Method and kit for evaluating in-vivo dispersion range of collagenase

Abstract

The invention discloses a method for evaluating a collagenase dispersion range, which comprises the following steps: labeling collagenase by using a fluorescent marker to prepare a fluorescently labeled collagenase injection; injecting the fluorescently-labeled collagenase injection into the subcutaneous fat layer of the skin tissue; carrying out fluorescence imaging on the skin tissue at a plurality of time points after administration by utilizing a fluorescence imaging system; and measuring the subcutaneous dispersion radius, diameter, perimeter or area of the collagenase at each time point according to the fluorescence imaging. The invention also discloses a kit for evaluating the dispersion range of collagenase for injection, the kit comprises a fluorescent marker and skin tissue, the fluorescent marker is CY7 fluorescent dye, and the skin tissue is artificial bionic skin. According to the method, the long-wave fluorescent probe and the in-vivo imaging technology are used for visual evaluation of the in-vivo local dispersion range of the collagenase for injection for the first time, and the blank in evaluation of the in-vivo action range of the collagenase after subcutaneous injection in the prior art is filled.

Description

Technical Field

[0001] The invention relates to the technical field of drug evaluation, and in particular to a method and a kit for evaluating the diffusion range of collagenase injection in vivo through fluorescent labeling. Background Art

[0002] With the improvement of living standards and changes in dietary structure, the number of overweight people in the world has increased significantly. Moderate to severe contour bulge or excessive plumpness caused by accumulation of submental fat (SMF) is usually manifested as a "double chin". The 2019 American Society of Dermatologic Surgeons' consumer survey on skin beauty showed that 73% of respondents were troubled by "excessive fat accumulation under the chin / neck". The waist and abdomen are one of the core parts of the human body. Many people have the habit of sitting for a long time and lack of exercise, which leads to fat accumulation in the waist and abdomen, forming an "apple-shaped figure". Moderate to severe contour bulge or excessive plumpness caused by fat accumulation in the waist and abdomen and SMF accumulation may cause social embarrassment and have a negative impact on the self-image perception of many patients.

[0003] Diet and exercise have limited effects on reducing localized fat accumulation. For many years, surgical liposuction has been the main means of reducing moderate to severe contour bulges or excessive fullness, and fat accumulation in the waist and abdomen caused by SMF accumulation. Although liposuction is an effective and reliable treatment, many patients may be reluctant to undergo surgery due to the high cost, long recovery time after surgery, and risks such as bruising, bleeding, infection, scarring, and neurological complications. Injectable collagenase can act on fat cells attached to the extracellular matrix (ECM) of adipose tissue, destroy the collagen network in fat cells to free fat cells, thereby inducing apoptosis of fat cells and achieving lipolysis, thereby improving the accumulation of fat under the chin and waist and abdomen to achieve the effect of body shaping. Therefore, injectable collagenase was developed for localized fat management.

[0004] Injectable collagenase is used to treat submental and abdominal fat accumulation and is currently under clinical research. After local injection, collagenase degrades the extracellular matrix collagen in the subcutaneous fat layer, causing apoptosis of adipocytes, which is expected to effectively reduce local fat accumulation. In order to achieve uniform degradation of subcutaneous fat, the injection dose and injection point interval of injectable collagenase need to be strictly controlled during injection. At present, adverse reactions during the treatment of local fat accumulation include bruising at the injection site, nodules at the injection site, hematoma at the injection site, etc., and most of the adverse reactions are related to the inability to effectively control the injection dose and injection interval. Therefore, obtaining the single-point diffusion range of injectable collagenase in the early clinical stage can effectively control the usage and dosage of injectable collagenase, and ensure the effectiveness and safety of clinical treatment of local fat accumulation.

[0005] The current method for evaluating fat thickness is ultrasound detection, which can evaluate the thickness change of the fat layer after local subcutaneous injection, but cannot provide the range of action of the drug after single-point injection. In vitro tissue injection method, a piece of freshly dissected pig back tissue is usually taken, and the collagenase for injection is injected into the subcutaneous fat layer. After incubation at 37°C, it is cut open and the range of action of different collagenase injection doses is determined by judging the size of the cavity formed in the fat layer. [1] This method is carried out in vitro and cannot fully simulate the form and scope of action of drugs entering the subcutaneous tissue in vivo. Currently, there is no suitable method to evaluate the effective scope of injectable collagenase products after subcutaneous injection to provide data support for the design of clinical injection doses and injection point intervals.

[0006] Non-patent literature [1]: Chen F, Du G, Shih M, Yuan H, Bao P, Shi S, Cang Y and ZhangZ 2019Safe and effective subcutaneous adipolysis in minipigs by acollagenasederivative ed H Nadeem PLOS ONE 14e0227202 Summary of the invention

[0007] Technical Problems to be Solved by the Invention

[0008] In order to achieve uniform degradation of subcutaneous fat, the injection dose and injection point interval of injectable collagenase need to be strictly controlled. Currently, there is no suitable method to evaluate the effective range of injectable collagenase products after subcutaneous injection to provide data support for the design of clinical injection dose and injection point interval.

[0009] The present invention adopts a long-wave fluorescent probe (CY7 fluorescent dye) to label the injectable collagenase, and injects the labeled collagenase into the subcutaneous fat layer of rats and Bama miniature pigs. The fluorescence imaging method is used to visualize the subcutaneous action range of the collagenase, intuitively obtain the action range of the collagenase in the body under different dosage conditions, obtain the action diameter, and provide a sufficient basis for the design of clinical injection schemes.

[0010] Technical means to solve technical problems

[0011] The present invention uses a long-wave fluorescent probe (such as CY7 fluorescent dye) to label injectable collagenase, injects the labeled collagenase into the subcutaneous fat layer of an animal through subcutaneous injection, and uses a fluorescent imaging system to detect the fluorescence range of the collagenase under the animal's skin to measure the diffusion radius, diameter, circumference or area.

[0012] The present invention includes the following technical solutions.

[0013] A method for evaluating the diffusion range of collagenase, characterized by comprising the following steps:

[0014] Labeling collagenase with a fluorescent marker to prepare a fluorescent labeled collagenase injection solution;

[0015] Inject fluorescently labeled collagenase injection into the subcutaneous fat layer of skin tissue;

[0016] Using a fluorescence imaging system, performing fluorescence imaging of the skin tissue at several time points after administration;

[0017] Based on the fluorescence imaging, the diffusion radius, diameter, circumference or area of ​​collagenase in the subcutaneous tissue at each time point was measured.

[0018] In one embodiment of the method for evaluating the diffusion range of collagenase of the present invention, the fluorescent marker is a cyanine dye.

[0019] In one embodiment of the method for evaluating the diffusion range of collagenase of the present invention, the cyanine dye is a CY7 type cyanine dye.

[0020] In one embodiment of the method for evaluating the diffusion range of collagenase of the present invention, the CY7-type cyanine dye is selected from one or more of CY7-COOH, CY7-NH2, CY7-NHS, and CY7-MAL.

[0021] In one embodiment of the method for evaluating the diffusion range of collagenase of the present invention, the reaction of labeling collagenase with a fluorescent marker is carried out at a temperature of 2-8° C. and a pH of 7.4±0.5.

[0022] In one embodiment of the method for evaluating the diffusion range of collagenase of the present invention, the skin tissue is selected from ex vivo skin of an animal or artificial bionic skin; and / or

[0023] The collagenase is selected from collagenase (type I, II, III, IV collagenase) extracted from Clostridium histolyticum or wild-type or mutant collagenase expressed by genetic engineering. It is also applicable to proteases from other sources that can degrade natural collagen fibers with triple-stranded superhelical structures and extracellular matrix ECM.

[0024] In one embodiment of the method for evaluating the diffusion range of collagenase of the present invention, the time point is 30 minutes, 24 hours, 48 ​​hours, or the 5th day.

[0025] In one embodiment of the method for evaluating the diffusion range of collagenase of the present invention, the dosage of the fluorescently labeled collagenase is 0.01 mg / dose or more.

[0026] In one embodiment of the method for evaluating the diffusion range of collagenase of the present invention, the excitation wavelength of the fluorescence imaging is greater than 700 nm, and more preferably the second near-infrared region, ie, a wavelength of 1000-1700 nm.

[0027] The present invention also provides a kit for evaluating the diffusion range of injectable collagenase, comprising: a fluorescent marker and skin tissue, wherein the fluorescent marker is a CY7 fluorescent dye; and / or

[0028] The skin tissue is selected from ex vivo skin of an animal or artificial bionic skin; and / or

[0029] The collagenase is selected from type I, II, III, IV and V collagenases extracted from Clostridium histolyticum, or wild-type or mutant collagenases expressed by genetic engineering, or proteases that can degrade natural collagen fibers with triple superhelical structures and extracellular matrix ECM.

[0030] Beneficial technical effects

[0031] The present invention is the first to use long-wave fluorescent probes and in vivo imaging technology to visualize the local action range of injectable collagenase, and intuitively obtain the action range of different active doses of collagenase after injection. This fills the technical gap that the existing technology cannot directly obtain the diffusion radius of injectable collagenase after subcutaneous injection and cannot evaluate the action range of collagenase in the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 : Schematic diagram of the subcutaneous injection point of collagenase in clinical treatment.

[0033] Figure 2 : Figures showing the results of fluorescence detection after subcutaneous injection of unlabeled collagenase and CY7-labeled collagenase into SD rats.

[0034] Figure 3 : A graph showing the fluorescence detection results 24 hours after subcutaneous injection of different doses of CY7-labeled collagenase in Bama miniature pigs in vitro.

[0035] Figure 4 : A graph showing the fluorescence detection results of Bama miniature pigs on the 7th day after subcutaneous injection of different doses of CY7-labeled collagenase.

[0036] Figure 5 : Shows the statistical analysis results of the frontal fluorescence diffusion area using the software of the fluorescence imaging system 24 hours and 7 days after subcutaneous injection of different doses of CY7-labeled collagenase in Bama miniature pigs. DETAILED DESCRIPTION

[0037] It is to be understood that as used herein, the singular forms "a," "an," "an," and "the" include plural forms unless the context clearly dictates otherwise.

[0038] The terms "above", "below", "within", and "at least" herein include the present number unless the context clearly dictates otherwise.

[0039] When the embodiments are described herein as "comprising", "containing", or "including", other similar embodiments described as "consisting of" and / or "consisting essentially of" are also provided. It should also be understood that when the embodiments are described herein as "consisting essentially of", other similar embodiments described as "consisting of" are also provided.

[0040] The term "and / or" as used herein in phrases such as "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0041] The "±" used in phrases such as "A±B" herein means all values ​​ranging from the value of AB to the value of A+B. For example, 7.4±0.5 means all values ​​from 6.9-7.9 and values ​​within the range of 6.9-7.9 after rounding.

[0042] Herein, "collagenase" and "collagenase protein" have the same meaning and are sometimes used interchangeably herein.

[0043] Fluorescence bioimaging technology

[0044] Fluorescence bioimaging is an advanced technology that uses fluorescent markers to observe and analyze biological samples. It has a wide range of applications in biomedical research, disease diagnosis, and drug development. Near-infrared fluorescence imaging is a widely used bioimaging technology that uses light in the near-infrared region to reduce the absorption and scattering of light by biological tissues, thereby achieving deep tissue imaging. It has the characteristics of deep tissue penetration, low background fluorescence interference, and minimal light damage to biological samples.

[0045] Method for determining the range of action of collagenase

[0046] The invention evaluates the action range of collagenase injected into human skin tissue by measuring the diffusion radius, diameter, perimeter or area of ​​fluorescently labeled collagenase injected into skin tissue of living or ex vivo animals.

[0047] The method for measuring the diffusion range of collagenase of the present invention comprises the following steps:

[0048] Labeling collagenase with a fluorescent marker to prepare a fluorescent labeled collagenase injection solution;

[0049] Inject fluorescently labeled collagenase injection into the subcutaneous fat layer of skin tissue;

[0050] Using a fluorescence imaging system, performing fluorescence imaging of the skin tissue at several time points after administration;

[0051] According to the fluorescence range, the diffusion radius, diameter, circumference or area of ​​collagenase under the skin at each time point was measured;

[0052] The maximum diffusion radius was determined as the diffusion radius of collagenase.

[0053] Choice of fluorescent marker

[0054] The fluorescent marker in the present invention is not particularly limited, as long as it can be fluorescently imaged under an imaging system, for example, near-infrared fluorescent dyes can be used. Near-infrared fluorescent dyes mainly include: cyanine dyes, rhodamines, squaric acids, porphyrins, etc. Among them, cyanine dyes (also called "polymethine cyanine dyes") are a class of conjugated organic small molecule systems composed of an odd number of carbon atoms to form a resonant methine (methine) conjugated chain and terminated by two nitrogen-containing heterocyclic rings. Cyanine dyes (Cyan i ne) can be exemplified by CY3, CY3.5, CY5, CY5.5, CY7, IR783, IR820, BOD IPY. The wavelength of CY7 is longer than that of CY3 and CY5, closer to the near-infrared region, and has less interference with fluorescence imaging. CY7 cyanine dyes have a variety of derivative forms, including CY7-carboxyl (CY7-COOH), CY7-amino (CY7-NH2), CY7-active ester (CY7-NHS) and CY7-maleimide (CY7-MAL), which can be used in the present invention. Among them, CY7-active ester has good water solubility and better stability, higher stability to light and heat, and can be stored for a long time and maintain its fluorescence properties. Therefore, CY7-active ester is most preferred.

[0055] Excitation wavelength for fluorescence imaging

[0056] In the present invention, the wavelength range of the imaging window of fluorescence imaging is not particularly limited. One embodiment of the present invention shows that compared with the visible light (360-760nm) and near infrared region I (760-900nm) windows, the tissue light absorption in the near infrared region II (900-1880nm, NI RI I) window is significantly improved, which significantly suppresses the scattering background in two-dimensional array imaging, thereby improving the imaging effect. Therefore, the excitation light wavelength in the present invention is preferably near infrared region II (900-1880nm, NI RI I), which has a deeper tissue penetration depth and smaller energy attenuation than near infrared region I excitation light.

[0057] Fluorescence labeling reaction

[0058] In the present invention, the fluorescent labeling reaction includes mixing reactants, adjusting pH, controlling reaction time and temperature, and purification and detection. In the present invention, the reaction temperature is preferably 2-8°C, the reaction pH is preferably 7.4±0.5, and it is preferably carried out under light-proof conditions. The reaction time is not particularly limited, and the binding of the marker to the collagenase can usually be completed in about 1 hour. After the labeling is completed, it is preferably removed by an appropriate purification method to remove unreacted dyes and impurities, and the fluorescent properties and purity of the labeled product are detected. The purification method is not particularly limited, and the collagenase protein purification method commonly used in the art can be used, for example, dialysis, gel filtration chromatography, ion exchange chromatography, affinity chromatography, molecular sieve purification, etc.

[0059] Collagenase activity assay

[0060] Among the methods for detecting the activity of collagenase, commonly used methods include suspension method, chemical analysis method, colorimetry, spectrophotometry, ninhydrin colorimetry, electrophoresis, FALGPA method, zymography, immunological method, etc. The present invention is not particularly limited and can be selected according to the situation of method development. The present invention uses ninhydrin colorimetry and FALGPA method.

[0061] Dosage of fluorescently labeled collagenase

[0062] The fluorescence intensity shows a positive dose-effect relationship with the dosage of the fluorescently labeled collagenase. One embodiment of the present invention shows that 0.01 mg / dose can clearly display fluorescence. Therefore, in the measurement method of the present invention, the dosage of the fluorescently labeled collagenase is not particularly limited, as long as it can be imaged in a fluorescent imaging system after being labeled.

[0063] Skin tissue

[0064] The present invention is applicable to skin tissue of live animals or isolated animals, and artificial bionic skin tissue. The animals are not particularly limited, and are preferably warm-blooded animals, such as dogs, cats, mice, rats, guinea pigs, rabbits, cows, horses, sheep, goats, pigs, dogs, etc. From the perspective of animal protection, artificial bionic skin tissue is preferred.

[0065] Fluorescence imaging time points

[0066] In some embodiments of the present invention, the selection of the time point is not necessarily limited to 30 minutes, 24h, 48h and the 5th day, and can be adjusted according to actual conditions. According to embodiments of the present invention, the diffusion area of ​​CY7 fluorescently labeled collagenase in subcutaneous fat changes with time and presents a parabolic correlation, which increases with time in the first 24 hours to 48 hours, and shows a weakening trend in the later period (e.g., the 4th and 5th days) due to the attenuation of fluorescein or the metabolism of the drug. Therefore, in order to shorten the cycle and simplify the procedure, the time point of fluorescence imaging is selected at 30 minutes, 24h, 48h and the 5th day.

[0067] Collagenase for injection

[0068] The collagenase of the present invention can be crude collagenase extracted from Clostridium histolyticum (containing collagenase I, II,

[0069] Type III, IV and V collagenase), or wild-type or mutant collagenase expressed by genetic engineering. Also applicable to other sources, it can degrade natural collagen fibers with triple-stranded superhelical structure and proteases of extracellular matrix ECM.

[0070] Example

[0071] The present invention is described in detail below with reference to the following examples. The examples provide detailed implementation methods and processes to fully disclose and demonstrate that the invention can be implemented. The examples do not represent limitations on the invention, and the protection scope of the present invention is not limited to the following examples.

[0072] The instruments, chemicals, biological products, reagents, materials, experimental animals, etc. whose sources are not specified in the examples of the specification are purchased through general commercial channels. The experimental methods without specific conditions in the examples of the specification are usually carried out according to conventional experimental conditions in the field or according to the conditions recommended by the manufacturer.

[0073] Example 1: Fluorescence labeling of collagenase protein

[0074] Selection of marker: In this example, Cyanine 7NHS ester (CY7-NHS ester for short) of the CY7-active ester (CY7-NHS) class was selected as a fluorescent marker. CY7-NHS ester has an N-hydroxysuccinimide (NHS) ester functional group.

[0075] Weigh 1.0 mg of CY7 NHS ester and dissolve it in 180 μL DMSO (dimethyl sulfoxide) to a 5.5 mg / mL solution. Take 65 μL of the prepared DMSO solution of CY7 NHS ester and slowly add it to the collagenase protein solution. Rotate the reaction solution at 4°C in the dark to react for 1 hour. After the reaction is completed, dialyze it with 1×PBS at 4°C overnight. The next day, replace it with new 1×PBS and continue dialysis for 5 hours. After the dialysis is completed, collect and aliquot it, store it at 4°C in the dark, and obtain the collagenase protein fluorescently labeled with CY7 NHS ester (hereinafter sometimes collectively referred to as "CY7 labeled collagenase").

[0076] Example 2: Detection of collagenase activity after fluorescence labeling

[0077] Biological activity: Detected by ninhydrin colorimetric method, the biological activity of the fluorescently labeled collagenase sample in Example 1 was 114 U per 1 mg of protein.

[0078] Biochemical activity: Detected by the FALGPA method, the biochemical activity of the fluorescently labeled collagenase sample in Example 1 was 0.7 U per 1 mg of protein.

[0079] The results showed that the collagenase labeled with CY7 fluorescence still maintained its biological and biochemical activity.

[0080] Example 3: Fluorescence intensity and action radius of CY7-labeled collagenase in vivo after subcutaneous injection in SD rats

[0081] SD rats were injected with 0.04 and 0.15 mg / dose of CY7-labeled collagenase samples on the right upper back and right lower back, respectively, and injected with 0.04 and 0.15 mg / dose of unlabeled collagenase for injection (blank control) on the left upper back and left lower back, respectively. Fluorescence imaging of the backs of SD rats was performed at 30 minutes, 24 hours, 48 ​​hours, the 5th day and the 9th day after administration, and the fluorescence intensity and diffusion area were calculated.

[0082] Figure 2 : is a graph showing the fluorescence imaging results of SD rats injected with 0.04 and 0.15 mg / dose of unlabeled collagenase and 0.04 and 0.15 mg / dose of CY7-labeled collagenase, respectively. Figure 2 As shown in the figure, no fluorescence signal was observed on the back of SD rats injected with unlabeled collagenase (blank control group) at 30 minutes, 24 hours, 48 ​​hours, 5 days and 9 days after administration, while obvious fluorescence signal was observed at two doses of 0.04 and 0.15 mg / dose CY7-labeled collagenase. The signal showed a dose-effect relationship, and a time-effect relationship trend was observed at 24 hours, 48 ​​hours, 5 days and 9 days.

[0083] Table 1 is a summary of the fluorescence diffusion area and diffusion radius at different time points after subcutaneous injection of different doses of CY7-labeled collagenase in SD rats. As shown in Table 1, the diffusion area of ​​the experimental group of CY7-labeled collagenase (0.04 mg / dose) at 30 minutes, 24 hours, 48 ​​hours, 5 days and 9 days after administration was 1.84 cm 2 、3.34cm 2 、3.76cm 2 、1.32cm 2 、0.68cm 2 The diffusion areas of the experimental group with 0.15 mg / dose were 3.65 cm at 30 min, 24 h, 48 h, 5 days and 9 days, respectively. 2 、10.87cm 2 、11.57cm 2 、7.21cm 2 、5.24cm 2 From the diffusion area at each time point, it can be seen that the diffusion area of ​​the 0.15mg / dose experimental group is higher than that of the 0.04mg / dose experimental group. The diffusion area of ​​the two dosages shows a parabolic correlation with time. It expands with the increase of time in the early stage, and then weakens due to the attenuation of fluorescein or the metabolism of the drug. The diffusion area of ​​the 0.04mg / dose experimental group and the 0.15mg / dose experimental group reached a peak at 48h after administration, which was 3.76cm 2 and 11.57cm 2 , and the corresponding diffusion radii are 1.09cm and 1.92cm respectively.

[0084] Table 1: Summary of fluorescence diffusion area and diffusion radius at different time points after subcutaneous injection in SD rats

[0085]

[0086] *Diffusion radius estimation method: The drug fluorescence diffusion area is calculated using the circle area calculation formula S = πr 2 Perform calculations.

[0087] Example 4: Fluorescence intensity and action radius of CY7-labeled collagenase after subcutaneous injection in Bama miniature pigs

[0088] Remove the pig skin from the back of the Bama miniature pig, rinse the pig skin with hot water to remove dirt, scrape off the hair roots, and then cut it into pieces of appropriate size and place it at a temperature of 2-8°C for later use.

[0089] Bama miniature pigs were subcutaneously injected with 0.04, 0.08, 0.10, and 0.15 mg / dose of CY7-labeled collagenase, and each dose was injected at two points. The fluorescence intensity and diffusion radius of each dose group were observed 24 hours and 7 days after administration. The same volume of 0.15 mg / dose of unlabeled collagenase was injected as a blank control group.

[0090] The results of the positive fluorescence test 24 hours after administration are as follows Figure 3 The results showed that the fluorescence intensity and diffusion area of ​​the CY7-labeled collagenase 0.15 mg / dose experimental group were generally higher than those of the other dose groups. The frontal fluorescence detection results on the 7th day after administration were as follows Figure 4 The results showed that the fluorescence intensity and diffusion area of ​​0.15 mg / dose were also higher than those of other dose groups. Based on the front fluorescence detection results at 24 hours and 7 days after administration, the fluorescence intensity and diffusion area of ​​each dose group at 24 hours after administration were stronger than those at 7 days after administration.

[0091] The statistical analysis of the positive fluorescence diffusion area was performed using the system software of PerkinElmer's fluorescence imaging system (IVIS LuminaⅢ). The results are shown in the figure. Figure 5 As shown in Table 2. At the same dosage, the fluorescence diffusion area on the 7th day of administration was smaller than that at 24 h after administration. At 24 h after administration, when the dosage of CY7-labeled collagenase was 0.04, 0.08, 0.10, and 0.15 mg / dose, the fluorescence diffusion areas were 4.5, 4.2, 6.5, and 6.9 cm, respectively. 2 When the dose was 0.04 mg / dose, the fluorescence diffusion area was smaller than that of other dose groups, but the overall fluorescence diffusion area showed an upward trend with the increase of the dose; on the 7th day of administration, when the dose of CY7-labeled collagenase was 0.04, 0.08, 0.10, and 0.15 mg / dose, the fluorescence diffusion areas were 0.7, 0.9, 1.4, and 2.9 cm, respectively. 2 ,The results showed that with the increase of the dosage, the fluorescence diffusion area gradually increased.

[0092] Table 2: The radius of action corresponding to the drug diffusion area after subcutaneous injection in Bama miniature pigs

[0093]

[0094]

[0095] *: Diffusion radius estimation method: Drug fluorescence diffusion area is calculated using the circle area calculation formula S = πr 2 Perform calculations.

[0096] It should be noted that the present invention is not limited to the above implementation modes and examples. The above implementation modes and examples are only examples, and implementation modes that have substantially the same structure as the technical idea and exert the same effect within the scope of the technical solution of the present application are all included in the technical scope of the present application. In addition, within the scope of the subject matter of the present application, various modifications and substitutions of the implementation modes / examples that can be thought of by those skilled in the art, and other methods formed by combining some of the constituent elements in the implementation modes are also included in the scope of the present application, and the scope of protection of the present invention is defined by the attached claims.

Claims

1. A method for evaluating the diffusion range of collagenase, characterized in that: The steps include: Labeling collagenase with a fluorescent marker to prepare a fluorescent labeled collagenase injection solution; Inject fluorescently labeled collagenase injection into the subcutaneous fat layer of skin tissue; Using a fluorescence imaging system, performing fluorescence imaging of the skin tissue at several time points after administration; Based on the fluorescence imaging, the diffusion radius, diameter, circumference or area of ​​collagenase in the subcutaneous tissue at each time point was measured.

2. The method for evaluating the diffusion range of collagenase according to claim 1, wherein: The fluorescent marker is a cyanine dye.

3. The method for evaluating the diffusion range of collagenase according to claim 2, wherein: The cyanine dye is a CY7 type cyanine dye.

4. The method for evaluating the diffusion range of collagenase according to claim 3, wherein: The CY7 cyanine dye is selected from one or more of CY7-COOH, CY7-NH2, CY7-NHS, and CY7-MAL.

5. The method for evaluating the diffusion range of collagenase according to claim 1, wherein the reaction of labeling collagenase with a fluorescent marker is carried out at a temperature of 2-8°C and a pH of 7.4±0.

5.

6. The method for evaluating the diffusion range of collagenase according to claim 1, wherein the skin tissue is selected from ex vivo skin of an animal or artificial bionic skin; and / or The collagenase is selected from type I, II, III, IV collagenase extracted from Clostridium histolyticum or wild-type or mutant collagenase expressed by genetic engineering, or a protease that can degrade natural collagen fibers with triple-stranded superhelical structures and extracellular matrix ECM.

7. The method for evaluating the diffusion range of collagenase according to claim 1, wherein: The time points are 30 minutes, 24 hours, 48 ​​hours, and the 5th day.

8. The method for evaluating the diffusion range of collagenase according to claim 1, wherein: The dosage of the fluorescently labeled collagenase is 0.01 mg / dose or more.

9. The method for evaluating the diffusion range of collagenase according to claim 1, wherein the excitation wavelength of the fluorescence imaging is greater than 700 nm.

10. A kit for evaluating the diffusion range of collagenase for injection, comprising: A fluorescent marker and skin tissue, wherein the fluorescent marker is a CY7 cyanine dye; and / or The skin tissue is selected from ex vivo skin of an animal or artificial bionic skin; and / or The collagenase is selected from type I, II, III, and IV collagenase extracted from Clostridium histolyticum, or wild-type or mutant collagenase expressed by genetic engineering, or a protease that can degrade natural collagen fibers with triple superhelical structures and extracellular matrix ECM.

Images