Determination method for concentration of < 125 > I labeled drug in whole blood

Abstract

The invention provides a method for measuring the concentration of a < 125 > I labeled drug in whole blood, which comprises the following steps: adding a trichloroacetic acid solution with the concentration of 250 g / L into a collected whole blood sample, uniformly mixing, centrifuging, removing supernate, and collecting a first precipitate; adding a trichloroacetic acid solution with the concentration of 100-250 g / L into the first precipitate, uniformly mixing, centrifuging, removing supernate, and collecting a second precipitate; adding trichloroacetic acid solutions with the concentration of 100-250 g / L into the second precipitate, uniformly mixing, centrifuging, removing supernate, collecting a third precipitate, and detecting the radioactivity count of the third precipitate by adopting a gamma-counter. The method provided by the invention can be used for removing free < 125 > I generated by deiodination and free < 125 > I adsorbed on the surface of a precipitate, so that errors caused by inaccurate detection concentration due to deiodination are avoided, the operation is simple and convenient, the sensitivity is high, and the test pretreatment and detection efficiency and accuracy are greatly improved.

Description

Technical Field

[0001] This invention relates to the field of pharmacokinetic technology, and particularly to a whole blood... 125 Methods for determining the concentration of I-labeled drugs. Background Technology

[0002] 125 I is a commonly used radioactive isotope with a half-life of 60.14 days. 125 I is often used to label biomolecules such as peptides, proteins, and enzymes containing tyrosine residues, and then the concentration of the analyte is estimated by measuring the intensity of the radioactive signal. 125 Methods for labeling I-type drugs mainly include the chloramine-T method, the lactoperoxidase method, and the electrophilic substitution reaction of the iodobenzene ring. These methods involve oxidation or enzyme-catalyzed reactions to... 125 I covalently binds to the side chain of tyrosine or histidine, but in vivo and in vitro biological matrices 125 I-labeled drugs undergo deiodination over time and under physiological conditions, meaning the labeled iodine detaches from its original position and becomes free iodine, thus affecting the biological matrix. 125 Direct concentration determination of I-labeled drugs can lead to biases and cannot accurately reflect the actual metabolism of drugs in vivo. Furthermore, electrophoresis is often used to detect the concentration of I-labeled drugs in samples after labeling. 125 I-labeled drug concentrations are cumbersome to test and take a long time to analyze.

[0003] The above problems urgently need to be addressed. Summary of the Invention

[0004] This invention discloses a whole blood medium 125 The method for determining the concentration of I-labeled drugs aims to solve the technical problems existing in the prior art.

[0005] The present invention adopts the following technical solution: This invention provides a whole blood medium 125 A method for determining the concentration of I-labeled drugs in whole blood. 125 The method for determining the concentration of I-labeled drugs includes the following steps: Add a 250 g / L trichloroacetic acid solution to the collected whole blood sample, mix well, centrifuge, remove the supernatant, and collect the first precipitate; Add a trichloroacetic acid solution with a concentration of 100-250 g / L to the first precipitate, mix well, centrifuge and remove the supernatant, and collect the second precipitate; Add a 100-250 g / L trichloroacetic acid solution to the second precipitate, mix well, centrifuge and remove the supernatant, collect the third precipitate, and use a gamma counter to detect the radioactivity count of the third precipitate.

[0006] In one possible implementation, the volume ratio of the 250 g / L trichloroacetic acid solution to the whole blood sample is 5:1.

[0007] In one possible implementation, the 250 g / L trichloroacetic acid solution is prepared by adding 50.0 mL of pure water to 12.5 g of ammonium acetate and mixing thoroughly.

[0008] In one possible implementation, the volume of the trichloroacetic acid solution with a concentration of 100-250 g / L added is 1 mL.

[0009] In one possible implementation, the trichloroacetic acid solution with a concentration of 100 g / L-250 g / L is prepared by adding 50.0 mL of pure water to 5.00 g-12.5 g of ammonium acetate and mixing thoroughly.

[0010] In one possible implementation, the centrifugal speed is 2000 rpm.

[0011] In one possible implementation, the centrifugation time is 5 minutes.

[0012] In one possible implementation, the whole blood sample contains 125 I-labeled proteoglycan molecules.

[0013] In one possible implementation, the 125 The labeling method for I-labeled proteoglycan molecules involves adding Na+ with a radioactivity of 4.8 mCi to a proteoglycan molecule solution. 125 I. Mix well, then add 1 μg / μL chloramine T solution and mix well. React for 30 seconds, then add 1 μg / μL sodium metabisulfite solution and mix well to obtain the desired product. 125 I-labeled proteoglycan molecules.

[0014] The technical solution adopted in this invention can achieve the following beneficial effects: This invention mainly provides a whole blood... 125 Methods for determining the concentration of labeled drugs, in the labeled 125 After step I, trichloroacetic acid solutions of different concentrations were added to whole blood samples for three precipitation cycles. Radioactivity counts were then determined using a gamma counter. The addition of trichloroacetic acid effectively removed free radioactive substances generated during deiodination. 125 I and free adsorbed on the surface of the precipitate 125 This technology avoids errors caused by inaccurate detection concentrations due to deiodination, is easy to operate, and has high sensitivity, greatly improving the efficiency and accuracy of pretreatment and detection. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments. In the description of this invention, it should be noted that the term "or" is generally used to include the meaning of "and / or," unless otherwise expressly indicated.

[0016] Application Overview: 125 I is a commonly used radioactive isotope with a half-life of 60.14 days. 125 I is commonly used for labeling biomolecules such as peptides and proteins containing tyrosine residues, as well as enzymes. Current technologies typically use... 125 After I-labeling, electrophoresis is often used to detect I in the sample. 125 I-labeled drug concentration, for example, the specific method for determining the relationship between the blood concentration of 125I-NGF in mice and time using a combination of isotope tracing and electrophoretic separation is as follows: Take... 125 185 kBq of I-NGF and 1 mg of NGF were dissolved in 1 mL of 20 mmol / L pH 7.4 phosphate buffer. 20 μL of sample was mixed with 20 μL of double-strength electrophoresis loading buffer and boiled for 5 min to prepare the electrophoresis sample. 20 μL of the electrophoresis sample was loaded onto a 12% polyacrylamide gel for electrophoresis. After electrophoresis, the polyacrylamide gel was stained with Coomassie Brilliant Blue for 3 h, destained for 3 h, and stored in 7% acetic acid solution. The NGF electrophoretic position was recorded. The gel was cut into 0.5 cm segments along the electrophoretic direction, and the radioactivity intensity of each segment was measured using a gamma counter. The radioactivity peak and the position of the NGF staining spot were compared.

[0017] Six mice were intravenously injected 125 I-NGF 10 ng / g (3.7 kBq / g); after administration, blood was collected at different time points, plasma was separated, 25 μL of plasma was taken, 25 μL of 2x electrophoresis loading solution was added, mixed well and boiled for 5 min to prepare electrophoresis sample, 40 μL of electrophoresis sample was loaded onto 12% polyacrylamide gel for electrophoresis, after electrophoresis, the polyacrylamide gel was stained with Coomassie brilliant blue for 3 h and the gel was stained for 3 h, and stored in 7% acetic acid solution; the gel corresponding to the NGF position was cut off and placed in a measuring tube to measure the radioactivity intensity and determine the NGF blood drug concentration.

[0018] Therefore, determining the concentration of the labeled substance using the above methods is cumbersome and time-consuming. Furthermore, the labeled drug undergoes deiodination over time and under physiological conditions, which can lead to changes in the concentration of the labeled substance in the biological matrix. 125 Directly measuring the concentration of I-labeled drugs can lead to biases and cannot accurately reflect the actual metabolism of drugs in vivo.

[0019] This invention removes free iodine produced during deiodization by adding trichloroacetic acid solutions of different concentrations for three precipitation processes. 125 I and free adsorbed on the surface of the precipitate 125 This method avoids errors caused by inaccurate concentration detection due to deiodination. Combined with a gamma counter to measure radioactivity counts, it is easy to operate, highly sensitive, and greatly improves the efficiency and accuracy of pretreatment and detection.

[0020] Example 1 Isolation and purification of proteoglycan molecules: Take an appropriate amount of proteoglycan (provided by the China Institute of Radiation Protection) sample, grind it thoroughly, weigh 170.0 mg, add 8.5 mL of water, mix well for 30 min to dissolve the sample; after the sample is fully dissolved, centrifuge at 12000 rpm for 10 min, remove the precipitate at 22℃, and keep the supernatant; cut a dialysis bag about 10 cm long, with a molecular weight cutoff of 3500 Da, and boil it with 10 mM EDTA solution for 1 min; put the supernatant into the dialysis bag, dialyze with water for 72 h, and collect the liquid; inject the collected liquid into a 10 mL sterile vial, put it in a freeze dryer, pre-cool at -20℃ for 10 min, dry for 48 h, and wait for the sample chamber to be in a vacuum state; after the temperature and pressure drop, loosen the vent valve, after 10 min, vent the gas and take out the sample, dissolve the dried proteoglycan molecules in 0.2 M, pH 7.5 PB buffer.

[0021] Proteoglycan molecules 125 I mark: Take 92 μL of a proteoglycan solution containing 200 μg of proteoglycan molecules; in a glove box, add 10 μL of Na₂ with a radioactivity of 4.8 mCi to the above system. 125 I. Mix well; add 20 μL of chloramine T solution with a concentration of 1 μg / μL, mix well and react for 30 s; then add 40 μL of sodium metabisulfite solution with a concentration of 1 μg / μL, mix well, and label the product.

[0022] Purification of the labeled product: The labeled product was added to the center of a G25 gel column, centrifuged at 12,000 rpm for 3 min, and the purified labeled product was collected.

[0023] Whole blood sample collection: Six SD rats, half male and half female, were selected and administered purified blood via gavage in a single dose. 125I-labeled proteoglycan molecules (50 μCi / rat). Blood was collected from the orbital region of each rat 1 hour before and 1 hour after drug administration and weighed. After collection, the blood samples were placed into EP tubes containing heparin anticoagulation, for a total of 6 EP tubes containing whole blood samples. An additional EP tube containing blank whole blood was added as a background. The tubes were stored at 4°C.

[0024] Preparation of a 250 g / L trichloroacetic acid solution: Weigh 12.5 g of ammonium acetate, add 50.0 mL of pure water, mix well, and store at room temperature. The shelf life is three days from the date of preparation.

[0025] Preparation of a 100 g / L trichloroacetic acid solution: Weigh 5.00 g of ammonium acetate, add 50.0 mL of pure water, mix well, and store at room temperature. The shelf life is three days from the date of preparation.

[0026] Add 5 times the volume of 250 g / L trichloroacetic acid solution to 6 EP tubes containing whole blood samples and 1 EP tube containing blank whole blood samples. Mix thoroughly using a vortex mixer (Vortex QL-901). Centrifuge the mixed rat whole blood samples at 2000 rpm for 5 min in an Eppendorf Centrifuge 5424R. Remove the supernatant and collect the first precipitate. Add 1 mL of 100 g / L trichloroacetic acid solution to the first precipitate and centrifuge at 2000 rpm for 5 min in the same refrigerated centrifuge. Remove the supernatant and collect the second precipitate. Add another 1 mL of 100 g / L trichloroacetic acid solution to the second precipitate and centrifuge at 2000 rpm for 5 min in the same refrigerated centrifuge. After min, the supernatant was removed, the third precipitate was collected, and the radioactivity counts of the blank sample (background) and the third precipitate were detected using a gamma counter (PerkinElmer; 2480 WIZARD2).

[0027] Example 2 Isolation and purification of proteoglycan molecules: Take an appropriate amount of proteoglycan (provided by the China Institute of Radiation Protection) sample, grind it thoroughly, weigh 170.0 mg, add 8.5 mL of water, mix well for 30 min to dissolve the sample; after the sample is fully dissolved, centrifuge at 12000 rpm for 10 min, remove the precipitate at 22℃, and keep the supernatant; cut a dialysis bag about 10 cm long, with a molecular weight cutoff of 3500 Da, and boil it with 10 mM EDTA solution for 1 min; put the supernatant into the dialysis bag, dialyze with water for 72 h, and collect the liquid; inject the collected liquid into a 10 mL sterile vial, put it in a freeze dryer, pre-cool at -20℃ for 10 min, dry for 48 h, and wait for the sample chamber to be in a vacuum state; after the temperature and pressure drop, loosen the vent valve, after 10 min, vent the gas and take out the sample, dissolve the dried proteoglycan molecules in 0.2 M, pH 7.5 PB buffer.

[0028] Proteoglycan molecules 125 I mark: Take 92 μL of a proteoglycan molecular solution, containing 200 μg of proteoglycan drug; in a glove box, add 10 μL of Na₂ with a radioactivity of 4.8 mCi to the above system. 125 I. Mix well; add 20 μL of chloramine T solution with a concentration of 1 μg / μL, mix well and react for 30 s; then add 40 μL of sodium metabisulfite solution with a concentration of 1 μg / μL, mix well, and label the product.

[0029] Purification of the labeled product: The labeled product was added to the center of a G25 gel column, centrifuged at 12,000 rpm for 3 min, and the purified labeled product was collected.

[0030] Whole blood sample collection: Six SD rats, half male and half female, were selected and administered purified blood via gavage in a single dose. 125 I-labeled proteoglycan molecules (50 μCi / rat). Blood was collected from the orbital region of each rat 1 hour before and 1 hour after administration and weighed. The blood samples were placed into EP tubes containing heparin anticoagulation, for a total of 6 EP tubes containing whole blood samples. An additional EP tube containing blank whole blood was added as a background sample and stored at 4°C.

[0031] Preparation of a 250 g / L trichloroacetic acid solution: Weigh 12.5 g of ammonium acetate, add 50.0 mL of pure water, mix well, and store at room temperature. The shelf life is three days from the date of preparation.

[0032] Add 5 times the volume of 250 g / L trichloroacetic acid solution to 6 EP tubes containing whole blood samples and 1 EP tube containing blank whole blood samples. Mix the mixture using a vortex mixer (Vortex QL-901). Centrifuge the mixed rat whole blood samples at 2000 rpm for 5 min in an Eppendorf Centrifuge 5424R. Remove the supernatant and collect the first precipitate. Add 1 mL of 250 g / L trichloroacetic acid solution to the first precipitate and centrifuge at 2000 rpm for 5 min in the same refrigerated centrifuge. Remove the supernatant and collect the second precipitate. Add another 1 mL of 250 g / L trichloroacetic acid solution to the second precipitate and centrifuge at 2000 rpm for 5 min in the same refrigerated centrifuge. After min, the supernatant was removed, the third precipitate was collected, and the radioactivity counts of the blank sample (background) and the third precipitate were detected using a gamma counter (PerkinElmer; 2480 WIZARD2).

[0033] Example 3 Isolation and purification of proteoglycan molecules: Take an appropriate amount of proteoglycan (provided by the China Institute of Radiation Protection) sample, grind it thoroughly, weigh 170.0 mg, add 8.5 mL of water, mix well for 30 min to dissolve the sample; after the sample is fully dissolved, centrifuge at 12000 rpm for 10 min, remove the precipitate at 22℃, and keep the supernatant; cut a dialysis bag about 10 cm long, with a molecular weight cutoff of 3500 Da, and boil it with 10 mM EDTA solution for 1 min; put the supernatant into the dialysis bag, dialyze with water for 72 h, and collect the liquid; inject the collected liquid into a 10 mL sterile vial, put it in a freeze dryer, pre-cool at -20℃ for 10 min, dry for 48 h, and wait for the sample chamber to be in a vacuum state; after the temperature and pressure drop, loosen the vent valve, after 10 min, vent the gas and take out the sample, dissolve the dried proteoglycan molecules in 0.2 M, pH 7.5 PB buffer.

[0034] Proteoglycan molecules 125 I mark: Take 92 μL of a proteoglycan solution containing 200 μg of proteoglycan molecules; in a glove box, add 10 μL of Na₂ with a radioactivity of 4.8 mCi to the above system. 125 I. Mix well; add 20 μL of chloramine T solution with a concentration of 1 μg / μL, mix well and react for 30 s; then add 40 μL of sodium metabisulfite solution with a concentration of 1 μg / μL, mix well, and label the product.

[0035] Purification of the labeled product: The labeled product was added to the center of a G25 gel column, centrifuged at 12,000 rpm for 3 min, and the purified labeled product was collected.

[0036] Whole blood sample collection: Six SD rats, half male and half female, were selected and administered purified blood via gavage in a single dose. 125 I-labeled proteoglycan molecules (50 μCi / rat). Blood was collected from the orbital region of each rat 1 hour before and 1 hour after drug administration and weighed. After collection, the blood samples were placed into EP tubes containing heparin anticoagulation, for a total of 6 EP tubes containing whole blood samples. An additional EP tube containing blank whole blood was added as a background. The tubes were stored at 4°C.

[0037] Preparation of a 250 g / L trichloroacetic acid solution: Weigh 12.5 g of ammonium acetate, add 50.0 mL of pure water, mix well, and store at room temperature. The shelf life is three days from the date of preparation.

[0038] Five times the volume of 250 g / L trichloroacetic acid solution was added to six EP tubes containing whole blood samples and one EP tube containing blank whole blood samples. The mixture was then vortexed (Vortex QL-901) and thoroughly mixed. The mixed rat whole blood samples were then centrifuged at 2000 rpm for 5 min in a high-speed refrigerated centrifuge (Eppendorf; Centrifuge 5424R). The supernatant was removed, and the precipitate was collected. The radioactivity counts of the blank sample (background) and the precipitate were detected using a gamma counter (PerkinElmer; 2480 WIZARD2).

[0039] Table 1. Radioactive count values ​​before and after administration in Examples 1-3

[0040] As shown in Table 1, in Example 1, when 5 times the volume of 250 g / L trichloroacetic acid solution was added to whole blood samples from rats for the first time, and the precipitate was collected by centrifugation, 1 mL of 100 g / L trichloroacetic acid solution was added for the second and third times. After centrifugation and collection of the precipitate, the radioactivity count of the precipitate was detected using a gamma counter. This process can precipitate the proteoglycan drug while free proteoglycans can be released. 125 I remains in the supernatant and is removed by centrifugation. This method can basically reflect the actual concentration of the prototype drug.

[0041] In Example 2, a 250 g / L trichloroacetic acid solution (5 times its volume) was added three times to a whole blood sample from rats. The precipitate was collected and placed in a gamma counter to detect its radioactivity. The count results showed that a significant portion of the precipitate remained free radioactive. 125The radioactive adsorption of I on the surface of the precipitate or its binding with plasma proteins is included in the radioactivity count, resulting in a higher count, but better than in Example 3.

[0042] In Example 3, five times the volume of a 250 g / L trichloroacetic acid solution was added to whole blood samples from rats. After collecting the precipitate, it was placed in a gamma counter to detect the radioactivity count. According to the count results, a large portion of the precipitate remained free. 125 Radioactive adsorption of I on the surface of precipitates or binding with plasma proteins can be included in the radioactivity count, leading to higher count results.

[0043] The above describes embodiments of the present invention. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of the present invention.

Claims

1. A type of whole blood 125 The method for determining the concentration of I-labeled drugs is characterized by, Includes the following steps: Add a 250 g / L trichloroacetic acid solution to the collected whole blood sample, mix well, centrifuge, remove the supernatant, and collect the first precipitate; Add a trichloroacetic acid solution with a concentration of 100-250 g / L to the first precipitate, mix well, centrifuge and remove the supernatant, and collect the second precipitate; Add a 100-250 g / L trichloroacetic acid solution to the second precipitate, mix well, centrifuge and remove the supernatant, collect the third precipitate, and use a gamma counter to detect the radioactivity count of the third precipitate.

2. The determination method according to claim 1, characterized in that, The volume ratio of the 250 g / L trichloroacetic acid solution to the whole blood sample was 5:

1.

3. The determination method according to claim 2, characterized in that, The 250 g / L trichloroacetic acid solution is prepared by adding 50.0 mL of pure water to 12.5 g of ammonium acetate and mixing well.

4. The determination method according to claim 1, characterized in that, Add 1 mL of a trichloroacetic acid solution with a concentration of 100-250 g / L.

5. The determination method according to claim 4, characterized in that, The method for preparing the trichloroacetic acid solution with a concentration of 100 g / L-250 g / L is to add 50.0 mL of pure water to 5.00 g-12.5 g of ammonium acetate and mix well.

6. The determination method according to claim 1, characterized in that, The centrifugal speed is 2000 rpm.

7. The determination method according to claim 1, characterized in that, The centrifugation time was 5 minutes.

8. The determination method according to claim 1, characterized in that, The whole blood sample contained 125 I-labeled proteoglycan molecules.

9. The determination method according to claim 8, characterized in that, The 125 The labeling method for I-labeled proteoglycan molecules involves adding Na+ with a radioactivity of 4.8 mCi to a proteoglycan molecule solution. 125 I. Mix well, then add 1 μg / μL chloramine T solution and mix well. React for 30 seconds, then add 1 μg / μL sodium metabisulfite solution and mix well to obtain the desired product. 125 I-labeled proteoglycan molecules.

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