CN107303271A - An injection containing small molecule hyaluronic acid and its application - Google Patents

Abstract

The invention discloses a kind of injection containing small molecule hyaluronic acid and its application, the preparation of described injection comprises the following steps：A. using animal cell or yeast or plant expression to produce saccharified recombinant human hyaluronidase PH20；B. using macromolecular hyaluronic acid raw material to be configured to macromolecular hyaluronic acid solution, introduce sodium chloride and magnesium ion, add recombinant human hyaluronidase PH20 to cut, obtain the reaction solution of hyaluronic acid fragment containing molecular weight 10 60KD；C. to residual recombinant human hyaluronidase PH20 inactivation treatment；D. carry out bacterial filtration, virus filtration inactivation and ultrafiltration concentration.Injection of the present invention not only has the effect of facial beauty, facial anti-aging, also unexpectedly shows rapid analgesia, antipruritic, increase physical strength and energy, reduce head and neck subcutaneous fat and to the therapeutic effect of multiple diseases and disease state.

Description

An injection containing small molecule hyaluronic acid and its application

technical field

The invention relates to the field of medicines for treating diseases, in particular to an injection containing small molecule hyaluronic acid and its use for treating various diseases.

Background technique

Hyaluronic acid, also known as hyaluronic acid, uronic acid, hyaluronic acid, English name hyaluronic acid (referred to as HA), is a high-level polysaccharide composed of D-glucuronic acid and N-acetylglucosamine, and the number of disaccharide units can reach 25,000 you. Tissues such as human subcutaneous tissue, epidermis and oropharyngeal mucosa contain a large amount of macromolecular hyaluronic acid. In other words, macromolecular hyaluronic acid is the basic building material of various tissues such as human skin, mucous membranes, and subcutaneous tissue, and has the functions of water retention and moisturizing. The existing hyaluronic acid products in the market are mainly facial care or facial beauty products with the addition of macromolecular hyaluronic acid as the main functional ingredient.

A series of studies in recent years have shown that small molecule hyaluronic acid and macromolecular hyaluronic acid, which are distinguished according to molecular weight, show functional differences in small animal experiments and cell-level studies. The mainstream literature shows that small molecule hyaluronic acid Acid is a degradation product of inflammation, which is consistent with the distribution of inflammation. Its function is to initiate and promote inflammatory responses. The main references include:

[1]. Jaime M. Cyphert, Carol S. Trempus, and Stavros Garantziotis, SizeMatters: Molecular Weight Specificity of Hyaluronan Effects in Cell Biology (Review Article), International Journal of Cell Biology, Volume 2015 (2015), Article ID 563818.

[2].Jiang D1,Liang J,Noble PW.Hyaluronan as an immune regulator inhuman diseases.Physiol Rev.2011Jan;91(1):221-64.PMID:21248167.

[3]. Zgheib C1, Xu J1, Liechty KW1. Targeting Inflammatory Cytokines and Extracellular Matrix Composition to Promote Wound Regeneration. Adv Wound Care (New Rochelle). 2014 Apr 1; 3(4): 344-355. PMID: 24757589.

[4].Voelcker V1,Gebhardt C,Averbeck M,Saalbach A,Wolf V,Weih F,Sleeman J,Anderegg U,Simon J.Hyaluronan fragments induce cytokine and metalloprotease upregulation in human melanoma cells in part by signaling via TLR4.Exp Dermatol. 2008 Feb;17(2):100-7.PMID:18031543.

[5]. Esser PR1, U, Dürr C, von Loewenich FD, Schempp CM, Freudenberg MA, Jakob T, Martin SF. Contact sensitizers induce skin inflammation via ROSproduction and hyaluronic acid degradation. PLoS One. 2012; 7(7): e41340. PMID: 22848468.

[6].Black KE1,Collins SL,Hagan RS,Hamblin MJ,Chan-Li Y,Hallowell RW,Powell JD,Horton MR.Hyaluronan fragments induce IFNβvia a novel TLR4-TRIF-TBK1-IRF3-dependent pathway.J Inflamm( Lond).2013 May 30;10(1):23.PMID:23721397.

[7]. Horton MR1, McKee CM, Bao C, Liao F, Farber JM, Hodge-DuFour J, Puré E, Oliver BL, Wright TM, Noble PW. Hyaluronan fragments synergize with interferon-gamma to induce the C-X-C chemokines mig and interferon-inducible protein-10in mouse macrophages. J Biol Chem. 1998 Dec 25; 273(52): 35088-94. PMID: 9857043.

[8]. Hodge-Dufour J1, Noble PW, Horton MR, Bao C, Wysoka M, Burdick MD, Strieter RM, Trinchieri G, Puré E. Induction of IL-12 and chemokines by hyaluronan requires adhesion-dependent priming of resident but not elicited macrophages. J Immunol. 1997 Sep 1; 159(5): 2492-500. PMID: 9278343.

[9].McKee CM1,Lowenstein CJ,Horton MR,Wu J,Bao C,Chin BY,Choi AM,Noble PW.Hyaluronan fragments induce nitric-oxide synthase in murine macrophages through a nuclear factor kappaB-dependent mechanism.J BiolChem.1997 Mar 21;272(12):8013-8.PMID:9065473.

[10].McKee CM1,Penno MB,Cowman M,Burdick MD,Strieter RM,Bao C,NoblePW.Hyaluronan(HA)fragments induce chemokine gene expression in alveolarmacrophages.The role of HA size and CD44.J Clin Invest.1996Nov 15 ;98(10):2403-13.PMID:8941660.

[11].Ghosh S1,Hoselton SA2,Wanjara SB2,Carlson J3,McCarthy JB4,DorsamGP2,Schuh JM2.Hyaluronan stimulates ex vivo B lymphocyte chemotherapy and cytokine production in a murine model of fungal allergicasthma.Immunobiology.27018PM 27015 .

[12].Ghosh S1,Samarasinghe AE,Hoselton SA,Dorsam GP,SchuhJM.Hyaluronan deposition and co-localization with inflammatory cells and collagen in a murine model of fungal allergic asthma.Inflamm Res.2014 Jun;63(6):475- 84. PMID: 24519432.

[13]. Nikitovic D1, Berdiaki A2, Galbiati V3, Kavasi RM2, Papale A3, Tsatsakis A4, Tzanakakis GN2, Corsini E3. Hyaluronan regulates chemicalallergen-induced IL-18 production in human keratinocytes. Toxicol Lett. ):89-97.PMID:25280773.

[14].Fieber C1,Baumann P,Vallon R,Termeer C,Simon JC,Hofmann M,AngelP,Herrlich P,Sleeman JP.Hyaluronan-oligosaccharide-induced transcription of metalloproteases.J Cell Sci.2004 Jan 15;117(Pt 2 ):359-67.PMID:14657275.

[15].Campo GM1,Avenoso A,D'Ascola A,Scuruchi M,Prestipino V,NastasiG,Calatroni A,Campo S.The inhibition of hyaluronan degradation reduced pro-inflammatory cytokines in mouse synovial fibroblasts subjected to collagen-induced arthritis. J Cell Biochem. 2012 Jun; 113(6):1852-67. PMID: 22234777.

[16].Campo GM1,Avenoso A,D'Ascola A,Prestipino V,Scuruchi M,NastasiG,Calatroni A,Campo S.4-mer hyaluronan oligosaccharides stimulate inflammation response in synovial fibroblasts in part via TAK-1 and in part via p38-MAPK .Curr Med Chem.2013;20(9):1162-72.PMID:23298137.

[17].Liang J1,Jiang D,Jung Y,Xie T,Ingram J,Church T,Degan S,LeonardM,Kraft M,Noble PW.Role of hyaluronan and hyaluronan-binding proteins in human asthma.J Allergy Clin Immunol.2011August ;128(2):403-411.PMID:21570715.

However, the applicant’s research results in recent years (patent application numbers 200780052196.7, 201310325056.X, 201310454955.X, 201410153593.5, 201510065499.9, 201510065498.4, 201510067326.0, 20151033352 have another possibility of inhibiting inflammation, namely hyaluronic acid), suggesting that 6 function. These literatures suggest that the anti-inflammatory function of small-molecule hyaluronic acid is related to its production method and the degree of molecular size, and the comparative results of animal experiments and human experiments also suggest that the function of small-molecule hyaluronic acid can be improved in small animals. It may be completely different from that in the human body. For example, the above literature shows that small molecule hyaluronic acid (molecular weight 10-60KD) can be made into in vitro preparations for local use (such as daily necessities, hygiene products, cosmetics, skin care products, disinfection products, etc.), which helps to treat skin and mucous membrane inflammation, and achieve Skin beauty and anti-aging effects, while small molecule hyaluronic acid less than 10KD basically has no such effects. However, the recent patent WO/2014/165713 of CEDARS-SINAI MEDICAL CENTER shows that the small molecule hyaluronic acid produced by bacterial streptococci hyaluronidase cutting less than 10KD does not cause inflammation, but the bacterial Streptomyces hyaluronidase produces more than 10KD However, the small molecule hyaluronic acid caused inflammation, suggesting that the function of small molecule hyaluronic acid may also be related to the type of cutting enzyme.

The above contradictory research results suggest that the exact function of the small-molecule hyaluronic acid fragment is currently difficult to determine, and the production method, molecular weight, type of cutting enzyme, and use of the small-molecule hyaluronic acid fragment may affect its functional performance. In addition to these known and possible factors, there are still many other unknown factors that researchers are not sure about. Therefore, although people in the industry pin their hopes on the medical value of small-molecule hyaluronic acid, it is still difficult to predict the success of clinical research and druggability of small-molecule hyaluronic acid, especially to determine clinical indications. Although our previous research obtained a feasible and effective small-molecule hyaluronic acid in vitro preparation, due to the complex internal environment of the human body, the in vivo injection preparation of small-molecule hyaluronic acid may be completely different from the in vitro preparation. The effects in humans and animals may not be the same, so it needs to be verified by clinical research. Moreover, the small molecule hyaluronic acid used as an in vivo injection preparation will have higher requirements at least in many aspects, such as high purity, no allergic reaction, clear indications and side effects, clear dosage of each injection, and injection route , injection dose, etc. Solving the above problems has great scientific significance and commercial value.

Contents of the invention

An object of the present invention is to provide an injection containing small molecule hyaluronic acid with a specific production method.

Another object of the present invention is to provide the use of the injection for treating various diseases unexpectedly discovered and for preparing medicines for treating the various diseases.

To achieve the above object, the present invention adopts the following technical solutions:

An injection containing small-molecule hyaluronic acid, the preparation of which comprises the following steps: A. using animal cells, yeast or plants to express and produce saccharified recombinant human hyaluronidase PH20; B. using macromolecular hyaluronidase The hyaluronic acid raw material is prepared into a macromolecular hyaluronic acid solution, sodium chloride and magnesium ions are introduced, and the recombinant human hyaluronidase PH20 is added to cut the macromolecular hyaluronic acid to obtain a hyaluronic acid fragment with a molecular weight of 10-60KD C. inactivate the recombinant human hyaluronidase PH20 remaining in the obtained reaction solution; D. carry out bacterial filtration, virus filtration inactivation and ultrafiltration concentration.

As a further improvement, the preparation process also includes the step of removing the inactivated recombinant human hyaluronidase.

The purity of the saccharified recombinant human hyaluronidase PH20 in the step A is >99.0%, and the purity of the macromolecular hyaluronic acid raw material in the step B is >99.9%.

In the step B, the molecular weight of the macromolecular hyaluronic acid raw material is 800-1200KD; the concentrations of the sodium chloride and magnesium ions in the macromolecular hyaluronic acid solution are 80-90mM and 1mM respectively; The macromolecular hyaluronic acid raw material corresponds to adding 20,000 units of the recombinant human hyaluronidase, and the enzyme cleavage reaction is carried out at 37 degrees for 6 hours.

Each 2-4ml injection contains 100mg of the hyaluronic acid fragment and 0-20ug of the inactivated recombinant human hyaluronidase residue, the concentration of the magnesium ion in the injection is 1mM, the chloride The concentration of sodium in the injection is 115-125mM.

The injection is used for treating floaters, vitreous turbidity and vitreous detachment and vision loss caused by vitreous turbidity.

The injection is used for treating pain from skin, mucous membrane, connective tissue, muscle, myofascia, tendon, joint, wound, and scar, and itching from skin, mucous membrane, wound, and scar.

The injection is used for treating diseases that can cause pain or itching, including large area burns, trauma scars, surgical wound scars, neurodermatitis, psoriasis, eczema, herpetic eczema, sweat herpes.

The injection is used for treating myofasciitis of the waist, back, neck and shoulders, and muscle and tendon injuries.

The injection is used for treating hyperosteogeny, bone spurs and intervertebral disc herniation.

The injection is used for treating ankylosing spondylitis.

The injection is used for treating endometriosis with dysmenorrhea.

The injection is used for treating abdominal mucosal adhesion and pelvic mucosal adhesion caused after operation or peritoneal dialysis.

The injection is used for treating metastasis and recurrence of solid tumors.

The injection is used for increasing physical strength and energy or recovering physical strength and energy after solid tumor chemotherapy.

The injection is used to promote functional recovery after cerebral infarction.

The injection is used for facial beautification and treatment of facial aging.

The injection is used for reducing the subcutaneous fat tissue of the head, face and neck or the subcutaneous fat tissue of the whole body.

The injection is used for rapid treatment of periodontal disease, oral ulcer and throat disease, including periodontal disease, oral ulcer and throat disease caused by chemotherapy and aging.

The injection is used for treating conjunctival diseases, including conjunctival diseases caused by chemotherapy and aging.

The injection is used for treating coronary heart disease with angina pectoris, treating complications after vascular stents, and treating cardiovascular diseases.

The injection is used in the subcutaneous fat layer of the abdomen or at painful/itchy points or at diseased sites.

The dose of the injection is 1-2 injections per day, and each injection dose contains 100 mg of the hyaluronic acid fragment with a molecular weight of 10-60KD.

Owing to adopting above-mentioned technical scheme, the present invention has following advantage at least:

(1) The injection is easy to produce, stable in preparation, does not require facial injection, does not cause pain and facial inflammation, and can quickly and effectively perform facial beautification and facial anti-aging through abdominal subcutaneous fat layer injection.

(2) The injection of the present invention unexpectedly has the effects of rapidly relieving pain, relieving itching, increasing physical strength and reducing subcutaneous fat of the head, face and neck.

(3) It is even more miraculous that the injection of the present invention has the effect of treating various diseases and disease states, including myofascitis, muscle and tendon injuries, painful bone spurs and herniated intervertebral discs, painful ankylosing spondylitis, Vitreous opacity, extensive burn scars with inflammation, itching and pain, trauma scars, surgical wound scars, peritoneal and pelvic mucosal adhesions after surgery or peritoneal dialysis, endometriosis with dysmenorrhea, itchy Neurodermatitis, psoriasis, itchy senile eczema, infantile eczema and herpetic eczema or sweat herpes, physical recovery after solid tumor chemotherapy and prevention of solid tumor metastasis, functional recovery after cerebral infarction, periodontal disease and oral ulcers, throat disease, Conjunctival diseases, aging and aging-related diseases, coronary heart disease, functional recovery after myocardial infarction, prevention and treatment of complications after vascular stents, and prevention and treatment of cardiovascular and cerebrovascular diseases.

Description of drawings

The above is only an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention, the present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

(a) and (b) in Fig. 1 are the scale-up and large-scale culture workshops of the rapid-flow animal cell reactor for producing high-purity glycosylated recombinant human hyaluronidase.

Fig. 2 is the enzyme of highly purified recombinant human hyaluronidase with glycosylation produced by animal cells (CHO cells).

Fig. 3 is the preparation form of the injection containing small molecule hyaluronic acid according to the present invention.

Figure 4 shows the antibacterial effect of hyaluronic acid fragments with a molecular weight of 10KD-60KD.

Figure 5 shows the anti-inflammatory and antibacterial effects of the injection solution of the present invention on small animal skin wounds.

Among Fig. 6 (a)-(e) shows respectively the injection of the present invention to the influence of IL-1, IL-6, IFN-beta, IFN-alpha, IL-10 (note: LPS=endotoxin; L+A= LPS+anti-human TLR4 single chain antibody; L+HA=LPS+B-HA).

Figure 7. Subject ophthalmic laboratory diagnosis with vitreous opacity and vitreous detachment.

Fig. 8 is a comparison of photos of some subjects before and after treatment, showing the therapeutic effect of the injection of the present invention on the facial beauty and anti-aging of the human body by injecting the subcutaneous fat layer of the abdomen.

Fig. 9 is the effect of using the injection of the present invention to treat a large area burn without skin grafting to continuously thicken, itching and scars with chronic inflammatory reactions.

Fig. 10 is the effect of injecting the injection solution of the present invention on abdominal subcutaneous fat layer to treat neurodermatitis.

Fig. 11 is the effect of treating psoriasis by injecting the injection solution of the present invention into the abdominal subcutaneous fat layer.

detailed description

First of all, it needs to be explained that the existing macromolecular hyaluronic acid injections in the market are mainly used for facial beauty, facial anti-aging and intra-articular injection to lubricate joints. The injection of the present invention is an injection containing small molecule hyaluronic acid. Based on the current research results in the industry, the exact function of small molecule hyaluronic acid is difficult to determine, and even based on the current scientific experiment results (see the previous background technology), there are contradictory functional phenomena, and the mechanism of this phenomenon It is not yet fully understood, and it may be necessary to define the production method of the small molecule hyaluronic acid to clarify its efficacy. Therefore, it is difficult for people in the industry to predict the prospects of clinical research and commercialization (that is, druggability) of small molecule hyaluronic acid.

The injection containing small molecule hyaluronic acid obtained by a specific production method provided by the present invention has been found through clinical research that the inventor has unexpectedly obtained the use of the injection for treating many new indications. This use can be achieved through more convenient injection Ways such as abdominal subcutaneous fat layer injection or painful / itchy point site injection or diseased site injection can be reflected.

The inventor used the method "outside the instruction manual" to carry out the cosmetic and anti-aging research of the injection, and found that the above-mentioned injection containing small molecule hyaluronic acid of the present invention can not only effectively promote facial beauty and anti-aging through the injection of the subcutaneous fat layer of the abdomen. Face anti-aging can also surprisingly increase physical strength, energy and reduce subcutaneous fat in the head, face and neck. According to the feedback from users, this injection of the present invention also shows the effect of quickly relieving pain and itching. More miraculously, Further in-depth studies have found that the injection of the present invention has the effect of treating various diseases and disease states, including myofascitis, muscle and tendon injuries, painful bone spurs and herniated intervertebral discs, painful ankylosing spondylitis, Vitreous opacity, extensive burn scars with inflammation, itching and pain, trauma scars, surgical wound scars, peritoneal and pelvic mucosal adhesions after surgery or peritoneal dialysis, endometriosis with dysmenorrhea, itchy Neurodermatitis, psoriasis, itchy senile eczema, infantile eczema and herpetic eczema or sweat herpes, physical recovery after solid tumor chemotherapy and prevention of solid tumor metastasis, functional recovery after cerebral infarction, periodontal disease and oral ulcers, throat disease, Conjunctival disease, aging and age-related diseases, coronary heart disease, prevention and treatment of post-stent complications, and cardiovascular disease.

The injection containing small molecule hyaluronic acid described in the present invention is obtained through a specific production method. The small-molecule hyaluronic acid is a hyaluronic acid fragment with therapeutic efficacy produced by recombinant human hyaluronidase PH20, sodium ions and magnesium ions to activate macromolecular hyaluronic acid; the hyaluronic acid injection contains hyaluronic acid The molecular weight of the hyaluronic acid fragment is 10KD-60KD; the hyaluronic acid fragment with a molecular weight of 10KD-60KD is a glycosylated recombinant human hyaluronidase produced by animal cells, Chinese hamster ovary cells, yeast or plants. It is obtained by cutting macromolecular hyaluronic acid for 5-6 hours in the presence of magnesium ions and sodium chloride; the recombinant human hyaluronidase in the injection is heat-inactivated at 80 degrees for 45-60 minutes. In order to ensure the safety of the preparation, the glycosylated recombinant human hyaluronidase PH20 is preferably of high purity (preferably >99.0% purity), and the purity of the macromolecular hyaluronic acid raw material is preferably medical grade >99.9%. In actual production, high-purity raw materials can be used directly, or the subsequent steps can be performed after purifying low-purity saccharified recombinant human hyaluronidase PH20 or macromolecular hyaluronic acid raw materials. Combined with clinical research, we found that hyaluronic acid fragments containing recombinant human hyaluronidase residual protein have no allergic reactions and no side effects when injected into the subcutaneous fat layer of the abdomen or pain points or diseased parts.

Further, the inactivated recombinant human hyaluronidase PH20 residual protein was separated by affinity column chromatography, precipitation, ultrafiltration and dialysis to obtain hyaluronic acid fragments containing only 10KD-60KD, sodium chloride and Injection of magnesium ions. Combined with human clinical research, we found that injections in the subcutaneous fat layer or pain points or injections in diseased areas containing or not containing the inactivated recombinant human hyaluronidase PH20 residual protein have therapeutic effects, and there is no allergic reaction and There is no side effect, suggesting that the hyaluronic acid fragment of 10KD-60KD is the main active ingredient.

Further, according to the above-mentioned production method, the injection preparation containing small molecule hyaluronic acid of the present invention can be made into at least the following two preparation forms, one preparation does not contain inactivated recombinant human hyaluronidase PH20 residual protein, The other contains an enzymatically inactive recombinant human hyaluronidase PH20 residual protein. Preferably, in order to make the therapeutic effect of the injection preparation containing the residual protein of recombinant human hyaluronidase PH20 without enzymatic activity more reliable, the residual protein is controlled to be less than 20 micrograms in actual production.

As a preferred embodiment, the present invention prepares the following injection 1 (every 2-4 ml contains 100 mg of hyaluronic acid fragments with a molecular weight of 10KD-60KD and inactivated recombinant human hyaluronic acid without enzymatic activity) according to the above two preparation forms Enzyme PH20 residual protein is less than 20 micrograms, the concentration of sodium chloride in the injection is 115-125mM, and the concentration of magnesium ions is 1mM) and injection 2 (each 2-4ml contains 100mg of hyaluronic acid fragments with a molecular weight of 10KD-60KD, injection The concentration of sodium chloride in the medium is 115-125mM, the concentration of magnesium ions is 1mM, and does not contain recombinant human hyaluronidase PH20 residual protein without enzymatic activity). The injection can be stored at 4 to 12 degrees or -8 to -70 degrees and room temperature and is stable within 2 years.

Further clinical verification of the above two preparations found that subcutaneous or painful/itch point injections or injections of said injections into diseased areas were injected 1-2 times a day, and each injection dose contained said hyaluronic acid fragments 100mg, convenient and effective, without side effects and allergic reactions, can be used to prepare biopharmaceuticals and Class 3 medical devices.

The present invention is described below through specific examples. It should be understood that the examples described here are only used to illustrate and explain the present invention, and are not intended to limit the present invention.

Example 1

Objective: To study the production process of injections, and to manufacture injections with and without recombinant human hyaluronidase PH20 residual protein.

Method: Using animal cells (CHO cells) to produce glycosylated recombinant human hyaluronidase PH20, including: artificially synthesizing the gene cDNA of glycosylated recombinant human hyaluronidase PH20, inserting GC-rich pMH3, pMH4, pMH5 Empty expression vector, build up pMH3-PH20, pMH4-PH20, pMH5-PH20 expression vector (adopting the technology construction expression vector described in the patent document of CN102124019A here to express recombinant protein so that pMH3-PH20, pMH4- The cDNA expression vectors of PH20 and pMH5-PH20 were transferred into CHO-S cell lines, and the CHO-S cell lines highly expressing PH20 were screened, and amplified and large-scale cultured in the rapid flow animal cell reactor shown in Figure 1; the cells containing PH20 Harvest liquid is filtered at 0.22um and separated by 2-3 steps such as ion column, and then after bacteria filtration, virus filtration inactivation, ultrafiltration concentration and other steps, the sterile and virus-free organic substance with a purity of >99.0% as shown in Figure 2 is produced. Glycated recombinant human hyaluronidase PH20.

Prepare injection-grade hyaluronic acid raw materials or hyaluronic acid injections with a molecular weight of 800-1200KD or purify non-injection-grade hyaluronic acid raw materials with a molecular weight of 800-1200KD to injection-grade hyaluronic acid raw materials; Add the hyaluronic acid raw material into injection-grade pure water once or several times, and then add sodium chloride 80-90mM, magnesium ion 1mM, recombinant human hyaluronidase 20000 units (about 20 micrograms) per gram of hyaluronic acid, Mix well, react at 37 degrees for 6 hours until the molecular weight of the hyaluronic acid fragment reaches 10KD-60KD, add sodium chloride 35-45mM to adjust the osmotic pressure to 280-300 milliosm/L (mOsm/L), and then heat 45-60 minutes at 80 degrees, bacteria filtration and virus filtration inactivation, ultrafiltration concentration and other steps to make hyaluronic acid injection 1 (each 2-4 ml contains 100 mg of hyaluronic acid fragments with a molecular weight of 10KD-60KD, and also contains free The recombinant human PH20 hyaluronidase that degrades hyaluronic acid activity remains below 20ug, and the concentration of sodium chloride in the injection is 115-125mM, and the concentration of magnesium ions is 1mM,). Using recombinant human hyaluronidase affinity column chromatography, column chromatography, precipitation, dialysis, ultrafiltration concentration, etc. sequentially or separately, the recombinant human hyaluronic acid fragment injection containing recombinant human hyaluronidase residual hyaluronic acid Removal of acidase residual protein, manufacture of hyaluronic acid injection 2 that does not contain recombinant human hyaluronidase residual protein (each 2-4 ml contains 100 mg of hyaluronic acid fragments with a molecular weight of 10KD-60KD, and the sodium chloride content in the injection 115-125mM, magnesium ion content 1mM).

result:

Fig. 2 is the electrophoresis figure of the glycosylated recombinant human hyaluronidase produced by animal cells (CHO cells) with purity >99%;

The hyaluronic acid injection 1 and hyaluronic acid injection 2 formulations manufactured were as follows:

Hyaluronic acid injection 1: 100mg/2ml of hyaluronic acid fragments with a molecular weight of 10KD-60KD, 115-125mM sodium chloride, 1mM magnesium ion, 20ug of recombinant human PH20 hyaluronidase residual protein without hyaluronic acid degradation activity Below (endotoxin less than 0.5IU/ml, sterile, virus-free).

Hyaluronic acid injection 2: 100mg/2ml of hyaluronic acid fragments with a molecular weight of 10KD-60KD, sodium chloride 115-125mM, magnesium ions 1mM (endotoxin less than 0.5IU/ml, sterile, virus-free).

Conclusion: The results show that hyaluronic acid injection 1 (Figure 3) and injection 2 containing and without hyaluronic acid fragments of recombinant human hyaluronidase residual protein were successfully produced, and the production process of the injection was clarified.

Example 2

Objective: To study the anti-inflammatory and antibacterial effects of Hyaluronic Acid Injection 1 of Example 1 in vitro experiments, molecular cytology experiments, and small animal experiments.

Method: The Hyaluronic Acid Injection 1 of Example 1 diluted 1-fold with normal saline was used to conduct in vitro antibacterial experiments, molecular cytology experiments, and small animal experiments to detect the anti-inflammatory and antibacterial effects of Hyaluronic Acid Injection.

Results: Figure 4 shows that hyaluronic acid injection inhibits the growth of Porphyromonas gingivalis clones in vitro experiments, suggesting that hyaluronic acid injection materials are not conducive to the growth of Porphyromonas gingivalis (Note: the negative control is normal saline, Positive control is antibiotic metronidazole); Figure 5 shows the anti-inflammatory and antibacterial effects of hyaluronic acid injection on small animal skin wounds; Figure 6 shows that hyaluronic acid injection promotes human TLR4 receptor positive cells to secrete IL-10 , while inhibiting the secretion of TNF, IL-1, IL-6 and interferon beta caused by endotoxin (note: LPS=endotoxin; L+A=LPS+anti-human TLR4 single chain antibody; L+HA= LPS+B-HA). Combined with literature research, there are articles (CuixiaYang, Manlin Cao, Hua Liu, Yiqing He, Jing Xu, Yan Du, Yiwen Liu, Wenjuan Wang, LianCui, Jiajie Hu, Feng Gao, The high and low molecular weight forms of hyaluronan have distinct effects on CD44 Clustering, Published on November 1, 2012asManuscript M112.349209) indicated that CD44 receptors may also be involved in the mechanism of action of hyaluronic acid injection.

Conclusion: Hyaluronic acid injection has obvious anti-inflammatory and anti-bacterial effects on human cells and small animal skin wounds. Its anti-inflammatory and anti-bacterial effects may be achieved through various channels, including endotoxin receptors, beta-defensin secretion, CD44 Mediated removal and accumulation of inflammatory cells.

Example 3

Purpose: To study the beauty and anti-aging effects of two kinds of hyaluronic acid injections containing (experimental group 1) and not (experimental group 2) hyaluronic acid fragments of recombinant human hyaluronidase PH20 residual protein in the above embodiment 1 efficacy and safety.

Method: Use the injection solution containing hyaluronic acid fragments of recombinant human hyaluronidase PH20 residual protein (100mg of hyaluronic acid fragments with a molecular weight of 10KD-60KD, 115-125mM sodium chloride, 1mM magnesium ions, no degraded hyaluronic acid The active recombinant human hyaluronidase residual protein is less than 20 micrograms) is the experimental group 1, which is used for various diseases or subclinical problems frequently occurring or aging and aging-related diseases frequently occurring in China's current air pollution, drinking water pollution and work pressure environment 38 subjects (average age 61±26 years old, 20 males, 18 females) for clinical research; using hyaluronic acid fragment injection (molecular weight 10KD-60KD) without recombinant human hyaluronidase residual protein 100mg of hyaluronic acid fragments, sodium chloride 115-125mM and magnesium ion 1mM) for experimental group 2, 100mg each time (referring to 100mg of hyaluronic acid fragments with a molecular weight of 10KD-60KD contained in the injection, the same below) Abdominal subcutaneous fat layer injection, 1-2 times a day, continuous injection for at least 7 days, for various diseases or subclinical problems frequently occurring or aging and aging-related diseases in China's current environment of air pollution, drinking water pollution and high work pressure Multiple 38 subjects (mean age 62±21 years old, 19 males, 19 females) were clinically studied;

The cosmetic effects of the two injections were compared by the method of face observation before and after treatment or photo comparison and scoring (score: invalid, uncertain, obviously effective), and the method of asking the doctor about his feelings, symptoms, physical examination and laboratory tests was used for evaluation The safety of the injection, that is, the presence or absence of side effects, including the presence or absence of pain at the injection site, physical discomfort before and after injection, physical and mental status, original pain in the body, existing diseases in the body, and changes in various subclinical problems in the body .

result:

Table 1: The effect of two kinds of injections in the treatment of facial beauty and anti-aging

invalid Not sure Obviously effective Number of samples Experimental group 1 0 0 38 38 Experimental group 2 0 0 38 38

Figure 8 shows the comparison of photos of some subjects before and after treatment, showing the therapeutic effect of hyaluronic acid injection on human facial beauty and facial aging. It also shows that hyaluronic acid injection can significantly reduce the head, face, neck, Thickness of subcutaneous adipose tissue in shoulder, back, abdomen and upper and lower extremities.

Table 2: The effect of two injections on reducing subcutaneous fat

invalid Not sure Obviously effective Number of samples Experimental group 1 2 6 30 38 Experimental group 2 1 6 31 38

Table 3: Presence or absence of pain, local redness and swelling, local and systemic allergies after deep injection into abdominal subcutaneous fat layer of two injections

none Not sure have Number of samples Experimental group 1 38 0 0 38 Experimental group 2 38 0 0 38

In addition to the above cosmetic effects, safety and side effects, we also found that 3 subjects felt comfortable and sleepy after injection in the abdominal subcutaneous fat layer due to fatigue; the sleepiness lasted for 0.5-1.5 hours, and one of them was injected for the first time After taking a nap for 1.0 hour, the mental and physical strength increased significantly after waking up; the other 2 cases no longer experienced post-injection drowsiness after 2 injections.

Table 4: Side effects of drowsiness after injection of two injections

none Not sure have Number of samples Experimental group 1 36 0 2 38 Experimental group 2 37 0 1 38

Unexpectedly, 71 of the 76 subjects felt that their energy and physical strength began to increase 30 minutes after the injection of the abdominal subcutaneous fat layer, showing that they were willing to talk, wash dishes, cook, take a walk, and plan to go out for a trip , working and not wanting to rest etc. for 3-4 days.

Table 5: Effects of two injections on increasing mental and physical strength

none Not sure Obviously Number of samples Experimental group 1 0 3 35 38 Experimental group 2 0 2 36 38

All 76 subjects (including 20 cases in experimental group 1 and 17 cases in experimental group 2) had 27 subjects with different shoulder, back, waist, leg, and arm pains. Unexpectedly, 30 cases after abdominal subcutaneous fat layer injection Minutes later, all felt pain relief, and 9 cases of them added 50 mg injection of local pain points, and the pain relief began to appear 2 minutes after the injection, which was equivalent to the effect of 4 cases of self-reported previous local lidocaine mixed prednisone injection closed treatment.

Table 6: Analgesic effect of hyaluronic acid injection

All 76 subjects (including 17 cases in experimental group 1 and 16 cases in experimental group 2) had 33 cases of itching in various degrees. Unexpectedly, all of them felt pruritus 1 day after abdominal subcutaneous fat layer injection. Pain lessened or disappeared.

Table 7: Antipruritic effect of hyaluronic acid injection

in conclusion:

1. Both hyaluronic acid injections containing and not containing hyaluronic acid fragments of recombinant human hyaluronidase residual protein have the effects of human facial beautification and facial anti-aging.

2. The two injections containing and not containing hyaluronic acid fragments of recombinant human hyaluronidase residual protein can significantly reduce the thickness of subcutaneous fat tissue in the head, face, neck, shoulder, back, abdomen, upper and lower extremities.

3. No pain, local redness, local and systemic allergies were observed in the abdominal subcutaneous fat layer injection of the two injections containing and not containing the hyaluronic acid fragment of the recombinant human hyaluronidase residual protein, and it is convenient to use.

3. Two kinds of injections containing and not containing hyaluronic acid fragments of recombinant human hyaluronidase residual protein After the first injection into the abdominal subcutaneous fat layer, I felt comfortable, sleepy, and sleepy, and the incidence rate was less than 4.0%. .

4. Unexpectedly, the abdominal subcutaneous fat layer injection of the two injections containing and not containing hyaluronic acid fragments of recombinant human hyaluronidase residual protein has obvious effects of increasing energy and physical strength.

5. Unexpectedly, two kinds of injections containing and not containing hyaluronic acid fragments of recombinant human hyaluronidase residual protein have rapid and obvious analgesic effects when injected into the abdominal subcutaneous fat layer.

6. Unexpectedly, two injections containing and not containing hyaluronic acid fragments of recombinant human hyaluronidase residual protein have obvious antipruritic effects when injected into the abdominal subcutaneous fat layer.

Example 4

Objective: To further analyze the changes in the diseases and subclinical problems of 76 subjects in "Example 3" before and after treatment with hyaluronic acid injection, and to clarify the new clinical indications of hyaluronic acid injection.

Methods: Further analysis of the changes of diseases and subclinical problems suffered by 76 subjects in "Example 3" before and after treatment with hyaluronic acid injection, including pre-injection medical history, laboratory test results and post-injection changes, To clarify the possible new clinical indications of hyaluronic acid injection.

result:

1. Quick and effective treatment of myofasciitis and old sports injuries: Myofascitis and old sports injuries are sports injuries and senile degenerative related diseases. The pathogenesis is not completely clear. There is a professional chiropractor in the United States who specializes in the treatment of this disease. There is currently no cure.

Among the 76 subjects, we analyzed 7 subjects with myofasciitis of lower back, neck, upper and lower extremities (4 cases in experimental group 1 and 3 cases in experimental group 2) and 4 cases of old sports injuries (experimental group 2 cases of 1 and 2 cases of experimental group 2), the results are shown in Table 8:

Table 8: Effects of hyaluronic acid injection on myofascitis and old sports injuries

Among them, a 57-year-old male subject suffered from old sports injuries for 17 years and suffered from neck and back myofasciitis for 3 years. The pain occurred every year when the weather was cold, and the pain was very tormenting. restricted. After one injection of 100mg in the subcutaneous fat layer of the abdomen, the pain was relieved after 30 minutes, and the effect of adding a 100mg deep injection to the local pain point on the back of the neck was better. After injecting 15x100mg within 2 months, the back pain point nodules disappeared, the thickness of the neck and back muscles became significantly thinner, and there was no recurrence in 12 months. The results showed that in addition to pain relief, hyaluronic acid injection finally solved the inflammatory lesions of old sports injuries and myofascial inflammation, and achieved the purpose of muscle and myofascial reorganization and regeneration.

2. Hyaluronic acid injection quickly and effectively treats bone spurs, herniated intervertebral discs or cervical spondylosis in the back: Bone spurs and herniated discs or cervical spondylosis in the lower back is a kind of senile degenerative and sports injury-related diseases. The pathogenesis is not completely clear, and it is still unclear There is no cure. Among the 76 subjects, we analyzed 11 subjects suffering from lumbar hyperosteogeny and bone spurs (6 examples of experimental group 1 and 5 examples of experimental group 2), and the results are shown in Table 9:

Table 9: Effect of Hyaluronic Acid Injection in Treating Bone Spurs and Herniated Intervertebral Disc or Low Back and Cervical Spondylosis

Among them, a 57-year-old female subject suffered from hyperosteogeny and bone spurs in the back and neck for 14 years. Decrease in ability. One day after 100mg injection of abdominal subcutaneous fat layer, the self-reported pain was relieved by more than 50%, and he could go to the kitchen to make dumplings and cook; the self-reported pain was relieved by more than 80% after the 100mg injection on the second day; the self-reported pain was relieved by 95% after the 100mg injection on the third day %above. No recurrence 2 months after injection of 15x100mg within 2 months. The results showed that hyaluronic acid injection, in addition to relieving pain, finally started the reorganization and regeneration of old damaged inflammatory lesions.

Another 57-year-old female subject suffered from cervical hyperostosis and bone spurs for 4 years. The range of motion of the neck and back was limited, and the pain occurred from time to time. The pain was excruciating and facial numbness. Certain muscle movements would also cause pain and numbness of the upper limbs. and reduced ability to work. One day after injection of 100 mg of abdominal subcutaneous fat layer, self-reported pain was relieved by more than 50%; after 100 mg injection the next day, self-reported pain was relieved by more than 80%. There was no recurrence within 2 months after injection of 15x100mg within 2 months, indicating that hyaluronic acid injection not only relieves pain, but also begins the process of reorganization and regeneration of old damaged inflammatory lesions. Note: All the subjects above continued the treatment, waiting for the results of relapse or non-relapse for more than 6 months.

3. Hyaluronic acid injection quickly and effectively treats vitreous opacity: Vitreous opacity is a degenerative disease of the elderly, the pathogenesis is unknown, and there is no effective treatment method yet. Vitreous opacity, also known as floaters, further develops into vitreous detachment, which damages the fundus and affects vision. We analyzed 9 cases (4 cases of experimental group 1 and 5 cases of experimental group 2) suffering from vitreous opacity and vitreous detachment among the 76 subjects, and the results are shown in Table 10:

Table 10: Effects of hyaluronic acid injection on vitreous opacity and vitreous detachment

A case of a 61-year-old female subject. Figure 7 shows the laboratory diagnosis of the subject suffering from vitreous opacity and vitreous detachment. Due to the need for facial cosmetic treatment, she received 100 mg injection of hyaluronic acid into the abdominal subcutaneous fat layer. It was found that the eye vision improved, and on the third day, it was found that the eye floaters became smaller; continue to inject 15x100 mg every three days, the vision continued to improve (from 0.2 to 0.5) and the floaters continued to become thinner and shallower, two There was no obvious recurrence within a month. The above results show that hyaluronic acid injection is surprisingly fast and effective in treating vitreous opacity (floaters) and vitreous detachment.

4. Hyaluronic acid injection treats wound scars with itching (chronic inflammation) and pain (inflammatory pain): minor wounds of people with scar constitution can gradually form large scars, which are constantly thickened and itchy. The goal of treatment is to relieve itching and Reduce scar thickening; after extensive burns, scars without skin grafting continue to thicken, itching, and have chronic inflammatory reactions (Figure 9). The goal of treatment is to relieve itching, eliminate inflammation, and reduce scar thickening; surgical wounds are mainly red, swollen, and painful , inflammatory response exudate and secondary infection, the goal of treatment is to accelerate healing and reduce scar formation. We analyzed a total of 8 subjects (4 subjects in experimental group 1 and 4 subjects in experimental group 2) suffering from itchy (chronic inflammation) and painful (inflammatory pain) wound scars among 76 subjects, and the results As shown in Table 11:

Table 11: Effect of hyaluronic acid injection on wound scars with itching (chronic inflammation) and pain (inflammatory pain)

A 63-year-old woman had extensive burns on her lower limbs 12 years ago, without skin grafting, severe itching, difficulty falling asleep, and inflammation around the scars. After hyaluronic acid injection of 2x100mg for 3 consecutive days, the scar became thinner, the inflammation around the scar disappeared, the itching was significantly relieved, and the pain was significantly relieved; after the continuous injection of hyaluronic acid 30x100mg, the scar and surrounding skin were significantly improved, including scar thinning, inflammation disappeared, and itching was relieved. But not completely disappeared (Figure 9).

5. Hyaluronic acid injection can quickly and effectively treat abdominal mucosal adhesions caused by surgery or peritoneal dialysis: surgical injuries, peritoneal dialysis, etc. almost all cause abdominal mucosal inflammation and adhesions, which can cause symptoms and pathological changes depending on the severity, including abdominal pain , abdominal tenderness, abdominal mass, and intestinal obstruction. Currently, there is no feasible way to eradicate intra-abdominal adhesions. We analyzed 3 cases (1 case of experimental group 1 and 2 cases of experimental group 2) suffering from abdominal mucosal adhesions after surgery or dialysis among the 76 subjects. The results are shown in Table 12:

Table 12: Hyaluronic acid injection quickly and effectively treats abdominal adhesions caused by surgery or peritoneal dialysis

One month after subtotal gastrectomy and peripheral lymph node removal, a 43-year-old female found lumps and pain in her abdomen after taking a non-liquid diet. After injecting 5x100mg of hyaluronic acid into the subcutaneous fat layer of the abdomen, all the above symptoms and signs disappeared, and there was no recurrence of intestinal obstruction after taking non-liquid food, and the mental state improved significantly. The results of the treatment indicated that the hyaluronic acid injection treatment may eliminate the symptoms and signs of abdominal adhesions by reducing the inflammation of the abdominal mucosa.

6. Hyaluronic acid injection treats endometriosis or pelvic mucosal adhesions with dysmenorrhea: endometriosis is often accompanied by infertility and dysmenorrhea; infertility and dysmenorrhea may be related to pelvic mucosal inflammation and Adhesion related; treatment of dysmenorrhea and endometriosis or pelvic mucosal adhesions and there is no effective method; we analyzed 3 cases of subjects suffering from pelvic mucosal adhesions and dysmenorrhea among 76 subjects (experimental group 1 2 Example and 1 example of experimental group 2), the results are shown in Table 13:

Table 13: Hyaluronic acid injection in the treatment of endometriosis or pelvic adhesions with dysmenorrhea

The above results show that hyaluronic acid injection can significantly reduce dysmenorrhea, indicating that hyaluronic acid injection can reduce the degree of inflammation of pelvic endometriosis and the degree of referred pain caused by pelvic adhesions, suggesting that hyaluronic acid injection may also be used for Treatment of infertility caused by endometriosis and poor tubal motility caused by pelvic adhesions.

7. Hyaluronic acid injection in the treatment of itchy neurodermatitis, psoriasis, itchy senile eczema, herpetic eczema or sweat herpes: We analyzed 76 subjects in the elderly suffering from neurodermatitis, psoriasis, and itching 19 cases of eczema, herpetic eczema or sweat herpes subjects (9 cases of experimental group 1 and 10 cases of experimental group 2), the results are shown in Table 14:

Table 14: The effect of hyaluronic acid injection on neurodermatitis and psoriasis with pruritus

One of the subjects with neurodermatitis in Table 14 (male, 57 years old) suffered from neurodermatitis for 8 years, with bilateral symmetrical multiple skin lesions, severe itching and local skin inflammatory lesions. The subject was not satisfied with the effect of Stelara (anti-IL-12/23 antibody) injection before. After injecting hyaluronic acid injection 20x100mg into the abdominal subcutaneous fat layer, the itching basically disappeared, and the skin inflammatory lesions basically recovered (Figure 10), and the subject was satisfied with the effect.

One of the subjects with psoriasis in Table 14 (male, 52 years old) suffered from psoriasis for 18 years, with multiple local skin inflammatory lesions and thickening of the skin on the abdomen, upper limbs and lower limbs, accompanied by itching symptoms. Two years ago, he used Stelara (anti-IL-12/23 antibody) injection treatment, and the effect of the subject was not satisfactory. After injecting hyaluronic acid injection 10x100mg into the abdominal subcutaneous fat layer, the itching basically disappeared, the thickened skin lesions were obviously thinned, and the inflammatory skin lesions of the skin recovered obviously (Figure 11), and the subjects were satisfied with the effect.

Table 15: The effect of hyaluronic acid injection on senile eczema and herpetiform eczema or sweat herpes with itching

One of the subjects with sweat herpes in Table 15 (female, 33 years old) suffered from sweat herpes for 9 years, with onset every summer. After injecting hyaluronic acid injection 20x100mg into the abdominal subcutaneous fat layer, sweat herpes did not occur again during the injection period and 1 year after the injection.

The results in Tables 14 and 15 show that the injection of hyaluronic acid injection into the abdominal subcutaneous fat layer can effectively treat itchy neurodermatitis, psoriasis, itchy senile eczema, herpetic eczema or sweat herpes.

8. Hyaluronic acid injection quickly treats periodontal inflammation, oral ulcer, throat inflammation, and conjunctival inflammation: periodontal inflammation, oral ulcer, throat inflammation, and conjunctival inflammation are common in the elderly, and they are aging-related diseases. Pollution, contaminated drinking water and a stressful work environment are linked. We analyzed 26 subjects (16 cases in experimental group 1 and 10 cases in experimental group 2) suffering from periodontal inflammation, oral ulcer, throat inflammation, and conjunctival inflammation among the 76 subjects. The results are shown in Table 16 Shown:

Table 16: The effect of hyaluronic acid injection on periodontal inflammation, oral ulcer, throat inflammation and conjunctival inflammation

The results in Table 16 show that hyaluronic acid injection has a faster effect on the treatment of periodontal inflammation, oral ulcer, throat inflammation and conjunctival inflammation than our previous topical use of hyaluronic acid injection spraying.

9. Hyaluronic acid injection is effective in treating ankylosing spondylitis: ankylosing spondylitis is a disease of inflammatory pain and severe deformation of the spine. We analyzed 2 cases of subjects suffering from ankylosing spondylitis among the 76 subjects (1 case in experimental group 1 and 1 case in experimental group 2), and the results are shown in Table 17:

Table 17: The effect of hyaluronic acid injection on ankylosing spondylitis

Ankylosing spondylitis is a disease of inflammatory pain. The analgesic effect of hyaluronic acid injection treatment indicates that the inflammation of ankylosing spondylitis is reduced or eliminated, suggesting that the severe deformation of the spine caused by inflammation will not continue to develop.

10. Hyaluronic acid injection for physical recovery and prevention of solid tumor metastasis after solid tumor chemotherapy: We analyzed a total of 76 subjects who needed physical recovery and prevention of solid tumor metastasis after solid tumor chemotherapy 4 examples (4 examples of experimental group 1 and 4 examples of experimental group 3), the results are as shown in table 18:

Table 18: The effect of hyaluronic acid injection on physical recovery and prevention of solid tumor metastasis after chemotherapy for solid tumors

Pancreatic head cancer is a very malignant tumor, which develops rapidly after metastasis and dies within 90 days at the fastest. Foreign studies have shown that inflammation may be an important factor in the development and metastasis of pancreatic head cancer. The subject (male, 46 years old) in Table 18 after chemotherapy for pancreatic head cancer found that the pancreatic head cancer had metastasized to the liver due to jaundice, which caused jaundice due to blockage of the bile duct. Chemotherapy and traditional Chinese medicine treatment. Hyaluronic acid injection 5x100mg was injected into abdominal subcutaneous fat layer to restore physical strength; Ultrasound examination after hyaluronic acid injection 20x100mg abdominal subcutaneous fat layer injection showed that the pancreatic head cancer and the mass metastasized to the liver began to shrink, and the subjects were normal work for more than 90 days. The above results show that hyaluronic acid injection can promote the recovery of physical strength and energy after chemotherapy in the treatment of solid tumors and may prevent the development and further metastasis of pancreatic head cancer by inhibiting inflammation. Foreign reports (Halozyme Receives Orphan Drug Designation For PEGylated Recombinant Human Hyaluronidase PH20 For Pancreatic Cancer at: http://www.halozyme.com/Investors/News-Releases/News-Release-Details/2014/Halozyme-Receives-Orphan-Drug-Designation -For-PEGylated-Recombinant-Huma n-Hyaluronidase-PH20-For-Pancreatic-Cancer/default.aspx#sthash.aLjvxToQ.dp uf) Long-acting hyaluronidase PEGPH20 combined with chemotherapy effectively prolongs the survival time of pancreatic ductal carcinoma patients, Its mechanism of action may be to use hyaluronic acid in the human body to produce injections containing small molecule hyaluronic acid similar to the present invention.

The subject (female, 62 years old) in Table 18 after ovarian cancer surgery and chemotherapy was extremely weak due to lymph node metastasis chemotherapy and radiotherapy, and was bedridden for 24 hours. Hyaluronic acid injection 20x100mg was injected into the subcutaneous fat layer of the abdomen, physical strength and energy began to recover the next day after the injection of 1x100mg, and after all injections of 20x100mg, CA125 was surprisingly reduced from 500U/ml before treatment to 50U/ml, and the tumor and metastatic mass shrank , indicating that hyaluronic acid injection can promote the recovery of physical strength and energy after chemotherapy in the treatment of ovarian cancer and may prevent the development and further metastasis of ovarian cancer by inhibiting inflammation.

The 64-year-old female subject in Table 18 was diagnosed with lung cancer in the right lung, accompanied by metastasis in the left lung, brain, bone, and mediastinal lymph nodes. Surgical treatment was no longer possible, and radiotherapy and Tarceva chemotherapy were used. The subject was weak after radiotherapy and chemotherapy, and 10x100mg of hyaluronic acid injection was injected into the subcutaneous fat layer of the abdomen, and his physical strength and energy began to recover on the first day after the injection of 1x100mg. It shows that hyaluronic acid injection can promote the recovery of physical strength and energy after radiotherapy and chemotherapy of lung cancer, and may also prevent the development and further metastasis of lung cancer by inhibiting inflammation.

XI. Hyaluronic acid injection in the treatment of meningioma recurrence after surgery: We analyzed 2 subjects (1 case in experimental group 1 and 1 case in experimental group 2) who had meningioma recurrence after surgery among 76 subjects. Example), the results are shown in Table 19:

Table 19: The effect of hyaluronic acid injection on the recurrence of meningioma after surgery

A female subject (61 years old) in Table 19 was diagnosed with meningioma by CT in 2007 due to frequent headaches. After surgery in the same year, the headaches disappeared. He had frequent headaches since 2015, and was diagnosed with meningioma recurrence by CT, that is, the meninges were obviously thickened. Hyaluronic acid injection 15x100mg was injected into the subcutaneous fat layer of the abdomen, the headache attacks were reduced, and finally stopped completely. The CT examination results showed that the previously proliferated meninges were significantly thinner, and there was no more headache attacks 90 days after treatment. The above results show that hyaluronic acid injection can be used to treat meningioma or meningioma recurrence after surgery.

12. Hyaluronic acid injection for functional recovery after cerebral embolism: We analyzed 4 subjects in need of functional recovery after cerebral embolism among 76 subjects (1 case of experimental group 1 and 1 case of experimental group 2). 3 examples), the results are shown in Table 20:

Table 20: Effect of hyaluronic acid injection on functional recovery after cerebral embolism

In Table 20, a male subject (67 years old) had half body weakness and walking weakness 30 days after cerebral embolism. After injecting hyaluronic acid injection 10x100mg into the abdominal subcutaneous fat layer, the half body softness improved significantly and walking was weak, indicating that hyaluronic acid The injection can be used for functional recovery after cerebral embolism.

Another male subject (59 years old) in Table 20 had impaired vision 4 days after cerebral embolism and could only see objects within 20cm. Objects within 40cm can be seen, indicating that hyaluronic acid injection can be used for functional recovery after cerebral embolism.

Thirteen. Hyaluronic acid injection is used for coronary heart disease, prevention of complications after vascular stenting and prevention of cardiovascular diseases: coronary heart disease, complications after vascular stenting and other cardiovascular diseases may be chronic inflammatory diseases in nature, and effective drugs are urgently needed Prevention methods. We analyzed 6 subjects (3 examples of experimental group 1 and 3 examples of experimental group 2) with complications after coronary heart disease and vascular stent in 76 subjects, and the results are shown in Table 21:

Table 21: The effect of hyaluronic acid injection on the treatment of coronary heart disease, prevention of complications after vascular stents and prevention of cardiovascular diseases

The results in Table 21 show that hyaluronic acid injection can be used to treat coronary heart disease, prevent complications after vascular stents, and possibly prevent cardiovascular diseases.

in conclusion:

1. Hyaluronic acid injection is used for the treatment of painful back, neck and shoulder myofasciitis and muscle and tendon injuries through abdominal subcutaneous fat layer injection or injection at pain points or diseased parts;

2. Hyaluronic acid injection is used to treat painful hyperosteogeny, bone spurs, and herniated intervertebral discs through abdominal subcutaneous fat layer injection or injection at pain points or diseased areas;

3. Hyaluronic acid injection is injected through the abdominal subcutaneous fat layer for the treatment of painful ankylosing spondylitis;

4. Hyaluronic acid injection is injected through the subcutaneous fat layer of the abdomen for the treatment of floaters or vitreous opacity and the vitreous detachment and vision loss caused by it;

5. Hyaluronic acid injection is injected through the subcutaneous fat layer of the abdomen for the treatment of large area burns, trauma scars and surgical wound scars with inflammation, itching and pain;

6. Hyaluronic acid injection is injected through the abdominal subcutaneous fat layer or used in the affected area to treat abdominal and pelvic mucosal adhesions after surgery and peritoneal dialysis;

7. Hyaluronic acid injection is used for the treatment of dysmenorrhea endometriosis through abdominal subcutaneous fat injection;

8. Hyaluronic acid injection is injected through the subcutaneous fat layer of the abdomen for the treatment of itchy neurodermatitis, psoriasis, itchy senile eczema and herpetic eczema or sweat herpes;

9. Hyaluronic acid injection is injected through the subcutaneous fat layer of the abdomen for the recovery of physical strength and energy after solid tumor chemotherapy and the prevention of solid tumor metastasis;

10. Hyaluronic acid injection is used to promote functional recovery after cerebral infarction through abdominal subcutaneous fat injection;

11. Hyaluronic acid injection is used for rapid treatment of periodontal diseases and oral ulcers through abdominal subcutaneous fat injection, including periodontal diseases and oral ulcers caused by chemotherapy and aging;

12. Hyaluronic acid injection is injected through the subcutaneous fat layer of the abdomen for rapid treatment of throat diseases, including throat diseases caused by chemotherapy and aging;

13. Hyaluronic acid injection is used for rapid treatment of conjunctival diseases, including conjunctival diseases caused by chemotherapy and aging, through abdominal subcutaneous fat injection;

14. Hyaluronic acid injection is injected through the abdominal subcutaneous fat layer for the treatment of coronary heart disease with angina pectoris, the prevention and treatment of complications after vascular stents, and the prevention and treatment of cardiovascular diseases;

Example 5

Objective: To investigate the activation and commercial production of hyaluronic acid injection 1 or 2 with the therapeutic effects shown in Examples 3-4 by enzymatic cleavage methods of other kinds of hyaluronic acid.

Methods: (1) Study the production methods of various hyaluronidases found in the existing literature and the results of enzymatic cleavage of macromolecular hyaluronic acid; (2) Produce different hyaluronidases independently, cooperatively or under contract, and verify The feasibility of enzymatically cutting and activating macromolecular hyaluronic acid; (3) research what kind of hyaluronidase can cut and activate macromolecular hyaluronic acid to what molecular weight? What kind of enzyme can cut the terminal structure to activate the small molecule hyaluronic acid to have the therapeutic effect shown by the hyaluronic acid injection in Example 3-4? What hyaluronidase residues do not cause allergic reactions in humans?

Results and discussion: Based on the existing literature, we have studied the production methods of various hyaluronidases and their methods of enzymatically cleaving macromolecular hyaluronic acid. The research results are reported as follows:

1. In addition to the neutral human sperm acrosomal hyaluronidase PH20 (or Hyal3 or SPAM1) studied in this paper, humans also have intracellular acid hyaluronidases Hyal1 and Hyal2. Through the literature "Recombinant human hyaluronidaseHyal-1: Insect cells versus Escherichia colias expression system and identification of low molecular weight inhibitors, Glycobiology, 17(4):444-53, 2007", we found that insect cells Drosophila Schneider-2 (DS-2) The specific activity of the produced recombinant human Hyal1 hyaluronidase is 8.6U/mg, and the optimal reaction pH value is 3.5-4.0, but the yield is too low, so it is not suitable for activation and commercial production of hyaluronic acid injection with a molecular weight of 10-60KD. In addition, without human trials, it is difficult to confirm that the hyaluronic acid injection with a molecular weight of 10-60KD formed by cutting macromolecular hyaluronic acid with recombinant human Hyal1 hyaluronidase will have the therapeutic effect shown in Examples 3-4. We used animal cells (CHO-S cells), GC-rich pMH3, pMH4, pMH5 expression vectors and human Hyal1 and Hyal2 cDNA to produce recombinant human Hyal1 and Hyal2 proteins, and found that the activity of hyaluronidase was much smaller than that of our current PH20 ( 1x105U/mg enzyme protein), suggesting that recombinant human Hyal1 and Hyal2 are not suitable for animal cell production.

2. The yield of neutral human sperm acrosome hyaluronidase PH20 (or Hyal3 or SPAM1) expressed by yeast can reach the commercial level, and the specific activity can reach 10,000 IU/mg enzyme protein, which is suitable for enzyme digestion activation and commercialization Production has the hyaluronic acid injection of the therapeutic effect shown in embodiment 3-4. We use neutral human sperm acrosomal hyaluronidase PH20 expressed by yeast, and use recombinant human hyaluronidase affinity column chromatography, precipitation, column chromatography, dialysis, ultrafiltration concentration, etc. sequentially or separately after enzyme digestion, to extract Containing recombinant human hyaluronidase residual protein removal, manufacturing hyaluronic acid injection 2 (100 mg/2 ml of hyaluronic acid fragments with a molecular weight of 10KD-60KD) similar to Example 1 that does not contain recombinant human hyaluronidase residual protein , sodium chloride 115-125mM, magnesium ions 1mM), and clinical research was carried out on 38 subjects with various aging-related diseases. The results in Table 21 show that the neutral human sperm acrosomal hyaluronidase PH20 expressed by yeast Although the specific activity is small, it has a certain impact on the purity of hyaluronic acid injection, and the residual protein containing recombinant human hyaluronidase can be removed, which can be used to manufacture the therapeutic effect shown in Examples 3-4 and no allergic side effects. Hyaluronic Acid Injection 2.

Table 22: The clinical effect of hyaluronic acid injection 2 produced by using yeast-expressed neutral human sperm acrosomal hyaluronidase PH20 on 38 subjects with multiple aging-related diseases

3. We use plant Arabidopsis thaliana to produce oilseed expression system to produce neutral human sperm acrosomal hyaluronidase PH20 fusion protein through our cooperative laboratory, and found that it can be used to produce the above-mentioned ones shown in Examples 3-4 See Table 23 for the specific effects of Hyaluronic Acid Injection 2 with therapeutic effects and no allergic side effects. However, compared with the production of recombinant hyaluronidase by animal cells, the cost of hyaluronidase PH20 fusion protein is low, the yield is large, and the activity is low. Compared with the high-purity glycosylated recombinant human hyaluronidase produced by cells, it is more suitable for the production of external hyaluronic acid nebulizers, sprays and gels with low purity requirements and therapeutic effects.

Table 23: The clinical effect of Hyaluronic Acid Injection 2 manufactured by plant-expressed neutral human sperm acrosomal hyaluronidase PH20 on 35 subjects with multiple aging-related diseases

4. The hyaluronidase extracted from goat or bovine testis is mainly neutral goat or bovine sperm acrosome hyaluronidase PH20. Skin allergy test is required before human use. Although the cost is low, the purity is also low, and the cost of removing sheep or bovine hyaluronidase protein residues is high. Compared with recombinant human hyaluronidase with glycosylation >99.0% purity produced by animal cells Chinese hamster ovary cells , think that it is not suitable for the production of hyaluronic acid injection 1 or 2 with high purity requirements and the therapeutic effect shown in Examples 3-4 and no allergic side effects. In addition, hyaluronidase extracted from sheep or bovine testes cuts macromolecular hyaluronic acid to form a hyaluronic acid fragment with a molecular weight between 10-60KD, which is difficult to determine without human clinical trials. It will be shown in Examples 3-4 therapeutic effect.

5. The patent WO/2014/165713 of CEDARS-SINAI MEDICAL CENTER shows that small molecule hyaluronic acid produced by bacterial streptococci hyaluronidase less than 10KD does not cause inflammation, but small molecules larger than 10KD produced by bacterial Streptomyces hyaluronidase Hyaluronic acid causes an inflammatory response, which is contradictory to the therapeutically effective hyaluronic acid fragments with a molecular weight of 10KD-60KD produced by recombinant human hyaluronidase and magnesium ion cleavage to activate macromolecular hyaluronic acid. The important thing is The residual protein of bacterial hyaluronidase may cause allergic reactions to the human body, so it is not suitable for the production of hyaluronic acid injection 1 or 2 with high purity requirements, the therapeutic effect shown in Examples 3-4 and no allergic side effects.

6. The hyaluronidase in bee venom is highly allergenic, and it not only promotes the absorption of bee venom, but also causes allergic reactions to humans. The hyaluronidase in the honeybee venom extracted and expressed by microorganisms is not suitable for the production of hyaluronic acid injection 1 or 2 with high purity requirements, the therapeutic effect shown in Examples 3-4 and no allergic side effects. In addition, hyaluronic acid fragments with a molecular weight between 10-60KD formed by the hyaluronidase of honeybees cutting macromolecular hyaluronic acid are difficult to confirm that they will have the therapeutic effect shown in Examples 3-4 without human clinical trials.

7. Blood-sucking leeches secrete leech hyaluronidase, which can dissolve the skin and help leeches enter the human body to suck blood. Documents "Enzymatic production of specifically distributed hyaluronanoligosaccharides, Carbohydr Polym, 2015, 129:194-200" and "High-yield novel leechhyaluronidase to expedite the preparation of specific hyaluronan oligomers (2014), Scientific Reports #4 transparent Article 47" The yield, enzymatic cleavage activity, and cost of the urnidase fusion protein expressed in yeast can all meet commercial requirements, and can cut macromolecular hyaluronic acid into small molecular hyaluronic acid with a molecular weight of 10-60KD. However, hyaluronic acid fragments with a molecular weight between 10-60KD formed by cleavage of macromolecular hyaluronic acid by leech hyaluronidase are difficult to confirm that they will have the therapeutic effect shown in Examples 3-4 without human clinical trials. The residue of leech hyaluronidase can cause an allergic reaction, and it needs to be completely removed before it can be used to produce hyaluronic acid injection 2 with the therapeutic effect shown in Examples 3-4 and no allergic side effects. We compared leech hyaluronidase with >99.0% pure glycosylated recombinant human hyaluronidase produced by animal cells Chinese hamster ovary cells. We think that leech hyaluronidase is not suitable for the production of high purity requirements. Example 3- 4 Hyaluronic Acid Injection 1 or 2 with curative effects and no allergic side effects.

Finally, it should be noted that: the above is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, it still The technical solutions described in the foregoing embodiments may be modified, or some technical features thereof may be equivalently replaced. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (23)

1. a kind of parenteral solution containing micromolecule hyaluronic acid, it is characterised in that the preparation of the parenteral solution comprises the following steps：

A. there is the recombined human hyaluronidase PH20 of saccharification using zooblast or yeast or plant Expression product；

B. macromolecular hyaluronic acid solution is configured to using macromolecular hyaluronic acid raw material, introduces sodium chloride and magnesium ion, added The recombined human hyaluronidase PH20 cuts the macromolecular hyaluronic acid, obtains the hyalomitome containing molecular weight 10-60KD The reaction solution of acid fragment；

C. recombined human hyaluronidase PH20 inactivation treatments to being remained in obtained reaction solution；

D. bacteriological filter, virus filtration inactivation are carried out and is concentrated by ultrafiltration.

2. a kind of parenteral solution containing micromolecule hyaluronic acid according to claim 1, it is characterised in that described to prepare The step of journey also includes the recombined human hyaluronidase being inactivated described in removal.

3. a kind of parenteral solution containing micromolecule hyaluronic acid according to claim 1, it is characterised in that the step A In have the recombined human hyaluronidase PH20 purity of saccharification>Macromolecular hyaluronic acid material purity in 99.0%, the step B >99.9%.

4. a kind of parenteral solution containing micromolecule hyaluronic acid according to claim 3, it is characterised in that the step B The molecular weight of middle macromolecular hyaluronic acid raw material is 800-1200KD；The sodium chloride and magnesium ion are in the macromolecular hyalomitome Concentration in acid solution is respectively 80-90mM and 1mM；Every gram of macromolecular hyaluronic acid raw material correspondence adds 20000 units The recombined human hyaluronidase, the endonuclease reaction in 37 degree carry out 6 hours.

5. a kind of parenteral solution containing micromolecule hyaluronic acid according to claim any one of 1-4, it is characterised in that institute State recombined human hyaluronidase residual 0 that every 2-4ml parenteral solutions contain the hyaluronic acid fragments 100mg and the inactivation~ 20ug, concentration of the magnesium ion in parenteral solution is 1mM, and concentration of the sodium chloride in parenteral solution is 115-125mM.

6. a kind of purposes of parenteral solution containing micromolecule hyaluronic acid according to claim any one of 1-5, its feature It is, the parenteral solution is used to treat under muscae volitantes, vitreous opacity and vitreum stripping and eyesight caused by vitreous opacity Drop.

7. a kind of purposes of parenteral solution containing micromolecule hyaluronic acid according to claim any one of 1-5, its feature It is, the parenteral solution, which is used to treat, comes from skin, mucous membrane, connective tissue, muscle, muscular fascia, tendon, joint, wound, scar Pain and from skin, mucous membrane, wound, scar itch.

8. a kind of purposes of parenteral solution containing micromolecule hyaluronic acid according to claim any one of 1-5, its feature It is, the parenteral solution is used to treat that the disease of pain or itch can be caused, including large-area burns, wound cicatrix, operation wound Mouth scar, neurodermatitis, psoriasis, eczema, Kaposi, pompholyx.

9. a kind of purposes of parenteral solution containing micromolecule hyaluronic acid according to claim any one of 1-5, its feature It is, the parenteral solution is used to treat back neck and shoulder fasciitis, injury of muscle and tendon.

10. a kind of purposes of parenteral solution containing micromolecule hyaluronic acid according to claim any one of 1-5, its feature It is, the parenteral solution is used to treat osteoproliferation, spur, disc herniation.

11. a kind of purposes of parenteral solution containing micromolecule hyaluronic acid according to claim any one of 1-5, its feature It is, the parenteral solution is used to treat ankylosing spondylitis.

12. a kind of purposes of parenteral solution containing micromolecule hyaluronic acid according to claim any one of 1-5, its feature It is, the parenteral solution is used to treat the endometriosis for having dysmenorrhoea.

13. a kind of purposes of parenteral solution containing micromolecule hyaluronic acid according to claim any one of 1-5, its feature It is, the parenteral solution is used to treat the abdominal cavity mucous membrane adhesion and pelvic cavity mucous membrane adhesion triggered after Post operation or peritoneal dialysis.

14. a kind of purposes of parenteral solution containing micromolecule hyaluronic acid according to claim any one of 1-5, its feature It is, the parenteral solution is used to treat solid tumor transfer and recurred.

15. a kind of purposes of parenteral solution containing micromolecule hyaluronic acid according to claim any one of 1-5, its feature It is, the parenteral solution is used to increase the recovery of muscle power and energy after muscle power and energy or solid tumor chemotherapy.

16. a kind of purposes of parenteral solution containing micromolecule hyaluronic acid according to claim any one of 1-5, its feature It is, the parenteral solution is used to promote functional rehabilitation after cerebral infarction.

17. a kind of purposes of parenteral solution containing micromolecule hyaluronic acid according to claim any one of 1-5, its feature It is, the parenteral solution is used for beautifying face, the facial aging for the treatment of.

18. a kind of purposes of parenteral solution containing micromolecule hyaluronic acid according to claim any one of 1-5, its feature It is, the parenteral solution is used to reduce head, face, neck subcutaneus adipose tissue or whole body subcutaneus adipose tissue.

19. a kind of purposes of parenteral solution containing micromolecule hyaluronic acid according to claim any one of 1-5, its feature It is, the parenteral solution is used for fast treating periodontal disease, canker sore and pharyngolaryngitis, including tooth caused by chemotherapy and aging All diseases, canker sore and pharyngolaryngitis.

20. a kind of purposes of parenteral solution containing micromolecule hyaluronic acid according to claim any one of 1-5, its feature It is, the parenteral solution is used to treat eye conjunctiva disease, including eye conjunctiva disease caused by chemotherapy and aging.

21. a kind of purposes of parenteral solution containing micromolecule hyaluronic acid according to claim any one of 1-5, its feature It is, the parenteral solution, which is used for treatment, anginal coronary heart disease, treats intravascular stent infectious-related complication, treats angiocardiopathy.

22. a kind of purposes of parenteral solution containing micromolecule hyaluronic acid according to claim 6-21, it is characterised in that The occupation mode of the parenteral solution is abdominal subcutaneous fat layer or pain/itchy points injection location or disease sites injection.

23. a kind of purposes of the parenteral solution containing micromolecule hyaluronic acid according to claim 22, it is characterised in that described Parenteral solution dosage is the daily hyaluronic acid fragments injected 1-2 times, the molecular weight 10-60KD is contained in per injection dosage 100mg。