CN105985441A - Deuterohaemin hexapeptide derivative, preparation method and application thereof - Google Patents
Deuterohaemin hexapeptide derivative, preparation method and application thereof Download PDFInfo
- Publication number
- CN105985441A CN105985441A CN201510041700.XA CN201510041700A CN105985441A CN 105985441 A CN105985441 A CN 105985441A CN 201510041700 A CN201510041700 A CN 201510041700A CN 105985441 A CN105985441 A CN 105985441A
- Authority
- CN
- China
- Prior art keywords
- fmoc
- peptide
- cpp
- disease
- secondary heme
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a deuterohaemin hexapeptide derivative which has a Dh-[beta]-AH-CPP structure, wherein the CPP refers to cytomembrane penetrating peptide, and the Dh-[beta]-AH is coupled to the CPP through a covalent bond. The invention relates to a preparation method of the deuterohaemin hexapeptide derivative, relates to a composition including the deuterohaemin hexapeptide derivative, and relates to an application of the deuterohaemin hexapeptide derivative in preparation of a medicine for preventing and/or treating diseases or preventing senility. The deuterohaemin hexapeptide derivative is especially suitable for oral delivery.
Description
Technical field
The present invention relates to secondary heme 6 peptide derivant and its production and use.Specifically, originally
Invention relates to secondary heme 6 peptide derivant Dh-β-AH-CPP, the compositions that comprises it, it prepares
Method and the purposes in preparation has the medicament of ascorbate peroxidase enzymatic activity thereof.
Background technology
Secondary heme 6 peptide (Deuterohaemin-β-Ala-His-Thr-Val-Glu-Lys,
Dh-β-AHTVEK, DhHP-6) as the peptide derivative of a kind of iron porphyrin, it is ascorbic acid mistake
The analogue enztme of oxide enzyme, internal/in vitro tests result shows that it has good antioxidant activity,
The most aobvious at prevention and/or treatment diabetes, cataract, cardiovascular and cerebrovascular disease, aging, aspect of inflammation
Show preferable effect.
But, secondary heme 6 peptide as a peptide species, owing to molecular weight is big, is difficult to through cell
Film, therefore conventional route of administration is injection.But, the convenience, safe and suitable used from patient
Answering property angle considers, the in particular, for example this kind of situation needing long-term prescription of diabetes such is given
Prescription formula is unsatisfactory.Compared to drug administration by injection, oral administration is more to manage in various administering mode
One of mode thought.Although at present by polypeptide chain modification technique (seeing CN 103450341 A)
Significantly improve the stability of polypeptide, thus the bioavailability of polypeptide can be improved, for oral administration
Lay a good foundation, but the intestinal cell that these modifications but can not significantly improve secondary heme 6 peptide is saturating
The property crossed, therefore, is still not suitable for being administered orally.
In order to solve the problems referred to above, need to transform, secondary heme 6 peptide to be adapted to further
Oral.
Summary of the invention
It is an object of the present invention to provide secondary heme 6 peptide derivant being suitable to be administered orally.
The inventors discovered that, by the sequence truncation of secondary heme 6 peptide, obtain that there is ascorbic acid mistake
The minimum unit Dh-β-AH of peroxidase activity, then this activity unit is penetrated with cell membrane
Peptide (cell penetrating peptide, CPP) chemical coupling, can obtain active being administered orally
Secondary heme 6 peptide derivant Dh-β-AH-CPP.Described secondary heme 6 peptide derivant
Dh-β-AH-CPP has and well wears film properties, increases across intestinal cell ability.
Therefore, in the first aspect of the invention, it is provided that have the secondary heme 6 of formula
Peptide derivant:
Dh-β-AH-CPP,
Wherein CPP represents penetratin, and described CPP and described Dh-β-AH is chemistry
Coupling.
Described chemical coupling refers to that described CPP and described Dh-β-AH is to be connected to one by covalent bond
Rise.
Preferably, described CPP be GRKKRRQRRRPPQ, RRRRRRRR,
KLALKLALKALKAALKLA、MVTVLFRRLRIRRACGPPRVRV、
RQIKIWFQNRRMKWKK, KKTWWKTWWTKWSQPKKKRKV or
AGYLLGKINLKALAALAKKIL。
In a second aspect of the present invention, it is provided that a kind of pharmaceutical composition, described pharmaceutical composition bag
Secondary heme 6 peptide derivant containing the present invention and pharmaceutically useful carrier.
In a third aspect of the present invention, it is provided that the preparation of secondary heme 6 peptide derivant of the present invention
Method, said method comprising the steps of:
1) on solid phase carrier, according to Fmoc solid-phase synthesis from C end (c-terminus) to N
Each aminoacid of CPP sequence that end (aminoterminal) sequentially coupling is protected through Fmoc and
Fmoc-His-OH, Fmoc-β-Ala-OH thus obtain through protection NH2-β-Ala-His-CPP-
Solid phase carrier;
2) coupling secondary heme;
3) deprotection group, cuts the Dh-β-AH-CPP of gained from solid phase carrier simultaneously
Get off, obtain the solution containing Dh-β-AH-CPP.
Preferably, wherein said CPP be GRKKRRQRRRPPQ, RRRRRRRR,
KLALKLALKALKAALKLA、MVTVLFRRLRIRRACGPPRVRV、
RQIKIWFQNRRMKWKK, KKTWWKTWWTKWSQPKKKRKV or
AGYLLGKINLKALAALAKKIL。
Preferably, carrier conventional during described solid phase carrier is Fmoc solid-phase synthesis.Preferably,
Described solid phase carrier is Rink amide mbha resin.
Preferably, to 3) in the solution containing Dh-β-AH-CPP that obtains be purified, purification
Method is such as washed, recrystallization and chromatography (such as HPLC) etc..Preferred color of choice spectrometry, more excellent
Select HPLC.
Owing to secondary heme 6 peptide derivant (Dh-β-AH-CPP) of the present invention has ascorbic acid mistake
Peroxidase activity, and permeability cell is good, therefore can using in the way of oral as ascorbic acid
Peroxidase is used for treatment and/or the prevention of disease.
Therefore, a fourth aspect of the present invention provides secondary heme 6 peptide derivant of the present invention in preparation
For preventing and/or treat disease or the purposes in pre-anti-aging medicament, described disease is permissible
Use ascorbate peroxidase enzyme or there is the compound of ascorbate peroxidase enzymatic activity control
Treating, specifically, described disease can be selected from diabetes, cataract, cardiovascular and cerebrovascular disease, inflammation
Disease, described medicament is particularly suitable for oral administration.
Detailed description of the invention
Disclosed herein the aminoacid sequence of a series of polypeptide, those skilled in the art can manage
Solve, when representing a certain sequence with triliteral amino acid residue or single letter, this sequence
Represent from left to right is this polypeptide sequence from N end (aminoterminal) to C end (c-terminus).
Such as when the sequence using " Ala-His-Thr-Val-Glu-Lys " to represent a certain polypeptide, mean
The sequence of this polypeptide is " N end-Ala-His-Thr-Val-Glu-Lys-C end ", and when using
When " AHTVEK " represents the sequence of a certain polypeptide, mean the sequence of this polypeptide for " N end-
AHTVEK-C end ".
When the most specifically indicating amino acid whose configuration, it refers to a-amino acid.
Used English in the present invention and abbreviation and implication thereof are described as follows:
In one embodiment of the invention, it is provided that there is secondary heme 6 peptide of formula
Derivant:
Dh-β-AH-CPP,
Wherein CPP represents penetratin, its be GRKKRRQRRRPPQ,
RRRRRRRR、KLALKLALKALKAALKLA、
MVTVLFRRLRIRRACGPPRVRV、RQIKIWFQNRRMKWKK、
KKTWWKTWWTKWSQPKKKRKV or
AGYLLGKINLKALAALAKKIL, described CPP with Dh-β-AH are connected by covalent bond.
Above-mentioned secondary heme 6 peptide derivant can as ascorbate peroxidase enzyme for prevention and/
Or treatment disease or pre-anti-aging, described disease be can use ascorbate peroxidase enzyme or
There is the disease of the compounds for treating of ascorbate peroxidase enzymatic activity, specifically, described disease
Sick selected from diabetes, cataract, cardiovascular and cerebrovascular disease, inflammation.
Present invention also offers a kind of pharmaceutical composition, described pharmaceutical composition includes one or many
Plant secondary heme 6 peptide derivant and one or more pharmaceutically useful carriers of the present invention.
" pharmaceutically useful carrier " refers to biologically or other aspects will not have ill effect
Material, i.e. together can give tested by secondary heme 6 peptide derivant of described material with the present invention
Person, will not cause simultaneously any bad biological impact or in harmful manner with containing its medicine
Any other component of compositions interacts.Obviously should select to make active component tested
Any degraded in person's body is preferably minimized so that the carrier that is minimized of any adverse side effect, and this is
Well-known to those skilled in the art.Suitably carrier includes but not limited to: antioxidant, anticorrosion
Agent, coloring agent, flavoring agent and diluent, emulsifying agent, suspension agent, solvent, filler, increment
Agent, buffer agent, carrier, diluent, excipient and/or medicinal adjuvant.
In another embodiment of the present invention, it is provided that secondary heme 6 peptide of the present invention spreads out
Biological preparation method, said method comprising the steps of:
1) on solid phase carrier, according to Fmoc solid phase synthesis process from C end (c-terminus) to N
Each aminoacid of CPP sequence that end (aminoterminal) sequentially coupling is protected through Fmoc and
Fmoc-His-OH, Fmoc-β-Ala-OH, thus obtain the NH through protection2-β-Ala-His-CPP-
Solid phase carrier;
2) coupling secondary heme;
3) deprotection group, cuts down Dh-β-AH-CPP from solid phase carrier simultaneously,
Obtain the solution containing Dh-β-AH-CPP.
Preferably, wherein said CPP be GRKKRRQRRRPPQ, RRRRRRRR,
KLALKLALKALKAALKLA、MVTVLFRRLRIRRACGPPRVRV、
RQIKIWFQNRRMKWKK, KKTWWKTWWTKWSQPKKKRKV or
AGYLLGKINLKALAALAKKIL。
Preferably, carrier conventional during described solid phase carrier is Fmoc solid-phase synthesis.Preferably,
Described solid phase carrier is Rink amide mbha resin.
Preferably, to step 3) in the solution containing Dh-β-AH-CPP that obtains be purified,
Purification process such as washs, recrystallization and chromatography (such as HPLC) etc., preferred color of choice spectrometry,
More preferably HPLC.
In another embodiment of the present invention, it is provided that secondary heme 6 peptide of the present invention derives
Thing is used for preventing and/or treat the purposes in disease or pre-anti-aging medicament, described disease in preparation
It is can to use ascorbate peroxidase enzyme or there is the change of ascorbate peroxidase enzymatic activity
The disease of compound treatment, specifically, described disease is selected from diabetes, cataract, cardiovascular and cerebrovascular vessel
Disease, inflammation, described medicament is particularly suitable for oral administration.
Can use when oral administration tablet, delaying type tablet, sublingual tablet, capsule,
Induction type aerosol, induction type solution, Foradil Aerolizer formoterol fumarate or liquid preparation (such as mixture,
Solution, elixir, syrup or suspension agent) form, all dosage forms all contain the present invention's
Secondary heme 6 peptide derivant;Described preparation can be prepared by means commonly known in the art.
If compositions is the form of tablet, can use and be commonly used for preparing any of solid preparation
Pharmaceutical carrier.The example of described carrier include magnesium stearate, Talcum, gel, arabic gum,
Stearic acid, starch, lactose and sucrose.
Tablet optionally can be suppressed or mould together with one or more auxiliary elements and prepare.Compacting
Tablet can by compacting in applicable machine optionally and binding agent, lubricant, inert diluent,
Lubricant, surfactant or dispersant in free-flowing form (such as powder or
Grain) active component prepare.Molded tablet can pass through will be by inert liquid in applicable machine
Prepared by the mixture of the powdered compounds of body diluent moistening.Described tablet can be optional
Cladding coating or allocate to provide slowly through indentation and can carrying out or discharge work therein with controlling
Property composition.
For Tabules, depending on dosage, described secondary heme 6 peptide derivant can form agent
1 weight % of type, to 80 weight %, more generally forms 5 weight % of dosage form to 60 weight %.
In addition to described secondary heme 6 peptide derivant, tablet usually contains disintegrating agent.Disintegrating agent
Example includes sodium starch glycollate, sodium carboxymethyl cellulose, carboxymethylcellulose calcium, crosslinking
Carmethose, polyvinylpolypyrrolidone, polyvinylpyrrolidone, methylcellulose, crystallite are fine
Dimension element, the substituted hydroxypropyl cellulose of low alkyl group, starch, pregelatinized starch and Sargassum
Acid sodium.Disintegrating agent would generally constitute 1 weight % to 25 weight % of dosage form, preferably 5 weight
% to 20 weight %.
Tablet also can contain binding agent.Binding agent is commonly used to give tablet formulation with tackness
Matter.Be suitable for binding agent include microcrystalline Cellulose, gel, saccharide, Polyethylene Glycol, natural and
Glue, polyvinylpyrrolidone, pregelatinized starch, hydroxypropyl cellulose and the hydroxypropyl of synthesis
Ylmethyl cellulose.The amount of binding agent is usually about 10 weight % of tablet to about 90 weight %.
Tablet also can contain diluent, such as lactose (monohydrate, a hydration of spray drying
Thing, anhydride etc.), mannitol, xylitol, dextrose, sucrose, Sorbitol, micro-
Crystalline cellulose, starch and calcium phosphate dibasic dihydrate.The amount of diluent is usually about the 0 of tablet
Weight % is to the diluent of about 85 weight %.
Tablet also can optionally comprise surfactant, such as sodium lauryl sulfate and polysorbate
Ester 80, and fluidizer, such as silicon dioxide and Talcum.If it does, surfactant
Amount be usually 0.2 weight % to 5 weight % of tablet, and the amount of fluidizer is usually tablet
0.2 weight % to 1 weight %.
Tablet the most also comprises lubricant, such as magnesium stearate, calcium stearate, zinc stearate,
Sodium stearyl fumarate, and the mixture of magnesium stearate and sodium lauryl sulfate.Lubricant leads to
Often with 0.25 weight % to 10 weight % of tablet, preferably 0.5 weight % is to 3 weight %
Amount exists.Other conventional ingredients include antioxidant, coloring agent, flavoring agent, preservative and
Odor mask.
Described secondary heme 6 peptide that exemplary tablet contains up to about 80 weight % derives
Thing, about 2 weight % are to the disintegrating agent of about 10 weight %, about 10 weight % to about 90 weight
The binding agent of %, about 0 weight % are to the diluent of about 85 weight %, and about 0.25 weight
The lubricant of % to about 10 weight %.Tablet mixture by directly compressing or can pass through roll
Carry out compressed shape piece agent.Or, the part of tablet mixture or mixture can before film-making wet method
Granulation, dry granulation or fusion method are pelletized, are melted condensation or extruding.Final preparation can comprise one
Layer or multilamellar and can being coated or uncoated;Or be encapsulated.
If medicament is the form of capsule, being encapsulated of any routine is suitable for, such as at hard gel
Capsule uses above-mentioned carrier.If medicament is soft gel capsule form, it is contemplated that any generally
For preparing pharmaceutical carrier (such as water-base cement, cellulose, the silicate of dispersant or suspension agent
Or oils) and included in soft gel capsule.
Solid preparation for oral administration can be formulated as i.e. releasing and/or modified release.Modification is released
Put preparation include postpone release, sustained release, pulsation release, control discharge, Targeting delivery with
And program release.
Liquid preparation includes suspension agent, solution, syrup and elixir.Described preparation can be used as
Filler in soft capsule or hard capsule and generally include carrier (such as water, ethanol, poly-second two
Alcohol, propylene glycol, methylcellulose or applicable oil), and one or more emulsifying agents and/
Or suspension agent.Solution can be water soluble salt or the combination of other derivants, the example of reactive compound
As, sucrose is to form syrup.Liquid preparation can also come by restoring the solid in such as deck
Preparation.
Embodiment
Below in conjunction with embodiment, the invention will be further described.Following example are to make ability
Territory those of ordinary skill better understood when the present invention, this solely for illustrative purposes, not
It is intended to limit the scope of the present invention.Make efforts to guarantee the standard about numeral (such as quantity, temperature etc.)
Really property, but it is also contemplated that attend the meeting some errors of existence and deviation.Except as otherwise noted, number is attached most importance to
Amount number, temperature with DEG C in units of or as ambient temperature, pressure be close or equal to normal pressure.
Main agents, instrument and source thereof:
Rink amide MBHA resin、Fmoc-β-Ala-OH、Fmoc-Ala-OH、
Fmoc-Asn(Trt)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Arg(Pbf)-OH、
Fmoc-Cys(Trt)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Gln(Trt)-OH、
Fmoc-Gly-OH、Fmoc-His(Trt)-OH、Fmoc-Ile-OH、Fmoc-Leu-OH、
Fmoc-Lys(Boc)-OH、Fmoc-Met-OH、Fmoc-Pro-OH、Fmoc-Phe-OH、
Fmoc-Ser(tBu)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Trp(Boc)-OH、
Fmoc-Tyr(tBu)-OH、Fmoc-Val-OH、DIC、HOBt、PyBop、DIEA、
Piperidines, trifluoroacetic acid, methyl phenyl ethers anisole, phenol: gill biochemical corp, Shanghai
DMEM culture medium, hyclone, HBBS, D-HBBS: Germany GIBCO
Invitrogen company
High performance liquid chromatograph: Agilent company of the U.S.
MALDI-TOF-TOF 5800 mass spectrograph: American AB Sciex company
NanoDrop 2000 UV detector: Thermo scientific company of the U.S.
CO2Incubator: Thermo company of the U.S.
SW-CJ-2F clean work station: the safe and sound company in Suzhou
YMC ODS-A chromatographic column: YMC company of Japan
Eclipse XDB C18 chromatographic column: Agilent company of the U.S.
Interpolation type 24 porocyte culture plate, Millicel-ERS2 resistance instrument: U.S. Millipore
Company
The preparation of embodiment 1.Dh-β-AHTVEK
Swelling: by the Rink amide mbha resin of 0.1mmol, (substitution value is
0.33mmol/g) it is placed in reactor, adds 4ml DMF, in 27 DEG C, 130rpm shaking
30min carries out swelling, uses 10ml DMF washing resin, washs 3min every time, wash altogether
Wash 6 times, drain after washing.
Deprotection: in reactor add 4ml 20% piperidines-DMF solution, 27 DEG C with
130rpm shakes 20min, washes resin with 10ml DMF, washs 3min every time, altogether washing
6 times, drain after washing.
Coupling: by Fmoc-Lys (Boc)-OH (0.3mmol), PyBop (0.3mmol),
HOBt (0.3mmol), base reagent NMM (0.6mmol) are dissolved in 4ml DMF,
It is subsequently adding in reactor, at 27 DEG C, shakes 1h with 130rpm.Wash with 10mlDMF
Resin, washs 3min every time, and washing 6 times, drain after washing, obtain Fmoc-Lys altogether
(Boc)-Rink amide MBHA。
Repeat above-mentioned deprotection and coupling step, successively by Fmoc-Glu (OtBu)-OH,
Fmoc-Val-OH、Fmoc-Thr(tBu)-OH、Fmoc-His(Trt)-OH、
Fmoc-β-Ala-OH is connected on Fmoc-Lys (Boc)-Rink amide MBHA,
Rear removing Fmoc protection group,
NH2-β-Ala-His(Trt)-Thr(tBu)-Val-Glu(OtBu)-Lys(Boc)-Rink amide
Mbha resin.
By secondary heme (0.3mmol), PyBop (0.3mmol), HOBt (0.3mmol),
Base reagent NMM (0.6mmol) is dissolved in 4ml DMF, is then added in reactor,
1h is shaken with 130rpm at 27 DEG C.Then drain, obtain
Dh-β-Ala-His(Trt)-Thr(tBu)-Val-Glu(OtBu)-Lys(Boc)-Rink amide
Mbha resin.
Resin peptide 10ml DMF and the 10ml methanol of gained are washed 3min/ time × 6 respectively
Secondary, it is vacuum dried 12-15h.
After each deprotection and coupling reaction, take a small amount of resin particle, add Kaiser ninhydrin
Reagent (A, the ethanol solution of 6% 1,2,3-indantrione monohydrate;B, the ethanol solution of 80% phenol;C,
Each 2 of the pyridine solution of 0.001M KCN), at 120 DEG C, react 5min, if resin is
Blue (primary amine) shows that amino exposes, and reaction completes.
Cutting: addition 4ml cutting liquid TFA: methyl phenyl ethers anisole: phenol in resin peptide: water (92.5:
2.5:2.5:2.5), at 27 DEG C, shake 1h with 130rpm, be filtered to remove resin, contained
There is the cutting liquid of thick peptide.
Precipitation: be added dropwise in 50ml cold diethyl ether by the above-mentioned cutting liquid containing thick peptide, first at-20 DEG C
Place 1h, then at 4 DEG C, be centrifuged 10min with 8000rpm, heavy with the washing of 20ml ether
Form sediment 2 times, centrifugal, collect thick peptide precipitation, be vacuum dried 12-15h.
Purification: dried thick peptide is dissolved in water, and it is described below that HPLC is prepared in use half
Chromatographic condition is purified.Fraction collection aim colour spectral peak, merges the purity group more than 95%
Point.Collect liquid 37 DEG C, 50mbar decompression under rotary evaporation to remove acetonitrile, by remaining
Aqueous solution lyophilization 24h, obtains pure peptide.Chromatographic condition: chromatographic column YMC ODS-A
(250 × 20mm, 10 μm), mobile phase A is water, and B is acetonitrile (all containing 0.1%TFA),
Gradient elution, in 60min, the ratio of B phase is increased to 70%, flow velocity 20ml/min by 10%,
Column temperature 25 DEG C, carries out ultraviolet detection under 386nm.
Structural Identification: sterling peptide is carried out with RP-HPLC and MALDI-TOF-TOF MS
Identify.Chromatographic condition: being dissolved by dried sterling peptide pure water, concentration is 1mg/ml.
Chromatographic column Eclipse XDB C18 (4.6 × 150mm, 5 μm), mobile phase A is 10% acetonitrile
-water, B is 90% acetonitrile-water (all containing 0.1%TFA), gradient elution, B in 5min
The ratio of phase is increased to 20% by 10%, then increases to 50% through 10min, and last 5min increases to
70%, carry out ultraviolet detection, column temperature 25 DEG C under flow velocity 1.0ml/min, 386nm.Mass spectrum bar
Part: dried sterling peptide purified water being dissolved, concentration is 0.1mg/ml.By 0.5 μ L
Dh-β-the AHTVEK of purification and 0.5 μ L substrate (concentration be 50mM be dissolved in acetonitrile
R-cyano group-4-hydroxycinnamic acid) mix homogeneously, point sample is on rustless steel test board, and room temperature is done
Dry.Reflective-mode, cation scans.Qualification result shows, the sterling peptide sequence of gained and reason
Opinion is consistent, purity 98.3%.
The preparation of embodiment 2Dh-β-AHTVE
Use synthetic method same as in Example 1 and purification process, successively will
Fmoc-Glu(OtBu)-OH、Fmoc-Val-OH、Fmoc-Thr(tBu)-OH、
Fmoc-His (Trt)-OH, Fmoc-β-Ala-OH and secondary red sanguinin are connected to Rink amide
On MBHA, then cut, precipitate, purification, obtain the Dh-pure peptide of β-AHTVE.And adopt
With method RP-HPLC same as in Example 1 and MALDI-TOF-TOF MS to pure
Peptide carries out Structural Identification.Qualification result shows, the sterling peptide sequence of gained is consistent with theory, pure
Degree 97.6%.
The preparation of embodiment 3Dh-β-AHTV
Use synthetic method same as in Example 1 and purification process, successively will
Fmoc-Val-OH、Fmoc-Thr(tBu)-OH、Fmoc-His(Trt)-OH、
Fmoc-β-Ala-OH and time red sanguinin are connected on Rink amide MBHA, then cutting,
Precipitation, purification, obtain the Dh-pure peptide of β-AHTV.And use method same as in Example 1
With RP-HPLC and MALDI-TOF-TOF MS, pure peptide is carried out Structural Identification.Identify knot
Fruit shows, the sterling peptide sequence of gained is consistent with theory, purity 98.7%.
The preparation of embodiment 4Dh-β-AHT
Use synthetic method same as in Example 1 and purification process, successively will
Fmoc-Thr (tBu)-OH, Fmoc-His (Trt)-OH, Fmoc-β-Ala-OH and secondary red blood
Element is connected on Rink amide MBHA, and then cutting, purification, precipitation, obtain
The pure peptide of Dh-β-AHT.And use method RP-HPLC same as in Example 1 and
MALDI-TOF-TOF MS carries out Structural Identification to pure peptide.Qualification result shows, gained
Sterling peptide sequence is consistent with theory, purity 98.0%.
The preparation of embodiment 5Dh-β-AH
Use synthetic method same as in Example 1 and purification process, successively will
Fmoc-His (Trt)-OH, Fmoc-β-Ala-OH and secondary red sanguinin are connected to Rink amide
On MBHA, then cutting, purification, precipitation, obtain the Dh-pure peptide of β-AH.And use with
Pure peptide is entered by method RP-HPLC and MALDI-TOF-TOF MS that embodiment 1 is identical
Row Structural Identification.Qualification result shows, the sterling peptide sequence of gained is consistent with theory, purity
97.3%.
The preparation of embodiment 6Dh-β-A
Use synthetic method same as in Example 1 and purification process, successively will
Fmoc-β-Ala-OH and time red sanguinin are connected on Rink amide MBHA, then cutting,
Purification, precipitation, obtain the Dh-pure peptide of β-A.And use method same as in Example 1 to use
RP-HPLC and MALDI-TOF-TOF MS carries out Structural Identification to pure peptide.Qualification result
Showing, the sterling peptide sequence of gained is consistent with theory, purity 97.7%.
The preparation of embodiment 7Dh-β-AH-GRKKRRQRRRPPQ
Use synthetic method same as in Example 1 and purification process, successively will
Fmoc-Gln(Trt)-OH、Fmoc-Pro-OH、Fmoc-Pro-OH、
Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、
Fmoc-Gln(Trt)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、
Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Arg(Pbf)-OH、
Fmoc-Gly-OH, Fmoc-His (Trt)-OH, Fmoc-β-Ala-OH, secondary red sanguinin connect
On Rink amide mbha resin, then cut, precipitate, purification, obtain
The pure peptide of Dh-β-AH-GRKKRRQRRRPPQ.And use method same as in Example 1
With RP-HPLC and MALDI-TOF-TOF MS, pure peptide is carried out Structural Identification.Identify knot
Fruit shows, the sterling peptide sequence of gained is consistent with theory, purity 98.3%.
The preparation of embodiment 8Dh-β-AH-RRRRRRRR
Use synthetic method same as in Example 1 and purification process, successively will
Fmoc-Arg (Pbf)-OH (repeat coupling 8 times), Fmoc-His (Trt)-OH,
Fmoc-β-Ala-OH, secondary red sanguinin are connected on Rink amide mbha resin, then
Cutting, precipitation, purification, obtain the Dh-pure peptide of β-AH-RRRRRRRR.And use with real
Execute identical method RP-HPLC of example 1 and pure peptide is carried out by MALDI-TOF-TOF MS
Structural Identification.Qualification result shows, the sterling peptide sequence of gained is consistent with theory, purity
97.2%.
The preparation of embodiment 9Dh-β-AH-KLALKLALKALKAALKLA
Use synthetic method same as in Example 1 and purification process, successively will
Fmoc-Ala-OH、Fmoc-Leu-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-OH、
Fmoc-Ala-OH、Fmoc-Ala-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-OH、
Fmoc-Ala-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Ala-OH、
Fmoc-Leu-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-OH、Fmoc-Ala-OH、
Fmoc-Leu-OH、Fmoc-Lys(Boc)-OH、Fmoc-His(Trt)-OH、
Fmoc-β-Ala-OH, secondary red sanguinin are connected on Rink amide mbha resin, then
Cutting, precipitation, purification, obtain Dh-β-AH-KLALKLALKALKAALKLA pure
Peptide.And use method RP-HPLC same as in Example 1 and MALDI-TOF-TOF
MS carries out Structural Identification to pure peptide.Qualification result shows, the sterling peptide sequence of gained is with theoretical
Unanimously, purity 97.9%.
The preparation of embodiment 10Dh-β-AH-MVTVLFRRLRIRRACGPPRVRV
Use synthetic method same as in Example 1 and purification process, successively will
Fmoc-Val-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、
Fmoc-Arg(Pbf)-OH、Fmoc-Pro-OH、Fmoc-Pro-OH、Fmoc-Gly-OH、
Fmoc-Cys(Trt)-OH、Fmoc-Ala-OH、Fmoc-Arg(Pbf)-OH、
Fmoc-Arg(Pbf)-OH、Fmoc-Ile-OH、Fmoc-Arg(Pbf)-OH、
Fmoc-Leu-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Arg(Pbf)-OH、
Fmoc-Phe-OH、Fmoc-Leu-OH、Fmoc-Val-OH、Fmoc-Thr(tBu)-OH、
Fmoc-Val-OH、Fmoc-Met-OH、Fmoc-His(Trt)-OH、Fmoc-β-Ala-OH、
Secondary red sanguinin is connected on Rink amide mbha resin, then cuts, precipitates, purification,
Obtain the Dh-pure peptide of β-AH-MVTVLFRRLRIRRACGPPRVRV.And use with real
Execute identical method RP-HPLC of example 1 and pure peptide is carried out by MALDI-TOF-TOF MS
Structural Identification.Qualification result shows, the sterling peptide sequence of gained is consistent with theory, purity
97.5%.
The preparation of embodiment 11Dh-β-AH-RQIKIWFQNRRMKWKK
Use synthetic method same as in Example 1 and purification process, successively will
Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Trp(Boc)-OH、
Fmoc-Lys(Boc)-OH、Fmoc-Met-OH、Fmoc-Arg(Pbf)-OH、
Fmoc-Arg(Pbf)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Gln(Trt)-OH、
Fmoc-Phe-OH、Fmoc-Trp(Boc)-OH、Fmoc-Ile-OH、
Fmoc-Lys(Boc)-OH、Fmoc-Ile-OH、Fmoc-Gln(Trt)-OH、
Fmoc-Arg (Pbf)-OH, Fmoc-His (Trt)-OH, Fmoc-β-Ala-OH are connected to Rink
On amide mbha resin, then cut, precipitate, purification, obtain
Dh-β-AH-RQIKIWFQNRRMKWKK.And use method same as in Example 1
With RP-HPLC and MALDI-TOF-TOF MS, pure peptide is carried out Structural Identification.Identify knot
Fruit shows, the sterling peptide sequence of gained is consistent with theory, purity 97.6%.
The system of embodiment 12Dh-β-AH-KKTWWKTWWTKWSQPKKKRKV
Standby
Use synthetic method same as in Example 1 and purification process, successively will
Fmoc-Val-OH、Fmoc-Lys(Boc)-OH、Fmoc-Arg(Pbf)-OH、
Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、
Fmoc-Pro-OH、Fmoc-Gln(Trt)-OH、Fmoc-Ser(tBu)-OH、
Fmoc-Trp(Boc)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Thr(tBu)-OH、
Fmoc-Trp(Boc)-OH、Fmoc-Trp(Boc)-OH、Fmoc-Thr(tBu)-OH、
Fmoc-Lys(Boc)-OH、Fmoc-Trp(Boc)-OH、Fmoc-Trp(Boc)-OH、
Fmoc-Thr(tBu)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、
Fmoc-His (Trt)-OH, Fmoc-β-Ala-OH, secondary red sanguinin are connected to Rink amide
On mbha resin, then cut, precipitate, purification, obtain
The pure peptide of Dh-β-AH-KKTWWKTWWTKWSQPKKKRKV.And use and implement
Pure peptide is tied by method RP-HPLC and MALDI-TOF-TOF MS that example 1 is identical
Structure is identified.Qualification result shows, the sterling peptide sequence of gained is consistent with theory, purity 98.0%.
The preparation of embodiment 13Dh-β-AH-AGYLLGKINLKALAALAKKIL
Use synthetic method same as in Example 1 and purification process, by Fmoc-Leu-OH,
Fmoc-Ile-OH、Fmoc-Lys(Boc)-OH、Fmoc-Lys(Boc)-OH、
Fmoc-Ala-OH、Fmoc-Leu-OH、Fmoc-Ala-OH、Fmoc-Ala-OH、
Fmoc-Leu-OH、Fmoc-Ala-OH、Fmoc-Lys(Boc)-OH、Fmoc-Leu-OH、
Fmoc-Asn(Trt)-OH、Fmoc-Ile-OH、Fmoc-Lys(Boc)-OH、
Fmoc-Gly-OH、Fmoc-Leu-OH、Fmoc-Leu-OH、Fmoc-Tyr(tBu)-OH、
Fmoc-Gly-OH、Fmoc-Ala-OH、Fmoc-His(Trt)-OH、Fmoc-β-Ala-OH、
Secondary red sanguinin is connected on Rink amide mbha resin, then cuts, precipitates, purification,
Obtain Dh-β-AH-AGYLLGKINLKALAALAKKIL.And use and embodiment 1
Identical method RP-HPLC and MALDI-TOF-TOF MS carry out structure mirror to pure peptide
Fixed.Qualification result shows, the sterling peptide sequence of gained is consistent with theory, purity 97.8%.
Embodiment 14 peroxidase activity
Pure peptide pure water obtained by embodiment 1-13 is set to the peptide that concentration is 200 μMs/L
Solution, then according to following method measures the peroxidase activity of the pure peptide of gained respectively:
Take 2.5ml 50mM sodium dihydrogen phosphate (pH7.4)-0.1mM EDTANa2Buffer,
20 μ l ascorbic acid (0.5mM), 10 μ l H2O2(0.3mM), 20 μ l peptide solutions, add
Enter in 1cm quartz colorimetric utensil, final volume 3ml, wherein it is not added with ascorbic acid and H2O2For sky
White comparison, room temperature 25 DEG C monitoring 290nm absorbance reduction in 60s (spot/5s).
Active unit is defined as the amount of the peptide needed for conversion 1 μm ol ascorbic acid per minute, than work
It is defined as the activity that every μm ol peptide is had.Computing formula is as follows, and U ratio lives (Unit/ μm ol),
△ A absorbance is poor, the extinction coefficient (2.8mM of ε ascorbic acid-1.cm-1), c peptide concentration (μM),
1000 Units conversion factor.
As shown in table 1 result, along with DhHP-6 sequence is by further truncate, its enzyme activity by
Gradually reducing (secondary heme 6 peptide derivant 2-6), wherein Dh-β-AH is minimum activity unit,
Activity is about the 50% of DhHP-6, and Dh-β-A is the most inactive.This shows His imidazoles
Ring and Dh iron atom, at axial coordination, play an important role for electron transmission.By Dh-β-AH
Coupling rich in (secondary heme 6 peptide derivant 7-13) after the cell-penetrating peptide of hydrophilic amino acid, its
Enzyme activity significantly improves, and reaches the level suitable with DhHP-6.
The peroxidase activity of the peptide of table 1 embodiment 1-13 gained
Embodiment 15Caco-2 permeability cell
Caco-2 cell is (in Shanghai Inst. of Life Science, CAS cellular resources
The heart) (add 10% hyclone, 1% non essential amino acid (moral with DMEM culture medium
State Gibco), 2mM glutamine) Secondary Culture (37 DEG C, 5%CO2).Take 60~66
Generation cell, by 2.5 × 105Cell/cm2Cover plant is (interpolation type 24 hole on polycarbonate membrane
Tissue Culture Plate, aperture 0.4 μm, surface area 0.6cm2).Within every 2 days, change above-mentioned DMEM
Culture medium, to be grown to 21~23 days, cell monolayer broke up completely, and cross-film resistance tends towards stability
(500~800 Ω .cm2), may be used for film through measuring.Cell with HBSS (Hank ' s
Balanced salt solution) wash 2 times, add 400 μ l HBSS, at 37 DEG C, hatch 30min.
The peptide sample of gained in embodiment is dissolved (peptide solubility is as 1mM) with HBSS, through 0.22 μm
Filter membrane is degerming, takes 200 μ l and joins cell top side, and 400 μ l HBSS join cell substrate side,
Hatch 120min for 37 DEG C.Collect substrate side HBSS, with the HBSS collected by HPLC mensuration
In peptide content, in the unit of account time, the transit dose of secondary heme peptide, i.e. transmission rates, enter
And calculate apparent permeability coefficients.Formula is as follows, PappApparent permeability coefficients (cm/s), C0
The initial concentration of peptide, A film surface area (0.6cm2), dQ/dt secondary heme peptide transmission rates:
Table 2 result shows, the apparent infiltration of minimum activity unit Dh-β-AH and DhHP-6
Coefficient is suitable, Papp3 × 10-8Cm/s scope, is the compound of intestinal malabsorption.To the greatest extent
The fat-soluble increase of pipe Dh-β-AH, molecular weight reduce, but do not improve its cell membrane and penetrate
Property, illustrate that Dh-β-AH is still the intercellular Passive diffusion relying on Concentraton gradient.By Dh-β-AH
After coupling cell-penetrating peptide (secondary heme 6 peptide derivant 7-13), coupling has each cell-penetrating peptide sequence
Dh-β-AH is significantly increased (P across the permeability of Caco-2 cellappIncrease 6-7 times),
Wherein rich in the cell-penetrating peptide effect of amphipathic aminoacid (in table 2 numbered 5,7,8 and 9)
More excellent.And by Dh-β-AH and cell-penetrating peptide physical mixed, it is impossible to improve the apparent of Dh-β-AH
(apparent permeability coefficients is 3.1 × 10 to infiltration coefficient-8Cm/s), illustrate that it must pass through chemical bond
It is connected with cell-penetrating peptide and could improve transcellular permeability.To sum up, Dh-β-AH-CPP is across carefully
Born of the same parents' performance is greatly improved, and can develop oral drugs as guide structure.
The Caco-2 permeability cell of the peptide of table 2 embodiment 1-13 gained
Embodiment 16. oral tablet
Composition:
Secondary heme 6 peptide derivant of 100mg embodiment 7 preparation
Dh-β-AH-GRKKRRQRRRPPQ, 50mg lactose (monohydrate), 50mg are beautiful
Rice starch (natural), 10mg polyvinylpyrrolidone (PVP 25) and 2mg magnesium stearate.
Tablet weight 212mg, diameter 8mm, radius of curvature 12mm.
Preparation:
Dh-β-AH-GRKKRRQRRRPPQ prepared by embodiment 7, newborn sugar and starch
Mixture pelletize with the aqueous solution of the PVP25 of 5% (w/w).By described particle drying,
Then mix with magnesium stearate 5 minutes.This mixture is compressed in conventional tablet presses.Pressure
The standard of contracting is the pressure of 15kN.
Embodiment 17. oral suspension agent
Composition:
Secondary heme 6 peptide derivant of 100mg embodiment 9 preparation
Dh-β-AH-KLALKLALKALKAALKLA, 100mg concentration are the ethanol water of 96%
Solution, 40mg(purchased from FMC, the xanthan gum of Pennsylvania, USA)
With 9.9g water.
The single dose of secondary heme 6 peptide derivant 9 of the 100mg present invention corresponds to 10ml
Oral suspension agent.
Preparation:
Rhodigel is suspended in the ethanol water of 96%, is prepared by embodiment 9
Dh-β-AH-KLALKLALKALKAALKLA joins in suspension.Under agitation add
Water.By described mixture stir about 6h until xanthan gumExpand completely.
Embodiment 18. oral solution
Composition:
Secondary heme 6 peptide derivant of 500mg embodiment 13 preparation
Dh-β-the AH-AGYLLGKINLKALAALAKKIL, (polymerization of 2.5g polysorbate
Degree is 80) and 97g PEG400.100mg
The single dose of Dh-β-AH-AGYLLGKINLKALAALAKKIL corresponds to 20g mouth
Take solution.
Preparation:
Under agitation, prepared by embodiment 13
Dh-β-AH-AGYLLGKINLKALAALAKKIL is suspended in PEG400 and gathers
In the mixture of sorbate.It is stirred continuously until
Dh-β-AH-AGYLLGKINLKALAALAKKIL is completely dissolved.
Claims (10)
1. secondary heme 6 peptide derivant, it has Dh-β-AH-CPP structure, wherein CPP
Representing penetratin, described Dh-β-AH and described CPP is by covalent bond coupling.
2. secondary heme 6 peptide derivant of claim 1, wherein CPP is
GRKKRRQRRRPPQ、RRRRRRRR、KLALKLALKALKAALKLA、
MVTVLFRRLRIRRACGPPRVRV、RQIKIWFQNRRMKWKK、
KKTWWKTWWTKWSQPKKKRKV or
AGYLLGKINLKALAALAKKIL。
3. a pharmaceutical composition, it includes the secondary heme of one or more claim 1 or 2
6 peptide derivants and pharmaceutically useful carrier.
4. the pharmaceutical composition of claim 3, wherein said pharmaceutically useful carrier selected from antioxidant,
Preservative, coloring agent, flavoring agent, diluent, emulsifying agent, suspension agent, solvent, filler,
Extender, buffer agent, carrier, diluent, excipient and/or medicinal adjuvant.
5. the method preparing secondary heme 6 peptide derivant of claim 1 or 2, described side
Method comprises the following steps:
1) on solid phase carrier, the most even to N end from C end according to Fmoc solid-phase synthesis
Join through protection described CPP in each aminoacid, Fmoc-His-OH and
Fmoc-β-Ala-OH, thus obtain the NH through protection2-β-Ala-His-CPP-solid phase carrier;
2) coupling secondary heme;
3) deprotection group, cuts down Dh-β-AH-CPP from solid phase carrier simultaneously,
Obtain the solution containing Dh-β-AH-CPP.
6. the method for claim 5, wherein said CPP be GRKKRRQRRRPPQ,
RRRRRRRR、KLALKLALKALKAALKLA、
MVTVLFRRLRIRRACGPPRVRV、RQIKIWFQNRRMKWKK、
KKTWWKTWWTKWSQPKKKRKV or
AGYLLGKINLKALAALAKKIL。
7. the method for claim 5 or 6, also includes in step 3) in the secondary heme that obtains
The step that 6 peptide derivants are purified, purification process is in washing, recrystallization and chromatography
Plant or multiple.
8. secondary heme 6 peptide derivant of claim 1 or 2 is used for preventing and/or treating disease in preparation
Disease or the purposes in pre-anti-aging medicament, described disease is for can use ascorbic acid peroxide
Compound enzyme or there is the disease of compounds for treating of ascorbate peroxidase enzymatic activity.
9. the purposes of claim 8, wherein said disease is selected from diabetes, cataract, heart and brain blood
Pipe disease, inflammation.
10. the purposes of claim 8 or 9, wherein said medicament is used for being administered orally.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201510041700.XA CN105985441A (en) | 2015-01-27 | 2015-01-27 | Deuterohaemin hexapeptide derivative, preparation method and application thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201510041700.XA CN105985441A (en) | 2015-01-27 | 2015-01-27 | Deuterohaemin hexapeptide derivative, preparation method and application thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
CN105985441A true CN105985441A (en) | 2016-10-05 |
Family
ID=57034138
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201510041700.XA Pending CN105985441A (en) | 2015-01-27 | 2015-01-27 | Deuterohaemin hexapeptide derivative, preparation method and application thereof |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN105985441A (en) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101157726A (en) * | 2007-11-14 | 2008-04-09 | 李惟 | Deuterohemin short-peptide compound and its application in preparation of anti-cataractogenesis drugs |
CN102311500A (en) * | 2010-07-02 | 2012-01-11 | 杭州师范大学 | Antiviral fusion protein and application thereof |
CN103965287A (en) * | 2014-05-07 | 2014-08-06 | 吉林大学 | Deuterohemin-beta-Ala-His-Lys(DhHP-3), and preparation method and application thereof |
-
2015
- 2015-01-27 CN CN201510041700.XA patent/CN105985441A/en active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101157726A (en) * | 2007-11-14 | 2008-04-09 | 李惟 | Deuterohemin short-peptide compound and its application in preparation of anti-cataractogenesis drugs |
CN102311500A (en) * | 2010-07-02 | 2012-01-11 | 杭州师范大学 | Antiviral fusion protein and application thereof |
CN103965287A (en) * | 2014-05-07 | 2014-08-06 | 吉林大学 | Deuterohemin-beta-Ala-His-Lys(DhHP-3), and preparation method and application thereof |
Non-Patent Citations (1)
Title |
---|
LIYAN LEI,等: "Deuterohemin-AlaHisLys mitigates the symptoms of rats with non-insulin dependent diabetes mellitus by scavenging reactive oxygen species and activating the PI3-K/AKT signal transduction pathway", 《CHEMICO-BIOLOGICAL INTERACTIONS》 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN110229216B (en) | Compstatin peptides with improved pharmacokinetic properties | |
KR100354897B1 (en) | Compounds with growth hormone releasing properties | |
AU2005305157B2 (en) | Adrenocorticotropic hormone analogs and related methods | |
CN102397558B (en) | Positioning pegylation modified compound of Exendin-4 analog and application thereof | |
TW201754B (en) | ||
KR101413051B1 (en) | Cytotoxic conjugates having neuropeptide y receptor binding compound | |
CZ291382B6 (en) | Peptide derivatives influencing growth hormone release, pharmaceutical composition containing them and their use | |
EP2922876A1 (en) | Improved peptide pharmaceuticals for insulin resistance | |
KR20080082672A (en) | Neuropeptide-2 receptor-agonists | |
CN109865142A (en) | For the improvement peptide medicine of insulin resistance | |
JPH07507330A (en) | Polypeptide bombesin antagonist | |
JP6800372B2 (en) | A long-acting palmitic acid-bound GnRH derivative and a pharmaceutical composition containing the same. | |
EP3162811B1 (en) | Peptide as absorption enhancer and composition containing same | |
CN105985441A (en) | Deuterohaemin hexapeptide derivative, preparation method and application thereof | |
CN105585611B (en) | Octapeptide modified dexamethasone, preparation, nanostructure and application thereof | |
US9393271B2 (en) | GHRH agonists for islet cell transplantation and function and the treatment of diabetes | |
KR20180020789A (en) | Conjugate of minoxidil and peptide | |
RU2056432C1 (en) | Peptide derivatives and their physiologically acceptable salts | |
EP1536808B1 (en) | Heterocarpin, a plant-derived protein with anti-cancer properties | |
KR102160566B1 (en) | Conjugate of minoxidil and peptide | |
CN102241757B (en) | Polypeptide analogue of human osteocalcin | |
KR20130046702A (en) | Novel whitening compound and whitening cosmetic composition comprising the same | |
AU2017399481B2 (en) | Conjugate of salicylic acid and peptide | |
US20190175688A1 (en) | Cyclic Peptides and Methods Using Same | |
KR102190495B1 (en) | Peptide for promoting mucous membrane permeation and composition comprising the same |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20161005 |
|
WD01 | Invention patent application deemed withdrawn after publication |