Application of radionuclide 177Lu labeled FAPI in preparation of targeted myocardial fibrosis drugs

Abstract

The invention provides an application of a radionuclide 177Lu labeled FAPI in preparation of a drug for targeting myocardial fibrosis. The therapeutic effect of the radionuclide 177Lu labeled FAPI is mainly that the activation of fibroblasts is inhibited through 177Lu, and due to the fact that the dosage used for treatment is low, the method can be a more economical and practical method for improving the heart function and reducing myocardial fibrosis after myocardial infarction.

Description

Technical Field

[0001] The present invention belongs to the technical field of nuclear medicine and relates to radionuclides 177 The application of Lu-labeled FAPI (fibroblast activation protein inhibitor) in the preparation of drugs targeting myocardial fibrosis Background Art

[0002] Cardiovascular diseases rank first in the composition ratio of disease deaths among urban and rural residents in China. According to the "Summary of the Report on Cardiovascular Health and Diseases in China 2022", the incidence and mortality of cardiovascular diseases among Chinese residents continue to rise. Treatment measures for myocardial infarction (MI) such as percutaneous coronary intervention and coronary artery bypass grafting can significantly reduce mortality, but cannot prevent myocardial fibrosis occurring after acute myocardial infarction. Due to the prevalence of unhealthy lifestyles among Chinese residents and the accelerating aging of the population, the incidence of myocardial infarction will still continue to rise, and the treatment of myocardial fibrosis after myocardial infarction will be a long-term problem in clinical practice

[0003] Myocardial infarction is a serious cardiovascular disease with a high mortality rate. Myocardial fibrosis occurring after myocardial infarction is a key factor leading to the development of heart failure and has an important impact on the prognosis of patients. Myocardial infarction causes myocardial cell death, activates the innate immune pathway, releases cytokines, chemokines and adhesion molecules, triggers a series of inflammatory reactions and activates myocardial fibroblasts, which play an important role in myocardial fibrosis and extracellular matrix deposition. During the injury process, fibrotic cytokines such as fibroblast growth factor and transforming growth factor are up-regulated, leading to the differentiation of cardiac fibroblasts into myofibroblasts, and forming scars by secreting a large amount of extracellular matrix. Although early reparative scars can prevent myocardial rupture and maintain normal myocardial structure, over-activation of myofibroblasts can lead to excessive secretion of extracellular matrix, damage the normal structure of the heart, promote systolic and diastolic dysfunction of cardiac function, and thus lead to the development of myocardial infarction into heart failure

[0004] After myocardial infarction, the continuous activation of myofibroblasts mediated by various fibrotic mediators is a key event related to the development of myocardial fibrosis. Killing FAP-positive fibroblasts through FAP-targeted chimeric antigen receptor T cells can prevent excessive ventricular remodeling and shows the potential to reduce cardiac fibrosis. However, the ablation of FAP-positive cells mediated by chimeric antigen receptor T cells can also cause serious side effects such as cachexia and anemia. Therefore, developing safer FAP-targeted drugs to inhibit the activation of myocardial fibroblasts has important clinical significance for the treatment of myocardial fibrosis after myocardial infarction Summary of the Invention

[0005] Aiming at the above technical problems, the purpose of the present invention is to provide the application of radionuclide 177 Lu-labeled FAPI in the preparation of drugs targeting myocardial fibrosis

[0006] The technical solution adopted by the present invention to achieve the technical purpose is as follows:

[0007] The present invention provides the application of radionuclide 177 Lu-labeled FAPI in the preparation of a drug for targeting myocardial fibrosis.

[0008] Preferably, the myocardial fibrosis is myocardial fibrosis after acute myocardial infarction.

[0009] Preferably, the radionuclide 177 Lu-labeled FAPI can significantly increase the left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS), and significantly decrease the left ventricular end-diastolic volume (LVEDV) and left ventricular end-diastolic diameter (LVEDD).

[0010] Preferably, the radionuclide 177 Lu-labeled FAPI can significantly reduce the myocardial fibrosis area and reduce the infarcted segments.

[0011] Preferably, the radionuclide 177 Lu-labeled FAPI can significantly reduce the protein levels of periostin and vimentin, thereby affecting fibroblast activation.

[0012] Preferably, the radionuclide 177 Lu-labeled FAPI is 177 Lu-FAPI-04.

[0013] More preferably, the drug for targeting myocardial fibrosis enables the dosage of FAPI-04 to be much lower than the dosage required to exert the therapeutic effect through the role of 177 Lu in inhibiting fibroblasts.

[0014] Preferably, the FAPI also includes FAPI-34, FAPI-46, FAPI-74, and FAP-2286. These FAPIs may also play a role in treating myocardial fibrosis after myocardial infarction by binding to the radionuclide.

[0015] The 177 Lu-FAPI-04 of the present invention has 177 the dual effects of 177 Lu and FAPI. 177Lu uses the direct or indirect action of β particles to break DNA strands, and at the same time uses the targeting effect of radiation combined with its ligand to achieve the purpose of precisely killing target cells. FAPI-04 is a specific inhibitor of FAP, which can bind to FAP with high specificity and be rapidly internalized. FAPI-04 is rapidly cleared from the blood, and since the expression of FAP in normal tissues is very low, the binding of FAP to FAPI is also very low, and the uptake of FAPI by normal tissues is limited. In this application, only one 177 Lu-FAPI-04 injection was performed on rats on the 7th day after myocardial infarction, and the dose of FAPI used was much lower than the dose required to exert a therapeutic effect. It can be considered that the FAPI used in this application has 177 no effect on the 177 treatment of Lu-FAPI-04, and its therapeutic effect is mainly achieved by 177 Lu inhibiting fibroblast activation. Since Description of the Drawings

[0016] Figure 1 177 Echocardiogram analysis after Lu-FAPI-04 treatment. Echocardiogram analysis of LVEF, LVFS, LVEDV, and LVEDD at 7, 14, 21, and 28 days after MI ( 177 Lu-FAPI group n = 11, vehicle group n = 9, sham operation group n = 9). 177 Lu-FAPI-04 was injected on the 7th day after MI. There was no significant difference in cardiac function parameters among the three groups before coronary artery ligation, 177 and there was no significant difference in cardiac function parameters between the Lu-FAPI group and the vehicle group 177 before Lu-FAPI-04 treatment. LVEF = left ventricular ejection fraction; LVFS = left ventricular fractional shortening; LVEDV = left ventricular end-diastolic volume; LVEDD = left ventricular end-diastolic diameter.

[0017] Figure 2 177 Fibrotic area after Lu treatment. (A) Representative cardiac sections from the apex to the base were stained with Masson at 28 days after MI. (B) Quantification of the fibrotic area at 28 days after MI (vehicle group n = 4, 177 Lu-FAPI group n = 6). (C) Counting of infarcted segments in MRI images (n = 5). (*P < 0.05)

[0018] Figure 3Pathological analysis of the heart tissues of MI rats. (A) Representative IHC images of FAP at 7 days after MI. 1 and 2 show high-power microscopic views of the infarcted area and the distal normal myocardium, respectively. (B) Quantitative analysis of the cell area of FAP + in the infarcted area and the distal normal myocardium at 7 days after MI (n = 5). (C) 177 Immunofluorescence staining of α-SMA, periostin, and vimentin (green, magnification: 400×) in the Lu-FAPI-04 injection group at 7 days after injection (14 days after MI), the Lu-FAPI group, the carrier group, and the sham operation group. The protein levels of α-SMA, periostin, and vimentin were quantitatively analyzed, and the fold changes compared with the sham operation group were statistically calculated. Sham operation group: Sham-operated rats treated with saline (n = 5). Carrier group: MI rats treated with saline (n = 5). 177 Lu-FAPI group: 177 MI rats treated with Lu-FAPI-04 (n = 5). (ns = no significant difference, *P < 0.05) 177

[0019] Figure 4 Radiopharmaceutical uptake and biochemical analysis of MI rats. (A) 177 Lu-FAPI-04 uptake at 3 hours and 24 hours after injection (n = 3). (B-E) 177 Biochemical analysis of the Lu-FAPI group, the carrier group, and the sham operation group (n = 3). ALB = albumin; ALT = alanine aminotransferase; BUN = blood urea nitrogen; CR = creatinine. Detailed implementation mode

[0020] The present invention will be described in detail below in combination with the detailed implementation mode. The following specific examples are helpful for those skilled in the art to further understand the present invention, but do not limit the present invention in any form.

[0021] Example

[0022] 1. 177 Preparation of Lu-FAPI-04:

[0023] 1. Weigh 1.84 ± 0.0056 g of sodium acetate, dissolve it in 50 ml of ultrapure water, adjust the pH to 4.5 with glacial acetic acid, and filter it through a 0.2 μm sterile filter;

[0024] 2. Take 2 μl of the FAPI-04 precursor solution (concentration: 10 mg / ml) and add it to 198 μl of the diluent (0.45 M pH 4.5 sodium acetate buffer solution);

[0025] 3. Turn on the thermostatic mixer and preheat it to 95 °C;

[0026] 4. Add 177 LuCl3 (88 μl, 2375 MBq) dissolved in 0.05 M HCl to a mixture of FAPI-04 (0.1 mg / ml, 800 μl) and 0.45 M sodium acetate buffer (476 μl, pH 4.5). Heat the reaction system to 95 °C and react for 20 min. Dilute and filter the final product with 0.9% normal saline.

[0027] II. 177 Effect of Lu-FAPI-04 on cardiac function after acute myocardial infarction

[0028] 1. Construction of MI rats by coronary artery ligation: Select normal rats with similar cardiac function parameters (LVEF (76.25 ± 4.25%), LVFS (40.46 ± 4.58%), LVEDV (304.56 ± 43.32 μl), and LVEDD (7.12 ± 0.52 mm)) for coronary artery ligation. Male Wistar rats (6 weeks old, 200 g; Charles River) were anesthetized by inhalation of isoflurane (5%, 0.25 MPa, 1 L / min). After thoracotomy on the left side, the left anterior descending coronary artery was ligated. Electrocardiogram examination was performed on the rats within 2 hours after ligation to verify the success of coronary artery ligation (ST segment elevation of more than 0.2 mV was considered successful). The rats were divided into 177 Lu-FAPI treatment groups as 177 Lu-FAPI group, vehicle group (Vehicle), and sham operation group (Sham). 177 In the Lu-FAPI group and vehicle group, MI rats were injected with 177 Lu-FAPI-04 (0.4 MBq / g) and 0.9% normal saline, respectively. Cardiac function was monitored weekly after injection. Echocardiography was performed on the three groups of rats on the 7th, 14th, 21st, and 28th days after myocardial infarction.

[0029] 2. Echocardiography: Before the examination, the rats were placed in an animal anesthesia box and anesthetized with 2% isoflurane. After anesthesia, the rats were tied to the operating table with tape, and the electrocardiogram was continuously monitored during echocardiography. In this application, a color Doppler device (model: Vinn06lab) was used to measure 5 cardiac cycles through the chest wall to evaluate cardiac structure and function. M-mode measurement: left ventricular ejection fraction (LVEF, %), left ventricular fractional shortening (LVFS, %), left ventricular end-diastolic volume (LVEDV, ml), and left ventricular end-diastolic diameter (LVDd, mm). Each parameter was measured three times, and the average value was taken as the final result.

[0030] 3. Experimental results: The results showed 177The LVEF of the Lu-FAPI group rats showed an increasing trend 14 days after MI (42.64±3.43% vs 43.69±3.54%). At 28 days after MI, the LVEF of the MI rats was significantly lower than that of the sham operation group. Compared with the vehicle control, the rats treated with 177 Lu-FAPI-04 showed significantly increased LVEF (45.74±5.37% vs 39.83±2.00%, p = 0.006) and LVFS (23.00±4.33% vs 18.77±1.28%, p = 0.011) ( Figure 1 A, B), while LVEDV (299.24±61.03 μl vs 428.52±134.84 μl, p = 0.01) and LVEDD (7.75±0.58 mm vs 8.79±0.99 mm, p = 0.009) were significantly decreased ( Figure 1 C, D). The above findings revealed 177 the positive effect of Lu-FAPI-04 treatment on the improvement of cardiac function. Three. 177 Detection of the therapeutic effect of Lu-FAPI-04 on myocardial fibrosis

[0031] After the echocardiogram examination on the 28th day after myocardial infarction, all three groups of rats were sacrificed and the hearts were removed. The heart tissues were washed clean with normal saline, fixed with 4% paraformaldehyde, embedded in paraffin and sectioned. Then the heart sections were stained with Masson and observed under an optical microscope for histological analysis. The experimental results showed that compared with the empty vector group (25.02±5.6%), 177 the fibrotic area of the Lu-FAPI group (16.27±4.3%) was significantly reduced ( Figure 2 A, B). MRI examination showed 177 that on average 9 out of 18 segments in the Lu-FAPI group had myocardial fibrosis, which was significantly less than 11 out of 18 segments in the empty vector group ( Figure 2 C). The above results all supported 177 that Lu-FAPI therapy could reduce cardiac fibrosis after MI. Four. 177 Detection of fibroblast activation after Lu-FAPI-04 treatment

[0032] After the echocardiogram examination on the 7th day after myocardial infarction, rats in the empty vector group were randomly selected, sacrificed, and their hearts were removed. The heart tissues were collected, washed clean with physiological saline, and subjected to immunohistochemical (IHC) staining. The heart tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned. Then, immunohistochemical staining was performed on the heart sections to detect the expression of FAP protein, and histological analysis was carried out by observing the sections under an optical microscope. After the echocardiogram examination on the 14th day after myocardial infarction, all three groups of rats were sacrificed and their hearts were removed. The heart tissues were collected and washed clean with physiological saline. After fresh tissue sampling for immunofluorescence staining, OCT embedding was performed quickly, and the tissue blocks were fixed on a cryostat. The freezing temperature regulator was adjusted to freeze the tissue blocks on the freezing table. After staying at room temperature for a while, sectioning and subsequent immunofluorescence staining were carried out.

[0033] The results of IHC staining showed that in the hearts of rats in the empty vector group 7 days after MI, FAP + cells mainly aggregated in the infarct area, and there were almost no FAP + cells in the distant normal myocardium ( Figure 3 A, B). The results of immunofluorescence staining showed that 177 in rats in the Lu-FAPI group and the empty vector group, the fibroblast activation markers α-SMA, periostin, and vimentin were all significantly upregulated 14 days after MI, but the expression of these markers was not prominent in the sham operation group ( Figure 3 C). Although 177 no obvious difference in α-SMA expression was observed between the Lu-FAPI group and the empty vector group, periostin and vimentin showed a significant decrease after injection of 177 Lu-FAPI. These observations showed that 177 Lu-FAPI-04 could affect fibroblast activation and play its therapeutic role. V. 177 Biodistribution and toxicity detection of Lu-FAPI-04

[0034] In this application, MI rats were injected with 177 Lu-FAPI-04 (n = 6, 0.4 MBq / g) on the 7th day after myocardial infarction. The rats were sacrificed and dissected at 3 hours and 24 hours after radionuclide injection (n = 3 for each time point), and the heart, liver, spleen, kidney, lung, and blood were removed. The radioactivity was quantified using a gamma counter (model: 2470Wizard). The radioactivity counts were corrected with 177 Lu standard solution to ensure accurate results. 177 The biodistribution of Lu-FAPI-04 is shown in Figure 4 A. Relatively high 177 radioactivity was observed in the heart 3 hours after injection of177 Lu-FAPI-04 accumulated and significantly decreased 24 h after administration, whereas 177 The accumulation of Lu-FAPI-04 was increased in the liver and kidney.

[0035] 177 The toxicity of Lu-FAPI-04 was evaluated by laboratory tests, including liver function tests (albumin and alanine aminotransferase) and renal function tests (blood urea nitrogen and creatinine), at 3, 24, and 168 hours after nuclide injection. Biochemical analysis showed that compared with the empty vector group, 177 The blood biochemical indices of the rats in the Lu-FAPI group showed no abnormal parameters 168 hours after radionuclide injection ( Figure 4 B, C, D, E), indicating 177 Lu-FAPI treatment did not produce significant hepatotoxicity or nephrotoxicity, these data suggest 177 Lu-FAPI-04 may be a safe drug for the treatment of MI.

[0036] To date, antifibrotic therapies targeting fibroblasts have been used in preclinical studies to improve left ventricular function. Elimination of activated fibroblasts expressing FAP by chimeric antigen receptor T cells has been shown to reduce fibrosis and improve cardiac function in mice. However, elimination of FAP-expressing stromal cells from the bone marrow may induce cachexia and anemia. In contrast, 177 Lu-FAPI has no obvious side effects on fibroblast inhibition at the current dose and may be a safer way to improve cardiac function and reduce myocardial fibrosis after myocardial infarction. 177 Lu-FAPI-04 injection, and the FAPI dose used is much lower than the dose required for the therapeutic effect. It can be considered that the FAPI used in this application is 177 Lu-FAPI-04 treatment had no effect; its therapeutic efficacy was mainly 177 Lu inhibits the effect of fibroblasts. 177 Lu-FAPI-04 treatment uses a lower dose and may therefore be a more cost-effective approach to improve cardiac function and reduce myocardial fibrosis after myocardial infarction.

[0037] Obviously, the above embodiments of the present invention are merely examples to more clearly illustrate the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. Radionuclide 177 Application of Lu-labeled FAPI in the preparation of drugs targeting myocardial fibrosis.

2. The application according to claim 1, wherein The myocardial fibrosis is myocardial fibrosis after acute myocardial infarction.

3. The application according to claim 1, wherein The radioactive nuclide 177 Lu-labeled FAPI can significantly increase the left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS), and significantly reduce the left ventricular end-diastolic volume (LVEDV) and left ventricular end-diastolic diameter (LVEDD).

4. The application according to claim 3, wherein The radioactive nuclide 177 177 Lu-labeled FAPI can significantly reduce the area of myocardial fibrosis and the number of infarcted segments.

5. The application according to claim 4, wherein The radioactive nuclide 177 Lu-labeled FAPI can significantly reduce the protein levels of periostin and vimentin, thereby affecting fibroblast activation.

6. The application according to any one of claims 1 to 5, characterized in that, The radioactive nuclide 177 Lu-labeled FAPI is 177 Lu-FAPI-04.

7. The application according to claim 6, wherein The targeted myocardial fibrosis drug enables the dosage of FAPI-04 to be much lower than the dosage required to achieve a therapeutic effect through the effect of 177 Lu in inhibiting fibroblasts.

8. The application according to claim 1, wherein The FAPI also includes FAPI-34, FAPI-46, FAPI-74, and FAP-2286.

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