CN114920741B - Iodine-labeled tumor KRAS G12C mutation targeting tracer agent, preparation method and application - Google Patents

Abstract

The invention relates to an iodine-marked tumor KRAS G12C mutation targeting tracer agent, a preparation method and application. The existing KRAS mutation detection means cannot completely meet the target requirement of accurate diagnosis and treatment. The invention provides an iodine-marked tumor KRAS G12C mutation targeting compound I-QZLO, which contains ¹²³ I、 ¹²⁴ I、 ¹²⁵ I or ¹³¹ The radiotracer of the compound I is used as a radioactive diagnostic probe for KRAS G12C mutation positive tumor of human or animals, ¹²⁷ the compound I was used as a stable standard. The radioactive tracer prepared by the invention has proper physicochemical and radiological properties, relatively ideal biological characteristics, good in-vivo biological distribution, and high targeting and high specificity for KRASG12C mutation proved by in-vitro cell and animal experiments, and can be used for KRASG12C mutation specific molecular imaging, molecular targeted therapeutic drug selection and curative effect judgment.

Description

Iodine-labeled tumor KRAS G12C mutation targeting tracer agent, preparation method and application

Technical Field

The invention relates to a radioactive imaging tracer, in particular to an iodine-marked murine sarcoma virus oncogene (kirsten rat sarcoma viral oncogene, KRAS) G12C mutation targeting tracer, a preparation method and application.

Background

Lung cancer is a serious disease that severely threatens human health, with morbidity and mortality leading to all tumors. In recent years, the therapeutic effect of EGFR mutant lung cancer is revolutionarily improved by molecular targeted drugs represented by EGFR-tyrosine kinase inhibitors (Epidermal growth factor receptor-tyrosine kinase inhibitor, EGFR-TKI), and the Progression-free survival (PFS) is greatly increased to 8.4-13.1 months from 4.6-6.7 months of traditional chemoradiotherapy. However, clinical studies have also shown that 29% -63% of EGFR mutant patients do not respond to EGFR-TKI treatment. Therefore, it is difficult to accurately evaluate and predict the therapeutic effect of EGFR-TKI of lung cancer by simply relying on EGFR mutation screening, and a new strategy for EGFR-TKI therapeutic effect prediction is urgently needed to be established.

KRAS mutation, one of the most common genetic mutations in lung cancer, can significantly reduce or even completely eliminate the therapeutic response of lung cancer patients to EGFR-TKI, and is an important efficacy predictive marker. Based on the important role of KRAS mutation in lung cancer molecular targeted therapy decisions and prognosis, several international organizations such as the american society for clinical oncology (American Society of Clinical Oncology, ASCO) recommend KRAS mutation screening as a routinely accepted clinical molecular examination for lung cancer patients; the national cancer network (National Comprehensive Cancer Network, NCCN) guidelines indicate that "KRAS mutation or not determines whether patients still have to detect other molecular therapeutic targets". Therefore, accurate detection of KRAS mutation is a significant clinical need for accurate diagnosis and treatment of lung cancer. However, in the existing KRAS mutation detection means in clinic, invasive puncture biopsy is limited by the limitation of a puncture part and cannot accurately reflect the heterogeneity of KRAS, and the problem of low sensitivity exists in circulating tumor DNA detection, so that the two problems cannot completely meet the target of accurate diagnosis and treatment. The molecular image can be used for real-time, quantitative and visual imaging of living biological molecules.

In recent years, KRAS mutation targeting molecules AMG510 and ARS1620 and the like are sequentially introduced, the former has been approved by FDA and marketed, and the KRAS mutation targeting molecules AMG510 and ARS1620 can inhibit the activity of KRAS G12C protein by irreversibly binding to the KRAS mutation targeting molecules AMG and ARS1620, so that a brand-new development opportunity is provided for molecular imaging of KRAS mutation. Patients with objective remission rate (Objective Response Rate, ORR, patient proportion with tumor volume reduction of > 30%) of 36% (95% CI: 28-45), 81% (95% CI: 73-87) achieved disease control (achieving complete remission, partial remission and patient proportion with stable disease condition exceeding 3 months) with once daily oral administration of 960mg AMG510 treatment. Median duration of remission (Duration of response, doR) was 10 months.

Disclosure of Invention

The invention aims to provide an iodine-marked tumor KRAS G12C mutation targeting tracer, a preparation method and application thereof, which are used for marking radionuclide I on the basis of AMG510 to construct the targeting tumor KRAS G12C mutation targeting tracer so as to solve the problem that the existing KRAS mutation detection means cannot completely meet the accurate diagnosis and treatment target.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

providing an iodine-labeled tumor KRAS G12C mutation targeting compound which is I-QZLO and has the chemical structure as follows:

wherein I is ¹²³ I、 ¹²⁴ I、 ¹²⁵ I、 ¹²⁷ I or ¹³¹ I。

In another aspect, there is provided a radiotracer comprising a compound as described, the tracer comprising an I-QZLO having the chemical structure:

wherein I is ¹²³ I、 ¹²⁴ I、 ¹²⁵ I or ¹³¹ I。

Further, the radiotracer also includes a solvent.

Further, the solvent is PBS containing ethanol or injection water or physiological saline solution.

In another aspect, there is provided the use of a radiotracer as described as a KRAS G12C mutation positive tumour radiodiagnostic probe for humans or animals.

In another aspect, there is provided a method for preparing a radiotracer as described, the method for preparing a tracer comprising I-QZLO by an automated synthesis module, comprising in particular the steps of:

dissolving the Iodogen and the AMG510 in dimethyl sulfoxide, diluting with normal saline to obtain a reagent No. 1, adding a reagent No. 2 and a radioactive sodium iodide solution to obtain a mixed solution, and reacting at room temperature; wherein iodine is ¹²³ I、 ¹²⁴ I、 ¹²⁵ I or ¹³¹ I；

Acetonitrile is taken as a mobile phase, and the mixed solution is separated by a preparation liquid chromatograph of a synthesizer;

and (3) passing the separated product through a C18 reversed-phase solid-phase extraction column, then flushing with injection water, and eluting adsorbates on the C18 reversed-phase solid-phase extraction column into a product bottle sequentially by using ethanol and PBS or injection water or normal saline to obtain the tracer containing the I-QZLO.

In another aspect, there is provided the use of a compound as described, wherein the compound is I-QZLO, having the chemical structure:

wherein I is ¹²⁷ I；

The compounds are useful as stabilizing standards.

In another aspect, there is provided a process for the preparation of the compound, I-QZLO, having the chemical structure:

wherein I is ¹²⁷ I；

The preparation method comprises the following steps:

dissolving the Iodogen and AMG510 in dimethyl sulfoxide, diluting with normal saline, adding into iodide solution to obtain mixed solution, reacting at room temperature, separating product by high performance liquid chromatography, and freeze drying overnight to obtain the compound.

Further, the iodide is potassium iodide or sodium iodide, and the iodine is ¹²⁷ I。

Compared with the prior art, the invention has the following beneficial effects:

the radioactive tracer prepared by the invention has proper physicochemical and radiological properties, relatively ideal biological characteristics, good in-vivo biological distribution, and high targeting and high specificity for KRAS G12C mutation proved by in-vitro cell and animal experiments, and can be used for KRAS G12C mutation specific molecular imaging, molecular targeted therapeutic drug selection and curative effect judgment.

The present invention selects nuclide iodine to label AMG 510. The dilute ketone structure in AMG510 is active group, and can be irreversibly condensed with sulfhydryl of KRAS protein G12C mutated Cys amino acid to inhibit KRAS protein activity, thereby achieving therapeutic effect. According to the invention, the iodine labeling of the AMG510 by the Iodogen method does not change the dilute ketone structure, so that the specificity and affinity of the I-QZLO to the KRAS G12C mutation are reserved, and the radioactive tracing of the KRAS G12C mutation can be realized. Meanwhile, iodine has a plurality of isotopes, including single photon nuclides such as iodine-123, iodine-125, iodine-131 and positron nuclides such as iodine-124, so that the requirements of different types of imaging, such as positron emission computed tomography (Positron Emission Computed Tomography, PET) and single photon emission computed tomography (Single Photon Emission Computed Tomography), can be met; the half-life of the radioisotope of the iodine is long, for example, the half-life of the iodine-124 is 4.17 days, and the half-life of the iodine-131 is 8.02 days, so that the radioisotope can be delivered in a medium-long distance and is convenient for clinical use.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other embodiments of the drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a block diagram of the present invention prepared in example 1 ¹²⁷ HRMS diagram of I-QZLO.

FIG. 2 is a schematic illustration of the process of example 2 ¹³¹ Allinone module labeling schematic diagram of I-QZLO.

FIG. 3 is a schematic illustration of the process of example 2 of the present invention ¹³¹ Radioactive high performance liquid chromatography (Radio-HPLC) co-injection identification chart of I-QZLO.

FIG. 4 is a photograph of a sample of the present invention prepared in example 2 ¹³¹ In vitro stability results for I-QZLO.

FIG. 5 is a photograph of an example 2 of the present invention ¹³¹ Cell uptake results for I-QZLO.

FIG. 6 is a photograph of an example 2 of the present invention ¹³¹ Results of I-QZLO in vivo distribution in normal mice.

Detailed Description

In order that the invention may be readily understood, a more complete description of the invention will be rendered by reference to the appended drawings. The drawings illustrate preferred embodiments of the invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

It should be noted that like reference numerals and letters refer to like items, and thus once an item is defined in one embodiment, no further definition or explanation thereof is necessary in subsequent embodiments. The particular implementation is described in terms of steps in some embodiments for clarity and accuracy of presentation and should not be construed as limiting the sequence. In addition, in the examples, the chemical used in the steps is an existing material or a commercially available product.

AMG510 (motorasib) is a small molecule, specific, irreversible KRAS G12C inhibitor that binds irreversibly to KRAS G12C protein to inhibit its activity, interferes with the dissociation of GDP on KRAS G12C protein, and inhibits KRAS-mediated signaling by locking KRAS G12C protein in an inactive GDP-bound state. The precedent of nuclide labeling is not seen at present, and the method is not used for the radiodiagnosis of KRAS G12C mutation positive tumors.

According to the invention, AMG510 is taken as a basis, radioiodination is carried out on the AMG510 to obtain an iodine-labeled tumor KRAS G12C mutation targeting compound, a radioactive molecular image probe is constructed, and a radioactive tracer containing the tumor KRAS G12C mutation targeting compound is prepared, so that the preparation of the radioactive tracer based on the AMG510 has feasibility, and the radioactive tracer is named as I-quinazolinone (quinazolinone), namely I-QZLO, and has the chemical structure:

the structure comprises KRAS G12C targeting ligand AMG510 and labeled radionuclide iodine, in particular ¹²³ I、 ¹²⁴ I、 ¹²⁵ I or ¹³¹ I。

Contains I-QZLO ¹²³ I、 ¹²⁴ I、 ¹²⁵ I or ¹³¹ I) Also included in the radiotracer of (2) is a solvent which may be added at relevant steps in the preparation process using ethanol-containing PBS or injectable water or physiological saline solution. The radiotracer can be used as a human or animal KRAS G12C mutation positive tumor radiodiagnostic probe.

The radioactive tracer is prepared by an automatic synthesis module and specifically comprises the following steps:

dissolving the Iodogen and the AMG510 in dimethyl sulfoxide, diluting with normal saline to obtain a reagent No. 1, adding a reagent No. 2 and a radioactive sodium iodide solution to obtain a mixed solution, and reacting at room temperature; wherein iodine is ¹²³ I、 ¹²⁴ I、 ¹²⁵ I or ¹³¹ I；

Acetonitrile is taken as a mobile phase, and the mixed solution is separated by a preparation liquid chromatograph of a synthesizer;

and (3) passing the separated product through a C18 reversed-phase solid-phase extraction column, then flushing with injection water, and eluting adsorbates on the C18 reversed-phase solid-phase extraction column into a product bottle sequentially by using ethanol and PBS or injection water or normal saline to obtain the radioactive tracer containing the I-QZLO.

The invention also provides another tumor KRAS G12C mutation targeting compound I-QZLO by ¹²⁷ The I mark can be used as a stable standard substance, and has the chemical structure as follows:

the preparation method of the stable standard substance comprises the following steps:

dissolving the Iodogen and AMG510 in dimethyl sulfoxide, diluting with normal saline, adding into iodide solution to obtain mixed solution, reacting at room temperature, separating product by high performance liquid chromatography, and freeze drying overnight to obtain stable standard product. The iodide is potassium iodide or sodium iodide, and the iodine is ¹²⁷ I。

The two preparation methods can be completed in the same synthesis route, and the synthesis route of the I-QZLO is specifically as follows:

step 1: AMG510 is stabilized by reaction condition "a" which is: dichloro diphenyl glycoluril (Iodogen), naI or KI, DMSO.

The step 1 specifically comprises the following steps:

dissolving equimolar dichlorodiphenyl glycoluril (Iodogen) and AMG510 in dimethyl sulfoxide, diluting with physiological saline, adding 1.5 times equivalent of non-radioactive sodium iodide or potassium iodide aqueous solution to obtain mixed solution, reacting at room temperature for 30 min, separating product by high performance liquid chromatography, and lyophilizing overnight to obtain stable standard substance ¹²⁷ I-QZLO。

Step 2: AMG510 is subjected to reaction condition "b" to obtain a radioactive tracer, wherein reaction condition "b" is: dichloro diphenyl glycoluril (Iodogen), naI, DMSO.

The step 2 is specifically as follows:

step 2.1: dissolving the Iodogen and the AMG510 in dimethyl sulfoxide, diluting with normal saline to obtain a reagent No. 1, adding a reagent No. 2 and a radioactive sodium iodide solution to obtain a mixed solution, and reacting for 10 minutes at room temperature; wherein iodine is ¹²³ I、 ¹²⁴ I、 ¹²⁵ I or ¹³¹ I；

Step 2.2: separating the mixed solution by using 50% acetonitrile as a mobile phase through a preparation liquid chromatograph of a synthesizer;

step 2.3: and (3) passing the separated product through a C18 reversed-phase solid-phase extraction column, then flushing with injection water, and eluting adsorbates on the C18 reversed-phase solid-phase extraction column into a product bottle sequentially by using ethanol and PBS or injection water or normal saline to obtain the radioactive tracer containing the I-QZLO.

Example 1:

by passing through ¹²⁷ I marking AMG510, preparing stable standard substance ¹²⁷ I-QZLO：

AMG510 (7.2 mg,0.013 mmol) and Iodogen (19.3 mg,0.045 mmol) were dissolved in 0.2mL anhydrous dimethyl sulfoxide (dimethyl sulfoxide, DMSO), and 0.8mL non-radioactive potassium iodide ([ solution) ¹²⁷ I]Aqueous solution of KI,23.2mg,0.14 mmol) was reacted at room temperature for 30 minutes to give a brown solution. Purifying by high performance liquid chromatography (High Performance Liquid Chromatography, HPLC) to obtain product, and lyophilizing overnight to obtain white powder stable standard compound ¹²⁷ I-QZLO (3.71 mg, 41.7%). FIG. 1 is a schematic view of ¹²⁷ HRMS diagram of I-QZLO, in which ESI-MS, [ M+H ]] ⁺ :m/z＝687.3。

Stable standard ¹²⁷ The I-QZLO is used for assisting the structural identification of the radioactive probe and the high-efficiency liquid phase purification.

Example 2:

by passing through ¹³¹ I labelling AMG510, preparation of the compositions containing ¹³¹ Tracer of I-QZLO:

step 1: mu.g of Iodogen and 50. Mu.g of AMG510 were dissolved in 100. Mu.L of anhydrous dimethyl sulfoxide (dimethyl sulfoxide, DMSO), diluted to 0.5mL with physiological saline, and added [ thereto ¹³¹ I]NaI solution (50 mCi,2.5 mL) was reacted at room temperature for 10min.

Step 2: and (3) taking 50% acetonitrile as a mobile phase, separating the mixed solution in the step (1) through a preparation liquid chromatograph of a synthesizer at a flow rate of 4mL/min, and collecting the separated product with the retention time of 9-11 min on a gamma chromatogram in a 50mL rotary bottle (containing 25mL injection water). The synthesizer is an automatic synthesizer of the models such as Trasis alinone or GE FXFN. FIG. 2 is a schematic view of ¹³¹ Allinone module labeling schematic diagram of I-QZLO.

Step 3: passing the separated product through C18 reversed phase solid phase extraction column, washing with 10mL of injection water, eluting the adsorbate on the C18 reversed phase solid phase extraction column with 1.0mL of absolute ethanol and 9.0mL of injection water, and filtering with a sterilizing filter (Merck Millipore MILLEX-GV 0.22 μm SLGVR33 RB) to obtain injectable product ¹³¹ I-QZLO solution (10% ethanol).

The preparation time of this example was about 40 minutes, with a total radiochemical yield of 24.0±5.66% (n=3, corrected for decay) and a radiochemical purity of greater than 99%.

The compositions prepared in this example contain ¹³¹ The performance of the I-QZLO tracer is measured as follows:

(1) High performance liquid chromatography (Radio-HPLC) identification:

HPLC conditions: the column was an octadecyl bonded silica gel reversed phase column (Inertsil ODS-SP,4.6 mm. Times.250 mm,5 μm, shimadzu Corp.) with acetonitrile and water (v: v=50:50) as mobile phases, isocratic elution, flow rate 1mL/min. ¹³¹ The retention time of the I-QZLO is 10.09min, the radiochemical purity is more than 99 percent and is higher than that of o-iodine in pharmacopoeia [ ¹³¹ I]The radiochemical purity of the sodium hippurate is more than 95 percent (Chinese pharmacopoeia 2020 edition, two parts). The Radio-HPLC results are shown in FIG. 3.

(2) Checking:

the pH value is 5.0-8.0 (Chinese pharmacopoeia 2020 edition, two parts, appendix VI H). Bacterial endotoxin detection: taking appropriate amount of the product (i.e. filtering with sterilizing filter to obtain product for injectionBy injection ¹³¹ I-QZLO solution), after 60-fold dilution with water for bacterial endotoxin test, the test was performed according to standard methods (Chinese pharmacopoeia 2020 edition, two parts, appendix XI E), the endotoxin content per 1mL of the product being less than 15EU. Sterile inspection: and detecting a proper amount of the product according to a standard method (Chinese pharmacopoeia 2020 edition, two parts and annex XI H), wherein the product meets the requirements.

(3) Radioactivity concentration:

accurately measuring a certain volume of the product, placing the product in an activity meter to measure the activity, and calculating the radioactive concentration according to the volume of the sample and the activity thereof. The radioactive concentration of the product is more than 37MBq/mL.

(4) Validity period: and calculating 48h from the calibration time.

(5) Fat-solubility: as measured by a shaking flask method, the liquid crystal display device, ¹³¹ I-QZLO lipid solubility log d=2.14±0.06.

(6) FIG. 4 is a diagram of ¹³¹ In vitro stability results of I-QZLO (I-QZLO) are shown, and after incubation for 2h at room temperature ¹³¹ The chemical purity of I-QZLO in normal saline is still more than 99%, which shows that ¹³¹ The I-QZLO has high physiological saline stability and is beneficial to clinical use.

FIG. 5 is a schematic view of a display ¹³¹ I-QZLO uptake results in H358 (KRAS G12C positive) cells, which showed that ¹³¹ I-QZLO is taken up in H358 cells to show that the protein can specifically bind KRAS G12C protein, and the AMG510 is marked ¹³¹ After I, the affinity and specificity for KRAS G12C protein are maintained.

FIG. 6 is a diagram of ¹³¹ I-QZLO normal mouse in vivo biodistribution result graph, ¹³¹ I-QZLO uptake in the liver was highest at 5 minutes, 40.4+ -3.9% ID/g, with time, ¹³¹ I-QZLO uptake is reduced in the liver and distribution is increased in the gastrointestinal tract, indicating probe intestinal hepatic metabolism. In addition, when injected into the tail vein for 15 minutes, the uptake of the kidney was higher than that of the liver, indicating that some of the liver metabolites were further metabolized by the kidney. The uptake of all other organs or tissues was relatively low, below 1% id/g at 15 minutes. These results indicate ¹³¹ Pharmacokinetics and safety of I-QZLO.

The foregoing description of the invention has been presented for purposes of illustration and description, and is not intended to be limiting. Several simple deductions, modifications or substitutions may also be made by a person skilled in the art to which the invention pertains, based on the idea of the invention.

Claims (5)

1. The application of the iodine-labeled tumor KRAS G12C mutation targeting compound in preparing a human or animal KRAS G12C mutation positive tumor radiodiagnosis probe is characterized in that:

the compound is I-QZLO, and the chemical structure is as follows:

wherein I is ¹³¹ I。

2. A radiotracer comprising a compound according to claim 1, wherein:

the radioactive tracer comprises I-QZLO, and the chemical structure is as follows:

wherein I is ¹³¹ I。

3. A radiotracer according to claim 2, wherein:

the radiotracer also includes a solvent.

4. A radiotracer according to claim 3, wherein:

the solvent is PBS containing ethanol or injection water or physiological saline solution.

5. A method of preparing a radiotracer according to claim 4, wherein:

the preparation method prepares the radioactive tracer containing the I-QZLO through an automatic synthesis module, and specifically comprises the following steps:

dissolving the Iodogen and the AMG510 in dimethyl sulfoxide, diluting with normal saline to obtain a reagent No. 1, adding a reagent No. 2 and a radioactive sodium iodide solution to obtain a mixed solution, and reacting at room temperature; wherein iodine is ¹³¹ I；

Acetonitrile is taken as a mobile phase, and the mixed solution is separated by a preparation liquid chromatograph of a synthesizer;

and (3) passing the separated product through a C18 reversed-phase solid-phase extraction column, then flushing with injection water, and eluting adsorbates on the C18 reversed-phase solid-phase extraction column into a product bottle sequentially by using ethanol and PBS or injection water or normal saline to obtain the radioactive tracer containing the I-QZLO.