CN114681595A - Application of hyaluronidase in preparation of medicine for improving blood circulation of any skin flap - Google Patents
Application of hyaluronidase in preparation of medicine for improving blood circulation of any skin flap Download PDFInfo
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- CN114681595A CN114681595A CN202210412740.0A CN202210412740A CN114681595A CN 114681595 A CN114681595 A CN 114681595A CN 202210412740 A CN202210412740 A CN 202210412740A CN 114681595 A CN114681595 A CN 114681595A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/08—Solutions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01035—Hyaluronoglucosaminidase (3.2.1.35), i.e. hyaluronidase
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Abstract
The invention relates to application of hyaluronidase in preparing a medicament for improving blood circulation of any flap. The invention discovers that the hyaluronidase can obviously improve the survival rate of any flap, reduce the area of a non-perfusion area of the blood flow of the flap, reduce the inflammatory expression of related factors in the flap, promote the formation of microvessels in any flap and reduce the formation of fibrosis in flap tissues, and shows that the hyaluronidase has wide clinical application prospect as a blood circulation improving agent of any flap.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to a medicine for improving blood circulation of any flap.
Background
Hyaluronic acid, also called hyaluronic acid, is a glycosaminoglycan naturally occurring in the body of an organism. Hyaluronic acid is a disaccharide unit formed by connecting D-glucuronic acid and N-acetyl-D-glucosamine by beta-1, 3 glycosidic bonds, and the disaccharide unit is connected by beta-1, 4 glycosidic bonds to form polysaccharide. Hyaluronic acid is widely present in animal connective tissues, such as joints, cartilage, skin, vitreous, umbilical cord, synovial fluid, cockscomb, wharton's jelly, and group a and C hemolytic streptococci, and has important functions of providing toughness to animal organisms, serving as a soft tissue support structure, regulating cellular metabolism, and the like.
Hyaluronic acid is generally a colorless, odorless, amorphous solid that is very soluble in water and insoluble in organic solvents. The hyaluronic acid molecule is in a rigid spiral column shape in spatial structure, and hydroxyl groups on the inner side of the column are continuously and directionally arranged, so that the hyaluronic acid molecule has both hydrophilic and hydrophobic properties, and can form a three-dimensional network structure when the concentration of the hyaluronic acid molecule is very low. Therefore, hyaluronic acid has super-strong hygroscopicity, water retention property and viscoelasticity, can be compatible with water 1000 times of the mass of hyaluronic acid, is an excellent natural moisturizing factor, and is commonly used in the fields of cosmetic ingredients, clinical surgery anti-adhesion protective agents and the like.
Hyaluronic acid can be classified into 3 types according to its molecular weight: high molecular weight (5X 10)3-2×108kD), low molecular weight (10-50kD) and oligomeric hyaluronic acid (below 10 kD). The biological activity of hyaluronic acid is closely related to the molecular weight, high molecular weight hyaluronic acid has stronger water-retaining property but lacks physiological activity, low molecular weight hyaluronic acid has the physiological functions of promoting bone and angiogenesis, promoting wound healing, enhancing immunoregulation and the like, and the hyaluronidase hydrolysis method is an important method for preparing low molecular weight hyaluronic acid with biological activity.
Hyaluronidase is a multifunctional endoglycosidase that improves vascularity, reduces connective tissue interstitial pressure, regulates inflammatory expression levels, and destroys normal extracellular matrix structures. Initially, FDA approval of hyaluronidase as an adjuvant increased the absorption and diffusion of other injected drugs. When the hyaluronidase is combined with the local anesthetic, the onset time of anesthesia can be accelerated, and the swelling caused by local infiltration can be reduced; in addition, the diffusion and distribution of local anesthetic in the body can be promoted, the absorption of the local anesthetic in the body is increased, and the probability of occurrence of systemic reaction is increased.
In the exposed parts of bones, blood vessels, nerves and tendons, the problems of lack of blood supply, difficult survival, incapability of filling dead cavities, poor anti-infection capability and incapability of reconstructing important tissue functions exist in skin grafting repair, and the skin soft tissue defects are covered by often cutting any overlength skin flap around. However, the cutting of such skin flap is strictly limited by the length-width ratio, and generally can not exceed 1.5-2: 1, even in the head and neck with abundant blood circulation, the length-width ratio cannot exceed 3-4: 1, cutting an excessively long flap often causes more severe necrosis at the distal end of the flap to varying degrees, and it remains a challenge for the clinician to reduce any distal necrosis of the flap and to ensure more blood flow. There is therefore a need for a method or medicament which is effective in improving blood flow in any flap.
No report on the use of hyaluronidase for improving blood transport in any skin flap has been retrieved.
Disclosure of Invention
In order to solve the above technical problems, the present invention includes the following aspects:
a first aspect of the invention provides a formulation for improving blood flow in any flap, the formulation comprising hyaluronidase and a pharmaceutically acceptable excipient.
Preferably, the pharmaceutically acceptable auxiliary material is distilled water.
Preferably, the concentration of hyaluronidase in the formulation is 500-2000 IU/ml.
More preferably, the concentration of hyaluronidase in the formulation is 1500 IU/ml.
Preferably, the unit dose of hyaluronidase in the formulation is 400-800 IU.
More preferably, the unit dose of hyaluronidase in the formulation is 600 IU.
Preferably, the formulation is a liquid formulation.
A second aspect of the invention provides a method of improving blood flow in any skin flap, the method comprising subcutaneously injecting a formulation comprising hyaluronidase into any skin flap site.
In a third aspect, the invention provides the use of hyaluronidase in the manufacture of a medicament for improving blood flow in any flap.
Preferably, the concentration of hyaluronidase in the drug is 500-2000 IU/ml.
More preferably, the concentration of hyaluronidase in the medicament is 1500 IU/ml.
Preferably, the unit dose of hyaluronidase in the drug is 400-800 IU.
More preferably, the unit dose of hyaluronidase in the medicament is 600 IU.
In a fourth aspect, the invention provides the use of hyaluronidase in the manufacture of a medicament having one or more of the following functions: (1) increasing the survival rate of any flap, (2) decreasing the area of the non-perfused area of blood flow in the flap, (3) decreasing the inflammatory expression of the relevant factor in the flap, (4) promoting the formation of microvessels in any flap, and (5) decreasing the formation of fibrosis in the tissue of the flap.
Preferably, the concentration of hyaluronidase in the drug is 500-2000 IU/ml.
More preferably, the concentration of hyaluronidase in the medicament is 1500 IU/ml.
Preferably, the unit dose of hyaluronidase in the drug is 400-800 IU.
More preferably, the unit dose of hyaluronidase in the medicament is 600 IU.
Preferably, the related factors are IL-6 and TNF-alpha.
Preferably, the medicament has the following five functions simultaneously: (1) increasing the survival rate of any flap, (2) decreasing the area of the non-perfused area of blood flow in the flap, (3) decreasing the inflammatory expression of the relevant factor in the flap, (4) promoting the formation of microvessels in any flap, and (5) decreasing the formation of fibrosis in the tissue of the flap.
The invention has the following beneficial effects:
the invention unexpectedly discovers that the hyaluronidase can obviously improve the survival rate of any skin flap, reduce the area of a non-perfusion area of the blood flow of the skin flap, reduce the inflammatory expression of related factors in the skin flap, promote the formation of microvessels in any skin flap, reduce the formation of fibrosis in the tissue of the skin flap, and indicate that the hyaluronidase has wide clinical application prospect as a blood circulation improving agent of any skin flap. In addition, the optimal concentration (1500IU/ml) and the optimal unit dose (600IU) of the hyaluronidase are confirmed by optimizing the prescription of the preparation, the performance indexes related to any skin flap are obviously improved, and the treatment effect of the hyaluronidase is further improved.
Drawings
FIG. 1 is a comparison graph of the ratio of the skin flap survival area of rats in a control group to the skin flap survival area of rats in a test group;
FIG. 2 is a chart comparing the areas of skin flaps in non-perfused areas of blood flow in ICG images of rats in a control group and a test group;
FIG. 3 is a graph comparing the IL-6 content in the skin flap tissues of rats in the control group and the test group;
FIG. 4 is a graph comparing TNF- α levels in rat flap tissues of control and test groups;
FIG. 5 is a graph comparing the random blood vessel counts in the skin flap tissues of rats in the control group and the test group;
FIG. 6 is a graph showing a comparison of the degree of fibrosis in the skin flap tissues of rats in the control group and the test group.
Detailed Description
Test example 1 Effect of Hyaluronidase on blood transport in any skin flap of SD rat
First, test method
1. Grouping and design of experiments
A total of 24 SD rats were used in the experiment and randomly assigned to the experimental group (group a, 12) and the control group (group B, 12). Two groups of rats are all made into any skin flap models, 600IU of hyaluronidase (the concentration is 1500IU/ml) is injected into the rats in the group A every day after 1 week of operation, physiological saline with the same volume is injected into the rats in the group B after the operation, and the survival rate of any skin flap of the rats, the area of a blood flow perfusion area, the tissue inflammation level and the pathological results of tissues are recorded and analyzed after 1 week of operation.
2. Animal model
Any skin flap model in rats was used with a modified McFarlane skin flap: after anesthesia, the back area of the rat is subjected to depilation treatment, after disinfection, a skin flap of 9cmx3cm is designed along the central axis of the back, and the skin flap base is positioned on the dorsal iliac crest connecting line of the tail end of the rat. Incising skin, separating subcutaneous tissue to superficial layer of deep fascia, performing electrocoagulation or ligation during bleeding to protect subdermal capillary vessel net, after completely lifting skin flap, identifying bilateral symmetrical iliac artery at basal side, performing electrocoagulation treatment, and suturing skin flap in situ with 4-0 silk thread.
3. Drug injection and tissue harvesting
Any skin flap on the back of the rat was divided into 3 sections: the head side to the tail side is divided into 3 regions, each of which is 3cmx3cm, region i, region ii and region iii. Each zone was divided into two subregions according to the midline, and the solution was injected uniformly at the midpoint of these 6 subregions. When the tissue was excised post-operatively, tissues adjacent to each subregion, approximately 1cmx0.5cm in area, were collected along the left side of the midline for ELISA analysis of inflammatory expression levels and the right side for pathological analysis of the tissues.
4. Gross observation of skin flaps
By means of shooting, the skin pictures of the target area of each rat are recorded for 7 days before and after operation, and reference objects such as a ruler and the like can be placed on one side of each rat for analyzing the survival area ratio of the skin flap on the back of each rat by using ImageJ software after the operation.
5. Skin flap near-infrared fluorescence imaging observation
After 7 days of operation, anesthetizing the rat, performing unhairing treatment on an observation area, placing the treated rat on a fluorescence imaging table, injecting 10 mu L of indocyanine green through tail vein, and recording or photographing brightness change of a target flap area in real time under near-infrared fluorescence so as to divide a perfusion area and a non-attention area of blood flow in the flap area.
6. Analysis of inflammation levels in flap tissue
Tissues collected on the left side of the midline were analyzed for inflammation levels by ELISA 7 days post-surgery. Rinsing the tissue block in ice-cold physiological saline, removing blood filter paper, wiping to remove attached fascia and hair tissue, cutting the large tissue block with ophthalmic scissors, homogenizing, and performing in ice-water bath. When in tissue homogenization, the ratio of the tissue quality to the homogenization medium is 1: 10, the homogenate medium is 0.86% of cold normal saline, and is ground into 10% of tissue homogenate liquid by a tissue crusher at 10000r/min, so that the grinding time of skin and muscle tissues can be prolonged. And centrifuging the prepared homogenate for 5 minutes in a centrifugal machine with the speed of 5000r/min, and reserving supernatant for detection. All standards in the kit were first diluted in the desired concentration gradient. Mu.l of the diluted standard sample is added into the test wells in sequence, and 20. mu.l of the protein sample and 20. mu.l of the reagent diluent are added into each residual well. Subsequently, 50. mu.l of biotinylated antibody working solution was added to each standard and sample well, and the reaction well was sealed with a sealing plate of gummed paper. After 2h incubation at room temperature, the plates were washed 5 times. Then 100 mul of the diluted working solution of the enzyme conjugate is added into each reaction hole, and the reaction holes are sealed by sealing plate gummed paper. Incubate with shaker at room temperature for 1h, and repeat washing the plate 5 times. Add chromogenic substrate 100 u l/hole, room temperature light protection incubation 10-15 minutes. Adding 100 μ l/well of stop solution, mixing, performing dual-wavelength detection with standard instrument within 30 min, measuring the maximum absorption wavelength at 450nm and OD value at 570mm or 630mm reference wavelength, and subtracting the measured value at 570nm or 630nm from the measured value at 450 nm.
7. Histological analysis
Tissues harvested on the right side of the midline were used for histopathological evaluation 7 days post-operatively. Microvessel density was observed using CD31 staining. The tissue slide was antigen-labeled specifically with the endothelial cell marker CD31, and from the 10-fold magnified image, the number of blood vessels (sum) was calculated in three randomly different regions per section.
The degree of fibrosis of the flap tissue was observed by masson trichrome staining. The tissue slides were washed at least twice with xylene for about 10 minutes, then with deionized water to remove paraffin, and then the deparaffinized sections were stained in weibert's iron hematoxylin solution for 10 fractions and washed in warm water for 5 minutes. The sections were then stained in Biebrich scarlet acid fuchsin solution for 15 minutes and briefly rinsed in deionized water. The slides were then placed in a 1% phosphomolybdic acid solution for 10 minutes, transferred to an aniline blue solution for 5 minutes, and a 1% acetic acid solution for 1 minute. The samples were finally dehydrated with 95% and 100% alcohol and clarified in xylene before installation. From the magnified 10-fold image (model BX 40; olympus, tokyo, japan), 3 different stained areas in each section were randomly selected. The extent of fibrosis was quantified by measuring the fiber area using Image-J software (NIH, besistar, maryland).
8. Statistical analysis
Statistical analyses were performed using GraphPadPrism9 software in unison and data were expressed as means ± standard deviation. Within and between groups were compared using one-way anova analysis of variance, with p <0.05 indicating that the difference was statistically significant.
Second, test results
1. Ratio of flap survival area
The test results show that the ratio of the survival area of the skin flap of the control group to that of the experimental group is (76.83 +/-7.89)% and (54.25 +/-6.42)%, the difference of each experimental group has statistical significance (p is less than 0.0001, see figure 1), and the result shows that the survival area of the skin flap of the rat of the experimental group is larger than that of the rat of the control group after the hyaluronidase is injected into the skin flap of the rat of the experimental group, and the hyaluronidase is proved to have obvious improvement on the survival of any skin flap.
2. Area of skin flap in non-perfused area of blood flow
The test results show that the ICG imaging of the control group and the experimental group has the skin flap area of the blood flow non-perfusion area of (11.31 +/-4.55) cm2And (5.72. + -. 3.46) cm2The differences between the test groups are statistically significant (p)<0.0001, see fig. 2), indicating that the area of the non-perfusion area of the blood flow of the skin flap under the ICG development is significantly smaller than that of the control group rats after the skin flap injection of the experimental group rats, and the hyaluronidase is proved to have significant improvement on the perfusion of any skin flap.
3. Level of tissue inflammation (IL-6/TNF-alpha)
The test results show that the IL-6 content in the skin flap tissues of the control group and the experimental group is 705.7 +/-32.44 pg/ml and 377.9 +/-45.25 pg/ml respectively, the difference of each test group is statistically significant (p <0.0001, see figure 3), the TNF-alpha content in the skin flap tissues of the control group and the experimental group is 1386 +/-145.85 pg/ml and 949.4 +/-223.62 pg/ml respectively, the difference of each test group is statistically significant (p <0.0001, see figure 4), the inflammation level of the skin flap tissues of the experimental group rats after the skin flaps are injected with hyaluronidase is obviously lower than that of the control group rats, and the hyaluronidase is proved to be capable of obviously reducing the inflammation expression of related factors in the skin flaps.
4. Density of microvessels
The test results show that the random blood vessel counts in the skin flap tissues of the control group and the experimental group are respectively 16.75 +/-6.83 and 32.75 +/-10.35, the difference of each experimental group has statistical significance (p is less than 0.0001, see figure 5), the result shows that after the skin flap of the rat in the experimental group is injected with hyaluronidase, the density of the microvasculature in the skin flap tissue is obviously higher than that of the control group, and the hyaluronidase is proved to be capable of obviously promoting the formation of the microvasculature in any skin flap.
5. Degree of fibrosis
The test results show that the ratio of the areas of fibrosis in the skin flap tissues of the control group and the experimental group is (42.49 +/-8.52)% and (26.59 +/-10.45)%, the difference of each experimental group has statistical significance (p is less than 0.0001, see figure 6), the result shows that after hyaluronidase is injected into the skin flap of the rat in the experimental group, the degree of fibrosis in the skin flap tissues is less than that of the control group, and the hyaluronidase is proved to be capable of remarkably reducing the formation of fibrosis in the skin flap tissues.
In conclusion, the hyaluronidase has a remarkable improvement effect on the blood circulation of any skin flap of a rat, and the hyaluronidase has a wide clinical application prospect as an improvement agent for the blood circulation of any skin flap.
Claims (10)
1. A formulation for improving blood flow in any flap, comprising hyaluronidase and a pharmaceutically acceptable excipient.
2. The formulation of claim 1, wherein the pharmaceutically acceptable excipient is distilled water.
3. The formulation of claim 1, wherein the hyaluronidase in the formulation is at a concentration of 500-2000 IU/ml.
4. The formulation of claim 3, wherein the hyaluronidase in the formulation is at a concentration of 1500 IU/ml.
5. A method of improving blood flow in any skin flap, the method comprising subcutaneously injecting a formulation comprising hyaluronidase into any skin flap site.
6. The application of hyaluronidase in the preparation of drugs for improving the blood circulation of any skin flap is characterized in that the unit dose of the hyaluronidase in the drugs is 400-800 IU.
7. The use according to claim 6, wherein the unit dose of hyaluronidase in the medicament is 600 IU.
8. Use of hyaluronidase in the manufacture of a medicament, wherein the medicament has one or more of the following functions: (1) increasing the survival rate of any flap, (2) decreasing the area of the non-perfused area of blood flow in the flap, (3) decreasing the inflammatory expression of the relevant factor in the flap, (4) promoting the formation of microvessels in any flap, and (5) decreasing the formation of fibrosis in the tissue of the flap.
9. The use of claim 8, wherein the unit dose of hyaluronidase in the medicament is 400-800 IU.
10. The use according to claim 9, wherein the unit dose of hyaluronidase in the medicament is 600 IU.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4820516A (en) * | 1985-02-16 | 1989-04-11 | Biopharm (Uk) Limited | Hyaluronidase |
| US20110184381A1 (en) * | 2007-08-14 | 2011-07-28 | Universitaet Bern | Repair of defects or lesions in cartilage and bone using a chondro-regulative matrix |
| CN105412918A (en) * | 2015-11-18 | 2016-03-23 | 上海交通大学医学院附属第九人民医院 | Drug formula capable of dissolving cross-linked hyaluronic acid thrombosis |
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2022
- 2022-04-19 CN CN202210412740.0A patent/CN114681595A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4820516A (en) * | 1985-02-16 | 1989-04-11 | Biopharm (Uk) Limited | Hyaluronidase |
| US20110184381A1 (en) * | 2007-08-14 | 2011-07-28 | Universitaet Bern | Repair of defects or lesions in cartilage and bone using a chondro-regulative matrix |
| CN105412918A (en) * | 2015-11-18 | 2016-03-23 | 上海交通大学医学院附属第九人民医院 | Drug formula capable of dissolving cross-linked hyaluronic acid thrombosis |
Non-Patent Citations (3)
| Title |
|---|
| NSINGI N ANTÓNIO等: "Viability of randomized skin flaps—an experimental study in rats", JOURNAL OF SURGICAL RESEARCH, vol. 207, pages 92 - 101, XP029846905, DOI: 10.1016/j.jss.2016.08.065 * |
| 上海第一医学院眼耳鼻喉科医院眼科教研组编著: "现代中药药理与临床应用手册", vol. 1, 28 February 1977, 人民卫生出版社, pages: 622 * |
| 侯春林等编: "《皮瓣外科学 第2版》", vol. 2, 31 January 2013, 上海科学技术出版社, pages: 159 - 164 * |
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