Use of bevacizumab in the preparation of a medicament for inhibiting hemorrhagic lesions of cerebral vascular malformations

Abstract

The application provides application of bevacizumab in preparation of a medicine for inhibiting hemorrhagic lesions of cerebral vascular malformations, and belongs to the technical field of medicine application. The application firstly discovers and proves by experiments that bevacizumab can be used for binding free VEGF molecules in serum of a patient with a cavernous cerebral vascular malformation, thereby inhibiting a VEGF path, and having obvious inhibiting effect on symptomatic hemorrhagic lesions of the cavernous cerebral vascular malformation, and developing a new use of bevacizumab, and providing a new selection of drug treatment for the patient with the cavernous cerebral vascular malformation.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical application technology, and in particular to the application of bevacizumab in the preparation of drugs for inhibiting hemorrhagic lesions of cerebral vascular malformations. Background Technology

[0002] Bevacizumab is a humanized anti-vascular endothelial growth factor (VEGF) monoclonal antibody. It is an inhibitor of VEGF, capable of binding to and neutralizing all human VEGF-A subtypes and biologically active proteolytic fragments. It is a best-selling cancer treatment drug from Roche and, as an anti-angiogenic agent, it inhibits tumor spread, growth, and enhances the efficacy of chemotherapy by binding to VEGF and blocking tumor angiogenesis. Currently, bevacizumab is widely used to treat lung cancer, colon cancer, rectal cancer, breast cancer, and glioblastoma.

[0003] VEGF pathway activation plays a crucial role in various solid tumors and benign vascular diseases. Currently, bevacizumab is a well-established anti-angiogenic drug for treating various solid tumors; however, there are no reports of its effectiveness in inhibiting symptomatic hemorrhage in familial cavernous malformation. Summary of the Invention

[0004] To address the aforementioned problems, this invention aims to provide the application of bevacizumab in the preparation of drugs for inhibiting hemorrhagic lesions caused by cerebral vascular malformations, particularly in the preparation of drugs for inhibiting symptomatic hemorrhage caused by familial cavernous malformations. This application is the first to discover that bevacizumab can inhibit hemorrhagic lesions caused by cerebral vascular malformations, and further confirms this conclusion in experiments, thus opening up new applications for bevacizumab.

[0005] In one embodiment, the hemorrhagic lesion includes symptomatic hemorrhagic lesions and punctate lesions.

[0006] In one embodiment, the inhibition of hemorrhagic lesions caused by cerebral vascular malformations includes preventing intracranial hemorrhage, reducing the formation of intracranial hemorrhagic lesions, and reducing the size of lesions exceeding 10,000 μm. 2 The number of lesions.

[0007] In one embodiment, the hemorrhagic lesion is caused by familial cavernous malformation.

[0008] In one embodiment, the drug is used to bind to free VEGF molecules in serum, thereby preventing the activation of the VEGF molecular pathway by proto-oncogene mutations.

[0009] In one embodiment, the proto-oncogene is the PIK3CA gene.

[0010] In one embodiment, the proto-oncogene mutation is the H1047R mutation of the PIK3CA gene.

[0011] In one embodiment, the effective dosage of the drug is 1–3 mg / kg.

[0012] Preferably, the effective dosage of the drug is 2 mg / kg.

[0013] In one embodiment, the drug is administered orally or via intravenous infusion.

[0014] In one embodiment, the dosage form of the drug includes injections, tablets, pills, powders, and capsules.

[0015] Compared with the prior art, the present invention has at least the following beneficial effects:

[0016] This application is the first to discover and experimentally demonstrate that bevacizumab can bind to free VEGF molecules in the serum of patients with cavernous vascular malformations, thereby inhibiting the VEGF pathway and significantly inhibiting symptomatic hemorrhage lesions of cavernous vascular malformations. This opens up new applications for bevacizumab and provides a new drug treatment option for patients with familial cavernous vascular malformations. Attached Figure Description

[0017] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. In the drawings:

[0019] Figure 1 The natural history differs between familial cavernous malformation (CCM) symptomatic hemorrhage (ICH) lesions and dot-sized lesions, among which:

[0020] A. Classification of multiple lesions in familial CCM: white arrows indicate ICH lesions, and black arrows indicate punctate lesions;

[0021] B. Total number of lesions, follow-up months, bleeding events, and surgical resection status of enrolled patients;

[0022] C. Kaplan-Meier curves show the incidence of bleeding events between ICH lesions and punctate lesions (Log-rank test p<0.001);

[0023] Representative follow-up MRI of D.ICH lesions (white arrows) and punctate lesions (black arrows);

[0024] Representative follow-up MRI of punctate lesions (black arrows) after surgical resection of E.ICH lesions.

[0025] Figure 2 This is a mutation map of familial CCM, in which:

[0026] A. Germ cell mutations and somatic cell mutations in 18 subgroup lesions of CCM (3 punctate lesions and 15 ICH lesions) were summarized by whole exome sequencing (WES) and / or droplet digital PCR (ddPCR);

[0027] B. ddPCR shows the allelic mutation frequency of PIK3CA mutations in the study, and the pie chart shows the incidence of PIK3CA mutations detected by ddPCR;

[0028] Genotype enrichment analysis of C.ICH and punctate lesions, *.P<0.05.

[0029] Figure 3 This diagram illustrates how PI3K activation in endothelial cells promotes the VEGF signaling pathway, leading to aggravated CCM hemorrhage in mice.

[0030] A. Western blot results, where the left image shows HUVECs transfected with siPTEN, and the right image shows simultaneous transfection with siPTEN and siKrit1;

[0031] B. Schematic diagram of the experimental method;

[0032] C. MRI and representative gross images of the four groups (CTRL: AAV-control+Krit1) fl / fl PIK3CA H1047R :AAV-PIK3CA H1047R +Krit1 fl / fl Krit1 iECKO AAV-control+Krit1 iECKO PIK3CA H1047R +Krit1 iECKO AAV-PIK3CA H1047R +Krit1 iECKO (n=6 per group), white arrows indicate ICH lesions in mice, bar, 1000um;

[0033] D. The number of ICH lesions on MRI in the four groups at P90 (n=6), ****, P<0.0001;

[0034] E. Compare the number of CCM lesions in the cerebrum and cerebellum in the four groups by histological examination (n=6), bar=200um;

[0035] F. Immunofluorescence staining expression of phosphorylated AKT, VEGFA, and TM in brain endothelial cells at P90, bar = 100 μm.

[0036] Figure 4 For bevacizumab to inhibit PIK3CA H1047R +Krit1 iECKO A schematic diagram illustrating the formation of CCM lesions in mice, where:

[0037] A. Schematic diagram of the experimental method;

[0038] B. MRI and representative gross images of the two groups (AAV-PIK3CA) H1047R +Krit1 iECKO + Bevacizumab, n=7, AAV-PIK3CA H1047R +Krit1 iECKO +PBS, n=6), white arrows show ICH lesions in mice, bar, 1000um;

[0039] C. Number of ICH lesions on MRI in both groups at P90 (AAV-PIK3CA) H1047R +Krit1 iECKO + Bevacizumab, n=7, AAV-PIK3CA H1047R +Krit1 iECKO +PBS, n=6), P<0.01;

[0040] D. Comparison of the number of CCM lesions located in the cerebrum and cerebellum between the two groups by histological examination (AAV-PIK3CA) H1047R +Krit1 iECKO + Bevacizumab, n=7, AAV-PIK3CA H1047R +Krit1 iECKO +PBS, n=6), bar=2000um;

[0041] E. and F. represent the total number and size of CCM lesions exceeding 10,000 μm as determined by histological examination in the two groups at P90, respectively. 2 The number of lesions (AAV-PIK3CA) H1047R +Krit1 iECKO + Bevacizumab, n=7, AAV-PIK3CAH1047R +Krit1 iECKO +PBS, n=6), P<0.01, ***, P<0.001. Detailed Implementation

[0042] To more clearly illustrate the overall concept of this application, a detailed description is provided below by way of embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of this application. However, it will be apparent to those skilled in the art that this application can be implemented without one or more of these details. In other instances, to avoid confusion with this application, some technical features well-known in the art have not been described.

[0043] Unless otherwise specified, all reagents or instruments used in the following embodiments, unless otherwise indicated by the manufacturer, are commercially available products. Where specific conditions are not specified in the embodiments, they are performed under standard conditions or conditions recommended by the manufacturer.

[0044] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all adopt conventional techniques in the fields of analytical chemistry, cell culture, and related fields.

[0045] Example 1: Genotypic Analysis of Hemorrhagic Lesions in Cerebral Vascular Malformations

[0046] This embodiment analyzes the genotype of hemorrhagic lesions in cerebral arteriovenous malformation (CCM), taking hemorrhagic lesions caused by familial multiple cavernous malformation (CCM) as an example. First, CCM lesions were divided into two types: symptomatic hemorrhagic lesions and punctate lesions. To explore the clinical characteristics of these two subtypes, 10 patients with multiple lesions carrying germline mutations in the CCM gene were retrospectively reviewed from 2008 to 2021. The results are as follows... Figure 1 As shown in AE.

[0047] Depend on Figure 1 The results of the AB study showed that among these 10 patients, there were 12 hemorrhagic lesions and 505 punctate lesions.

[0048] Depend on Figure 1 The results of CE showed that after a follow-up period of 3230.7 lesion years, 11 lesions experienced bleeding events. Kaplan-Meier analysis showed that the average annual incidence of rebleeding events in the initial hemorrhagic lesions (22.0 / 100 lesion years) was significantly higher than that in the punctate lesions (0.03 / 100 lesion years) (p<0.001).

[0049] The above results indicate that the two types of lesions have drastically different natural histories.

[0050] Furthermore, the genotypes of the two types of lesions were analyzed through gene sequencing, and the results are as follows: Figure 2 As shown in AC.

[0051] Depend on Figure 2 The results of AC showed that, through genetic sequencing analysis of 15 hemorrhagic lesions and 3 punctate lesions, 86.6% (13 / 15) of the hemorrhagic lesions carried somatic mutations in the PIK3CA gene and germline mutations in the CCM gene, while 100% (3 / 3) of the punctate lesions carried only germline mutations in the CCM gene; these findings were statistically significant (p = 0.012).

[0052] The data above indicate that the two types of lesions have different genotypes.

[0053] Example 2: Study on signaling pathways related to hemorrhagic lesions in cerebral vascular malformations

[0054] Furthermore, the function of the PIK3CA mutation was explored in vitro on umbilical vein endothelial cells (HUVECs). The results are as follows: Figure 3 As shown in AF.

[0055] The PI3K signaling was activated by transfecting PTEN with small interfering RNA (siPTEN), and the results were as follows: Figure 3 As shown in Figure A. Figure 3 Western blot analysis (A) revealed that transfection of HUVECs with siPTEN and co-transfection with siPTEN and siKrit1 increased the expression of phosphorylated AKT, VEGFA, ROCK2, and TM proteins. Furthermore, TM expression was reversed by MK-2206 (a PI3K signaling inhibitor) and Fasudil (a ROCK signaling inhibitor). This indicates that, compared to the control group, PTEN knockout HUVECs showed increased levels of phosphorylated AKT (p-AKT), VEGFA, phosphorylated ROCK2 (p-ROCK2), and thrombomodulin (TM). MK-2206 and Fasudil reversed the increased TM expression. Additionally, activation of the PI3K pathway was found to increase the expression of p-AKT, VEGFA, p-ROCK2, and TM in Krit1 knockout HUVECs. MK-2206 and Fasudil also reversed TM expression. Increased TM expression is considered a cause of recurrent bleeding in CCM.

[0056] The genotype-phenotype correlation between the two disease subgroups was further validated in Krit1 knockout mice. The experimental procedures are as follows: Figure 3As shown in B, Krit1 knockout in the endothelium was induced in mice on day 1 of birth (P1) via injection of 4-hydroxytamoxifen (4OHT). iECKO 7T magnetic resonance imaging (MRI) was performed at P30 and P60, and then at P60, mice were injected via the retroorbital vein with control adeno-associated virus (AAV-control) and PIK3CA mutant adeno-associated virus (AAV-PIK3CA). H1047R Mice underwent brain MRI and histological examination at P90. The results are as follows: Figure 3 As shown in CF.

[0057] Depend on Figure 3 C indicates that, on MRI, AAV-PIK3CA H1047R +Krit1 iECKO The group observed typical subacute hemorrhagic lesions, while AAV-control+Krit1 iECKO Only chronic hemorrhagic small lesions were observed in the group.

[0058] Furthermore, by Figure 3 From D and 3E, we know that AAV-PIK3CA H1047R +Krit1 iECKO Group and AAV-control+Krit1 iECKO The mean number of lesions in the groups were 98.2 and 44.5, respectively, in AAV-PIK3CA. H1047R +Krit1 iECKO More large lesions (>10000μm) were observed in the group. 2 ).

[0059] In addition, by Figure 3 As can be seen from F, immunofluorescence staining revealed that it was related to Krit1. fl / fl +AAV-PIK3CA H1047R Or AAV-control+Krit1 iECKO Compared to mice, AAV-PIK3CA at 90 days of age H1047R +Krit1 iECKO The expression of VEGFA and TM was significantly increased in the endothelial cells of the mouse brain.

[0060] The above experimental results indicate that PIK3CA mutations increase lesion bleeding by activating the VEGF pathway.

[0061] Example 3: Inhibitory effect of bevacizumab on hemorrhagic lesions of cerebral vascular malformations

[0062] Both in vitro and in vivo experiments have demonstrated that activation of the PI3K-VEGF pathway plays a crucial role in the genotype-phenotype correlation between the two lesion subgroups. It is hypothesized that VEGF inhibitors may limit bleeding in cavernous malformations. Bevacizumab, a monoclonal antibody that binds to VEGFA isoforms, is an effective treatment for various cancers.

[0063] To investigate the therapeutic effect of bevacizumab on cavernous malformations, such as... Figure 4 As shown in Figure A, the following experimental method was used: Krit1 knockout in mouse endothelium was induced by injection of 4-hydroxytamoxifen (4OHT) on the first day of mouse birth (P1). iECKO Mice underwent MRI at P30 and P60, and then AAV-PIK3CA was injected via the retroorbital vein at P60. H1047R Mice were simultaneously administered bevacizumab 2 mg / kg or PBS twice weekly via tail vein injection for three weeks. Finally, MRI and histological examinations were performed on the mice at P90. The results are as follows: Figure 4 As shown in BF.

[0064] Depend on Figure 4 The results of the BC study showed that, compared with the control group, the mice injected with bevacizumab had almost no hemorrhagic lesions in their tissues, indicating that bevacizumab treatment can effectively inhibit the formation of hemorrhagic lesions.

[0065] At the same time, by Figure 4 The results from DF showed that hematoxylin and eosin staining indicated that bevacizumab treatment significantly reduced the number and size of CCM lesions exceeding 10,000 μm. 2 The number of lesions. Experimental data indicate that bevacizumab is a potential drug for effectively inhibiting hemorrhagic lesions caused by cavernous malformations.

[0066] The above description is merely an embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of this application should be included within the scope of the claims of this application.

Claims

1. The use of bevacizumab in the preparation of a drug for inhibiting hemorrhagic lesions in familial cavernous malformation, wherein the hemorrhagic lesions include symptomatic hemorrhagic lesions and punctate lesions; wherein the drug is used to bind to free VEGF molecules in serum to prevent activation of the VEGF molecular pathway by proto-oncogene mutations, wherein the proto-oncogene mutation is the H1047R mutation of the PIK3CA gene.

2. The application according to claim 1, characterized in that, The suppression of hemorrhagic lesions in familial cavernous malformations includes preventing intracranial hemorrhage, reducing the formation of intracranial hemorrhagic lesions, and reducing lesions larger than 10,000 µm. 2 The number of lesions.

3. The application according to claim 1, characterized in that, The effective dosage of the drug is 1~3 mg / kg.

4. The application according to claim 1, characterized in that, The drug can be administered orally or via intravenous infusion.

5. The application according to claim 1, characterized in that, The dosage forms of the drug include injections, tablets, pills, powders, or capsules.

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