WO2017010845A1 - Composition pour la prévention ou le traitement de maladies infectieuses à staphylocoques - Google Patents

Composition pour la prévention ou le traitement de maladies infectieuses à staphylocoques Download PDF

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WO2017010845A1
WO2017010845A1 PCT/KR2016/007748 KR2016007748W WO2017010845A1 WO 2017010845 A1 WO2017010845 A1 WO 2017010845A1 KR 2016007748 W KR2016007748 W KR 2016007748W WO 2017010845 A1 WO2017010845 A1 WO 2017010845A1
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composition
pgn
wta
treatment
prevention
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이복률
안동호
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재단법인 목암생명과학연구소
부산대학교 산학협력단
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/14—Peptides containing saccharide radicals; Derivatives thereof, e.g. bleomycin, phleomycin, muramylpeptides or vancomycin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)

Definitions

  • the present invention relates to a pharmaceutical composition for the prevention or treatment of Staphylococcal infection disease.
  • Staphylococcus aureus is a human skin, soft tissue (soft tissue) and can lead to severe bloodstream infection (Lowy FD, The New England Journal of Medicine, 339: 520-532, 1998).
  • Staphylococcus aureus may be transformed into methicillin-resistant strain (methsaillin-resistant S. aureus , MRSA) resistant to beta-lactam family of methicillin. MRSA infections are difficult to treat and have a poor prognosis, which is a social problem.
  • CA-MRSA community-associated MRSA strains
  • H-MRSA hospital-associated MRSA strains
  • USA300 MRSA strain which is spreading in the United States, causes serious diseases in children or those with reduced immune function. Therefore, new vaccines or treatments having a prophylactic and therapeutic effect on MRSA infection are required.
  • the cell walls of Gram-positive bacteria are four components, including peptidoglycan (PGN), wall teichoic acid (WTA), lipoteicoic acid (LTA), and capsular polysaccharides (CP) It consists of.
  • PPN peptidoglycan
  • WTA wall teichoic acid
  • LTA lipoteicoic acid
  • CP capsular polysaccharides
  • the cell wall component of staphylococci is a glycopolymer, and its structure is very complicated, and it is difficult to separate and purify each as a single substance.
  • various cell wall components are exposed to the outside, which makes it difficult to identify which component acts as a ligand in the host's immune system.
  • WTA-PGN wall teichoic acid-attached peptidoglycan
  • Another object of the present invention to provide a method for preventing or treating staphylococcal infection disease using the composition.
  • Still another object of the present invention is to provide a method for preparing soluble dumpeichoic acid-attached peptidoglycan (WTA-PGN) which can be used as an active ingredient in the composition.
  • WTA-PGN soluble dumpeichoic acid-attached peptidoglycan
  • the present invention is a composition for the prevention or treatment of staphylococcal infection disease, which contains the wall teichoic acid-attached peptidoglycan (WTA-PGN) represented by the general formula 1 as an active ingredient to provide:
  • WTA-PGN wall teichoic acid-attached peptidoglycan
  • n is an integer of 10 to 50
  • m is an integer of 1 to 3
  • A is N-acetylmannosamine (ManNAc)
  • B is N-acetylglucosamine (GlcNAc)
  • O is An integer from 1 to 5
  • R 1 and R 2 are each independently hydroxy, tetrapeptide or pentapeptide
  • R 3 is hydroxy or N-acetylmuramic acid (MurNAc).
  • the present invention also provides a method for preventing or treating Staphylococcal infection disease, comprising administering the composition to a subject in need thereof.
  • the present invention provides: 1) to obtain a double-mutant strain is lgt (lipoprotein diacylglycerol transferase) and oatA (O-acetyl transferase) gene deletions from the wild type Staphylococcus aureus; 2) heating the double mutant strain to a temperature of 40 to 70 ° C; 3) adding ⁇ -lytic enzyme to the heated strain; 4) obtaining a soluble WTA-PGN containing fraction from the enzyme additive of step 3); 5) adding lysozyme or mutanolysin to the soluble WTA-PGN containing fractions; And 6) obtaining a soluble WTA-PGN from the enzyme additive of step 5).
  • lgt lipoprotein diacylglycerol transferase
  • oatA O-acetyl transferase
  • Soluble WTA-PGN represented by Formula 1 according to the present invention increases the production of IL-17A in the mouse body when administered to mice, the soluble WTA-PGN can be usefully used for the prevention and treatment of staphylococci have.
  • FIG. 1 is a graph showing the elution pattern of S. aureus T384 strain ( ⁇ lgt ⁇ oatA ) prepared in one embodiment of the present invention after treatment with ⁇ -lytic enzyme and HPLC separation.
  • Figure 2 is a graph showing the elution pattern separated by HPLC treatment of lysozyme to each fraction obtained by treatment with insoluble ⁇ -lytic enzyme to the heat-treated S. aureus T384 strain and separated by HPLC:
  • F An elution pattern separated by HPLC after treating lysozyme to the F fraction.
  • Figure 3 shows the results of the production of IL-17A after injection into the mouse each peak fraction obtained after the enzyme treatment:
  • FIG. 4 is a graph showing an elution pattern of Sephacryl-B-TSK-B fractions separated by a C18 reversed phase column.
  • FIG. 5 is a graph comparing the amount of IL-17A induced when mice were injected with each of the six fractions in which Sephacryl-B-TSK-B fractions were separated by a C18 reversed phase column (Con: Sephacryl-B-TSK).
  • -B fraction A: A fraction
  • B B fraction
  • C C fraction
  • D D fraction
  • E E fraction
  • F F fraction
  • FIG. 6 is a graph showing an elution pattern in which the C to E fractions separated from the C18 reverse phase column in Example 5 were re-separated into a C18 reverse phase column.
  • Figure 7 is a graph comparing the production of IL-17A induced when mice were injected with C-E fractions re-separated in a C18 reversed phase column (C: C fraction, D: D fraction, E: E fraction).
  • wall teichoic acid is one of the cell wall components of S. aureus , combined with a glycerol phosphate repeat unit and a ribitol phosphate repeat unit (N-acetyl refers to a glycopolymer consisting of mannosamine)-( ⁇ -1,3)-(N-acetylglucosamine).
  • peptidoglycan refers to a repeat glycopolymer of N-acetylmuriamic acid (MurNAc) and N-acetylglucosamine (GlcNAc) linked by stem-peptide bonds.
  • wall teichoic acid-attached peptidoglycan refers to a structure in which the wall teichoic acid and peptidoglycan are covalently bonded.
  • the present invention provides a composition for the prevention or treatment of Staphylococcal infection disease, which comprises aste active acid-attached peptidoglycan (WTA-PGN) represented by the following general formula (1):
  • n is an integer of 10 to 50
  • m is an integer of 1 to 3
  • A is N-acetylmannosamine (ManNAc)
  • B is N-acetylglucosamine (GlcNAc)
  • O is An integer from 1 to 5
  • R 1 and R 2 are each independently hydroxy, tetrapeptide or pentapeptide
  • R 3 is hydroxy or N-acetylmuramic acid (MurNAc).
  • n is an integer from 35 to 45
  • m is 3
  • A is ManNAc
  • B is GlcNAc
  • O is an integer from 1 to 5
  • R 1 and Each R 2 is independently hydroxy, tetrapeptide or pentapeptide and R 3 is hydroxy or MurNAc.
  • n 40, m is 3, A is ManNAc, B is GlcNAc, O is an integer from 1 to 5, R 1 and R 2 is Each independently is a tetrapeptide or pentapeptide and R 3 is hydroxy or MurNAc.
  • a and B may be linked to a ⁇ -position to each other.
  • the tetrapeptide is -X 1 -X 2 -X 3 -X 4 (SEQ ID NO: 1)
  • the pentapeptide is (SEQ ID NO: 2).
  • X 1 may be Ala or Gly
  • X 2 may be Glu or Asp
  • X 3 may be Lys, Arg, or His
  • X 4 may be Ala or Gly.
  • the tetrapeptide is-(L-Ala)-(D-Glu)-(L-Lys)-(D-Ala) (SEQ ID NO: 3), and the pentapeptide is (SEQ ID NO: 4).
  • the PGN structure of Formula 1 may be represented by the following formula (1).
  • the R 1 or R 2 of the WTA-PGN may form the R cross-linking with each other and any one of 1 or R 2 of other WTA-PGN,
  • the WTA-PGN along the two WTA-PGN may also be present in the form of a dimer combined with it.
  • the staphylococcus may be methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive Staphylococcus aureus (MSSA) or pathogenic staphylococcus.
  • MRSA methicillin-resistant Staphylococcus aureus
  • MSSA methicillin-sensitive Staphylococcus aureus
  • infectious diseases caused by Staphylococcus aureus include soft tissue infection, purulent arthritis, purulent osteomyelitis, otitis media, pneumonia, sepsis, acute respiratory tract infection, infection due to the use of a catheter, postoperative wound infection, bacteremia, Endocarditis and food poisoning.
  • composition according to the present invention may further comprise a pharmaceutically acceptable carrier, diluent and adjuvant in addition to the WTA-PGN.
  • Carriers used in the compositions according to the invention are selected based on the method and route of administration, and standard drug compositions, and include, for example, carrier proteins (ie, bovine serum albumin (BSA), egg white albumin (OVA), human serum albumin). (HSA) and keyhole limpet hemocyanin (KLH)), solubilizers (ie ethanol, polysorbate and Cremophor EL TM), isotonic agents, preservatives, antioxidants, excipients (ie lactose, starch, crystalline cellulose, mannitol) , Maltose, calcium hydrogen phosphate, light anhydrous silicic acid and calcium carbonate), binder (ie starch, polyvinylpyrrolidone, hydroxypropyl cellulose, ethyl cellulose, carboxymethyl cellulose and gum arabic), lubricant (ie magnesium stearate) , Talc, and cured oils, and the like) and stabilizers (ie, lactos
  • composition according to the present invention can be combined with a known KLH solution (Calbiotec, dissolving 125 mg per ml of 50% glycerol solution) as a carrier protein to enhance antigenicity.
  • KLH solution Calbiotec, dissolving 125 mg per ml of 50% glycerol solution
  • Diluents used in the compositions according to the invention can be selected based on the method and route of administration and the actual standard drug composition.
  • examples of diluents include water, physiological saline, phosphate buffered physiological saline, and bicarbonate solutions.
  • Adjuvants used in the compositions according to the invention may be selected based on the method and route of administration and the actual standard drug composition.
  • adjuvant include cholera toxin, E. coli dipyrotoxin (LT), liposomes and immune stimulatory complex (ISCOM).
  • the present invention also provides a method for preventing or treating Staphylococcal infection disease, comprising administering the composition to a subject in need thereof.
  • the subject may be a mammal, specifically a human.
  • the method can prevent or treat staphylococcal infections by simultaneously inducing opsonophagocytosis and phagocytosis in a subject.
  • the method may also increase the amount of IL-17A produced within 24 hours after administration of the composition according to the invention in a subject.
  • the administration may vary depending on the age, weight, sex and general state of health of the subject. Routes for such administration include oral and parenteral administration (eg, intravenous administration, arterial administration and topical administration), preferably parenteral administration.
  • parenteral administration eg, intravenous administration, arterial administration and topical administration
  • Formulations for oral and parenteral administration and methods for their preparation are known to those skilled in the art.
  • Formulations for oral and parenteral administration may be prepared by conventional procedures, eg, by admixing with the aforementioned pharmaceutically acceptable carriers.
  • Examples of formulations for oral administration include solid or liquid formulations such as solvents, tablets, granules, powders or capsules.
  • Examples of formulations for parenteral administration include solvents, suspensions, ointments, creams, suppositories, eye drops, nasal drops and ear drops.
  • biodegradable polymers eg, poly-D, L-lactide-co-glycoside or polyglycoside
  • buck base e.g, US patents
  • 5,417,986, 4,675,381 and 4,450,150 e.g., US patents
  • flavors and colorings may be added.
  • Suitable pharmaceutical carriers, diluents and pharmaceutically necessary materials for their use are described in Remington's Pharmaceutical Sciences.
  • the dosage of the composition according to the present invention is determined based on the type of adjuvant, the method and frequency of administration, and the desired effect and may generally be from 1 ⁇ g to 100 mg of WTA-PGN in a single adult dose.
  • the dosage may generally be from 1 ⁇ g to 1 mg of WTA-PGN in a single adult dose.
  • the administration can be administered several times if necessary.
  • the composition may be administered again by supplementing three times after the composition has been initially administered at regular intervals.
  • the compositions for the first and second reinforcements may be administered 8-12 weeks and 16-20 weeks after the first administration using the same formulation, respectively.
  • the present invention provides: 1) to obtain a double-mutant strain is lgt (lipoprotein diacylglycerol transferase) and oatA (O-acetyl transferase) gene deletions from the wild type Staphylococcus aureus; 2) heating the double mutant strain to a temperature of 40 to 70 ° C; 3) adding ⁇ -lytic enzyme to the heated strain; 4) obtaining a soluble WTA-PGN containing fraction from the enzyme additive of step 3); 5) adding lysozyme or mutanolysin to the soluble WTA-PGN containing fractions; And 6) obtaining a soluble WTA-PGN from the enzyme additive of step 5).
  • lgt lipoprotein diacylglycerol transferase
  • oatA O-acetyl transferase
  • Step 1) there is provided a method comprising obtaining a double-mutant strains and oatA lgt gene deletion from the wild-type staphylococci.
  • Double mutant strain ( ⁇ lgt ⁇ oatA obtained in the above step 1)) whereby the lgt gene deletion not likely to be contaminated by the lipoprotein can be obtained pure WTA-PGN.
  • the deletion of the oatA gene eliminates the acetyl group in the MurNAc residue of the PGN, and the resulting WTA-PGN can be easily degraded by the ⁇ -lytic enzyme of step 2).
  • Such double mutant strains can be obtained by known methods from wild type staphylococci, for example methicillin-resistant staphylococci (MRSA), methicillin-sensitive staphylococci (MSSA) or pathogenic staphylococci.
  • MRSA methicillin-resistant staphylococci
  • MSSA methicillin-sensitive staphylococci
  • the double mutant strain T363 strain of the lgt gene while having a resistance to play Oh, my God (phleomycin) defect (Nakayama M et al, Journal of Immunology 189:. 5903-591, 2012) and resistant to erythromycin T0003 strain (Park KH et al. , Journal of Biological Chemistry 285, 27167-27175, 2010) with oatA gene deficient can be prepared by transduction through phage 80.
  • Step 2) provides a step of heating the double mutant strain to a temperature of 40 to 70 °C.
  • the temperature may be 45 to 68 °C, 50 to 65 °C or 55 to 63 °C, in one embodiment of the present invention the temperature may be 60 °C.
  • step 2) may comprise the step of culturing the double mutant strain obtained in step 1), followed by heat treatment.
  • Step 3) provides a step of treating ⁇ -lytic enzyme on the heated strain.
  • the ⁇ -lytic enzyme plays a role in degrading pentaglycine ((Gly) 5) linkages linking the stem peptides present at the MurNAc residue of the insoluble WTA-PGN obtained in step 2), thereby insoluble WTA contained in the heated strain. Change PGN to soluble WTA-PGN.
  • Such ⁇ -lytic enzymes are commercially available or described in Li et al. , Journal of Biochemitry 122, 772-778, 1997, can be isolated and purified.
  • An example of a commercially available ⁇ -lytic enzyme is lysostaphin.
  • Step 3) may be carried out by suspending the strain heated in step 2) in a buffer, and then adding ⁇ -lytic enzyme and reacting with stirring at 30 to 40 ° C. for 10 to 14 hours.
  • Step 4) provides a step of obtaining a soluble WTA-PGN containing fraction from the enzyme additive of step 3).
  • This step can be carried out by passing the ⁇ -lytic enzyme additive through HPLC to obtain a fraction, and then selecting the fraction containing soluble WTA-PGN in the fraction. At this time, the fraction containing WTA-PGN can be confirmed by PAGE or silver nitrate staining method.
  • the column used in the HPLC purification may be HiTrap Q FF (GE Healthcare), which is an anion exchange resin that binds with the anion of WTA ribitol phosphate.
  • HiTrap Q FF GE Healthcare
  • Step 5 provides the step of adding lysozyme or mutanolysine to the soluble WTA-PGN containing fractions.
  • the lysozyme or mutanolysine transforms the polymeric PGN into an oligomeric PGN by breaking down the bond between MurNAc and GlcNAc of the PGN.
  • Step 5) may be carried out by suspending the soluble WTA-PGN obtained in step 4) in a buffer, and then adding lysozyme or mutanolysine for 10 to 14 hours with stirring at 30 to 40 ° C.
  • Step 6) provides a step of obtaining soluble WTA-PGN from the enzyme additive of step 5).
  • the step can be carried out by passing lysozyme or mutanolysine enzyme additive through HPLC to obtain a fraction, and then selecting the fraction containing soluble WTA-PGN from the fraction. At this time, the fraction containing WTA-PGN can be confirmed by PAGE or silver nitrate staining method.
  • the column used for HPLC purification may be HiTrap-Q (GE Healthcare).
  • the method according to the invention may further comprise further purifying the WTA-PGN after step 6).
  • Said further purification can be carried out by gel filtration chromatography or reverse phase liquid chromatography.
  • the soluble WTA-PGN prepared in step 6) can be further purified via gel filtration chromatography using Sephacryl S-200 HR column or reverse phase liquid chromatography using Symmetry Shield TM RP18 column. have.
  • the soluble WTA-PGN prepared in step 6) is further purified by gel filtration chromatography using Sephacryl S-200 HR column and reverse phase liquid chromatography using two Symmetry Shield TM RP18 columns. Can be.
  • the active fraction can be selected based on the amount of IL-17A produced after injection of each fraction obtained in the chromatography into the mouse abdominal cavity.
  • the strain can be used to isolate WTA, WTA-PGN and PGN without contamination by lipoproteins by deletion of the lgt gene.
  • the PGN MurNAc residue 6 position oxygen does not have an acetyl group, so that the isolated PGN can be easily degraded by lysozyme.
  • the stored strain was centrifuged at 6,000 rpm at 4 ° C. for 30 minutes to obtain pellets.
  • 20 ml of saline was added to resuspend the pellet, which was centrifuged for 5 minutes at 3,000 ⁇ g, 4 ° C. to obtain pellet.
  • the flask was stirred every 5 minutes during the heat treatment process so that the strain was uniformly heat treated.
  • the diluted solution of the strain was cooled to 30 ° C., which was centrifuged at 3,000 ⁇ g, 20 ° C. for 10 minutes to obtain pellets.
  • the pellet was suspended in 10 ml saline and centrifuged for 5 minutes under the same conditions as above to obtain the pellet.
  • the obtained pellet was washed three times with sterile distilled water, and the pellet was suspended in 3 ml of distilled water and lyophilized.
  • the concentrated sample was subjected to size exclusion chromatography on a Sephacryl S-100 column (1.6 cm x 87 cm) using 10 mM sodium citrate buffer (pH 6.0) containing 200 mM NaCl.
  • the eluate was measured for absorbance at 280 nm, and the fractions showing lytic activity among the fractions with high absorbance were collected and concentrated again.
  • Li et al. Based on the results of (1997), ⁇ -lytic enzyme fractions were selected from the concentrated fractions.
  • the selected fractions were subjected to size exclusion chromatography on a Superdex-75 column (1 cm ⁇ 30 cm) using 10 mM sodium citrate buffer (pH 6.0) containing 200 mM NaCl. As a result, ⁇ -lytic enzyme was finally obtained.
  • the obtained ⁇ -lytic enzyme was incubated with PGN suspension derived from Micrococcus luteus (ATCC 9341) or insoluble staphylococcus, and the lytic activity or lytic activity of the enzyme was confirmed.
  • enzymes obtained by Procise® Protein sequencer Cat. # 491-0, Applied Biosystems, USA
  • electrophoresis were identified.
  • the obtained enzyme was a ⁇ -lytic enzyme by confirming that the N-terminal sequence was S-P-N-G-L-L-Q-F-P-F (SEQ ID NO: 5) and the size was about 20 kDa.
  • the supernatant obtained was filtered with a filter having a pore size of 0.45 ⁇ m and the filtrate was HPLC (805 MANOMETRIC MODULE, 811C) equipped with a HiTrap Q FF column equilibrated with 20 mM Tris-HCl buffer (pH 7.0) (buffer A). DYNAMIC MIXER, 305 PUMP, 306 PUMP, 151 UV / VIS Detector, Gilson, USA). Thereafter, 20 mM Tris-HCl buffer (pH 7.0) (buffer B) containing 1 M NaCl was eluted by performing a linear gradient from 0 M to 1 M NaCl concentration. The concentration gradient of the buffer B was performed for 100 minutes in a gradient up to 80%, 1 minute in a gradient up to 100%, and then eluted at 100% for 10 minutes.
  • each fraction was named A, B, C, D, E and F, and each of these fractions was precipitated with acetone and then lyophilized.
  • Each lyophilized A to F fraction was suspended in 20 mM Tris-HCl buffer (pH 7.0). 12.5 ⁇ g of lysozyme (Cat. # 62970, Sigma-Aldrich Co. LLC., USA) was added to 1 mg of the lyophilized fraction and reacted with stirring at 180 rpm and 37 ° C. for 12 hours. The subsequent procedure was performed under the same conditions and methods as in Example ⁇ 3-2>, except that the HiTrap Q column was used.
  • Each of the peaks identified above were named as AL-A for the A peak obtained in the A fraction, and the same as the BL-A for the A peak obtained in the B fraction, and AL-A, BL-A, CL-B, and DL.
  • the -B, DL-C, EL-C and FL-B peaks were respectively precipitated with acetone and then lyophilized.
  • the mixed peaks (EL-C + FL-B) in which each of the CL-B, DL-B and DL-C peaks and the EL-C and FL-B peaks were mixed were prepared by lyophilization, respectively. It was. Meanwhile, C57BL / 6J female mice weighing 15 ⁇ 0.5 g at 5 weeks of age were purchased from Korea Research Institute of Bioscience and Biomedical Research Center (Ochang Campus, Korea) and were commercially available solid feeds (Cat. # 5L79, Orient Bio, Inc.). Korea) was adapted to the environment (20-25 ° C., humidity 55%) for one week while feeding. Six to twelve animals per group were divided into six groups per group in a completely randomized design to provide diet and drinking water by ad libitum. Body weight and dietary intake of each mouse were measured once daily and the lights were turned on and off at 12 hour intervals.
  • the amount of IL-17A is ELISA Ready-SET-Go!
  • the kit (Cat. # 88-7371-88, eBioscience, USA) was used to measure according to the manufacturer's protocol. As a result, a value obtained by correcting the absorbance value measured at 450 nm to the absorbance value measured at 550 nm was determined as the amount of final IL-17A.
  • Example ⁇ 3-3> The lyophilized fractions in Example ⁇ 3-3> were further purified using various columns.
  • the solvent was allowed to flow until the UV detector was stabilized under conditions of a flow rate of 0.3 ml / min, a sensitivity of 2, a peak width of 10.0 sec, and a UV absorbance of 220 nm so that there was no error due to impurity inflow during the experiment.
  • the injector was converted to the loaded state to inject the suspended peak fractions.
  • the eluent was obtained by converting the injector into the injection state before elution, and the elution conditions were the same as the equilibration conditions.
  • the obtained eluate was named 'Sepacryl-B fraction' and stored by lyophilization.
  • the Sephacryl-B fraction was further purified using a TSK G2000SW column. Specifically, 3.5 mg of the lyophilized Sephacryl-B fraction was suspended in 5 mM sodium phosphate buffer (pH 6.0) containing 100 mM NaCl to prepare a sample. On the other hand, the column was prepared by washing and equilibrating with the buffer. Equilibration was performed until the UV detector stabilized under conditions of flow rate 3 ml / min, sensitivity 0.5, peak width 10 seconds and UV absorbance 202 nm. After equilibration, the inlet was changed to a rod state to slowly inject the sample, and when the elution started, the inlet was changed to infusion to obtain an eluate. In this case, 5 mM sodium phosphate buffer (pH 6.0) containing 100 mM NaCl was used as the elution buffer.
  • 5 mM sodium phosphate buffer (pH 6.0) containing 100 mM NaCl was used as the e
  • the B fraction which most significantly increased the production of IL-17A was named 'Sepacryl-B-TSK-B fraction'. Lyophilized and stored.
  • a C18 reversed phase column was used to further purify the lyophilized Sephacryl-B-TSK-B fraction.
  • Example 5 Each fraction obtained in Example 5 was used to confirm IL-17A inducing activity in mice under the same conditions and methods as in Example 4, and the results are shown in FIG. 5.
  • Example 5 Using a fraction of 2 mg of the lyophilized C to E peak obtained in Example 5, a C18 reversed phase column was used in the same conditions and methods as in Example 5 to purify it more purely.
  • Example 7 WTA was first removed to confirm the structure of Fraction C, which showed a production increase activity of IL-17A.
  • lyophilized C fraction was dissolved in 50 ⁇ l of sterile distilled water. 50 ⁇ l of 25% TFA was added thereto and incubated with stirring at a temperature of 37 ° C. for 12 hours. After incubation, 100 ⁇ l of sterile distilled water was added to prepare a final 6.25% concentration of TFA. Thereafter, the Symmetry Shield TM RP18 column was used in the same conditions and methods as in Example 5 to purify only PGN free of WTA. At this time, the flow rate was carried out under the conditions of 1 mL / min, a sensitivity of 0.5, a column temperature of 40 ° C., and a UV absorbance of 203 nm. The concentration gradient of the mobile phase for elution was performed for 10 minutes at 0%, 50 minutes at gradient to 43.8%, 2 minutes at gradient to 100%, and then eluted at 100% for 10 minutes.
  • Example ⁇ 8-1> The eluate obtained in Example ⁇ 8-1> was loaded onto the column via an autoinjector in a Nano-flow (n) LC-MS / MS system.
  • nLC-MS / MS systems are high resolution hybrid masses with nLC (EASY-nLC 1000, Thermo Fisher Scientific, USA) with fritless electrospray (ESI) columns (150 ⁇ m ⁇ 100 mm, Nikkyo Technos Co. Ltd, Japan) It consists of a spectrometer (Q-Exactive mass spectrometer, Thermo Fisher Scientific, USA).
  • nLC separation was eluted using a 32% (v / v) acetonitrile solution containing 0.1% (v / v) formic acid as elution buffer for 30 minutes at a flow rate of 300 nL / min.
  • the eluate was sprayed online with a mass spectrometer at a pressure of 2.0 kPa in the bipolar state, and the analyzer was operated in data-dependent mode to automatically switch between MS and MS / MS acquisition.
  • a mass resolution of 35,000 was obtained at m / z 400 through full-scan mass spectra (m / z 300 to 2,000) measurements.
  • PGN having a structure represented by the following formula 1 was confirmed. It had five monosaccharide chains represented by GlcNAc-MurNAc-GlcNAc-MurNAc-GlcNAc and had one phosphate group bound to the fourth MurNAc residue.
  • the PGN of the C fraction had a molecular weight of 2167.91.

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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne une composition pharmaceutique pour la prévention ou le traitement de maladies infectieuses à staphylocoques. Plus particulièrement, le WTA-PGN soluble représenté par la formule générale 1 selon la présente invention augmente la production d'IL-17A dans le corps de souris lorsqu'il est administré auxdites souris, et, par conséquent, le WTA-PGN soluble peut être utilisé avantageusement dans la prévention et le traitement de staphylocoques.
PCT/KR2016/007748 2015-07-15 2016-07-15 Composition pour la prévention ou le traitement de maladies infectieuses à staphylocoques WO2017010845A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040247605A1 (en) * 2002-12-02 2004-12-09 Kokai-Kun John Fitzgerald Wall teichoic acid as a target for anti-staphylococcal therapies and vaccines
KR20100056510A (ko) * 2007-09-11 2010-05-27 몬도바이오테크 래보래토리즈 아게 치료제로서의 유로딜라틴의 용도
KR101062525B1 (ko) * 2002-11-12 2011-09-06 더 브리검 앤드 우먼즈 하스피털, 인크. 포도상구균 감염에 대한 다당류 백신
KR20110124060A (ko) * 2010-05-10 2011-11-16 부산대학교 산학협력단 Ｗｔａ를 유효성분으로 함유하는 백신 조성물
WO2013168965A2 (fr) * 2012-05-07 2013-11-14 목암생명공학연구소 Composition de vaccin pour prévenir une infection par staphylococcus aureus

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101062525B1 (ko) * 2002-11-12 2011-09-06 더 브리검 앤드 우먼즈 하스피털, 인크. 포도상구균 감염에 대한 다당류 백신
US20040247605A1 (en) * 2002-12-02 2004-12-09 Kokai-Kun John Fitzgerald Wall teichoic acid as a target for anti-staphylococcal therapies and vaccines
KR20100056510A (ko) * 2007-09-11 2010-05-27 몬도바이오테크 래보래토리즈 아게 치료제로서의 유로딜라틴의 용도
KR20110124060A (ko) * 2010-05-10 2011-11-16 부산대학교 산학협력단 Ｗｔａ를 유효성분으로 함유하는 백신 조성물
WO2013168965A2 (fr) * 2012-05-07 2013-11-14 목암생명공학연구소 Composition de vaccin pour prévenir une infection par staphylococcus aureus

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