Method for detecting plasma protein binding rate of siRNA (small interfering ribonucleic acid) medicine in human body and experimental animal

Abstract

The invention provides a method for rapidly and accurately detecting the plasma protein binding rate of siRNA (small interfering Ribonucleic Acid) drugs in human bodies and experimental animals, which is used for detecting the plasma protein binding rate by adopting the change of electrophoresis mobility and comprises the following steps: a, preparing a drug stock solution; b, preparing a sample; c, electrophoresis; d, dyeing; e, gel imaging; and f, data analysis. The binding rate measured by the method is more accurate, and the error is smaller; in the prior art, the strip is clear, and under general conditions, due to the existence of plasma, when a high-concentration sample is applied by using a page adhesive, a serious trailing phenomenon occurs in the strip, so that the subsequent calculation is influenced. According to the invention, after a dilution process before sample loading is added, a high-concentration sample can also run out of a clear strip, so that subsequent calculation is facilitated; the repeatability is good.

Description

Technical Field

[0001] This invention relates to a method for detecting the plasma protein binding rate of siRNA drugs in humans and experimental animals. Background Technology

[0002] Plasma protein binding (PPB) refers to the degree to which a drug binds to proteins in the blood or plasma. In pharmacology, drugs exist in two forms: unbound and bound. Unbound drug molecules can diffuse across cell membranes, reach their sites of action, and be eliminated. In contrast, drugs bound to plasma proteins are mostly retained in the vascular lumen, providing a reservoir of drugs that are released into circulation as the concentration of free drug decreases. PPB is a key parameter routinely measured in drug development, used to establish pharmacokinetic / pharmacodynamic (PK / PD) relationships, perform interspecies parameter conversions, and calculate the therapeutic index of a drug. Commonly used methods for PPB detection include balanced dialysis, ultrafiltration, and ultracentrifugation. siRNA drugs, with their high electronegativity, large molecular weight, and rigid double-stranded structure, are difficult to detect using conventional PPB detection methods. Electrophoretic mobility shift analysis (EMSA) has gradually become a universal method for determining the plasma protein binding rate of siRNA drugs, replacing traditional methods by separating siRNA-protein complexes from free siRNA based on differences in migration rates and indirectly estimating binding rates by analyzing free siRNA bands. However, EMSA is not a classic quantitative method for PPB detection due to its lower accuracy, reproducibility, and standardization, and its sensitivity to operating conditions. In particular, the quality of gel imaging directly determines the accuracy of subsequent data analysis. Therefore, establishing a standardized electrophoresis and pretreatment procedure to obtain clear, easily analyzable, reproducible gel images that accurately reflect drug plasma protein binding is crucial for determining the PPB of siRNA drugs using EMSA.

[0003] Application No. 202310125839.7, Invention Title: A Method for Rapidly Detecting the Binding Rate of Oligonucleotide Drugs to Human and Experimental Animal Plasma Proteins, discloses a method based on changes in electrophoretic mobility and comparing the gray values ​​of electrophoretic bands to detect the binding rate of oligonucleotide drugs to human and experimental animal plasma proteins. Specifically, it includes the following steps: 1) preparation of drug samples, 2) preparation of plasma sample T, 3) preparation of free sample F, 4) electrophoresis, and 5) data analysis. This invention is convenient and rapid, and can detect the plasma protein binding rate of oligonucleotide drugs in a short time. However, the plasma protein binding rate of Oxiumo disclosed in this patent document still has a significant error compared to the plasma protein binding rate of Oxiumo reported in other literature. Summary of the Invention

[0004] This invention provides a rapid and accurate method for detecting the plasma protein binding rate of siRNA drugs in humans and experimental animals.

[0005] This invention provides a rapid and accurate method for detecting the plasma protein binding rate of siRNA drugs in humans and experimental animals. It utilizes electrophoretic mobility changes to detect plasma protein binding rate and includes the following steps: a. Preparation of siRNA stock solutions: Dissolve siRNA powder in enzyme-free water to prepare stock solutions of 2.5 mg / mL, 0.5 mg / mL, and 0.05 mg / mL; b. Sample preparation: Take the siRNA stock solution from step a and add it to the experimental group sample prepared at the test concentration in the plasma to be tested. Take the siRNA stock solution from step a and add it to the control group sample prepared at the test concentration in 1×PBS. Incubate the above samples. c. Electrophoresis: Load the precast gel onto the gel electrophoresis tank, take the incubated sample from step b, add 1×PBS to dilute and mix well, then add 10×DNA loading buffer and mix well, then spot the sample onto the precast gel wells for electrophoresis. d. Staining: Remove the gel and soak it in SYBR GOLD nucleic acid dye; e. Gel imaging: After staining, the gel is removed and placed in a chemiluminescence imager to acquire images; f. Data Analysis: Analyze imaging images and calculate plasma protein binding rates.

[0006] The concentrations of the siRNA stock solution in step a are 2.5 mg / mL, 0.5 mg / mL, and 0.05 mg / mL, respectively.

[0007] The method for incubating the samples was to incubate the samples in a 37 °C water bath for 2 h.

[0008] In step b, before loading the incubated sample, add 1×PBS at a ratio of 1:5 and mix well.

[0009] The electrophoresis conditions used in step c are as follows: the gel is 12%~16% TBE PAGE gel; the electrophoresis buffer is 0.5×TBE electrophoresis buffer; and the electrophoresis apparatus is set to a constant voltage of 150 V.

[0010] The soaking time in step d is 30 minutes.

[0011] The analysis method described in step e involves using ImageJ software to quantify the free bands in the electrophoretic imaging image and calculate their integrated gray value IntDen.

[0012] The formula for calculating the plasma protein binding rate is as follows: .

[0013] The method for detecting the plasma protein binding rate of siRNA drugs in humans and experimental animals presented in this invention yields accurate results. Compared to the method disclosed in patent application 202310125839.7, the binding rate measured by this invention is more accurate and has smaller errors. The bands are clear; generally, due to the presence of plasma, severe band tailing occurs when using Page gel to spot high-concentration samples, affecting subsequent calculations. This invention incorporates a dilution process before sample loading, enabling clear bands to be obtained even for high-concentration samples, facilitating subsequent calculations. Furthermore, it exhibits good repeatability. Attached Figure Description

[0014] Figure 1 Electrophoresis images of Givosiran after incubation with 1×PBS, human plasma, and blank human plasma (without oligonucleotide drugs) at test concentrations of 50 μg / mL, 10 μg / mL, and 1 μg / mL; Figure 2 for Figure 1 The corresponding electrophoretic bands were converted into peak area maps by ImageJ software after calculating the gray values ​​of the bands. After integration, the relative concentration of oligonucleotide drugs in the corresponding bands could be obtained. Figure 3 The effect of whether or not a dilution process is performed before sample loading on the electrophoresis results. Detailed Implementation

[0015] Example 1: Method for detecting the plasma protein binding rate of Givosiran in humans and experimental animals according to the present invention. 1. Experimental Design

[0016]

[0017] 2. Experimental Grouping Experimental group (TS): The test substance was added to the plasma of various genera and incubated at 37 °C before electrophoresis.

[0018] Control group (NC): The test substance was added to PBS buffer and incubated at 37 °C before electrophoresis.

[0019] Blank control group (BL): Enzyme-free water was added to the plasma of various genera and incubated at 37 °C before electrophoresis.

[0020] 3. Experimental Procedure 3.1 Preparation of stock solution Givosiran stock solution: Dissolve Givosiran powder in enzyme-free water to prepare a 2.5 mg / mL stock solution, and then dilute to prepare 0.5 mg / mL and 0.05 mg / mL stock solutions respectively.

[0021] 3.2 Sample preparation Experimental group (TS): 2 μL of Givosiran stock solution was added to 98 μL of various plasmas to prepare plasma samples of the test concentration, and then the samples were incubated in a 37 °C water bath for 2 h.

[0022] Control group (NC): 2 μL of Givosiran stock solution was added to 98 μL of PBS buffer to prepare a PBS buffer sample of the test concentration, and then the sample was incubated in a 37 °C water bath for 2 h.

[0023] Blank control group (BL): 2 μL of enzyme-free water was added to 98 μL of plasma of various species, and the samples were then incubated in a 37 °C water bath for 2 h.

[0024] 3.3 Electrophoresis Dilute 5×TBE buffer to 0.5× with pure water. Pre-chill the 0.5×TBE buffer at 4 ℃. After sample incubation, load the precast gel into the gel electrophoresis tank. Take an incubated sample and dilute it with 1×PBS at a ratio of 1:5. Mix well, then add 10×DNA loading buffer and mix well. Spot the sample onto the precast gel wells for electrophoresis, with a duplicate well count of 2. Stop electrophoresis when bromophenol blue has migrated to approximately 2 / 3 of the gel.

[0025] The electrophoresis conditions were as follows: the gel was 12%~16% TBE PAGE gel; the electrophoresis buffer was 0.5×TBE electrophoresis buffer; and the electrophoresis apparatus was set to a constant voltage of 150 V.

[0026] 3.4 Staining Dilute SYBR GOLD nucleic acid dye at a ratio of 1:10000 in 0.5×TBE buffer to prepare 1× gel staining solution. After electrophoresis, remove the gel and place it in 1× gel staining solution for staining in the dark for about 30 minutes.

[0027] 3.5 Gel Imaging After staining, the gel is removed and placed in a chemiluminescence imager. The nucleic acid gel imaging mode is selected, and the appropriate exposure time is adjusted for imaging.

[0028] 4. Data Processing (1) The bands that are in the same position as the control group bands are free bands (unbound bands). ImageJ software was used to quantify the free bands in the electrophoresis images and calculate their integrated gray value IntDen.

[0029] (2) Calculation of plasma protein binding rate

[0030] (3) Evaluation criteria The plasma protein binding rates of the siRNA drug Givosiran used in this experiment are shown in the following table:

[0031]

[0032] 5. Results At test concentrations of 1, 10, and 50 μg / mL, the protein binding rates of the positive control drug Givosiran in SD rat plasma were 96.3%, 85.9%, and 26.7%, respectively; in cynomolgus monkey plasma, they were 93.7%, 76.8%, and 15.9%, respectively; and in human plasma, they were 93.4%, 61.5%, and 13.9%, respectively. Figure 1 Electrophoresis images of Givosiran after incubation with 1×PBS, human plasma, and blank human plasma (without oligonucleotide drugs) at test concentrations of 50 μg / mL, 10 μg / mL, and 1 μg / mL (two parallel bands for each sample).

[0033] Figure 2 for Figure 1 The corresponding electrophoretic bands are converted into peak area maps by calculating the gray values ​​of the bands using ImageJ software. After integration, the relative concentration of oligonucleotide drugs in the corresponding bands can be obtained.

[0034] Example 2: Selection of Gel and Electrophoresis Solution in the Detection Method of the Present Invention The effects of two different gels on protein binding rate were tested.

[0035] 1. The human plasma protein binding rates of 1, 10, and 50 μg / mL Givosiran were determined according to the method described in patent application number 202310125839.7, and were 64.57%, 33.79%, and 6.09%, respectively, which differed significantly from the literature values. Gel and electrophoresis conditions: 12%~16% Tris-glycine Page gel and Tris-glycine-natural electrophoresis buffer; 2. The human plasma protein binding rates of 1, 10, and 50 μg / mL Givosiran were determined according to the method described in this invention, and were 93.4%, 61.5%, and 13.9%, respectively, which are more accurate and close to the literature values. The gel and electrophoresis buffer used in this invention are: 12%~16% TBE PAGE gel and 0.5×TBE electrophoresis buffer.

[0036] As can be seen, the plasma protein binding rate measured by this invention is more accurate after optimizing the gel and electrophoresis conditions.

[0037] Example 3: Optimization of the pretreatment for electrophoresis using the detection method of the present invention In general, when using Page gel to spot high-concentration samples, severe band tailing occurs, affecting subsequent calculations. This method, by adding a dilution process before sample loading, can produce clear bands even in high-concentration samples (50 μg / mL), facilitating subsequent calculations.

[0038] The gel was measured according to the method described in this invention, but without step c, which involves "diluting the sample incubated in step b with 1×PBS at a ratio of 1:5 and mixing well." The gel results were as follows: Figure 3 As shown in a; the gel was measured according to the method described in this invention, but when step c was added, "take the sample incubated in step b and dilute it with 1×PBS at a ratio of 1:5 and mix well", the gel results were as follows. Figure 3 As shown in b (each gel image from left to right is siRNA+PBS (control group), siRNA+plasma (experimental group), n=2). It can be seen that after adding the dilution process before loading, the obtained electrophoretic bands are clearer and there is no dragging phenomenon, which facilitates subsequent analysis.

[0039] Example 4: Repeatability test of the detection method of the present invention The steps described in Example 1 were repeated multiple times. The average plasma protein binding rates of various species are shown in the table below. The repeatability was good, and the error was within ±10%.

[0040]

Claims

1. A rapid and accurate method for detecting the plasma protein binding rate of siRNA drugs in humans and experimental animals, characterized in that: It uses changes in electrophoretic mobility to detect plasma protein binding rate, and includes the following steps: a. Preparation of drug stock solution: Dissolve siRNA drug powder in enzyme-free water to prepare drug stock solution; b. Sample preparation: Take the drug stock solution from step a and add it to plasma to prepare the experimental group sample at the test concentration; take the drug stock solution from step a and add it to 1×PBS to prepare the control group sample at the test concentration, and incubate the above samples. c. Electrophoresis: Load the precast gel onto the gel electrophoresis tank, take the sample after incubation in step b, add 1×PBS to dilute and mix well, then add 10×DNA loading buffer and mix well, then spot the sample onto the precast gel wells for electrophoresis. d. Staining: Remove the gel and soak it in SYBR GOLD nucleic acid dye; e. Gel imaging: After staining, the gel is removed and placed in a chemiluminescence imager to acquire images; f. Data Analysis: Analyze imaging images and calculate plasma protein binding rates.

2. The method for detecting the plasma protein binding rate of siRNA drugs in humans and experimental animals according to claim 1, characterized in that: The concentrations of the drug stock solutions described in step a are 2.5 mg / mL, 0.5 mg / mL, and 0.05 mg / mL, respectively.

3. The method for detecting the plasma protein binding rate of siRNA drugs in humans and experimental animals according to claim 1, characterized in that: Step b involves incubating the sample in a 37 °C water bath for 2 hours.

4. The method for detecting the plasma protein binding rate of siRNA drugs in humans and experimental animals according to claim 1, characterized in that: In step c, the incubated sample is diluted with PBS at a ratio of 1:

5.

5. The method for detecting the plasma protein binding rate of siRNA drugs in humans and experimental animals according to claim 1, characterized in that: The electrophoresis conditions used in step c are as follows: the gel is 12%~16% TBE PAGE gel; the electrophoresis buffer is 0.5×TBE electrophoresis buffer; and the electrophoresis apparatus is set to a constant voltage of 150 V.

6. The method for detecting the plasma protein binding rate of siRNA drugs in humans and experimental animals according to claim 1, characterized in that: Step d involves soaking the gel in SYBR GOLD nucleic acid dye for 30 minutes.

7. The method for detecting the plasma protein binding rate of siRNA drugs in humans and experimental animals according to claim 1, characterized in that: The analysis method described in step f is to use ImageJ software to quantify the free bands in the electrophoretic imaging image and calculate their integrated gray value IntDen.

8. The method for detecting the plasma protein binding rate of siRNA drugs in humans and experimental animals according to claim 1, characterized in that: The formula for calculating the plasma protein binding rate in step f is: 。

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