CN105294852A - Conjugate of polyethylene glycol and tumor necrosis factor alpha or analogue of polyethylene glycol and tumor necrosis factor alpha and medical application of conjugate - Google Patents
Conjugate of polyethylene glycol and tumor necrosis factor alpha or analogue of polyethylene glycol and tumor necrosis factor alpha and medical application of conjugate Download PDFInfo
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Abstract
The invention relates to conjugate of polyethylene glycol and a tumor necrosis factor alpha or analogue of the polyethylene glycol and the tumor necrosis factor alpha and medical application of the conjugate, in particular to conjugate of polyethylene glycol-tumor necrosis factor alpha or analogue of the polyethylene glycol-tumor necrosis factor alpha, a preparation method of the conjugate and application of the conjugate serving as a therapeutic agent in preventing or treating tumor or cancer. The conjugate of polyethylene glycol-tumor necrosis factor alpha or analogue of the polyethylene glycol-tumor necrosis factor alpha can lower acute toxicity of medicine, reduce the intravenous dosing frequency, reduce the dosage of applied medicine, increase the anti-tumor therapeutic effect, solve the problem that efficacy half-life of the tumor necrosis factor alpha protein is short through parenteral medicine application, achieve the target of preventing and treating tumor or cancer and thus has beneficial medical prospect.
Description
Technical field
The present invention relates to the conjugate of polyoxyethylene glycol-tumor necrosis factor alpha (TNF α) or polyoxyethylene glycol-tumor necrosis factor alpha analogue, and preparation method thereof, and it is as the purposes of medicine in prevention or treatment tumour or cancer.
Background technology
Tumor necrosis factor alpha (TNF α) is the multifunctional cytokine albumen that can cause malignant tumor cell apoptosis.TNF α is secreted by the mononuclear macrophage activated, it is the cytokine that anti-tumor activity found up to now is the strongest, it has Several Kinds of Malignancy cell significantly kills cell (tumoricidal) and inhibiting tumour cells (tumoristatic) effect (Carswell, the people such as EA, 1975, Anendotoxininducedserumfactorthatcausesnecrosisoftumors. Proc.Natl.Acad.Sci.USA72; 3666-3670).
TNF α found by American scientist in latter stage in the 1970's, TNF α is by combining with the acceptor (TNFR2) of a high molecular acceptor (TNFR1) and a lower molecular weight, conduct all stechiologies, individual physiology, pathology, and kill pharmacotoxicological effect (people such as AndrewsJS, 1990.Characterizationofthereceptorsfortumornecrosisfacto r (TNF) andlymphotoxin (LT) onhumanlymphocytes.J.Immunol.144 of malignant cell; The people such as 2582-2591.DembicZ, 1990.TwohumanTNFreceptorshavesimilarextracellular, butdistinctintracellular, domainsequences.Cytokine2; 231-237).The effect experiment data presentation of the clinical treatment delivered early stage melanochrome cancer and treatment soft tissue malignant sarcomas: TNF α is too short at people's circulation time in vivo, and blood circulation organ toxicity is too high, therefore it is for malignant melanoma, not good (the people such as BartlettDL of clinical efficacy of the parenteral injection form of soft tissue sarcoma, Aphase1trialofcontinuoushyperthermicperitonealperfusionw ithtumornecrosisfactorandCisplatininthetreatmentofperito nealcarcinomatosis, Cancer, 83:1251-1261.).But in a kind of clinical trial model of body local treatment malignant tumour, namely in limbs ex vivo perfusion model (isolatedperfusedlimbmodel), TNF α can pour into from the local intra-arterial of patient's single limb, melanoma on limbs or sarcoma focus can be exposed to the TNF α medicine of local intra-arterial injection in the long period, and the TNF α then in limbs can be directed to a pump receptacle from the reflux veins blood vessel of the other end.This limbs ex vivo perfusion model produces systemic organs's toxicity when applying to avoid TNF α to be back to systemic circulation system, show the superior clinical therapeutic efficacy (Christoforidis killing malignant cell, the people such as D, Isolatedlimbperfusion:distincttourniquetandtumornecrosis factoreffectsontheearlyhemodynamicresponse.2003.ArchSurg, 138:17-25.Eggermont, the people such as AM, 1996.Isolatedlimbperfusionwithhigh-dosetumornecrosisfact or-alphaincombinationwithinterferongammaandmephalanforno n-resectableextremitysofttissuesarcomas:amulticentertria l, J.Clin.Oncol, 14:2653-2665.).In the malignant tumour clinical trial in latter stage of this treatment body local, the TNF α medicine of regional perfusion can increase considerably tumor focus drug exposure levels, then medicine can be refluxed by vein blood vessel and import in a pump receptacle, thus reduces the general toxicity of the systemic circulation system of patient.Can find from these body locals treatment clinical laboratory data, TNF α is for the very surprising (Eggermont of result for the treatment of of the melanoma on limbs or soft tissue sarcoma cell, the people such as AM, 2003.Tumornecrosisfactor-basedisolatedlimbperfusionwithf orSoftTissueSarcomasandMelanoma:TenyearsofsuccessfulAnti-vascularTherapy.CurrOncolRep, 5:79-80.FrakerDL Deng people, BiologicaltherapywithTNF:systemicadministrationandisolat edlimbperfusion.1995.In:V.T.Devita, S.HellmanandS.A.RosenbergSA.Philadelphia:J.B.LippincottC o.pp295-328.VanEtten, B. people is waited, 2003.Fiftytumornecrosisfactor-basedisolatedlimbperfusion sforlimbsalvageinpatientsolderthan75yearswithlimb-threat eningsofttissuesarcomasandotherextremitytumors.Ann.SurgO ncol.10:32-37.).Clinical test results confirm TNF α latter stage malignant tumor patient people interior curative effect be characterized as focal tumor cell hemorrhagic necrosis, the complete incidence graph treatment rate of local tumor focus even can reach more than 90%.
For overcoming TNF α in the short shortcoming of people's circulation time in vivo, thus the general clinical treatment of malignancy disease can be applied more broadly in, recent domestic scholar is making great efforts research TNF α pharmaceutical carrier transfer system, i.e. polymer chemistry modifying method, so that the prolong drug transformation period, improve the targeting of medicine, and reduce the untoward reaction of medicine.The present inventor proposes, utilize polyethyleneglycol modified technology, solve parenterai administration TNF α protein drug transformation period short shortcoming, the prolong drug transformation period, thus can be used on human body, utilize parenterai administration mode fully to present and kill (tumoricidalactivity) malignant cell, but not suppress the specific bioactivity preventing (tumoristaticactivity) malignant cell growth.
Summary of the invention
In order to overcome the defect of prior art, the object of the present invention is to provide the conjugate of a kind of new polyoxyethylene glycol-tumor necrosis factor alpha or polyoxyethylene glycol-tumor necrosis factor alpha analogue.The present invention utilizes the activated polyethylene glycol of different polymerization methods or different polymeric chain length to carry out chemically modified to tumor necrosis factor alpha or its analogue, thus produces the medicine for preventing or treat various malignant tumour of a series of new chemical constitution and chemical structure.
Tumor necrosis factor alpha analogue of the present invention can be the mutain of TNF α, A21K, G31K simple point mutation TNF α albuminoid of preferred TNF α.
In the present invention's preferred embodiment, described polyoxyethylene glycol is linear polyethylene glycol, and the length range of described polyoxyethylene glycol linear chain is 5,000-20,000, be preferably 20,000.
In another preferred embodiment of the present invention, described polyoxyethylene glycol is the polyoxyethylene glycol of activation, the polyoxyethylene glycol of described activation is the methoxysuccinyl imines glutarate NHS ester (MethoxySuccinimidylGlutarateNHSester of polyoxyethylene glycol, or succinimide carboxymethyl NHS ester (SuccinimidylCarboxymethylNHSester, SCM-PEG) SG-PEG).
In a particularly preferred embodiment according to the invention, the conjugate of described polyoxyethylene glycol-tumor necrosis factor alpha or polyoxyethylene glycol-tumor necrosis factor alpha analogue 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 the preparation method of the conjugate of polyoxyethylene glycol-tumor necrosis factor alpha as above or polyoxyethylene glycol-tumor necrosis factor alpha analogue, it comprises the polyoxyethylene glycol of activation and TNF α or the catalytic step of TNF alpha analog.Wherein, described polyoxyethylene glycol is linear polyethylene glycol, and the length range of described polyoxyethylene glycol linear chain is 5,000-20,000, preferably 20,000.The polyoxyethylene glycol of described activation is preferably methoxysuccinyl imines glutarate NHS ester or the succinimide carboxymethyl NHS ester of polyoxyethylene glycol.
The amount ratio scope of described TNF α or TNF alpha analog and polyoxyethylene glycol is 1:20 – 1:30, preferred 1:20 by weight in a preferred embodiment in accordance with this invention.
In another preferred embodiment of the present invention, in above-mentioned preparation method, the pH scope control of described reaction, 8.5 to 9.5, is preferably 9.0 ± 0.1.The temperature range of described reaction controls at 25 to 37 DEG C, and preferred temperature controls at 30 ± 0.1 DEG C.
Polyoxyethylene glycol chemistry modification reaction
The reaction of polyoxyethylene glycol chemistry is the important control program in the present invention.The polyoxyethylene glycol of activation is 9.0 ± 0.1 by the pH value in reaction of control and optimize to the protein modified reaction of TNF α, reaction times controls be shorter than 30 minutes, the amount ratio of albumen and polyoxyethylene glycol is 1:20 – 1:30 (w:w), temperature of reaction controls at 25 to 37 DEG C, enable the Methionin of TNF α protein surface fully and activated polyethylene glycol react, to obtain the polyethyleneglycol modified coupled product of specified molecular weight.
As above, if employing different molecular weight, or the different activated polyethylene glycol of polymerization methods carries out chemically modified to biomacromolecule albumen, then can obtain polyoxyethylene glycol high purity, narrow unidirectional distribution (molecular weight distribution is between 1.01-1.05), molecular-weight average be 5, the polyoxyethylene glycol of 000 – 20,000 and the stable conjugate (TNF α PEG) of tumor necrosis factor alpha or its analogue.
After polyoxyethylene glycol chemistry has reacted, can with through molecular weight 50, the filter membrane device of 000, does not complete the albumen of chemical reaction by residue, and remain unreacted linear polyethylene glycol high speed centrifugation and be separated, the conjugate of highly purified polyoxyethylene glycol-albumen thing can be obtained.
This is a kind of biochemical reaction of gentleness, by forming ester bond or amido linkage and the conjugate obtained with acid anhydrides or carboxylic acid reaction, relatively stable under common physiological condition, can't rupture.
The invention further relates to a kind of pharmaceutical composition, it contains the conjugate of polyoxyethylene glycol-tumor necrosis factor alpha as above or polyoxyethylene glycol-TNF (Tumor Necrosis Factor) alpha analogue, and pharmaceutically acceptable carrier.
Those skilled in the art can understand, pharmaceutical composition of the present invention can be formulated into various dosage form well known in the art according to concrete method of application, such as oral 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, sprays, aerosol etc.), or injection formulations (solution, suspension agent, emulsion).Be preferably injection formulations within the scope of the present invention.
Pharmaceutically acceptable carrier, adjuvant or thinner can be comprised, such as: weighting agent, disintegrating agent, lubricant, suspending agent, tackiness agent, sweeting agent, correctives, sanitas, matrix etc. according to pharmaceutical composition of the present invention.Weighting agent is such as: starch, pregelatinized Starch, lactose, N.F,USP MANNITOL, chitin, Microcrystalline Cellulose, sucrose etc.; Disintegrating agent is such as: starch, pregelatinized Starch, Microcrystalline Cellulose, sodium starch glycolate, crosslinked polyethylene pyrroles, low-substituted hydroxypropyl cellulose, croscarmellose sodium etc.; Lubricant is such as: Magnesium Stearate, sodium lauryl sulphate, talcum powder, silicon-dioxide etc.; Suspending agent is such as: polyvinylpyrrolidone, Microcrystalline Cellulose, sucrose, agar, Vltra tears etc.; Tackiness agent such as, starch slurry, polyvinylpyrrolidone, Vltra tears etc.Composition of the present invention can be made by utilizing any currently known methods in this area, can provide activeconstituents that is quick, lasting or slow releasing after making patient's medication.
The conjugate that the invention further relates to polyoxyethylene glycol-tumor necrosis factor alpha as above or polyoxyethylene glycol-tumor necrosis factor alpha analogue or the pharmaceutical composition comprising it are for the preparation of the purposes prevented and/or treated in the medicine of tumour or cancer.Wherein said tumour or cancer can be lymphatic cancer, cellule type or lung cancer in non-cellule type, prostate cancer, kidney, liver cancer, cancer of colon, melanoma, mammary cancer.
The dosage of inventive compound can be different from the difference of the severity of the situation of individuality and weight, the state of an illness, medicament forms, route of administration and dosage period, and it also can be selected by those skilled in the art.Dosage can be single-dose or the every day of administration several times every day.The clinical active drug dosage of the tumor necrosis factor alpha that the present invention is polyethyleneglycol modified is at least 10 to 3000ng/m
2/ 7-14 days.
Pharmaceutical composition of the present invention is administered to individual animals by all means as Mammals (rat, mouse, domesticate animals or the mankind), all administering modes are all expections, such as, administration can be subcutaneous, intracutaneous, intermembranous, suppository and aerosol form of medication.
In the present invention, term polyoxyethylene glycol-TNF α conjugate is a general proper noun, is typically expressed as " (TNF α PEG) ".It represents the polyoxyethylene glycol chemistry reaction by the special groups of TNF α and activation, connects and the polymer of formation through covalent linkage.It comprises all polyoxyethylene glycol-TNF α conjugates of the present invention and polyoxyethylene glycol-TNF alpha analog conjugate.Such as, TNF α SGPEG5, TNF α SGPEG10, TNF α SGPEG20, TNF α SCMPEG5, TNF α SCM10PEG, TNF α SCM20PEG can be comprised.
Tumor necrosis factor alpha (TNF α) described in the present invention, also contains the similar gene protein of the single-point of this protein molecular gene-correlation, double mutant and multipoint mutation.
Being meant to of " Pegylation tumour necrosis factor (albumen) " of the present invention, " polyethyleneglycol modified tumour necrosis factor (albumen) " is equal to, and it is equal to the conjugate of polyoxyethylene glycol-tumor necrosis factor alpha conjugate or polyoxyethylene glycol-tumor necrosis factor alpha analogue.
The difference of polymeric unit, polymerization methods pressed by polyoxyethylene glycol described in the present invention, is divided into straight chain and branched chain type; The polyoxyethylene glycol of different molecular weight size can be marked off, as PEG5000, PEG10000, PEG20000 etc. by the difference of the polymerization degree.
The present invention can according to by the macromolecular feature of modified biological, the surface amino groups acid of the polyoxyethylene glycol of chemical reaction, employing unimodal molecular weight or a different molecular weight to TNF α and tumor necrosis factor alpha mutation expression protein derivatives (TNF α mutin) is utilized to carry out different modes and modification in various degree, thus extend the biological activity transformation period of this medicine, increase water-soluble, and reduce the toxic side effect of biomacromolecule, under parenterai administration condition, obtain the long-acting treatment effect of expection.
Below will by the present invention of specific embodiments and the drawings circumstantial letter.
Accompanying drawing explanation
Fig. 1 is the figure of the structure of display pET28a (+) expression plasmid.
Fig. 2 is the figure that SDS-polyacrylamide coagulates burnt cataphoretic determination TNF α albumen and TNF α SGPEG20 purity.
Embodiment
Further illustrate the present invention below in conjunction with embodiment, but these embodiments are only used for the present invention is described, and the scope do not limited the present invention in any way.
The genetic expression of embodiment 1TNF α albumen
Use full chemical synthesis process, following TNF α gene is compiled into the special expression of amino acids password (Pennica of intestinal bacteria, D.Nedwin, G.E., Hayflick, J.S., atal, Nature, 1984, 312 (20/27), 724-729, Humantumournecrosisfactor:precursorstructure, expressionandhomologytolymphotoxin) expression plasmid pET28a (+) (pETSystemNovagenManual11thEdition is inserted, UserProtocolTB055Rev.C0611JN) in, then according to the conventional procedure (NovagenUserProtocolTB055Rev.C0611JN) in Novagen company present method handbook, plasmid is penetrated e. coli bl21 (DE3), in BL21 (DE3) sS (NovagenpETSystemManual11thEdition), then single bacterium colony is selected from by the colibacillary agar plates of above-mentioned expression plasmid, insert in aseptic LB (Luria-Bertani) substratum of 5mL (purchased from Chemical Reagent Co., Ltd., Sinopharm Group), in 37 DEG C, cultivate according to Standard Operations Manual mode.Then the substratum of 5 liters is progressively amplified to according to 1% ratio, to strain density OD
260during for 0.6-0.8, be cooled to 18 DEG C, add certain density isopropyl-β-D-thiogalactoside(IPTG) (IPTG) and induce TNF α protein expression 16 hours.The 5000rpm centrifugation of intestinal bacteria body is collected, and then uses precooling (4 DEG C) lysis buffer, broken bacterium in Ultrasonic Cell Disruptor.The plasmid construct that pET28a (+) expresses is shown in Fig. 1.
TNF α gene amino acid sequence
MHHHHHHVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVEL
RDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNL
LSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLD
FAESGQVYFGIIAL
SEQIDNO：1
The genetic expression of embodiment 2TNF α albumen analogue (mutant)
Use full chemical synthesis process, following TNF α A21K, TNF α G31K, TNF α A21KG31K gene are compiled into the special expression of amino acids coding of intestinal bacteria, insert among expression plasmid pET28a (+).When full chemosynthesis TNF α genes encoding: 1) by the acid propyl password of the 21st sequence, make Methionin password into, 2) the 31st glycine password is changed into Methionin password, 3) by the acid propyl password of the 21st sequence, 31st glycine password makes Methionin password into simultaneously, then according to the program (NovagenUserProtocolTB055Rev.C0611JN) in Novagen company method handbook, implant pET28a (+) plasmid (pETSystemNovagenManual11thEdition, UserProtocolTB055Rev.C0611JN), above-mentioned TNF alpha analog mutant protein can be expressed.Relevant above-mentioned TNF α albumen analogue is cultivated at e. coli bl21 (DE3), BL21 (DE3) sS agar plates, sterile LB medium amplifies, broken bacterium, supernatant collection, protein purification supervisor, all according to the method handbook of Novagen company, as above-described embodiment 1, cultivate according to Standard Operations Manual mode.
TNF α mutator gene aminoacid sequence
TNFαA21K
MHHHHHHVRSSSRTPSDKPVKHVVANPQAEGQLQWLNRRANALLANGVEL
RDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNL
LSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLD
FAESGQVYFGIIAL
SEQIDNO：2
TNFαG31K
MHHHHHHVRSSSRTPSDKPVAHVVANPQAEKQLQWLNRRANALLANGVEL
RDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNL
LSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLD
FAESGQVYFGIIAL
SEQIDNO：3
TNFαA21KG31K
MHHHHHHVRSSSRTPSDKPVKHVVANPQAEKQLQWLNRRANALLANGVEL
RDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNL
LSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLD
FAESGQVYFGIIAL
SEQIDNO：4
The purifying of embodiment 3TNF α albumen and mutant thereof
As above the supernatant liquor after the lysis buffer obtained in embodiment 1 and 2 breaks bacterium contains a large amount of water soluble expression albumen.Supernatant liquor after broken bacterium is slow transitted through speed circulation nickel ion (Ni-Sepharose with 1.0 milliliters/per minute flow velocity
tM6FastFlow) affinity column (GEHealthcareBioscienceAB, Sweden), by the indwelling of TNF α albumen on nickel ion post, then with special nickel ion post elute soln, 0.6M imidazoles histamine (imidazole) aqueous solution wash-out, (>95%) TNF α albumen of preliminary purification.By the albumen obtained thus by the fast negatively charged ion (SPSepharose that circulates
tMfastFlow) resin-column (GEHealthcareBioscienceAB, Sweden), by albumen indwelling on this resin ion exchange column, then with phosphate buffered saline buffer (pH8.5) wash-out containing 0.5MNaCl, highly purified (>99.5%) TNF α albumen is further obtained.Sudden change derived protein (TNF α A21K, TNF α G31K, TNF α A21KG31K) the in kind purifying of TNF α.
Embodiment 4TNF α protein concentration, purity check (SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of protein)
Protein concentration analytical procedure: adopt Bradford-Lowry method to measure the concentration of protein.The data of the dyestuff Xylene Brilliant Cyanine G that the method combines based on the protein of different concns are also different and set up.First with the amount of the standard protein of known different concns institute combination dye for foundation, draw typical curve.Then, the dyestuff binding capacity by measuring albumen infers this protein example concentration.
Purity of protein analytical procedure: protein biology macromole, because molecular weight is different, under same current field condition, can by SDS-polyacrylamide gel electrophoresis (SDSPAGE) by different molecular weight, different types of protein biology macromole separately.While determining whether foreign protein exists, also can determine the purity of agnoprotein matter and the size of protein molecular.
Fig. 2 is the figure that SDS-polyacrylamide coagulates burnt cataphoretic determination TNF α albumen and TNF α SGPEG20 purity.As shown in Figure 2, in embodiment 1, the supernatant liquor after lysis buffer breaks bacterium contains a large amount of water soluble expression albumen, after nickel ion affinity column purifying, can obtain a large amount of low-purity (>95%) TNF α albumen.Spent ion exchange resin purifying can obtain the TNF α albumen of high purity (>99%) further again.After methoxyl group PEG Succinimidyl glutarate is modified, the TNF α SGPEG20 molecular weight of gained increases and is detained in high molecular weight region.
The purity of each TNF α protein mutant (TNF α A21K, TNF α G31K, TNF α A21KG31K) according to as above methods analyst, result all obtains highly purified TNF α protein mutant.
The preparation of embodiment 5 Pegylation tumor necrosis factor alpha (TNF α SCMPEG5)
SCM-PEG5000, SCM-PEG10000, SCM-PEG20000, SG-PEG5000, SG-PEG10000 and SG-PEG20000 in following examples are all purchased from Beijing Jian Kai scientific & technical corporation.
Get the sodium phosphate buffer of 1.0ml0.1MpH8.5, in water-bath, be preheated to 25 DEG C.Get the TNF α albumen (embodiment 1) of 2.5 micrograms after purifying, be slowly added in sodium phosphate buffer, make it fully dissolve.Get the methoxyl group PEG succinimide carboxymethyl ester (MethoxyPEGsuccinimidylcarboxymethylester of 50.0 micromolar amounts 5000, SCM-PEG5000) (the triumphant science and technology of Beijing key, Jenkemtechnology), it is added to rapidly in above-mentioned solution, and agitation as appropriate and activated polyethylene glycol was dissolved completely in 30 seconds, a large amount of bubble will be avoided to produce simultaneously.Now TNF α and polyoxyethylene glycol amount ratio are 1:20 (w/w).Then, carry out reaction 30 minutes under stirring in 25 DEG C, then add 100.0 microgram glycine termination reactions.After having reacted, reaction soln is inserted and possesses molecular weight 50, in the centrifuge tube device of the filter membrane device of 000, do not complete albumen and the unreacted linear polyethylene glycol of residue of reaction by high speed rotating centrifugal (3000xG30 minute) filtering residue, obtain highly purified polyoxyethylene glycol-tumor necrosis factor alpha conjugate (TNF α SCMPEG5).The filtrate of TNF α SCMPEG5 carries out sterile filtration on the sterilised membrane filter of 0.2 μm, obtains final aseptic protein injection liquid medicine, is placed in 4 DEG C of sealed, steriles and keeps in Dark Place.
The preparation of embodiment 6 Pegylation tumor necrosis factor alpha (TNF α SGPEG5)
Get the sodium phosphate buffer of 1.0ml0.1MpH8.5, in water-bath, be preheated to 25 DEG C.Get the TNF α albumen that 2.5 micrograms are expressed through purifying, be slowly added in sodium phosphate buffer, make it fully dissolve.Get 50.0 micromolar amounts 5, methoxy poly (ethylene glycol) Succinimidyl glutarate (the MethoxyPEGsuccinimidylglutarateester of 000, SG-PEG5000), it is added to rapidly in above-mentioned solution, and agitation as appropriate and activated polyethylene glycol was dissolved completely in 30 seconds, a large amount of bubble will be avoided to produce simultaneously.Now TNF α and polyoxyethylene glycol amount ratio are 1:20 (w/w).Then, carry out reaction 30 minutes under stirring in 25 DEG C, then add 100.0 microgram glycine termination reactions.After having reacted, reaction soln is inserted and possesses molecular weight 50, in the centrifuge tube device of the filter membrane device of 000, do not complete albumen and the unreacted linear polyethylene glycol of residue of reaction by high speed rotating centrifugal (3000xG30 minute) filtering residue, obtain highly purified polyoxyethylene glycol-tumor necrosis factor alpha conjugate (TNF α SGPEG5).The filtrate of TNF α SGPEG5 carries out sterile filtration on the sterilised membrane filter of 0.2 μm, obtains final aseptic protein injection liquid medicine, is placed in 4 DEG C of sealed, steriles and keeps in Dark Place.
The preparation of embodiment 7 Pegylation tumor necrosis factor alpha (TNF α SCMPEG10)
Get the sodium phosphate buffer of 1.0ml0.1MpH8.5, in water-bath, be preheated to 25 DEG C.Get the TNF α albumen (embodiment 1) of 2.5 micrograms after purifying, be slowly added in sodium phosphate buffer, make it fully dissolve.Get 50.0 micromolar amounts 10, the methoxyl group PEG succinimide carboxymethyl ester (MethoxyPEGsuccinimidylcarboxymethylester of 000, SCM-PEG10000), it is added to rapidly in above-mentioned solution, and agitation as appropriate and activated polyethylene glycol was dissolved completely in 30 seconds, a large amount of bubble will be avoided to produce simultaneously.Now TNF α and polyoxyethylene glycol amount ratio are 1:20 (w/w).Then, carry out reaction 30 minutes under stirring in 25 DEG C, then add 100.0 microgram glycine termination reactions.After having reacted, reaction soln is inserted and possesses molecular weight 50, in the centrifuge tube device of the filter membrane device of 000, do not complete albumen and the unreacted linear polyethylene glycol of residue of reaction by high speed rotating centrifugal (3000xG30 minute) filtering residue, obtain highly purified polyoxyethylene glycol-tumor necrosis factor alpha conjugate (TNF α SCMPEG10).The filtrate of TNF α SCMPEG10 carries out sterile filtration on the sterilised membrane filter of 0.2 μm, obtains final aseptic protein injection liquid medicine, is placed in 4 DEG C of sealed, steriles and keeps in Dark Place.
The preparation of embodiment 8 Pegylation tumor necrosis factor alpha (TNF α SGPEG10)
Get the sodium phosphate buffer of 1.0ml0.1MpH8.5, in water-bath, be preheated to 25 DEG C.Get the TNF α albumen that 2.5 micrograms are expressed through purifying, be slowly added in sodium phosphate buffer, make it fully dissolve.Get 50.0 micromolar amounts 10, methoxy poly (ethylene glycol) Succinimidyl glutarate (the MethoxyPEGsuccinimidylglutarateester of 000, SG-PEG10000), it is added to rapidly in above-mentioned solution, and agitation as appropriate and activated polyethylene glycol was dissolved completely in 30 seconds, a large amount of bubble will be avoided to produce simultaneously.Now TNF α and polyoxyethylene glycol amount ratio are 1:20 (w/w).Then, carry out reaction 30 minutes under stirring in 25 DEG C, then add 100.0 microgram glycine termination reactions.After having reacted, reaction soln is inserted and possesses molecular weight 50, in the centrifuge tube device of the filter membrane device of 000, do not complete albumen and the unreacted linear polyethylene glycol of residue of reaction by high speed rotating centrifugal (3000xG30 minute) filtering residue, obtain highly purified polyoxyethylene glycol-tumor necrosis factor alpha conjugate (TNF α SGPEG10).The filtrate of TNF α SGPEG10 carries out sterile filtration on the sterilised membrane filter of 0.2 μm, obtains final aseptic protein injection liquid medicine, is placed in 4 DEG C of sealed, steriles and keeps in Dark Place.
The preparation of embodiment 9 Pegylation tumor necrosis factor alpha (TNF α SCMPEG20)
Get the sodium phosphate buffer of 1.0ml0.1MpH8.5, in water-bath, be preheated to 25 DEG C.Get the TNF α albumen that 2.5 microgram purifying are expressed, be slowly added in sodium phosphate buffer, make it fully dissolve.Get 50.0 micromolar amounts 20, the methoxyl group PEG succinimide carboxymethyl ester (MethoxyPEGsuccinimidylcarboxymethylester of 000, SCM-PEG20000), it is added to rapidly in above-mentioned solution, and agitation as appropriate and activated polyethylene glycol was dissolved completely in 30 seconds, a large amount of bubble will be avoided to produce simultaneously.Now TNF α and polyoxyethylene glycol amount ratio are 1:20 (w/w).Then, carry out reaction 30 minutes under stirring in 25 DEG C, then add 100.0 microgram glycine termination reactions.After having reacted, reaction soln is inserted and possesses molecular weight 50, in the centrifuge tube device of the filter membrane device of 000, do not complete albumen and the unreacted linear polyethylene glycol of residue of reaction by high speed rotating centrifugal (3000xG30 minute) filtering residue, obtain highly purified polyoxyethylene glycol-tumor necrosis factor alpha conjugate (TNF α SCMPEG20).The filtrate of TNF α SCMPEG20 carries out sterile filtration on the sterilised membrane filter of 0.2 μm, obtains final aseptic protein injection liquid medicine, is placed in 4 DEG C of sealed, steriles and keeps in Dark Place.
The preparation of embodiment 10 Pegylation tumor necrosis factor alpha (TNF α SGPEG20)
Get the sodium phosphate buffer of 1.0ml0.1MpH8.5, in water-bath, be preheated to 25 DEG C.Get the TNF α that 2.5 micrograms are expressed through purifying, be slowly added in sodium phosphate buffer, make it fully dissolve.Get 50.0 micromolar amounts 20, the methoxyl group PEG Succinimidyl glutarate (MethoxyPEGsuccinimidylglutarateester of 000, SG-PEG20000), it is added to rapidly in above-mentioned solution, and agitation as appropriate and activated polyethylene glycol was dissolved completely in 30 seconds, a large amount of bubble will be avoided to produce simultaneously.Now TNF α and polyoxyethylene glycol amount ratio are 1:20 (w/w).Then, carry out reaction 30 minutes under stirring in 25 DEG C, then add 100.0 microgram glycine termination reactions.After having reacted, reaction soln is inserted and possesses molecular weight 50, in the centrifuge tube device of the filter membrane device of 000, do not complete albumen and the unreacted linear polyethylene glycol of residue of reaction by high speed rotating centrifugal (3000xG30 minute) filtering residue, obtain highly purified polyoxyethylene glycol-tumor necrosis factor alpha conjugate (TNF α SGPEG20).The filtrate of TNF α SGPEG20 carries out sterile filtration on the sterilised membrane filter of 0.2 μm, obtains final aseptic protein injection liquid medicine, is placed in 4 DEG C of sealed, steriles and keeps in Dark Place.
Embodiment 11 Pegylation tumor necrosis factor alpha (TNF α SGPEG20) preparation method amplifies
Get the sodium phosphate buffer of 100ml0.1MpH8.5, in water-bath, be preheated to 30 DEG C.Get 300 micrograms through the circulation negatively charged ion (SPSepharose that overruns
tMfastFlow) resin-column, highly purified and TNF α albumen, dilution be dissolved in sodium phosphate buffer.Get 7500.0 micromolar amounts 20, the methoxyl group PEG Succinimidyl glutarate (MethoxyPEGsuccinimidylglutarateester of 000, SG-PEG20000), it is added to rapidly in above-mentioned reaction soln, and agitation as appropriate and activated polyethylene glycol was dissolved completely in 30 seconds, a large amount of bubble will be avoided to produce simultaneously.Now TNF α and polyoxyethylene glycol amount ratio are 1:25 (w/w).Then, carry out reaction 30 minutes under stirring in 30 DEG C, then add 300.0 microgram glycine termination reactions.After having reacted, by the tubular fibre separator column ultrafiltration (HolofiberfiltrationC06-E050-10-SmPES of reaction soln by tool molecular weight 50,000 filtering membrane
spectrumLABS.USA) be concentrated into 1/10 (10ml) of original volume, and TNF α SGPEG20 is separated with unreacted polyoxyethylene glycol, glycine.In this 10ml concentrated solution, again add the sodium phosphate buffer of 0.1MpH7.5 to 150ml, by same tubular fibre separator column, again by volume concentration to 10ml.So same operation repeats 9 times, the sodium phosphate buffer of 0.1MpH7.5 is made to replace the sodium phosphate buffer of the 0.1MpH8.5 in polyoxyethylene glycol reaction completely, filtering residue does not complete the albumen of reaction, the glycine of filtering residue stopped reaction, and filtering remains unreacted linear polyethylene glycol.After concentrating for 10th time, get sodium phosphate buffer cleaning tubular fibre separator column and the separator tube of 50ml0.1MpH7.5.After the concentrated solution of scavenging solution and 15ml merges, gained filtrate carries out sterile filtration through the sterilised membrane filter of 0.2 μm, obtains final product TNF α PEG20.Final product is packed as 1.0 milliliters of aseptic ampere bottles of sterilizing, and is placed in 4 DEG C of sealed, steriles and keeps in Dark Place.
The preparation of embodiment 12TNF α A21KSGPEG20
Get the sodium phosphate buffer of 1.0ml0.1MpH8.5, in water-bath, be preheated to 25 DEG C.Get the TNF α A21K that 2.5 micrograms are expressed through purifying, be slowly added in sodium phosphate buffer, make it fully dissolve.Get 50.0 micromolar amounts 20, the methoxyl group PEG Succinimidyl glutarate (MethoxyPEGsuccinimidylglutarateester of 000, SG-PEG20000), it is added to rapidly in above-mentioned solution, and agitation as appropriate and activated polyethylene glycol was dissolved completely in 30 seconds, a large amount of bubble will be avoided to produce simultaneously.Now TNF α A21K and polyoxyethylene glycol amount ratio are 1:20 (w/w).Then, carry out reaction 30 minutes under stirring in 25 DEG C, then add 100.0 microgram glycine termination reactions.After having reacted, reaction soln is inserted and possesses molecular weight 50, in the centrifuge tube device of the filter membrane device of 000, do not complete albumen and the unreacted linear polyethylene glycol of residue of reaction by high speed rotating centrifugal (3000xG30 minute) filtering residue, obtain highly purified polyoxyethylene glycol-tumor necrosis factor alpha analogue conjugate (TNF α A21KSGPEG20).The filtrate of TNF α A21KSGPEG20 carries out sterile filtration on the sterilised membrane filter of 0.2 μm, obtains final aseptic protein injection liquid medicine, is placed in 4 DEG C of sealed, steriles and keeps in Dark Place.
The preparation of embodiment 13TNF α G31KSGPEG20
Get the sodium phosphate buffer of 1.0ml0.1MpH8.5, in water-bath, be preheated to 25 DEG C.Get the TNF α G31K that 2.5 micrograms are expressed through purifying, be slowly added in sodium phosphate buffer, make it fully dissolve.Get 50.0 micromolar amounts 20, the methoxyl group PEG Succinimidyl glutarate (MethoxyPEGsuccinimidylglutarateester of 000, SG-PEG20000), it is added to rapidly in above-mentioned solution, and agitation as appropriate and activated polyethylene glycol was dissolved completely in 30 seconds, a large amount of bubble will be avoided to produce simultaneously.Now TNF α G31K and polyoxyethylene glycol amount ratio are 1:20 (w/w).Then, carry out reaction 30 minutes under stirring in 25 DEG C, then add 100.0 microgram glycine termination reactions.After having reacted, reaction soln is inserted and possesses molecular weight 50, in the centrifuge tube device of the filter membrane device of 000, do not complete albumen and the unreacted linear polyethylene glycol of residue of reaction by high speed rotating centrifugal (3000xG30 minute) filtering residue, obtain highly purified polyoxyethylene glycol-tumor necrosis factor alpha analogue conjugate (TNF α G31KSGPEG20).The filtrate of TNF α G31KSGPEG20 carries out sterile filtration on the sterilised membrane filter of 0.2 μm, obtains final aseptic protein injection liquid medicine, is placed in 4 DEG C of sealed, steriles and keeps in Dark Place.
The preparation of embodiment 14TNF α A21KSCMPEG20
Get the sodium phosphate buffer of 1.0ml0.1MpH8.5, in water-bath, be preheated to 25 DEG C.Get the TNF α A21K albumen that 2.5 microgram purifying are expressed, be slowly added in sodium phosphate buffer, make it fully dissolve.Get 50.0 micromolar amounts 20, the methoxyl group PEG succinimide carboxymethyl ester (MethoxyPEGsuccinimidylcarboxymethylester of 000, SCM-PEG20000), it is added to rapidly in above-mentioned solution, and agitation as appropriate and activated polyethylene glycol was dissolved completely in 30 seconds, a large amount of bubble will be avoided to produce simultaneously.Now TNF α A21K and polyoxyethylene glycol amount ratio are 1:20 (w/w).Then, carry out reaction 30 minutes under stirring in 25 DEG C, then add 100.0 microgram glycine termination reactions.After having reacted, reaction soln is inserted and possesses molecular weight 50, in the centrifuge tube device of the filter membrane device of 000, do not complete albumen and the unreacted linear polyethylene glycol of residue of reaction by high speed rotating centrifugal (3000xG30 minute) filtering residue, obtain highly purified polyoxyethylene glycol-tumor necrosis factor alpha analogue conjugate (TNF α A21KSCMPEG20).The filtrate of TNF α A21KSCMPEG20 carries out sterile filtration on the sterilised membrane filter of 0.2 μm, obtains final aseptic protein injection liquid medicine, is placed in 4 DEG C of sealed, steriles and keeps in Dark Place.
The preparation of embodiment 15TNF α G31KSCMPEG20
Get the sodium phosphate buffer of 1.0ml0.1MpH8.5, in water-bath, be preheated to 25 DEG C.Get the TNF α G31K albumen that 2.5 microgram purifying are expressed, be slowly added in sodium phosphate buffer, make it fully dissolve.Get 50.0 micromolar amounts 20, the methoxyl group PEG succinimide carboxymethyl ester (MethoxyPEGsuccinimidylcarboxymethylester of 000, SCM-PEG20000), it is added to rapidly in above-mentioned solution, and agitation as appropriate and activated polyethylene glycol was dissolved completely in 30 seconds, a large amount of bubble will be avoided to produce simultaneously.Now TNF α G31K and polyoxyethylene glycol amount ratio are 1:20 (w/w).Then, carry out reaction 30 minutes under stirring in 25 DEG C, then add 100.0 microgram glycine termination reactions.After having reacted, reaction soln is inserted and possesses molecular weight 50, in the centrifuge tube device of the filter membrane device of 000, do not complete albumen and the unreacted linear polyethylene glycol of residue of reaction by high speed rotating centrifugal (3000xG30 minute) filtering residue, obtain highly purified polyoxyethylene glycol-tumor necrosis factor alpha analogue conjugate (TNF α G31KSCMPEG20).The filtrate of TNF α G31KSCMPEG20 carries out sterile filtration on the sterilised membrane filter of 0.2 μm, obtains final aseptic protein injection liquid medicine, is placed in 4 DEG C of sealed, steriles and keeps in Dark Place.
Test example 1 conjugate of the present invention becomes the killing activity analysis of fiber oncocyte L929 to mouse
Mouse becomes fiber oncocyte L929 purchased from the American Type Culture Collection council of Chinese Academy of Sciences cell bank, vitro culture is in DMEM (Dulbecco ' sModifiedEagleMedium) substratum, the Cell culture invitro liquid (being called for short DMEM+10%FCS) of additional 10% calf serum preparation, hatches and is grown on 37 DEG C, 5%CO
2(HumphreysDT1, WilsonMRCytokine.1999Oct in incubator; 11 (10): 773-82.ModesofL929celldeathinducedbyTNF-alphaandothercyt otoxicagents).Be in the L929 cell of logarithmic phase, count with after 1% tryptic digestion, adjustment cell concn is 5x10
4/ ml, adds 100ul/ hole in 96 orifice plates by L929 cell, often organize 4 multiple holes, every plate sets 6 drug level groups, returns and is placed in 37 DEG C, 5%CO
2in incubator.Cultivate after 24 hours, use DMEM nutrient solution, the TNF α albumen obtained by embodiment 1 by following concentration successively doubling dilution: 0, (control wells), 0.001 μ g/mL, 0.01 μ g/mL, 0.1 μ g/mL, 0.3 μ g/mL, 1.0 μ g/ml, 10.0 μ g/mL.After adding medicine, 96 orifice plates are returned and be placed in incubator, continue cultivation 72 hours.Next day, take out 96 orifice plates, according to following literature method (MonoclonalantibodytargetingofN-cadherininhibitsprostatec ancergrowth, metastasisandcastrationresistance.NatureMedicine, 2010,16 (12), 1414-1420) 10 μ LCCK8 enzyme substrate solutions are added to every hole, and by culture plate at 37 DEG C, 5%CO
2cultivate 1 hour in constant incubator.Then, be determined at the absorbancy at 450nm place by microplate reader (TECANinfinite200PRO), then calculate, and curve plotting obtain IC
50data.
In addition, method as described above, measures TNF α A21K, TNF α G31K, TNF α SGPEG5, TNF α SGPEG10, TNF α SGPEG20, TNF α SCMPEG5, TNF α SCMPEG10, TNF α SCMPEG20, TNF α A21KSGPEG20, TNF α G31KSGPEG20 become fibroma target L929 cell L929 killing activity to mouse.
The Activity Results obtained is as shown in following table 1 and table 2.
Table 1 conjugate of the present invention becomes the killing activity of fiber oncocyte L929 to mouse
From upper table 1, the cell-lethal that the wild-type TNF α albumen after highly purified has height for target test L929 cell is active, cancer cell in vitro killing activity IC
50value is at 25 ± 14ng/mL.The L929 cell-lethal that coupling protein after the chemically modified of the activated polyethylene glycol ester of polyoxyethylene glycol chain length 20,000 still maintains high strength is active, the IC of TNF α SGPEG20
50value is 45 ± 23ng/mL, and the polyoxyethylene glycol chemistry modification fully showing long-chain significantly can not reduce the tumour cell killing activity of TNF α.Versus wild type TNF α, replaces the 21st L-Ala with Methionin and have dropped 2 times and 6 times respectively, IC with the cancer cell in vitro killing activity that two purifying TNF α that Methionin replaces the 31st glycine derive mutain
50value is respectively 50.3ng/mL and 150ng/mL.Pass through with after same polyoxyethylene glycol chain length 20,000 activated polyethylene glycol ester chemically modified, the TNF α of two purifying derives the cancer cell in vitro killing activity all slightly decline of mutain conjugate TNF α A21KSGPEG20 and TNF α G31KSGPEG20, IC
50value is respectively 58 ± 23ng/mL and 450ng/mL.
Above-mentioned external activity experimental data display, when employing polyoxyethylene glycol chain length 20,000 the chemically modified of activated polyethylene glycol ester when, the TNF α of wild-type TNF α albumen and TNF α A21K, TNF α G31K two purifying derives the high ira vitro killing activity that mutain can keep target cancer cells L929 equally.TNF α-polyethylene glycol conjugation thing TNF α SGPEG20 after the modification of polyoxyethylene glycol chemistry can reach the highest tumour cell killing activity.
The conjugate of the present invention of table 2 different polyoxyethylene glycol chain length becomes the killing activity of fiber oncocyte L929 to mouse
From upper table 2, polyoxyethylene glycol chain length becomes the killing activity of fiber oncocyte L929 to have conclusive effect for mouse.When the chain length of SCM polyoxyethylene glycol or SG polyoxyethylene glycol equals 5, when 000, the TNF α-polyethylene glycol conjugation thing after modification loses the cell-lethal activity of nearly 50 times.When the chain length of SCM polyoxyethylene glycol or SG polyoxyethylene glycol equals 10, when 000, it is active that the TNF α-polyethylene glycol conjugation thing after the modification of polyoxyethylene glycol chemistry loses nearly 1.3-12 cell-lethal doubly.As can be seen here, the polyoxyethylene glycol chain length of optimal selection is 20,000, in this chain length modification situation, polyoxyethylene glycol chemistry modify after TNF α-polyethylene glycol conjugation thing there is no and reduce active, and bottom line inactivation can be reached become the external killing activity of fiber oncocyte L929 with the mouse of topnotch.
Test example 2 conjugate of the present invention is to the killing activity analysis of human malignant tumor cell
Target cell is pernicious human body malignant melanoma A375 cell, hepatocellular carcinoma H22 cell, colorectal carcinoma born of the same parents HCT116 cell, nonsmall-cell lung cancer A549 and mammary cancer MDA-MB231 cell, all purchased from the American Type Culture Collection council of Chinese Academy of Sciences cell bank.Each target cell, vitro culture is in DMEM substratum, and the DMEM+10%FCS Cell culture invitro liquid of additional 10% calf serum preparation, hatches and be grown on 5%CO
2, in the constant incubator of 37 DEG C.The target cell being in logarithmic phase counts with after 1% tryptic digestion, and adjustment cell concn is 5x10
4/ ml, every hole 100 μ l adds 96 orifice plates, and often organize 4 multiple holes, every plate sets 6 drug level groups, returns and is placed in 37 DEG C, 5%CO
2in incubator.Cultivate after 24 hours, use DMEM nutrient solution, the TNF α albumen obtained by embodiment 1 by following concentration successively doubling dilution: 0 (control wells), 0.001 μ g/mL, 0.01 μ g/mL, 0.1 μ g/mL, 0.3 μ g/mL, 1.0 μ g/ml, 10.0 μ g/mL.After adding medicine, 96 orifice plates are returned and be placed in incubator, continue cultivation 72 hours.Next day, take out 96 orifice plates, add 10 μ LCCK8 enzyme substrate solutions to every hole, then by culture plate at 37 DEG C, 5%CO
2cultivate 1 hour in constant humidity incubator.Then, be determined at the absorbancy at 450nm place by microplate reader (TECANinfinite200PRO), and the method as following document calculates, and curve plotting, obtain IC
50data.
In addition, method as described above, measures TNF α A21K, TNF α G31K, TNF α SCMPEG20 to the IC50 value of each target cell.
Measurement result is as shown in table 3 below.
Table 3 conjugate of the present invention is to the killing activity IC of malignant cell
50zhi Spectrum
As above shown in table 3, TNF α, TNF α SCMPEG20 be lethal cytoactive in various degree to human malignant tumor cell display, but relative TNF α, TNF α PEG20 is all compared to mouse for the active susceptibilitys of four kinds of malignant cells becomes fibroma L929 cell lower.IC
50value display human malignant colon cancer cell susceptibility is the highest, can become clinical first object indication.
Anti-tumor activity analysis in test example 3 nude mice
Set up model of nude mice bearing tumor
Mouse is become fiber oncocyte L929 (11) incubation growth in the DMEM base nutrient solution of the foetal calf serum of additional 10%, reach and be in logarithmic phase.Count with after 1% tryptic digestion, adjustment cell concn is 1.0 × 10
7/ ml, then by 0.3mL cell suspending liquid (3.0 × 10
6cell) vaccinal injection enters Balb/c nude mouse (purchased from Hunan Si Laike Jing Da laboratory animal company limited) back.L929 target cell is at nude mice tumor growth, and regularly use vernier caliper measurement gross tumor volume, volume reaches 100 – 150mm
3after, for active testing.Above-mentioned nude mice is divided into contrast or treatment group, often organize 5, tail vein injection PBS (solvent control group) and TNF α or TNF α SGPEG20 (administration group) respectively, inject once every day with 0.3 μ g and 1.0 μ g, successive administration 21 days (qdx21), or inject weekly once with 0.3 μ g and 1.0 μ g, successive administration 3 weeks (q7dx3).Day by day record the toxic reaction situation of animal after administration, measure a gross tumor volume weekly, measure nude mouse body weight change twice.Meter record animal dead situation, calculates meta survival number of days, and makes body weight change curve and animals survived curve (Kaplan-Meierplot).Testing drug is calculated to medium lethal dose (50%LethalDose, the LD of nude mice with improvement karber's method
50), the activity of killing malignant tumour of research TNF α, the anti-tumour effect of quantitative analysis TNF α and TNF α SGPEG20.
The result obtained is as shown in table 4 below.
Anti-tumor activity in table 4 nude mice L929 tumor model
As above, shown in table 4, TNF α, in this experimental animal model, with in tail intravenously administrable 0.3 μ g successive administration every day 21 days situations, can suppress 45%L929 tumor volume growth, can extend the tumor bearing nude mice survival time 176%.When the dosage of TNF α administration is increased to 1.0 μ g, continuous 21 days under tail vein injection administrations, although 80% tumour L929 volume can be suppressed, but can not extend tumor bearing nude mice life time (-4%), sufficient proof high dosage (1.0 μ g) TNF α can cause nude mice general toxicity and shorten the survival time of tumor bearing nude mice.In this experimental animal model, with (7 days) weekly, tail intravenously administrable once, each 0.3 μ g, successive administration 3 weeks (three times altogether, totally 21 days) in situation, TNF α only can suppress L929 tumor volume growth in 1.5% nude mouse, also can not extend the tumor bearing nude mice survival time (-8%).In addition, with TNF α PEG20 through tail intravenously administrable, (7 days) tail intravenously administrable once weekly, per injection administration 0.3 μ g, successive administration 3 weeks (three times altogether, totally 21 days) in situation, TNF α PEG20 can suppress L929 tumor volume growth in 48% nude mouse, and simultaneously also can the significant prolongation tumor bearing nude mice survival time (218%).This Per-Hop behavior experimental data fully shows, in middle dosage (0.3 μ g) tail intravenously administrable situation, TNF α PEG20 can avoid high dosage (1.0 μ g//every day) to inject the toxicity in vivo of TNF α for nude mice, and demonstrate optimal inhibition tumor growth, extend the result for the treatment of of the survival time of tumor bearing nude mice.This Per-Hop behavior experimental result shows, and is the TNF α coupling protein that 20,000 activated polyethylene glycol is modified, can extends effective drug duration, and can reach the object of Per-Hop behavior treatment malignant tumour once by optimized choice chain length.
Test example 4 conjugate of the present invention reduces normal mouse toxicity in vivo
Laboratory animal: normal Balb/c mouse, purchased from Hunan Si Laike Jing Da laboratory animal company limited
Mouse experiment divides into groups: dosage 0.2,0.6,2,6,20,30,40,50,60,80,120,160, the 200 μ g of TNF α, TNF α SCMPEG5, TNF α SCMPEG10, TNF α SCMPEG20.
Experimental technique: mouse vein is administered once, then Continuous Observation record acute toxic symptoms, the death condition of animal in 7 days.According to each treated animal death toll, adopt the Probit Return Law, calculate acute toxicity LD with IBMSPSSStatistics19
50statistical software calculate acute toxicity LD
50value, calculates the medium lethal dose (LD that mouse vein gives TNF α and TNF α PEG
50) and 95% credibility interval (Asimplifiedmethodtoevaluatetheacutetoxicityofricinandric inusagglutinin.ZhanJ, ZhouP.Toxicology.2003Apr15; 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 table 5 conjugate of the present invention
From upper table 5, TNF α through different chain length degree polyethyleneglycol modified after, acute toxicity LD
50value raises, and acute toxicity declines.This phenomenon becomes the external killing activity of fibroma cancer cells L929 similar with mouse.The external killing activity of TNF α SCMPEG5 declines, and in the Mice Body of this albumen, acute toxicity also declines about 238 times.The laboratory animal death on the same day upon administration of TNF α group, and the death time of TNF α SCMPEG20 administration group obviously postpones, dead successively in 7 days upon administration.Preferred TNF α SCMPEG20 coupling protein is compared with the LD of prototype TNF α
50add 1.9 times, the acute toxicity of display TNF α SCMPEG20 declines 1.9 times, increases treatment window, is conducive to repeatedly, intravenously administrable weekly, has the prospect being developed as long-term treatment malignancy disease medicine.
Claims (13)
1. the conjugate of polyoxyethylene glycol-tumor necrosis factor alpha or polyoxyethylene glycol-tumor necrosis factor alpha analogue, wherein said tumor necrosis factor alpha (TNF α) analogue is A21K, G31K simple point mutation analogue albumen of TNF α.
2. the conjugate of polyoxyethylene glycol-tumor necrosis factor alpha according to claim 1 or polyoxyethylene glycol-tumor necrosis factor alpha analogue, wherein said polyoxyethylene glycol is linear polyethylene glycol, and the length range of described polyoxyethylene glycol linear chain is 5,000-20,000, be preferably 20,000.
3. the conjugate of polyoxyethylene glycol-tumor necrosis factor alpha according to claim 1 and 2 or polyoxyethylene glycol-tumor necrosis factor alpha analogue, wherein said polyoxyethylene glycol is the polyoxyethylene glycol of activation, and the polyoxyethylene glycol of described activation is preferably methoxysuccinyl imines glutarate NHS ester or the succinimide carboxymethyl NHS ester of polyoxyethylene glycol.
4. the conjugate of the polyoxyethylene glycol-tumor necrosis factor alpha according to any one of claims 1 to 3 or polyoxyethylene glycol-tumor necrosis factor alpha analogue, it 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.
5. the preparation method of the conjugate of polyoxyethylene glycol-tumor necrosis factor alpha according to claim 1 or polyoxyethylene glycol-tumor necrosis factor alpha analogue, it is characterized in that, comprise the polyoxyethylene glycol of activation and TNF α or the catalytic step of TNF alpha analog.
6. preparation method according to claim 5, wherein, described polyoxyethylene glycol is linear polyethylene glycol, and the length range of described polyoxyethylene glycol linear chain is 5,000-20,000, preferably 20,000.
7. preparation method according to claim 5, the polyoxyethylene glycol of wherein said activation, the polyoxyethylene glycol of described activation is methoxysuccinyl imines glutarate NHS ester or the succinimide carboxymethyl NHS ester of polyoxyethylene glycol.
8. the preparation method according to any one of claim 5 to 7, wherein, the amount ratio scope of described TNF α or TNF alpha analog and polyoxyethylene glycol is 1:20 – 1:30 by weight.
9. the preparation method according to any one of claim 5 to 8, is characterized in that, the pH scope control of described reaction, 8.5 to 9.5, is preferably 9.0 ± 0.1.
10. the preparation method according to any one of claim 5 to 9, is characterized in that, the temperature range of described reaction controls at 25 to 37 DEG C, and preferred temperature controls at 30 ± 0.1 DEG C.
11. 1 kinds of pharmaceutical compositions, it is containing the conjugate of polyoxyethylene glycol-tumor necrosis factor alpha described in good grounds any one of claim 1-4 or polyoxyethylene glycol-tumor necrosis factor alpha analogue, and pharmaceutically acceptable carrier.
The conjugate of 12. polyoxyethylene glycol-tumor necrosis factor alphas according to any one of claim 1-4 or polyoxyethylene glycol-tumor necrosis factor alpha analogue or pharmaceutical composition according to claim 10, for the preparation of the purposes prevented and/or treated in the medicine of tumour or cancer.
13. purposes according to claim 12, wherein said tumour or cancer are selected from lymphatic cancer, cellule type or lung cancer in non-cellule type, prostate cancer, kidney, liver cancer, colorectal carcinoma, melanoma, mammary cancer.
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