WO2024002192A1 - Response marker for anti-vascular endothelial growth factor therapy of age-related macular degeneration and use thereof - Google Patents

Abstract

Provided in the present invention are a response marker for an anti-vascular endothelial growth factor therapy of age-related macular degeneration and the use thereof. Specifically, provided in the present invention is the use of an anti-VEGF responsive marker detection reagent in the preparation of a diagnostic reagent or a kit for judging the anti-VEGF responsiveness. Studies show that the anti-VEGF responsive marker can be used as a marker for judging the anti-VEGF responsiveness and evaluating the therapeutic effect of the anti-VEGF, and has a high sensitivity and specificity.

Description

Response markers to anti-vascular endothelial growth factor therapy in age-related macular degeneration and their applications Technical field

The present invention relates to the field of biomedicine, and specifically to response markers for anti-vascular endothelial growth factor therapy in age-related macular degeneration and their applications.

Background technique

Age-related macular degeneration (AMD) is the leading cause of visual impairment and blindness in people over 50 years old worldwide. As the aging population increases, more than 20% of the world's population may suffer from AMD. The 2010 Global Burden of Disease Study reported that the number of healthy life years lost due to vision-related disability caused by AMD increased by 160%. AMD is divided into dry AMD and neovascular AMD (nAMD). nAMD is characterized by the occurrence of choroidal neovascularization (CNV) under the fovea of the macula, which can cause structural damage to the macula, leading to irreversible central vision damage or even blindness. Polypoidal chorioretinopathy (PCV) is a subtype of nAMD that is more common in Asian populations and manifests as serous or hemorrhagic retinal pigment epithelial detachment, subretinal hemorrhage, and orange-red subretinal nodular lesions.

The common pathological mechanism of nAMD and PCV is pathological neovascularization mediated by vascular endothelial growth factor (VEGF). Therefore, intravitreal injection of anti-VEGF drugs is recommended by various international guidelines as the first-line treatment for such diseases. However, although multiple randomized double-blind controlled clinical trials (RCTs) have reported the efficacy and safety of anti-VEGF drugs in the treatment of nAMD and PCV, about 30% of patients still have no response or poor response to anti-VEGF treatment in the real world. . Recent studies have shown that individual differences in response to anti-VEGF therapy depend on a variety of factors, including the patient's age, disease duration, lifestyle, local anatomy of the lesion, and genetic polymorphisms. At the same time, although intravitreal injections have been clinically proven to be safe, the risk of infection from repeated intraocular injections remains. In addition, anti-VEGF drugs are biological drugs and are expensive. The high cost of monthly treatment (approximately 4,000 yuan/month) also brings a heavy financial burden to AMD patients.

Therefore, there is an urgent need in this field to develop an effective, simple, and economical method to predict an individual's response to intraocular injection of anti-VEGF drugs for the treatment of AMD, guide precise medication, and formulate personalized treatment plans.

Contents of the invention

The purpose of the present invention is to provide a specific marker and detection method for early evaluation of the response to intraocular injection of anti-VEGF drugs for the treatment of AMD with high sensitivity and specificity. Specifically, it relates to response markers for anti-vascular endothelial growth factor therapy in age-related macular degeneration and their applications.

In the first aspect of the present invention, there is provided the use of an anti-VEGF responsive marker detection reagent for preparing a diagnostic reagent or kit, which is used to (a) determine whether a certain subject adopts Responsiveness to anti-VEGF therapy, and/or (b) evaluating the therapeutic efficacy of anti-VEGF therapy for AMD in a subject;

Wherein, the anti-VEGF responsive marker is selected from the following group:

(A) Any marker selected from A1 to A24, or a combination thereof: (A1) LPC 18:0 sn-1; (A2) PC 38:6; (A3) SM 34:1; (A4) PC 34 :0; (A5)PC 34:1; (A6)PC 38:5; (A7)PC 36:3; (A8)PC O-38:6; (A9)PC 36:2; (A10)LPE 22 :6 sn-1; (A11)PC O-38:5; (A12)PE O-38:7; (A13)PC O-36:5; (A14)LPC O-16:1; (A15)PC 38:4; (A16)LPC 16:1 sn-1; (A17)PE O-36:5; (A18)LPC 15:0 sn-2; (A19)PC 34:2; (A20)PC O- 36:4; (A21)PC 36:4; (A22)LPC 16:1sn-2; (A23)PC 33:1; (A24)PC 38:7.

In another preferred example, the anti-VEGF responsive marker also includes any marker selected from the following group B, or a combination thereof: (B1) LPC 16:0 sn-1; (B2) LPC 16:0 sn-2; (B3)LPC 18:1 sn-1; (B4)LPC 18:2sn-1; (B5)LPC 22:6 sn-1.

In another preferred embodiment, each biomarker is identified by mass spectrometry, preferably by chromatography-mass spectrometry, such as gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS). Law.

In another preferred example, the anti-VEGF responsive markers further include at least 2 selected from A1 to A24.

In another preferred embodiment, the anti-VEGF responsive marker is selected from a combination of one or more markers from A1 to A24 and one or more markers from B1 to B5.

In another preferred embodiment, the diagnostic reagent or kit is used to detect anti-VEGF responsive markers in serum.

In another preferred example, the A1-A7 markers are selected from Table A:

Table A

In another preferred example, the B1-B5 markers are selected from Table B:

Table B

In another preferred embodiment, the AMD includes neovascular AMD.

In another preferred embodiment, the neovascular AMD also includes polypoidal chorioretinopathy (PCV).

In a second aspect of the present invention, a kit is provided. The kit contains a detection reagent, and the detection reagent is used to detect anti-VEGF responsive markers in the sample to be tested,

Wherein, the anti-VEGF responsive marker is selected from the following group:

(A) A combination of two or more markers selected from A1 to A24;

(B) A combination of one or more markers selected from A1 to A24 and one or more markers from B1 to B5.

In another preferred embodiment, the sample to be tested is from a subject selected from the following group: non-AMD subjects, AMD subjects who have not used anti-VEGF therapy, and subjects who have used anti-VEGF therapy.

In another preferred embodiment, the object is a human.

In a third aspect of the present invention, a detection method is provided, including the steps:

(a) Provide a test sample, the test sample is selected from a serum sample;

(b) detecting the concentration of the anti-VEGF responsive marker in the test sample, recorded as C1; and

(c) comparing the concentration C1 of the anti-VEGF responsive marker with the control reference value C0,

Wherein, the anti-VEGF responsive marker is selected from the following group:

(A) Any marker selected from A1 to A24, or a combination thereof: (A1) LPC 18:0 sn-1; (A2) PC 38:6; (A3) SM 34:1; (A4) PC 34 :0; (A5)PC 34:1; (A6)PC 38:5; (A7)PC 36:3; (A8)PC O-38:6; (A9)PC 36:2; (A10)LPE 22 :6 sn-1; (A11)PC O-38:5; (A12)PE O-38:7; (A13)PC O-36:5; (A14)LPC O-16:1; (A15)PC 38:4; (A16)LPC 16:1 sn-1; (A17)PE O-36:5; (A18)LPC 15:0 sn-2; (A19)PC 34:2; (A20)PC O- 36:4; (A21)PC 36:4; (A22)LPC 16:1sn-2; (A23)PC 33:1; (A24)PC 38:7;

If the test result of the anti-VEGF responsive marker of the test subject meets the following conditions, it will indicate that the AMD patient is suitable for anti-VEGF treatment and has good results:

(1) When a certain marker is an up-regulated marker in Table A in the test subject, and the expression level of the marker is higher than the reference value or standard value C0, the test subject will have a good effect of anti-VEGF treatment. ;

(2) When a certain marker is a down-regulated marker in Table A in the test subject, and the expression level of the marker is lower than the reference value or standard value C0, the test subject will have a good effect of anti-VEGF treatment. .

In a fourth aspect of the present invention, a diagnostic device is provided, the device comprising:

(a) Input module, the input module is used to input anti-VEGF marker data of a certain subject's serum sample;

The response markers to anti-VEGF therapy include those selected from the group consisting of:

(A1)LPC 18:0 sn-1; (A2)PC 38:6; (A3)SM 34:1; (A4)PC 34:0; (A5)PC 34:1; (A6)PC 38:5 ;(A7)PC 36:3;(A8)PC O-38:6;(A9)PC 36:2;(A10)LPE 22:6 sn-1;(A11)PC O-38:5;(A12 )PE O-38:7; (A13)PC O-36:5; (A14)LPC O-16:1; (A15)PC 38:4; (A16)LPC 16:1 sn-1; (A17) PE O-36:5; (A18)LPC 15:0 sn-2; (A19)PC 34:2; (A20)PC O-36:4; (A21)PC 36:4; (A22)LPC 16: 1 sn-2; (A23) PC 33:1; (A24) PC 38:7;

(b) A processing module that compares the input marker concentration C1 with the control reference value C0 to obtain a discrimination result, wherein when the response marker concentration conforms to the response pattern, it is prompted that the object is suitable for anti- A responder to VEGF therapy; when the marker concentration does not conform to a response pattern, it indicates that the subject is a non-responder to anti-VEGF therapy; and

(c) Output module, the output module is used to output the diagnosis result.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described one by one here.

Description of drawings

Figure 1 shows the study flow chart.

Figure 2 shows the metabolic profile changes in patients with neovascular age-related macular degeneration (nAMD) and polypoidal chorioretinopathy (PCV) before and after anti-VEGF treatment: (A) Principal component analysis (PCA) model of nAMD patients before and after anti-VEGF treatment , no separation trend. (B) Partial least squares discriminant analysis (PLS-DA) model of nAMD patients before and after anti-VEGF treatment. There is no separation trend in the PLS-DA model. (C) Replacement test of the PLS-DA model in nAMD patients before and after anti-VEGF treatment. The PLSDA model of nAMD may be overfitting. (D) Principal component analysis (PCA) model of PCV patients before and after anti-VEGF treatment, with no separation trend.

Figure 3 shows the metabolic differences between responders and non-responders to anti-VEGF therapy after combining neovascular age-related macular degeneration (nAMD) and polypoidal chorioretinopathy (PCV): (A) nAMD and PCV The principal component analysis (PCA) model of metabolic differences between patients who were responders and non-responders to anti-VEGF treatment showed a clear trend of separation. (B) Partial least squares discriminant analysis (PLS-DA) model of metabolic differences between responders and non-responders to anti-VEGF treatment in patients with nAMD and PCV, showing an obvious trend of separation. (C) PLS-DA model permutation test of metabolic differences between responders and non-responders to anti-VEGF therapy in nAMD and PCV patients. (D) Volcano plot shows the differential metabolites in responders and non-responders to anti-VEGF treatment (fold change>1.2, FDR<0.01). Blue and red points represent down-regulated and up-regulated differential metabolites, respectively. (E) Venn diagram of differential metabolites between responders and non-responders to anti-VEGF treatment in patients with nAMD and PCV.

Figure 4 shows the metabolic differences between responders and non-responders to anti-VEGF treatment in neovascular age-related macular degeneration (nAMD) and polypoidal chorioretinopathy (PCV): (A) Anti-VEGF in nAMD patients The principal component analysis (PCA) model of metabolic differences between responders and non-responders to treatment showed a clear trend of separation. (B) Partial least squares discriminant analysis (PLS-DA) model of metabolic differences between responders and non-responders to anti-VEGF treatment in nAMD patients, showing an obvious trend of separation. (C) Principal component analysis (PCA) model of metabolic differences between responders and non-responders to anti-VEGF treatment in PCV patients, showing an obvious trend of separation. (D) Partial least squares discriminant analysis (PLS-DA) model of metabolic differences between responders and non-responders to anti-VEGF treatment in PCV patients, showing an obvious trend of separation.

Figure 5 shows differential metabolite subclass and metabolic pathway enrichment associated with anti-VEGF treatment response in patients with neovascular age-related macular degeneration (nAMD) and polypoidal chorioretinopathy (PCV): (A) Compared with nAMD and PCV Pie chart of differential metabolite subclasses in patients who were responders and non-responders to anti-VEGF therapy; (B) Differential metabolites in patients who were responders and non-responders to anti-VEGF therapy with nAMD and PCV Bar chart of metabolic pathway enrichment; (C) ROC curve of discovery set serum LPC 18:0 sn-1 to distinguish patients with treatment response/non-response; (D) Discovery set cutoff value of serum LPC 18:0 sn-1 The accuracy of predicting treatment response/non-response in the validation set was 72.4%; (E) Using public data analysis, the LPC18:0-related gene was determined to be the von Willebrand Factor gene at 125.554Mbp on chromosome 6, marked in red.

Detailed ways

After extensive and in-depth research, the present inventor discovered for the first time serum metabolic markers of AMD's response to intraocular injection of anti-VEGF drugs. Specifically, the present invention discovered a biomarker set, which includes a variety of serum metabolic markers related to the response of AMD patients to intraocular injection of anti-VEGF drugs, and can be used to evaluate the ocular response of test subjects to anti-VEGF drugs. The response to intra-injection treatment of AMD has the advantages of high sensitivity and specificity, and has important application value. On this basis, the present invention was completed.

The inventors identified serum biomarkers related to anti-VEGF treatment response in AMD patients through metabolomics detection and analysis methods, and distinguished responders and non-responders to anti-VEGF treatment through peripheral blood detection. The metabolomics research results of the present invention show that disorders of some metabolic pathways play an important role in the occurrence and development of nAMD and PCV, thereby providing a basis for precise treatment and classification of such diseases, and also further elucidating the occurrence of such diseases. Lay the foundation for the development mechanism.

the term

The terms used in the present invention have the meanings commonly understood by those of ordinary skill in the relevant art. However, in order to better understand the present invention, some definitions and related terms are explained as follows:

As used herein, the term "response" or "response" refers to a test subject's response or response to anti-VEFG therapy.

As used herein, the term "anti-VEGF responsive marker of the invention" refers to one or more markers shown in Table 3A and/or Table 3B.

As used herein, the term "sample" or "sample" refers to material specifically associated with a subject from which specific information about the subject can be determined, calculated, or inferred. The sample may consist in whole or in part of biological material from the subject. A sample may also be a material that has come into contact with a subject in such a way that tests performed on the sample provide information about the subject. The sample may also be a material that has been in contact with other materials that are not the subject's but that enable the first material to be subsequently tested to determine information about the subject, for example the sample may be a probe or dissection Knife cleaning fluid. The sample may be a source of biological material external to the subject, as long as a person skilled in the art can still determine information about the subject from the sample.

As used herein, the terms "reference value,""referencevalue" and "control reference value" are used interchangeably. A value that is statistically related to a specific result when compared to the results of an analysis. In a preferred embodiment, the reference value is determined based on statistical analysis of studies comparing proliferative diabetic retinopathy marker concentrations C11 and C12 with known clinical outcomes. Some of these studies are shown in the Examples section of this article. However, studies from the literature and user experience with the methods disclosed herein can also be used to produce or adjust reference values. Reference values may also be determined by considering conditions and outcomes that are particularly relevant to the patient's medical history, genetics, age, and other factors.

age-related macular degeneration

Age-related macular degeneration (AMD) is the leading cause of visual impairment in older adults worldwide. AMD is divided into dry AMD and neovascular AMD (nAMD). nAMD is characterized by the occurrence of choroidal neovascularization (CNV) under the fovea of the macula, which can cause structural damage to the macula, leading to irreversible central vision damage or even blindness. Polypoidal chorioretinopathy (PCV) is a subtype of nAMD that is more common in Asian populations and manifests as serous or hemorrhagic retinal pigment epithelial detachment, subretinal hemorrhage, and orange-red subretinal nodular lesions.

The common pathological mechanism of nAMD and PCV is pathological neovascularization mediated by vascular endothelial growth factor (VEGF). Therefore, intravitreal injection of anti-VEGF drugs can inhibit abnormal new blood vessels and is the first-line treatment for such diseases internationally.

Detection reagents and detection methods

The present invention relates to the quantitative and qualitative detection of metabolite levels in human serum. These tests are well known in the art. The levels of metabolites in the human body detected in the test can be used to determine the response to anti-VEGF therapy.

In the present invention, a variety of methods can be used to conduct qualitative or quantitative detection of the reaction between the detection reagent and the sample, and the preferred method is gas chromatography-mass spectrometry (GC-MS). In the present invention, the detection reagents for metabolites include mass spectrometry detection reagents (such as 1,2-dihenarachidoyl-sn-glycero-3-phosphocholine ), methylene chloride, methanol, concentrated sulfuric acid, isooctane).

One way to detect the content of metabolites in a sample is to use the chromatographic analysis method mentioned above to conduct qualitative and quantitative analysis of metabolites in the human body.

Reagent test kit

The present invention also provides a reagent for judging the responsiveness of anti-VEGF therapy and/or the efficacy of anti-VEGF therapy. box. Kits useful in the present invention generally include detection reagents, and/or instructions for responsiveness to anti-VEGF therapy and/or efficacy of anti-VEGF therapy.

Among them, the instructions describe the detection method and relevant instructions for judging the responsiveness of anti-VEGF therapy and/or the efficacy of anti-VEGF therapy based on the measured values C1 of different samples.

A typical kit of the present invention can be used to detect human blood samples and serum samples.

It should be understood that after the present invention first reveals the correlation between the specific human metabolite of the present invention and the responsiveness to anti-VEGF therapy, those skilled in the art can easily make early judgments on the responsiveness to anti-VEGF therapy based on the above correlation and guide subsequent More precise treatment to avoid repeated intraocular injections.

diagnostic equipment

The invention also provides a diagnostic device, which includes:

(a) Input module, the input module is used to input anti-VEGF marker data of a certain subject's serum sample;

The response markers to anti-VEGF therapy include those selected from the group consisting of:

(A1)LPC 18:0 sn-1; (A2)PC 38:6; (A3)SM 34:1; (A4)PC 34:0; (A5)PC 34:1; (A6)PC 38:5 ;(A7)PC 36:3;(A8)PC O-38:6;(A9)PC 36:2;(A10)LPE 22:6 sn-1;(A11)PC O-38:5;(A12 )PE O-38:7; (A13)PC O-36:5; (A14)LPC O-16:1; (A15)PC 38:4; (A16)LPC 16:1 sn-1; (A17) PE O-36:5; (A18)LPC 15:0 sn-2; (A19)PC 34:2; (A20)PC O-36:4; (A21)PC 36:4; (A22)LPC 16: 1 sn-2; (A23) PC 33:1; (A24) PC 38:7;

(b) A processing module that compares the input marker concentration C1 with the control reference value C0 to obtain a discrimination result, wherein when the response marker concentration conforms to the response pattern, it is prompted that the object is suitable for anti- A responder to VEGF therapy; when the marker concentration does not conform to a response pattern, it indicates that the subject is a non-responder to anti-VEGF therapy; and

(c) Output module, the output module is used to output the diagnosis result.

The main advantages of the present invention include:

1. Through the method of detecting serum metabolites, the present invention can early identify people who respond well to anti-VEGF intraocular injection treatment and provide individualized and precise treatment.

2. Through the detection of serum metabolites, we can early identify the response to anti-VEGF intraocular injection treatment, avoid serious infectious complications such as endophthalmitis that may be caused by repeated intraocular injection, and reduce the economic burden of AMD patients.

3. Provide a theoretical basis for the development of new drugs and mechanism research related to AMD.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are for illustration only This invention is not intended to limit the scope of the invention. Experimental methods without specifying specific conditions in the following examples usually follow conventional conditions or conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

Example 1

1.1 Study population

This study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Shanghai First People's Hospital (Approval Number: 2016KY115-2). On March 9, 2017, this study was registered on the official website of www.ClinicalTrial.gov (registration number: NCT03128463). All subjects provided written informed consent.

1.2 Inclusion and exclusion criteria

The discovery set of all participants in this study was patients who visited the Ophthalmology Clinic of Shanghai First People's Hospital from March 2017 to March 2019. The validation set consists of patients who visited the ophthalmology outpatient clinics of 12 ophthalmology centers across the country from April 2017 to December 2018. Inclusion criteria: (1) age ≥50 years old; (2) diagnosed with nAMD or PCV; (3) received at least one intravitreal injection of Conbercept Ophthalmic Injection (English name: Conbercept). Exclusion criteria: (1) intravitreal injection or systemic anti-VEGF drug treatment within 3 months; (2) other treatments within 3 months (such as photodynamic therapy, retinal laser photocoagulation, vitrectomy, etc.); (3) ) There are other causes of subretinal or intraretinal fluid accumulation/hemorrhage, such as diabetic retinopathy, retinal vein occlusion, uveitis, etc.; (4) There are other serious systemic diseases that may affect the serum metabolic profile.

1.3 Eye examination and evaluation

At visit 1 (V1), the patient's age, gender, any medical/surgical medical history and concomitant medication use were recorded. All participants underwent a comprehensive ophthalmic examination, including slit-lamp biomicroscopy, best-corrected visual acuity (BCVA) using the Early Treatment Diabetic Retinopathy Study (ETDRS) alphabet, fundus color photography, intraocular pressure (IOP) examination, optical Coherence tomography (SD-OCT) and other examinations. When the differential diagnosis of the disease is difficult, fundus fluorescein angiography (FFA) and indocyanine green fundus angiography (ICGA) are added to help confirm the diagnosis. The fundus specialist confirmed the patient's diagnosis based on fundus changes and imaging examinations, and administered intravitreal Conbercept Ophthalmic Injection under topical anesthesia in the operating room. One month after treatment, all patients will undergo routine follow-up, that is, the second visit (V2), and BCVA, IOP, SD-OCT results, etc. of all patients will be collected.

1.4 Evaluation of response to anti-VEGF treatment

According to the response of patients after anti-VEGF treatment, they were divided into responders and non-responders. The main basis for judging the discovery set is the patient's SD-OCT retinal anatomical morphological changes from V1 to V2. Two experienced fundus specialists independently judged the response, and then a senior fundus doctor confirmed the final response grouping. If there are any differences of opinion, all judges will reach a consensus after discussion. Respondent definition: from V1 to V2 on SD-OCT Patients with intramembranous fluid (IRF) or subretinal fluid (SRF) that have mostly resolved after treatment. Non-responders were defined as patients whose IRF, SRF and central retinal thickness (CRT) on SD-OCT increased after treatment or had no significant changes from V1 to V2. The evaluation basis of the validation set: According to the value of CRT, responders are defined as: patients whose CRT decreases by ≥10% on SD-OCT from V1 to V2. Non-responder definition: patients with <10% decrease in CRT on SD-OCT from V1 to V2.

1.5 Serum sample collection and preprocessing

Collect 10 ml of peripheral fasting blood from the patient before intraocular injection of anti-VEGF drugs at V1 and during follow-up at V2, and centrifuge (1500 rpm, 10 min, 20°C) within 30 minutes after blood collection. Take the upper serum and store it immediately in a -80°C refrigerator. During the analysis process, take 100 μL of serum, add 400 μL of methanol extractant containing internal standard, vortex, centrifuge, and take two 180 μL portions to freeze-dry for positive and negative ion analysis. Before analysis, reconstitute with 50 μL of 25% acetonitrile and wait for liquid chromatography-mass spectrometry (LC-MS) analysis. Before analyzing the actual sample, a blank sample is used to balance the system; during the analysis of the actual sample, quality control (QC) is used to monitor the stability of the instrument operation and the repeatability of the analysis. The preparation method of QC samples is the same as that of actual samples. For every 10 actual samples, one QC sample needs to be run.

1.6LC-MS detection instruments and parameters

The instrument used was Vanquish UPLC-Q Exactive (Thermo Fisher Scientific, Rockford, IL, USA). Chromatographic separation conditions: positive ion mode, chromatographic column: Waters BEH C8 column, column temperature: 60°C, flow rate: 0.4ml/min. Mobile phase: water plus 0.1% formic acid (Phase A) and acetonitrile plus 0.1% formic acid (Phase B). Gradient: the initial gradient is 5% B, maintained for 0.5 minutes, then linearly increased to 40% B within 1.5 minutes, linearly increased to 100% B within 6 minutes and maintained for 2 minutes, and dropped back to the initial gradient of 5% at 10.1 minutes B, Equilibrate for 2 minutes. Negative ion mode, column: ACQUITY UPLC HSS T3, column temperature: 60°C, flow rate: 0.4ml/min. Mobile phase: Phase A is water with 6.5mM NH4HCO3 added; phase B is an aqueous solution containing 95% methanol and 6.5mM NH4HCO3. Gradient: starting gradient is 2% B, maintained for 0.5 min, increased to 40% B within 2 min, then linearly increased to 100% B within 6 min and maintained for 2 min, dropped back to the initial gradient of 2% B at 10.1 min, balanced 1.9 minutes. Mass spectrometry data acquisition parameters: full scanning range positive ions m/z 80-1200, spray voltage 3.50kV; negative ions 80-1200, spray voltage 3.00kV. The capillary temperature is 300°C, the auxiliary heating gas temperature is 350°C, the sheath gas and auxiliary gas flow rates are 45 and 10 (arbitrary units) respectively, and the resolution is set to 7e ⁴ .

1.7 Data processing and statistical analysis

Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were performed using SIMCA-P software version 14.0 (Umetrics AB, Umea, Sweden) to study differences in metabolic profiles between responders and non-responders. , calculate the fold difference (FC) and false discovery rate (FDR) to express the significance of metabolite changes, and define metabolites with FC>1.2 and FDR<0.01 as differential metabolites. The area under the receiver operating characteristic curve (AUC) was calculated to screen biomarkers to distinguish responders from non-responders. Using MetaboAnalyst5.0 software (http://www.metaboanalyst.ca/) Draw volcano plots, Venn diagrams, pie charts, and metabolite enrichment pathway bar charts of differential metabolites. Statistical analysis was performed using SPSS 22.0 software.

1.8 Results

General information of study subjects: 132 eyes of 132 patients in the discovery set (nAMD, n=62; PCV, n=70) were included in the study. In the validation set, 132 eyes of 87 patients (nAMD, n=64; PCV, n=23) were included in the study.

The average CRT of the responder group in the discovery set was significantly reduced from 502.8 (458.8 to 546.8) μm to 342.9 (315.0 to 370.8) μm, P<0.001.

The average CRT of the non-responder group increased from 524.5 (451.2 to 597.8) μm to 530.3 (455.0 to 605.7) μm. The difference was not statistically significant, P=0.348. The weighted kappa value of the evaluation consistency test for the response to anti-VEGF treatment in the discovery set was 0.862 (P<0.001), indicating that the evaluation method has good reliability. The average CRT of the responder group in the validation set was significantly reduced from 397.5 (351.8 to 443.3) μm to 260.7 (233.3 to 288.2) μm, P<0.001. The average CRT of the non-responder group increased from 348.9 (304.0-393.7) μm to 364.7 (319.1-410.4) μm. The difference was not statistically significant, P=0.076.

Table 1. General information of research subjects

Abbreviations: BCVA, best corrected visual acuity; ETDRS, Early Treatment Diabetic Retinopathy Study; IOP, intraocular pressure; CRT, central retinal thickness.

Note: a: independent-samples t test, b: Chi-square test, c: Mann-Whitney U test.

Changes in metabolic profiles before and after anti-VEGF treatment: There is no significant separation trend in the PCA and PLS-DA models of the serum metabolic profiles of nAMD patients before and after anti-VEGF treatment. At the same time, the PCA model of PCV patients also showed no significant separation trend before and after anti-VEGF treatment. The PLS-DA model of PCV before and after treatment could not be modeled because the difference was not significant (Figure 2).

Analysis of metabolic differences between responders and non-responders to anti-VEGF therapy in patients with nAMD and PCV: A total of 248 metabolites were detected through untargeted LC-MS metabolomic analysis, and the stability and stability of the instrument operation were verified through QC samples Analytical reproducibility. Among them, the relative standard deviation (RSD) of 226 metabolites (91.1%) was less than 30%, indicating that the detection method has good stability. When nAMD and PCV were combined, both PCA and PLS-DA results showed (Figure 3) that the overall metabolic profiles of responders and non-responders showed an obvious separation trend, and a total of 85 differential metabolites were identified (FC >1.2 and FDR<0.01). Among them, lysophosphatidylcholine (LPC) 18:0 sn-1 had the highest AUC in distinguishing responders from non-responders (AUC=0.896, 95%CI)=0.833~0.949). The top 20 differential metabolites are listed in descending order of AUC, as shown in Table 2. The first among them is LPC 18:0 sn-1. The concentration of serum LPC 18:0 sn-1 is significantly increased in the non-responsive group. In the discovery set, LPC 18:0 sn-1 distinguishes the boundary of response/non-response. The value is 11.4. This boundary value was verified in the validation set, and the accuracy of distinguishing response/non-response was 72.4%. Using public data analysis, the LPC 18:0-related gene was determined to be the 125.554Mbp von Willebrand Factor gene on chromosome 6 (Figure 5). The AUCs of other differential metabolites are detailed in Table 2, Table 3A and Table 3B.

In addition, when PCA and PLS-DA models were established for nAMD and PCV patients respectively, it was found that the overall metabolic profiles of responders and non-responders showed a clear separation trend (Figure 4).

Table 2. The top 20 differential metabolites between responders and non-responders to anti-VEGF treatment (arranged by AUC from large to small)

Abbreviations: FDR, false discovery rate; AUC, area under the receiver operating characteristic curve; FC: fold change

By screening the AUC values of each differential metabolite, the top 29 differential metabolites with AUC values were selected as biomarkers for evaluating anti-VEGF response.

In addition, the inventors unexpectedly discovered 24 new differential metabolites that have not been reported, as shown in Table 3A. The five reported differential metabolites are shown in Table 3B.

Table 3A

Table 3B

Enrichment of metabolic pathways related to anti-VEGF treatment response in patients with nAMD and PCV: differential metabolite subcategories related to treatment response mainly include diacylglycerolcholine, LPC, branched-chain fatty acids, unsaturated fatty acids, phosphocholine (PC ), etc. The enrichment of metabolic pathways is shown in Figure 5. The enriched metabolite pathways are shown in Figure 5. The diacylglycerolcholine and LPC metabolic pathways are the two most significantly different metabolic pathways.

Example 2

The first differential metabolite LPC 18:0 sn-1 in Table A and the top 10 differential metabolites in the validation set population (n=84) were used to predict the accuracy of distinguishing response/non-response Make an evaluation.

The results show that the accuracy of LPC 18:0 sn-1 alone is 72.4%. The accuracy of individual differential metabolites at positions 2-10 in Table A is approximately 60-70%.

In addition, as the number of differential metabolites involved in the evaluation increases, the accuracy of the evaluation results also increases accordingly. The top three differential metabolites ((A1)LPC 18:0 sn-1; (A2)PC 38:6; (A3)SM 34:1) are used for prediction, and the accuracy is greater than approximately 78%.

discuss

Conbercept Ophthalmic Injection is a new anti-VEGF drug independently developed in my country. In current clinical practice, there is still a lack of reliable and accurate methods to predict its response to the treatment of AMD and guide precise medication. The inventor first found through analysis of serum metabolic profiles before and after treatment that there was no significant change in the serum metabolic profiles before and after intravitreal anti-VEGF treatment, indicating that intraocular anti-VEGF administration has little impact on serum metabolic profiles, and the difference in patient treatment responses mainly depends on Individual differences, independent of treatment-induced metabolic changes.

Secondly, the inventor found that the serum metabolic profiles of responders and non-responders were significantly separated, thus inferring that metabolic differences are one of the important factors affecting the response to anti-VEGF drugs. Metabolic pathways currently recognized to be related to the occurrence and development of AMD include abnormalities in lipid metabolism, nucleotide metabolism, carbohydrate metabolism, and amino acid metabolism pathways. The differential metabolites we identified are mainly enriched in lipids, including LPC, PC, carnitine, fatty acids, etc. Among the 89 identified differential metabolites, the AUC of each metabolite in distinguishing responders and non-responders was calculated and can be used as a potential biomarker to predict treatment response. Among them, LPC 18:0 sn-1 The highest diagnostic efficiency. The research of the present invention shows that one important biomarker or a combination of multiple important biomarkers can be used to predict the treatment response of AMD patients to Conbercept.

AMD has serious harm to visual function. When anti-VEGF drugs are used in the real world, 30% of non-responders exist. There is a lack of effective identification methods. The present invention has discovered a set of biomarkers related to the response to the treatment of AMD by Conbercept. , can be used to evaluate the response of test subjects to Conbercept for the treatment of AMD. It has the advantages of high sensitivity and high specificity, and has important application value. It is conducive to early clinical identification of AMD patients who have a good response to Conbercept treatment and can benefit significantly, and early effective anti-VEGF intervention to save visual function; early use of drugs with other mechanisms for targeted treatment of non-responders , reducing unnecessary repeated intraocular injections and the economic burden associated with anti-VEGF.

All documents mentioned in this application are incorporated by reference in this application to the same extent as if each individual document was individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims (10)

1. The use of an anti-VEGF responsive marker detection reagent, which is characterized in that it is used to prepare a diagnostic reagent or kit, and the diagnostic reagent or kit is used to (a) determine the responsiveness of a certain subject to anti-VEGF therapy. , and/or (b) evaluate the therapeutic effect of anti-VEGF treatment for AMD in a certain subject;

   Wherein, the anti-VEGF responsive marker is selected from the following group:

   (A) Any marker selected from A1 to A24, or a combination thereof: (A1) LPC 18:0 sn-1; (A2) PC 38:6; (A3) SM 34:1; (A4) PC 34 :0; (A5)PC 34:1; (A6)PC 38:5; (A7)PC 36:3; (A8)PC O-38:6; (A9)PC 36:2; (A10)LPE 22 :6 sn-1; (A11)PC O-38:5; (A12)PE O-38:7; (A13)PC O-36:5; (A14)LPC O-16:1; (A15)PC 38:4; (A16)LPC 16:1 sn-1; (A17)PE O-36:5; (A18)LPC 15:0 sn-2; (A19)PC 34:2; (A20)PC O- 36:4; (A21)PC 36:4; (A22)LPC 16:1sn-2; (A23)PC 33:1; (A24)PC 38:7.
2. The use according to claim 1, wherein the anti-VEGF responsive marker also includes any marker selected from the following group B, or a combination thereof: (B1) LPC 16:0 sn-1; (B1) LPC 16:0 sn-1; B2)LPC 16:0 sn-2; (B3)LPC 18:1 sn-1; (B4)LPC 18:2 sn-1; (B5)LPC 22:6 sn-1.
3. The use according to claim 1, wherein the anti-VEGF responsive markers further include at least 2 selected from A1 to A24.
4. The use according to claim 1, wherein the anti-VEGF responsive marker is selected from a combination of one or more markers from A1 to A24 and one or more markers from B1 to B5.
5. The use according to claim 1, wherein the diagnostic reagent or kit is used to detect anti-VEGF responsive markers in serum.
6. The use of claim 1, wherein the AMD includes neovascular AMD.
7. A kit, characterized in that the kit contains a detection reagent, and the detection reagent is used to detect anti-VEGF responsive markers in the sample to be tested,

   Wherein, the anti-VEGF responsive marker is selected from the following group:

   (A) A combination of two or more markers selected from A1 to A24;

   (B) A combination of one or more markers selected from A1 to A24 and one or more markers from B1 to B5.
8. The kit according to claim 7, wherein the sample to be tested is from a subject selected from the following group: non-AMD subjects, AMD subjects who have not used anti-VEGF therapy, and AMD subjects who have used anti-VEGF therapy. .
9. A detection method, characterized by including the steps:

   (a) Provide a test sample, the test sample is selected from a serum sample;

   (b) detecting the concentration of the anti-VEGF responsive marker in the test sample, recorded as C1; and

   (c) comparing the concentration C1 of the anti-VEGF responsive marker with the control reference value C0,

   Wherein, the anti-VEGF responsive marker is selected from the following group:

   (A) Any marker selected from A1 to A24, or a combination thereof: (A1) LPC 18:0 sn-1; (A2) PC 38:6; (A3) SM 34:1; (A4) PC 34 :0; (A5)PC 34:1; (A6)PC 38:5; (A7)PC 36:3; (A8)PC O-38:6; (A9)PC 36:2; (A10)LPE 22 :6 sn-1; (A11)PC O-38:5; (A12)PE O-38:7; (A13)PC O-36:5; (A14)LPC O-16:1; (A15)PC 38:4; (A16)LPC 16:1 sn-1; (A17)PE O-36:5; (A18)LPC 15:0 sn-2; (A19)PC 34:2; (A20)PC O- 36:4; (A21)PC 36:4; (A22)LPC 16:1sn-2; (A23)PC 33:1; (A24)PC 38:7;

   If the test result of the anti-VEGF responsive marker of the test subject meets the following conditions, it will indicate that the AMD patient is suitable for anti-VEGF treatment and has good results:

   (1) When a certain marker is an up-regulated marker in Table A in the test subject, and the expression level of the marker is higher than the reference value or standard value C0, the test subject will have a good effect of anti-VEGF treatment. ;

   (2) When a certain marker is a down-regulated marker in Table A in the test subject, and the expression level of the marker is lower than the reference value or standard value C0, the test subject will have a good effect of anti-VEGF treatment. .
10. A diagnostic device, characterized in that the device includes:

    (a) Input module, the input module is used to input anti-VEGF marker data of a certain subject's serum sample;

    The response markers to anti-VEGF therapy include those selected from the group consisting of:

    (A1)LPC 18:0 sn-1; (A2)PC 38:6; (A3)SM 34:1; (A4)PC 34:0; (A5)PC 34:1; (A6)PC 38:5 ;(A7)PC 36:3;(A8)PC O-38:6;(A9)PC 36:2;(A10)LPE 22:6 sn-1;(A11)PC O-38:5;(A12 )PE O-38:7; (A13)PC O-36:5; (A14)LPC O-16:1; (A15)PC 38:4; (A16)LPC 16:1 sn-1; (A17) PE O-36:5; (A18)LPC 15:0 sn-2; (A19)PC 34:2; (A20)PC O-36:4; (A21)PC 36:4; (A22)LPC 16: 1 sn-2; (A23) PC 33:1; (A24) PC 38:7;

    (b) A processing module that compares the input marker concentration C1 with the control reference value C0 to obtain a discrimination result, wherein when the response marker concentration conforms to the response pattern, it is prompted that the object is suitable for anti- A responder to VEGF therapy; when the marker concentration does not conform to a response pattern, it indicates that the subject is a non-responder to anti-VEGF therapy; and

    (c) Output module, the output module is used to output the diagnosis result.