68Ga labeled probe capable of visualizing hepatocyte functions and preparation method of 68Ga labeled probe

Abstract

The invention provides a < 68 > Ga-labeled small molecule compound as well as a preparation method and application thereof, and belongs to the technical field of radiopharmaceuticals and nuclear medicine. The [68Ga] Ga-DTPA-EOB probe marked by 68Ga is suitable for positron emission tomography, has better temporal-spatial resolution and dynamic imaging capability, and can quantify tracer distribution. Under the condition that the pH value of the < 68 > Ga is 3.8-4.0, < 68 > Ga labeling can be completed only within 5-10 minutes at room temperature, the labeling efficiency is high (the labeling rate is greater than or equal to 95%), and the amount of EOB-DTPA required for labeling is extremely small. The radioactive activity needed by PET imaging is low, the radiation burden on a patient is small, and the gadolinium deposition risk is avoided. The [68Ga] Ga-DTPA-EOB probe is particularly suited for scenes requiring rapid diagnosis, avoiding MRI contraindications, or requiring multi-modal imaging.

Description

Technical Field

[0001] The present invention belongs to the technical field of radiopharmaceuticals and nuclear medicine, and specifically relates to a method for visualizing liver cell function. 68 Ga-labeled probe, preparation method and application thereof. Background Art

[0002] Gadolinium ethoxybenzyldiethylenetriaminepentaacetic acid (Gd-EOB-DTPA, trade name: Promec) is a hepatobiliary-specific MRI contrast agent. It is the disodium salt of a chelate of gadolinium (Gd) and ethoxybenzyl (EOB). Gd-EOB-DTPA is used not only for the diagnosis of liver diseases but also for the evaluation of bile duct morphology and function.

[0003] However, the existing Gd-EOB-DTPA, as the gold standard for MRI hepatobiliary imaging, still has the following problems: (1) Single functional assessment: It can only reflect the uptake function of hepatocytes and is not sensitive enough to assess the biliary excretion capacity under pathological conditions (such as cirrhosis); (2) Limited dynamic monitoring capability: MRI has low temporal resolution, making it difficult to track metabolic processes in real time and unable to quantitatively analyze pathological changes at the molecular level; (3) Gadolinium is highly toxic. The high dose of Gd-EOB-DTPA used as a nuclear magnetic resonance contrast agent may cause an increase in free gadolinium. In addition, the contrast agent is excreted through the kidneys. For patients with renal insufficiency, the excretion rate of the contrast agent is slowed, which may lead to a prolonged retention time of gadolinium in the body. For patients with existing kidney disease, it may aggravate renal impairment and even lead to acute kidney injury. In addition, for patients with existing liver insufficiency, large-scale or long-term use may place a greater burden on the liver. Studies have shown that long-term use of gadolinium-containing contrast agents may be associated with the occurrence of liver fibrosis.

[0004] Therefore, imaging markers for evaluating hepatobiliary function need to be further developed. Summary of the Invention

[0005] The present invention is to solve the above technical problems, thereby providing a nuclear medicine professional field 68 Ga-labeled small molecule compounds, their preparation methods, and their applications in visualizing hepatocyte function in liver diseases.

[0006] In order to achieve the above technical objectives, the technical solutions adopted by the present invention are as follows:

[0007] The present invention first provides a 68 Ga-labeled small molecule compound, the structural formula of the compound is shown in the following formula <Ⅰ>:

[0008]

[0009] The second object of the present invention is to provide a 68 The preparation method of a Ga-labeled small molecule compound comprises the following steps:

[0010] (1) Dissolve disodium benzoate (EOB-DTPA) in water and then mix with sodium acetate;

[0011] (2) using hydrochloric acid to elute the germanium gallium generator, discarding the front and back sections, taking the eluent in the middle section, adding it to the mixed solution obtained in step (1), and adjusting the pH to 3.8-4.2;

[0012] (3) The product obtained in step (2) is heated to 35-45°C for reaction. After the reaction is complete, the 68 Ga-labeled small molecule compounds.

[0013] Furthermore, the concentration of disodium thiocyanate in step (1) is 1 mg / mL.

[0014] Furthermore, the concentration of sodium acetate in step (1) is 1 mol / L.

[0015] Furthermore, the concentration of the hydrochloric acid in step (2) is 0.1 mol / L.

[0016] Furthermore, the reaction time in step (3) is 10 minutes.

[0017] Furthermore, after the reaction in step (3) is complete, the pH of the solution is adjusted to 5-7 to obtain an injection solution.

[0018] The third object of the present invention is to provide a nuclear medicine probe for visualizing liver cell function for the purpose of non-disease diagnosis and treatment, the probe comprising the above formula 68 Ga-labeled small molecule compounds.

[0019] The fourth object of the present invention is to provide a 68 Application of Ga-labeled small molecules as nuclear medicine probes for visualizing hepatocyte function.

[0020] The beneficial effects of the present invention are as follows:

[0021] (1) The present invention adopts 68 Ga-labeled nuclear medicine probes[ 68Ga]Ga-DTPA-EOB can be well used for the detection of liver disease by visualizing liver cell function and is applicable to positron emission tomography (PET). It has higher spatiotemporal resolution and dynamic imaging capabilities and can quantify tracer distribution. The existing Gd-EOB-DTPA can only be used for magnetic resonance imaging (MRI).

[0022] (2) PET does not rely on a magnetic field environment and is more user-friendly to patients with claustrophobia or those with metal implants (such as pacemakers). Therefore, the probe of the present invention has better application prospects. 68 Ga 3+ With Gd 3+ As a trivalent cation, it has the same charge characteristics after binding to EOB-DTPA and may retain a hepatocyte-specific uptake mechanism similar to that of Gd-EOB-DTPA.

[0023] (3) 68 Ga is completed in just 5-10 minutes at room temperature under pH=3.8-4.2 conditions. 68 Ga-labeled, and the labeling efficiency is high (labeling rate ≥ 95%), the amount of EOB-DTPA required for labeling is extremely small; while the clinical dose of Gd-EOB-DTPA is usually very large.

[0024] (4) 68 Ga has a short half-life (only 68 minutes), and the radioactivity required for PET imaging is low, which reduces the radiation burden on patients and eliminates the risk of gadolinium deposition. However, Gd-EOB-DTPA carries the potential risk of long-term gadolinium deposition in the body (especially in patients with renal insufficiency).

[0025] (5)[ 68 Ga]Ga-DTPA-EOB combines the high-resolution, fast dynamic imaging advantages of PET with the hepatobiliary specificity of EOB-DTPA. It is particularly suitable for scenarios that require rapid diagnosis, avoid MRI contraindications, or require multimodal imaging, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 for[ 68 Synthesis route and quality control of Ga]Ga-DTPA-EOB; A, [ 68 Synthesis route of Ga]Ga-DTPA-EOB; B. Radiochemical purity test; C. [ 68 Stability curves of Ga]Ga-DTPA-EOB in PBS and serum from 0 to 240 min.

[0027] Figure 2 for[ 68Distribution of Ga]Ga-DTPA-EOB in normal rats and rats with chronic liver injury; A, normal control group and chronic liver injury group (liver fibrosis model induced by CCl4 for 5 weeks) injected with [ 68 Dynamic whole-body PET / CT images from 0 to 28.5 min after Ga]Ga-DTPA-EOB; BG, SUVmax time-radioactivity uptake curves of the heart, blood vessels, intestine, kidney, muscle, and liver from 0 to 28.5 min in the normal control group and the chronic liver injury group, respectively (n = 3 for each group, error bars represent mean ± SD).

[0028] Figure 3 Chronic liver injury group (CCl4-induced liver fibrosis model) [ 68 Ga]Ga-DTPA-EOB PET / CT dynamic imaging: A, [ 68 Axial and coronal CT, PET, and PET / CT images of the livers treated with Ga]Ga-DTPA-EOB in the normal control group and the chronic liver injury group induced by CCl4 for 4 and 5 weeks; BC, time-radioactivity uptake curves of liver SUVmax and SUVR in the normal control group and the chronic liver injury group (liver fibrosis model induced by CCl4 for 4 weeks) (n=3 in each group); DE, time-radioactivity uptake curves of liver SUVmax and SUVR in the normal control group and the chronic liver injury group (liver fibrosis model induced by CCl4 for 5 weeks) (n=3 in each group). Figure 4 The liver of rats in the chronic liver injury group (CCl4-induced liver fibrosis model for 7 weeks) 68 Ga]Ga-DTPA-EOB PET / CT, liver[ 68 Comparison of Ga]Ga-FAPI PET / CT and liver plain scan MRI (T2WI sequence): A, Transverse and coronal images of the liver in the normal control group and the chronic liver injury group (liver fibrosis model induced by CCl4 for 7 weeks) 68 Ga]Ga-DTPA-EOB PET / CT, [ 68 Ga]Ga-FAPIPET / CT, and MRI plain scan (T2WI sequence); BC, [ 68 Ga]Ga-DTPA-EOB at 13.5 min in rats' livers (n=3 in each group, error bars represent mean ± SD), *P < 0.05; DE, [ 68 Ga]Ga-FAPI at 13.5 min in the liver SUVmax and SUVR of rats in the normal control group and chronic liver injury group (n=3 in each group, error bars represent mean±SD), **P<0.01.

[0029] Figure 5The acute liver injury rat model (CCl4-induced acute liver injury ALI model and ischemia-reperfusion IRI model) 68 Ga]Ga-DTPA-EOB PET / CT dynamic imaging: A, acute liver injury group (ALI model and IRI model) and normal control rats [ 68 A, Axial and coronal CT, PET, and PET / CT images of the liver obtained by dynamic imaging with Ga]Ga-DTPA-EOB PET / CT; B, Time-radioactivity uptake curves of the liver SUVmax within 0-28.5 min in the acute liver injury group, the chronic liver injury group (liver fibrosis model induced by CCl4 for 5 weeks), and the normal control group (n=3 in each group); C, Comparison of the liver SUVmax and SUVR at 13.5 min in each group (n=3 in each group, error bars represent mean ± SD), *P < 0.05, **P < 0.01.

[0030] Figure 6 To verify the results of liver pathology and serology: A, HE (200×) and Masson (100×) staining results of livers in each group; BC, the percentage of liver collagen fibers in Masson staining of livers in each group (%) and the corresponding liver [ 68 SUVmax of Ga]Ga-DTPA-EOB uptake. D. Serum ALT statistical results for each group. E. Serum AST statistical results for each group. (Normal control group, n = 5; chronic liver injury group, n = 4; acute liver injury group, ALI model, n = 3; IRI model, n = 3) *P < 0.05, **: P < 0.01, ***: P < 0.001.

[0031] Figure 7 HE and Masson staining of the brain, heart, lung, liver, stomach, spleen, kidney, large intestine, testis, small intestine and pancreas of normal rats in the conventional imaging dose control group and the simulated toxic dose group (HE 200×, brain coronal 10×, brain coronal local magnification 400×, Masson 100×).

[0032] Figure 8 Prepared by the method of Comparative Example 1 68 HPLC detection spectrum of Ga]Ga-DTPA-EOB.

[0033] Figure 9 Prepared by the method of Comparative Example 2 68 HPLC detection spectrum of Ga]Ga-DTPA-EOB. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is described in detail below with reference to the following embodiments. It is necessary to point out that the following embodiments are only used to explain and illustrate the present invention and are not intended to limit the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above invention content still fall within the scope of protection of the present invention.

[0035] Example 1

[0036] 1. Synthesis and Characterization of Probes

[0037] 1. 68 Synthesis of Ga]Ga-DTPA-EOB Probe

[0038] [ 68 The synthetic route of Ga]Ga-DTPA-EOB is as follows Figure 1 As shown in A, the specific steps are as follows:

[0039] (1) First, dissolve 1 mg of DTPA-EOB in 1 mL of ultrapure water, take 100 μL (100 μg) and add it to a reaction flask. Then, add 210 μL of 1 mol / L sodium acetate to the reaction flask.

[0040] (2) Take 6 mL of 0.1 mol / L hydrochloric acid to rinse the Eckert & Ziegler gallium germanium generator, discard the front section and the back section 2 mL, take the end with the highest gallium ion concentration 2 mL and add it to the reaction bottle, so that the final pH of the reaction solution is about 4;

[0041] (3) Heat the reaction flask to 40°C. The reaction will be complete in about 10 minutes. After the reaction, adjust the pH to 5-7 without purification and it is ready for injection. Take 100 μL for product quality control.

[0042] 2. 68 Characterization of Ga]Ga-DTPA-EOB

[0043] (1) Take 20 μL of the reaction solution and perform radiochemical purity test using an Agilent 1260 HPLC instrument equipped with a radioactivity detector ( Figure 1 Middle B), the detection results showed that the radiochemical purity was ≥95%.

[0044] (2) 68 The stability of Ga]Ga-DTPA-EOB was tested by adding it to PBS and PBS containing 10% FBS ( Figure 1 C), the results show [ 68 Ga]Ga-DTPA-EOB has good stability in both PBS and serum.

[0045] 2. Performance testing of probes

[0046] 1. 68 Distribution of Ga]Ga-DTPA-EOB in rats

[0047] [ 68 The distribution of Ga]Ga-DTPA-EOB in rats is as follows Figure 2 As shown in Figure A, the probe is mainly excreted through the kidneys and intestines. In the control group and the chronic liver injury group (CCl4-induced liver fibrosis model), [ 68 There was no significant difference in the uptake of Ga]Ga-DTPA-EOB in the two groups, while the time-radioactivity uptake trends of the other organs were consistent. Except for the liver, there was no significant difference in the SUVmax of other organs between the two groups (P>0.05). At the beginning of the experiment, all organs showed uptake, with the highest uptake in the kidney (average SUVmax during the imaging period of 14.98±4.05) and the lowest uptake in muscle (average SUVmax during the imaging period of 0.60±0.04). Subsequently, uptake in the heart, blood vessels, and muscles gradually decreased; the intestinal tract was uptaken by [ 68 The excretion of Ga]Ga-DTPA-EOB through the hepatobiliary pathway showed a trend of increasing first and then decreasing. The uptake by the kidneys also showed a pattern of increasing first and then decreasing, and reached the maximum uptake value at 4.5 minutes ( Figure 2 (Medium BG).

[0048] 2. 68 Imaging experiment of Ga]Ga-DTPA-EOB in chronic liver injury group (CCl4-induced liver fibrosis model)

[0049] [ 68 Imaging of liver fibrosis using Ga]Ga-DTPA-EOB Figure 3 As shown in A, the chronic liver injury group started to show liver [ 68 The uptake of Ga]Ga-DTPA-EOB PET was lower than that of the normal control group. Figure 3 BE showed that the time-radioactivity uptake of the liver of the normal control group showed a trend of gradually increasing to a stable state from 0 to 28.5 minutes. The liver uptake of the normal control group was higher than that of the chronic liver injury group at different induction time periods. At the fourth week of induction, there was no statistically significant difference in liver SUVmax and SUVR at each time point between the chronic liver injury group and the control group (P>0.05). The time-radioactivity uptake curve of the chronic liver injury group also showed a trend of gradually increasing to a stable state ( Figure 3B and C). At the 5th week of induction, the time-radioactivity uptake curve of the chronic liver injury group showed a gradual downward trend. Starting from 10.5 minutes, the difference in liver SUVmax between the two groups was statistically significant (P < 0.05), but there was no statistically significant difference in SUVR between the two groups at any time point (P > 0.05) ( Figure 3 D and E).

[0050] 3. 68 Ga]Ga-DTPA-EOB, [ 68 Imaging comparison between Ga]Ga-FAPI and T2WI MRI

[0051] In the seventh week of CCl4 induction, [ 68 Ga]Ga-DTPA-EOB, [ 68 Ga]Ga-FAPIPET / CT imaging and liver plain MRI imaging (T2WI sequence). 68 The uptake of Ga]Ga-DTPA-EOB in the liver reached its peak between 4.5 and 19.5 minutes, so we chose to perform static imaging and analysis at 13.5 minutes. The results showed that the liver of the chronic liver injury group [ 68 The liver uptake on Ga]Ga-DTPA-EOB PET images was lower than that in the normal control group, and the uptake value difference trend was synchronized. The difference between the two groups was statistically significant, which was consistent with the previous imaging results ( Figure 4 AC, SUVmax P=0.03, SUVR P=0.02). 68 Ga]Ga-FAPI showed slight uptake (SUVmax 0.51±0.07), which was significantly different from the normal control group (SUVmax 0.19±0.07) (SUVmax P=0.009, SUVR P=0.002). Figure 4 DE), but the visual contrast on PET images is not as good as [ 68 Ga]Ga-DTPA-EOB was obvious. Liver plain scan MRI imaging (T2WI sequence) showed mild fibrosis imaging changes in the chronic liver injury group (CCl4-induced liver fibrosis model for 7 weeks), manifested as linear high T2 signals in the liver parenchyma, indicating liver interstitial hyperplasia, but the visual difference was not obvious compared with the normal control group ( Figure 4 A).

[0052] 4. Acute liver injury 68 Ga]Ga-DTPA-EOB PET / CT liver imaging

[0053] The liver uptake of the two models in the acute liver injury group was lower than that in the normal control group, showing a unilateral downward trend, and the decrease was more obvious than that in the chronic liver injury group ( Figure 5 A and B). We selected the 13.5-min time point for analysis and found that the uptake in the ALI model induced by CCl4 (SUVmax 0.72±0.34) and the IRI model induced by liver ischemia-reperfusion (SUVmax 0.76±0.23) was almost the same, and the uptake in the liver fibrosis group was slightly higher (SUVmax 1.30±0.38). Compared with the normal group, the liver uptake in the ALI model (SUVmax P=0.0012, SUVR P=0.0014) and the IRI model (SUVmax P=0.0014, SUVR P=0.0017) were significantly different. The difference in liver SUVmax (P=0.03) in the liver fibrosis group was statistically significant, but the difference in SUVR was not statistically significant (P>0.05) ( Figure 5 CD).

[0054] 5. Verification of liver pathology and serology results

[0055] After imaging, whole blood was collected from the heart of rats in the acute liver injury group (ALI model and IRI model) and chronic liver injury group (CCl4-induced liver fibrosis model for 5 weeks and 7 weeks), and serum was centrifuged to obtain ALT and AST test for liver function. At the same time, specimens of the right lobe of the liver were taken, fixed, and paraffin blocks and white slides were prepared for HE and Masson staining. HE staining showed that some hepatocytes in the chronic liver injury group (CCl4-induced liver fibrosis model for 5 weeks and 7 weeks) had fatty degeneration and ballooning; in the acute liver injury group (CCl4-induced ALI model), there was obvious hepatocyte necrosis and diffuse fatty degeneration in the liver lobules, accompanied by mild inflammatory cell infiltration; HE staining of the liver of rats in the IRI model showed hepatocyte degeneration and necrosis, a large number of sinusoids dilated, and red blood cell infiltration ( Figure 6 A).

[0056] Masson staining showed that fibrotic septa were formed in the liver tissue of the chronic liver injury group at 5 weeks of induction, and the liver structure was destroyed. At 7 weeks of induction, the fibrotic bands in the liver lobules and portal areas increased significantly. The fibrotic bands in the liver lobules and portal areas in the acute liver injury group were not as obvious as those in the chronic group ( Figure 6 A). Quantitative analysis of collagen fibers by Masson staining showed that the liver collagen fibers in both the chronic and acute groups increased compared with the normal group, and the difference in the chronic group was more statistically significant (P<0.05, Figure 6 B), but 68 The uptake of Ga]Ga-DTPA-EOB was not proportional to the amount of collagen fibers ( Figure 6 C), presumably liver tissue [ 68The uptake of Ga]Ga-DTPA-EOB may be more related to the functional status of liver cells. Serological results showed that the ALT and AST in the acute liver injury group were significantly higher than those in the chronic liver injury group and the normal control group, and the trend was consistent with the liver tissue [ 68 The uptake of Ga]Ga-DTPA-EOB was inversely proportional to the Figure 6 D and E).

[0057] 6. 68 Biosafety Testing of Ga]Ga-DTPA-EOB

[0058] The normal rats were injected with a dose higher than the previous imaging dose [ 68 Ga]Ga-DTPA-EOB (100MBq / kg) was used to simulate the toxic dose. 24 hours later, the brain, heart, lungs, liver, stomach, spleen, kidneys, large intestine, testicles, small intestine and pancreas were taken for HE staining, and the liver was also stained with Masson staining. Compared with the normal dose group (20MBq / kg), the HE staining results of each organ in the simulated toxic dose group showed that the tissue structure was intact and no obvious pathological abnormalities were observed. The Masson staining results of the liver also did not show abnormal deposition of collagen fibers ( Figure 7 ). Preliminary Notes[ 68 Safety of Ga]Ga-DTPA-EOB PET imaging.

[0059] Comparative Example 1

[0060] [ 68 The synthesis method of Ga]Ga-DTPA-EOB is as follows:

[0061] First, 1 mg of DTPA-EOB was dissolved in 1 mL of ultrapure water, and 100 μL (100 μg) was added to the reaction flask. Then 4 mL of pH = 7.4 PBS buffer was added to the reaction flask. 6 mL of 0.1 mol / L hydrochloric acid was used to rinse the Eckert & Ziegler gallium germanium generator, and 2 mL of the front and back sections were discarded. 2 mL of the middle section with the highest gallium ion concentration was added to the reaction flask, and the reaction flask was heated to 100 ° C and reacted for 10 minutes. 100 μL was taken for product quality control, and the radiochemical purity was 38.5% (by HPLC). Figure 8 ).

[0062] Comparative Example 2

[0063] [ 68 The synthesis method of Ga]Ga-DTPA-EOB is as follows:

[0064] First, 1 mg of DTPA-EOB was dissolved in 1 mL of ultrapure water, and 100 μL (100 μg) was added to the reaction flask. Then 4 mL of 0.5 mol / L potassium hydrogen phthalate (KHP) buffer was added to the reaction flask. 6 mL of 0.1 mol / L hydrochloric acid was used to rinse the Eckert & Ziegler gallium germanium generator, and 2 mL of the front and back sections were discarded. 2 mL of the middle section with the highest gallium ion concentration was added to the reaction flask, and the reaction flask was heated to 100 ° C and reacted for 10 minutes. 100 μL was taken for product quality control, and the radiochemical purity was 43.7% (by HPLC). Figure 9 ).

Claims

1. A 68 Ga-labeled small molecule compound, characterized in that The structural formula of the compound is shown in the following formula:

2. The method according to claim 1 68 The method for preparing a Ga-labeled small molecule compound is characterized in that: The following steps are involved: (1) dissolving disodium thiocarbate in water and then mixing with sodium acetate; (2) using hydrochloric acid to elute the germanium gallium generator, discarding the front and back sections, taking the eluent in the middle section, adding it to the mixed solution obtained in step (1), and adjusting the pH to 3.8-4.2; (3) The product obtained in step (2) is heated to 35-45°C for reaction. After the reaction is complete, the 68 Ga-labeled small molecule compounds.

3. The preparation method according to claim 1, characterized in that The concentration of disodium thiocyanate in step (1) is 1 mg / mL.

4. The preparation method according to claim 1, characterized in that The concentration of sodium acetate in step (1) is 1 mol / L.

5. The preparation method according to claim 1, characterized in that The concentration of hydrochloric acid in step (2) is 0.1 mol / L.

6. The preparation method according to claim 1, characterized in that The reaction time in step (3) is 10 min.

7. The preparation method according to claim 1, characterized in that After the reaction is complete in step (3), the pH of the solution is adjusted to 5-7.

8. A nuclear medicine probe for visualizing liver cell function for the purpose of non-disease diagnosis and treatment, characterized in that: The probe comprises the probe according to claim 1 68 Ga-labeled small molecule compounds.

9. The method according to claim 1 68 Application of Ga-labeled small molecule compounds in the preparation of nuclear medicine probes for visualizing hepatocyte function.

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