CN105294852B - The conjugate and its medical usage of polyethylene glycol and tumor necrosis factor α or its analog - Google Patents
The conjugate and its medical usage of polyethylene glycol and tumor necrosis factor α or its analog Download PDFInfo
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Abstract
The present invention relates to the conjugates and its medical usage of polyethylene glycol and tumor necrosis factor α or its analog.Specifically, the present invention relates to a kind of polyethylene glycol-tumor necrosis factor α or polyethylene glycol-tumor necrosis factor α analog conjugate, and preparation method thereof as well as therapeutic agent preventing or treating the purposes in tumour or cancer.The conjugate of polyethylene glycol-tumor necrosis factor α or its analog of the invention can reduce the acute toxicity of drug, reduce intravenously administrable frequency, reduce the dosage of application drug, increase antineoplaston effect, and solve the problems, such as parenterai administration tumor necrosis factor α albumen drug effect half-life short, reach prevention or treat the target of tumour or cancer, therefore there is advantageous medical prospect.
Description
Technical field
The present invention relates to polyethylene glycol-tumor necrosis factor α (TNF α) or polyethylene glycol-tumor necrosis factor α analogs
Conjugate, and preparation method thereof as well as therapeutic agent preventing or treating the purposes in tumour or cancer.
Background technique
Tumor necrosis factor α (TNF α) is the multifunctional cytokine egg that can cause malignant tumor cell apoptosis
It is white.TNF α is secreted by the mononuclear macrophage activated, is the strongest cell factor of anti-tumor activity found so far,
There is apparent killing cell (tumoricidal) and inhibiting tumour cells (tumoristatic) to Several Kinds of Malignancy cell
Act on (Carswell, EA et al., 1975, An endotoxin induced serum factor that causes
necrosis of tumors.Proc.Natl.Acad.Sci.USA 72；3666-3670).
TNF α found in latter stage in the 1970's by American scientist, TNF α can by with a high molecular weight receptor
(TNFR1) and the receptor of a lower molecular weight (TNFR2) combines, to conduct all stechiologies, individual physiology, disease
Neo-Confucianism, and kill pharmacotoxicological effect (Andrews JS et al., the 1990.Characterization of of malignant cell
the receptors for tumor necrosis factor(TNF)and lymphotoxin(LT)on
humanlymphocytes.J.Immunol.144；2582-2591.Dembic Z et al., 1990.Two human TNF
receptors have similar extracellular,but distinct intracellular,domain
sequences.Cytokine 2；231-237).The clinical treatment early stage melanin cancer and treatment soft tissue delivered are disliked
The effect experiment data of property sarcoma are shown: TNF α is too short in people's circulation time in vivo, and blood circulation system organ toxicity's mistake
Height, therefore its bad (Bartlett of clinical efficacy for malignant mela noma, the parenteral injection form of soft tissue sarcoma
DL et al., A phase 1trial of continuous hyperthermic peritoneal perfusion with
tumor necrosis factor and Cisplatin in the treatment of peritoneal
carcinomatosis,Cancer,83:1251-1261.).But in a kind of clinical test of body local treatment malignant tumour
In model, i.e., in limbs ex vivo perfusion model (isolated perfused limb model), TNF α can be single from patient
The local intra-arterial of limbs is perfused, and the melanoma or sarcoma lesion on limbs can be exposed to local intra-arterial injection in the long period
TNF α drug, then the TNF α in limbs can be directed in a pump receptacle from the reflux veins blood vessel of the other end.It is this
Limbs ex vivo perfusion model in the case where applying to avoid TNF α from being back to systemic circulation system and generate systemic organs' toxicity,
Show superior clinical therapeutic efficacy (Christoforidis, D et al., the Isolated limb for killing malignant cell
perfusion:distinct tourniquet and tumor necrosis factor effects on the early
Hemodynamic response.2003.Arch Surg, 138:17-25.Eggermont, AM et al., 1996.Isolated
limb perfusion with high-dose tumor necrosisfactor-alpha in combination with
interferon gamma and mephalan for non-resectable extremity soft tissue
sarcomas:a multicenter trial；J.Clin.Oncol,14:2653-2665.).The end of body local is treated herein
In phase malignant tumour clinical test, the TNF α drug of regional perfusion can increase considerably tumor focus drug exposure levels, then
Drug can be flowed back by vein blood vessel and be imported into a pump receptacle, thus the general toxicity of the systemic circulation system of reduction patient.
From these body locals treat clinical laboratory data it can be found that TNF α on limbs melanoma or soft tissue sarcoma it is thin
The therapeutic effect of born of the same parents very surprising (Eggermont, AM et al., 2003.Tumor necrosis factor-based
isolated limb perfusion with for Soft Tissue Sarcomas and Melanoma:Ten years
Of successful Anti-vascular Therapy.CurrOncol Rep, 5:79-80.Fraker DL et al.,
Biological therapy with TNF:systemic administration and isolated limb
perfusion.1995.In:V.T.Devita,S.Hellman and S.A.Rosenberg SA.Philadelphia:
J.B.Lippincott Co.pp 295-328.Van Etten, B. et al., 2003.Fifty tumor necrosis
factor-based isolated limb perfusions for limb salvage inpatients older than
75years with limb-threatening soft tissue sarcomas and other extremity
tumors.Ann.SurgOncol.10:32-37.).Clinical test results confirm TNF α in latter stage malignant tumor patient human body
Curative effect feature is focal tumor cell hemorrhagic necrosis, the complete incidence graph treatment rate of local tumor lesion even up to 90% with
On.
For the disadvantage for overcoming TNF α short in people's circulation time in vivo, so that malignant tumour can be applied more broadly in
The systemic clinical treatment of disease, recent domestic scholar are making great efforts to study TNF α pharmaceutical carrier transmission system, i.e. high score
Sub- chemical modification method improves the targeting of drug, and reduce the adverse reaction of drug to extend drug half-life.This
Inventor proposes, is prolonged using polyethyleneglycol modified technology to solve the disadvantage that parenterai administration TNF α protein drug half-life short
Kill is sufficiently presented so as to be used on human body in long drug half-life in the way of parenterai administration
(tumoricidalactivity) malignant cell, and non-inhibited prevention (tumoristatic activity) malignant tumour
The specific bioactivity of cell growth.
Summary of the invention
In order to overcome the drawbacks of the prior art, the purpose of the present invention is to provide a kind of new polyethylene glycol-neoplasm necrosis
Factor-alpha or polyethylene glycol-tumor necrosis factor α analog conjugate.The present invention utilizes different polymerization methods or different polymerizations
The activated polyethylene glycol of chain length is chemically modified tumor necrosis factor α or its analog, to generate a series of newization
Composition is learned with chemical structure for preventing or treating the drug of various malignant tumours.
Tumor necrosis factor α analog of the present invention can be TNF α mutain, the preferably A21K of TNF α,
G31K simple point mutation TNF α albuminoid.
In a preferred embodiment of the present invention, the polyethylene glycol is linear polyethylene glycol, and the poly- second two
The length range of alcohol linear chain is 5,000-20,000, preferably 20,000.
In another preferred embodiment of the present invention, the polyethylene glycol is the polyethylene glycol of activation, the activation
Polyethylene glycol be polyethylene glycol methoxysuccinyl imines glutarate NHS ester (Methoxy Succinimidyl
Glutarate NHS ester, SG-PEG) or succinimide carboxymethyl NHS ester (Succinimidyl Carboxymethyl
NHS ester, SCM-PEG).
In a particularly preferred embodiment according to the invention, the polyethylene glycol-tumor necrosis factor α or poly- second two
Alcohol-tumor necrosis factor α analog conjugate is TNF α SCMPEG5, TNF α SGPEG10, TNF α SCMPEG20, TNF α
SGPEG20, TNF α A21KSGPEG20, TNF α G31KSGPEG20, TNF α A21KSCMPEG20 or TNF α G31KSCMPEG20.
The present invention further provides polyethylene glycol-tumor necrosis factor α as described above or polyethylene glycol-tumor necrosis factors
The preparation method of the conjugate of sub- alpha analog comprising by the polyethylene glycol of activation and TNF α or TNF α analog haptoreaction
The step of.Wherein, the polyethylene glycol is linear polyethylene glycol, and the length range of the polyethylene glycol linear chain is 5,000-
20,000, preferably 20,000.The polyethylene glycol of the activation is preferably the methoxysuccinyl imines glutarate of polyethylene glycol
NHS ester or succinimide carboxymethyl NHS ester.
The amount ratio of the TNF α or TNF α analog and polyethylene glycol in a preferred embodiment in accordance with this invention
Range is 1:20-1:30, preferably 1:20 by weight.
In another preferred embodiment of the present invention, in above-mentioned preparation method, the pH scope control of the reaction exists
8.5 to 9.5, preferably 9.0 ± 0.1.The temperature range of the reaction is controlled at 25 to 37 DEG C, preferred temperature control 30 ±
0.1℃。
Polyethylene glycol chemistry modification reaction
The reaction of polyethylene glycol chemistry is the important control program in the present invention.The polyethylene glycol of activation is protein modified to TNF α
Reaction is the pH value in reaction by control optimization 9.0 ± 0.1, and reaction time control was at shorter than 30 minutes, albumen and poly- second two
The amount ratio of alcohol is 1:20-1:30 (w:w), and reaction temperature is controlled at 25 to 37 DEG C, enables the lysine of TNF α protein surface
It is adequately reacted with activated polyethylene glycol, to obtain the polyethyleneglycol modified coupled product of specified molecular weight.
As above, if using the different activated polyethylene glycol of different molecular weight or polymerization methods to large biological molecule albumen
Be chemically modified, then can get polyethylene glycol high-purity, narrow unidirectional distribution (molecular weight distribution is between 1.01-1.05),
Stabilization conjugate (the TNF α of polyethylene glycol and tumor necrosis factor α or its analog that average molecular weight is 5,000-20,000
PEG)。
Polyethylene glycol chemistry after the reaction was completed, can use the filter membrane device through molecular weight 50,000, residue not completed
The albumen of chemical reaction, and remaining unreacted linear polyethylene glycol can be obtained the poly- second of high-purity with being centrifuged at a high speed
Glycol-albumen object conjugate.
This is a kind of mild biochemical reaction, is obtained and forming ester bond or amido bond with acid anhydrides or carboxylic acid reaction
Conjugate, it is relatively stable under common physiological condition, can't be broken.
The invention further relates to a kind of pharmaceutical compositions, contain polyethylene glycol-tumor necrosis factor α as described above
Or polyethylene glycol-TNF (Tumor Necrosis Factor) alpha analog conjugate and pharmaceutically acceptable carrier.
It will be appreciated by those skilled in the art that pharmaceutical composition of the invention can be formulated according to specific method of application
At various dosage forms well known in the art, for example, peroral dosage form (pulvis, tablet, capsule, soft capsule, liquid medicine, syrup,
Wine made of broomcorn millet ball, powder, wafer, granula) or epidermis preparation (emulsifiable paste, ointment, lotion, gel, face cream, plaster, paste, spray, gas
Mist agent etc.) or ejection preparation (solution, suspending agent, emulsion).It is within the scope of the present invention preferably ejection preparation.
Pharmaceutical composition according to the present invention may include pharmaceutically acceptable carrier, adjuvant or diluent, such as: it fills out
Fill agent, disintegrating agent, lubricant, suspending agent, adhesive, sweetener, corrigent, preservative, matrix etc..Filler is for example: starch,
Pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose, sucrose etc.；Disintegrating agent is for example: starch, pregelatinized starch, micro-
Crystalline cellulose, sodium carboxymethyl starch, crosslinked polyethylene pyrroles, low-substituted hydroxypropyl cellulose, croscarmellose sodium etc.；Profit
Lubrication prescription is for example: magnesium stearate, lauryl sodium sulfate, talcum powder, silica etc.；Suspending agent is for example: polyvinylpyrrolidone,
Microcrystalline cellulose, sucrose, agar, hydroxypropyl methyl cellulose etc.；Adhesive is for example, starch slurry, polyvinylpyrrolidone, hydroxypropyl
Ylmethyl cellulose etc..Composition of the invention can be by being made, so that patient's medication using any known method in this field
Quick, lasting or slow release active constituent can be provided afterwards.
The invention further relates to polyethylene glycol-tumor necrosis factor α as described above or polyethylene glycol-tumor necrosis factors
The conjugate of sub- alpha analog is preparing the medicine for preventing and/or treating tumour or cancer comprising its pharmaceutical composition
Purposes in object.Wherein the tumour or cancer can be lymph cancer, cellule type or lung cancer in non-cellule type, prostate
Cancer, kidney, liver cancer, cancer of colon, melanoma, breast cancer.
The dosage of inventive compound can be according to the situation and weight, the severity of the state of an illness, drug of individual
Form, the difference of administration route and dosage period and it is different, can also be selected by those skilled in the art.Dosage can
To be daily single-dose or be administered multiple times daily.The clinic of the polyethyleneglycol modified tumor necrosis factor α of the present invention is effectively
Pharmaceutical quantities are at least 10 to 3000ng/m2/ 7-14 days.
Pharmaceutical composition of the invention is administered to individual animals such as mammal (rat, mouse, domestication by all means
Animals or humans), all administration modes be it is contemplated that for example, administration can be between subcutaneous, intradermal, film, suppository and aerosol
Agent form of medication.
In the present invention, term polyethylene glycol-TNF α conjugate is a general proper noun, is typically expressed as " (TNF α
PEG)".It indicates to be reacted by the special groups of TNF α and the polyethylene glycol chemistry of activation, the polymerization formed through being covalently keyed
Body.It includes all polyethylene glycol of the invention-TNF α conjugates and polyethylene glycol-TNF α analog conjugate.For example, can be with
Include TNF α SGPEG5, TNF α SGPEG10, TNF α SGPEG20, TNF α SCMPEG5, TNF α SCM10PEG, TNF α SCM20PEG.
Heretofore described tumor necrosis factor α (TNF α) also contains the single-point, double of the protein molecular gene-correlation
The similar gene protein of point mutation and multipoint mutation.
" Pegylation tumor necrosis factor (albumen) " of the present invention, " polyethyleneglycol modified tumor necrosis factor
Sub (albumen) " means equivalent, is equal to polyethylene glycol-tumor necrosis factor α conjugate or polyethylene glycol-tumour is bad
The conjugate of necrosis factor alpha analog.
Heretofore described polyethylene glycol is divided into straight chain and branched chain type by the difference of polymeric unit, polymerization methods；By poly-
Right difference can mark off the polyethylene glycol of different molecular weight size, such as PEG 5000, PEG 10000, PEG 20000
Deng.
The characteristics of present invention can be according to large biological molecule be modified, using primary chemical reaction, using unimodal molecular weight
Or the polyethylene glycol of different molecular weight is to TNF α and tumor necrosis factor α mutation expression protein derivatives (TNF α mutin)
Surface amino groups acid carries out different modes and different degrees of modification, to extend the bioactivity half-life period of the drug, increases water
Dissolubility, and the toxic side effect of large biological molecule is reduced, expected long-acting treatment effect is obtained under the conditions of parenterai administration.
The specific embodiments and the drawings detail specifications present invention will be passed through below.
Detailed description of the invention
Fig. 1 is the figure for showing the structure of pET28a (+) expression plasmid.
Fig. 2 is the figure of SDS- polyacrylamide solidifying burnt electrophoretic determination TNF α albumen and TNF α SGPEG20 purity.
Specific embodiment
The present invention is further illustrated with reference to embodiments, but these embodiments are only used to illustrate the present invention, without to appoint
Where formula limits the scope of the invention.
The gene expression of 1 TNF α albumen of embodiment
With full chemistry synthetic method, following TNF α gene is compiled into the special expression of amino acids password of Escherichia coli
(Pennica,D.Nedwin,G.E.,Hayflick,J.S.,at al,Nature,1984,312(20/27),724-729,
Human tumour necrosis factor:precursor structure,expression and homology to
Lymphotoxin) it is placed in expression plasmid pET28a (+) (pET System Novagen Manual 11th Edition, User
Protocol TB055Rev.C 0611JN) in, then according to the conventional program in Novagen company this method handbook
(Novagen User Protocol TB055Rev.C 0611JN), penetrates e. coli bl21 (DE3), BL21 for plasmid
(DE3) in sS (Novagen pET System Manual 11th Edition), then from by the large intestine of above-mentioned expression plasmid
Single bacterium colony is selected in the agar plates of bacillus, is placed in sterile LB (Luria-Bertani) culture medium of 5mL (purchased from state
Chemical reagent Co., Ltd, medicine group), in 37 DEG C, according to Standard Operations Manual mode culture.Then it is gradually put according to 1% ratio
Greatly to 5 liters of culture medium, until strain density OD260When for 0.6-0.8,18 DEG C are cooled to, certain density isopropyl-β-D- is added
Thiogalactoside (IPTG) induces TNF α protein expression 16 hours.Escherichia coli body is collected with 5000rpm centrifugation, then
With pre-cooling (4 DEG C) lysis buffer, bacterium is broken in Ultrasonic Cell Disruptor.The plasmid construct of pET28a (+) expression is shown in Fig. 1.
TNF α gene amino acid sequence
MHHHHHHVRS SSRTPSDKPV AHVVANPQAE GQLQWLNRRA NALLANGVEL
RDNQLVVPSE GLYLIYSQVL FKGQGCPSTH VLLTHTISRI AVSYQTKVNL
LSAIKSPCQR ETPEGAEAKP WYEPIYLGGV FQLEKGDRLS AEINRPDYLD
FAESGQVYFG IIAL
SEQ ID NO:1
The gene expression of 2 TNF α albumen analog (mutant) of embodiment
With full chemistry synthetic method, following TNF α A21K, TNF α G31K, TNF α A21KG31K gene are compiled into large intestine
The special expression of amino acids coding of bacillus, is placed among expression plasmid pET28a (+).In full chemistry synthesis TNF α gene coding
When: 1) by the propyl acid code of 21st sequence, be changed to lysine password, 2) that the 31st glycine password is changed to lysine is close
Code, 3) by the propyl acid code of the 21st sequence, the 31st glycine password is changed to lysine password simultaneously, then basis
Program (Novagen User Protocol TB055Rev.C 0611JN) in Novagen company method handbook, implantation
PET28a (+) plasmid (pET System Novagen Manual 11th Edition, User Protocol TB055Rev.C
0611JN), above-mentioned TNF α analog mutant protein can be expressed.Related above-mentioned TNF α albumen analog is in e. coli bl21
(DE3), BL21 (DE3) sS agar plates culture, sterile LB medium amplification, breaks bacterium, supernatant collection, the journeys such as protein purification
Sequence, according to the method handbook of Novagen company, such as above-described embodiment 1, according to Standard Operations Manual mode culture.
TNF α mutated gene amino acid sequence
TNFαA21K
MHHHHHHVRS SSRTPSDKPV KHVVANPQAE GQLQWLNRRA NALLANGVEL
RDNQLVVPSE GLYLIYSQVL FKGQGCPSTH VLLTHTISRI AVSYQTKVNL
LSAIKSPCQR ETPEGAEAKP WYEPIYLGGV FQLEKGDRLS AEINRPDYLD
FAESGQVYFG IIAL
SEQ ID NO:2
TNFαG31K
MHHHHHHVRS SSRTPSDKPV AHVVANPQAE KQLQWLNRRA NALLANGVEL
RDNQLVVPSE GLYLIYSQVL FKGQGCPSTH VLLTHTISRI AVSYQTKVNL
LSAIKSPCQR ETPEGAEAKP WYEPIYLGGV FQLEKGDRLS AEINRPDYLD
FAESGQVYFG IIAL
SEQ ID NO:3
TNFαA21KG31K
MHHHHHHVRS SSRTPSDKPV KHVVANPQAE KQLQWLNRRA NALLANGVEL
RDNQLVVPSE GLYLIYSQVL FKGQGCPSTH VLLTHTISRI AVSYQTKVNL
LSAIKSPCQR ETPEGAEAKP WYEPIYLGGV FQLEKGDRLS AEINRPDYLD
FAESGQVYFG IIAL
SEQ ID NO:4
The purifying of 3 TNF α albumen of embodiment and its mutant
Supernatant after the broken bacterium of lysis buffer obtained in Examples 1 and 2 as above includes a large amount of water soluble expression
Albumen.By the supernatant after broken bacterium with 1.0 milliliters/per minute flow velocity slow transit through speed circulation nickel ion (Ni-SepharoseTM
6FastFlow) affinity column (GE Healthcare Bioscience AB, Sweden), by the indwelling of TNF α albumen in nickel ion column
On, solution then is eluted with special nickel ion column, 0.6M imidazoles histamine (imidazole) aqueous solution elutes, preliminary purification (>
95%) TNF α albumen.Thus obtained albumen is passed through into speed circulation anion (SPSepharoseTMFast Flow) resins exchange
Column (GE Healthcare Bioscience AB, Sweden), by albumen indwelling on this resin ion exchange column, is then used
The phosphate buffer (pH 8.5) of the NaCl containing 0.5M elutes, and obtains further highly purified (> 99.5%) TNF α albumen.
The mutation derived protein (TNF α A21K, TNF α G31K, TNF α A21KG31K) of TNF α in kind purifies.
4 TNF α protein concentration of embodiment, purity analysis (SDS- polyacrylamide gel electrophoresis (SDS-PAGE) albumen point
Analysis)
Protein concentration analysis method: the concentration of protein is measured using Bradford-Lowry method.This method is based on
The protein of various concentration in conjunction with dyestuff Coomassie brilliant blue data it is also different and establish.It is dense with known difference first
The amount of the standard protein institute combination dye of degree is foundation, draws standard curve.Then, it is pushed away by measuring the dyestuff binding capacity of albumen
Disconnected protein example concentration out.
Purity of protein analysis method: protein biology macromolecular, since molecular weight difference can be by under same current field condition
SDS- polyacrylamide gel electrophoresis (SDSPAGE) separates different molecular weight, different types of protein biology macromolecular.True
While determining foreign protein whether there is, the purity of agnoprotein matter and the size of protein molecular can also be determined.
Fig. 2 is the figure of SDS- polyacrylamide solidifying burnt electrophoretic determination TNF α albumen and TNF α SGPEG20 purity.It can by Fig. 2
Know, in embodiment 1, the supernatant after the broken bacterium of lysis buffer includes a large amount of water soluble expression albumen, by nickel ion parent
After column purification, a large amount of low-purity (> 95%) TNF α albumen can be obtained.Further spent ion exchange resin purifying can obtain
To the TNF α albumen of high-purity (> 99%).The resulting TNF α after the modification of methoxyl group PEG Succinimidyl glutarate
SGPEG20 molecular weight increases and is detained in high molecular weight region.
Each TNF α protein mutant (TNF α A21K, TNF α G31K, TNF α A21KG31K) is analyzed according to method as above
Purity, as a result obtained the TNF α protein mutant of high-purity.
The preparation of 5 Pegylation tumor necrosis factor α (TNF α SCMPEG5) of embodiment
SCM-PEG5000, SCM-PEG10000, SCM-PEG20000, SG-PEG5000, SG- in following embodiment
PEG10000 and SG-PEG20000 is purchased from Beijing Jian Kai scientific & technical corporation.
The sodium phosphate buffer for taking 1.0ml 0.1M pH 8.5, is preheated to 25 DEG C in water-bath.Take 2.5 micrograms by pure
TNF α albumen (embodiment 1) after change, is slowly added into sodium phosphate buffer, dissolves it sufficiently.Take 50.0 micromolars
Methoxyl group PEG succinimide carboxymethyl ester (the MethoxyPEG succinimidyl carboxymethyl of amount 5000
Ester, SCM-PEG5000) (the triumphant science and technology of Beijing key, Jenkem technology), it is rapidly added into above-mentioned solution,
And it suitably stirs and is completely dissolved activated polyethylene glycol in 30 seconds, while a large amount of bubbles to be avoided to generate.At this time TNF α with
Polyethylene glycol amount ratio is 1:20 (w/w).Then, reaction 30 minutes is carried out under 25 DEG C of stirrings, and it is sweet that 100.0 micrograms are then added
Propylhomoserin terminates reaction.After the reaction was completed, reaction solution merging is had to the centrifugation pipe device of the filter membrane device of molecular weight 50,000
It is interior, the remaining albumen for not completing reaction is filtered out with high speed rotation centrifugation (3000x G30 minute) and remaining unreacted is linearly gathered
Ethylene glycol obtains polyethylene glycol-tumor necrosis factor α conjugate (TNF α SCMPEG5) of high-purity.The filter of TNF α SCMPEG5
Liquid is sterile filtered on 0.2 μm of sterilised membrane filter, obtains finally sterile protein injection liquid drug, is placed in 4 DEG C of sealed, steriles and keeps away
Light saves.
The preparation of 6 Pegylation tumor necrosis factor α (TNF α SGPEG5) of embodiment
The sodium phosphate buffer for taking 1.0ml 0.1M pH 8.5, is preheated to 25 DEG C in water-bath.Take 2.5 micrograms by pure
The TNF α albumen for changing expression, is slowly added into sodium phosphate buffer, dissolves it sufficiently.Take 50.0 micromolar amounts 5,000
Methoxy poly (ethylene glycol) Succinimidyl glutarate (Methoxy PEG succinimidyl glutarate ester,
SG-PEG5000), it is rapidly added into above-mentioned solution, and suitably stirs and keep activated polyethylene glycol completely molten in 30 seconds
Solution, while a large amount of bubbles to be avoided to generate.TNF α and polyethylene glycol amount ratio are 1:20 (w/w) at this time.Then, it is stirred in 25 DEG C
Under carry out reaction 30 minutes, be then added 100.0 microgram glycine terminate reaction.After the reaction was completed, reaction solution is placed in and is had
In the centrifugation pipe device of the filter membrane device of standby molecular weight 50,000, residue is filtered out with high speed rotation centrifugation (3000x G30 minutes)
The albumen and remaining unreacted linear polyethylene glycol for not completing reaction, obtain polyethylene glycol-tumor necrosis factor α of high-purity
Conjugate (TNF α SGPEG5).The filtrate of TNF α SGPEG5 is sterile filtered on 0.2 μm of sterilised membrane filter, obtains finally sterile
Protein injection liquid drug is placed in 4 DEG C of sealed, steriles and is kept in dark place.
The preparation of 7 Pegylation tumor necrosis factor α (TNF α SCMPEG10) of embodiment
The sodium phosphate buffer for taking 1.0ml 0.1M pH 8.5, is preheated to 25 DEG C in water-bath.Take 2.5 micrograms by pure
TNF α albumen (embodiment 1) after change, is slowly added into sodium phosphate buffer, dissolves it sufficiently.Take 50.0 micromolars
Methoxyl group PEG succinimide carboxymethyl ester (the MethoxyPEG succinimidyl carboxymethyl of amount 10,000
Ester, SCM-PEG10000), it is rapidly added into above-mentioned solution, and suitably stir and make activated polyethylene glycol at 30 seconds
It is inside completely dissolved, while a large amount of bubbles to be avoided to generate.TNF α and polyethylene glycol amount ratio are 1:20 (w/w) at this time.Then, in
Reaction 30 minutes is carried out under 25 DEG C of stirrings, 100.0 microgram glycine are then added and terminate reaction.After the reaction was completed, it will react molten
In the centrifugation pipe device for the filter membrane device that liquid merging has molecular weight 50,000, (3000x G30 minutes) is centrifuged with high speed rotation
The remaining albumen for not completing reaction and remaining unreacted linear polyethylene glycol are filtered out, polyethylene glycol-tumour of high-purity is obtained
Necrosin & conjugate (TNF α SCMPEG10).The filtrate of TNF α SCMPEG10 carries out sterile mistake on 0.2 μm of sterilised membrane filter
Filter, obtains finally sterile protein injection liquid drug, is placed in 4 DEG C of sealed, steriles and is kept in dark place.
The preparation of 8 Pegylation tumor necrosis factor α (TNF α SGPEG10) of embodiment
The sodium phosphate buffer for taking 1.0ml 0.1M pH 8.5, is preheated to 25 DEG C in water-bath.Take 2.5 micrograms by pure
The TNF α albumen for changing expression, is slowly added into sodium phosphate buffer, dissolves it sufficiently.Take 50.0 micromolar amounts 10,000
Methoxy poly (ethylene glycol) Succinimidyl glutarate (Methoxy PEG succinimidyl glutarate ester,
SG-PEG10000), it is rapidly added into above-mentioned solution, and suitably stirs and keep activated polyethylene glycol complete in 30 seconds
Dissolution, while a large amount of bubbles to be avoided to generate.TNF α and polyethylene glycol amount ratio are 1:20 (w/w) at this time.Then, it is stirred in 25 DEG C
Reaction 30 minutes is carried out under mixing, and 100.0 microgram glycine are then added and terminate reaction.After the reaction was completed, reaction solution is placed in
In the centrifugation pipe device for having the filter membrane device of molecular weight 50,000, filtered out with high speed rotation centrifugation (3000x G30 minutes) surplus
The albumen of remaining unfinished reaction and remaining unreacted linear polyethylene glycol, obtain polyethylene glycol-tumor necrosis factor of high-purity
Sub- α conjugate (TNF α SGPEG10).The filtrate of TNF α SGPEG10 is sterile filtered on 0.2 μm of sterilised membrane filter, obtains most
Sterile protein injection liquid drug eventually, is placed in 4 DEG C of sealed, steriles and is kept in dark place.
The preparation of 9 Pegylation tumor necrosis factor α (TNF α SCMPEG20) of embodiment
The sodium phosphate buffer for taking 1.0ml 0.1M pH 8.5, is preheated to 25 DEG C in water-bath.Take 2.5 microgram purifying tables
The TNF α albumen reached, is slowly added into sodium phosphate buffer, dissolves it sufficiently.Take the first of 50.0 micromolar amounts 20,000
Oxygroup PEG succinimide carboxymethyl ester (Methoxy PEG succinimidyl carboxymethyl ester, SCM-
PEG20000), it is rapidly added into above-mentioned solution, and suitably stirs and keep activated polyethylene glycol completely molten in 30 seconds
Solution, while a large amount of bubbles to be avoided to generate.TNF α and polyethylene glycol amount ratio are 1:20 (w/w) at this time.Then, it is stirred in 25 DEG C
Under carry out reaction 30 minutes, be then added 100.0 microgram glycine terminate reaction.After the reaction was completed, reaction solution is placed in and is had
In the centrifugation pipe device of the filter membrane device of standby molecular weight 50,000, residue is filtered out with high speed rotation centrifugation (3000x G30 minutes)
The albumen and remaining unreacted linear polyethylene glycol for not completing reaction, obtain polyethylene glycol-tumor necrosis factor α of high-purity
Conjugate (TNF α SCMPEG20).The filtrate of TNF α SCMPEG20 is sterile filtered on 0.2 μm of sterilised membrane filter, obtains finally
Sterile protein injection liquid drug is placed in 4 DEG C of sealed, steriles and is kept in dark place.
The preparation of 10 Pegylation tumor necrosis factor α (TNF α SGPEG20) of embodiment
The sodium phosphate buffer for taking 1.0ml 0.1M pH 8.5, is preheated to 25 DEG C in water-bath.Take 2.5 micrograms by pure
The TNF α for changing expression, is slowly added into sodium phosphate buffer, dissolves it sufficiently.Take the first of 50.0 micromolar amounts 20,000
Oxygroup PEG Succinimidyl glutarate (Methoxy PEG succinimidyl glutarate ester, SG-
PEG20000), it is rapidly added into above-mentioned solution, and suitably stirs and keep activated polyethylene glycol completely molten in 30 seconds
Solution, while a large amount of bubbles to be avoided to generate.TNF α and polyethylene glycol amount ratio are 1:20 (w/w) at this time.Then, it is stirred in 25 DEG C
Under carry out reaction 30 minutes, be then added 100.0 microgram glycine terminate reaction.After the reaction was completed, reaction solution is placed in and is had
In the centrifugation pipe device of the filter membrane device of standby molecular weight 50,000, residue is filtered out with high speed rotation centrifugation (3000x G30 minutes)
The albumen and remaining unreacted linear polyethylene glycol for not completing reaction, obtain polyethylene glycol-tumor necrosis factor α of high-purity
Conjugate (TNF α SGPEG20).The filtrate of TNF α SGPEG20 is sterile filtered on 0.2 μm of sterilised membrane filter, obtain finally without
Mycoprotein injection drug is placed in 4 DEG C of sealed, steriles and is kept in dark place.
The amplification of 11 Pegylation tumor necrosis factor α (TNF α SGPEG20) preparation method of embodiment
The sodium phosphate buffer for taking 100ml 0.1M pH 8.5, is preheated to 30 DEG C in water-bath.Take 300 micrograms through overrunning
Circulate anion (SPSepharoseTMFastFlow) resin-column, it is highly purified and TNF α albumen, dilution be dissolved into
In sodium phosphate buffer.Take the methoxyl group PEG Succinimidyl glutarate (Methoxy of 7500.0 micromolar amounts 20,000
PEG succinimidyl glutarate ester, SG-PEG20000), it is rapidly added into above-mentioned reaction solution, and
It suitably stirs and is completely dissolved activated polyethylene glycol in 30 seconds, while a large amount of bubbles to be avoided to generate.TNF α and poly- at this time
Ethylene glycol amount ratio is 1:25 (w/w).Then, reaction 30 minutes is carried out under 30 DEG C of stirrings, and the sweet ammonia of 300.0 micrograms is then added
Acid terminates reaction.After the reaction was completed, reaction solution is passed through into the doughnut splitter ultrafiltration of tool 50,000 filter membrane of molecular weight
(Holofiber filtration C06-E050-10-S mPESSpectrum LABS.USA) it is concentrated into original
1/10 (10ml) of volume, and TNF α SGPEG20 is separated with unreacted polyethylene glycol, glycine.To the 10ml concentrate
In the sodium phosphate buffer of 0.1MpH7.5 is added again to 150ml, it is again that volume is dense by same doughnut splitter
It is reduced to 10ml.So same operation is repeated 9 times, so that the sodium phosphate buffer of 0.1MpH7.5 replaces polyethylene glycol anti-completely
The sodium phosphate buffer of 0.1M pH8.5 in answering filters out the remaining albumen for not completing reaction, filters out remaining stopped reaction
Glycine, and filter out remaining unreacted linear polyethylene glycol.After 10th concentration, the sodium phosphate of 50ml 0.1M pH7.5 is taken
Buffer solution for cleaning doughnut splitter and separating pipe.Gained filtrate is through 0.2 μm after cleaning solution merges with the concentrate of 15ml
Sterilised membrane filter be sterile filtered, obtain final product TNF α PEG20.Final product is packed as 1.0 milliliters of sterile amperes of sterilizing
Bottle, is placed in 4 DEG C of sealed, steriles and is kept in dark place.
The preparation of 12 TNF α A21KSGPEG20 of embodiment
The sodium phosphate buffer for taking 1.0ml 0.1M pH 8.5, is preheated to 25 DEG C in water-bath.Take 2.5 micrograms by pure
The TNF α A21K for changing expression, is slowly added into sodium phosphate buffer, dissolves it sufficiently.Take 50.0 micromolar amounts 20,000
Methoxyl group PEG Succinimidyl glutarate (Methoxy PEG succinimidyl glutarate ester, SG-
PEG20000), it is rapidly added into above-mentioned solution, and suitably stirs and keep activated polyethylene glycol completely molten in 30 seconds
Solution, while a large amount of bubbles to be avoided to generate.TNF α A21K and polyethylene glycol amount ratio are 1:20 (w/w) at this time.Then, in 25 DEG C
Reaction 30 minutes is carried out under stirring, and 100.0 microgram glycine are then added and terminate reaction.After the reaction was completed, reaction solution is set
In the centrifugation pipe device for entering to have the filter membrane device of molecular weight 50,000, filtered out with high speed rotation centrifugation (3000x G30 minutes)
Residue does not complete the albumen and remaining unreacted linear polyethylene glycol of reaction, obtains polyethylene glycol-neoplasm necrosis of high-purity
Factor alpha analog conjugate (TNF α A21KSGPEG20).The filtrate of TNF α A21KSGPEG20 is enterprising in 0.2 μm of sterilised membrane filter
Row is sterile filtered, and obtains finally sterile protein injection liquid drug, is placed in 4 DEG C of sealed, steriles and is kept in dark place.
The preparation of 13 TNF α G31KSGPEG20 of embodiment
The sodium phosphate buffer for taking 1.0ml 0.1M pH 8.5, is preheated to 25 DEG C in water-bath.Take 2.5 micrograms by pure
The TNF α G31K for changing expression, is slowly added into sodium phosphate buffer, dissolves it sufficiently.Take 50.0 micromolar amounts 20,000
Methoxyl group PEG Succinimidyl glutarate (Methoxy PEG succinimidyl glutarate ester, SG-
PEG20000), it is rapidly added into above-mentioned solution, and suitably stirs and keep activated polyethylene glycol completely molten in 30 seconds
Solution, while a large amount of bubbles to be avoided to generate.TNF α G31K and polyethylene glycol amount ratio are 1:20 (w/w) at this time.Then, in 25 DEG C
Reaction 30 minutes is carried out under stirring, and 100.0 microgram glycine are then added and terminate reaction.After the reaction was completed, reaction solution is set
In the centrifugation pipe device for entering to have the filter membrane device of molecular weight 50,000, filtered out with high speed rotation centrifugation (3000x G30 minutes)
Residue does not complete the albumen and remaining unreacted linear polyethylene glycol of reaction, obtains polyethylene glycol-neoplasm necrosis of high-purity
Factor alpha analog conjugate (TNF α G31KSGPEG20).The filtrate of TNF α G31KSGPEG20 is enterprising in 0.2 μm of sterilised membrane filter
Row is sterile filtered, and obtains finally sterile protein injection liquid drug, is placed in 4 DEG C of sealed, steriles and is kept in dark place.
The preparation of 14 TNF α A21KSCMPEG20 of embodiment
The sodium phosphate buffer for taking 1.0ml 0.1M pH 8.5, is preheated to 25 DEG C in water-bath.Take 2.5 microgram purifying tables
The TNF α A21K albumen reached, is slowly added into sodium phosphate buffer, dissolves it sufficiently.Take 50.0 micromolar amounts 20,000
Methoxyl group PEG succinimide carboxymethyl ester (Methoxy PEG succinimidyl carboxymethyl ester,
SCM-PEG20000), it is rapidly added into above-mentioned solution, and suitably stirs and keep activated polyethylene glycol complete in 30 seconds
Dissolution, while a large amount of bubbles to be avoided to generate.TNF α A21K and polyethylene glycol amount ratio are 1:20 (w/w) at this time.Then, in 25
DEG C stirring under carry out reaction 30 minutes, be then added 100.0 microgram glycine terminate reaction.After the reaction was completed, by reaction solution
It is placed in the centrifugation pipe device for the filter membrane device for having molecular weight 50,000, with high speed rotation centrifugation (3000x G30 minutes) filter
Except the remaining albumen for not completing reaction and remaining unreacted linear polyethylene glycol, the polyethylene glycol-tumour for obtaining high-purity is bad
Necrosis factor alpha analog conjugate (TNF α A21KSCMPEG20).Sterilised membrane filter of the filtrate of TNF α A21KSCMPEG20 at 0.2 μm
On be sterile filtered, obtain finally sterile protein injection liquid drug, be placed in 4 DEG C of sealed, steriles and be kept in dark place.
The preparation of 15 TNF α G31KSCMPEG20 of embodiment
The sodium phosphate buffer for taking 1.0ml 0.1M pH 8.5, is preheated to 25 DEG C in water-bath.Take 2.5 microgram purifying tables
The TNF α G31K albumen reached, is slowly added into sodium phosphate buffer, dissolves it sufficiently.Take 50.0 micromolar amounts 20,000
Methoxyl group PEG succinimide carboxymethyl ester (Methoxy PEG succinimidyl carboxymethyl ester,
SCM-PEG20000), it is rapidly added into above-mentioned solution, and suitably stirs and keep activated polyethylene glycol complete in 30 seconds
Dissolution, while a large amount of bubbles to be avoided to generate.TNF α G31K and polyethylene glycol amount ratio are 1:20 (w/w) at this time.Then, in 25
DEG C stirring under carry out reaction 30 minutes, be then added 100.0 microgram glycine terminate reaction.After the reaction was completed, by reaction solution
It is placed in the centrifugation pipe device for the filter membrane device for having molecular weight 50,000, with high speed rotation centrifugation (3000x G30 minutes) filter
Except the remaining albumen for not completing reaction and remaining unreacted linear polyethylene glycol, the polyethylene glycol-tumour for obtaining high-purity is bad
Necrosis factor alpha analog conjugate (TNF α G31KSCMPEG20).Sterilised membrane filter of the filtrate of TNF α G31KSCMPEG20 at 0.2 μm
On be sterile filtered, obtain finally sterile protein injection liquid drug, be placed in 4 DEG C of sealed, steriles and be kept in dark place.
The conjugate of the present invention of test example 1 analyzes mouse at the killing activity of fiber oncocyte L929
Mouse is purchased from Chinese Academy of Sciences's American Type Culture Collection committee cell bank, in vitro culture at fiber oncocyte L929
In the cell in vitro that DMEM (Dulbecco ' s Modified Eagle Medium) culture medium, additional 10% calf serum are prepared
Culture solution (abbreviation DMEM+10%FCS), incubation are grown on 37 DEG C, 5%CO2(Humphreys DT1, Wilson in incubator
MRCytokine.1999Oct；11(10):773-82.Modes of L929cell death induced by TNF-alpha
and other cytotoxic agents).L929 cell in logarithmic growth phase, with being counted after 1% trypsin digestion,
Adjustment cell concentration is 5x104The hole 100ul/ in 96 orifice plates, every group of 4 multiple holes is added in L929 cell by/ml, and every plate sets 6
Drug concentration group returns and is placed in 37 DEG C, 5%CO2In incubator.After culture 24 hours, with DMEM culture solution, successively by following concentration
The TNF α albumen that doubling dilution is made by embodiment 1: 0, (control wells), 0.001 μ g/mL, 0.01 μ g/mL, 0.1 μ g/mL, 0.3 μ
g/mL,1.0μg/ml,10.0μg/mL.96 orifice plates time are placed in incubator after drug is added, are persistently cultivated 72 hours.Next day,
96 orifice plates are taken out, according to following literature methods (Monoclonal antibody targeting of N-cadherin
inhibits prostate cancer growth,metastasis and castration resistance.Nature
Medicine, 2010,16 (12), 1414-1420) to 10 μ L CCK8 enzyme substrate solutions of every hole addition, and by culture plate 37
DEG C, 5%CO2It is cultivated 1 hour in constant incubator.Then, it is measured with microplate reader (TECANinfinite200PRO) in 450nm
The absorbance at place, then calculates, and draws curve and obtain IC50Data.
In addition, method as described above, measurement TNF α A21K, TNF α G31K, TNF α SGPEG5, TNF α SGPEG10,
TNFαSGPEG20、TNFαSCMPEG5、TNFαSCMPEG10、TNFαSCMPEG20、TNFαA21KSGPEG20、TNFα
G31KSGPEG20 is to mouse at the killing activity of fibroma target L929 cell L929.
Shown in the obtained following Tables 1 and 2 of Activity Results.
The conjugate of the present invention of table 1 is to mouse at the killing activity of fiber oncocyte L929
By upper table 1 it is found that the wild type TNF α albumen after highly purified has height for target test L929 cell
Cell-lethal activity, cancer cell in vitro killing activity IC50Value is in 25 ± 14ng/mL.By the work of polyethylene glycol chain length 20,000
The chemically modified coupling protein of polyethylene glycol ester still maintains high-intensitive L929 cell-lethal activity, TNF α
The IC of SGPEG2050Value is 45 ± 23ng/mL, sufficiently shows that the polyethylene glycol chemistry modification of long-chain will not be significantly
Reduce the tumour cell killing activity of TNF α.For versus wild type TNF α, with lysine replace the 21st alanine and with rely
Propylhomoserin replaces two cancer cell in vitro killing activities for purifying the derivative mutain of TNF α of the 31st glycine to decline respectively
2 times and 6 times, IC50Value is respectively 50.3ng/mL and 150ng/mL.By being activated with same polyethylene glycol chain length 20,000
After the modification of macrogol ester chemistry, the derivative mutain conjugate TNF α A21KSGPEG20 of the TNF α of two purifying and TNF α
The cancer cell in vitro killing activity of G31KSGPEG20 slightly declines, IC50Value is respectively 58 ± 23ng/mL and 450ng/mL.
Above-mentioned external activity experimental data is shown, is learned when the activated polyethylene glycol using polyethylene glycol chain length 20,000 is esterified
In the case where modification, the derivative mutain of the TNF α of wild type TNF α albumen and TNF α A21K, TNF α G31K two purifying is same
It can keep the high ira vitro killing activity to target cancer cell L929.TNF α-polyethylene glycol after the modification of polyethylene glycol chemistry
Conjugate TNF α SGPEG20 can reach highest tumour cell killing activity.
The conjugate of the present invention of the different polyethylene glycol chain lengths of table 2 is to mouse at the killing activity of fiber oncocyte L929
By upper table 2 it is found that polyethylene glycol chain length for mouse at fiber oncocyte L929 killing activity have it is decisive
Effect.TNF α-polyethylene glycol conjugation object when the chain length of SCM polyethylene glycol or SG polyethylene glycol is equal to 5,000, after modification
Lose nearly 50 times of cell-lethal activity.When the chain length of SCM polyethylene glycol or SG polyethylene glycol is equal to 10,000, poly- second two
The chemically modified TNF α of alcohol-polyethylene glycol conjugation object loses nearly 1.3-12 times of cell-lethal activity.It can be seen that best
The polyethylene glycol chain length of selection is 20,000, the poly- second of TNF α-in the modification of this chain length, after the modification of polyethylene glycol chemistry
Glycol conjugate has no reduction activity, and the mouse that can reach bottom line inactivation and topnotch is external at fiber oncocyte L929
Killing activity.
The conjugate of the present invention of test example 2 analyzes the killing activity of human malignant tumor cell
Target cell is pernicious human body black cancer A375 cell, hepatocellular carcinoma H22 cell, colon cancer born of the same parents HCT116 thin
Born of the same parents, non-small cell lung cancer A549 and breast cancer MDA-MB231 cell, are purchased from Chinese Academy of Sciences American Type Culture Collection committee member
It can cell bank.Each target cell, the DMEM+10%FCS body that in vitro culture is prepared in DMEM culture medium, additional 10% calf serum
Outer cell culture fluid, incubation are grown on 5%CO2, in 37 DEG C of constant incubator.Target cell in logarithmic growth phase is used
It is counted after 1% trypsin digestion, adjustment cell concentration is 5x104/ ml, 96 orifice plates of every 100 μ l of hole addition, every group of 4 multiple holes,
Every plate sets 6 drug concentration groups, returns and is placed in 37 DEG C, 5%CO2In incubator.After culture 24 hours, with DMEM culture solution, press
The following concentration TNF α albumen that successively doubling dilution is made by embodiment 1: 0 (control wells), 0.001 μ g/mL, 0.01 μ g/mL,
0.1μg/mL,0.3μg/mL,1.0μg/ml,10.0μg/mL.96 orifice plates time are placed in incubator after drug is added, continue to train
It supports 72 hours.Next day takes out 96 orifice plates, 10 μ L CCK8 enzyme substrate solutions is added to every hole, then by culture plate in 37 DEG C, 5%
CO2It is cultivated 1 hour in constant humidity incubator.Then, the suction at 450nm is measured with microplate reader (TECANinfinite200PRO)
Luminosity, and the method for such as following documents calculates, and draws curve, obtains IC50Data.
In addition, method as described above, measurement TNF α A21K, TNF α G31K, TNF α SCMPEG20 are to each target
The IC50 value of cell.
Measurement result is as shown in table 3 below.
Killing activity IC of the conjugate of the present invention of table 3 to malignant cell50Value spectrum
Shown in table 3 as above, lethal thin different degrees of to human malignant tumor cell display of TNF α, TNF α SCMPEG20
Cytoactive, but opposite TNF α, TNF α PEG20 for four kinds of malignant cell activity susceptibilitys than for mouse at fibroma
L929 cell is lower.IC50Value display human malignant colon cancer cell sensibility highest, can become clinical first object indication.
Anti-tumor activity is analyzed in 3 nude mice of test example
Establish model of nude mice bearing tumor
Mouse is cultivated into life at fiber oncocyte L929 (11) in the DMEM base culture solution of additional 10% fetal calf serum
It is long, reach in logarithmic growth phase.With counting after 1% trypsin digestion, adjustment cell concentration is 1.0 × 107/ ml, then
By 0.3mL cell suspending liquid (3.0 × 106Cell) vaccinal injection enter Balb/c nude mouse (purchased from Hunan Si Laike scape up to experiment
Company of Animals Ltd.) back.L929 target cell periodically uses vernier caliper measurement gross tumor volume, volume in nude mice tumor growth
Reach 100-150mm3Afterwards, it is used for active testing.Above-mentioned nude mice is divided into control or treatment group, every group 5, tail vein is infused respectively
PBS (solvent control group) and TNF α or TNF α SGPEG20 (administration group) are penetrated, is injected daily once, continuously with 0.3 μ g and 1.0 μ g
21 days (qdx 21) is administered, or is injected weekly once with 0.3 μ g and 1.0 μ g, successive administration 3 weeks (q7d x 3).After administration by
The toxic reaction situation of day entry animal measures weekly a gross tumor volume, measures nude mouse changes of weight twice.Meter record animal
Death condition, position is survived number of days in calculating, and makes changes of weight curve and animal survival curve (Kaplan-Meier
plot).Testing drug is calculated to median lethal dose (the 50%Lethal Dose, LD of nude mice with improvement karber's method50), study TNF
The activity of the kill malignant tumour of α, the anti-tumor effect of quantitative analysis TNF α and TNF α SGPEG20.
Obtained result is as shown in table 4 below.
Anti-tumor activity in 4 nude mice L929 tumor model of table
Shown in table 4 as above, TNF α was in this experimental animal model, with daily tail vein administration 0.3 μ g successive administration 21 days
In the case of, it is able to suppress 45%L929 tumor volume growth, is able to extend the tumor bearing nude mice time-to-live 176%.When TNF α is administered
Dosage improve to 1.0 μ g, continuous 21 days through under tail vein injection administrations, although being able to suppress 80% tumour L929 body
Product, but tumor bearing nude mice life time (- 4%) can not be extended, it is naked sufficiently to prove that high dose (1.0 μ g) TNF α can cause
Mouse general toxicity and the time-to-live for shortening tumor bearing nude mice.In this experimental animal model, with the administration of (7 days) tail vein weekly one
Secondary, 0.3 μ g every time, in the case of successive administration 3 weeks (in total three times, totally 21 days), TNF α is merely capable of inhibiting in 1.5% nude mouse
L929 tumor volume growth can not also extend the tumor bearing nude mice time-to-live (- 8%).In addition, quiet through tail with TNF α PEG20
Arteries and veins administration, (7 days) tail vein is administered once weekly, per injection be administered 0.3 μ g, successive administration 3 weeks (in total three times, totally 21 days)
In the case of, TNF α PEG20 is able to suppress L929 tumor volume growth in 48% nude mouse, while it is naked also can significantly to extend lotus knurl
The mouse time-to-live (218%).This weekly administration experimental data is sufficiently shown, under middle dosage (0.3 μ g) tail vein administrations,
TNF α PEG20 can be to avoid high dose (1.0 μ g//daily) injection TNF α for the toxicity in vivo of nude mice, and shows best
Inhibit tumour growth, extends the therapeutic effect of the time-to-live of tumor bearing nude mice.This weekly administration experimental result is shown, is passed through
Optimum choice chain length is the TNF α coupling protein of 20,000 activated polyethylene glycol modification, is able to extend efficacy time, and can
Achieve the purpose that the primary treatment malignant tumour of weekly administration.
The conjugate of the present invention of test example 4 reduces normal mouse toxicity in vivo
Experimental animal: normal Balb/c mouse is purchased from Hunan SJA Laboratory Animal Co. , Ltd
Mouse experiment grouping: TNF α, TNF α SCMPEG5, TNF α SCMPEG10, the dosage 0.2 of TNF α SCMPEG20,0.6,
2、6、20、30、40、50、60、80、120、160、200μg。
Experimental method: mouse vein is administered once, be then observed continuously and record the acute toxic symptoms of animal in 7 days,
Death condition.It is calculated with IBM SPSS Statistics19 acute according to groups of animals death toll using the Probit Return Law
Toxicity LD50Statistical software calculate acute toxicity LD50Value, calculates the median lethal that mouse vein gives TNF α and TNF α PEG
Measure (LD50) and 95% credibility interval (A simplified method to evaluate the acute toxicity of
ricin and ricinus agglutinin.Zhan J,Zhou P.Toxicology.2003Apr 15；186(1-2):
119-23)。
The In vivotoxicity result of conjugate of the present invention is as shown in table 5 below.
The In vivotoxicity result of the conjugate of the present invention of table 5
By upper table 5 it is found that TNF α by different chain length it is polyethyleneglycol modified after, acute toxicity LD50Value increases, acute
Toxicity decline.This phenomenon is similar at the fibroma cancer cell external killing activity of L929 with mouse.TNF α SCMPEG5's is external
Killing activity declines, and acute toxicity also declines about 238 times in the Mice Body of this albumen.The experimental animal of TNF α group upon administration when
It is dead, and the death time obvious postpone of TNF α SCMPEG20 administration group, dead successively in 7 days upon administration.Preferred TNF
LD of the α SCMPEG20 coupling protein compared with prototype TNF α501.9 times are increased, the acute toxicity decline 1.9 of display TNF α SCMPEG20
Times, increase therapeutic window, be conducive to repeatedly, intravenously administrable weekly, before there is exploitation to be long-term treatment malignancy disease drug
Scape.
Claims (10)
1. polyethylene glycol-tumor necrosis factor α analog conjugate, wherein the polyethylene glycol is the first of molecular weight 20,000
Oxygroup polyethylene glycol Succinimidyl glutarate, the tumor necrosis factor α analog are the A21K simple point mutation class of TNF α
Like object albumen, and its amino acid sequence is as shown in SEQ ID NO:2.
2. the preparation method of polyethylene glycol according to claim 1-tumor necrosis factor α analog conjugate, special
Sign is, includes the steps that the polyethylene glycol and the tumor necrosis factor α analog haptoreaction.
3. preparation method according to claim 2, wherein the tumor necrosis factor α analog and the polyethylene glycol
Amount ratio range be 1:20-1:30 by weight.
4. preparation method according to claim 2, which is characterized in that the pH scope control of the reaction is 8.5 to 9.5.
5. the preparation method according to claim 4, which is characterized in that the pH scope control of the reaction is 9.0 ± 0.1.
6. preparation method according to claim 2, which is characterized in that the temperature range of the reaction is controlled 25 to 37
℃。
7. preparation method according to claim 6, which is characterized in that the temperature range of the reaction is controlled 30 ± 0.1
℃。
8. a kind of pharmaceutical composition contains polyethylene glycol according to claim 1-tumor necrosis factor α analog
Conjugate and pharmaceutically acceptable carrier.
9. polyethylene glycol according to claim 1-tumor necrosis factor α analog conjugate or according to claim
Pharmaceutical composition described in 8 is preparing the purposes in the drug for preventing and/or treating tumour.
10. purposes according to claim 9, wherein the tumour is selected from lymph cancer, cellule type or non-cellule type
Lung cancer, prostate cancer, kidney, liver cancer, colorectal cancer, melanoma, breast cancer.
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Citations (2)
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|---|---|---|---|---|
| CN1243444A (en) * | 1997-01-15 | 2000-02-02 | 凤凰药理学公司 | Modified tumor necrosis factor |
| EP1354893A2 (en) * | 2002-03-25 | 2003-10-22 | Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo | Physiologically active complex |
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|---|---|---|---|---|
| CN1243444A (en) * | 1997-01-15 | 2000-02-02 | 凤凰药理学公司 | Modified tumor necrosis factor |
| EP1354893A2 (en) * | 2002-03-25 | 2003-10-22 | Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo | Physiologically active complex |
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Denomination of invention: Polyethylene glycol and tumor necrosis factor a Coupling compounds of or their analogues and their pharmaceutical uses Effective date of registration: 20230316 Granted publication date: 20190607 Pledgee: Guangdong Development Bank Co.,Ltd. Yueyang Branch Pledgor: YUEYANG XINHUA PHARMACEUTICAL CO.,LTD. Registration number: Y2023980035121 |