public databases combined with in vitro experiments revealed that the endogenous creatine pathway is highly expressed in DLBCL.
[0030] Figure 1 The public database combined with in vitro experiments revealed that DLBCL upregulated the endogenous creatine production pathway. In the figure: AC shows the expression of GAMT, GATM, and CKB, the rate-limiting enzymes in the endogenous creatine synthesis pathway of DLBCL and adjacent tumors in the public database; D shows the expression of key enzymes and creatine transporters in the endogenous creatine production pathway of DLBCL cell lines verified by qPCR; E shows the expression of GAMT in DLBCL by Western-blot; F shows the creatine level in DLBCL cell lines; G shows the creatine phosphate level in DLBCL cell lines; and H shows the ATP level in DLBCL cell lines. (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).
[0031] from Figure 1 AC found that compared with adjacent adjacent tissues, the expression of GAMT, GATM, and CKB, the rate-limiting enzymes in the endogenous creatine synthesis pathway in DLBCL, was upregulated; Figure 1 The qPCR validation results of D showed that compared with normal B lymphocytes, the expression of key enzymes of endogenous creatine production pathway, GAMT, GATM, and CKB, were upregulated in DLBCL cell lines (Farage, SUDHL8, KIS1, and U2932), with GAMT being the most significantly upregulated, and there was no difference in the expression of creatine transporter SLC6A8; Figure 1 The Western-blot results in middle E demonstrated that GAMT expression was significantly upregulated in DLBCL cell lines compared with normal B lymphocytes; Figure 1 Middle (F) shows that creatine levels in DLBCL cell lines are elevated compared with normal B cells; Figure 1 Middle G shows that DLBCL cell lines have elevated levels of creatine phosphate compared with normal B cells; Figure 1 Figure H shows that the ATP level in DLBCL cell lines is increased compared with normal B cells. The above results suggest that the endogenous creatine pathway plays an important role in the occurrence and development of DLBCL.
[0032] Example 2: GAMT, the rate-limiting enzyme of the endogenous creatine pathway, is upregulated in DLBCL and is associated with poor prognosis; apoptosis of DLBCL cells increases after GAMT knockout.
[0033] This example detects the immunohistochemistry of GAMT in newly diagnosed (ND) and relapsed (RR) DLBCL patients; GAMT expression and survival are analyzed from the GEO database (GSE10846); CRISPR-Cas9 technology is used to knock out the GAMT gene in the DLBCL cell line (Farage), and the knockout efficiency is detected by Western-blot. CCK8 is then used to detect the result of cell proliferation for 24-48 hours, and flow cytometry is used to detect cell apoptosis.
[0034] Figure 2 The expression of GAMT, the rate-limiting enzyme of the endogenous creatine pathway, in DLBCL, and the results of cell proliferation and apoptosis after knocking out the GAMT gene are shown in Figure 1, where: AB are the immunohistochemistry of GAMT in newly diagnosed (ND) and relapsed (RR) DLBCL patients; C is the GAMT expression and survival in the GEO database (GSE10846), (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001); D is a schematic diagram of the knockout efficiency of GAMT gene knockout in DLBCL cell lines; E is a diagram showing the results of CCK8 detection of cell proliferation; Figure 2 F in the figure is the result of flow cytometry detection of cell apoptosis.
[0035] from Figure 2 In the AB of relapsed patients, the expression level of GAMT was found to be higher than that of newly diagnosed patients. Figure 2 It can be seen from the C that the GAMT expression level in the GEO database (GSE10846) is negatively correlated with survival.
[0036] from Figure 2 It can be seen from E that after knocking out GAMT, cell proliferation slowed down; Figure 2 It can be seen from Figure F that cell apoptosis increased after GAMT knockout. Knocking down GAMT can reduce the level of creatine in tumor cells, inhibit cell proliferation, and induce cell apoptosis.
[0037] Example 3: Cyclocreatine kills DLBCL cells and creatine phosphate antagonizes the cell inhibition caused by cyclocreatine.
[0038] In this example, the proliferation capacity of two DLBCL cell lines (Farage, SUDHL8) under different concentration treatment conditions was detected. DLBCL cells in the logarithmic growth phase were co-incubated with cyclocreatine (0 mM, 5 mM, 10 mM) for 1-9 days, and the relative proliferation rate was calculated by cell counting method. After the DLBCL cell lines (Farage, SUDHL8) in the logarithmic growth phase were co-incubated with cyclocreatine (10 mM), phosphocreatine (10 mM) was added or not added to the system;
[0039] The results are as follows Figure 3 As shown, Figure 3 The figures are the results of the test on killing DLBCL cells by cyclocreatine (Sigma, #377627), wherein: A is the result of the cell counting method to detect the inhibition rate of different concentrations of cyclocreatine on DLBCL cell line (Farage); B is the result of the cell counting method to detect the inhibition rate of different concentrations of cyclocreatine on DLBCL cell line (SUDHL8); C is the result of the recovery experiment to detect the addition or absence of phosphocreatine (10 mM) in the system after the DLBCL cell line (Farage) was co-incubated with cyclocreatine (10 mM); D is the result of the recovery experiment to detect the addition or absence of phosphocreatine (Sigma, #10621714001, 10 mM) in the system after the DLBCL cell line (SUDHL8) was co-incubated with cyclocreatine (10 mM); (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).
[0040] Figure 3 A and B in the figure show that after cyclocreatine treatment, the survival rate of DLBCL cell lines was significantly downregulated within a certain range, and the killing effect of cyclocreatine on DLBCL cells was time- and concentration-dependent. With the delay of time and the increase of concentration, the killing effect of cyclocreatine on DLBCL cells increased.
[0041] Figure 3 As shown in C and D, DLBCL cells were treated with cyclocreatine alone in vitro, and cyclocreatine had a killing effect on the DLBCL cell line; adding phosphocreatine to cyclocreatine could partially restore cell viability; the results showed that phosphocreatine could antagonize the inhibitory effect of cyclocreatine on cells.
[0042] Example 4: Cyclocreatine inhibits the growth of DLBCL cells in mice.
[0043] In this study, DLBCL cell lines were transplanted into NOD SCID mice to establish a subcutaneous tumor model of DLBCL mice. The mice were randomly divided into two groups: a negative control group (ddH2O, 50 mg / kg / d) and a cyclocreatine group (50 mg / kg / d). Tumors and body weight were tested every 3 days for 9 consecutive days. After the 9th day, the tumors were removed, photographed, and weighed to determine the antitumor efficacy of the test substance. The results are shown in Figure 4 As shown.
[0044] Figure 4The results of the experiment of cyclocreatine inhibiting the growth of DLBCL tumors are shown in Figure 1, where A is the animal model diagram; B is the DLBCL subcutaneous tumor after treatment with cyclocreatine and placebo ddH2O; C is the size of the DLBCL subcutaneous tumor after treatment with cyclocreatine and placebo ddH2O. (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). It was found that compared with placebo ddH2O, cyclocreatine can significantly inhibit the growth of DLBCL subcutaneous tumors.
[0045] Diffuse large B-cell lymphoma relies on endogenous creatine synthesis to meet the needs of malignant proliferation of tumor cells. DLBCL cell lines have higher levels of creatine, creatine phosphate and ATP than normal B cells. Creatine deprivation inhibits tumor cell growth and promotes tumor cell apoptosis. GAMT is a key rate-limiting enzyme in endogenous creatine synthesis. Knocking down GAMT can reduce creatine levels in tumor cells, inhibit cell proliferation, and induce cell apoptosis. Cyclic creatine interferes with creatine energy metabolism, inhibits the proliferation of diffuse large B-cell lymphoma, and has a killing effect on diffuse large B-cell lymphoma.
[0046] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and details may be made without departing from the spirit and scope of the present invention.
Claims
1. Application of cyclocreatine in the preparation of therapeutic drugs for diffuse large B-cell lymphoma.
2. The use according to claim 1, characterized in that: Cyclocreatine as a creatine metabolism inhibitor in the treatment of diffuse large B-cell lymphoma.
3. The use according to claim 1, characterized in that: Cyclic creatine is used to interfere with the phosphorylation of creatine kinase to form phosphocreatine in diffuse large B-cell lymphoma cells.
4. The use according to claim 1, characterized in that: Cyclocreatine is used to interfere with creatine energy metabolism in diffuse large B-cell lymphoma cells.
5. The use according to claim 1, characterized in that: Cyclocreatine is used to inhibit cell proliferation in diffuse large B-cell lymphoma.
6. The use according to claim 1, characterized in that: Cyclocreatine is used to induce apoptosis in diffuse large B-cell lymphoma.
7. The use according to claim 1, characterized in that: The concentration range of cyclocreatine killing diffuse large B-cell lymphoma cells is 5 to 10 mM.