Method for determining < 99m > Tc-DOTA-FAPI-46 and metabolites thereof in biological sample

Abstract

The invention provides a method for determining < 99m > Tc-DOTA-FAPI-46 and metabolites of < 99m > Tc-DOTA-FAPI-46 in a biological sample. The determination method comprises the following steps: after performing intravenous injection on < 99m > Tc-DOTA-FAPI-46, collecting the sample; adding an organic solvent into the collected sample, uniformly mixing, centrifuging, taking supernate, blow-drying the supernate, dissolving the supernate in normal saline, and concentrating by 1-10 times to obtain a sample injection solution, and the volume ratio of the collected sample to the added organic solvent is 1: (1-3); the sample injection solution is taken and put into high performance liquid chromatography for determination, a detector is a radioactive detector, a chromatographic column is a reversed-phase chromatographic column, and the elution mode is gradient elution; and calculating the proportion of the peak areas of < 99m > Tc-DOTA-FAPI-46, radioactive metabolites and radioactive impurities in the sample in the total radioactive components according to the determined peak area containing the radioactive components. By setting proper pretreatment conditions and liquid chromatogram detection conditions, radioactive metabolites can be detected in a biological sample, the detection sensitivity and accuracy are improved, and the in-vivo stability of drugs can be accurately and efficiently evaluated.

Description

Technical Field

[0001] The present invention relates to the technical field of radiopharmaceutical analysis, and in particular to a method for determining 99m Methods for Tc-DOTA-FAPI-46 and its metabolites. Background Art

[0002] Fibroblast Activation Protein (FAP) is a type II transmembrane serine protease that is highly expressed in cancer-associated fibroblasts (CAFs) and is basically not expressed in healthy adult organs or tissues. FAP is associated with tumor invasion, angiogenesis, and metastasis, and is therefore considered a specific marker for CAFs, becoming a potential target for tumor diagnosis and treatment. Fibroblast activation protein inhibitor (FAPI) is a small molecule inhibitor that can specifically bind to FAP and is one of the promising molecular probes for precise tumor chemodiagnosis and treatment. Among them, FAPI-21 and FAPI-46 have received widespread attention due to their good tumor-to-background ratio.

[0003] 99m Tc is the most widely used single-photon radionuclide in single-photon emission computed tomography (SPECT / CT). 99m Tc-DOTA-FAPI-46 molecular probe can be used for early diagnosis of various tumors and has high potential clinical value. 99m The detection method of Tc-FAPI drugs is mainly through 99m The gamma signal emitted by Tc is detected. After biological samples such as blood, urine, tissues and organs are collected, a gamma counter is used for detection, or SPECT / CT is used for imaging and tumor diagnosis.

[0004] 99m Tc-DOTA-FAPI-46 is used as a molecular probe to specifically bind to tumor markers in vivo for diagnosis, and its drug stability in vivo is crucial. 99m The detection method of Tc radioactive labeled drugs generally uses a gamma counter to detect the total radioactive substances, which cannot distinguish 99m Tc and its impurities or possible radioactive metabolites are 99m The in vivo evaluation of Tc radiolabeled drugs is not comprehensive enough to fully demonstrate 99mThe in vivo stability and in vivo processes (absorption, metabolism, distribution, excretion, etc.) of Tc radiolabeled drugs urgently require an accurate, rapid, and simple method to detect them in biological samples. 99m Analytical detection methods for Tc-DOTA-FAPI-46 and its metabolites / impurities. Summary of the invention

[0005] The present invention discloses a method for determining 99m The method of Tc-DOTA-FAPI-46 and its metabolites is intended to solve the technical problems existing in the prior art.

[0006] The present invention adopts the following technical solutions:

[0007] The present invention provides a method for determining 99m The method of Tc-DOTA-FAPI-46 and its metabolites comprises: collecting samples, intravenously injecting 99m Tc-DOTA-FAPI-46 was added to the sample for pretreatment, and an organic solvent was added to the sample for mixing, and the supernatant was taken after centrifugation. The supernatant was blown dry and then dissolved in physiological saline again. The concentration after dissolution was 1 / 2-1 / 10 of the concentration of the supernatant to obtain an injection solution. The ratio of the sample to the organic solvent was 1:(1-3). Radioactivity detection was performed by taking the concentrated injection solution and placing it into a high performance liquid chromatography for determination. The detector was an ultraviolet detector connected in series with a radioactivity detector, the chromatographic column was a reverse phase chromatographic column, and the elution method was gradient elution. Data processing was performed by calculating the peak area of ​​the radioactive component in the sample according to the high performance liquid chromatography. 99m The ratio of the peak area of ​​Tc-DOTA-FAPI-46, radioactive metabolites, and radioactive impurities to the total radioactive components.

[0008] Preferably, 99m Tc-DOTA-FAPI-46 is a product of DOTA-FAPI-46 and sodium pertechnetate ( 99m Tc) reaction is obtained.

[0009] Preferably, 30 μg of DOTA-FAPI-46 is dissolved in 0.5 mL of sodium chloride injection, and then 1 mL of sodium pertechnetate injection is added, and the mixture is shaken thoroughly to dissolve the solid matter completely until it becomes clear. Under sealed conditions, the mixture is heated in a metal bath at 100° C. for 30 min, taken out, and cooled to room temperature to obtain a clear and transparent liquid. 99m Tc-DOTA-FAPI-46.

[0010] Preferably, the structural formula of DOTA-FAPI-46 is:

[0011]

[0012] Preferably, the sample pretreatment further comprises: adding the sample to the organic solvent in a volume ratio of 1:(1-3), centrifuging at 10000-15000 rpm for 5-15 min at 4°C after mixing, taking the supernatant, drying it with N2 at 40-90°C, and then dissolving it again in physiological saline.

[0013] Preferably, the sample pretreatment further comprises: the organic solvent comprises methanol, acetonitrile or a mixed solution of methanol and acetonitrile, and preferably, the volume ratio of methanol to acetonitrile in the mixed solution of methanol and acetonitrile is 1:1.

[0014] Preferably, the radioactivity detector is a Gabi Nova radioactivity detector.

[0015] Preferably, the chromatographic conditions are: mobile phase A is 0.01 M PBS buffer at pH 7.4; mobile phase B is acetonitrile; gradient elution conditions are: 0 min, 90% mobile phase A, 10% mobile phase B; 20 min, 60% mobile phase A, 40% mobile phase B; 25 min, 90% mobile phase A, 10% mobile phase B; 30 min, 90% mobile phase A, 10% mobile phase B; flow rate is 0.5-1.5 mL / min.

[0016] Preferably, the reverse phase chromatography column is 5 μm, 4.6×250 mm C 18 The column temperature of the reverse phase column is 30-45°C, the temperature of the injector is 4-15°C, and the injection volume is 10-100 μL.

[0017] Preferably, the sample collection further comprises: the intravenous injection 99m Blood, urine, and tissue homogenate samples were collected at different time points after Tc-DOTA-FAPI-46.

[0018] Preferably, the collection of the blood sample further comprises: the intravenous injection 99m At different time points after Tc-DOTA-FAPI-46, 0.5-5 ml of blood was collected through the vein using a syringe, and then the collected blood was placed in an anticoagulant blood collection tube.

[0019] Preferably, the collection of the urine sample comprises: the intravenous injection 99m 4 h after Tc-DOTA-FAPI-46, urine was collected by compressing the bladder, inserting a urinary catheter, or using a metabolic cage.

[0020] Preferably, the collection of tissue homogenate samples includes: intravenous injection 99mAt different time points after Tc-DOTA-FAPI-46, tissues and organs were removed after anesthesia, and physiological saline was added at a mass concentration of 1:(1-3) and the tissues were homogenized. The supernatant was collected by centrifugation at 3000-4000 rpm for 5-10 min.

[0021] The technical solution adopted by the present invention can achieve the following beneficial effects:

[0022] The present invention provides a method for determining 99m The method for determining Tc-DOTA-FAPI-46 and its metabolites comprises the following steps: intravenous injection 99m Tc-DOTA-FAPI-46 was added to the sample; an organic solvent was added to the sample to mix well, and the supernatant was taken after centrifugation. The supernatant was blown dry and then dissolved in physiological saline and concentrated 1-10 times to obtain an injection solution, and the volume ratio of the sample to the organic solvent was 1:(1-3); the injection solution was put into a high performance liquid chromatography for determination, the detector was a radioactive detector, the chromatographic column was a reverse phase chromatographic column, and the elution method was gradient elution; according to the peak area of ​​the radioactive component measured, the amount of radioactive components in the sample was calculated. 99m The peak area ratio of Tc-DOTA-FAPI-46, radioactive metabolites, and radioactive impurities to the total radioactive components. By setting appropriate pretreatment conditions and liquid chromatography detection conditions, radioactive metabolites can be detected in biological samples, which improves the sensitivity and accuracy of detection. The determination method of the present invention is effective for 99m The study of Tc-DOTA-FAPI-46 and its metabolites is helpful to analyze the absorption, distribution, metabolism, excretion and other processes of drugs in the body, and can accurately and efficiently evaluate the in vivo stability of drugs. 99m Requirements for biological sample analysis of Tc-DOTA-FAPI-46 and its metabolites. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The figure shows the blood Radio-HPLC detection chromatogram 5 minutes after administration provided in Example 1 of the present invention;

[0024] Figure 2 The figure shows the blood Radio-HPLC detection chromatogram 20 minutes after administration provided in Example 1 of the present invention;

[0025] Figure 3 Shown is a blood Radio-HPLC detection chromatogram 1 hour after administration provided in Example 1 of the present invention;

[0026] Figure 4 Shown is a blood Radio-HPLC detection chromatogram 2 hours after administration provided in Example 1 of the present invention;

[0027] Figure 5 Shown is a blood Radio-HPLC detection chromatogram 4 hours after administration provided in Example 1 of the present invention;

[0028] Figure 6 Shown is the Radio-HPLC detection chromatogram of urine 4 hours after administration provided in Example 1 of the present invention;

[0029] Figure 7 Shown is a chromatogram of kidney Radio-HPLC detection after administration provided in Example 1 of the present invention;

[0030] Figure 8 Shown is a chromatogram of Radio-HPLC detection of the small intestine after administration provided in Example 1 of the present invention;

[0031] Fig. 9 Shown is a chromatogram of heart Radio-HPLC detection after drug administration provided in Example 1 of the present invention;

[0032] Fig.10 Shown is the Radio-HPLC detection chromatogram of free technetium provided in Example 1 of the present invention;

[0033] Fig.11 The embodiment 1 of the present invention is shown as follows 99m Radio-HPLC detection chromatogram of Tc-DOTA-FAPI-46;

[0034] Fig.12 Shown is a Radio-HPLC detection chromatogram of blood pre-treated with acetonitrile and redissolved with physiological saline provided in Example 2 of the present invention;

[0035] Fig.13 Shown is a Radio-HPLC detection chromatogram of blood pre-treated with acetonitrile and redissolved with acetonitrile provided in Example 3 of the present invention;

[0036] Fig.14 Shown is a Radio-HPLC detection chromatogram of blood pre-treated with a mixed solution of acetonitrile and methanol and redissolved with physiological saline provided in Example 4 of the present invention;

[0037] Fig.15 Shown is a chromatogram of Radio-HPLC detection of blood without drug injection provided in Example 5 of the present invention;

[0038] Fig.16 The results are shown in Example 5 of the present invention, which are placed at room temperature for 0 hours. 99m Blood Radio-HPLC detection chromatogram of Tc-DOTA-FAPI-46;

[0039] Fig.17 The results are shown in Example 5 of the present invention, which are placed at room temperature for 6 hours. 99m Blood Radio-HPLC detection chromatogram of Tc-DOTA-FAPI-46. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the specific embodiments of the present invention and the corresponding drawings. In the description of the present invention, it should be noted that the term "or" is usually used in the sense of including "and / or", unless the content clearly indicates otherwise.

[0041] Example 1

[0042] Experimental system: 18 6-week-old ICR mice, half male and half female, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0043] 99m Synthesis of Tc-DOTA-FAPI-46: 30 μg of DOTA-FAPI-46 was dissolved in 0.5 mL of sodium chloride injection, and then 1 mL of sodium pertechnetate injection was added. The mixture was shaken thoroughly to dissolve the solid completely until it became clear. The mixture was heated in a metal bath at 100°C for 30 min under sealed conditions, and then taken out and cooled to room temperature. The obtained clear and transparent liquid was 99m Tc-DOTA-FAPI-46.

[0044] Dosage and dosage: Single injection via tail vein 99m Tc-DOTA-FAPI-46, injection dose is 2.5mCi / mouse.

[0045] Biological sample collection:

[0046] Blood sample collection: injection 99m 5min, 20min, 1h, 2h, and 4h after Tc-DOTA-FAPI-46, 50μL of blood was collected from the orbital vein using a syringe under isoflurane anesthesia, and the collected blood was then placed in an anticoagulated blood collection tube.

[0047] Urine Sample Collection: Injection 99m 4 h after Tc-DOTA-FAPI-46, the mouse was gently grasped and the abdomen at the bladder position was moderately pressed to stimulate urination and collect urine.

[0048] Collection of tissue homogenate samples: injection 99m5min, 20min, 1h, 2h, and 4h after Tc-DOTA-FAPI-46, the abdominal cavity was bled after isoflurane anesthesia, and tissues and organs (heart, kidney, liver, bladder, femur (hind limb), lung, large intestine, small intestine, etc.) were taken after bled, gently squeezed, and residual blood was absorbed with absorbent paper.

[0049] Sample pretreatment: 50 μL of the collected blood was added with 150 μL of methanol and mixed, then centrifuged at 14,000 rpm for 10 min at 4°C. The supernatant was taken and dried with N2 using an Arica V96 nitrogen concentrator. The supernatant was then placed in physiological saline and redissolved. The concentration after redissolution was 1 / 4 of the concentration of the supernatant.

[0050] Methanol was added to the collected urine sample at a volume ratio of 1:3, and the sample was mixed and centrifuged at 14,000 rpm for 10 min at 4°C. The supernatant was taken and dried with N2 using an Arica V96 nitrogen concentrator. The supernatant was then dissolved in physiological saline. The concentration after redissolution was 1 / 4 of the concentration of the supernatant.

[0051] Kidney, small intestine, and heart tissues were collected, and physiological saline was added at a ratio of 1:3 (w:v). The tissues were homogenized using a tissue grinder, and centrifuged at 3000 rpm for 10 min. The supernatant was taken and dried with N2 using an Arica V96 nitrogen concentrator. The supernatant was then dissolved in physiological saline. The concentration after redissolution was 1 / 4 of the concentration of the supernatant.

[0052] Radio-HPLC detection: Take 50 μL of the injection solution after re-dissolution (concentration) and inject it for detection. The chromatographic conditions are as follows:

[0053] Mobile phase A: 0.01 M PBS buffer, pH 7.4;

[0054] Mobile phase B: acetonitrile;

[0055] Chromatographic column: Shimadzu VP-ODS column (4.6×250mm, 5μm)

[0056] UV detector: 210nm;

[0057] Radioactivity detector: Raytest (model: Gabi)

[0058] Flow rate: 1.0 mL / min;

[0059] Column temperature: 40°C;

[0060] Injector temperature: 4 °C;

[0061] Injection volume: 50 μL;

[0062] Elution gradient: 0 min, 90% mobile phase A, 10% mobile phase B; 20 min, 60% mobile phase A, 40% mobile phase B; 25 min, 90% mobile phase A, 10% mobile phase B; 30 min, 90% mobile phase A, 10% mobile phase B.

[0063] Figure 1 The figure shows the blood Radio-HPLC detection chromatogram 5 minutes after administration provided in Example 1 of the present invention; Figure 2 The figure shows the blood Radio-HPLC detection chromatogram 20 minutes after administration provided in Example 1 of the present invention; Figure 3 Shown is a blood Radio-HPLC detection chromatogram 1 hour after administration provided in Example 1 of the present invention; Figure 4 Shown is a blood Radio-HPLC detection chromatogram 2 hours after administration provided in Example 1 of the present invention; Figure 5 Shown is a blood Radio-HPLC detection chromatogram 4 hours after administration provided in Example 1 of the present invention; Figure 6 Shown is the Radio-HPLC detection chromatogram of urine 4 hours after administration provided in Example 1 of the present invention; Figure 7 Shown is a chromatogram of kidney Radio-HPLC detection after administration provided in Example 1 of the present invention; Figure 8 Shown is a chromatogram of Radio-HPLC detection of the small intestine after administration provided in Example 1 of the present invention; Fig. 9 Shown is a chromatogram of heart Radio-HPLC detection after drug administration provided in Example 1 of the present invention; Fig.10 Shown is the Radio-HPLC detection chromatogram of free technetium provided in Example 1 of the present invention; Fig.11 The embodiment 1 of the present invention is shown as follows 99m Radio-HPLC detection chromatogram of Tc-DOTA-FAPI-46; Figure 1-Figure 11 As shown, the horizontal axis in the figure is the retention time. 99m The retention time of Tc-DOTA-FAPI-46 is about 20.5min ( Fig.11 ), the retention time of free technetium is about 4.5min ( Fig.10 ).

[0064] In blood tests, such as Figure 1-5 As shown, it can be detected from 5min to 2h 99m Tc-DOTA-FAPI-46 accounts for about 80% of the total peak area. No radioactive peak was detected at 4h. As time goes by, it reacts with substances in the body for metabolism, so no radioactive peak is detected. From 5min to 1h, except for the radioactive peak introduced by the drug (18min), the peak at 18min is 99mTc-DOTA-FAPI-46 itself contains impurities, and there is also a metabolite (14.6 min) that accounts for approximately 3% to 11% of the total peak area. The peak at 14.6 min is considered to be the possible major metabolite.

[0065] In urine testing, Figure 6 As shown, 99m Tc-DOTA-FAPI-46 accounts for about 50% of the total peak area, which indicates that there may be multiple radioactive metabolites. 99m After intravenous administration of Tc-DOTA-FAPI-46 injection, the drug mainly exists in the blood in its original form, while multiple radioactive metabolites exist in the urine.

[0066] In tissue homogenate assays, Figure 7-Figure 9 As shown, 99m The retention time of Tc-DOTA-FAPI-46 was about 20.4 min, and it was detected in the kidney and small intestine. 99m Tc-DOTA-FAPI-46 and its possible metabolite signals showed basically no response in heart and other tissues and organs.

[0067] Data analysis: Calculate the 99m The peak areas of Tc-DOTA-FAPI-46, free technetium and major metabolites accounted for 99m The ratio of the total peak area of ​​Tc-DOTA-FAPI-46.

[0068] Table 1 Blood 99m The ratio of the peak areas of Tc-DOTA-FAPI-46, free technetium and major metabolites

[0069] Time <![CDATA[ 99m Tc-DOTA-FAPI-46]]> Free technetium Main metabolites 5min 90.11％ 0.42％ 3.08％ 20min 82.45％ 1.12％ 6.96％ 1h 85.78％ - 11.37％ 2h 100％ - - 4h - - -

[0070] As shown in Table 1, after the pretreatment method and Radio-HPLC detection, 99m Tc-DOTA-FAPI-46 accounts for the largest proportion of the total peak area, while free technetium accounts for the smallest proportion. The proportion of the main metabolites increases with time. This is because 99m Tc-DOTA-FAPI-46 reacts with substances in the body to metabolize, so the larger the proportion of the peak area of ​​the main metabolite, the longer the time. 99m The smaller the proportion of the peak area of ​​Tc-DOTA-FAPI-46, the less likely it is that only 99m Tc-DOTA-FAPI-46 and its main metabolites were detected, but free technetium was not detected; and 2 hours after administration, no peak area of ​​free technetium and its main metabolites appeared, that is, free technetium and its main metabolites were not detected.

[0071] Example 2

[0072] Experimental system: Two 6-week-old ICR mice, half male and half female, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0073] 99m Synthesis of Tc-DOTA-FAPI-46: 30 μg of DOTA-FAPI-46 was dissolved in 0.5 mL of sodium chloride injection, and then 1 mL of sodium pertechnetate injection was added. The mixture was shaken thoroughly to dissolve the solid completely until it became clear. The mixture was heated in a metal bath at 100°C for 30 min under sealed conditions, and then taken out and cooled to room temperature. The obtained clear and transparent liquid was 99m Tc-DOTA-FAPI-46.

[0074] Dosage and dosage: Single injection via tail vein 99m Tc-DOTA-FAPI-46, injection dose is 2.5mCi / mouse.

[0075] Biological sample collection:

[0076] Blood sample collection: injection 99m Immediately after isoflurane anesthesia, Tc-DOTA-FAPI-46 was injected 99m 2 min after Tc-DOTA-FAPI-46, blood was collected from the orbital vein using a capillary tube and then placed in an anticoagulant blood collection tube.

[0077] Sample pretreatment: 50 μL of the collected blood was added to 150 μL of acetonitrile and mixed, then centrifuged at 14000 rpm at 4°C for 10 min. The supernatant was taken and dried with N2 using an Arica V96 nitrogen concentrator. Then, it was put into physiological saline to be redissolved. The concentration after redissolution was 1 / 4 of the concentration of the supernatant.

[0078] Radio-HPLC detection: Take 50 μL of the injection solution after re-dissolution (concentration) and inject it for detection. The chromatographic conditions are as follows:

[0079] Mobile phase A: 0.01 M PBS buffer, pH 7.4;

[0080] Mobile phase B: acetonitrile;

[0081] Chromatographic column: Shimadzu VP-ODS column (4.6×250mm, 5μm)

[0082] UV detector: 210nm;

[0083] Radioactivity detector: Raytest (model: Gabi)

[0084] Flow rate: 1.0 mL / min;

[0085] Column temperature: 40°C;

[0086] Injector temperature: 4 °C;

[0087] Injection volume: 30 μL;

[0088] Elution gradient: 0 min, 90% mobile phase A, 10% mobile phase B; 20 min, 60% mobile phase A, 40% mobile phase B; 25 min, 90% mobile phase A, 10% mobile phase B; 30 min, 90% mobile phase A, 10% mobile phase B.

[0089] Fig.12 The figure shows the Radio-HPLC chromatogram of the blood pre-treated with acetonitrile and redissolved with saline provided in Example 2 of the present invention. Fig.12 As shown, pretreatment with acetonitrile and redissolution with saline can detect

[0090] 99m Tc-DOTA-FAPI-46, its retention time is about 17min.

[0091] Example 3

[0092] Experimental system: Two 6-week-old ICR mice, half male and half female, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0093] 99m Synthesis of Tc-DOTA-FAPI-46: 30 μg of DOTA-FAPI-46 was dissolved in 0.5 mL of sodium chloride injection, and then 1 mL of sodium pertechnetate injection was added. The mixture was shaken thoroughly to dissolve the solid completely until it became clear. The mixture was heated in a metal bath at 100°C for 30 min under sealed conditions, and then taken out and cooled to room temperature. The obtained clear and transparent liquid was 99m Tc-DOTA-FAPI-46.

[0094] Dosage and dosage: Single injection via tail vein 99m Tc-DOTA-FAPI-46, injection dose is 2.5mCi / mouse.

[0095] Biological sample collection:

[0096] Blood sample collection: injection 99m After isoflurane anesthesia, Tc-DOTA-FAPI-46 was injected 99m2 min after Tc-DOTA-FAPI-46, blood was collected from the orbital vein using a capillary tube and then placed in an anticoagulant blood collection tube.

[0097] Sample pretreatment: 50 μL of the collected blood was added to 150 μL of acetonitrile and mixed, then centrifuged at 14000 rpm at 4°C for 10 min. The supernatant was taken and dried with N2 using an Arica V96 nitrogen concentrator. Then, it was put into acetonitrile to be redissolved. The concentration after redissolution was 1 / 4 of the concentration of the supernatant.

[0098] Radio-HPLC detection: Take 50 μL of the injection solution after re-dissolution (concentration) and inject it for detection. The chromatographic conditions are as follows:

[0099] Mobile phase A: 0.01 M PBS buffer, pH 7.4;

[0100] Mobile phase B: acetonitrile;

[0101] Chromatographic column: Shimadzu VP-ODS column (4.6×250mm, 5μm)

[0102] UV detector: 210nm;

[0103] Radioactivity detector: Raytest (model: Gabi)

[0104] Flow rate: 1.0 mL / min;

[0105] Column temperature: 40°C;

[0106] Injector temperature: 4 °C;

[0107] Injection volume: 100 μL;

[0108] Elution gradient: 0 min, 90% mobile phase A, 10% mobile phase B; 20 min, 60% mobile phase A, 40% mobile phase B; 25 min, 90% mobile phase A, 10% mobile phase B; 30 min, 90% mobile phase A, 10% mobile phase B.

[0109] Fig.13 The figure shows the Radio-HPLC detection chromatogram of the blood pre-treated with acetonitrile and redissolved with acetonitrile provided in Example 3 of the present invention, as shown in FIG. Fig.13 As shown, the pretreatment with acetonitrile and the redissolution with acetonitrile can detect 99m Tc-DOTA-FAPI-46, but it will cause solvent effects, resulting in 99m The retention time of Tc-DOTA-FAPI-46 shifted to 2.7 min.

[0110] Example 4

[0111] Experimental system: 188 6-week-old ICR mice, half male and half female, were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0112] 99m Synthesis of Tc-DOTA-FAPI-46: 30 μg of DOTA-FAPI-46 was dissolved in 0.5 mL of sodium chloride injection, and then 1 mL of sodium pertechnetate injection was added. The mixture was shaken thoroughly to dissolve the solid completely until it became clear. The mixture was heated in a metal bath at 100°C for 30 min under sealed conditions, and then taken out and cooled to room temperature. The obtained clear and transparent liquid was 99m Tc-DOTA-FAPI-46.

[0113] Dosage and dosage: Single injection via tail vein 99m Tc-DOTA-FAPI-46, injection dose is 2mCi / mouse.

[0114] Biological sample collection:

[0115] Blood sample collection: injection 99m After 2 min of isoflurane anesthesia, Tc-DOTA-FAPI-46 was injected 99m 25 min after Tc-DOTA-FAPI-46, blood was collected from the orbital vein using a syringe capillary, and then the collected blood was placed in an anticoagulant blood collection tube.

[0116] Sample pretreatment: 80 μL of the collected blood was added with 240 μL of a 1:1 (v:v) mixed solution of acetonitrile and methanol, and centrifuged at 14,000 rpm for 10 min at 4°C. The supernatant was taken and dried with N2 using an Arica V96 nitrogen concentrator. The supernatant was then placed in physiological saline and dissolved again. The concentration after redissolution was 1 / 4 of the concentration of the supernatant.

[0117] Radio-HPLC detection: Take 50 μL of the injection solution after re-dissolution (concentration) and inject it for detection. The chromatographic conditions are as follows:

[0118] Mobile phase A: 0.01 M PBS buffer, pH 7.4;

[0119] Mobile phase B: acetonitrile;

[0120] Chromatographic column: Shimadzu VP-ODS column (4.6×250mm, 5μm)

[0121] UV detector: 210nm;

[0122] Radioactivity detector: Raytest (model: Gabi)

[0123] Flow rate: 1.0 mL / min;

[0124] Column temperature: 40°C;

[0125] Injector temperature: 4 °C;

[0126] Injection volume: 50 μL;

[0127] Elution gradient: 0 min, 90% mobile phase A, 10% mobile phase B; 20 min, 60% mobile phase A, 40% mobile phase B; 25 min, 90% mobile phase A, 10% mobile phase B; 30 min, 90% mobile phase A, 10% mobile phase B.

[0128] Fig.14 The figure shows the Radio-HPLC detection chromatogram of the blood pre-treated with the mixed solution of acetonitrile and methanol and redissolved with physiological saline provided in Example 4 of the present invention; Fig.14 As shown, 99m The retention time of Tc-DOTA-FAPI-46 is about 20.4min, and the retention time of free technetium is about 4.0min. In addition to the radioactive peak introduced by the drug (18min), there may be a metabolite with a retention time of about 14.5min. This shows that the acetonitrile and methanol 1:1 (v:v) mixed solution is used for pretreatment and then dissolved again in saline. 99m Tc-DOTA-FAPI-46.

[0129] Example 5

[0130] The blood samples of ICR mice were measured 99m The analytical method for Tc-DOTA-FAPI-46 and its metabolites was methodologically validated.

[0131] Selectivity verification: Blood (blank sample) was collected from 6 ICR mice without drug injection. 50 μL of the collected blood was added to 150 μL of methanol and mixed, then centrifuged at 14000 rpm at 4°C for 10 min. The supernatant was taken and dried with N2 in an Arica V96 nitrogen concentrator. Then, it was put into physiological saline to be redissolved. The concentration after redissolution was 1 / 4 of the concentration of the supernatant.

[0132] Radio-HPLC detection: Take 50 μL of the injection solution after re-dissolution (concentration) and inject it for detection. The chromatographic conditions are as follows:

[0133] Mobile phase A: 0.01 M PBS buffer, pH 7.4;

[0134] Mobile phase B: acetonitrile;

[0135] Chromatographic column: Shimadzu VP-ODS column (4.6×250mm, 5μm)

[0136] UV detector: 210nm;

[0137] Radioactivity detector: Raytest (model: Gabi)

[0138] Flow rate: 1.0 mL / min;

[0139] Column temperature: 40°C;

[0140] Injector temperature: 4 °C;

[0141] Injection volume: 50 μL;

[0142] Elution gradient: 0 min, 90% mobile phase A, 10% mobile phase B; 20 min, 60% mobile phase A, 40% mobile phase B; 25 min, 90% mobile phase A, 10% mobile phase B; 30 min, 90% mobile phase A, 10% mobile phase B.

[0143] Fig.15 The figure shows the Radio-HPLC chromatogram of blood without drug injection provided in Example 5 of the present invention. Fig.15 As shown, no radioactive components were detected in the blood samples of the 6 mice that were not injected, and none of them interfered with 99m Determination after Tc-DOTA-FAPI-46 was added to blood samples.

[0144] Detection limit verification: add 0.1μCi of 99m Tc-DOTA-FAPI-46 was detected by Radio-HPLC, and the signal-to-noise ratio (S / N) of the radioactive signal peak was between 3 and 10, and it could be measured continuously for 3 times.

[0145] Sample stability verification: 0.1 μCi of 99m Tc-DOTA-FAPI-46 was placed at room temperature for 6 hours and then tested by Radio-HPLC. Fig.16 The results are shown in Example 5 of the present invention, which are placed at room temperature for 0 hours. 99m Blood Radio-HPLC detection chromatogram of Tc-DOTA-FAPI-46; Fig.17 The results are shown in Example 5 of the present invention, which are placed at room temperature for 6 hours. 99m The blood Radio-HPLC chromatogram of Tc-DOTA-FAPI-46, Figure 16-Figure 17As shown, compared with 0 h at room temperature, there is no new radioactive peak in the Radio-HPLC detection after 6 h at room temperature, which indicates that it does not affect the determination of metabolites in the blood and the sample has good stability.

[0146] Current biological samples 99m The detection method of Tc radioactive labeled drugs generally uses a gamma counter to detect the total radioactive substances. 99m 100 μL of ICR mouse blood containing Tc-DOTA-FAPI-46 was added with methanol as a precipitant, mixed and centrifuged at 14000 rpm for 10 min at 4°C. The radioactivity counts of the supernatant and precipitate were detected using a gamma counter, which were 288636.28 and 91908.23, respectively. The gamma counter can only measure the total content of radioactive substances, but cannot distinguish 99m Tc and its impurities or possible radioactive metabolites, which are 99m The in vivo evaluation of Tc radiolabeled drugs is not comprehensive enough to fully demonstrate 99m The in vivo stability and in vivo process of Tc radiolabeled drugs. The radio-HPLC separation and detection method can accurately distinguish and determine 99m Appropriate pretreatment methods can achieve lower detection sensitivity for Tc and its impurities or possible radioactive metabolites. This method can be used to accurately and efficiently evaluate the stability of drugs in vivo.

[0147] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for determining the 99m The method of Tc-DOTA-FAPI-46 and its metabolites, characterized in that The steps include: Sample collection, intravenous injection 99m Samples were collected after Tc-DOTA-FAPI-46; Pretreatment of the sample: adding an organic solvent to the collected sample, mixing, centrifuging, taking the supernatant, blowing the supernatant dry, and then dissolving it in physiological saline again. The concentration after dissolution is 1 / 2-1 / 10 of the concentration of the supernatant to obtain the injection solution. The volume ratio of the collected sample to the organic solvent is 1:(1-3); Radioactivity detection: the sample solution is placed in a high performance liquid chromatography for determination, the detector is a UV detector connected in series with a radioactivity detector, the chromatographic column is a reverse phase chromatographic column, and the elution method is gradient elution; Data processing, based on the peak area of ​​the measured radioactive components, calculate the 99m The ratio of the peak area of ​​Tc-DOTA-FAPI-46, radioactive metabolites, and radioactive impurities to the total radioactive components.

2. The method of claim 1 for determining the 99m The method of Tc-DOTA-FAPI-46 and its metabolites, characterized in that The sample pretreatment also includes: adding the sample to the organic solvent in a volume ratio of 1:(1-3), centrifuging at 10000-15000 rpm for 5-15 minutes at 4°C after mixing, taking the supernatant, drying it with N2 at 40-90°C, and then dissolving it again in physiological saline.

3. The method of claim 2 for determining the 99m The method of Tc-DOTA-FAPI-46 and its metabolites, characterized in that The sample pretreatment further includes: the organic solvent includes methanol, acetonitrile or a mixed solution of methanol and acetonitrile. Preferably, the volume ratio of methanol to acetonitrile in the mixed solution of methanol and acetonitrile is 1:

1.

4. The method of claim 1 for determining the 99m The method of Tc-DOTA-FAPI-46 and its metabolites, characterized in that The radioactivity detector is a Gabi Nova radioactivity detector.

5. The method of claim 1 for determining the 99m The method of Tc-DOTA-FAPI-46 and its metabolites, characterized in that The chromatographic conditions are as follows: mobile phase A is 0.01M PBS buffer with a pH of 7.4; mobile phase B is acetonitrile; gradient elution conditions are as follows: 0 min, 90% mobile phase A, 10% mobile phase B; 20 min, 60% mobile phase A, 40% mobile phase B; 25 min, 90% mobile phase A, 10% mobile phase B; 30 min, 90% mobile phase A, 10% mobile phase B; and the flow rate is 0.5-1.5 mL / min.

6. The method of claim 5 for determining the 99m The method of Tc-DOTA-FAPI-46 and its metabolites, characterized in that The reverse phase chromatographic column is a 5 μm, 4.6×250 mm C 18 The column temperature of the reverse phase column is 30-45°C, the temperature of the injector is 4-15°C, and the injection volume is 10-100 μL.

7. The method of claim 1 for determining the 99m The method of Tc-DOTA-FAPI-46 and its metabolites, characterized in that The sample collection also includes: intravenous injection 99m Blood, urine, and tissue homogenate samples were collected at different time points after Tc-DOTA-FAPI-46.

8. The method of claim 6 for determining the 99m The method of Tc-DOTA-FAPI-46 and its metabolites, characterized in that The blood sample collection includes: intravenous injection 99m At different time points after Tc-DOTA-FAPI-46, 0.5-5 ml of blood was collected through the vein using a syringe, and then the collected blood was placed in an anticoagulant blood collection tube.

9. The method of claim 6 for determining the 99m The method of Tc-DOTA-FAPI-46 and its metabolites, characterized in that The collection of urine samples includes: 99m 4 h after Tc-DOTA-FAPI-46, compress the bladder and collect urine, insert a urinary catheter to collect urine, or use a metabolic cage to collect urine.

10. The method of claim 6 for determining the 99m The method of Tc-DOTA-FAPI-46 and its metabolites, characterized in that The collection of tissue homogenate samples includes: intravenous injection 99m At different time points after Tc-DOTA-FAPI-46, tissues and organs were removed after anesthesia, and physiological saline was added at a mass concentration of 1:(1-3) and the tissues were homogenized. The supernatant was collected by centrifugation at 3000-4000 rpm for 5-10 min.

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