CN104945487A - Radioactive halogen marked micromolecule cyclic peptide, composition and application of radioactive halogen marked micromolecule cyclic peptide - Google Patents

Abstract

The present invention provides a radioactive halogen-labeled small molecule cyclic peptide, characterized in that it is a chemical compound represented by formula (I): c(X ₁ HWGFTLX ₂ )-YZ (I), wherein, c(X ₁ HWGFTLX ₂ ) A cyclic peptide formed by the combination of 8-10 amino acids, X ₁ and X ₂ both have at least one amino acid residue; Y is a chelating ligand; Z is a metal complex containing radioactive halogen, which is chelated with Y . The present invention also provides a composition containing the above-mentioned small molecular cyclic peptide and the application of the small molecular cyclic peptide as a tumor imaging agent. According to the radioactive halogen-labeled small molecule cyclic peptide provided by the present invention, it can not only reach tumor cells in a targeted manner, but also has strong penetrability, can perform localized expression in deep tissues, has good clinical application prospects, and can be used for medical work. Provides a better basis for follow-up studies of the patient or diagnosis of cancer.

Description

Radioactive halogen-labeled small molecule cyclic peptide, composition and application thereof

technical field

The invention belongs to the field of medicine, and in particular relates to a radioactive halogen-labeled small molecular cyclic peptide, a composition containing the small molecular cyclic peptide and the application of the small molecular cyclic peptide as a tumor imaging agent.

Background technique

Tumor invasion and metastasis seriously affect the prognosis of patients, how to make an early diagnosis is a key clinical problem to be solved urgently.

Matrix metalloproteinases (matrixmetalloproteinases, MMPs) is a group of Zn ⁺ containing, involved in extracellular matrix (extra-cellular matrix, ECM) degradation of the most important class of proteases. Because MMPs can decompose the basement membrane and extracellular matrix, causing tumor invasion and metastasis, and MMPs can also regulate many components in the tumor microenvironment, thereby promoting the occurrence and development of tumors ^[1] , therefore, MMPs Preparing molecular probes for target sites for diagnosis is a hot spot in current research.

Gelatinase (MMP-2, 9) is an important subtype in the MMPs family. A large number of studies have found that it is highly expressed in various tumors such as ovarian cancer, prostate cancer, breast cancer, colon cancer, and kidney cancer ^[2] . MMP-2 is particularly closely related to the biological behavior of ovarian tumors, and its expression is related to the invasion, metastasis, malignancy and prognosis of ovarian cancer.

Small molecule cyclic peptide compounds have attracted the attention of many medical researchers as specific gelatinase inhibitors because of their good targeting and good stability in vivo. At present, there are research reports ^[3,4] using Cy5.5 to label the cyclic peptide c(KAHWGFTLD) NH2, and using the marker to perform optical imaging on a variety of living tumor models, but the fluorescence imaging distance of the marker is short and the tissue penetration The permeability is poor, the sensitivity is not high, and it is difficult to localize expression in deep tissues. It can only be used as a simple animal model for basic research, and lacks clinical application prospects.

[1]. Scherer RL, Mcintyre JO, Matrisian LM. Cancer Metastasis Rev, 2008, 27(4): 679-690.

[2]. Karakiulakis G, Papanikolaou C, Jankovic S Metal. Invasion Metastasis, 1997, 17:158–168.

[3].WangW, ShaoR, WuQ, KeS, McMurrayJ, LangFFJr, CharnsangavejC, GelovaniJG, LiC.MolImagingBiol.2009Nov-Dec;11(6):424-33.

[4]. LeeCM, JangD, CheongSJ, JeongMH, KimEM, KimDW, LimST, SohnMH, JeongHJ. IntJCancer. 2012Oct15;131(8):1846-53.

Contents of the invention

The purpose of the present invention is to provide a radioactive halogen-labeled small molecule cyclic peptide, a composition containing the radioactive halogen-labeled small molecule cyclic peptide and the application of the radioactive halogen-labeled small molecule cyclic peptide as a tumor imaging agent to solve above question.

In order to achieve the above object, the technical scheme adopted in the present invention is:

The radioactive halogen-labeled small molecule cyclic peptide is characterized in that it is a compound represented by formula (I):

c(X ₁ HWGFTLX ₂ )-YZ (I)

Among them, c(X ₁ HWGFTLX ₂ ) is a cyclic peptide formed by combining 8-10 amino acids, both X ₁ and X ₂ are at least one amino acid group; Y is a chelating ligand; Z is a metal complex containing radioactive halogen The substance is chelated with Y, wherein the cyclic peptide contains free amino groups, the amino groups are combined with the chelating ligand, and the radioactive halogen is ¹⁸ F, which is chelated with Y through metals.

In addition, the radioactive halogen-labeled small molecule cyclic peptide involved in the present invention may also have the following characteristics: wherein, c(X ₁ HWGFTLX ₂ ) is c(KAHWGFTLD)NH ₂ , that is, C6, and its structure is:

In addition, the radioactive halogen-labeled small molecule cyclic peptide involved in the present invention may also have the following characteristics: wherein, Y is 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) or 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA).

In addition, the radioactive halogen-labeled small molecule cyclic peptide involved in the present invention may also have the following characteristics: wherein, Z is an Al ¹⁸ F complex.

Furthermore, the present invention discloses a composition characterized by comprising the above radioactive halogen-labeled small molecule cyclic peptide and a medically acceptable carrier.

Moreover, the present invention also discloses the application of the radioactive halogen-labeled small molecule cyclic peptide as a tumor imaging agent.

Function and Effect of Invention

According to the radioactive halogen-labeled small molecule cyclic peptide provided by the present invention, since c(X ₁ HWGFTLX ₂ ) can specifically act on MMP-2 and 9, the radioactive halogen-labeled small molecule cyclic peptide can target the tumor cell.

In addition, due to the presence of ¹⁸ F, it is possible to use positron emission tomography (PET) to scan and image the distribution of radioactive halogen-labeled small molecule cyclic peptides in vivo, showing that the radioactive halogen-labeled small molecule cyclic peptides have high The uptake area is the tumor cell area. Using PET for imaging not only has high sensitivity and accurate results, but also can be detected non-invasively and dynamically.

In addition, due to the strong penetrability of ¹⁸ F, it can localize and express deep tissues, and the imaging outline is clear. It has a good clinical application prospect and can provide a good basis for medical workers' follow-up research or cancer diagnosis.

In addition, due to the chelating ligand, the chelating ligand can chelate with the metal complex containing halogen, and the formed chelate has high thermodynamic and dynamic stability, and can be kept complete in the body, so it can be better imaging.

Description of drawings

Fig. 1 is the HPLC spectrogram of the radioactive halogen-labeled small molecule cyclic peptide involved in the present invention in an embodiment;

Fig. 2 is the HPLC spectrogram of the in vitro stability test of the radioactive halogen-labeled small molecule cyclic peptide involved in the present invention;

Fig. 3 is a PET/CT whole-body static scanning image of the radioactive halogen-labeled small molecule cyclic peptide involved in the present invention;

Fig. 4 is the tumor time-radioactive counting curve of the radioactive halogen-labeled small molecule cyclic peptide involved in the present invention; and

Fig. 5 is an immunohistochemical staining diagram of the radioactive halogen-labeled small molecule cyclic peptide involved in the present invention in the embodiment of SKOV-3 ovarian cancer.

Detailed ways

The radioactive halogen-labeled small molecule cyclic peptides, compositions and applications of the present invention will be further described in conjunction with the accompanying drawings.

<Example>

In this example, the selected chelating ligand is NOTA, the metal complex containing radioactive halogen is Al ¹⁸ F complex, and the small molecule cyclic peptide is c(KAHWGFTLD)NH2, which is C6. In this example, c(KAHWGFTLD)NH-NOTA (C6-NOTA) was selected for labeling. C6-NOTA was synthesized by Hangzhou Zhongpei Biochemical Co., Ltd. by solid-phase synthesis.

1. Preparation of C6-NOTA-Al- ¹⁸ F

Step 1: producing [ ¹⁸ F]F ^- by nuclear reaction ¹⁸ O(P,n) ¹⁸ F on a cyclotron;

Step 2: Add sterilized water for injection to C6-NOTA to dissolve, and prepare a 1mg/ml solution;

Step 3: Take 30 μl of C6-NOTA sterile aqueous solution for injection, add 400 μl of acetonitrile, 6 μl of 2mmol/L aluminum chloride aqueous solution, 10 μl of acetic acid and 60 μl of ¹⁸ F ^- (20mCi) physiological saline solution, and react at 100°C for 15 minutes , add 15mL water for injection to dilute, pass through the C18 column, wash the C18 column twice with 25ml water for injection to remove excess fluoride ions and non-radioactive impurities, then rinse the C18 column with 300μl, 10mmol hydrogen chloride ethanol solution, and collect the markers The eluent was used to obtain a 9.7mCi hydrogen chloride ethanol solution of the marker.

The markers were analyzed by HPLC using a dedicated radioactive detector. The chromatographic analysis column is a C18 column (4.6×250mm), the mobile phase A is water (containing 0.1v% trifluoroacetic acid), and the mobile phase B is acetonitrile (containing 0.1v% trifluoroacetic acid). Gradient analysis conditions: flow rate is 1ml/min; 0~2min, A/B=95/5, 32minA/B=35/65. The detection wavelengths are 254nm and 218nm.

Fig. 1 is the HPLC spectrogram of the radiohalogen-labeled small molecule cyclic peptide involved in the present invention in an embodiment.

The HPLC analysis results shown in Figure 1 show that the radioactive peak time of C6-NOTA-Al- ¹⁸ F is 19.3 minutes, the radiochemical purity is >95%, and the labeling rate is 46.4%.

2. In vitro stability determination

Take the marker and dilute it with physiological saline, place it at 37°C for 0h, 2h and 4h, and take samples to determine the radiochemical purity.

Fig. 2 is the HPLC spectrogram of the in vitro stability test of the radioactive halogen-labeled small molecule cyclic peptide involved in the present invention.

The HPLC test results are shown in Figure 2, a is the HPLC spectrum when the storage time is 0h, b is the HPLC spectrum when the storage time is 2h, and c is the HPLC spectrum when the storage time is 4h. The results showed that the radiochemical purity of C6-NOTA-Al- ¹⁸ F did not change much when placed in normal saline for 0h, 2h and 4h, and its radiochemical purity was still greater than 95% after being placed in 4h. It shows that the chemical properties of the C6-NOTA-Al- ^18F provided in this example are relatively stable, and the radiochemical purity basically remains unchanged even if the standing time is prolonged.

3. In vivo PET imaging

3.1 PET/CT Whole Body Static Imaging

Four nude mice bearing SKOV-3 human ovarian cancer were placed on a MicroPET scanner (Inveon; Siemens). After anesthesia, 0.2ml of 50μCi marker was injected into the tail vein under the conditions of excessive C6 blocking and non-blocking, respectively, and static imaging was performed for 10 minutes at 30 minutes, 60 minutes and 120 minutes after injection. Tissue uptake values were analyzed using the region of interest technique (ROI).

Fig. 3 is a PET/CT whole-body static scanning image of the radioactive halogen-labeled small molecule cyclic peptide in an embodiment of the present invention.

As shown in Figure 3, a is the unblocked scanning image. In the absence of C6 blocking, there is obvious imaging 30 minutes after injection. At 120 min after injection, the uptake of markers was significantly reduced.

b is the scanning image of blocking, in the case of excessive C6 blocking, the uptake of C6-NOTA-Al- ^18F by tumor cells is significantly reduced, indicating that the C6-NOTA-Al- ^18F provided in this example can targeted to tumor cells.

3.2PET/CT Whole Body Dynamic Imaging

Two SKOV tumor-bearing mice were placed on a MicroPET scanner (Inveon; Siemens), anesthetized by isoflurane inhalation, and 0.2ml of 50μCi marker was injected through the tail vein, and dynamic imaging was performed at 0min-60min after injection, 5min/frame, Outline the region of interest to measure the time-radioactivity counting curves of tumors, heart, liver, spleen, lung, kidney, blood, muscle and other tissues and organs.

Fig. 4 is a tumor time-radioactive counting curve of the radioactive halogen-labeled small molecule cyclic peptide in an embodiment of the present invention.

As shown in Figure 4, in the case of excessive C6 blockade, the uptake of C6-NOTA-Al- ^18F by tumor tissue was significantly reduced.

4. In vivo distribution and competitive inhibition test

Using normal saline as the carrier, take 0.2ml of C6-NOTA-Al- ^18F normal saline solution and place it in a 50ml volumetric flask, add water for injection to make up the volume, and then take 0.1ml in a counting tube to measure the in vitro activity as a standard. Another 24 tumor-bearing mice were selected and divided into 4 groups, 6 in each group. After injecting 0.2ml of the diluted marker into the tail vein, the in vivo distribution test and competition inhibition test were performed respectively, and the experimental results are shown in Table 1.

Drug distribution experiment in vivo:

Eighteen SKOV tumor-bearing mice were anesthetized with isoflurane inhalation, and 30 minutes, 60 minutes, and 120 minutes after tail vein injection of ¹⁸ F-Al-NOTA-C60.74MBq (6 tumor-bearing mice at each time point), the mice were dissected. Tumors, hearts, livers, spleens, lungs, kidneys, muscles, intestines, stomachs, brains, etc. were weighed and counted. After radioactive decay correction, the dose of radiopharmaceuticals absorbed by each tissue was calculated (%ID/g).

Competitive inhibition experiments:

Six SKOV tumor-bearing mice were anesthetized as before. Excessive C6 was administered intravenously in advance, and 18F-Al-NOTA-C60.74MBq was injected intravenously 1 hour later. The mice were dissected 1 hour later, and the doses of radiopharmaceuticals absorbed by each tissue were measured and calculated ( %ID/g), the method is the same as the distribution experiment in vivo.

Table 1 In vivo distribution of ¹⁸ F-Al-NOTA-C6 in SKOV tumor-bearing mice

organize 30min 60min 120min Block 60min Blood 1.84±0.29 0.33±0.10 0.35±0.11 0.07±0.01 brain 0.08±0.05 0.03±0.02 0.03±0.02 0.01±0.00 Heart 0.46±0.08 0.09±0.03 0.11±0.07 0.04±0.01 liver 1.79±0.42 1.21±0.53 1.20±0.25 0.19±0.02 spleen 2.09±1.02 1.69±0.51 1.07±0.29 0.06±0.00 lung 1.09±0.16 0.31±0.07 0.31±0.03 0.10±0.02 kidney 7.14±0.05 4.55±1.38 4.72±0.31 3.51±0.32 Stomach 0.49±0.31 0.21±0.19 0.07±0.02 0.05±0.03 intestinal 0.47±0.09 0.13±0.07 0.12±0.07 0.04±0.01 muscle 0.57±0.15 0.08±0.04 0.21±0.08 0.12±0.02 pancreatic 0.35±0.01 0.16±0.14 0.09±0.06 0.04±0.02 gonad 0.50±0.12 0.05±0.03 0.09±0.02 0.05±0.10 thyroid 0.88±0.13 0.16±0.07 0.30±0.16 0.17±0.03 Fat 0.44±0.08 0.18±0.10 0.12±0.07 0.04±0.01 bone 0.57±0.04 0.41±0.13 0.65±0.07 0.32±0.09 the tumor 1.20±0.24 0.75±0.25 0.27±0.14 0.26±0.14

It can be seen from Table 1 that most of C6-NOTA-Al- ^18F is excreted by the kidneys, and the uptake of C6-NOTA-Al- ^18F by tumor cells reaches the highest at 30 minutes. Compared with tumor cells not blocked by C6, the uptake of C6-NOTA-Al- ¹⁸ F by tumor cells blocked by C6 was significantly reduced (p<0.05), which further illustrates the C6- NOTA-Al- ¹⁸ F can target tumor cells.

5. Histopathological test and MMP-2 immunohistochemical test

The tumor tissues obtained after microPET/CT imaging were dissected for pathological and MMP-2 immunohistochemical detection.

Fig. 5 is an immunohistochemical staining diagram of the radioactive halogen-labeled small molecule cyclic peptide involved in the present invention in the embodiment of SKOV-3 ovarian cancer.

As shown in Figure 5, MMP-2 was highly expressed in tumor tissues.

Function and effect of embodiment

According to the C6-NOTA-Al- ^18F provided in this example, since the cyclic peptide C6 is used for labeling, the IC50 value of the cyclic peptide C6 is 7.97 μM, which has good stability in vivo and can specifically act on MMP-2.

Therefore, C6-NOTA-Al- ¹⁸ F can target tumor cells and has good clinical application prospects.

In addition, because of the radioactive element ¹⁸ F, the distribution of C6-NOTA-Al- ¹⁸ F in the body can be scanned and imaged by positron emission tomography (PET), showing that the radioactive halogen-labeled small molecule cyclic peptide The high uptake area is the tumor cell area. Using PET for imaging not only has high sensitivity and accurate results, but also can be detected non-invasively and dynamically.

In addition, ¹⁸ F has a short half-life, is easy to metabolize, and has strong penetrability. It can localize and express deep tissues, and has clear imaging contours. better basis.

In addition, the C6-NOTA-Al- ^18F provided in this example has a short detection time and fast imaging, and PET imaging can be performed within 30 minutes. Moreover, the metabolism of C6-NOTA-Al- ¹⁸ F is fast, and the imaging is obviously weakened after 120 minutes.

In addition, the preparation method of C6-NOTA-Al- ¹⁸ F is simple and relatively stable, and it still shows good stability after being placed for 4 hours.

In addition, the C6-NOTA-Al- ¹⁸ F assay provided in this example has high sensitivity, compared with Cy5.5-C6, although both C6-NOTA-Al- ¹⁸ F and Cy5.5-C6 can be used to detect tumors in vivo. However, for optical imaging, the amount of Cy5.5-C6 injected per nude mouse was 15nmol, while for PET imaging, the dose of C6-NOTA-Al- ^18F injected per nude mouse was 0.74MBq (approximately 11× 10 ^-13 nmol).

Of course, the radioactive halogen-labeled small molecule cyclic peptide involved in the present invention is not limited to the content in the above examples. The above content is only a basic description of the concept of the present invention, and any equivalent transformation made according to the technical solution of the present invention shall fall within the scope of protection of the present invention.

In addition, the small molecule cyclic peptide used in the above examples is c(KAHWGFTLD)NH ₂ , and the c(X ₁ HWGFTLX ₂ ) involved in the present invention can also be c(KTAHWGFTLD)NH ₂ .

In addition, the above examples use NOTA as the chelating ligand, and the chelating ligand involved in the present invention can also be DOTA.

In addition, the radioactive halogen-labeled small molecule cyclic peptide involved in the present invention may also be a medically acceptable salt of c(X ₁ HWGFTLX ₂ )NH-YZ.

In addition, the radioactive halogen-labeled small-molecule cyclic peptides in the above examples use physiological saline as a carrier for biological imaging. The radioactive halogen-labeled small-molecule cyclic peptides involved in the present invention can also be selected from other medically acceptable carriers , the carrier can be a pharmaceutical excipient or a pharmaceutical solvent.

In addition, the radioactive halogen-labeled small molecule cyclic peptide provided by the present invention is used to prepare a tumor imaging agent, and the imaging agent can image tumor cells during cancer diagnosis or monitoring.

In addition, the radioactive halogen-labeled small molecule cyclic peptide provided by the present invention, in addition to being used as a tumor imaging agent to image tumors, can also be used as an imaging agent for other highly expressed MMP-2 receptors, such as Used as an atherosclerotic plaque imaging agent to visualize atherosclerotic plaque.

Claims (6)

1. The radioactive halogen-labeled small molecular cyclic peptide, characterized in that it is a compound shown in formula (I): c(X ₁ HWGFTLX ₂ )-YZ (I) c(X ₁ HWGFTLX ₂ ) is a cyclic peptide formed by combining 8-10 amino acids, and both X ₁ and X ₂ have at least one amino acid group; Y is a chelating ligand; Z is a metal complex containing a radioactive halogen, chelated with said Y, Wherein, the cyclic peptide contains free amino groups, the free amino groups are combined with the Y, and the radioactive halogen is ¹⁸ F. 2. the radioactive halogen-labeled small molecule cyclic peptide according to claim 1, characterized in that: Wherein, the c(X ₁ HWGFTLX ₂ ) is c(KAHWGFTLD)NH ₂ . 3. the radioactive halogen-labeled small molecule cyclic peptide according to claim 1, characterized in that: Wherein, the Y is 1,4,7-triazacyclononane-1,4,7-triacetic acid or 1,4,7,10-tetraazacyclododecane-1,4,7,10 - tetracarboxylic acid. 4. The radioactive halogen-labeled small molecule cyclic peptide according to claim 1, characterized in that: Wherein, the Z is Al ¹⁸ F complex. 5. A composition, characterized in that it comprises the radioactive halogen-labeled small molecule cyclic peptide according to any one of claims 1-4 and a medically acceptable carrier. 6. The use of the radioactive halogen-labeled small molecule cyclic peptide as described in any one of claims 1-4 as a tumor imaging agent.