WO2015190849A1 - Melittin-polyethylene glycol conjugate and pharmaceutical composition containing same - Google Patents
Melittin-polyethylene glycol conjugate and pharmaceutical composition containing same Download PDFInfo
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- WO2015190849A1 WO2015190849A1 PCT/KR2015/005892 KR2015005892W WO2015190849A1 WO 2015190849 A1 WO2015190849 A1 WO 2015190849A1 KR 2015005892 W KR2015005892 W KR 2015005892W WO 2015190849 A1 WO2015190849 A1 WO 2015190849A1
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- WIPO (PCT)
- Prior art keywords
- melittin
- polyethylene glycol
- mel
- formula
- compound
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- 229920001223 polyethylene glycol Polymers 0.000 title claims abstract description 138
- 239000002202 Polyethylene glycol Substances 0.000 title claims abstract description 102
- 239000008194 pharmaceutical composition Substances 0.000 title claims abstract description 12
- VDXZNPDIRNWWCW-JFTDCZMZSA-N melittin Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(N)=O)CC1=CNC2=CC=CC=C12 VDXZNPDIRNWWCW-JFTDCZMZSA-N 0.000 claims abstract description 98
- 108010036176 Melitten Proteins 0.000 claims abstract description 97
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- 230000004054 inflammatory process Effects 0.000 claims abstract description 7
- 125000003277 amino group Chemical group 0.000 claims abstract description 6
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- 239000000203 mixture Substances 0.000 claims description 30
- -1 polyethylene Polymers 0.000 claims description 20
- 238000000034 method Methods 0.000 claims description 15
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 claims description 14
- 239000003729 cation exchange resin Substances 0.000 claims description 12
- 239000004698 Polyethylene Substances 0.000 claims description 10
- 229920000573 polyethylene Polymers 0.000 claims description 10
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- 125000000217 alkyl group Chemical group 0.000 claims description 4
- BEOOHQFXGBMRKU-UHFFFAOYSA-N sodium cyanoborohydride Chemical compound [Na+].[B-]C#N BEOOHQFXGBMRKU-UHFFFAOYSA-N 0.000 claims description 4
- 125000002057 carboxymethyl group Chemical group [H]OC(=O)C([H])([H])[*] 0.000 claims description 3
- 229910052739 hydrogen Inorganic materials 0.000 claims description 3
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- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 3
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 3
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- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 22
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- HNSDLXPSAYFUHK-UHFFFAOYSA-N 1,4-bis(2-ethylhexyl) sulfosuccinate Chemical compound CCCCC(CC)COC(=O)CC(S(O)(=O)=O)C(=O)OCC(CC)CCCC HNSDLXPSAYFUHK-UHFFFAOYSA-N 0.000 description 2
- 108010030544 Peptidyl-Lys metalloendopeptidase Proteins 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
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- ZTQSAGDEMFDKMZ-UHFFFAOYSA-N butyric aldehyde Natural products CCCC=O ZTQSAGDEMFDKMZ-UHFFFAOYSA-N 0.000 description 2
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- 125000004177 diethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 2
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- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 2
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- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 description 2
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- OVHQWOXKMOVDJP-UHFFFAOYSA-N melitin Chemical compound OC1C(O)C(O)C(C)OC1OC1C(O)C(OC=2C=C3C(C(C(OC4C(C(O)C(O)C(COC5C(C(O)C(O)C(CO)O5)O)O4)O)=C(C=4C=CC(O)=CC=4)O3)=O)=C(O)C=2)OC(C)C1O OVHQWOXKMOVDJP-UHFFFAOYSA-N 0.000 description 2
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- 231100000419 toxicity Toxicity 0.000 description 2
- HMUNWXXNJPVALC-UHFFFAOYSA-N 1-[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperazin-1-yl]-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethanone Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)N1CCN(CC1)C(CN1CC2=C(CC1)NN=N2)=O HMUNWXXNJPVALC-UHFFFAOYSA-N 0.000 description 1
- LDXJRKWFNNFDSA-UHFFFAOYSA-N 2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)-1-[4-[2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidin-5-yl]piperazin-1-yl]ethanone Chemical compound C1CN(CC2=NNN=C21)CC(=O)N3CCN(CC3)C4=CN=C(N=C4)NCC5=CC(=CC=C5)OC(F)(F)F LDXJRKWFNNFDSA-UHFFFAOYSA-N 0.000 description 1
- GOZMBJCYMQQACI-UHFFFAOYSA-N 6,7-dimethyl-3-[[methyl-[2-[methyl-[[1-[3-(trifluoromethyl)phenyl]indol-3-yl]methyl]amino]ethyl]amino]methyl]chromen-4-one;dihydrochloride Chemical compound Cl.Cl.C=1OC2=CC(C)=C(C)C=C2C(=O)C=1CN(C)CCN(C)CC(C1=CC=CC=C11)=CN1C1=CC=CC(C(F)(F)F)=C1 GOZMBJCYMQQACI-UHFFFAOYSA-N 0.000 description 1
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- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
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Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
- C07K17/02—Peptides being immobilised on, or in, an organic carrier
- C07K17/08—Peptides being immobilised on, or in, an organic carrier the carrier being a synthetic polymer
Definitions
- the present invention relates to a melittin-polyethylene glycol combination having a low cytotoxicity and an excellent anti-inflammatory effect, a method for preparing the same, and a pharmaceutical composition for treating or preventing inflammation including the same as an active ingredient.
- Melittin is a natural physiologically active substance contained in bee venom and is a major component of bee venom. Melittin consists of 26 amino acids and has a helical structure (T C Terwilliger and D Eisenberg, J Biol Chem 257: 6016-6022 (1982)). Based on the proline amino acid No. 14, it has a bipolar property with hydrophobicity of the N-terminal part and hydrophilicity of the C-terminal part.
- Melitin is known to be able to inhibit the growth of 20-30 types of bacteria, and particularly shows stronger antimicrobial activity in gram positive than gram negative. In addition, it is known to have antifungal action. Reduces interfacial tension of cell membranes and increases cell permeability. In addition, melittin has anti-inflammatory action by increasing cortisol in plasma. In addition, the efficacy of anti-cancer, anti-inflammatory, anti-pain has been reported.
- melittin is toxic and has a short in vivo half-life.
- the present inventors have made diligent research efforts to reduce the toxicity of melittin, and found that the melittin-polyethylene glycol combination reduces the cytotoxicity of melittin and maintains various biological activities such as anti-inflammatory and anticancer of melittin.
- the present invention has been completed.
- an object of the present invention is to provide a melittin-polyethylene glycol combination having a low cytotoxicity and an excellent anti-inflammatory effect.
- Another object of the present invention is to provide a method for preparing the melittin-polyethylene glycol blend.
- Still another object of the present invention is to provide a pharmaceutical composition for treating or preventing inflammation including the melittin-polyethylene glycol combination.
- One embodiment of the present invention relates to a melittin-polyethylene glycol combination in which polyethylene glycol is bonded to the N-terminal amino group of melittin.
- the melittin-polyethylene glycol combination according to one embodiment of the present invention may be a compound represented by the following Chemical Formula 1.
- R is hydrogen or an alkyl group of C 1 -C 6 ,
- n is an integer from 10 to 1000
- n is an integer from 0 to 10
- the alkyl group of C 1 -C 6 means a straight or branched hydrocarbon having 1 to 6 carbon atoms, for example, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, or the like. Include but are not limited to.
- R is methyl
- n is an integer from 100 to 500
- n is an integer from 0 to 3
- Mel is a compound of Formula 1, which is melittin.
- One embodiment of the present invention relates to a method for producing the melittin-polyethylene glycol blend, the production method of the present invention as shown in Scheme 1 below,
- R, n, m and Mel are as defined in the formula (1).
- step (i) the melittin of Formula 2 and polyethylene glycol-alkylaldehyde of Formula 3 are combined to obtain a compound of Formula 4.
- melittin purified from bee venom or commercially available melittin can be used without limitation. Purified melittin can be sterilized over a 0.2 um filter, and for further purification, salt removal may be added through dialysis.
- Polyethyleneglycol-alkylaldehyde of the formula (3) is commercially available, the molecular weight is preferably 2 kDa to 40 kDa, more preferably 5 kDa to 20 kDa.
- the coupling reaction may be performed in an acidic buffer solution of pH 3.5 to 6, and the buffer solution may be used as acetate buffer, phosphate buffer and the like.
- the molar ratio of polyethyleneglycol-alkylaldehyde to melittin in the coupling reaction is preferably 1 to 10.
- 0.5 to 2 molar ratio of polyethylene glycol-alkylaldehyde may be additionally added.
- step (ii) the compound of formula 4 is reduced to prepare a melittin-polyethylene glycol compound of formula 1.
- the reduction reaction may be performed using a reducing agent such as sodium cyanoborohydride (NaCNBH 3 ).
- a reducing agent such as sodium cyanoborohydride (NaCNBH 3 ).
- the preparation method according to an embodiment of the present invention may further comprise the step of purifying the prepared melittin-polyethylene glycol formulation.
- the purification may be performed using a pH 7 to 8 buffer containing a salt such as sodium chloride on the cation exchange resin as an elution buffer.
- a salt such as sodium chloride
- the 1: 1 melittin-polyethylene glycol blend can be isolated purely.
- Polyethyleneglycol-alkylaldehyde that does not bind to melittin does not bind to the cation exchange resin, and the binding of the polyethyleneglycol-alkylaldehyde to the cation exchange resin becomes weaker.
- a strong binding melittin-polyethylene glycol mixture can be fractionated from the weak binding melittin-polyethylene glycol mixture, and finally the melittin unreacted when the highest salt concentration or sodium hydroxide is flown. It is fractionated.
- the cation exchange resin is a polymer into which a functional group having a cation exchange capacity is introduced, and as the polymer matrix, for example, polystyrene, polyphenolic resin, cellulose, polyacrylamide , Test column, Sepharose (Sepharose) and the like can be used, and as a functional group having a cation exchange capacity, for example, RCH 2 SO 3 - H + , C 6 H 5 SO 3 - H + , C 6 H 5 PO 3 2- (Na + ) 2 , RCOO - Na + , C 6 H 5 CH 2 N (CH 2 COO - H + ) 2 , carboxymethyl, phosphate, sulfoethyl, sulfopropyl ( sulfopropyl), diethyl (2-hydroxypropyl quaternary amino) (diethyl (2-hydroxylpropyl quaternary amino)) and the like can be used.
- Fractions containing only the melittin-polyethylene glycol combination of Formula 1 can be taken separately and lyophilized. If the final desired buffer solution is lyophilized, the composition may be determined using diafiltration such as concentration and dialysis.
- the melittin-polyethylene glycol combination of the present invention can reduce the toxicity possessed by melittin, and can increase the anti-inflammatory effect.
- the present invention provides a pharmaceutical composition for the treatment or prevention of inflammation, specifically rheumatoid arthritis, lupus, psoriasis, enteritis or osteoarthritis, comprising the melittin-polyethylene glycol combination of Formula 1 together with a pharmaceutically acceptable carrier It is about.
- the pharmaceutical composition according to the invention may be administered orally (eg, by taking or inhaling) or parenterally (eg by injection, deposition, transplantation, suppository), and the injection may for example be intravenous injection. , Intracutaneous injection, intramuscular injection or intraperitoneal injection.
- the pharmaceutical composition according to the present invention may be used as tablets, capsules, granules, fine subtilae, powders, sublingual tablets, suppositories, ointments, injections, emulsions, suspensions, syrups, sprays and the like. Can be formulated.
- the pharmaceutical compositions according to the present invention in various forms can be prepared by known techniques using pharmaceutically acceptable carriers commonly used in each formulation.
- Examples of pharmaceutically acceptable carriers include excipients, binders, disintegrating agents, lubricants, preservatives, antioxidants, isotonic agents, buffers, coatings, sweeteners, solubilizers, bases, dispersants, wetting agents , Suspending agents, stabilizers, coloring agents and the like.
- the pharmaceutical composition according to the invention comprises from about 0.01 to 95% by weight of the compound of the invention or a pharmaceutically acceptable salt thereof.
- the specific dosage of the pharmaceutical composition of the present invention may vary depending on the type of mammal including the person being treated, weight, sex, degree of disease, judgment of a doctor, and the like.
- 0.01 to 50 mg of active ingredient per kg of body weight is administered per day
- 0.01 to 10 mg of active ingredient per kg of body weight per day are administered.
- the total daily dose may be administered at one time or divided into several times depending on the extent of the disease, the judgment of the doctor.
- the melittin-polyethyleneglycol copolymer in which polyethyleneglycol is bonded to the N-terminal amino group of the melittin according to the present invention is lower in cytotoxicity and excellent in anti-inflammatory effect than melittin.
- a melittin-polyethylene glycol compound in which polyethylene glycol is bonded to the N-terminal amino group of melittin can be prepared in high yield and high selectivity.
- Example 1 is a diagram showing the results of Iodine staining and Coomassie staining after electrophoresis of the melittin-polyethylene glycol compound preparation product obtained in Example 1.
- FIG. 2 is an LC chromatograph after treatment of melittin (MEL) and melittin-polyethylene glycol compound (PEG-MEL) with Lys-C.
- MEL melittin
- PEG-MEL melittin-polyethylene glycol compound
- MEL melittin
- PEG-MEL melittin-polyethylene glycol combination
- MEL 4 is a graph showing cytotoxicity of melittin (MEL) and 1: 1 melittin-polyethylene glycol combination (mono-PEG (5k) -MEL, mono-PEG (20k) -MEL).
- MEL melittin
- 1 melittin-polyethylene glycol combination mono-PEG (5k) -MEL, mono-PEG (20k) -MEL.
- MEL melittin
- mono-PEG (5k) -MEL melittin-polyethylene glycol combination
- melittin 2.5 mg was dissolved in 50 mM acetate buffer, pH 3.5, 4.0, 5.0, 6.0, respectively.
- sodium cyanoborohydride (NaCNBH 3 ) was added to induce a reduction reaction of the compound. The reaction was carried out at room temperature for 6 hours. Then, 100 times more glycine (Glycine) than the molar ratio of methoxy polyethyleneglycol-propionaldehyde was added to stop the reaction.
- PEG-MEL melittin-polyethylene glycol compound
- a melittin-polyethyleneglycol blend was prepared in the same manner as in Example 1 except that the molar ratio of melittin and polyethylene glycol-propionaldehyde was 1: 7 or 1:10 at pH 3.5.
- the product was analyzed using HPLC and the results are shown in Table 1 below.
- melittin 2.5 mg was dissolved in 50 mM acetate buffer, pH 4.0.
- linear methoxy polyethyleneglycol-propionaldehyde PEG, molecular weight 20 kDa
- PEG linear methoxy polyethyleneglycol-propionaldehyde
- NaCNBH 3 sodium cyanoborohydride
- the obtained melittin-polyethylene glycol mixture preparation product was analyzed using HPLC, and the results are shown in Table 4 below.
- the reaction solution obtained in Example 1 was prepared by 0.2um filter.
- the cation exchange resin column SP capto TM Impres, GE
- the filtered reaction solution was flowed out.
- an additional buffer solution was sufficiently flowed. Only 1: 1 melittin-polyethyleneglycol blend (mono-PEG (5k) -MEL) was purified from the melittin and melittin-polyethyleneglycol blends bound to the cation exchange resin.
- the eluted fractions were subjected to electrophoresis and purified purely 1: 1 melittin-polyethylene glycol compound (mono-PEG (5k) -MEL) through Coomassie staining and Iodine staining. The more polyethylene glycol bound to one molecule melittin, the weaker the binding force with the ion exchange resin was confirmed.
- the reaction solution obtained in Example 4 was prepared by 0.2um filter.
- the cation exchange resin column SP capto TM Impres, GE
- the filtered reaction solution was flowed out.
- an additional buffer solution was sufficiently flowed. Only 1: 1 melittin-polyethyleneglycol combination (mono-PEG (20k) -MEL) was purified in the melittin and melittin-polyethyleneglycol combinations bound to the cation exchange resin.
- the eluted fractions were electrophoresed to purify 1: 1 melittin-polyethylene glycol compound (mono-PEG (20k) -MEL) through Coomassie staining and Iodine staining. The more polyethylene glycol bound to one molecule melittin, the weaker the binding force with the ion exchange resin was confirmed.
- Example 5 Peptide mapping was performed to determine whether the melittin-polyethylene glycol single compound (mono-PEG (5k) -MEL) obtained in Example 5 was specifically conjugated to the N-terminus. Melitin-polyethyleneglycol single combination was treated with endoproteinase Lys-C followed by LC-MS / MS analysis.
- the melittin-polyethyleneglycol formulation obtained in Example 5 was further subjected to Superdex 200 gel filtration. Peptide mapping was performed by collecting only the fractions except the front and back fractions of the melittin-polyethylene glycol peak. The results are shown in FIG.
- L1-PEG (5793 Da) was detected at retention time similar to L2. This is theoretically 137 Da that is greater than 5656 Da. This is the fraction of polyethylene glycol poly-dispersity (average molecular weight 4985 Da, molecular weight dispersibility: 4029 ⁇ 6041), except the long tailing portion of the melittin-polyethylene glycol peak in the tablet using Superdex 200 gel filtration The average molecular weight is expected to increase because it was obtained as a sample.
- Absorbance A Absorbance of the sample at 415 nm wavelength
- Absorbance B Absorbance of phosphate buffer at 415 nm
- Absorbance C Absorbance of 1% Triton X-100 at 415 nm
- the erythrocyte destructive capacity of 1: 1 melittin-polyethylene glycol copolymer (mono-PEG (5k) -MEL) was lower than that of melittin (MEL).
- Cell growth inhibition activity was measured according to the XTT assay (Cell Proliferation Kit II (XTT) assay (Roche)) method.
- XTT Cell Proliferation Kit II
- XTT Cell Proliferation Kit II assay
- RAW 264.7 cells were seeded in a 96-well plate at a concentration of 5 ⁇ 10 5 cells / ml at 200 ul per well, and then the melittin and 1: 1 melittin-polyethylene glycol blends were added at each concentration of lipopolysaccaride (LPS). After treatment with, incubated in 37 °C, 5% CO 2 incubator. As inflammation markers during inflammation, NO (nitrite oxide) and TNF- ⁇ produced by LPS were measured. JSH, Bay11-1272 anti-inflammatory reagent was used as a TNF- ⁇ inhibitor positive control.
- the measurement of NO was performed at 37 ° C. after simultaneous treatment with melittin (MEL) or 1: 1 melittin-polyethylene glycol compound (mono-PEG (5k) -MEL, mono-PEG (20k) -MEL) and LPS. in 5% CO 2 incubator and cultured for 24 hours. 50 ul of the supernatant was mixed with the same amount of Griess reagent (1% Sulfarunilamide, 0.1% naphthyethylenediamine dihydrochloride, and 2% phosphoric acid), and the absorbance was measured at 540 nm after standing at room temperature for 10 minutes. The results are shown in FIG.
- melittin (MEL) and 1: 1 melittin-polyethylene glycol combination inhibit NO production in a concentration-dependent manner. It was. However, in the case of melittin, a sharp decrease in NO at a concentration of 5 ug / ml or more is expected to be a cytotoxic effect.
- the 1: 1 melittin-polyethylene glycol combination was shown to inhibit NO production in a concentration dependent manner even at concentrations that do not exhibit cytotoxicity.
- TNF- ⁇ was performed after simultaneous treatment with melittin (MEL) or 1: 1 melittin-polyethylene glycol compound (mono-PEG (5k) -MEL, mono-PEG (20k) -MEL) and LPS, followed by 37 C was incubated for 6 hours in a 5% CO 2 incubator. Supernatant was taken and TNF- ⁇ ELISA was performed. The results are shown in FIG. 6 and Table 7.
- melittin (MEL) and 1: 1 melittin-polyethylene glycol combination (mono-PEG (5k) -MEL) were shown to inhibit the production of TNF- ⁇ in a concentration dependent manner.
- melittin (MEL) and 1: 1 melittin-polyethylene glycol combination (mono-PEG (5k) -MEL) were shown to inhibit the production of TNF- ⁇ in a concentration dependent manner.
- the inhibitory effect of melittin appeared more urgent at the concentration showing cytotoxicity.
- melittin inhibited the production of NO and TNF- ⁇ by 34% and 31%, respectively, at a concentration of 2.5 ug / ml, which showed no cytotoxicity
- a 1: 1 melittin-polyethylene glycol compound (mono -PEG (5k) -MEL) showed inhibitory effects of 72% and 57% at 20 ug / ml concentrations without cytotoxicity, respectively.
- the 1: 1 melittin-polyethylene glycol combination (mono-PEG (20k) -MEL) showed 45% and 42% inhibition at 20 ug / ml concentrations without cytotoxicity, respectively.
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Abstract
The present invention provides a melittin-polyethylene glycol conjugate in which polyethylene glycol is coupled to an N-terminal amino group of melittin, a method of manufacturing the same, and a pharmaceutical composition for treatment or prevention of an inflammation comprising the same as an active ingredient. The melittin-polyethylene glycol conjugate according to the present invention is less cytotoxic and has an excellent anti-inflammatory effect compared to melittin.
Description
본 발명은 세포독성이 낮고 항염 효과가 우수한 멜리틴-폴리에틸렌글리콜 배합체, 그의 제조방법 및 그를 유효성분으로 포함하는 염증의 치료 또는 예방용 약제학적 조성물에 관한 것이다.The present invention relates to a melittin-polyethylene glycol combination having a low cytotoxicity and an excellent anti-inflammatory effect, a method for preparing the same, and a pharmaceutical composition for treating or preventing inflammation including the same as an active ingredient.
멜리틴은 봉독에 포함되어 있는 천연생리활성물질로서, 봉독의 주요 성분이다. 멜리틴은 26개의 아미노산으로 구성되어 있으며, 나선형 구조를 가진 형태를 띄고 있다[T C Terwilliger and D Eisenberg, J Biol Chem 257:6016-6022 (1982)]. 14번 프롤린 아미노산을 기준으로 N-말단 부위의 소수성과 C-말단 부위의 친수성을 가지고 있는 양극성 성질을 가지고 있다.Melittin is a natural physiologically active substance contained in bee venom and is a major component of bee venom. Melittin consists of 26 amino acids and has a helical structure (T C Terwilliger and D Eisenberg, J Biol Chem 257: 6016-6022 (1982)). Based on the proline amino acid No. 14, it has a bipolar property with hydrophobicity of the N-terminal part and hydrophilicity of the C-terminal part.
멜리틴은 20-30 종류의 세균들의 성장을 억제할 수 있는 것으로 알려졌으며, 특히 그람 음성균(gram negative)보다 그람 양성균(gram positive)에서 강력한 항균 활성을 나타낸다. 그 외에도 항진균 작용이 있다고 알려져 있다. 세포막의 계면 장력을 감소시키고, 세포의 투과성을 증가시킨다. 뿐만 아니라 멜리틴은 혈장내의 코티솔(cortisol)을 증가시킴으로써 항염증 작용이 있다. 또한 항암, 항염증, 항통증 등의 효능이 보고되고 있다. Melitin is known to be able to inhibit the growth of 20-30 types of bacteria, and particularly shows stronger antimicrobial activity in gram positive than gram negative. In addition, it is known to have antifungal action. Reduces interfacial tension of cell membranes and increases cell permeability. In addition, melittin has anti-inflammatory action by increasing cortisol in plasma. In addition, the efficacy of anti-cancer, anti-inflammatory, anti-pain has been reported.
대한민국 공개특허 제10-2006-0034961호에서 봉독 및 봉독의 주성분인 멜리틴의 NF-κB의 억제효능이 in vivo와 in vitro에서 확인되었으며, 멜리틴의 항암효능에 대한 연구는 혈관형성인자(VEGF)에 관련된 pathway에 대한 유전자 발현의 억제를 통하여 이루어진다고 알려져 있다. 또한 많은 연구결과를 통해 멜리틴의 경우 NO(Nitric oxide), TNF-α의 세포내 수준을 낮추며, 류마티스 관절염에 대한 항염 효과에 대한 연구가 진행된 바 있다 [Joint Bone Spine 78:471-477 (2011), Arthritis and Rheumatism 50(11) 3504-3515 (2004), J Biol Chem 284(6) 3804-3838 (2009)]. 이외의 멜리틴의 항바이러스 효능과 약물 및 유전자 전달에 대한 가능성 등이 시사된 바 있다[BioSci Rep 27:189-223, 2007].Inhibitory effects of NF- κ B in the major component of bee venom and bee venom melittin in the Republic of Korea Patent Publication No. 10-2006-0034961 call has been identified in in vivo and in vitro, study of the anticancer efficacy of melittin is angiogenic factors ( VEGF) is known to be through the inhibition of gene expression for pathways associated with. In addition, many studies have shown that melittin lowers intracellular levels of nitric oxide (NO) and TNF- α , and studies on anti-inflammatory effects on rheumatoid arthritis [Joint Bone Spine 78: 471-477 (2011) ), Arthritis and Rheumatism 50 (11) 3504-3515 (2004), J Biol Chem 284 (6) 3804-3838 (2009)]. Other antiviral effects of melittin and the potential for drug and gene delivery have been suggested [BioSci Rep 27: 189-223, 2007].
그러나, 멜리틴은 독성이 있고, 생체내 반감기가 짧은 문제점이 있었다. However, melittin is toxic and has a short in vivo half-life.
본 발명자들은 멜리틴(melittin)의 독성을 감소시키고자 예의 연구 노력한 결과, 멜리틴-폴리에틸렌글리콜 배합체가 멜리틴의 세포독성을 감소시키고, 멜리틴의 항염, 항암 등 다양한 생리활성을 유지함을 알아내고 본 발명을 완성하게 되었다.The present inventors have made diligent research efforts to reduce the toxicity of melittin, and found that the melittin-polyethylene glycol combination reduces the cytotoxicity of melittin and maintains various biological activities such as anti-inflammatory and anticancer of melittin. The present invention has been completed.
따라서 본 발명의 목적은 세포독성이 낮고 항염 효과가 우수한 멜리틴-폴리에틸렌글리콜 배합체를 제공하는 것이다.Accordingly, an object of the present invention is to provide a melittin-polyethylene glycol combination having a low cytotoxicity and an excellent anti-inflammatory effect.
본 발명의 다른 목적은 상기 멜리틴-폴리에틸렌글리콜 배합체의 제조방법을 제공하는 것이다.Another object of the present invention is to provide a method for preparing the melittin-polyethylene glycol blend.
본 발명의 또 다른 목적은 상기 멜리틴-폴리에틸렌글리콜 배합체를 포함하는 염증의 치료 또는 예방용 약제학적 조성물을 제공하는 것이다. Still another object of the present invention is to provide a pharmaceutical composition for treating or preventing inflammation including the melittin-polyethylene glycol combination.
본 발명의 일 실시형태는 멜리틴의 N-말단 아미노기에 폴리에틸렌글리콜이 결합된 멜리틴-폴리에틸렌글리콜 배합체에 관한 것이다.One embodiment of the present invention relates to a melittin-polyethylene glycol combination in which polyethylene glycol is bonded to the N-terminal amino group of melittin.
본 발명의 일 실시형태에 따른 멜리틴-폴리에틸렌글리콜 배합체는 하기 화학식 1의 화합물일 수 있다.The melittin-polyethylene glycol combination according to one embodiment of the present invention may be a compound represented by the following Chemical Formula 1.
[화학식 1] [Formula 1]
상기 식에서,Where
R은 수소 또는 C1-C6의 알킬기이고,R is hydrogen or an alkyl group of C 1 -C 6 ,
n은 10 내지 1000의 정수이며,n is an integer from 10 to 1000,
m은 0 내지 10의 정수이고,m is an integer from 0 to 10,
Mel은 멜리틴이다.Mel is a melittin.
본 명세서에서 C1-C6의 알킬기는 탄소수 1 내지 6개로 구성된 직쇄형 또는 분지형 탄화수소를 의미하며, 예를 들어 메틸, 에틸, n-프로필, n-부틸, n-펜틸, n-헥실 등이 포함되나 이에 한정되는 것은 아니다.In the present specification, the alkyl group of C 1 -C 6 means a straight or branched hydrocarbon having 1 to 6 carbon atoms, for example, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, or the like. Include but are not limited to.
본 발명의 일 실시형태에 따른 멜리틴-폴리에틸렌글리콜 배합체는Melitin-polyethylene glycol blend according to an embodiment of the present invention
R은 메틸이고,R is methyl,
n은 100 내지 500의 정수이며,n is an integer from 100 to 500,
m은 0 내지 3의 정수이고,m is an integer from 0 to 3,
Mel은 멜리틴인 상기 화학식 1의 화합물이다.Mel is a compound of Formula 1, which is melittin.
본 발명의 일 실시형태는 상기 멜리틴-폴리에틸렌글리콜 배합체의 제조방법에 관한 것으로, 본 발명의 제조방법은 하기 반응식 1에 나타낸 바와 같이, One embodiment of the present invention relates to a method for producing the melittin-polyethylene glycol blend, the production method of the present invention as shown in Scheme 1 below,
(i) 하기 화학식 2의 멜리틴과 하기 화학식 3의 폴리에틸렌글리콜-알킬알데하이드를 결합반응시켜 하기 화학식 4의 화합물을 수득하는 단계; 및(i) combining the melittin of Formula 2 with polyethylene glycol-alkylaldehyde of Formula 3 to obtain a compound of Formula 4; And
(ii) 하기 화학식 4의 화합물을 환원반응시키는 단계를 포함한다. (ii) reducing the compound of formula (4).
[반응식 1] Scheme 1
상기 식에서, R, n, m 및 Mel은 상기 화학식 1에서 정의한 바와 같다.Wherein R, n, m and Mel are as defined in the formula (1).
상기 단계 (i)에서는 화학식 2의 멜리틴과 화학식 3의 폴리에틸렌글리콜-알킬알데하이드를 결합반응시켜 화학식 4의 화합물을 수득한다.In step (i), the melittin of Formula 2 and polyethylene glycol-alkylaldehyde of Formula 3 are combined to obtain a compound of Formula 4.
상기 멜리틴으로는 봉독으로부터 정제한 멜리틴 또는 상업적으로 판매하고 있는 멜리틴을 제한 없이 사용할 수 있다. 정제한 멜리틴은 0.2 um 필터를 걸쳐 제균을 할 수 있으며, 추가 정제를 위해서는 투석을 통하여 염을 제거하는 과정을 부가할 수 있다. As the melittin, melittin purified from bee venom or commercially available melittin can be used without limitation. Purified melittin can be sterilized over a 0.2 um filter, and for further purification, salt removal may be added through dialysis.
상기 화학식 3의 폴리에틸렌글리콜-알킬알데하이드는 상업적으로 입수할 수 있으며, 분자량은 2 kDa 내지 40 kDa인 것이 바람직하며, 5 kDa 내지 20 kDa인 것이 보다 바람직하다.Polyethyleneglycol-alkylaldehyde of the formula (3) is commercially available, the molecular weight is preferably 2 kDa to 40 kDa, more preferably 5 kDa to 20 kDa.
상기 결합반응은 pH 3.5 내지 6의 산성 완충용액에서 수행될 수 있으며, 상기 완충용액으로는 아세트산염 완충액, 인산염 완충액 등을 사용할 수 있다.The coupling reaction may be performed in an acidic buffer solution of pH 3.5 to 6, and the buffer solution may be used as acetate buffer, phosphate buffer and the like.
상기 결합반응에서 멜리틴에 대한 폴리에틸렌글리콜-알킬알데하이드의 몰비율은 1 내지 10이 바람직하다.The molar ratio of polyethyleneglycol-alkylaldehyde to melittin in the coupling reaction is preferably 1 to 10.
본 발명의 일 실시형태에 따른 제조방법은 멜리틴과 폴리에틸렌글리콜의 1:1 배합체의 수율을 증가시키기 위해서, 0.5 내지 2 몰비율의 폴리에틸렌글리콜-알킬알데하이드를 추가적으로 첨가할 수 있다.In the production method according to an embodiment of the present invention, in order to increase the yield of the 1: 1 blend of melittin and polyethylene glycol, 0.5 to 2 molar ratio of polyethylene glycol-alkylaldehyde may be additionally added.
상기 단계 (ii)에서는 화학식 4의 화합물을 환원반응시켜 화학식 1의 멜리틴-폴리에틸렌글리콜 배합체를 제조한다.In step (ii), the compound of formula 4 is reduced to prepare a melittin-polyethylene glycol compound of formula 1.
상기 환원반응은 소디움 시아노보로하이드라이드(Sodium cyanoborohydride: NaCNBH3)와 같은 환원제를 사용하여 수행할 수 있다.The reduction reaction may be performed using a reducing agent such as sodium cyanoborohydride (NaCNBH 3 ).
본 발명의 일 실시형태에 따른 제조방법은 제조된 멜리틴-폴리에틸렌글리콜 배합체를 정제하는 단계를 추가로 포함할 수 있다. The preparation method according to an embodiment of the present invention may further comprise the step of purifying the prepared melittin-polyethylene glycol formulation.
상기 정제는 양이온 교환 수지 상에서 염화나트륨과 같은 염을 포함하는 pH 7 내지 8의 완충용액을 전개액(elution buffer)으로 사용하여 수행할 수 있다. 염의 농도를 조절하면서 기울기 용리하여 1:1 멜리틴-폴리에틸렌글리콜 배합체를 순수하게 분리할 수 있다. 멜리틴과 결합하지 않은 폴리에틸렌글리콜-알킬알데하이드는 양이온 교환 수지에 결합하지 않으며, 폴리에틸렌글리콜-알킬알데하이드가 결합할수록 양이온 교환 수지에 대한 결합력은 약하게 된다. 따라서 염 농도를 높힘으로써 결합력이 약한 멜리틴-폴리에틸렌글리콜 배합체로부터 결합력이 강한 멜리틴-폴리에틸렌글리콜 배합체가 분획될 수 있으며, 마지막으로 가장 높은 염 농도 또는 수산화나트륨을 흘려주었을 때 반응하지 않은 멜리틴이 분획된다.The purification may be performed using a pH 7 to 8 buffer containing a salt such as sodium chloride on the cation exchange resin as an elution buffer. By gradient elution while adjusting the salt concentration, the 1: 1 melittin-polyethylene glycol blend can be isolated purely. Polyethyleneglycol-alkylaldehyde that does not bind to melittin does not bind to the cation exchange resin, and the binding of the polyethyleneglycol-alkylaldehyde to the cation exchange resin becomes weaker. Thus, by increasing the salt concentration, a strong binding melittin-polyethylene glycol mixture can be fractionated from the weak binding melittin-polyethylene glycol mixture, and finally the melittin unreacted when the highest salt concentration or sodium hydroxide is flown. It is fractionated.
상기 양이온 교환 수지는 양이온 교환능이 있는 작용기가 도입된 폴리머로서, 폴리머 매트릭스로는 예를 들어, 폴리스틸렌(polystyrene), 폴리페놀릭(polyphenolic) 레진, 셀룰로우스(cellulose), 폴리아크릴아마이드(polyacrylamide), 테스트란, 세파로스(Sepharose) 등이 사용될 수 있고, 양이온 교환능이 있는 작용기로는 예를 들어, RCH2SO3
-H+, C6H5SO3
-H+, C6H5PO3
2-(Na+)2, RCOO-Na+, C6H5CH2N(CH2COO-H+)2, 카복시메틸(carboxymethyl), 포스페이트(phosphate), 설포에틸(sulfoethyl), 설포프로필(sulfopropyl), 디에틸(2-히드록시프로필 4차 아미노)(diethyl(2-hydroxylpropyl quaternary amino)) 등이 사용될 수 있다. 바람직하게는 세파로스 포스페이트(sepharose phosphate) 또는 세파로스 카복시메틸(sepharose carboxymethyl)을 사용할 수 있다.The cation exchange resin is a polymer into which a functional group having a cation exchange capacity is introduced, and as the polymer matrix, for example, polystyrene, polyphenolic resin, cellulose, polyacrylamide , Test column, Sepharose (Sepharose) and the like can be used, and as a functional group having a cation exchange capacity, for example, RCH 2 SO 3 - H + , C 6 H 5 SO 3 - H + , C 6 H 5 PO 3 2- (Na + ) 2 , RCOO - Na + , C 6 H 5 CH 2 N (CH 2 COO - H + ) 2 , carboxymethyl, phosphate, sulfoethyl, sulfopropyl ( sulfopropyl), diethyl (2-hydroxypropyl quaternary amino) (diethyl (2-hydroxylpropyl quaternary amino)) and the like can be used. Preferably, sepharose phosphate or sepharose carboxymethyl may be used.
화학식 1의 멜리틴-폴리에틸렌글리콜 배합체만이 함유된 분획물은 별도로 취한 후 동결건조할 수 있다. 동결건조 전 최종적으로 원하는 완충용액이 있을 경우 농축과 투석 등 디아필트레이션(diafiltration)을 이용하여 조성을 결정할 수도 있다.Fractions containing only the melittin-polyethylene glycol combination of Formula 1 can be taken separately and lyophilized. If the final desired buffer solution is lyophilized, the composition may be determined using diafiltration such as concentration and dialysis.
본 발명의 멜리틴-폴리에틸렌글리콜 배합체는 멜리틴이 가지고 있는 독성을 감소시킬 수 있으며, 항염 효과를 증가시킬 수 있다.The melittin-polyethylene glycol combination of the present invention can reduce the toxicity possessed by melittin, and can increase the anti-inflammatory effect.
따라서 본 발명은 상기 화학식 1의 멜리틴-폴리에틸렌글리콜 배합체를 약제학적으로 허용되는 담체와 함께 포함하는 염증, 구체적으로는 류마티스 관절염, 루푸스, 건선, 장염증 또는 골관절염의 치료 또는 예방용 약제학적 조성물에 관한 것이다. Accordingly, the present invention provides a pharmaceutical composition for the treatment or prevention of inflammation, specifically rheumatoid arthritis, lupus, psoriasis, enteritis or osteoarthritis, comprising the melittin-polyethylene glycol combination of Formula 1 together with a pharmaceutically acceptable carrier It is about.
본 발명에 따른 약제학적 조성물은 경구적으로(예를 들면, 복용 또는 흡입) 또는 비경구적으로(예를 들면, 주사, 침착, 이식, 좌약) 투여될 수 있으며, 주사는 예를 들면, 정맥주사, 피하주사, 근육내주사 또는 복강내주사일 수 있다. 본 발명에 따른 약제학적 조성물은 투여 경로에 따라, 정제, 캡슐제, 과립제, 파인 서브틸래(fine subtilae), 분제, 설하 정제, 좌약, 연고, 주사제, 유탁액제, 현탁액제, 시럽제, 분무제 등으로 제형화될 수 있다. 상기 여러 가지 형태의 본 발명에 따른 약제학적 조성물은 각 제형에 통상적으로 사용되는 약제학적으로 허용되는 담체(carrier)를 사용하여 공지기술에 의해 제조될 수 있다. 약제학적으로 허용되는 담체의 예는 부형제, 결합제, 붕해제(disintegrating agent), 윤활제, 방부제, 항산화제, 등장제(isotonic agent), 완충제, 피막제, 감미제, 용해제, 기제(base), 분산제, 습윤제, 현탁화제, 안정제, 착색제 등을 포함한다. The pharmaceutical composition according to the invention may be administered orally (eg, by taking or inhaling) or parenterally (eg by injection, deposition, transplantation, suppository), and the injection may for example be intravenous injection. , Intracutaneous injection, intramuscular injection or intraperitoneal injection. According to the route of administration, the pharmaceutical composition according to the present invention may be used as tablets, capsules, granules, fine subtilae, powders, sublingual tablets, suppositories, ointments, injections, emulsions, suspensions, syrups, sprays and the like. Can be formulated. The pharmaceutical compositions according to the present invention in various forms can be prepared by known techniques using pharmaceutically acceptable carriers commonly used in each formulation. Examples of pharmaceutically acceptable carriers include excipients, binders, disintegrating agents, lubricants, preservatives, antioxidants, isotonic agents, buffers, coatings, sweeteners, solubilizers, bases, dispersants, wetting agents , Suspending agents, stabilizers, coloring agents and the like.
본 발명에 따른 약제학적 조성물은 약제의 형태에 따라 다르지만, 본 발명의 화합물 또는 그의 약제학적으로 허용되는 염을 약 0.01 내지 95 중량%로 포함한다. The pharmaceutical composition according to the invention, depending on the form of the medicament, comprises from about 0.01 to 95% by weight of the compound of the invention or a pharmaceutically acceptable salt thereof.
본 발명의 약제학적 조성물의 구체적인 투여량은 치료되는 사람을 포함한 포유동물의 종류, 체중, 성별, 질환의 정도, 의사의 판단 등에 따라 다를 수 있다. 바람직하게는, 경구투여의 경우에는 하루에 체중 1kg당 활성성분 0.01 내지 50 mg이 투여되고, 비경구투여의 경우에는 하루에 체중 1kg당 활성성분 0.01 내지 10 mg이 투여된다. 상기 총 일일 투여량은 질환의 정도, 의사의 판단 등에 따라 한번에 또는 수회로 나누어 투여될 수 있다.The specific dosage of the pharmaceutical composition of the present invention may vary depending on the type of mammal including the person being treated, weight, sex, degree of disease, judgment of a doctor, and the like. Preferably, in the case of oral administration, 0.01 to 50 mg of active ingredient per kg of body weight is administered per day, and in the case of parenteral administration, 0.01 to 10 mg of active ingredient per kg of body weight per day are administered. The total daily dose may be administered at one time or divided into several times depending on the extent of the disease, the judgment of the doctor.
본 발명에 따른 멜리틴의 N-말단 아미노기에 폴리에틸렌글리콜이 결합된 멜리틴-폴리에틸렌글리콜 배합체는 멜리틴에 비해 세포독성이 낮고 항염 효과가 우수하다. 또한, 본 발명의 제조방법에 따르면, 멜리틴의 N-말단 아미노기에 폴리에틸렌글리콜이 결합된 멜리틴-폴리에틸렌글리콜 배합체를 고수율 및 고선택성으로 제조할 수 있다.The melittin-polyethyleneglycol copolymer in which polyethyleneglycol is bonded to the N-terminal amino group of the melittin according to the present invention is lower in cytotoxicity and excellent in anti-inflammatory effect than melittin. In addition, according to the production method of the present invention, a melittin-polyethylene glycol compound in which polyethylene glycol is bonded to the N-terminal amino group of melittin can be prepared in high yield and high selectivity.
도 1은 실시예 1에서 수득한 멜리틴-폴리에틸렌글리콜 배합체 제조 생성물의 전기영동 후, Iodine 염색과 Coomassie 염색 결과를 나타낸 도면이다. 1 is a diagram showing the results of Iodine staining and Coomassie staining after electrophoresis of the melittin-polyethylene glycol compound preparation product obtained in Example 1.
도 2는 멜리틴(MEL) 및 멜리틴-폴리에틸렌글리콜 배합체(PEG-MEL)를 Lys-C로 처리한 후의 LC 크로마토그래프이다.FIG. 2 is an LC chromatograph after treatment of melittin (MEL) and melittin-polyethylene glycol compound (PEG-MEL) with Lys-C.
도 3은 멜리틴(MEL) 및 멜리틴-폴리에틸렌글리콜 배합체(PEG-MEL)의 적혈구 파괴능을 나타낸 그래프이다.3 is a graph showing the erythrocyte destructive ability of melittin (MEL) and melittin-polyethylene glycol combination (PEG-MEL).
도 4는 멜리틴(MEL) 및 1:1 멜리틴-폴리에틸렌글리콜 배합체(mono-PEG(5k)-MEL, mono-PEG(20k)-MEL)의 세포독성을 나타낸 그래프이다. 4 is a graph showing cytotoxicity of melittin (MEL) and 1: 1 melittin-polyethylene glycol combination (mono-PEG (5k) -MEL, mono-PEG (20k) -MEL).
도 5는 멜리틴(MEL) 및 1:1 멜리틴-폴리에틸렌글리콜 배합체(mono-PEG(5k)-MEL, mono-PEG(20k)-MEL)의 NO 생성 억제 효과를 나타낸 그래프이다.5 is a graph showing the NO production inhibitory effect of melittin (MEL) and 1: 1 melittin-polyethylene glycol combination (mono-PEG (5k) -MEL, mono-PEG (20k) -MEL).
도 6은 멜리틴(MEL) 및 멜리틴-폴리에틸렌글리콜 배합체(mono-PEG(5k)-MEL)의 TNF-α 발현 억제 효과를 나타낸 그래프이다.6 is a graph showing the effect of inhibiting TNF-α expression of melittin (MEL) and melittin-polyethylene glycol combination (mono-PEG (5k) -MEL).
이하, 실시예에 의해 본 발명을 보다 구체적으로 설명하고자 한다. 이들 실시예는 오직 본 발명을 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 국한되지 않는다는 것은 당업자에게 있어서 자명하다. Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it is apparent to those skilled in the art that the scope of the present invention is not limited to these examples.
실시예 1: 멜리틴-폴리에틸렌글리콜 배합체의 제조Example 1 Preparation of Melitin-Polyethylene Glycol Blend
멜리틴(MEL) 2.5 mg을 50 mM 아세트산염 완충액, pH 3.5, 4.0, 5.0, 6.0에 각각 용해하였다. 상기 멜리틴 용액에 직선형의 메톡시 폴리에틸렌글리콜-프로피온알데하이드(PEG, 분자량 5 kDa)를 멜리틴과의 몰 비율(molar ratio)이 1:1 또는 1:3이 되도록 첨가한 후 교반하였다. 메톡시 폴리에틸렌글리콜-프로피온알데하이드가 완전히 녹은 후, 소디움 시아노보로하이드라이드(Sodium cyanoborohydride: NaCNBH3)를 넣어 배합체의 환원반응을 유도하였다. 상기 반응을 실온에서 6 시간 동안 수행하였다. 그런 다음, 상기 반응을 정지시키기 위해 메톡시 폴리에틸렌글리콜-프로피온알데하이드의 몰비율보다 100 배 많은 글리신(Glycine)를 첨가하였다. 2.5 mg of melittin (MEL) was dissolved in 50 mM acetate buffer, pH 3.5, 4.0, 5.0, 6.0, respectively. A linear methoxy polyethylene glycol propionaldehyde (PEG, molecular weight 5 kDa) was added to the melittin solution such that the molar ratio with melittin was 1: 1 or 1: 3 and then stirred. After methoxy polyethylene glycol-propionaldehyde was completely dissolved, sodium cyanoborohydride (NaCNBH 3 ) was added to induce a reduction reaction of the compound. The reaction was carried out at room temperature for 6 hours. Then, 100 times more glycine (Glycine) than the molar ratio of methoxy polyethyleneglycol-propionaldehyde was added to stop the reaction.
멜리틴-폴리에틸렌글리콜 배합체(PEG-MEL)의 형성 유무를 전기영동 후, Iodine 염색과 Coomassie 염색을 통해서 분석하여, 그 결과를 도 1에 나타내었다. The formation of melittin-polyethylene glycol compound (PEG-MEL) was analyzed by Iodine staining and Coomassie staining after electrophoresis, and the results are shown in FIG. 1.
도 1로부터, 첨가된 메톡시 폴리에틸렌글리콜-프로피온알데하이드가 많을수록 배합 반응이 잘 이루어지며, pH 4.0, 5.0, 6.0 조건에서 멜리틴-폴리에틸렌글리콜 배합체가 형성됨을 확인할 수 있었다.From Figure 1, the more methoxy polyethylene glycol-propionaldehyde was added, the better the compounding reaction, it can be seen that the melittin-polyethylene glycol mixture is formed at pH 4.0, 5.0, 6.0 conditions.
실시예 2: 멜리틴-폴리에틸렌글리콜 배합체의 제조Example 2: Preparation of Melitin-Polyethylene Glycol Blend
pH 3.5 조건에서 멜리틴과 폴리에틸렌글리콜-프로피온알데히드의 몰비율이 1:7 또는 1:10이 되도록 하는 것을 제외하고는, 실시예 1과 동일한 방법으로 멜리틴-폴리에틸렌글리콜 배합체를 제조하였다. 생성물을 HPLC를 이용하여 분석하여, 그 결과를 하기 표 1에 나타내었다.A melittin-polyethyleneglycol blend was prepared in the same manner as in Example 1 except that the molar ratio of melittin and polyethylene glycol-propionaldehyde was 1: 7 or 1:10 at pH 3.5. The product was analyzed using HPLC and the results are shown in Table 1 below.
표 1 
Table 1
상기 표 1로부터, 몰 비율이 1:7 또는 1:10일 때 1:1 멜리틴-폴리에틸렌글리콜 배합체(mono-PEG-MEL)가 각각 65% 및 78% 형성됨을 확인할 수 있었다. From Table 1, it can be seen that when the molar ratio is 1: 7 or 1:10, 1: 1 melittin-polyethylene glycol compound (mono-PEG-MEL) is formed 65% and 78%, respectively.
실시예 3: 멜리틴-폴리에틸렌글리콜 배합체의 제조Example 3 Preparation of Melitin-Polyethylene Glycol Blend
pH 4.0 조건에서 멜리틴과 폴리에틸렌글리콜-프로피온알데히드의 몰 비율이 1:1이 되도록 하여 실시예 1과 동일한 방법으로 반응시켰을 때 하기 표 2에서 볼 수 있듯이, 61%의 멜리틴이 반응에 참여하지 않고 남아 있었다.When the molar ratio of melittin and polyethylene glycol propionaldehyde is 1: 1 at pH 4.0, the reaction is carried out in the same manner as in Example 1, and as shown in Table 2 below, 61% of melittin does not participate in the reaction. Remained without.
표 2 
TABLE 2
반응에 참여하지 않은 멜리틴을 반응시켜 1:1 멜리틴-폴리에틸렌글리콜 배합체(mono-PEG-MEL)의 추가적인 수율을 높이기 위해, 멜리틴에 대해 각각 0.5, 1.0, 2.0의 몰비율에 해당하는 메톡시 폴리에틸렌글리콜-프로피온알데하이드를 추가적으로 첨가하여 15시간 동안 반응시켰다. 생성물을 HPLC를 이용하여 분석하여, 그 결과를 하기 표 3에 나타내었다.In order to increase the yield of the 1: 1 melittin-polyethylene glycol compound (mono-PEG-MEL) by reacting the melittin not participating in the reaction, the molar ratios of 0.5, 1.0, and 2.0, respectively, correspond to the melittin. Methoxy polyethyleneglycol-propionaldehyde was further added and reacted for 15 hours. The product was analyzed using HPLC and the results are shown in Table 3 below.
표 3 
TABLE 3
상기 표 3으로부터, 추가된 메톡시 폴리에틸렌글리콜-프로피온알데하이드(PEG)의 양에 의존하여 1:1 멜리틴-폴리에틸렌글리콜 배합체 (mono-PEG-MEL)의 양이 각각 53%, 65% 및 75%임을 확인할 수 있었다. 한편 1:2, 1:3 멜리틴-폴리에틸렌글리콜 배합체(di,tri-PEG-MEL)는 거의 생성되지 않음을 알 수 있었다. From Table 3, the amount of 1: 1 melittin-polyethyleneglycol compound (mono-PEG-MEL) is 53%, 65% and 75, respectively, depending on the amount of added methoxy polyethyleneglycol-propionaldehyde (PEG) It was confirmed that%. On the other hand, it was found that 1: 2, 1: 3 melittin-polyethylene glycol compound (di, tri-PEG-MEL) is hardly produced.
실시예 4: 멜리틴-폴리에틸렌글리콜 배합체의 제조Example 4 Preparation of Melitin-Polyethylene Glycol Blend
멜리틴 2.5 mg을 50 mM 아세트산염 완충액, pH 4.0에 용해하였다. 상기 멜리틴 용액에 직선형의 메톡시 폴리에틸렌글리콜-프로피온알데하이드(PEG, 분자량 20 kDa)를 멜리틴과의 몰비율(molar ratio)이 1:1, 1:3 또는 1:5가 되도록 첨가한 후 교반하였다. 메톡시 폴리에틸렌글리콜-프로피온알데하이드가 완전히 녹은 후, 소디움 시아노보로하이드라이드(Sodium cyanoborohydride: NaCNBH3)를 넣어 배합체의 환원반응을 유도하였다. 상기 반응을 실온에서 6 시간 동안 수행하였다. 그런 다음, 상기 반응을 정지시키기 위해 메톡시 폴리에틸렌글리콜-프로피온알데하이드의 몰비율보다 100 배 많은 글리신(Glycine)를 첨가하였다. 2.5 mg of melittin was dissolved in 50 mM acetate buffer, pH 4.0. To the melittin solution, linear methoxy polyethyleneglycol-propionaldehyde (PEG, molecular weight 20 kDa) was added so that the molar ratio with melittin was 1: 1, 1: 3 or 1: 5, followed by stirring. It was. After methoxy polyethylene glycol-propionaldehyde was completely dissolved, sodium cyanoborohydride (NaCNBH 3 ) was added to induce a reduction reaction of the compound. The reaction was carried out at room temperature for 6 hours. Then, 100 times more glycine (Glycine) than the molar ratio of methoxy polyethyleneglycol-propionaldehyde was added to stop the reaction.
수득한 멜리틴-폴리에틸렌글리콜 배합체 제조 생성물을 HPLC를 이용하여 분석하여, 그 결과를 하기 표 4에 나타내었다.The obtained melittin-polyethylene glycol mixture preparation product was analyzed using HPLC, and the results are shown in Table 4 below.
표 4 
Table 4
상기 표 4로부터, 몰 비율이 1:1, 1:3, 1:5인 모든 조건에서 멜리틴-폴리에틸렌글리콜 배합체가 형성됨을 확인할 수 있었다. From Table 4, it can be seen that the melittin-polyethylene glycol mixture is formed under all conditions of the molar ratio of 1: 1, 1: 3, 1: 5.
실시예 5: 멜리틴-폴리에틸렌글리콜 배합체의 정제Example 5 Purification of Melitin-Polyethylene Glycol Blend
실시예 1에서 얻은 반응 용액을 0.2um 필터하여 준비하였다. 멜리틴-폴리에틸렌글리콜을 정제하기 위하여 양이온 교환 수지 컬럼(SP captoTM Impres, GE)을 20 mM 인산염, pH 7.0의 완충용액으로 평형화시킨 후, 상기 필터한 반응 용액을 흘려 주었다. 멜리틴과 반응하지 않은 메톡시 폴리에틸렌글리콜-프로피온알데하이드를 제거하기 위해 추가로 완충용액을 충분히 흘려 주었다. 양이온 교환 수지에 결합한 멜리틴 및 멜리틴-폴리에틸렌글리콜 배합체 중에서 1:1 멜리틴-폴리에틸렌글리콜 배합체(mono-PEG(5k)-MEL)만을 정제하였다. 1:2, 1:3 멜리틴-폴리에틸렌글리콜 배합체(di,tri-PEG(5k)-MEL)를 제거하기 위하여 1 M NaCl 용액의 9% gradient 완충용액으로 충분히 흘려 주었으며, 1:1 멜리틴-폴리에틸렌글리콜 배합체를 분리하기 위하여 30% gradient 완충용액을 흘려 주었다. 마지막으로 멜리틴은 100% 1 M NaCl로 흘려 주었다.The reaction solution obtained in Example 1 was prepared by 0.2um filter. In order to purify melittin-polyethylene glycol, the cation exchange resin column (SP capto TM Impres, GE) was equilibrated with a buffer of 20 mM phosphate, pH 7.0, and the filtered reaction solution was flowed out. In order to remove methoxy polyethyleneglycol-propionaldehyde which did not react with melittin, an additional buffer solution was sufficiently flowed. Only 1: 1 melittin-polyethyleneglycol blend (mono-PEG (5k) -MEL) was purified from the melittin and melittin-polyethyleneglycol blends bound to the cation exchange resin. In order to remove the 1: 2, 1: 3 melittin-polyethyleneglycol compound (di, tri-PEG (5k) -MEL), a sufficient flow of 1 M NaCl solution in 9% gradient buffer, 1: 1 melittin -30% gradient buffer solution was flowed to separate the polyethylene glycol compound. Finally, melittin was flowed into 100% 1 M NaCl.
용출된 분획물들을 전기영동 후 Coomassie 염색과 Iodine 염색을 통하여 1:1 멜리틴-폴리에틸렌글리콜 배합체(mono-PEG(5k)-MEL)를 순수하게 정제하였다. 1개 분자 멜리틴에 결합된 폴리에틸렌글리콜이 많을수록 이온교환 수지와의 결합력이 약한 것을 확인하였다.The eluted fractions were subjected to electrophoresis and purified purely 1: 1 melittin-polyethylene glycol compound (mono-PEG (5k) -MEL) through Coomassie staining and Iodine staining. The more polyethylene glycol bound to one molecule melittin, the weaker the binding force with the ion exchange resin was confirmed.
실시예 6: 멜리틴-폴리에틸렌글리콜 배합체의 정제Example 6: Purification of Melitin-Polyethylene Glycol Blend
실시예 4에서 얻은 반응 용액을 0.2um 필터하여 준비하였다. 멜리틴-폴리에틸렌글리콜을 정제하기 위하여 양이온 교환 수지 컬럼(SP captoTM Impres, GE)을 20 mM 인산염, pH 7.0의 완충용액으로 평형화시킨 후, 상기 필터한 반응 용액을 흘려 주었다. 멜리틴과 반응하지 않은 메톡시 폴리에틸렌글리콜-프로피온알데하이드를 제거하기 위해 추가로 완충용액을 충분히 흘려 주었다. 양이온 교환 수지에 결합한 멜리틴 및 멜리틴-폴리에틸렌글리콜 배합체 중에서 1:1 멜리틴-폴리에틸렌글리콜 배합체(mono-PEG(20k)-MEL)만을 정제하였다. 1:2, 1:3 멜리틴-폴리에틸렌글리콜 배합체(di,tri-PEG(20k)-MEL)를 제거하기 위하여 1 M NaCl 용액의 5% gradient 완충용액으로 충분히 흘려 주었으며, 1:1 멜리틴-폴리에틸렌글리콜 배합체를 분리하기 위하여 30% gradient 완충용액을 흘려 주었다. 마지막으로 멜리틴은 100% 1 M NaCl로 흘려 주었다.The reaction solution obtained in Example 4 was prepared by 0.2um filter. In order to purify melittin-polyethylene glycol, the cation exchange resin column (SP capto TM Impres, GE) was equilibrated with a buffer of 20 mM phosphate, pH 7.0, and the filtered reaction solution was flowed out. In order to remove methoxy polyethyleneglycol-propionaldehyde which did not react with melittin, an additional buffer solution was sufficiently flowed. Only 1: 1 melittin-polyethyleneglycol combination (mono-PEG (20k) -MEL) was purified in the melittin and melittin-polyethyleneglycol combinations bound to the cation exchange resin. In order to remove the 1: 2, 1: 3 melittin-polyethyleneglycol compound (di, tri-PEG (20k) -MEL), 1 M NaCl solution was sufficiently flowed with 5% gradient buffer, and 1: 1 melittin -30% gradient buffer solution was flowed to separate the polyethylene glycol compound. Finally, melittin was flowed into 100% 1 M NaCl.
용출된 분획물들을 전기영동 후 Coomassie 염색과 Iodine 염색을 통하여 1:1 멜리틴-폴리에틸렌글리콜 배합체(mono-PEG(20k)-MEL)를 정제하였다. 1개 분자 멜리틴에 결합된 폴리에틸렌글리콜이 많을수록 이온교환 수지와의 결합력이 약한 것을 확인하였다.The eluted fractions were electrophoresed to purify 1: 1 melittin-polyethylene glycol compound (mono-PEG (20k) -MEL) through Coomassie staining and Iodine staining. The more polyethylene glycol bound to one molecule melittin, the weaker the binding force with the ion exchange resin was confirmed.
실시예 7: N-말단 특이적 페길레이션 위치 확인Example 7: N-terminal specific pegylation position identification
상기 실시예 5에서 얻은 멜리틴-폴리에틸렌글리콜 단일 배합체(mono-PEG(5k)-MEL)가 N-말단에 특이적으로 접합된 형태인지를 확인하기 위하여 peptide mapping을 수행하였다. 멜리틴-폴리에틸렌글리콜 단일 배합체를 엔도프로테나제(endoproteinase) Lys-C로 처리한 후 LC-MS/MS 분석을 수행하였다.Peptide mapping was performed to determine whether the melittin-polyethylene glycol single compound (mono-PEG (5k) -MEL) obtained in Example 5 was specifically conjugated to the N-terminus. Melitin-polyethyleneglycol single combination was treated with endoproteinase Lys-C followed by LC-MS / MS analysis.
고순도의 멜리틴-폴리에틸렌글리콜을 확보하고자 실시예 5에서 수득한 멜리틴-폴리에틸렌글리콜 배합체를 추가적으로 Superdex 200 겔 필트레이션을 수행하였다. 멜리틴-폴리에틸렌글리콜 피크의 앞부분과 뒷부분의 분획을 제외한 분획만을 모아서 peptide mapping을 수행하였다. 그 결과를 도 2에 나타내었다.To ensure high purity melittin-polyethyleneglycol, the melittin-polyethyleneglycol formulation obtained in Example 5 was further subjected to Superdex 200 gel filtration. Peptide mapping was performed by collecting only the fractions except the front and back fractions of the melittin-polyethylene glycol peak. The results are shown in FIG.
엔도프로테나제 Lys-C 효소를 멜리틴에 처리할 경우, 이론적으로 4개의 펩타이드가 생성되고, 각 펩타이드의 분자량은 하기 표 5에 정리하였다. When endoprotein Lys-C enzyme is treated with melittin, theoretically four peptides are produced, and the molecular weight of each peptide is summarized in Table 5 below.
표 5
Table 5
| 펩타이드 단편 | 잔기 번호 | 서열 | 이론적 질량 |
| L1 | 1-7 | GIGAVLK | 656.42 |
| L2 | 8-21 | VLTTGLPALISWIK | 1510.91 |
| L3 | 22-23 | RK | 302.21 |
| L4 | 24-26 | RQQ | 430.23 |
| Peptide fragment | Residue number | order | Theoretical mass |
| L1 | 1-7 | GIGAVLK | 656.42 |
| L2 | 8-21 | VLTTGLPALISWIK | 1510.91 |
| L3 | 22-23 | RK | 302.21 |
| L4 | 24-26 | RQQ | 430.23 |
멜리틴 및 멜리틴-폴리에틸렌클리콜 배합체 각각 25ug에 0.5ug의 엔도프로테나제 Lys-C를 처리한 후 37℃에서 16시간 반응시켰다. 그런 다음, DTT(Dithiothreitol)를 상온에서 1시간 동안 처리한 후, LC-MS 크로마토그래피(UliMate 300 system, Dionex, US, Column: Acclaim RSLC 120 C18, Dionex)를 수행하였으며, 각 펩타이드 피크의 분자량(MicroQ-TOF III mass spectrometer, Bruker Daltonics, 255748 Germany, Mode: ESI+)을 확인하였다.25 ug of melittin and melittin-polyethylene glycol formulations were treated with 0.5 ug of endoproteinase Lys-C and reacted at 37 ° C. for 16 hours. Then, after treatment with DTT (Dithiothreitol) for 1 hour at room temperature, LC-MS chromatography (UliMate 300 system, Dionex, US, Column: Acclaim RSLC 120 C18, Dionex) was performed, the molecular weight of each peptide peak ( MicroQ-TOF III mass spectrometer, Bruker Daltonics, 255748 Germany, Mode: ESI +).
도 2는 멜리틴(MEL) 및 멜리틴-폴리에틸렌글리콜 배합체(mono-PEG(5k)-MEL)를 Lys-C로 처리한 후의 LC 크로마토그래프를 나타낸다. 각각의 크로마토그래프에서 3개(L1, L2, L3)의 펩타이드가 확인되었으며, L4의 펩타이드는 검출되지 않았다. L4의 아미노산 서열의 에너지 강도(intensity)가 낮기 때문에 확인되지 않은 것으로 예상된다. 나머지 3개의 펩타이드는 MicroQ-TOF 장비를 이용하여 분자량을 측정하여 각 펩타이드를 확인하였다. 2 shows LC chromatographs after treatment of melittin (MEL) and melittin-polyethyleneglycol combinations (mono-PEG (5k) -MEL) with Lys-C. Three peptides (L1, L2, L3) were identified in each chromatograph, and no peptide of L4 was detected. It is expected that this has not been confirmed because of the low energy intensity of the amino acid sequence of L4. The remaining three peptides were identified by measuring the molecular weight using a MicroQ-TOF instrument.
표 6 
Table 6
도 2 및 표 6에서 보듯이, 멜리틴과 mono-PEG(5k)-MEL의 L1 peptide peak 크기를 비교해 보면, mono-PEG(5k)-MEL의 L1이 멜리틴의 L1보다 상당히 감소되었으며, L1+PEG peak(분자량 5656Da)는 52.7min에 검출되었다. 이는 PEG가 멜리틴의 L1 peptide의 N-말단의 아미노기에 결합(conjugation)되었음을 말해준다.As shown in Figure 2 and Table 6, when comparing the L1 peptide peak size of the melittin and mono-PEG (5k) -MEL, L1 of mono-PEG (5k) -MEL was significantly reduced than L1 of the melittin, L1 + PEG peak (molecular weight 5656 Da) was detected at 52.7 min. This suggests that PEG is conjugated to the N-terminal amino group of L1 peptide of melittin.
L1-PEG(5793 Da)가 L2와 비슷한 머무름 시간(retention time)에서 검출되었다. 이는 이론상 분자량 5656Da보다 137Da이 큰 분자량이다. 이는 폴리에틸렌글리콜이 다분산성(poly-dispersity) (평균 분자량 4985Da, 분자량 분산성: 4029 ~ 6041)를 가지며, Superdex 200 겔 필트레이션을 이용한 정제에서 멜리틴-폴리에틸렌글리콜 피크의 길게 tailing되는 부분을 제외한 분획을 시료로 확보했기 때문에 평균 분자량이 증가한 것으로 예상된다.L1-PEG (5793 Da) was detected at retention time similar to L2. This is theoretically 137 Da that is greater than 5656 Da. This is the fraction of polyethylene glycol poly-dispersity (average molecular weight 4985 Da, molecular weight dispersibility: 4029 ~ 6041), except the long tailing portion of the melittin-polyethylene glycol peak in the tablet using Superdex 200 gel filtration The average molecular weight is expected to increase because it was obtained as a sample.
이러한 결과는 정제시, 멜리틴-폴리에틸렌글리콜 피크의 분획마다 다를 수 있음을 확인하였다. 즉, 멜리틴-폴리에틸렌글리콜 배합체의 이론적 분자량은 7846Da이지만, 양이온 교환수지를 이용한 정제에서 평균 분자량은 7892.38Da이었으며, 피크의 분자량 분산성은 6795부터 9070Da까지 퍼져 있었다. 대신에 양이온 교환수지 이후 Superdex 200 겔 필트레이션을 추가 실시한 경우, 멜리틴-폴리에틸렌글리콜 배합체의 평균 분자량은 7940Da이었으며, 분자량 분산성은 7323 ~ 8555Da로 퍼져 있는 것을 확인하였다. It was confirmed that these results may vary depending on the fraction of the melittin-polyethylene glycol peak at the time of purification. That is, the theoretical molecular weight of the melittin-polyethylene glycol mixture was 7846 Da, but the average molecular weight was 7892.38 Da in the purification using a cation exchange resin, the molecular weight dispersibility of the peak spread from 6795 to 9070 Da. Instead, when the Superdex 200 gel filtration was further performed after the cation exchange resin, the average molecular weight of the melittin-polyethylene glycol compound was 7940 Da and the molecular weight dispersibility was confirmed to be spread to 7323 ~ 8555 Da.
실험예 1: 멜리틴-폴리에틸렌글리콜 배합체의 적혈구 세포독성Experimental Example 1: Erythrocyte cytotoxicity of melittin-polyethylene glycol formulation
상기 실시예 5에서 얻은 1:1 멜리틴-폴리에틸렌글리콜 배합체(mono-PEG(5k)-MEL)의 세포독성을 측정하였다.Cytotoxicity of the 1: 1 melittin-polyethylene glycol combination (mono-PEG (5k) -MEL) obtained in Example 5 was measured.
멜리틴 및 1:1 멜리틴-폴리에틸렌글리콜 배합체를 웰(well) 당 100 uM부터 순차적으로 1/2씩 희석하였으며, 각 웰에 8% 양의 적혈구(Sheep’s Blood cell)를 첨가한 후 37 oC에서 1시간 동안 배양하였다. 1 시간 후, 배양액을 1000 g로 원심분리하였으며, 상등액을 취하여 415 nm 파장에서 흡광도를 측정함으로써, 헤모글로빈의 양을 조사하였다. 100% 세포 파괴능은 1% 트리톤 X-100을 처리한 후 측정된 흡광도로 하였다. 멜리틴 및 1:1 멜리틴-폴리에틸렌글리콜 배합체의 적혈구 파괴능은 하기 수학식 1에 의해 계산하였다.Melittin and 1: 1 melittin-polyethylene glycol-fold after the polymer was diluted to 100 uM per well from the (well) by sequentially 1/2, was added to red blood cells (Sheep's Blood cell) of 8% the amount in each well 37 o Incubated at C for 1 hour. After 1 hour, the culture was centrifuged at 1000 g, and the amount of hemoglobin was examined by taking the supernatant and measuring the absorbance at 415 nm. 100% cell disruption was determined by absorbance measured after treatment with 1% Triton X-100. Erythrocyte destructive capacity of melittin and 1: 1 melittin-polyethylene glycol combination was calculated by the following equation.
[수학식 1][Equation 1]
흡광도 A : 415 nm 파장에서 시료의 흡광도Absorbance A: Absorbance of the sample at 415 nm wavelength
흡광도 B : 415 nm 파장에서 인산염 완충액의 흡광도Absorbance B: Absorbance of phosphate buffer at 415 nm
흡광도 C : 415 nm 파장에서 1% 트리톤 X-100의 흡광도Absorbance C: Absorbance of 1% Triton X-100 at 415 nm
도 3에서 보듯이, 1:1 멜리틴-폴리에틸렌글리콜배합체(mono-PEG(5k)-MEL)의 적혈구 파괴능은 멜리틴(MEL)의 적혈구 파괴능보다 감소하였다.As shown in FIG. 3, the erythrocyte destructive capacity of 1: 1 melittin-polyethylene glycol copolymer (mono-PEG (5k) -MEL) was lower than that of melittin (MEL).
실험예 2: 세포독성 Experimental Example 2: Cytotoxicity
세포성장 저해활성 측정은 XTT assay(Cell Proliferation Kit II(XTT) assay (Roche)) 방법에 따라서 시행하였다. RAW 264.7 세포를 96-웰 플레이트에 5x105 세포/ml의 농도로 각 웰 당 200 ul 분주(seeding)하고, 멜리틴 및 1:1 멜리틴-폴리에틸렌글리콜 배합체를 각 농도별로 처리한 후, 37℃, 5% CO2 배양기에서 24 시간 동안 배양하였다. 배양 후 각 웰에 XTT solution(Cell Proliferation Kit II(XTT) assay (Roche))를 첨가하여 37℃, 5% CO2 배양기에서 2시간 반응 후 490 nm에서 흡광도를 측정하였다. 그 결과를 도 4에 나타내었다.Cell growth inhibition activity was measured according to the XTT assay (Cell Proliferation Kit II (XTT) assay (Roche)) method. At a concentration of 5x10 5 cells / ml for RAW 264.7 cells in 96-well plate 200 ul dispensed (seeding) per each well, melittin and 1: 1 melittin-after processing the copolymer of polyethylene glycol times for each concentration, 37 Incubated for 24 hours in a 5% CO 2 incubator. After incubation, XTT solution (Cell Proliferation Kit II (XTT) assay (Roche)) was added to each well, and the absorbance was measured at 490 nm after 2 hours in 37 ° C. and 5% CO 2 incubator. The results are shown in FIG.
도 4에서 보듯이, LPS 200 ng/ml만을 첨가하였을 때 세포독성에 거의 영향이 없었다(UN vs VH). 한편, 멜리틴(MEL)은 2.5 ug/ml 농도 이하에서 RAW 264.7 세포에 세포독성(cytotoxicity)이 없으며, 그 이상의 농도에서는 세포독성이 증가하는 것으로 나타났다. 반면, 1:1 멜리틴-폴리에틸렌글리콜 배합체(mono-PEG(5k)-MEL, mono-PEG(20k)-MEL)를 처리하였을 경우에는, 20 ug/ml 이상 농도까지 세포독성을 나타내지 않았다. 즉, 폴리에틸렌글리콜이 결합됨으로써 멜리틴의 세포독성이 감소됨을 확인할 수 있었다.As shown in Figure 4, the addition of only 200 ng / ml LPS had little effect on cytotoxicity (UN vs VH). On the other hand, melittin (MEL) has no cytotoxicity to RAW 264.7 cells below the concentration of 2.5 ug / ml, the cytotoxicity was increased at higher concentrations. On the other hand, when treated with 1: 1 melittin-polyethylene glycol combination (mono-PEG (5k) -MEL, mono-PEG (20k) -MEL), it did not show cytotoxicity to a concentration of 20 ug / ml or more. That is, it was confirmed that the cytotoxicity of the melittin is reduced by the combination of polyethylene glycol.
실험예 3: 항염 효과 Experimental Example 3: Anti-inflammatory Effect
멜리틴과 1:1 멜리틴-폴리에틸렌글리콜 배합체의 항염 효과를 확인하였다. RAW 264.7 세포를 96-웰 플레이트에 5x105 세포/ml의 농도로 각 웰 당 200 ul 분주(seeding)한 후, 멜리틴과 1:1 멜리틴-폴리에틸렌글리콜 배합체를 각 농도별로 LPS(Lipopolysaccaride)와 함께 처리한 후, 37℃, 5% CO2 배양기에서 배양하였다. 염증 시 염증지표로서 LPS에 의해 생성되는 NO(nitrite oxide)와 TNF-α를 측정하였다. TNF-α 억제물질 양성 대조군으로서 JSH, Bay11-1272 항염 시약을 사용하였다.The anti-inflammatory effect of melittin and 1: 1 melittin-polyethylene glycol combination was confirmed. RAW 264.7 cells were seeded in a 96-well plate at a concentration of 5 × 10 5 cells / ml at 200 ul per well, and then the melittin and 1: 1 melittin-polyethylene glycol blends were added at each concentration of lipopolysaccaride (LPS). After treatment with, incubated in 37 ℃, 5% CO 2 incubator. As inflammation markers during inflammation, NO (nitrite oxide) and TNF-α produced by LPS were measured. JSH, Bay11-1272 anti-inflammatory reagent was used as a TNF-α inhibitor positive control.
실험예 3-1: NO 생성 억제 효과 Experimental Example 3-1: NO production inhibitory effect
NO의 측정은 멜리틴(MEL) 또는 1:1 멜리틴-폴리에틸렌글리콜 배합체 (mono-PEG(5k)-MEL, mono-PEG(20k)-MEL)와 LPS를 동시 처리한 후, 37℃, 5% CO2 배양기에서 24 시간 배양하였다. 상등액 50 ul를 취하여 동량의 Griess reagent(1% Sulfarunilamide, 0.1% naphthyethylenediamine dihydrochloride, 및 2% phosphoric acid)와 혼합하여 상온에서 10분간 방치한 후 540nm에서 흡광도를 측정하였다. 그 결과를 도 5에 나타내었다. The measurement of NO was performed at 37 ° C. after simultaneous treatment with melittin (MEL) or 1: 1 melittin-polyethylene glycol compound (mono-PEG (5k) -MEL, mono-PEG (20k) -MEL) and LPS. in 5% CO 2 incubator and cultured for 24 hours. 50 ul of the supernatant was mixed with the same amount of Griess reagent (1% Sulfarunilamide, 0.1% naphthyethylenediamine dihydrochloride, and 2% phosphoric acid), and the absorbance was measured at 540 nm after standing at room temperature for 10 minutes. The results are shown in FIG.
도 5에서 보듯이, 멜리틴(MEL) 및 1:1 멜리틴-폴리에틸렌글리콜 배합체(mono-PEG(5k)-MEL, mono-PEG(20k)-MEL)는 농도 의존적으로 NO의 생성을 억제하였다. 다만, 멜리틴의 경우 5 ug/ml 농도 이상에서 NO가 급격하게 줄어든 것은 세포독성 효과로 예상된다. 1:1 멜리틴-폴리에틸렌글리콜 배합체의 경우 세포독성을 나타내지 않는 농도에서도 농도 의존적으로 NO 생성을 억제하는 것으로 나타났다. As shown in FIG. 5, melittin (MEL) and 1: 1 melittin-polyethylene glycol combination (mono-PEG (5k) -MEL, mono-PEG (20k) -MEL) inhibit NO production in a concentration-dependent manner. It was. However, in the case of melittin, a sharp decrease in NO at a concentration of 5 ug / ml or more is expected to be a cytotoxic effect. The 1: 1 melittin-polyethylene glycol combination was shown to inhibit NO production in a concentration dependent manner even at concentrations that do not exhibit cytotoxicity.
실험예 3-2: TNF-α 발현 억제 효과Experimental Example 3-2: TNF-α Expression Inhibitory Effect
TNF-α의 측정은 멜리틴(MEL) 또는 1:1 멜리틴-폴리에틸렌글리콜 배합체(mono-PEG(5k)-MEL, mono-PEG(20k)-MEL)와 LPS를 동시 처리한 후, 37℃, 5% CO2 배양기에서 6 시간 배양하였다. 상등액을 취하여 TNF-α ELISA를 수행하였다. 그 결과를 도 6 및 표 7에 나타내었다. The measurement of TNF-α was performed after simultaneous treatment with melittin (MEL) or 1: 1 melittin-polyethylene glycol compound (mono-PEG (5k) -MEL, mono-PEG (20k) -MEL) and LPS, followed by 37 C was incubated for 6 hours in a 5% CO 2 incubator. Supernatant was taken and TNF-α ELISA was performed. The results are shown in FIG. 6 and Table 7.
도 6에서 보듯이, 멜리틴(MEL) 및 1:1 멜리틴-폴리에틸렌글리콜 배합체(mono-PEG(5k)-MEL)는 농도 의존적으로 TNF-α의 생성을 억제하는 것으로 나타났다. 다만, 멜리틴의 억제 효과는 세포독성을 보이는 농도에서 더욱 급하게 나타났다. As shown in FIG. 6, melittin (MEL) and 1: 1 melittin-polyethylene glycol combination (mono-PEG (5k) -MEL) were shown to inhibit the production of TNF-α in a concentration dependent manner. However, the inhibitory effect of melittin appeared more urgent at the concentration showing cytotoxicity.
멜리틴 및 1:1 멜리틴-폴리에틸렌글리콜 배합체의 항염 효과를 정리하여 하기 표 7에 나타내었다. 이때 항염 효과는 세포독성을 나타내지 않는 농도에서의 항염 효과를 나타내었다. The anti-inflammatory effects of melittin and 1: 1 melittin-polyethylene glycol blends are summarized in Table 7 below. At this time, the anti-inflammatory effect showed an anti-inflammatory effect at a concentration that does not exhibit cytotoxicity.
표 7
TABLE 7
| MIC (세포성장의 최소 저해농도) | 항염 효과 | ||
| NO 억제 | TNF-α 억제 | ||
| MEL | 2.5 ug/ml | 34% | 31% |
| mono-PEG(5k)-MEL | 20 ug/ml 이상 | 72% | 57% |
| mono-PEG(20k)-MEL | 20 ug/ml 이상 | 45% | 42% |
| JSH | 30 uM | 100% | 48% |
| Bay11-1272 | 3 uM | 67% | 45% |
| MIC (minimum inhibitory concentration of cell growth) | Anti-inflammatory effect | ||
| NO suppression | TNF-α inhibition | ||
| MEL | 2.5 ug / ml | 34% | 31% |
| mono-PEG (5k) - | 20 ug / ml or more | 72% | 57% |
| mono-PEG (20k) - | 20 ug / ml or more | 45% | 42 |
| JSH | |||
| 30 | 100% | 48% | |
| Bay11-1272 | 3 uM | 67% | 45% |
상기 표 7에서 보듯이, 멜리틴은 세포독성을 나타내지 않는 농도 2.5 ug/ml에서 NO 및 TNF-α의 생성을 각각 34% 및 31% 억제하였으며, 1:1 멜리틴-폴리에틸렌글리콜 배합체(mono-PEG(5k)-MEL)는 세포독성을 나타내지 않는 20 ug/ml 농도에서 각각 72% 및 57% 억제하는 효과를 나타내었다. 1:1 멜리틴-폴리에틸렌글리콜 배합체(mono-PEG(20k)-MEL)는 세포독성을 나타내지 않는 20 ug/ml 농도에서 각각 45% 및 42% 억제하는 효과를 나타내었다.As shown in Table 7, melittin inhibited the production of NO and TNF-α by 34% and 31%, respectively, at a concentration of 2.5 ug / ml, which showed no cytotoxicity, and a 1: 1 melittin-polyethylene glycol compound (mono -PEG (5k) -MEL) showed inhibitory effects of 72% and 57% at 20 ug / ml concentrations without cytotoxicity, respectively. The 1: 1 melittin-polyethylene glycol combination (mono-PEG (20k) -MEL) showed 45% and 42% inhibition at 20 ug / ml concentrations without cytotoxicity, respectively.
Claims (15)
- 멜리틴의 N-말단 아미노기에 폴리에틸렌글리콜이 결합된 멜리틴-폴리에틸렌글리콜 배합체.A melittin-polyethylene glycol compound in which polyethylene glycol is bonded to the N-terminal amino group of melittin.
- 제1항에 있어서, 하기 화학식 1의 화합물인 멜리틴-폴리에틸렌글리콜 배합체:The melittin-polyethylene glycol formulation of claim 1, which is a compound of Formula 1:[화학식 1][Formula 1]상기 식에서,WhereR은 수소 또는 C1-C6의 알킬기이고,R is hydrogen or an alkyl group of C 1 -C 6 ,n은 10 내지 1000의 정수이며,n is an integer from 10 to 1000,m은 0 내지 10의 정수이고,m is an integer from 0 to 10,Mel은 멜리틴이다. Mel is a melittin.
- 제2항에 있어서, The method of claim 2,R은 메틸이고,R is methyl,n은 100 내지 500의 정수이며,n is an integer from 100 to 500,m은 0 내지 3의 정수이고,m is an integer from 0 to 3,Mel은 멜리틴인 화학식 1의 멜리틴-폴리에틸렌글리콜 배합체.Mel is a melittin melittin-polyethylene glycol compound of formula (1).
- (i) 하기 화학식 2의 멜리틴과 하기 화학식 3의 폴리에틸렌글리콜-알킬알데하이드를 결합반응시켜 하기 화학식 4의 화합물을 수득하는 단계; 및(i) combining the melittin of Formula 2 with polyethylene glycol-alkylaldehyde of Formula 3 to obtain a compound of Formula 4; And(ii) 하기 화학식 4의 화합물을 환원반응시키는 단계를 포함하는 하기 화학식 1의 멜리틴-폴리에틸렌글리콜 배합체의 제조방법:(ii) a method of preparing a melittin-polyethylene glycol compound of formula (1) comprising the step of reducing a compound of formula (4):상기 식에서, WhereR은 수소 또는 C1-C6의 알킬기이고,R is hydrogen or an alkyl group of C 1 -C 6 ,n은 10 내지 1000의 정수이며,n is an integer from 10 to 1000,m은 0 내지 10의 정수이고,m is an integer from 0 to 10,Mel은 멜리틴이다. Mel is a melittin.
- 제4항에 있어서, 단계 (i)에서 결합반응은 pH 3.5 내지 6의 산성 완충용액에서 수행되는 제조방법. The method of claim 4, wherein the binding reaction in step (i) is performed in an acidic buffer solution having a pH of 3.5 to 6. 6.
- 제5항에 있어서, 완충용액은 아세트산염 완충액 또는 인산염 완충액인 제조방법.The method of claim 5, wherein the buffer is acetate buffer or phosphate buffer.
- 제4항에 있어서, 단계 (i)에서 멜리틴에 대한 폴리에틸렌글리콜-알킬알데하이드의 몰비율은 1 내지 10인 제조방법. The process according to claim 4, wherein the molar ratio of polyethyleneglycol-alkylaldehyde to melittin in step (i) is 1 to 10.
- 제7항에 있어서, 단계 (i)에서 0.5 내지 2 몰비율의 폴리에틸렌글리콜-알킬알데하이드를 추가적으로 첨가하여 반응시키는 제조방법.The process according to claim 7, wherein in step (i), 0.5 to 2 molar ratio of polyethylene glycol-alkylaldehyde is additionally added to react.
- 제4항에 있어서, 단계 (ii)에서 환원반응은 소디움 시아노보로하이드라이드(Sodium cyanoborohydride: NaCNBH3)를 사용하여 수행되는 제조방법.The process according to claim 4, wherein the reduction in step (ii) is carried out using sodium cyanoborohydride (NaCNBH 3 ).
- 제4항에 있어서, 제조된 멜리틴-폴리에틸렌글리콜 배합체를 정제하는 단계를 추가로 포함하는 제조방법.The method of claim 4, further comprising purifying the prepared melittin-polyethylene glycol combination.
- 제10항에 있어서, 정제는 양이온 교환 수지 상에서 염을 포함하는 pH 7 내지 8의 완충용액을 전개액으로 사용하여 수행되는 제조방법.The method of claim 10, wherein the purification is performed using a buffer solution having a pH of 7 to 8 containing a salt on the cation exchange resin as a developing solution.
- 제11항에 있어서, 염의 농도를 조절하면서 기울기 용리하여 1:1 멜리틴-폴리에틸렌글리콜 배합체를 순수하게 분리하는 제조방법.12. The process according to claim 11, wherein the 1: 1 melittin-polyethylene glycol blend is purely separated by gradient elution while adjusting the salt concentration.
- 제11항에 있어서, 양이온 교환 수지가 세파로스 포스페이트(sepharose phosphate) 또는 세파로스 카복시메틸(sepharose carboxymethyl)인 제조방법.The method of claim 11, wherein the cation exchange resin is sepharose phosphate or sepharose carboxymethyl.
- 제1항 내지 제3항 중 어느 한 항에 따른 멜리틴-폴리에틸렌글리콜 배합체 및 약제학적으로 허용되는 담체를 포함하는 염증의 치료 또는 예방용 약제학적 조성물.A pharmaceutical composition for the treatment or prophylaxis of inflammation comprising the melittin-polyethylene glycol combination according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier.
- 제1항 내지 제3항 중 어느 한 항에 따른 멜리틴-폴리에틸렌글리콜 배합체 및 약제학적으로 허용되는 담체를 포함하는 류마티스 관절염, 루푸스, 건선, 장염증 또는 골관절염의 치료 또는 예방용 약제학적 조성물.A pharmaceutical composition for the treatment or prophylaxis of rheumatoid arthritis, lupus, psoriasis, enteritis or osteoarthritis, comprising the melittin-polyethylene glycol combination according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier.
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| CN113827703A (en) * | 2020-06-08 | 2021-12-24 | 沈阳药科大学 | Preparation and application of a kind of melittin-polysialic acid electrostatic complex |
| CN114437193A (en) * | 2022-01-29 | 2022-05-06 | 陕西未来多肽生物科技有限公司 | Gold-melittin nano hybrid and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111110856A (en) * | 2020-02-28 | 2020-05-08 | 苏州大学 | Antibacterial drug based on antibacterial peptide and hydrophilic polymer and preparation method thereof |
| CN111821466A (en) * | 2020-02-28 | 2020-10-27 | 苏州大学 | A kind of preparation method of antibacterial drug |
| CN111888483A (en) * | 2020-02-28 | 2020-11-06 | 苏州大学 | An antibacterial drug based on melittin and flexible polyethylene glycol |
| CN111888483B (en) * | 2020-02-28 | 2022-10-18 | 苏州大学 | Antibacterial drug based on melittin and flexible polyethylene glycol |
| CN111821466B (en) * | 2020-02-28 | 2022-10-18 | 苏州大学 | Preparation method of antibacterial drug |
| CN111110856B (en) * | 2020-02-28 | 2022-12-16 | 苏州大学 | Antibacterial drug based on antibacterial peptide and hydrophilic polymer and preparation method thereof |
| CN113827703A (en) * | 2020-06-08 | 2021-12-24 | 沈阳药科大学 | Preparation and application of a kind of melittin-polysialic acid electrostatic complex |
| CN114437193A (en) * | 2022-01-29 | 2022-05-06 | 陕西未来多肽生物科技有限公司 | Gold-melittin nano hybrid and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| KR101770664B1 (en) | 2017-08-23 |
| KR20150143346A (en) | 2015-12-23 |
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