CN107303271A - A kind of parenteral solution containing micromolecule hyaluronic acid and application thereof - Google Patents

A kind of parenteral solution containing micromolecule hyaluronic acid and application thereof Download PDF

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CN107303271A
CN107303271A CN201610264221.9A CN201610264221A CN107303271A CN 107303271 A CN107303271 A CN 107303271A CN 201610264221 A CN201610264221 A CN 201610264221A CN 107303271 A CN107303271 A CN 107303271A
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hyaluronic acid
parenteral solution
solution containing
acid according
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惠觅宙
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Priority to CN201610264221.9A priority Critical patent/CN107303271A/en
Priority to PCT/CN2016/080948 priority patent/WO2017185383A1/en
Priority to PCT/CN2017/081796 priority patent/WO2017186088A1/en
Priority to CA3049286A priority patent/CA3049286C/en
Priority to AU2017255833A priority patent/AU2017255833B2/en
Priority to US16/754,756 priority patent/US11576925B2/en
Priority to EP17788740.3A priority patent/EP3479830B1/en
Publication of CN107303271A publication Critical patent/CN107303271A/en
Priority to US18/073,604 priority patent/US11839625B2/en
Priority to US18/073,601 priority patent/US11826381B2/en
Priority to US18/073,596 priority patent/US11826380B2/en
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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
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/728—Hyaluronic acid
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/59—Biological synthesis; Biological purification

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  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
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  • Pharmacology & Pharmacy (AREA)
  • Dermatology (AREA)
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  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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Abstract

The invention discloses a kind of parenteral solution containing micromolecule hyaluronic acid and application thereof, the preparation of the parenteral solution comprises the following steps：A. using zooblast or the recombined human hyaluronidase PH20 for having saccharification of 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 recombined human hyaluronidase PH20 and cut, obtain the reaction solution of the hyaluronic acid fragments containing the 60KD of molecular weight 10；C. to the recombined human hyaluronidase PH20 inactivation treatments of residual；D. bacteriological filter, virus filtration inactivation are carried out and is concentrated by ultrafiltration.Parenteral solution of the present invention not only has beautifying face, the effect of facial anti-aging, also unexpectedly shows quickly alleviating pain, antipruritic, increase muscle power and energy, reduces head and neck subcutaneous fat and to a variety of diseases and the therapeutic effect of morbid state.

Description

A kind of parenteral solution containing micromolecule hyaluronic acid and application thereof
Technical field
The present invention relates to disease therapeuticing medicine field, more particularly to a kind of parenteral solution containing micromolecule hyaluronic acid and It is used for the purposes for treating a variety of diseases.
Background technology
Hyaluronic acid, also known as Hyaluronic Acid, uronic acid, sodium hyaluronate, English name hyaluronic acid (abbreviation HA), be by The higher polysaccharides of D-Glucose aldehydic acid and N-acetyl-glucosamine composition, dissacharide units are up to 25000 dongles.Subcutis, The tissue such as epidermis and mucous membrane of oropharynx contains substantial amounts of macromolecular hyaluronic acid.In other words, macromolecular hyaluronic acid is mankind's skin The basic architecture material of the various tissues such as skin, mucous membrane, subcutaneous, with water conservation and moisture-keeping functions.The existing hyaluronic acid production in market Product are mainly to add facial-care or beautifying face product of the macromolecular hyaluronic acid as main composition.
A series of research shows in recent years, and the micromolecule hyaluronic acid and macromolecular according to the size discrimination of molecular weight are saturating Bright matter acid shows difference functionally in toy tests the research with cellular level, and main flow document shows that small molecule is saturating Bright matter acid is the catabolite of inflammation, consistent with inflammation distribution, and its function is initiation and promotes inflammatory reaction, leading reference Including：
[1] .Jaime M.Cyphert, Carol S.Trempus, and Stavros Garantziotis, Size Matters: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 in human 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 signalling 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 ROS production 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-10 in 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 Biol Chem.1997 Mar 21；272(12):8013-8.PMID:9065473.
[10].McKee CM1,Penno MB,Cowman M,Burdick MD,Strieter RM,Bao C,Noble PW.Hyaluronan(HA)fragments induce chemokine gene expression in alveolar macrophages.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,Dorsam GP2,Schuh JM2.Hyaluronan stimulates ex vivo B lymphocyte chemotaxis and cytokine production in a murine model of fungal allergic asthma.Immunobiology.2015 Feb 7.PMID:25698348.
[12].Ghosh S1,Samarasinghe AE,Hoselton SA,Dorsam GP,Schuh JM.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 chemical allergen-induced IL-18 production in human keratinocytes.Toxicol Lett.2014 Oct 1；232(1):89-97.PMID:25280773.
[14].Fieber C1,Baumann P,Vallon R,Termeer C,Simon JC,Hofmann M,Angel P,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,Nastasi G,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,Nastasi G,Calatroni A,Campo S.4-mer hyaluronan oligosaccharides stimulate inflammation response in synovial fibroblasts in part via TAK-1and 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,Leonard M,Kraft M,Noble PW.Role of hyaluronan and hyaluronan-binding proteins in human asthma.J Allergy Clin Immunol.2011Aug；128(2):403-411.PMID:21570715.
And applicant in recent years achievement in research (number of patent application 200780052196.7,201310325056.X, 201310454955.X、201410153593.5、201510065499.9、201510065498.4、201510067326.0、 201510333526.6) but also point out another possibility, i.e. micromolecule hyaluronic acid that there is the function of suppressing inflammation.These texts The function of offering this suppression inflammation of prompting micromolecule hyaluronic acid is relevant with its production method, and also the degree with molecular size has Close, also, the comparing result of zoopery and human experimentation also points out the function of micromolecule hyaluronic acid in toy and human body It is middle possible entirely different.For example, document above shows that preparation in vitro can be made in micromolecule hyaluronic acid (molecular weight 10-60KD) It is local to use (such as commodity, amenities, cosmetics, skin care item, sterilizing article), contribute to treatment skin and catarrh Disease, realize beautifying skin and anti-aging effects, and the micromolecule hyaluronic acid less than 10KD does not have above-mentioned effect substantially.However, Nearest CEDARS-SINAI MEDICAL CENTER patent WO/2014/165713 shows saturating using bacterium streptococci The micromolecule hyaluronic acid less than 10KD that the sour cleavage of bright matter is produced does not cause inflammatory reaction, but bacterium Streptomyces is saturating The micromolecule hyaluronic acid more than 10KD that the sour enzyme of bright matter is produced but causes inflammatory reaction, points out the function of micromolecule hyaluronic acid It is also possible to relevant with the species of nickase.
The exact function of the conflicting result of study prompting micromolecule hyaluronic acid fragment of the above is difficult to really at present It is fixed, and the mode of production of micromolecule hyaluronic acid fragment, molecular size range, nickase species, occupation mode may all have influence on Its function is showed, and at present in addition to these known possible factors, still have that many scientific research personnel can not be certainly other not Master factor.Also therefore, although industry personage places hope on the medical value of micromolecule hyaluronic acid, but remains difficult to small point of prediction The clinical research of sub- hyaluronic acid and the success or not of druggability, are especially difficult to determine clinical indication.And we are first grinds A kind of feasible effective micromolecule hyaluronic acid preparation in vitro is obtained although studying carefully, because the interior environment of human body is sufficiently complex, The internal injection liquid formulation of micromolecule hyaluronic acid may also be entirely different with preparation in vitro, the effect in humans and animals body May not be consistent, therefore, more need the checking of clinical research.Also, the small molecule used as internal injection liquid formulation is transparent Matter acid at least will have higher requirement at many aspects, for example need to meet purity it is high, without allergic reaction, the clear and definite indication of needs And side effect, specify per injection dosage, injecting pathway, injection dosage etc..Solving problems above has great scientific research meaning Justice and commercial value.
The content of the invention
It is an object of the present invention to provide a kind of injection containing micromolecule hyaluronic acid with specific production method Liquid.
It is used to treat a variety of diseases having been surprisingly found that and preparation it is a further object to provide the parenteral solution Treat the purposes of the medicine of a variety of diseases.
To achieve the above object, the present invention is adopted the following technical scheme that：
A kind of parenteral solution containing micromolecule hyaluronic acid, the preparation of the parenteral solution comprises the following steps：A. using dynamic Thing cell or yeast or plant Expression product have the recombined human hyaluronidase PH20 of saccharification；B. it is former using macromolecular hyaluronic acid Material is configured to macromolecular hyaluronic acid solution, introduces sodium chloride and magnesium ion, adds the recombined human hyaluronidase PH20 and cuts The macromolecular hyaluronic acid is cut, the reaction solution of the hyaluronic acid fragments containing molecular weight 10-60KD is obtained；C. to obtaining reaction The recombined human hyaluronidase PH20 inactivation treatments remained in liquid；D. bacteriological filter, virus filtration inactivation are carried out and is concentrated by ultrafiltration.
As a further improvement, the preparation process also includes the recombined human hyaluronidase being inactivated described in removal Step.
There is the recombined human hyaluronidase PH20 purity of saccharification in the step A>Big point in 99.0%, the step B Sub- hyaluronic acid material purity>99.9%.
The molecular weight of macromolecular hyaluronic acid raw material is 800-1200KD in the step B；The sodium chloride and magnesium ion Concentration in the macromolecular hyaluronic acid solution is respectively 80-90mM, 1mM；Every gram of macromolecular hyaluronic acid raw material Correspondence adds the recombined human hyaluronidase of 20000 units, and the endonuclease reaction is carried out 6 hours in 37 degree.
The recombined human hyalomitome for containing the hyaluronic acid fragments 100mg and the inactivation per 2-4ml parenteral solutions Sour enzyme remains 0~20ug, and concentration of the magnesium ion in parenteral solution is 1mM, and concentration of the sodium chloride in parenteral solution is 115-125mM。
The parenteral solution is used to treat muscae volitantes, vitreous opacity and vitreum caused by vitreous opacity is peeled off and eyesight Decline.
The parenteral solution be used for treat from skin, mucous membrane, connective tissue, muscle, muscular fascia, tendon, joint, wound, The pain of scar and from skin, mucous membrane, wound, scar itch.
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 scar, neurodermatitis, psoriasis, eczema, Kaposi, pompholyx.
The parenteral solution is used to treat back neck and shoulder fasciitis, injury of muscle and tendon.
The parenteral solution is used to treat osteoproliferation, spur, disc herniation.
The parenteral solution is used to treat ankylosing spondylitis.
The parenteral solution is used to treat the endometriosis for having dysmenorrhoea.
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.
The parenteral solution is used to treat solid tumor transfer and recurred.
The parenteral solution is used to increase the recovery of muscle power and energy after muscle power and energy or solid tumor chemotherapy.
The parenteral solution is used to promote functional rehabilitation after cerebral infarction.
The parenteral solution is used for beautifying face, the facial aging for the treatment of.
The parenteral solution is used to reduce head, face, neck subcutaneus adipose tissue or whole body subcutaneus adipose tissue.
The parenteral solution is used for fast treating periodontal disease, canker sore and pharyngolaryngitis, including chemotherapy and aging cause Periodontal disease, canker sore and pharyngolaryngitis.
The parenteral solution is used to treat eye conjunctiva disease, including eye conjunctiva disease caused by chemotherapy and aging.
The parenteral solution, which is used for treatment, anginal coronary heart disease, treats intravascular stent infectious-related complication, the cardiovascular disease for the treatment of Disease.
The occupation mode of the parenteral solution is abdominal subcutaneous fat layer or pain/itchy points injection location or disease sites injection.
The parenteral solution dosage is injected 1-2 times to be daily, contains the molecular weight 10-60KD's in per injection dosage Hyaluronic acid fragments 100mg.
Due to using above-mentioned technical proposal, the present invention at least has advantages below：
(1) parenteral solution is convenient for production, preparation stabilization, is not required to facial injection, and pain and facial inflammation are not caused, is passed through The injection of abdominal subcutaneous fat layer can quickly effective beautifying face and facial anti-aging.
(2) parenteral solution of the invention unexpectedly has quickly alleviating pain, the physical energy of antipruritic, increase and reduces head neck The effect of portion's subcutaneous fat.
(3) it is that the parenteral solution of the present invention has the effect for treating a variety of diseases and morbid state, including flesh more magically Fascitis, injury of muscle and tendon, the spur and disc herniation that have pain, the ankylosing spondylitis for having pain, vitreous opacity, Have and trigger after inflammation, the large-area burns scar of scabies itching and pain, wound cicatrix, surgical wound scar, operation or peritoneal dialysis Abdominal cavity mucous membrane adhesion and the adhesion of pelvic cavity mucous membrane, have the endometriosis of dysmenorrhoea, the neurodermatitis for having itch, ox-hide Physical energy recovers and anti-after tinea, old eczema, infantile eczema and the Kaposi of itch or pompholyx, solid tumor chemotherapy Only solid tumor transfer, functional rehabilitation, periodontal disease and canker sore after cerebral infarction, pharyngolaryngitis, eye conjunctiva disease, aging and Diseases associated with senescence, coronary heart disease, myocardial infarction functional rehabilitation, prevention and treatment intravascular stent infectious-related complication and prevention and treatment Cardiovascular and cerebrovascular disease.
Brief description of the drawings
Above-mentioned is only the general introduction of technical solution of the present invention, in order to better understand the technological means of the present invention, below With reference to accompanying drawing, the present invention is described in further detail with embodiment.
(a) (b) is the rip current type zooblast reaction for the recombined human hyaluronidase for having saccharification for producing high-purity in Fig. 1 Device amplifies and large-scale culture workshop.
Fig. 2 is the enzyme of the recombined human hyaluronidase for having saccharification of the high-purity of zooblast (Chinese hamster ovary celI) production.
Fig. 3 is the dosage form of the parenteral solution of the present invention containing micromolecule hyaluronic acid.
Fig. 4 shows the fungistatic effect of molecular weight 10KD-60KD hyaluronic acid fragments.
Fig. 5 shows anti-inflammatory and fungistatic effect of the parenteral solution of the present invention to toy skin wounds.
In Fig. 6 (a)-(e) show respectively the present invention parenteral solution to IL-1, IL-6, IFN-β, IFN-α, IL-10 influence (note:LPS=endotoxin；L+A=LPS+anti-human TLR4single chain antibody；L+HA=LPS+B- HA)。
Fig. 7 is the subject's Ophthalmic laboratory diagnosis peeled off with vitreous opacity and vitreum.
Fig. 8 is that photo comparison shows abdominal subcutaneous fat layer injection parenteral solution of the present invention to people before and after the subject of part The therapeutic effect of body beautifying face and facial anti-aging.
Fig. 9 be use injection for treating large-area burns of the present invention after do not have skin-grafting formed constantly thicken, itch with have The effect of the scar of chronic inflammatory reaction.
Figure 10 is the effect of abdominal subcutaneous fat layer injection injection for treating neurodermatitis of the present invention.
Figure 11 is the abdominal subcutaneous fat layer injection psoriasic effect of injection for treating of the present invention.
Embodiment
Firstly the need of explanation, the existing macromolecular hyaluronic acid injection liquid in market is mainly used in beautifying face, face Anti-aging and joint cavity injection lubricating joint.Parenteral solution of the present invention is the parenteral solution containing micromolecule hyaluronic acid.Base Achievement in research in current industry, the exact function of micromolecule hyaluronic acid is difficult to determine, is based even on current science real Result (seing above described in background technology) is tested, conflicting function phenomenon is occurred in that, and the mechanism of action of this phenomenon is not yet Completely clearly, it may be necessary to which the production method for limiting micromolecule hyaluronic acid carrys out its clear and definite effect.Therefore, micromolecule hyaluronic acid Clinical research and commercialization (i.e. druggability) prospect industry personage also be difficult to prediction.
The parenteral solution containing micromolecule hyaluronic acid that a kind of specific production method that the present invention is provided is obtained, grinds through clinic Study carefully the purposes for finding that inventor unexpectedly obtains many new indications of the injection for treating, this purposes is by more easily noting Penetrate the mode such as injection of abdominal subcutaneous fat layer or pain/itchy points injection location or disease sites injection and emerge from.
Inventor carries out beauty and the research on anti-senescence of the parenteral solution using the method used " outside specification ", anticipates very much It is outer to find that the above-mentioned parenteral solution containing micromolecule hyaluronic acid of the present invention not only effectively be promoted by abdominal subcutaneous fat layer injection Enter beautifying face and facial anti-aging, moreover it is possible to increase muscle power, energy in surprise and reduce head and neck subcutaneous fat, according to using The feedback of person, this parenteral solution of the invention also shows quickly alleviating pain, antipruritic effect, is more magically, further deep The research discovery entered, effect of the parenteral solution of the present invention with a variety of diseases for the treatment of and morbid state, including myofascitis, Injury of muscle and tendon, the spur and disc herniation that have pain, the ankylosing spondylitis for having pain, vitreous opacity, have inflammation, Glue in the abdominal cavity triggered after large-area burns scar, wound cicatrix, surgical wound scar, operation or the peritoneal dialysis of scabies itching and pain Film coalescence and the adhesion of pelvic cavity mucous membrane, there are the endometriosis of dysmenorrhoea, the neurodermatitis for having itch, psoriasis, itch Physical energy recovers and prevents solid from swelling after old eczema, infantile eczema and Kaposi or pompholyx, solid tumor chemotherapy Functional rehabilitation, periodontal disease are related to aging to canker sore, pharyngolaryngitis, eye conjunctiva disease, aging after tumor metastasis, cerebral infarction Disease, coronary heart disease, prevention and treatment intravascular stent infectious-related complication and angiocardiopathy.
Parenteral solution of the present invention containing micromolecule hyaluronic acid, is obtained by specific production method.Institute Stating micromolecule hyaluronic acid is given birth to by recombined human hyaluronidase PH20, sodium ion and magnesium ion activated macromolecules hyaluronic acid The hyaluronic acid fragments for having therapeutic efficiency of production；Hyaluronic acid fragments molecular weight contained by the hyaluronic acid injection liquid is 10KD- 60KD；The molecular weight is by zooblast Chinese hamster ovary cells for 10KD-60KD hyaluronic acid fragments Or yeast or plant Expression product has the recombined human hyaluronidase of saccharification when 36-37 degree has magnesium ion and sodium chloride Cut what macromolecular hyaluronic acid was obtained for 5-6 hours；The recombined human hyaluronidase of the parenteral solution is by 80 degree of 45-60 minutes warm Inactivation.To ensure the security of preparation, the preferred high-purity (purity of recombined human hyaluronidase PH20 for having a saccharification> 99.0% is preferred), the macromolecular hyaluronic acid material purity uses medical grade purity>99.9% is preferred., can during actual production Directly to use the raw material of high-purity, the recombined human hyaluronidase PH20 or big for having saccharification to low-purity can also be passed through Molecular weight hyaluronic acid raw material is carried out subsequent step again after purification.With reference to clinical research, it has been found that transparent containing recombined human The hyaluronic acid fragments abdominal subcutaneous fat layer or pain spot injection location or disease sites of matter acid enzyme residual protein are injected without allergy React and have no side effect.
Further, using affinity column chromatography, precipitation, ultrafiltration and dialysis process, by the recombined human hyaluronidase of inactivation PH20 residual proteins are separated, and obtain comprising only the injection of 10KD-60KD hyaluronic acid fragments, sodium chloride and magnesium ion Liquid.With reference to Human Clinical Study, it has been found that subcutaneous layer of fat or the injection of pain spot injection location or disease sites contain and be free of The parenteral solution of the recombined human hyaluronidase PH20 residual proteins of the inactivation has therapeutic action, all without allergic reaction and without pair Effect, the hyaluronic acid fragments for pointing out 10KD-60KD are main active.
Further, according to aforementioned production method, the injection formulation of the invention containing micromolecule hyaluronic acid is at least The recombined human hyaluronidase PH20 residual proteins without inactivation in following two dosage forms, a kind of preparation can be made, separately A kind of then recombined human hyaluronidase PH20 residual proteins containing non-enzymatic activity.It is preferred that to make the weight containing non-enzymatic activity The injection formulation therapeutic effect of group people's hyaluronidase PH20 residual proteins is more reliable, and this residual egg is controlled in actual production It is less than 20 micrograms in vain.
As preferred embodiment, the present invention prepares following parenteral solution 1 (per 2-4ml according to above two dosage form Recombined human hyaluronidase containing the hyaluronic acid fragments 100mg that molecular weight is 10KD-60KD and the non-enzymatic activity of inactivation PH20 residual proteins are less than 20 micrograms, and sodium chloride concentration is that 115-125mM, magnesium ion concentration are 1mM in parenteral solution) and parenteral solution 2 (contain the hyaluronic acid fragments 100mg that molecular weight is 10KD-60KD per 2-4ml, sodium chloride concentration is 115- in parenteral solution 125mM, magnesium ion concentration are 1mM, the recombined human hyaluronidase PH20 residual proteins without non-enzymatic activity).The parenteral solution Can be in 4 to 12 degree or -8 to -70 degree and room temperature storage 2 years stably.
Further clinical verification to above two preparation finds that subcutaneous or pain/itchy points injection location or disease sites are noted The parenteral solution is penetrated, it is daily to inject 1-2 times, the hyaluronic acid fragments 100mg is contained in per injection liquor amount, easily and effectively, Have no side effect and allergic reaction, available for preparing biologics and 3 class medicine equipments.
Below by way of specific embodiment, the present invention will be described, it will be appreciated that embodiment described herein is only used In the description and interpretation present invention, it is not intended to limit the present invention.
Embodiment 1
Purpose：The production technology of parenteral solution is studied, manufacture respectively contains and without recombined human hyaluronidase PH20 residuals The parenteral solution of albumen.
Method：The recombined human hyaluronidase PH20 for having saccharification is produced using zooblast (Chinese hamster ovary celI), including：To have The recombined human hyaluronidase PH20 of saccharification gene cDNA is artificial synthesized, pMH3, pMH4, pMH5 null representation of the insertion rich in GC Carrier, builds up pMH3-PH20, pMH4-PH20, pMH5-PH20 expression vector and (uses notification number here for CN102124019A The technique construction expression vector recorded of patent document highly to express recombinant protein)；Again by pMH3-PH20, pMH4- PH20, pMH5-PH20 cDNA expression vectors are transferred to CHO-S cell lines, the high expression PH20 of screening CHO-S cell lines, in Fig. 1 Shown rip current type zooblast scaling -up and large-scale culture；It will be used after good harvest liquid 0.22um filterings containing PH20 The 2-3 such as ion column step separation, then after the step such as bacteriological filter and virus filtration inactivation, ultrafiltration concentration, purity shown in Fig. 2 is made >The 99.0% sterile virus-free recombined human hyaluronidase PH20 for having saccharification.
Prepare injection stage molecular weight for 800-1200KD hyaluronic acid raw material or hyaluronic acid injection liquid or will be non-injection Level molecular weight 800-1200KD hyaluronic acid feed purification is to injection stage hyaluronic acid raw material；By injection stage molecular weight 800- 1200KD hyaluronic acid raw material adds the pure water of injection stage once or several times, sequentially add sodium chloride 80-90mM, magnesium from Sub- 1mM, the unit of recombined human hyaluronidase 20000 (about 20 microgram)/every gram of hyaluronic acid, are sufficiently mixed, small in 37 degree of reactions 6 Molecular weight up to hyaluronic acid fragments reaches that osmotic pressure is adjusted to 280-300 millis and oozed by 10KD-60KD, plus sodium chloride 35-45mM Amount/liter (mOsm/L), then it is heated 80 degree 45-60 minutes, bacteriological filter and virus filtration inactivation, be concentrated by ultrafiltration etc. step be made Hyaluronic acid injection liquid 1 is (per 2-4 milliliter containing hyaluronic acid fragments 100mg of the molecular weight for 10KD-60KD, also containing whetheing there is drop Recombined human PH20 hyaluronidases residual below the 20ug of hyaluronic acid activity is solved, sodium chloride concentration 115-125mM in parenteral solution, Magnesium ion concentration 1mM).Sequentially or separately use recombined human hyaluronidase affinity column chromatography, column chromatography, precipitation, dialysis, super Filter concentration etc., the recombined human hyaluronidase of the hyaluronic acid fragments parenteral solution remained containing recombined human hyaluronidase is remained Albumen is removed, and the hyaluronic acid injection liquid 2 that manufacture does not contain recombined human hyaluronidase residual protein (contains per 2-4 milliliters and divided Sodium chloride content 115-125mM, magnesium ion content in the hyaluronic acid fragments 100mg that son amount is 10KD-60KD, parenteral solution 1mM)。
As a result：
Fig. 2 is purity>The electricity of the recombined human hyaluronidase for having saccharification of 99% zooblast (Chinese hamster ovary celI) production Swimming figure；
The hyaluronic acid injection liquid 1 and the preparation of hyaluronic acid injection liquid 2 of manufacture are as follows：
Hyaluronic acid injection liquid 1：Molecular weight is 10KD-60KD 100mg/2 milliliters of hyaluronic acid fragments, sodium chloride 115- 125mM, magnesium ion 1mM, below the recombined human PH20 hyaluronidase residual proteins 20ug (endogenous toxic materials without degraded hyaluronic acid activity Element is less than 0.5IU/ml, sterile, virus-free).
Hyaluronic acid injection liquid 2：Molecular weight is 10KD-60KD 100mg/2 milliliters of hyaluronic acid fragments, sodium chloride 115- 125mM, magnesium ion 1mM (endotoxin be less than 0.5IU/ml, sterile, virus-free).
Conclusion：As a result show successfully to have manufactured the hyaluronic acid piece contained and without recombined human hyaluronidase residual protein The hyaluronic acid injection liquid 1 (Fig. 3) and parenteral solution 2 of section, specify the production technology of parenteral solution.
Embodiment 2
Purpose：Experiment in vitro, molecular cytology experiment, the toy for studying the hyaluronic acid injection liquid 1 of embodiment 1 are tested Anti-inflammatory and fungistatic effect.
Method：Using 1 times of the hyaluronic acid injection liquid 1 of embodiment 1 of normal saline dilution, the antibacterial reality of experiment in vitro is done respectively Test, molecular cytology is tested, toy experiment, detects the anti-inflammatory and fungistatic effect of hyaluronic acid injection liquid.
As a result：Fig. 4 shows that hyaluronic acid injection liquid inhibits suppression gum puff quinoline monad clonal growth in experiment in vitro, Prompting hyaluronic acid injection liquid material is unfavorable for gum puff quinoline unit cell bacteria growing (note：Negative control is physiological saline, positive control For antibiotic metronidazole)；Fig. 5 shows anti-inflammatory and fungistatic effect of the hyaluronic acid injection liquid to toy skin wounds；Fig. 6 shows Show that hyaluronic acid injection liquid promotes people TLR4 receptor positive cells secretion IL-10, while inhibiting endotoxin (endotoxin) Caused TNF, IL-1, IL-6 and interferon beta secretion (note：LPS=endotoxin；L+A=LPS+anti- human TLR4 single chain antibody；L+HA=LPS+B-HA).With reference to literature research, there is article (Cuixia Yang,Manlin Cao,Hua Liu,Yiqing He,Jing Xu,Yan Du,Yiwen Liu,Wenjuan Wang,Lian Cui,Jiajie Hu,Feng Gao,The high and low molecular weight forms of hyaluronan have distinct effects on CD44 Clustering,Published on November 1,2012as Manuscript M112.349209) show that CD44 acceptors may also take part in the mechanism of action of hyaluronic acid injection liquid.
Conclusion：Hyaluronic acid injection liquid has obvious human body cell anti-inflammatory, toy skin wounds anti-inflammatory and antibacterial effect, Its anti-inflammatory is antibacterial to be realized by number of ways, including Endotoxin Receptors, beta-defensin secretion, the inflammation of CD44 mediations Disease cell is removed and assembled.
Embodiment 3
Purpose：Study in above example 1 containing (experimental group 1) and without (experimental group 2) recombined human hyaluronidase The human body face beautifying and anti-aging effect and safety of two kinds of hyaluronic acid injection liquid of the hyaluronic acid fragments of PH20 residual proteins Property.
Method：Use the parenteral solution (molecule of the hyaluronic acid fragments containing recombined human hyaluronidase PH20 residual proteins Measure the hyaluronic acid fragments 100mg for 10KD-60KD, sodium chloride 115-125mM, magnesium ion 1mM, without degraded hyaluronic acid activity Recombined human hyaluronidase residual protein be less than 20 micrograms) be experimental group 1, to Chinese current air pollution, contaminated drinking water and In the big environment of operating pressure various diseases subclinical problem is multiple or aging and diseases associated with senescence multiple 38 by Examination person (average age 61 ± 26 years old, man 20, female 18) carries out clinical research；Remained using without recombined human hyaluronidase (molecular weight is 10KD-60KD hyaluronic acid fragments 100mg, sodium chloride 115-125mM to the hyaluronic acid fragments parenteral solution of albumen With magnesium ion 1mM) it is experimental group 2, (molecular weight contained in pointed injection liquid is 10KD-60KD hyalomitome to each 100mg Acid fragment 100mg, similarly hereinafter) abdominal subcutaneous fat layer injection, daily 1-2 times, continuous injection at least more than 7 days, to Chinese current Various diseases or subclinical problem is multiple or aging and aging phase in air pollution, contaminated drinking water and the big environment of operating pressure 38 multiple subjects of related disorders (average age 62 ± 21 years old, man 19, female 19) carry out clinical research；
Two kinds of parenteral solution cosmetic results are contrasted using the method for face observation or photo comparison's scoring before and after subject (scoring：It is invalid, not can determine that, significant effective), using the side of doctor's inquiry impression, symptom, physical examination and laboratory examination Uncomfortable before and after the presence or absence of the security of method evaluation parenteral solution, i.e. the presence or absence of side effect, including injection site pain, injection, Energy muscle power situation, the original pain of body, the change situation of the original disease of body and the original various subclinical problems of body.
As a result：
Table 1：The effect of two kinds of injection for treating beautifying face anti-agings
It is invalid It not can determine that Significant effective Sample number
Experimental group 1 0 0 38 38
Experimental group 2 0 0 38 38
Photo comparison before and after Fig. 8 display portion subjects, display hyaluronic acid injection liquid to human body face beauty and The therapeutic effect of facial aging, also show hyaluronic acid injection liquid by abdominal subcutaneous fat layer injection significantly reduce head, face, Neck, shoulder, the back of the body, abdomen and upper and lower extremities position subcutaneus adipose tissue thickness.
Table 2：Two kinds of parenteral solutions reduce the effect of subcutaneous fat
It is invalid It not can determine that Significant effective Sample number
Experimental group 1 2 6 30 38
Experimental group 2 1 6 31 38
Table 3：Pain after the injection of two kinds of parenteral solution abdominal subcutaneous fat layer depth portions, local red and swollen, locally and systemically allergy There is nil case
Nothing It not can determine that Have Sample number
Experimental group 1 38 0 0 38
Experimental group 2 38 0 0 38
In addition to above cosmetic result, safety and side effect, it has been found that 3 subjects are because of fatigue, in subcutaneous abdomen fat Feel comfortable after fat layer injection and have confusing；Confusing continues 0.5-1.5 hours, wherein nap 1.0 is small after 1 injection for the first time When, spirit and muscle power substantially increase after waking up；It is another 2 injection 2 times after no longer inject after confusing.
Table 4：The side effect profile of confusing after two kinds of parenteral solution injections
Nothing It not can determine that Have Sample number
Experimental group 1 36 0 2 38
Experimental group 2 37 0 1 38
Out of a clear sky, 71 subjects are equal after 30 minutes after abdominal subcutaneous fat layer injection in all 76 subjects Feel that energy muscle power starts increase, show as being ready to speak, wash the dishes, cook, taking a walk, planning to travel outdoors, work and be not desired to stop Breath etc., continues 3-4 days.
Table 5：The spiritual and physical increased effect of two kinds of parenteral solutions
Nothing It not can determine that Substantially have Sample number
Experimental group 1 0 3 35 38
Experimental group 2 0 2 36 38
There are 27 subjects there are different shoulders in all 76 subjects (including 20 of experimental group 1,17 of experimental group 2) Waist-leg arm pain is carried on the back, out of a clear sky, pain relief is felt after 30 minutes after abdominal subcutaneous fat layer injection, wherein 9 chase after Plus local pain spot position 50mg injections, pain relief starts to occur after injection for 2 minutes, and wherein 4 readmes former part The effect of lidocaine mixing prednisone injection closed treating is suitable.
Table 6：The analgesic effect of hyaluronic acid injection liquid
There are 33 subjects there are different journeys in all 76 subjects (including 17 of experimental group 1,16 of experimental group 2) The itch of degree, out of a clear sky, pain relief or disappearance is felt after abdominal subcutaneous fat layer injection after 1 day.
Table 7：The antipruritic effect of hyaluronic acid injection liquid
Conclusion：
1st, contain and hyaluronic acid fragments without recombined human hyaluronidase residual protein two kinds of hyaluronic acid injections Liquid has the effect of human body face beauty and facial anti-aging.
2nd, contain and two kinds of parenteral solutions of hyaluronic acid fragments without recombined human hyaluronidase residual protein are all obvious Reduce head neck shoulder back of the body abdomen upper and lower extremities position subcutaneus adipose tissue thickness.
3rd, contain and hyaluronic acid fragments without recombined human hyaluronidase residual protein two kinds of parenteral solution belly skins Pain after injection of skin, local redness, locally and systemically allergy are not observed in lower fat deposit injection, easy to use.
3rd, contain and hyaluronic acid fragments without recombined human hyaluronidase residual protein two kinds of parenteral solution belly skins Lower fat deposit feels comfortable after being injected at injection for the first time, there is confusing, and sleepy is slept, and incidence is less than 4.0%.
4th, out of a clear sky, contain and hyaluronic acid fragments without recombined human hyaluronidase residual protein two kinds of notes Penetrating the layer injection of liquid abdominal subcutaneous fat has the effect of obvious increase energy muscle power.
5th, out of a clear sky, contain and hyaluronic acid fragments without recombined human hyaluronidase residual protein two kinds of notes Penetrating the layer injection of liquid abdominal subcutaneous fat has quick obvious analgesic effect.
6th, out of a clear sky, contain and hyaluronic acid fragments without recombined human hyaluronidase residual protein two kinds of notes Penetrating the layer injection of liquid abdominal subcutaneous fat has obvious antipruritic effect.
Embodiment 4
Purpose：" the disease that totally 76 subjects are suffered from of embodiment 3 " before and after further analysis hyaluronic acid injection liquid is treated The change situation of sick and subclinical problem, specifies the new clinical indication of hyaluronic acid injection liquid.
Method：" the disease that totally 76 subjects are suffered from of embodiment 3 " before and after further analysis hyaluronic acid injection liquid is treated Change before the change situation of sick and subclinical problem, including injection after medical history, laboratory examination results and injection, is specified transparent The possible new clinical indication of matter acid injection.
As a result：
First, rapid, effective treatment myofascitis and outmoded injury gained in sports：Myofascitis and outmoded injury gained in sports are that motion is damaged Wound and old degenerative are diseases related, and pathogenesis not exclusively understands that there is the occupation for specifically treating this disease in the U.S. Chiropractor, there is presently no the method for healing.
We analyze altogether suffers from back 7 (4 of experimental group 1 of neck upper and lower extremities myofascitis subject in 76 subjects 3 of example and experimental group 2) and outmoded injury gained in sports 4 (2 of experimental group 1 and 2 of experimental group 2), as a result as shown in table 8：
Table 8：Hyaluronic acid injection liquid treats myofascitis and the effect of outmoded injury gained in sports
Wherein 1 57 years old male subject suffered from nape myofascitis 3 years because of outmoded injury gained in sports 17 years, and annual weather is trembled with fear Broken out when cold, pain torments people very much, and certain muscular movement, which is involved, can also trigger pain, nape scope of activities is limited.Belly skin Lower 100mg of fat deposit feels pain relief after injecting 30 minutes, later additional nape local pain spot position 100mg deeps note Penetrate better.Injected in 2 months after 15x100mg, back pain spot tubercle disappears, nape part muscle thickness is substantially thinning, 12 The moon does not recur again.As a result show, hyaluronic acid injection liquid finally solves outmoded injury gained in sports and myofascitis in addition to analgesic Inflammatory disorderses, have reached the purpose of muscle and muscular fascia restructuring regeneration.
2nd, hyaluronic acid injection liquid rapid, effective treatment spur and disc herniation or back cervical spondylopathy：Spur and intervertebral Disk is protruded or back cervical spondylopathy is that a kind of old degenerative and injury gained in sports are diseases related, and pathogenesis not exclusively understands, mesh The preceding method do not cured also.We analyze altogether suffers from back neck osteoproliferation and spur subject 11 in 76 subjects (6 of experimental group 1 and 5 of experimental group 2), as a result as shown in table 9：
Table 9：Hyaluronic acid injection liquid treats the effect of spur and disc herniation or back cervical spondylopathy
Wherein 1 57 years old female subjects suffer from back neck osteoproliferation and spur 14 years, when break out, pain is tormented very much People, certain muscular movement, which is involved, can also trigger pain, and nape scope of activities is limited, and upper limbs is numb and ability to work declines.Belly Readme pain mitigates more than 50% after subcutaneous layer of fat 100mg is injected one day, and can arrive kitchen and make dumplings and cook；Second day Readme pain mitigates more than 80% after 100mg injections；Readme pain mitigates more than 95% after 100mg injections in 3rd day.2 Do not recurred again within 2 months after injection 15x100mg in month.As a result show, hyaluronic acid injection liquid finally starts outmoded in addition to analgesic The inflammatory disorderses restructuring regeneration of damage.
Wherein 57 years old female subjects of another example suffer from neck osteoproliferation and spur 4 years, and nape scope of activities is limited, Shi Fa Make, pain, which is tormented, people and has facial numbness sense, and certain muscular movement, which is involved, can also trigger pain, upper limbs is numb and ability to work under Drop.Readme pain mitigates more than 50% after abdominal subcutaneous fat layer 100mg is injected one day；Readme after 100mg injections in second day Pain mitigates more than 80%.Do not recurred again within 2 months after injecting 15x100mg in 2 months, show hyaluronic acid injection liquid except only Pain is outer, also begins to the restructuring regenerative process of the inflammatory disorderses of old injury.Note：All of above subject's continual cure, waits 6 The result for recurring or not recurring more than month.
3rd, hyaluronic acid injection liquid rapid, effective treatment vitreous opacity：Vitreous opacity is a kind of old degenerative disease Disease, pathogenesis fails to understand that there is presently no effective treatment method.Vitreous opacity is also known as muscae volitantes, is further developed into glass Glass body is peeled off, and is damaged eyeground and is affected one's power of vision.We analyze in 76 subjects with vitreous opacity and vitreum stripping altogether From subject 9 (4 of experimental group 1 and 5 of experimental group 2), as a result as shown in table 10：
Table 10：Hyaluronic acid injection liquid treats the effect that vitreous opacity and vitreum are peeled off
61 years old female subjects one, Fig. 7 is the subject room diagnosis peeled off with vitreous opacity and vitreum, Because needing face-beauty therapeutic, receive the 100 milligrams of injection treatment of hyaluronic acid abdominal subcutaneous fat layer, accidentally send out within the 2nd day after injection Lose face portion's eyesight improving, find within the 3rd day that eye flies mosquito spot and diminished；Continue using 15x100 milligrams of injections in every three days once, depending on Power continue to improve (being improved to 0.5 by 0.2) and winged mosquito spot continue it is thinning shoal, in two months without it is obvious repeatedly.Result above Show that the surprised rapid, effective treatment vitreous opacity (muscae volitantes) of hyaluronic acid injection liquid and vitreum are peeled off.
4th, the treatment of hyaluronic acid injection liquid has the wound scar of itch (chronic inflammation) and pain (inflammatory pain)：Have The slight wound of scar constitution people can gradually form large-scale scar, constantly thicken and itch, and therapeutic purpose is antipruritic and reduction scar Trace is thickened；The scar for not having skin-grafting to be formed after large-area burns is constantly thickened, and itch and has chronic inflammatory reaction (Fig. 9), is treated Target is antipruritic, diminishing inflammation and reducing scar thickens；Surgical wound be mainly redness, pain, inflammatory reaction ooze out and after Hair infection, therapeutic purpose is that healing acceleration and reduction scar are formed.We analyze (chronic with itch in 76 subjects altogether Inflammation) and pain (inflammatory pain) wound scar subject 8 (4 of experimental group 1 and 4 of experimental group 2), as a result As shown in table 11：
Table 11：The treatment of hyaluronic acid injection liquid has the wound scar of itch (chronic inflammation) and pain (inflammatory pain) Effect
Women lower limb large-area burns before 12 years in 63 years old, do not make skin graft, there is inflammation on serious itch, difficulty falling asleep, scar periphery. Rear scar is thinning for three days on end, scar periphery inflammation disappears, itch substantially mitigates, pain substantially subtracts by hyaluronic acid injection 2x100mg Gently；Hyaluronic acid is continuously injected scar and surrounding skin after 30x100mg and is obviously improved, including scar is thinning, inflammation disappears, scabies Itch mitigation, but thoroughly do not disappear (Fig. 9).
5th, the abdominal cavity mucous membrane adhesion that triggers after the operation of hyaluronic acid injection liquid rapid, effective treatment or peritoneal dialysis：Operation Damage, abdominal dialysis etc. almost cause abdominal cavity mucosal inflammation and adhesion, according to the difference of light and heavy degree can trigger symptom and Pathological change, including stomachache, abdominal tenderness, abdominal mass and intestinal obstruction.At present, also abdominal cavity is eradicated without feasible method to glue Even.We analyze 3 (1 of experimental group 1 of subject with pneumoretroperitoneum mucous membrane adhesion of performing the operation or dialyse in 76 subjects altogether 2 of example and experimental group 2), as a result as shown in table 12：
Table 12：The abdominal cavity adhesion triggered after the operation of hyaluronic acid injection liquid rapid, effective treatment or peritoneal dialysis
The big portion's excision of 43 years old women stomaches and peripheral lymph node are cleared away latter month, are started into after non-liquid food, it is found that belly has Lump and pain, vomiting after eating, seriously become thin with it is in poor health, through clinical diagnosis be abdominal cavity adhesion trigger intestinal obstruction.It is transparent After matter acid abdominal subcutaneous fat layer injection 5x100mg, above sings and symptoms all disappears, and enters after non-liquid food and to be sent out again without intestinal obstruction Raw, the state of mind is clearly better.Treatment results show that hyaluronic acid injection treatment may be by reducing the inflammation at abdominal cavity mucous membrane Disease, has been finally reached elimination abdominal cavity adhesion sings and symptoms.
6th, the treatment of hyaluronic acid injection liquid has endometriosis or the pelvic cavity mucous membrane adhesion of dysmenorrhoea：Endometrium is different Position disease is often accompanied by infertile and dysmenorrhoea；It is infertile may be relevant with adhesion with pelvic cavity mucosal inflammation with dysmenorrhoea；Treatment pain Through with endometriosis or the adhesion of pelvic cavity mucous membrane and there is no effective ways；We analyze altogether suffers from basin in 76 subjects The subject 3 (2 of experimental group 1 and 1 of experimental group 2) of transmucosal adhesion and dysmenorrhoea, as a result as shown in table 13：
Table 13：Hyaluronic acid injection liquid treats endometriosis or the pelvic cavity mucous membrane adhesion for having dysmenorrhoea
Result above shows that hyaluronic acid injection substantially alleviates dysmenorrhoea, shows that hyaluronic acid injection is reduced sub in pelvic cavity The degree of referred pain caused by the degree of inflammation and pelvic adhesion of Endometriosis, points out hyaluronic acid injection liquid to use Come treat tubal peristalsis caused by endometriosis and pelvic adhesion it is bad cause it is infertile.
7th, the treatment of hyaluronic acid injection liquid has the old eczema of the neurodermatitis, psoriasis, itch of itch, bleb sample Eczema or pompholyx：We analyze altogether suffers from neurodermatitis, psoriasis, old eczema, the blister of itch in 76 subjects Rash sample eczema or pompholyx subject 19 (9 of experimental group 1 and 10 of experimental group 2), as a result as shown in table 14：
Table 14：Hyaluronic acid injection liquid treats the neurodermatitis and psoriasic effect for having itch
Wherein one neurodermatitis subject (man, 57 years old) in table 14 suffers from neurodermatitis 8 years, and bilateral symmetric property is more Locate cutaneous lesions, serious itch and local dermal inflammatory skin damaged.Controlled before using Stelara (anti-IL-12/23 antibody) injections Treat, effect subject is unsatisfied with.Disappeared substantially using itch after the layer injection of hyaluronic acid injection liquid 20x100mg abdominal subcutaneous fats Lose, dermal inflammatory skin damaged is recovered (Figure 10) substantially, effect subject satisfaction.
Wherein one psoriasis subject (man, 52 years old) in table 14 suffers from psoriasis 18 years, belly, upper limbs and lower limb many places Thickened at the inflammatory skin damaged of local skin and skin damaged, with pruritis.Once using Stelara, (anti-IL-12/23 resists before 2 years Body) treatment is injected, effect subject is unsatisfied with.After the layer injection of hyaluronic acid injection liquid 10x100mg abdominal subcutaneous fats, Itch disappears substantially, thickens that skin damaged is substantially thinning, and dermal inflammatory skin damaged is substantially recovered (Figure 11), effect subject satisfaction.
Table 15：The treatment of hyaluronic acid injection liquid has the old eczema of itch and the effect of Kaposi or pompholyx
Wherein one pompholyx subject (female, 33 years old) in table 15 suffers from pompholyx 9 years, annual summer morbidity.Using saturating After bright matter acid injection 20x100mg abdominal subcutaneous fats layer injection, pompholyx does not occur again within 1 year during injection and after injecting.
The result of table 14,15 shows that the layer injection of hyaluronic acid injection liquid abdominal subcutaneous fat can effectively treat the god of itch Through property dermatitis, psoriasis, itch old eczema, Kaposi or pompholyx.
8th, hyaluronic acid injection liquid fast treating periodontal disease, canker sore, laryngopharyngitis, eye conjunctiva inflammation：Periodontitis Disease, canker sore, laryngopharyngitis, eye conjunctiva inflammation the elderly are multiple, are diseases associated with senescence, also dirty with Chinese air at present Dye, contaminated drinking water and the big environmental correclation connection of operating pressure.We analyze altogether suffers from periodontal disease, oral cavity in 76 subjects Ulcer, laryngopharyngitis, eye conjunctiva inflammation subject 26 (16 of experimental group 1 and 10 of experimental group 2), as a result such as the institute of table 16 Show：
Table 16：Hyaluronic acid injection liquid treatment periodontal disease, canker sore, laryngopharyngitis, the effect of eye conjunctiva inflammation
The result of table 16 shows that our former local use hyaluronic acid injection liquid of hyaluronic acid injection liquor ratio are coated with more Quickly treat periodontal disease, canker sore, laryngopharyngitis, the effect of eye conjunctiva inflammation.
9th, hyaluronic acid injection liquid effectively treats ankylosing spondylitis：Ankylosing spondylitis is inflammatory pain and backbone The disease of serious modification.We analyze 2 (1 of experimental group 1 of subject that ankylosing spondylitis is suffered from 76 subjects altogether 1 of example and experimental group 2), as a result as shown in table 17：
Table 17：Hyaluronic acid injection liquid treats the effect of ankylosing spondylitis
Ankylosing spondylitis is the disease of inflammatory pain, and the analgesic effect of hyaluronic acid injection liquid treatment shows tatanic The inflammation decline of rachitis is eliminated, and the serious modification of backbone caused by prompting inflammation will not continue to development.
Tenth, hyaluronic acid injection liquid recovers for physical energy after solid tumor chemotherapy and prevents solid tumor from shifting：I Analyze altogether have in 76 subjects needed after solid tumor chemotherapy physical energy recover and prevent that solid tumor from shifting by Examination person 4 (4 of experimental group 1 and 4 of experimental group 3), as a result as shown in table 18：
Table 18：Physical energy recovers and prevents what solid tumor from shifting after hyaluronic acid injection liquid treatment solid tumor chemotherapy Effect
Carcinoma of head of pancreas is a kind of very pernicious tumour, is quickly grown after transfer, dead in most fast 90 days.External research It is probably the important promoting factor of carcinoma of head of pancreas development and transfer to show inflammation.Subject (man, 46 after head of pancreas cancer chemotherapy in table 18 Year) because jaundice finds that carcinoma of head of pancreas is transferred to liver, block bile duct and cause jaundice, underwent operative is put into support and nasobiliary drainage solution Jaundice of having determined problem, monthly chemotherapy, while using treatment by Chinese herbs.Using hyaluronic acid injection liquid 5x100mg abdominal subcutaneous fats Layer injection, physical energy recovers；Ultrasonic examination shows pancreas after the layer injection of hyaluronic acid injection liquid 20x100mg abdominal subcutaneous fats Head cancer is started zooming out with the enclosed mass for being transferred to liver, and subject patient's normal work was more than 90 days.Result above shows hyalomitome Acid injection can promote physical energy after treatment solid tumor chemotherapy to recover and may prevent carcinoma of head of pancreas by suppressing inflammation Development and further transfer.Foreign countries report (Halozyme Receives Orphan Drug Designation For PEGylated Recombinant Human Hyaluronidase PH20For Pancreatic Cancer at： http：//www.halozyme.com/Investors/News-Releases/News-Release-Details/2014/Hal ozyme-Receives-Orphan-Drug-Designation-For-PEGylated-Recombinant-Huma n- Hyaluronidase-PH20-For-Pancreatic-Cancer/default.aspx#sthash.aLjvxToQ.dp uf) Long-acting hyaluronidase PEGPH20 combinations chemotherapy effectively extends the life span of Pancreatic neoplasms patient, and its mechanism of action may It is to produce the similar parenteral solution of the present invention containing micromolecule hyaluronic acid using hyaluronic acid in human body.
Subject (female, 62 years old) is because of lymphatic metastasis chemotherapy and radiation, body pole after oophoroma operation chemotherapy in table 18 Degree is weak, lies in bed within 24 hours.Using the layer injection of hyaluronic acid injection liquid 20x100mg abdominal subcutaneous fats, physical energy injection Start within second day after 1x100mg to recover, all injected CA125 after 20x100mg and dropped in surprise from 500U/ml before treatment 50U/ml, tumour and transfer enclosed mass reduce, and show that hyaluronic acid injection liquid can promote physical energy after treatment chemotherapy in ovarian cancer Recover and the development and further transfer of oophoroma may be prevented by suppressing inflammation.
64 years old female subjects in table 18 are diagnosed as right lung lung cancer, with left lung, brain, bone, vertical phrenic lymph nodes transfer, Can not surgical resection therapy, employ radiotherapy and Erlotinib chemotherapy.In poor health, hyaluronic acid after subject radiation therapy and chemotherapy Parenteral solution 10x100mg abdominal subcutaneous fats layer injection, physical energy daystart after injection 1x100mg recovers, all note Shoot away and find tumour through CT examination after 10x100mg and shift enclosed mass diminution, show that hyaluronic acid injection liquid can promote lung cancer to put Treat and recover with physical energy after chemotherapy, it is also possible to prevent the development of lung cancer by suppressing inflammation and further shift.
11, recurred after hyaluronic acid injection liquid treatment meningiomas surgery：We analyze in 76 subjects altogether brain The subject 2 (1 of experimental group 1 and 1 of experimental group 2) of film knurl recurrence after operation, as a result as shown in table 19：
Table 19：The effect recurred after hyaluronic acid injection liquid treatment meningiomas surgery
A female subjects (61 years old) in table 19, takes place frequently, meningioma, the same year hand is diagnosed as through CT for 2007 because of headache Postoperative, headache disappears.Start headache within 2015 to take place frequently, being diagnosed as recurrence of Meningiomas, i.e. meninx through CT substantially thickens.Hyaluronic acid Parenteral solution abdominal subcutaneous fat layer injection 15x100mg, Headache attacks are reduced, last to terminate completely, before CT examination result shows The meninx of hyperplasia is substantially thinning, and there is no headache breaking-out within 90 days after treatment.Result above shows that hyaluronic acid injection liquid can be used In recurrence after treatment meningioma or meningiomas surgery.
12, hyaluronic acid injection liquid is used for functional rehabilitation after cerebral embolism：We analyze in 76 subjects altogether brain The subject 4 (1 of experimental group 1 and 3 of experimental group 2) that functional rehabilitation needs after embolism, as a result as shown in table 20：
Table 20：Hyaluronic acid injection liquid is used for the effect of functional rehabilitation after cerebral embolism
30 days after male subject (67 years old) cerebral embolism in table 20, half body is soft, powerless on foot, hyaluronic acid injection After the layer injection of liquid 10x100mg abdominal subcutaneous fats, half body is soft to be clearly better, walks effectively, shows that hyaluronic acid injection liquid can use In the functional rehabilitation after cerebral embolism.
4 days, visual impairment, the thing that can only be seen in 20cm after another male subject (59 years old) cerebral embolism in table 20 Body, after the layer injection of hyaluronic acid injection liquid 10x100mg abdominal subcutaneous fats, eyesight is clearly better, it can be seen that the thing in 40cm Body, shows the functional rehabilitation that hyaluronic acid injection liquid can be used for after cerebral embolism.
13, hyaluronic acid injection liquid is used for coronary heart disease, prevents intravascular stent infectious-related complication and prevention of cardiovascular disease Disease：Coronary heart disease, intravascular stent infectious-related complication and other angiocardiopathies are substantially probably chronic inflammation disease, are badly in need of having The prevention and controls of effect.It is (real that we analyze the subject 6 for having coronary heart disease and intravascular stent infectious-related complication in 76 subjects altogether Test group 3 of 1 and experimental group 2 3), as a result as shown in table 21：
Table 21：Hyaluronic acid injection liquid is used to treat coronary heart disease, prevent intravascular stent infectious-related complication and prevention of cardiovascular The effect of disease
The result of table 21 show hyaluronic acid injection liquid can be used for treatment coronary heart disease, prevent intravascular stent infectious-related complication and There may be the effect of prevention of cardiovascular disease.
Conclusion：
1st, hyaluronic acid injection liquid passes through the injection of abdominal subcutaneous fat layer or pain spot injection location or disease sites injection There are the back neck and shoulder fasciitis and injury of muscle and tendon of pain in treatment；
2nd, hyaluronic acid injection liquid passes through the injection of abdominal subcutaneous fat layer or pain spot injection location or disease sites injection There are the osteoproliferation, spur, disc herniation of pain in treatment；
3rd, hyaluronic acid injection liquid injects the ankylosing spondylitis for treating pain by abdominal subcutaneous fat layer；
4th, hyaluronic acid injection liquid is injected for treating muscae volitantes or vitreous opacity and its by abdominal subcutaneous fat layer Caused vitreum is peeled off and visual impairment；
5th, hyaluronic acid injection liquid by abdominal subcutaneous fat layer inject for treat have inflammation, scabies itching and pain it is big Area Burn, wound cicatrix, surgical wound scar；
6th, hyaluronic acid injection liquid uses treatment Post operation, peritoneal dialysis by the injection of abdominal subcutaneous fat layer or site of disease The abdominal cavity mucous membrane adhesion and pelvic cavity mucous membrane adhesion triggered afterwards；
7th, hyaluronic acid injection liquid injects the endometriosis for treating dysmenorrhoea by abdominal subcutaneous fat layer；
8th, hyaluronic acid injection liquid by abdominal subcutaneous fat layer inject for treat itch neurodermatitis, ox-hide Tinea, the old eczema of itch and Kaposi or pompholyx；
9th, hyaluronic acid injection liquid is injected by abdominal subcutaneous fat layer and recovered for physical energy after solid tumor chemotherapy With prevent solid tumor from shifting；
10th, hyaluronic acid injection liquid is injected for promoting functional rehabilitation after cerebral infarction by abdominal subcutaneous fat layer；
11st, hyaluronic acid injection liquid is injected by abdominal subcutaneous fat layer and burst for fast treating periodontal disease and oral cavity Ulcer, including periodontal disease and canker sore caused by chemotherapy and aging；
12nd, hyaluronic acid injection liquid is injected for fast treating pharyngolaryngitis, including chemotherapy by abdominal subcutaneous fat layer With pharyngolaryngitis caused by aging；
13rd, hyaluronic acid injection liquid is injected for fast treating eye conjunctiva disease by abdominal subcutaneous fat layer, including is changed Treat and eye conjunctiva disease caused by aging；
14th, hyaluronic acid injection liquid is injected by abdominal subcutaneous fat layer has anginal coronary heart disease, prevention for treatment With treatment intravascular stent infectious-related complication, prevention and treatment angiocardiopathy；
Embodiment 5
Purpose：Inquire into other species hyaluronic acid enzymatic cleavage methods activation and commercially produced embodiment 3-4 and shown The hyaluronic acid injection liquid 1 or 2 of the therapeutic action come.
Method：(1) production method and digestion macromolecular of a variety of hyaluronidases being had found in research existing literature are saturating The result of bright matter acid；(2) different hyaluronidases are produced using autonomous or cooperation or contract, verifies its digestion and activated macromolecules The feasibility of hyaluronic acid；(3) study which type of hyaluronidase can with cutter activation macromolecular hyaluronic acid to what The molecular weight of sizeWhich type of cleavage end structure can activated micromolecular hyaluronic acid have embodiment 3-4 hyalomitome The therapeutic action that acid injection is showedWhich type of hyaluronidase residual does not cause people's anaphylactoid reaction
As a result and discuss：We combine the production method that existing literature have studied a variety of hyaluronidases, and their enzymes Result of study, is now reported as follows by the method for cutting macromolecular hyaluronic acid respectively：
1st, in addition to the gender bender's perforatorium hyaluronidase PH20 (or Hyal3 or SPAM1) studied herein, the mankind also have Intracellular acid hyaluronidase Hyal1 and Hyal2.Pass through document " Recombinant human hyaluronidase Hyal-1:Insect cells versus Escherichia colias expression system and Identification of low molecular weight inhibitors, Glycobiology, 17 (4):444-53, 2007 ", it has been found that the recombined human Hyal1 hyaluronidases of insect cell Drosophila Schneider-2 (DS-2) productions Specific activity 8.6U/mg, optimum response pH value 3.5-4.0, but yield is too low, is not suitable for activating and commercially producing molecular weight 10-60KD hyaluronic acid injection liquid.In addition, being difficult to determine that recombined human Hyal1 hyaluronic acid cleavages are big without normal individual trial The molecular weight 10-60KD of molecular weight hyaluronic acid formation hyaluronic acid injection liquid has the treatment that embodiment 3-4 is showed Effect.We using zooblast (CHO-S cells), pMH3, pMH4, pMH5 expression vector rich in GC and people Hyal1 and Hyal2cDNA produces recombined human Hyal1 and Hyal2 albumen, it is found that the activity of hyaluronidase is much smaller than what we produced at present PH20 (1x105U/mg zymoproteins), points out recombined human Hyal1 and Hyal2 not to be suitable for zooblast production.
2nd, gender bender's perforatorium hyaluronidase PH20 (or Hyal3 or SPAM1) yield of Yeast expression can reach business Industry level, specific activity can reach 10,000 IU/mg zymoproteins, is suitable for digestion activation and has commercially produced embodiment 3-4 institutes The hyaluronic acid injection liquid of the therapeutic action showed.We use gender bender's perforatorium hyaluronidase of Yeast expression PH20, sequentially or separately uses recombined human hyaluronidase affinity column chromatography, precipitation, column chromatography, dialysis, ultrafiltration concentration after digestion Deng, will be containing the removal of recombined human hyaluronidase residual protein, manufacture does not contain the class of recombined human hyaluronidase residual protein Like the hyaluronic acid injection liquid 2 in embodiment 1, (molecular weight is 10KD-60KD 100mg/2 milliliters of hyaluronic acid fragments, chlorination Sodium 115-125mM, magnesium ion 1mM), and clinical research, table 21 are carried out to 38 multiple subjects of various diseases associated with senescence Result show that although gender bender's perforatorium hyaluronidase PH20 specific activities of Yeast expression are small, to hyaluronic acid injection liquid Purity have certain influence, will containing recombined human hyaluronidase residual protein remove, can be used for manufacture has embodiment 3-4 institutes The therapeutic action showed and the hyaluronic acid injection liquid 2 without allergy side effect.
Table 22：The hyaluronic acid injection liquid 2 manufactured using gender bender's perforatorium hyaluronidase PH20 of Yeast expression The clinical effectiveness of 38 subject multiple to various diseases associated with senescence
3rd, we produce oily seed table by collaboration laboratory using plant Arabidopsis thaliana Arabidopsis thaliana Gender bender's perforatorium hyaluronidase PH20 fusion proteins are produced up to system, discovery, which can be used for production, above-mentioned has embodiment 3- 4 therapeutic actions showed and the hyaluronic acid injection liquid 2 without allergy side effect, specific effect are shown in Table 23.But compared to Using zooblast production restructuring hyaluronidase, hyaluronidase PH20 fusion protein costs are low, and yield is big, and activity is low, goes Except containing hyaluronidase PH20 fusion proteins residual cost it is high, with zooblast Chinese hamster ovary cells The recombined human hyaluronidase for having saccharification of the high-purity of production compares, and is particularly suited for the low external application of production purity requirement and controls Hyaluronic acid atomizer, spray and the gel for the treatment of effect.
Table 23：The hyaluronic acid injection liquid 2 for the gender bender's perforatorium hyaluronidase PH20 manufactures expressed using plant The clinical effectiveness of 35 subject multiple to various diseases associated with senescence
4th, the hyaluronidase that sheep or bull testis are extracted is mainly neutral sheep or ox perforatorium hyaluronidase PH20, people Need to do allergic experiment before use.Although cost is low, purity is also low, removes the sheep contained or ox hyaluronidase protein is residual The cost stayed is high, through what is produced with zooblast Chinese hamster ovary cells>The weight for having saccharification of 99.0% purity Group people's hyaluronidase compare, it is believed that be not suitable for production purity requirement it is high have the therapeutic action that embodiment 3-4 is showed With the hyaluronic acid injection liquid 1 or 2 without allergy side effect.In addition, the hyaluronic acid cleavage macromolecular that sheep or bull testis are extracted Hyaluronic acid fragments of the molecular weight of hyaluronic acid formation between 10-60KD are difficult that determination has reality without human clinical trial Apply the therapeutic action that a 3-4 is showed.
5th, CEDARS-SINAI MEDICAL CENTER patent WO/2014/165713 shows bacterium streptococci The micromolecule hyaluronic acid less than 10KD that hyaluronidase is produced does not cause inflammatory reaction, but bacterium Streptomyces is transparent The micromolecule hyaluronic acid more than 10KD that matter acid enzyme is produced causes inflammatory reaction, this and we use recombined human hyaluronidase with Magnesium ion cutter activation macromolecular hyaluronic acid and produce have therapeutic efficiency molecular weight be 10KD-60KD hyaluronic acid piece Section is conflicting, it is important that bacterial hyaluronidase residual protein may produce allergic reaction to human body, be not suitable for production The high therapeutic action for thering is embodiment 3-4 to be showed of purity requirement and hyaluronic acid injection liquid 1 without allergy side effect or 2。
6th, the hyaluronidase allergenicity in honey bee venom is very strong, in addition to promoting bee venom and absorbing, also causes allergy to people Reaction.Extract and microbial expression honey bee venom in hyaluronidase be unsuitable for produce purity requirement it is high have embodiment The therapeutic action that 3-4 is showed and the hyaluronic acid injection liquid 1 or 2 without allergy side effect.In addition, the hyaluronic acid of honeybee Hyaluronic acid fragments of the molecular weight between 10-60KD of cleavage macromolecular hyaluronic acid formation without human clinical trial very Hardly possible determines to have the therapeutic action that embodiment 3-4 is showed.
7th, the leech secretion leech hyaluronidase sucked blood, can dissolve skin, help leech to be sucked blood into human body.Document “Enzymatic production of specifically distributed hyaluronan Oligosaccharides, Carbohydr Polym, 2015,129:194-200 " and " High-yield novel leech hyaluronidase to expedite the preparation of specific hyaluronan oligomers (2014), Scientific Reports 4, Article#4471 " show leech hyaluronic acid enzyme fusion proteins in Yeast expression Yield, digestion activity, cost be attained by commercialization and require, and macromolecular hyaluronic acid can be cut into molecular weight 10- Micromolecule hyaluronic acid between 60KD.But, the molecular weight of leech hyaluronic acid cleavage macromolecular hyaluronic acid formation exists Hyaluronic acid fragments between 10-60KD are difficult to determine to have that embodiment 3-4 showed controls without human clinical trial Treatment is acted on.Leech hyaluronidase residual can cause allergic reaction, and thoroughly need to may be used to production and have embodiment 3-4 in the side of removing The therapeutic action showed and the hyaluronic acid injection liquid 2 without allergy side effect.We compare leech hyaluronidase Produced with zooblast Chinese hamster ovary cells>The recombined human hyaluronic acid for having saccharification of 99.0% purity Enzyme, it is believed that leech hyaluronidase be not suitable for the high therapeutic action for thering is embodiment 3-4 to be showed of production purity requirement and Hyaluronic acid injection liquid 1 or 2 without allergy side effect.
Finally it should be noted that：The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention, Although the present invention is described in detail with reference to the foregoing embodiments, for those skilled in the art, it still may be used To be modified to the technical scheme described in previous embodiment, or equivalent substitution is carried out to which part technical characteristic.It is all Within the spirit and principles in the present invention, any modification, equivalent substitution and improvements made etc., should be included in the guarantor of the present invention Within the scope of shield.

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。
CN201610264221.9A 2016-04-25 2016-04-25 A kind of parenteral solution containing micromolecule hyaluronic acid and application thereof Pending CN107303271A (en)

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PCT/CN2016/080948 WO2017185383A1 (en) 2016-04-25 2016-05-04 Hyaluronic injection and applications thereof
US16/754,756 US11576925B2 (en) 2016-04-25 2017-04-25 Application of low-molecular-weight hyaluronic acid (LMW-HA) fragments
CA3049286A CA3049286C (en) 2016-04-25 2017-04-25 Application of low-molecular-weight hyaluronic acid (lmw-ha) fragments
AU2017255833A AU2017255833B2 (en) 2016-04-25 2017-04-25 Application of small-molecule hyaluronic acid fragment
PCT/CN2017/081796 WO2017186088A1 (en) 2016-04-25 2017-04-25 Application of small-molecule hyaluronic acid fragment
EP17788740.3A EP3479830B1 (en) 2017-04-25 Application of small-molecule hyaluronic acid fragment
US18/073,604 US11839625B2 (en) 2016-04-25 2022-12-02 Application of low-molecular-weight hyaluronic acid (LMW-HA) fragments
US18/073,601 US11826381B2 (en) 2016-04-25 2022-12-02 Application of low-molecular-weight hyaluronic acid (LMW-HA) fragments
US18/073,596 US11826380B2 (en) 2016-04-25 2022-12-02 Application of low-molecular-weight hyaluronic acid (LMW-HA) fragments

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108210689A (en) * 2017-11-27 2018-06-29 南京天纵易康生物科技股份有限公司 A kind of canker sore film containing hyaluronic acid and preparation method thereof
CN108721320A (en) * 2017-04-24 2018-11-02 惠觅宙 The application of micromolecule hyaluronic acid segment
CN111249302A (en) * 2020-03-12 2020-06-09 李鑫荣 New application and stable manufacturing method of hyaluronic acid fragments
WO2021180252A1 (en) * 2020-03-12 2021-09-16 青岛农业大学 Novel application and manufacturing method for hyaluronic acid fragment
CN115397403A (en) * 2020-02-13 2022-11-25 剑桥企业有限公司 Use of compounds capable of cross-linking extracellular matrix to prevent or inhibit cancer cell migration
CN115429750A (en) * 2022-09-19 2022-12-06 长春嘉和外科医院 Application and method for preparing medicine from liquid preparation containing hyaluronidase

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002009728A1 (en) * 2000-07-31 2002-02-07 Dermal Research Laboratories, Inc. Methods of preventing or treating diseases and conditions using complex carbohydrates
CN103468662A (en) * 2013-09-29 2013-12-25 惠觅宙 Recombined human hyaluronidase, production and purification method and preparations thereof, use method and application
CN104548106A (en) * 2015-02-09 2015-04-29 惠觅宙 Bioactive hyaluronic acid syrup preparation
CN104666112A (en) * 2015-02-09 2015-06-03 惠觅宙 Biological-activity hyaluronic acid toothpaste
CN105018547A (en) * 2014-04-16 2015-11-04 惠觅宙 Bioactive hyaluronic acid fragment, production method, application, preparation and object containing preparation

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100594029C (en) * 2006-07-24 2010-03-17 凌沛学 Medicinal composition for injections in articular cavity

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002009728A1 (en) * 2000-07-31 2002-02-07 Dermal Research Laboratories, Inc. Methods of preventing or treating diseases and conditions using complex carbohydrates
CN103468662A (en) * 2013-09-29 2013-12-25 惠觅宙 Recombined human hyaluronidase, production and purification method and preparations thereof, use method and application
CN105018547A (en) * 2014-04-16 2015-11-04 惠觅宙 Bioactive hyaluronic acid fragment, production method, application, preparation and object containing preparation
CN104548106A (en) * 2015-02-09 2015-04-29 惠觅宙 Bioactive hyaluronic acid syrup preparation
CN104666112A (en) * 2015-02-09 2015-06-03 惠觅宙 Biological-activity hyaluronic acid toothpaste

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
惠觅宙等: ""小分子透明质酸在老年皮肤粘膜保健及炎症防御中的应用"", 《皮肤性病诊疗学杂志》 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108721320A (en) * 2017-04-24 2018-11-02 惠觅宙 The application of micromolecule hyaluronic acid segment
CN108210689A (en) * 2017-11-27 2018-06-29 南京天纵易康生物科技股份有限公司 A kind of canker sore film containing hyaluronic acid and preparation method thereof
CN115397403A (en) * 2020-02-13 2022-11-25 剑桥企业有限公司 Use of compounds capable of cross-linking extracellular matrix to prevent or inhibit cancer cell migration
CN111249302A (en) * 2020-03-12 2020-06-09 李鑫荣 New application and stable manufacturing method of hyaluronic acid fragments
WO2021180252A1 (en) * 2020-03-12 2021-09-16 青岛农业大学 Novel application and manufacturing method for hyaluronic acid fragment
CN115429750A (en) * 2022-09-19 2022-12-06 长春嘉和外科医院 Application and method for preparing medicine from liquid preparation containing hyaluronidase

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