Method for determining systemic biological distribution of protein polypeptide drugs

Abstract

The invention discloses a method for determining whole-body biological distribution of protein polypeptide drugs, and belongs to the technical field of in-vivo tracking of drugs. Comprising the following steps: mixing a protein polypeptide drug and a fluorescent dye with an NHS group, adding a pH buffer agent without a free amino group, maintaining the pH value of the mixed solution at 7.5-8.5, incubating, and purifying to remove redundant dye so as to obtain a fluorescence labeled product; imaging after in-vivo administration of the fluorescence labeling product, and obtaining the whole-body biological distribution condition of the protein polypeptide drug according to the fluorescence intensity obtained by imaging; or after the fluorescence labeling product is administrated in vivo, blood samples are collected at multiple different time points, fluorescence scanning is carried out, the blood concentration of the protein polypeptide medicine is obtained through calculation, and the whole-body biological distribution condition of the protein polypeptide medicine is obtained. According to the method provided by the invention, the determination process does not need radioactive substances, pollution reagents are not generated or used, and the method is safe and environment-friendly.

Description

Technical Field

[0001] The present invention relates to the technical field of in vivo drug tracking, and more particularly to a method for determining the systemic biodistribution of protein and polypeptide drugs. Background Art

[0002] Thrombolytic therapy plays a key role in the treatment of cardiovascular diseases. Plasminogen activators mediate thrombolysis by activating inactive plasminogens, which can degrade fibrin blood clots. Although the specificity, stability, safety, efficacy, etc. of plasminogen activators have been improved through several generations of development, there are still some limitations. Novel molecules from microorganisms, plants or animals, such as nattokinase (NK), snake venom plasminogen activator, etc., have become research hotspots due to their safety and ease of use. Among them, snake venom plasminogen activator has the advantages of a long half-life and high selectivity, and is one of the most valuable active components, providing a new source of natural products for the research of antithrombotic drugs.

[0003] Understanding the systemic biodistribution of drugs can provide important information on safety and dose parameters, and provide a basis for drug safety and improvement. Snake venom is a mixture of various proteins and polypeptides. Currently, the commonly used methods for analyzing the pharmacokinetic parameters of protein and polypeptide drugs at home and abroad include isotope-labeled tracer method, in vivo imaging technology, immunoassay (RIA, RIMA, ELISA, FPIA), chromatography (HPLC, HPCE, LC / MS, LC / MS / MS), etc.

[0004] Currently, the main method for studying the blood drug concentration and in vivo distribution of plasminogen activator from Gloydius brevicaudus venom (GBV-PA) is the isotope-labeled tracer method, and the application of the isotope-labeled tracer method in clinical pharmacokinetic research is restricted due to radioactive contamination. Summary of the Invention

[0005] The present invention provides a method for determining the systemic biodistribution and blood drug concentration of protein and polypeptide drugs, so as to solve the problem of high pollution in the determination of the in vivo distribution and blood drug concentration of protein and polypeptide drugs in the prior art.

[0006] In a first aspect, the present invention provides a method for determining the systemic biodistribution of protein polypeptide drugs for non-diagnostic and non-therapeutic purposes, including: mixing a protein polypeptide drug and a fluorescent dye with an NHS group, adding a pH buffer without free amino groups, maintaining the pH of the mixture at 7.5 - 8.5, incubating, purifying to remove excess dye, and obtaining a fluorescently labeled product; performing imaging after in vivo administration of the fluorescently labeled product, and obtaining the systemic biodistribution of the protein polypeptide drug according to the fluorescence intensity obtained from the imaging; or after in vivo administration of the fluorescently labeled product, collecting blood samples at multiple different time points, performing fluorescence scanning, referring to the concentration-fluorescence value standard curve of the fluorescently labeled product solution to obtain the concentration of the fluorescently labeled product in the blood sample, calculating to obtain the blood drug concentration of the protein polypeptide drug, and obtaining the systemic biodistribution of the protein polypeptide drug.

[0007] As a possible implementation, the fluorescent dye with an NHS group is a near-infrared fluorescent dye with an NHS group and a conjugated structure. The spectral range of near-infrared fluorescence is 700 - 1000 nm. In this range, the autofluorescence interference of organisms is small, and the penetration distance into tissues is high; the conjugated structure present in the dye can absorb and emit fluorescence in the near-infrared spectral range, improving the accuracy and sensitivity of imaging.

[0008] As a possible implementation, the dye is 680XL, 645, CF680 or Cyanine5.5 NHS ester, and the protein polypeptide drug is brevicaudus venom plasminogen activator. The primary amine on the molecule of brevicaudus venom plasminogen activator is positively charged under physiological conditions, with its electron pair facing outwards, making it easier to bind to the dye without changing the protein structure. The primary amine binds to the carboxyl group on the dye to form a stable amide bond, thereby labeling the dye molecule onto the brevicaudus venom plasminogen activator molecule.

[0009] As a possible implementation, the addition ratio of the brevicaudus venom plasminogen activator and the 680XL is 0.5 - 5 mg: 1 - 10 μL.

[0010] As a possible implementation, the incubation is carried out under dark shaking at room temperature until the labeling degree of the protein polypeptide drug is between 2 and 3.

[0011] As a possible implementation, the purification is carried out by dialysis with PBS buffer solution for 20 to 30 h, and the molecular weight cut-off is 7 to 25 kDa. The purpose of the dialysis operation is to remove the unbound free fluorescent dye. The purification column with a molecular weight cut-off of 7 kDa can quickly remove the unconjugated fluorophore. The molecular weight of the protein is about 25 kDa. A molecular weight cut-off in the range of 7 to 25 kDa can remove the free fluorescent dye without affecting the protein molecules.

[0012] As a possible implementation, the time point of the in vivo imaging is within the time period when the radiation efficiency of the fluorescently labeled product is greater than 0.5×10 7 (p / s) / (μW / cm 2 ). During this time period, the in vivo content of the fluorescently labeled product is relatively high, the imaging effect is good, and it can better reflect the in vivo distribution of the protein polypeptide drug.

[0013] As a possible implementation, the fluorescence scanning is performed using an in vivo imaging device. The detection range of the in vivo imaging device is 0 to 64000 photons, with higher detection sensitivity and more accurate detection results.

[0014] As a possible implementation, the steps for preparing the concentration-fluorescence value standard curve of the fluorescently labeled product solution include: preparing multiple fluorescently labeled product solutions with known concentrations using plasma as the solvent; measuring the fluorescence values of the fluorescently labeled product solutions at each concentration; and plotting the concentration-fluorescence value standard curve of the fluorescently labeled product solution.

[0015] In the present invention, a fluorescent dye with an NHS group is used to label the protein polypeptide drug. The carboxyl group in the NHS-activated dye is activated, enabling it to bind to the amino group on the protein polypeptide molecule to form a stable amide bond, thereby labeling the dye molecule onto the protein polypeptide drug molecule. Combining with in vivo imaging technology to study the in vivo distribution and determination of blood drug concentration after administration, the determination process does not involve radioactive substances and does not generate or use polluting reagents, with high safety.

[0016] The in vivo imaging technology in the present invention obtains the whole-body biodistribution of the protein polypeptide drug according to the fluorescence intensity, or collects blood samples at multiple different time points to obtain the blood drug concentration. This process visualizes the in vivo distribution process of the protein polypeptide drug, and detects the pharmacokinetic process in real-time and dynamically; it can also non-invasively obtain data continuously in the same experimental subject, avoiding the problem that the detection results are inaccurate due to individual differences. The operation steps are simple, with high stability and more accurate detection results.

[0017] Enzyme-linked immunosorbent assay (ELISA) is one of the most sensitive immunoassay methods in the prior art. Its typical detection range is 0.01 ng to 0.1 ng, and its sensitivity depends on the special characteristics of the antibody-antigen interaction. The detection limit for drugs is relatively high. In contrast, the method provided by the present invention uses a in vivo imaging device with a detection range of 0 to 64,000 photons. The detection range is not limited by the special characteristics of antigens and antibodies, the detection process is not easily interfered by endogenous substances, the detection sensitivity is high, and the detection results are accurate.

[0018] The present invention provides a method for detecting the whole-body biodistribution of GBV-PA. The primary amine on the GBV-PA molecule is more likely to bind to the carboxyl group on the NHS-activated dye to form a stable amide bond. GBV-PA is labeled without affecting its molecular structure, enabling more accurate determination of the whole-body biodistribution of GBV-PA, providing preliminary pharmacokinetic data for subsequent studies on GBV-PA, and further providing research ideas and theoretical basis for thrombolytic therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0020] Figure 1 It is a schematic diagram of the process for determining the in vivo distribution and blood drug concentration of protein drugs provided by the embodiments of the present invention.

[0021] Figure 2 It is the absorption spectra of GBV-PA-680XL, GBV-PA, and VT680XL provided by the embodiments of the present invention.

[0022] Figure 3 It is the SDS-PAGE electrophoresis results of GBV-PA and GBV-PA-680XL provided by the embodiments of the present invention. Among them, A is the Coomassie brilliant blue staining result. In A, a is GBV-PA and b is GBV-PA-680XL; B is the fluorescence scanning result of the small animal optical imaging system. In B, a is 20 μg GBV-PA-680XL, b is 20 μg GBV-PA, c is 10 μg GBV-PA-680XL, and d is 10 μg GBV-PA.

[0023] Figure 4This is the evaluation of the stability of GBV-PA-680XL in PBS and plasma by gel electrophoresis provided by the embodiments of the present invention. Among them, A is the SDS-PAGE result of GBV-PA-680XL in plasma, and B is the SDS-PAGE result of GBV-PA-680XL in PBS.

[0024] Figure 5 This is the representative image of in vivo imaging of BALB / c nude mice injected with GBV-PA-680XL or VT680XL via the tail vein at different times provided by the embodiments of the present invention. Among them, A is the back of the GBV-PA-680XL group, B is the back of the VT680XL group, C is the abdomen of the GBV-PA-680XL group, and D is the abdomen of the VT680XL group.

[0025] Figure 6 This is the distribution of GBV-PA-680XL in various organs and tissues of ICR mice at different times provided by the embodiments of the present invention. Among them, A is the brain, B is the heart, C is the lung, D is the kidney, E is the liver, F is the fat, and G is the muscle.

[0026] Figure 7 This is the distribution change curve of GBV-PA-680XL in various organs and tissues over time provided by the embodiments of the present invention.

[0027] Figure 8 This is the determination result of the blood drug concentration of ICR mice provided by the embodiments of the present invention. Among them, a is the standard curve of plasma GBV-PA-680XL provided by the embodiments of the present invention, and b is the standard color fluorescence image of the 96-well plate of plasma GBV-PA-680XL.

[0028] Figure 9 This is the blood drug concentration-time curve of ICR mice after intravenous injection of GBV-PA-680XL via the tail vein provided by the embodiments of the present invention. Among them, the logarithm of the GBV-PA-680XL concentration is the ordinate, and the time is the abscissa.

[0029] Figure 10 This is the standard curve of GBV-PA-680XL in the plasma of SD rats provided by the embodiments of the present invention. Among them, a is the standard curve read by the multifunctional microplate reader SpectraMax iD3, and b is the standard curve read by the small animal in vivo imaging system IVIS SPECTRUM.

[0030] Figure 11 This is the blood drug concentration-time curve of SD rats after intravenous injection of GBV-PA-680XL via the tail vein provided by the embodiments of the present invention. Among them, the logarithm of the GBV-PA-680XL concentration is the ordinate, and the time is the abscissa. Detailed implementation manners

[0031] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0032] To solve the problems of high pollution, complex operation and low measurement efficiency in the determination of the in vivo distribution and blood drug concentration of protein drugs in the prior art, the present invention provides a method for determining the systemic biodistribution of protein polypeptide drugs.

[0033] Figure 1 Exemplarily, a schematic diagram of the determination process of the in vivo distribution and blood drug concentration of protein drugs provided by the embodiments of the present invention is shown. As Figure 1 shown, the method for determining the systemic biodistribution of protein polypeptide drugs provided by the present invention includes: mixing a protein polypeptide drug and a fluorescent dye with an NHS group, adding a pH buffer without free amino groups, maintaining the pH of the mixed solution at 7.5 - 8.5, incubating, and purifying to obtain a fluorescently labeled product; after in vivo administration of the fluorescently labeled product, imaging is performed, and the in vivo distribution of the protein polypeptide drug is obtained according to the fluorescence intensity obtained from the imaging; or after in vivo administration of the fluorescently labeled product, blood samples are collected at multiple different time points, fluorescence scanning is performed, the concentration of the fluorescently labeled product in the blood sample is obtained by referring to the concentration-fluorescence value standard curve of the fluorescently labeled product solution, and the blood drug concentration of the protein polypeptide drug is calculated. Protein modification is often carried out under the pH condition of 7.5 - 8.5, and a buffer solution containing free amino groups cannot be used to avoid the combination of the buffer with the dye and reduce the labeling degree. Sodium bicarbonate buffer with a pH of 8.5 is commonly used.

[0034] The technical solutions of the present invention will be further elaborated below with reference to specific embodiments.

[0035] Example 1

[0036] This example provides an experiment for determining the labeling degree of VT680XL-labeled GBV-PA.

[0037] Take The lyophilized VT680XL (0.25 mg) in the 680XL (VT680XL) Protein Labeling Kit (PerkinElmer) was dissolved in 10 μL of dry DMSO (dimethyl sulfoxide) to obtain an activated VT680XL solution; 0.5 mL of GBV-PA with a concentration of 3.993 mg / mL, 50 μL of sodium bicarbonate solution (supplied with the VT680XL Protein Labeling Kit), and 4 μL of VT680XL solution were added to a 1.5 mL EP tube, and the mixture was incubated with shaking at room temperature in the dark for 2 h; after the incubation, the entire mixed solution was transferred into a dialysis cassette and dialyzed in PBS buffer for 24 h to remove the unbound free fluorescent dye, obtaining the fluorescently labeled product GBV-PA-680XL. The ratio of the addition amounts of the protein polypeptide drug and VT680XL can be selected from 0.5 - 5 mg:1 - 10 μL, the duration of incubation with shaking can be selected from 1.5 - 2.5 h, and the duration of the dialysis operation can be selected from 20 - 30 h. Specific operations are selected according to actual needs, and the present invention does not make specific limitations.

[0038] The absorbances of the prepared GBV-PA-680XL, protein GBV-PA, and fluorescent dye VT680XL were measured at 280 nm and 668 nm respectively using a NanoDrop 2000 spectrophotometer (Thermo Scientific). Figure 2 The absorption spectra of GBV-PA-680XL, GBV-PA, and VT680XL provided in this example are as Figure 2 shown. GBV-PA has only one absorption peak at 280 nm, VT680XL has only one absorption peak at 668 nm, and GBV-PA-680XL has one absorption peak at both 280 nm and 668 nm, which are the maximum absorbances of protein GBV-PA and fluorescent dye VT680XL respectively. It was preliminarily determined from the absorption spectral characteristics of the labeled product that VT680XL successfully labeled GBV-PA.

[0039] The prepared GBV-PA-680XL was diluted 3.3 times with PBS solution to obtain a GBV-PA-680XL sample solution. 2 μL of the GBV-PA-680XL sample solution was placed in an ultraviolet spectrophotometer (Thermo Scientific NanoDrop 2000) for detection, and the absorbances of the labeled product were measured at 280 nm and 668 nm respectively. The measured A 280 absorbance was 1.2, and the A 668 absorbance was 2.19. According to the following formula, the concentration of the successfully labeled product GBV-PA-680XL was calculated to be 2.6 mg / mL, and the recovery rate was 86.7%.

[0040] DOL (moles of dye per mole of protein) = MVT680XL (Dye concentration) / M GBV-PA (Protein concentration),

[0041] M VT680XL = A 668 / ε VT680XL , M GBV-PA = (A 280 - A 668 × 0.16) / ε GBV-PA ,

[0042] where ε is the molar extinction coefficient, the ε of VT680XL is 210000 L·mol -1 ·cm -1 , the ε of GBV-PA is 275482 L·mol -1 ·cm -1 .

[0043] It shows that VT680XL-labeled GBV-PA has a good labeling degree and can be used as a dye for fluorescent labeling of GBV-PA.

[0044] Example 2

[0045] This example provides a quantitative detection experiment of VT680XL-labeled GBV-PA.

[0046] Prepare an SDS gel, specifically composed of 10 mL of 15% separating gel and 4 mL of 5% stacking gel. Among them, the composition of the separating gel is: 1 mL of high-purity water + 5 mL of 30% acrylamide + 3.8 mL of Tris-HCl buffer with a concentration of 1.0 mol / L and a pH of 8.8 + 0.10 mL of 10% SDS + 0.10 mL of 10% ammonium persulfate + 4 μL of TEMED. The composition of the stacking gel is: 2.8 mL of high-purity water + 0.66 mL of 30% acrylamide + 0.5 mL of Tris-HCl buffer with a concentration of 1 mol / L and a pH of 6.8 + 0.04 mL of 10% ammonium persulfate + 4 μL of TEMED. After the gel preparation is completed, load 10 μg of GBV-PA-680XL prepared in Example 1 and 10 μg of GBV-PA into each well of Gel Plate A; load 10 μg and 20 μg of GBV-PA, and 10 μg and 20 μg of GBV-PA-680XL prepared in Example 1 into each well of Gel Plate B. During electrophoresis, the voltage of the stacking gel is 80 V and the electrophoresis time is 30 min; the voltage of the separating gel is 120 V, and stop electrophoresis after the bromophenol blue reaches the bottom of the gel, about 2.5 h, and keep it away from light throughout the process. After electrophoresis is completed, stain Gel Plate A with Coomassie Brilliant Blue. After the bands are clear, pour out the staining solution, add the decolorizing solution overnight, then discard the decolorizing solution, and take pictures and analyze with a gel imager (BIO-RAD). Gel Plate B is not stained with Coomassie Brilliant Blue, and fluorescence scanning is performed with a small animal fluorescence three-dimensional in vivo imaging system IVIS SPECTRUM (PerkinElmer).

[0047] Figure 3 The SDS-PAGE electrophoresis results of GBV-PA and GBV-PA-680XL provided in this example are shown. Among them, A is the result of Coomassie Brilliant Blue staining. In A, a is GBV-PA and b is GBV-PA-680XL; B is the result of fluorescence scanning with a small animal optical imaging system. In B, a is 20 μg of GBV-PA-680XL, b is 20 μg of GBV-PA, c is 10 μg of GBV-PA-680XL, and d is 10 μg of GBV-PA. It can be Figure 3 seen that the positions of GBV-PA-680XL and GBV-PA are the same, and their bands are similar, both are single protein bands, indicating that GBV-PA-680XL maintains the structural integrity of GBV-PA; the average fluorescence intensity of 10 μg of GBV-PA-680XL is 1.25×10 9 , and the average fluorescence intensity of 5 μg of GBV-PA-680XL is 6.33×10 8 . GBV-PA without labeled fluorescent dye shows no fluorescence, indicating that the fluorescence intensity of GBV-PA-680XL is proportional to the protein concentration.

[0048] Example 3

[0049] This example provides a detection experiment on the incubation stability of VT680XL-labeled GBV-PA in plasma.

[0050] The GBV-PA-680XL prepared in Example 1 was incubated in blank plasma and PBS at 37°C for 72 h, 48 h, and 24 h, and at 4°C in blank plasma and PBS for 72 h. After the incubation ended, using VT680XL, blank plasma, GBV-PA-680XL prepared in Example 1, and GBV-PA as controls, SDS-PAGE detection was performed, and fluorescence scanning was carried out using a small animal fluorescence three-dimensional in vivo imaging system IVIS SPECTRUM (PerkinElmer). The excitation wavelength was set to 668 nm and the emission wavelength was set to 688 nm.

[0051] Figure 4 For evaluating the stability of GBV-PA-680XL in PBS and plasma by the gel electrophoresis method provided in this example, where A is the SDS-PAGE result of GBV-PA-680XL in plasma, A1: Maker, A2: GBV-PA, A3: GBV-PA-680XL, A4: blank plasma, A5: GBV-PA-680XL incubated at 4°C for 72 h, A6 - A8: GBV-PA-680XL incubated at 37°C for 24 h, 48 h, and 72 h respectively; A9: VT680XL; B is the SDS-PAGE result of GBV-PA-680XL in PBS, B1: Marker, B2: GBV-PA, B3: GBV-PA-680XL incubated at 4°C for 72 h, B4 - B6: GBV-PA-680XL incubated at 37°C for 24 h, 48 h, and 72 h respectively, B7: VT680XL. As can be seen from Figure 4 A in it, the lanes without the labeled fluorescent dye, namely Maker and GBV-PA, do not show color. The fluorescent dye VT680XL has a small molecular weight and a faster electrophoresis speed, and the electrophoresis band position is close to the bottom of the gel plate. The GBV-PA-680XL incubated in plasma at 4°C for 72 h and the GBV-PA-680XL incubated in plasma at 37°C for 24 h, 48 h, and 72 h are in the same position as the unincubated GBV-PA-680XL, with similar fluorescence intensities, and there is no fluorescence intensity at the position of the fluorescent dye, indicating that GBV-PA-680XL is stable when incubated in plasma at 4°C and 37°C for 72 h. As can be seen from Figure 4As shown in B, the lanes without fluorescent dye labeling, namely Maker and GBV-PA, show no color, the electrophoresis position of fluorescent dye VT680XL is close to the bottom of the gel plate, the GBV-PA-680XL incubated in 4℃PBS for 72h and in 37℃PBS for 24h, 48h and 72h have the same fluorescence position and similar fluorescence intensity, and there is no fluorescence intensity at the fluorescent dye position, indicating that GBV-PA-680XL is stable after incubation in 4℃ and 37℃PBS for 72h.

[0052] Example 4

[0053] This example provides an experiment to detect the in vivo distribution of GBV-PA-680XL after administration.

[0054] Sixteen BALB / c nude mice with the same growth condition were selected and randomly divided into two groups, namely, GBV-PA-680XL group and VT680XL group. Each mouse in GBV-PA-680XL group was injected with GBV-PA-680XL prepared in Example 1 at a dose of 300 μg / kg through the tail vein, and each mouse in VT680XL group was injected with dye VT680XL at a dose of 300 μg / kg GBV-PA-680XL corresponding to VT680XL through the tail vein. Each group of mice was subjected to gas anesthesia at 5, 15, 30 min and 1, 6, 24, 48, 72 h after injection, and placed in Imaging was performed in Spectrum (PerkinElmer, USA).

[0055] Figure 5 The representative images of the in vivo imaging of BALB / c nude mice injected with GBV-PA-680XL or VT680XL at different times by tail vein provided in this example, wherein A is the back of the GBV-PA-680XL group, B is the back of the VT680XL group, C is the abdomen of the GBV-PA-680XL group, and D is the abdomen of the VT680XL group; Figure 5 It can be seen that a strong fluorescence signal can be seen in the whole body of BALB / c nude mice 5 minutes after tail vein injection of GBV-PA-680XL, and as time goes by, the fluorescence signal accumulates in the bladder, showing the characteristics of urine excretion. The fluorescence signals in the kidney and liver areas are still obvious at 24h and 48h, and the fluorescence signal in the kidney area can still be observed at 72h; however, in the mice injected with VT680XL through the tail vein, no fluorescence signal could be detected after 24h.

[0056] Forty-five ICR mice with the same growth status were randomly and evenly divided into 9 groups, namely the PBS group, the 1-min group, the 3-min group, the 10-min group, the 30-min group, the 1-h group, the 6-h group, the 24-h group, and the 48-h group. The mice in the PBS group were injected with PBS at a dose of 300 μg / kg. After 1 min, blood was collected by enucleating the eyeballs, and then the mice were quickly sacrificed by cervical dislocation and dissected. In the remaining 8 groups except the PBS group, each group of mice was injected with GBV-PA-680XL prepared in Example 1 via the tail vein at a dose of 300 μg / kg. In the remaining 8 groups except the PBS group, the mice in the corresponding groups were sacrificed by enucleating the eyeballs and quickly dislocating the cervical vertebrae at 1, 3, 10, 30 min and 1, 6, 24, 48 h after injection, and then dissected. The brain, heart, lung, kidney, liver, fat, and muscle of each group of mice were taken and placed in Spectrum for imaging. The imaging excitation wavelength was 668 nm and the emission wavelength was 688 nm. The luminescence or fluorescence intensity of the region of interest (roi) was quantified using Living Image 4.5 software (PerkinElmer, USA).

[0057] Figure 6 This shows the distribution of GBV-PA-680XL in various organs and tissues of the ICR mice provided in this example at different times. Among them, A is the brain, B is the heart, C is the lung, D is the kidney, E is the liver, F is the fat, and G is the muscle. It can be Figure 6 seen that 1 min after tail vein injection, GBV-PA-680XL was rapidly distributed in the heart, lung, kidney, liver, fat, and muscle, and the fluorescence signal in the brain tissue was low at this time. 10 min after administration, after the concentration of GBV-PA-680XL in the blood rapidly decreased, the fluorescence signal in the heart also decreased, while the fluorescence signals in the kidney and liver were enhanced. As time went by, 24 h after administration, the fluorescence signal of GBV-PA-680XL in the muscle gradually decreased, and there were still strong fluorescence signals in the lung, kidney, and liver, and the fluorescence signal intensity gradually weakened over time. Figure 7 This is the distribution change curve of GBV-PA-680XL in various organs and tissues over time provided in this example. It can be Figure 7 seen that the fluorescence intensities of the brain and heart of the mice injected with GBV-PA-680XL were higher than those of the mice injected with PBS in a short time, while the fluorescence intensities of the lung, kidney, liver, and muscle were significantly higher than those of the mice injected with PBS within 48 h. Among them, the fluorescence intensities of the liver and kidney of the mice injected with GBV-PA-680XL first increased and then decreased, and the fluorescence intensities of other organs and tissues gradually decreased.

[0058] Example 5

[0059] This example provides a blood drug concentration detection experiment after administration of GBV-PA-680XL.

[0060] For the mice in the PBS group, 1 min group, 3 min group, 10 min group, 30 min group, 1 h group, 6 h group, 24 h group and 48 h group in Example 4, 100 μL of blood was collected by enucleating the eyeballs and stored in a tube containing heparin sodium solution. Samples of 5 mice were collected at each time point.

[0061] Twenty SD rats with the same growth status were selected and randomly divided into four groups, namely group A, group B, group C and group D. The dosage of GBV-PA-680XL administered to group A and group B was 150 μg / kg, and the dosage of GBV-PA-680XL administered to group C and group D was 300 μg / kg. The blood sampling time for group A and group C was 0 - 6 h, and the blood sampling time for group B and group D was 24 - 48 h. After anesthetizing the rats in group A and group C with 20% urethane solution at a dose of 0.6 mL / 100 g by intraperitoneal injection, they were fixed supine on the rat operating table. A carotid artery catheter was inserted, a three-way valve was connected, and local heparin anticoagulation was performed. Before administration, 100 μL of blank blood sample was taken, and then GBV-PA-680XL was injected into the tail vein according to the dosage of each group as described above. After administration, 100 μL of blood was taken from the carotid artery three-way valve of the rats in group A and group C at 1 min, 3 min, 10 min, 30 min, 1 h and 6 h respectively, and stored in a tube containing heparin sodium solution in the dark; 100 μL of blood was taken from the orbital venous plexus of the rats in group B and group D at 24 h and 48 h respectively, and stored in a tube containing heparin sodium solution in the dark. The blood samples of the four groups were centrifuged at a speed of 3000 rpm for 10 min in the dark environment. The upper plasma was diluted with DMSO at a volume ratio of 1:2 to ensure capturing the maximum fluorescence signal, and a small animal fluorescence three-dimensional in vivo imaging system Spectrum (PerkinElmer) was used for fluorescence scanning. The excitation wavelength was set at 668 nm and the emission wavelength was set at 688 nm. The measured data was normalized to the standard curve prepared with reagents of known concentration to quantify the blood concentration (μg / mL).

[0062] Figure 8 The results of the determination of the blood drug concentration of ICR mice provided in this example. Among them, a is the standard curve of GBV-PA-680XL in mouse plasma provided in this example, and b is the standard color fluorescence image of the 96-well plate of GBV-PA-680XL in plasma. The linear equation of the standard curve of GBV-PA-680XL in mouse plasma is y = 1626.6x - 2E+07, R 2= 0.9974. By combining the fluorescence scanning results of this example with the standard curve, the content of GBV-PA-680XL in plasma was determined, and the plasma concentration-time curve of GBV-PA-680XL after single intravenous injection in mice was plotted. The results showed that after single intravenous injection of GBV-PA-680XL, the plasma concentration decreased rapidly within 3 min, the rate of decrease slowed down after 60 min, and at 48 h, the fluorescence signal of GBV-PA-680XL was still detected in the blood. Figure 9 This is the plasma concentration-time curve of ICR mice after tail vein injection of GBV-PA-680XL provided in this example. Among them, the logarithm of the GBV-PA-680XL concentration is the ordinate, and time is the abscissa. The curve characteristics conform to the kinetic characteristics of the two-compartment model. The pharmacokinetic parameters were calculated by DAS 2.0 software. The average value of the GBV-PA concentration measured at each time point was input into DAS 2.0 software for intelligent analysis. According to the analysis results, the best compartment model was selected as the two-compartment model, and the best weight was 1 / cc. The pharmacokinetic parameters of single intravenous injection of GBV-PA in mice are shown in Table 1.

[0063] Table 1 Pharmacokinetic parameters of ICR mice after single intravenous injection of GBV-PA-680XL

[0064]

[0065] The parameter equation was obtained from Table 1: C t = 5761.238e -28.918t + 1128.801e -0.038t , t 1 / 2α (Distribution half-life) was 1.4 min, t 1 / 2β (Elimination half-life) was 18.18 h.

[0066] Standard plasma containing different concentrations of GBV-PA-680XL was prepared. One 384-well plate was taken, and the indicated reagents were added to different wells according to the drug addition amounts shown in Table 2. Each group of drug addition was repeated in 3 wells. After mixing, the fluorescence values were read on a multifunctional microplate reader and a small animal in vivo imaging system, analyzed, and the standard curve of GBV-PA-680XL in SD rat plasma as shown in Figure 10 was prepared.

[0067] Table 2 Preparation of the standard curve of GBV-PA-680XL in SD rat plasma

[0068]

[0069]

[0070] Figure 10In it, a is the standard curve read by the multi-functional microplate reader SpectraMax iD3, and the linear equation is y = 495.34x - 131181, R 2 = 0.996, and the concentration range is 135.4 - 8666.7 μg / L; b is the standard curve read by the small animal in vivo imaging system IVIS SPECTRUM, and the linear equation is y = 19527x + 3E+06, R 2 = 0.9764, and the concentration range is 33.9 - 8666.7 μg / L.

[0071] By comparing the two detection methods, the small animal in vivo imaging system has higher sensitivity and can be used to detect lower-dose drug concentrations. The method of using the small animal in vivo imaging system to detect fluorescence intensity is selected to calculate the drug content in plasma and draw the Figure 11 blood concentration-time curve of SD rats after tail vein injection of GBV-PA-680XL as shown.

[0072] It can be Figure 11 seen that the trends of the blood concentration-time curves of rats intravenously injected with two doses of 150 μg / kg and 300 μg / kg of GBV-PA are basically the same, and both conform to the characteristics of the two-compartment model. It shows that within the range of 150 - 300 μg / kg of GBV-PA dose, it is eliminated according to the same-order kinetics. Using DAS2.0 software to calculate the pharmacokinetic parameters, it is suggested that after a single intravenous injection of 150 μg / kg or 300 μg / kg of GBV-PA in rats, the best compartment model of its drug-time curve is the two-compartment model, and the best weight is 1 / cc. The pharmacokinetic parameters of SD rats after tail vein injection of GBV-PA are shown in Table 3.

[0073] Table 3 Pharmacokinetic parameters of SD rats after a single intravenous injection of GBV-PA

[0074]

[0075]

[0076] As can be seen from Table 3, the parameter equation for tail vein injection of 150 μg / kg GBV-PA is: C t = 2522.347e -2.518t + 1449.921e -0.037t , t 1 / 2α is 16.5 min, and t 1 / 2β is 18.66 h; the parameter equation for tail vein injection of 300 μg / kg GBV-PA is: C t = 4870.802e -2.07t + 1836.972e -0.037t , t1 / 2α was 20.1 min, t 1 / 2β was 18.50 h. After intravenous injection of GBV-PA-680XL at 150 μg / kg and 300 μg / kg in rats, t 1 / 2β were 18.66 h and 18.50 h respectively, which were extremely close, indicating that within this dose range, GBV-PA-680XL was eliminated in rats according to first-order kinetics.

[0077] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments as well as all changes and modifications that fall within the scope of the present invention.

[0078] Obviously, those skilled in the art can make various changes and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A method for determining the systemic biodistribution of protein polypeptide drugs, characterized in that: include: Mixing protein peptide drugs and fluorescent dyes with NHS groups, adding a pH buffer without free amino groups, maintaining the pH of the mixture at 7.5-8.5, incubating, purifying and removing excess dyes, and obtaining a fluorescently labeled product; After administering the fluorescently labeled product in vivo, imaging is performed, and the systemic biodistribution of the protein polypeptide drug is obtained according to the fluorescence intensity obtained by imaging; or After the fluorescently labeled product is administered in vivo, blood samples are collected at multiple different time points, and fluorescence scanning is performed. The concentration of the fluorescently labeled product in the blood sample is obtained by referring to the concentration-fluorescence value standard curve of the fluorescently labeled product solution, and the blood concentration of the protein polypeptide drug is calculated to obtain the systemic biodistribution of the protein polypeptide drug.

2. The method according to claim 1, characterized in that: The fluorescent dye with NHS group is a near-infrared fluorescent dye with NHS group and conjugated structure.

3. The method according to claim 2, characterized in that The dye is 680XL, 645, CF680 or Cyanine5.5NHS ester, the protein polypeptide drug is a plasminogen activator from Agkistrodon acutus venom.

4. The method according to claim 3, characterized in that The agkistrodon acutus venom plasminogen activator and the The addition ratio of 680XL is 0.5-5 mg:1-10 μL.

5. The method according to claim 1, characterized in that The incubation is carried out in a dark shaking state at room temperature until the labeling degree of the protein polypeptide drug is between 2 and 3.

6. The method according to claim 1, characterized in that The purification is performed by dialysis with a PBS buffer solution for 20 to 30 hours, with a molecular weight cutoff of 7 to 25 kda.

7. The method according to claim 1, characterized in that The time point of in vivo imaging is when the radiation efficiency of the fluorescent labeled product is greater than 0.5×10 7 (p / s) / (μW / cm 2 ) time period.

8. The method according to claim 1, characterized in that The fluorescence scanning is performed using a living body imaging device.

9. The method according to claim 1, characterized in that: The steps of preparing the concentration-fluorescence value standard curve of the fluorescent labeled product solution include: Using plasma as solvent to prepare a plurality of solutions of the fluorescently labeled product with known concentrations; Measuring the fluorescence value of the fluorescent labeled product solution at each concentration respectively; A concentration-fluorescence value standard curve of the fluorescent labeled product solution is drawn.

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