WO2008076003A1 - Composition de ferments protéolytiques individuels - Google Patents

Composition de ferments protéolytiques individuels Download PDF

Info

Publication number
WO2008076003A1
WO2008076003A1 PCT/RU2007/000677 RU2007000677W WO2008076003A1 WO 2008076003 A1 WO2008076003 A1 WO 2008076003A1 RU 2007000677 W RU2007000677 W RU 2007000677W WO 2008076003 A1 WO2008076003 A1 WO 2008076003A1
Authority
WO
WIPO (PCT)
Prior art keywords
composition
proteolytic enzymes
individual
proteases
iicd
Prior art date
Application number
PCT/RU2007/000677
Other languages
English (en)
Russian (ru)
Inventor
Olga Anatolievna Klimova
Original Assignee
Joint-Stock Company 'high Tech'
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Joint-Stock Company 'high Tech' filed Critical Joint-Stock Company 'high Tech'
Publication of WO2008076003A1 publication Critical patent/WO2008076003A1/fr
Priority to US12/487,463 priority Critical patent/US20120058103A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to biotechnology, namely, to obtain a composition of individual enzymes with a wide spectrum of proteolytic activity, which makes the composition capable of hydrolyzing protein substrates up to individual amino acids.
  • the composition of the composition includes proteolytic enzymes (endo- and exopeptidases), which are clearly individualized, including the N-terminal sequence and molecular weight, and are characterized by high activity against many peptide and protein substrates, including collagen of various types.
  • proteolytic enzymes endo- and exopeptidases
  • proteolytic enzymes perform many physiological functions, from protein digestion to more specific ones, such as activation of zymogens and complement systems, are involved in the process of protein coagulation and lysis of fibrin clots, the maturation of hormones and biologically active peptides from precursor proteins and the transport of proteins through cell membranes. Comparison of amino acid sequences, three-dimensional structures and mechanisms of enzymatic reactions allows us to divide proteolytic enzymes into several groups.
  • proteolytic enzymes based on their catalysis mechanism allows us to distinguish 4 classes: serine proteases; metalloproteases; thiol (cysteine) and acid (aspartic) proteases. These proteases differ in sensitivity to various inhibitors, for example, serine proteases are inhibited by phenylmethylsulfonyl fluoride (PMSF) and diisopropyl fluorophosphate (DFP); metalloproteases — chelating agents such as EDTA, EGTA, o-phenanthroline; cysteine proteases with iodoacetamide and heavy metals and asparagine with pepstatin.
  • PMSF phenylmethylsulfonyl fluoride
  • DFP diisopropyl fluorophosphate
  • metalloproteases — chelating agents such as EDTA, EGTA, o-phenanthroline
  • Serine proteases usually have a slightly alkaline optimum pH, metalloproteases are neutral, and cysteine and aspartic proteases are acidic.
  • the rate of hydrolysis of a particular peptide bond by most proteases depends, as a rule, on the substrate specificity of the enzyme, the spatial availability of this peptide bond (especially if the substrate is a native rather than denatured protein) and does not depend on the size of the substrate molecule (on the length of the polypeptide chain) .
  • Substrate specificity lies in the ability of a given enzyme to hydrolyze peptide bonds between specific amino acid residues at the highest rate.
  • the representative representatives of the largest class of serine proteases are mammalian digestive proteases, which have similar amino acid sequences and spatial structures that can hydrolyze substrates to small fragments and differ in the type of cleavable peptide bond.
  • chymotrypsin hydrolyzes peptide bonds involving amino acids with aromatic side chains, trypsin - positively charged, elastase - amino acid residues of glycine, and to a lesser extent - leucine.
  • the mechanism of observed specificity is due to differences in the structure of the active centers of proteolytic enzymes.
  • proteases with a narrow substrate specificity are used in molecular biology as tools for studying the primary structure of proteins and peptides, and the proteases themselves are convenient objects for studying the structure of proteins and the mechanisms of their functioning.
  • Many proteases, such as trypsin, chymotrypsin, papain, and others serve as the active principle of many drugs used in cosmetology and veterinary medicine.
  • Serine proteases isolated from the digestive organs of invertebrates and fish are known (Zwillipg R., et al, 1975, FEBS Let. 60, 247-9; Gran G. A., et al, 1981, Methods 80, 722-734 ; Reesk GR and H. Neurth. 1972, Biochem. 1 1: 503-510, O.A. Klimova, et al, 1990, BBRC, v.166, N 3, 141 1-1420).
  • True collagenases (tissue and microbial origin), belonging to the class of metalloproteases, cleave native collagen at one point in one of the three polypeptide chains of the main structural element of native collagen - tropocollagen.
  • invertebrate serine proteases cleave all three tropocollagen polypeptide chains, and the resulting peptides undergo further hydrolysis up to individual amino acids, which are either included in the processes of anabolism or are rapidly excreted without causing intoxication.
  • serine proteases of invertebrates and fish to hydrolyze a wide range of substrates is associated with the structural features of not only their active centers, but also with the structure of the substrate of the binding sites - the secondary interactions of the substrate with the substrate-binding site make a significant contribution to the increase in catalytic activity.
  • the task is to total proteolysis of various protein substrates, up to individual amino acids, so that the amino acids obtained in this process can be used as building blocks in the processes of anabolism.
  • Trypsin cod have N-terminal sequence I-V-G-G-Y-Q / E-C-E / T-K / R-H-S-Q-A-N-Q-V-S-L-N-S, whereas trypsin mammals such as bovine trypsin have the N-terminal sequence I-V-G-G- Y-T-C-G-A-N-T-V-P-Y-Q-V-S-L-N-S. All three cod trypsin isoforms have a similar molecular weight of about 24 kDa.
  • serine proteases are endopeptidases and hydrolyze polypeptide substrates to the state of small peptides, but not individual amino acids, in addition, they are not able to hydrolyze a number of substrates, for example, native collagen, which limits the possibility of further use of such an enzyme composition.
  • An object of the present invention is to provide a composition consisting of proteolytic enzymes that is active, stable in a wide temperature and pH range and resistant to autolysis and is intended for widespread use for diagnostic, therapeutic purposes, in cosmetology and pharmacology, as well as in biotechnology .
  • proteolytic enzymes proteolytic enzymes
  • molecular weights of 100-11 kilodaltons were obtained from the digestive organs of hydrobionts.
  • individual components with molecular weights of 23-26 kilodaltons with the following N-terminal amino acid sequences were isolated and purified to a homogeneous state from proteolytic complexes:
  • New proteolytic compositions created on the basis of complexes of proteases and / or individual components with molecular weights of 23-36 kilodaltons have higher enzymatic activity and hydrolysis depth compared to previously known ones with respect to many peptide and protein substrates (including collagen of various types).
  • compositions with the above components The components of the composition with a molecular weight of 100-37 and 22-11 kDaltons were identified by polyacryamide gel electrophoresis (PAGE) under denaturing conditions (in the presence of sodium dodecyl sulfate); N-terminal amino acid sequences were not determined due to the low content of components in the composition.
  • the percentage of components in the composition was determined by the color of the electrophoregram in PAAG with subsequent scanning of the gel. For this, upon completion of electrophoresis, the gel was fixed in a 5% solution of trichloroacetic acid, stained with a solution of Compassion R-250 according to a standard method (L. Osterman.
  • compositions based on individual proteolytic enzymes are examples of the composition of compositions based on individual proteolytic enzymes.
  • compositions of proteolytic enzymes in the research process showed a higher enzymatic activity and the absence of allergic reactions with prolonged use.
  • the advantages of the claimed composition are confirmed by the results of comparative tests of compositions with different content of components (according to examples 1 and 3 of table 1) when exposed to type I collagen (table T).
  • Tests of the composition according to example 2 of table 1 yielded results, similar to the results of the composition of example 1, therefore, in table 2 they are not included.
  • a 50% (WVV) trichloroacetic acid solution was frozen at -70 ° C for 1 hour, thawed and centrifuged at 5,000 g for 5 minutes.
  • Hepatocytes from the liver of an adult and an embryo (5-8 weeks) were obtained according to standard methods.
  • the resulting precipitate was collected and resuspended in a 1% albumin hydrolyzate.
  • To count living cells 1 ml of cell suspension was taken, the content of intact cells was determined by staining with 0.2% trypan blue solution, followed by microscopy.
  • an ointment the active principle of which includes an elastase preparation and an acrylic acid-based polymer (US Pat. No. 4,276,281).
  • Elastase was obtained from the pancreas of mammals (specifically horses). This is a serine protease with a molecular weight of 25,900 daltons, consisting of 240 amino acid residues, with a known amino acid sequence (Schottep, D. M., Nartlew, B. S .; Biochem J. 131, 643, 1973).
  • elastase is based on its high efficiency for removing scab and other macromolecular debris (residues of damaged cells, partially or completely denatured collagen, etc.) from the surface of burned mammalian skin, thereby accelerating the cleaning of the affected surface and its preparation for granulation.
  • the main disadvantage of using elastase in this capacity is its low activity against a number of polypeptide substrates, including collagen of various types, and the shallow hydrolysis of polypeptide substrates (to large fragments), which must be removed from the affected surfaces by other methods (mechanical or chemical) further injuring the patient.
  • elastase In addition, with frequent application to the skin of a component such as elastase, the latter can cause some diseases due to its toxic and allergic effects.
  • this elastase-based therapeutic agent requires a limitation on the duration of use, and for some users prone to allergies, it can be completely contraindicated.
  • the closest drug to the claimed one is the agent proposed in the invention, “Method for the treatment of diseases accompanied by the formation of pus and / or necrotic tissue” according to the patent of the Russian Federation.
  • the disadvantage of this tool is that the main biologically active component is a complex that includes enzymes with non-specific activity as minor components, as a result of which the damaging effect of the protease complex in relation to living cells is significant.
  • composition of the protease complex is highly dependent on the physiological cycle of aquatic organisms from which this complex was obtained.
  • content of the components of the complex and, therefore, its properties vary widely, which complicates the use of the drug.
  • a drug containing the proposed composition of proteolytic enzymes as an active principle allows to reduce the concentration of the composition when used due to higher enzymatic activity and does not cause allergic reactions with prolonged use.
  • the composition of the composition can be optimized (including standardized) in relation to the object of influence, which increases its effectiveness and reduces side effects from the use.
  • Patient K. 25 years old, applied for scarring of the forehead and eyelids. From the anamnesis it is known that 3 months ago she was in a car accident, during which she received multiple injuries of soft tissues of the face with shards of glass. The wound of the left upper eyelid is sutured. The patient is concerned: the presence of itching in the area of scars, the presence of palpable small encapsulated foreign bodies under the skin, disfigurement.

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Dermatology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Cosmetics (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne la biotechnologie et notamment la fabrication de composition de ferments individuels possédant un large spectre d'activité protéolytique ce qui rend la composition capable d'hydrolyser des substrats protéiques jusqu'au niveau d'acides aminés individuels. La composition comprend au moins deux protéases avec une masse moléculaire entre 23 et 36 kilodalton et des séquences d'acides aminés terminales sont : I V G G T E V T P G E I P Y Q L S L Q D - I V G E V T P G E I P Y Q L S F Q D - I V G G Q E A S P G S W P X Q V G L F F - E A T S G Q F P Y Q X S F Q D - I V G G Q E A T P H T W V H Q V A L F E A T P H T X V H Q A L F I - A M D X T A Y X D Y D E I Q A X L K N T F E E I N S I L D G V - A A I L G D E Y L X S G G V V P Y V F G -Selon l'invention, les produits médicamenteux et cosmétiques comprenant en tant que principe actif cette composition à base de ferments protéolytiques individuels à degré de purification élevé permettent de réduire la concentration de la composition lors de l'utilisation grâce à une activité plus intense des ferments et ne provoquent pas de réactions allergiques pendant une utilisation prolongée. En outre, la teneur de la composition peut être optimisée (y.c. standardisée) par rapport l'objet du traitement, ce qui augmente son efficacité et réduit les effets secondaires dus à l'utilisation.
PCT/RU2007/000677 2006-12-20 2007-12-03 Composition de ferments protéolytiques individuels WO2008076003A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US12/487,463 US20120058103A1 (en) 2006-12-20 2009-06-18 Composition of individual proteolytic enzymes

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
RU2006147331 2006-12-20
RU2006147331 2006-12-20

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US12/487,463 Continuation US20120058103A1 (en) 2006-12-20 2009-06-18 Composition of individual proteolytic enzymes

Publications (1)

Publication Number Publication Date
WO2008076003A1 true WO2008076003A1 (fr) 2008-06-26

Family

ID=39537089

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/RU2007/000677 WO2008076003A1 (fr) 2006-12-20 2007-12-03 Composition de ferments protéolytiques individuels

Country Status (1)

Country Link
WO (1) WO2008076003A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2141310C1 (ru) * 1997-11-28 1999-11-20 Открытое акционерное общество "СПб-Технология" Активное начало косметического средства и косметическое средство (варианты)
RU2161503C2 (ru) * 1997-11-28 2001-01-10 Открытое акционерное общество "СПб-Технология" Лечебное средство
US20060134641A1 (en) * 1992-05-22 2006-06-22 Franklin Richard L Treating viral infections with krill enzymes

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060134641A1 (en) * 1992-05-22 2006-06-22 Franklin Richard L Treating viral infections with krill enzymes
RU2141310C1 (ru) * 1997-11-28 1999-11-20 Открытое акционерное общество "СПб-Технология" Активное начало косметического средства и косметическое средство (варианты)
RU2161503C2 (ru) * 1997-11-28 2001-01-10 Открытое акционерное общество "СПб-Технология" Лечебное средство

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BIOCHEM. BIOPHYS. RES. COMMUN., vol. 166, no. 3, 14 February 1990 (1990-02-14), pages 1411 - 1420 *
BMC STRUCTURAL BIOLOGY, vol. 42, 20 January 2004 (2004-01-20), pages 1 - 9 *
DATABASE MEDLINE [online] KLIMOVA O.A. ET AL.: "Isolation and characteristics of collagenolytic enzymes from the hepatopancreas of the crab Chionoecetes opiho", Database accession no. (1663026) *
DATABASE MEDLINE [online] KLIMOVA O.A. ET AL.: "The isolation and properties of collagenolytic proteases from crab hepatopancreas", Database accession no. (2154979) *
DATABASE MEDLINE [online] RUDENSKAYA G.N. ET AL.: "Collagenolytic serine protease PC and trypsin PC from king crab Paralithodes camtschaticus cDNA cloning and primary structure of the enzymes", Database accession no. (14731305) *
DOKL AKAD NAUK SSSR, vol. 317, no. 2, 1991, pages 482 - 484 *

Similar Documents

Publication Publication Date Title
Swenson et al. Snake venom fibrin (ogen) olytic enzymes
Berton et al. Analysis of the ex vivo specificity of human gelatinases A and B towards skin collagen and elastic fibers by computerized morphometry
EP0810875A1 (fr) Enzyme a fonctions multiples
Homsy et al. Characterization of human skin fibroblasts elastase activity
Shekhter et al. Collagenolytic enzymes and their applications in biomedicine
Weldon et al. Alsophinase, a new P-III metalloproteinase with α-fibrinogenolytic and hemorrhagic activity from the venom of the rear-fanged Puerto Rican Racer Alsophis portoricensis (Serpentes: Dipsadidae)
Shankar et al. Protease enzymes: highlights on potential of proteases as therapeutics agents
Wu et al. Purification and characterization of gelatinase-like proteinases from the dark muscle of common carp (Cyprinus carpio)
Bolger et al. Three-dimensional structure of fibrolase, the fibrinolytic enzyme from southern copperhead venom, modeled from the X-ray structure of adamalysin II and atrolysin C
CA2777650C (fr) Procede d'analyse electrophoretique
Yin et al. Gelatinolytic proteinase activities in human seminal plasma
RU2365623C2 (ru) Композиция индивидуальных протеолитических ферментов
WO2008076003A1 (fr) Composition de ferments protéolytiques individuels
US7947270B2 (en) Removing dental plaque with krill enzymes
Rudenskaya Brachyurins, serine collagenolytic enzymes from crabs
Wagner et al. Calf lens neutral proteinase activity using calf lens crystallin substrates
Bylander et al. Human and mouse homo-oligomeric meprin A metalloendopeptidase: substrate and inhibitor specificities
Dinu et al. Isolation and Characterization of Two Cathepsins from Muscle of Carassius auratus gibelio.
RU2610669C1 (ru) Способ получения протеолитического препарата для медицинского применения
Rutter et al. Varidase: the science behind the medicament
JP3113901B2 (ja) 血栓溶解剤
US20060134641A1 (en) Treating viral infections with krill enzymes
Klimova et al. Collagenolytic protease preparations from invertebrates: biochemical aspects of medical and cosmetological applications
RU2712528C1 (ru) Способ получения препарата коллагеназы в геле на основе пищевого хитозана и сукцината хитозана
RU2346983C1 (ru) Способ получения активатора плазминогена

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07861077

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC, EPO FORM 1205A DATED 13.11.2009

122 Ep: pct application non-entry in european phase

Ref document number: 07861077

Country of ref document: EP

Kind code of ref document: A1