CN1323714C - PEGlyated Chinese trichosannthes root protein medicine and its preparation - Google Patents
PEGlyated Chinese trichosannthes root protein medicine and its preparation Download PDFInfo
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- CN1323714C CN1323714C CNB200310112768XA CN200310112768A CN1323714C CN 1323714 C CN1323714 C CN 1323714C CN B200310112768X A CNB200310112768X A CN B200310112768XA CN 200310112768 A CN200310112768 A CN 200310112768A CN 1323714 C CN1323714 C CN 1323714C
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Abstract
The present invention relates to a polyethyleneglycolized trichosanthin medicine which is favorable to industrialized production, has the advantages of homogenization and high stability of a product, and very little generalized anaphylaxis, and can be repeatedly used, and a preparation method for the medicine. In the present invention, the polyethyleneglycolized trichosanthin is formed by that polyethyleneglycol and the N terminal of trichosanthin are connected through an amine bond, and a TCS molecule is combined with a polyethyleneglycol molecule. In the preparation method, polyethyleneglycol and trichosanthin carry out a reducible alkylation reaction according to a weight proportion (W/W) between 0.5 and 20, the pH value of a buffer system processed through the reducible alkylation reaction is between 3 and 8, and the reducible alkylation reaction has a temperature between 4 DEG C and 37 DEG C, and time of 4 to 48 hours. A product obtained by using the method of the present invention has good unicity and uniformity.
Description
Technical field
The claimed technical scheme of the present invention belongs to PEG-TCS medicine and this technical field of production technology thereof.
Background technology
Trichosanthin (Trichosanthin, TCS) be from cucurbitaceous plant Fructus Trichosanthis (Trichosanthes kirilowii Maxim) tuber, extract a kind of protein.It has various biological and pharmacy function, and clinical practice is in the treatment of induced labor and ectopic pregnancy, hydatidiform mole, choriocarcinoma.Discovery China such as American scholar McGrath in 1989 tradition induced labor Chinese medicine TCS can not only suppress duplicating of HIV in the T lymphocyte external, and the HIV in the Monocytes is duplicated inhibitory action is also arranged.The TCS that earlier 1990s derives from China in I phase that the U.S. carries out and II clinical trial phase proof can not only suppress the duplicating of HIV in the T lymphocyte effectively, and the HIV in the Monocytes is duplicated significant inhibitory effect is also arranged.Kill the Monocytes that is infected by HIV, can eliminate the depots of HIV, thereby really reduce the gradient of infection of body.
TCS does not have lethal effect to the cell that is not infected by HIV, does not suppress the hemopoietic function of bone marrow, thereby does not influence the recovery of immunologic function.These characteristics are importances that it is better than ucleosides such as the inverase of present clinical use such as AZT and non-nucleoside reverse transcriptase inhibitor, are representing the research direction of new class inverase.Yet TCS is a foreign protein, has strong immunogenicity, easily causes systemic anaphylaxis, in the body half-life shorter, these drawbacks limit its extensive use clinically.
Polyethylene Glycol (PEG) is a kind of water-soluble high-molecular substance of nontoxic, non-immunogenicity, can pass through covalent bonds mode modifying protein.Polyethyleneglycol modified pharmaceutical grade protein not only can reduce immunogenicity and toxicity but also can obviously prolong plasma half-life, improves the stability of pharmaceutical grade protein.Utilize PEG that it is modified, can reduce the drainage of protein molecule by kidney, the half-life in the extension body, reduce the medication number of times, increase the effectiveness of treatment.Polyethyleneglycol modified protein technology has been widely used in biomedicine field, kind surplus PEG modifying protein and other chemical compound have 40 at least so far.The PEGization product of FDA approved has Pegylation ADA Adenosine deaminase, Pegylation Radix Asparagi umbilicus amine enzyme, glycol interferon, Pegylation granulocyte colony-stimulating factor etc.
The patent No. be PCT/US94/01618 (Ribosome inactivating proteincompositions having reduced antigenicity) patent disclosure a kind of molecular weight that adopts be the method that 5000 mPEG-SC modifies TCS, resulting modified outcome unicity and homogeneity are all relatively poor, be difficult to separation and purification, and yield is also very low, poor stability can't obtain to meet the product of clinical practice.
Summary of the invention
Technical problem to be solved by this invention provides a kind of suitability for industrialized production that helps, and product is even, good stability, and has extremely low systemic anaphylaxis, can repeat nonexpondable PEG-TCS medicine and preparation method.
The scheme of technical solution problem of the present invention is: a kind of PEG-TCS medicine, be connected by the amine key by the N-terminal of Polyethylene Glycol and trichosanthin, and a TCS molecule is formed in conjunction with a peg molecule.
The solution of the present invention is based on that such understanding proposed: the PEG-TCS medicine of prior art is that to adopt the patent No. be that the disclosed technology of PCT/US94/01618 utilizes mPEG-SC to modify TCS, since mPEG-SC be enough to protein molecule in free amino combination the in the lysine residue, so the modified outcome that this patent obtains is the mixture of each TCS in conjunction with 3-5 PEG molecule, so resulting modified outcome unicity and homogeneity are poor, be difficult to separation and purification, and yield is also very low, can't obtain to meet the product of clinical practice.The molecular structure of medicine of the present invention is that a peg molecule combines with the N-terminal of TCS molecule, therefore its unicity and homogeneity height also be easy to purification, and the activity of purified product can keep preferably, and have only extremely low systemic anaphylaxis, can repeat repeatedly to use.
The molecule that is connected with the N-terminal of trichosanthin is the methoxy poly (ethylene glycol) anakmetomeres.Be specially a mono methoxy polyethylene glycol aldehyde.
The technical scheme that the invention provides the PEG-TCS of producing said structure is: the part by weight (W/W) of Polyethylene Glycol and the reaction of trichosanthin generation standard reductive alkylation is between 0.5-20, the pH value of the buffer system of generation standard reductive alkylation reaction is between 3-8, the temperature of standard reductive alkylation reaction is between 4-37 ℃, and the time of standard reductive alkylation reaction is 4-48 hour.Product according to this kind method gained all has unicity and homogeneity preferably.
Above-mentioned Polyethylene Glycol is molecular weight mono methoxy polyethylene glycol anakmetomeres between 10000-40000Da.Specifically can be: the mono methoxy polyethylene glycol anakmetomeres can be mono methoxy polyethylene glycol succinimido succinates or be methoxy poly (ethylene glycol) succinimidyl carbonate or mono methoxy polyethylene glycol aldehyde, mono methoxy polyethylene glycol aldehyde most preferably, its molecular weight can be between 5000-40000Da.
It is as follows to analyze TCS sequence (247 aminoacid, theoretical molecular 27143.73): DVSFRLSGATSSSYGVFISNLRKALPNERKLYDIPLLRSSLPGSQRYALIHLTNYA DETISVAIDVTNVYIMGYRAGDTSYFFNEASATEAAKYVFKDAMRKVTLPYSGNYE RLQTAAGKIRENIPLGLPALDSAITTLFYYNANSAASALMVLIQSTSEAARYKFIE QQIGKRVDKTFLPSLAIISLENSWSALSKQIQIASTNNGQFESPVVLINAQNQRVT ITNVDAGVVTSNIALLLNRNNMA
Mono methoxy polyethylene glycol (mPEG) anakmetomeres are all higher to the N end selectivity of TCS, so determine that the molecule that is connected with the N-terminal of trichosanthin in the above-mentioned PEG-TCS medicine is mono methoxy polyethylene glycol (mPEG) anakmetomeres of molecular weight between 10000-40000Da, the mono methoxy polyethylene glycol anakmetomeres can be mono methoxy polyethylene glycol succinimido succinates (mPEG-SS), it also can be methoxy poly (ethylene glycol) succinimidyl carbonate (mPEG-SC), also can be mono methoxy polyethylene glycol aldehyde (mPEG-ALD), mPEG anakmetomeres between this molecular weight are to the N-terminal selectivity height of trichosanthin, thereby can guarantee that they have unicity and homogeneity preferably.But mono methoxy polyethylene glycol aldehyde most preferably.This be since the N-terminal of TCS to the selectivity of these anakmetomeres apparently higher than other the N end of TCS to the selectivity of these anakmetomeres, so the homogeneity of product is greatly enhanced, and the molecular weight ranges that can use also can expand to 5000-40000Da.
Polyethylene Glycol and trichosanthin generation standard reductive alkylation reaction prioritization scheme are: the ratio (W/W) of Polyethylene Glycol and the reaction of trichosanthin generation standard reductive alkylation is between 4-10, pH value is between 4-7, temperature is between 8-25 ℃, and the time is between 16-24 hour.The catalyst that adds in above PEG-TCS generation standard reductive alkylation reaction is Na CNBH
3
The concrete modification reaction process of mono methoxy polyethylene glycol aldehyde is:
(1) providing ionic strength is the buffer system of 10-500mmol/L, pH4.5-6.5;
(2) trichosanthin and mono methoxy polyethylene glycol aldehyde molecule reacted 18-22 hour in the system that step (1) provides, and temperature is controlled at 18-25 ℃; The use consumption is 5～100mMNaCNBH
3Be catalyst;
(3) after the product process cationic exchange resin adsorption that step (2) reaction obtains, use eluting salt, collect and mainly contain the PEG-TCS eluting peak that a peg molecule connects a trichosanthin molecule, after concentration, go up the separation and purification of molecular sieve gel chromatographic column, collect the PEG-TCS that a peg molecule connects a trichosanthin molecule.
The technical scheme that the present invention proposes can be utilized the Polyethylene Glycol method of modifying high to the N-terminal selectivity of trichosanthin preferably, the N-terminal that is polyethylene active molecule and trichosanthin is connected by amine key mode, not only make the unicity of product, and its homogeneity all is greatly enhanced, thereby easily purification with separate, have higher yield, make suitability for industrialized production become easy; Simultaneously the activity of purified product can keep preferably, has possessed high induced labor activity, HIV (human immunodeficiency virus)-resistant activity and anti-tumor activity and extremely low systemic anaphylaxis simultaneously and can repeat nonexpondable characteristics.
Description of drawings
Fig. 1 and Fig. 2 are that embodiment 7 resulting PEG-TCS are under SDS-PAGE and SE-HPLC mensuration, 1,2 is PEG-TCS among shown Fig. 1,3 is the reactant liquor of polyethyleneglycol modified trichosanthin, 4 is trichosanthin, 5 is the protein electrophoresis molecular weight standard: molecular weight ranges (KD): 14.2,20.1,24.0,29.0,36.0,45.0,66.0.SE-HPLC condition among shown Fig. 2: chromatographic column: TSK G3000SW (7.8mm * 30cm), detect wavelength: 280nm, elution buffer: 0.9%NaCl flow velocity: 1.0mL/min.
Fig. 3 is trichosanthin MALDI-TOF-MS molecular weight determination figure.
Fig. 4 is PEG-TCS MALDI-TOF-MS molecular weight determination figure.
Fig. 5 is trichosanthin trypsin digestion peptide MALDI-TOF-MS molecular weight determination figure.
Fig. 6 is PEG-TCS trypsin digestion peptide MALDI-TOF-MS molecular weight determination figure.
Fig. 7 is the antibody binding capacity comparison diagram of trichosanthin, PEG-TCS and rabbit variola amyloid proteins.
The specific embodiment
The trichosanthin that each embodiment uses be from big right plant radices trichosanthis, obtain, purity is greater than 99%, and maintains higher biological activity.The mPEG-ALD molecular weight is 20000Da.TCS is dissolved in the phosphate buffer of pH6.0,100mmol/L, the weight ratio ratio shown in the according to the form below adds mPEC-ALD, after the dissolving, adds Na CNBH
3, making its final concentration is 10mM, reaction is 24 hours under 20 ℃ of conditions of room temperature.
The separation and purification process that mPEG-ALD modifies the trichosanthin medicine is: after the product that will obtain with the modification reaction of trichosanthin adsorbs through SP Sepharose FF, NaCl gradient elution with 0-0.5M, through going up Sephacryl S200 separation and purification after ultrafiltration (the MILLPORE film of the 10KD) concentration, the purity of resulting PEG-TCS medicine scans the yield that peg molecule of method examination connects the PEG-TCS of a trichosanthin molecule greater than 99% by the SDS-PAGE gel.
| Numbering | mPEG-ALD/TCS(w/w) | Target product PEG-TCS yield (%) |
| | 0.4 | |
| Embodiment | ||
| 2 | 0.5 | Relatively |
| Embodiment | ||
| 3 | 1.0 | Can |
| | 4.0 | Good |
| Embodiment 5 | 6.0 | Good |
| Embodiment 6 | 7.0 | Best |
| Embodiment 7 | 8.0 | Best |
| Embodiment 8 | 10.0 | Good |
| Embodiment 9 | 13.0 | Better |
| Embodiment 10 | l6.0 | Can |
| Embodiment 11 | 20.0 | Can |
| Embodiment 12 | 21.0 | Difference |
By last table explanation, mPEC-ALD/TCS part by weight difference, the yield of gained target product PEG-TCS is different, wherein between 0.5-20, especially the ratio between 4-10 is suitable.
According to all the other conditions identical with the condition of identical mPEG-ALD/TCS (w/w) match ratio of embodiment 7 under, the target product PEG-TCS yield (%) of different molecular weight is as follows:
| Numbering | Molecular weight Da | Target product PEG-TCS yield (%) |
| Embodiment 7-1 | 2000 | Difference |
| Embodiment 7-2 | 5000 | Better |
| Embodiment 7-3 | 10000 | Good |
| Embodiment 7-4 | 12000 | Best |
| Embodiment 7-5 | 20000 | Best |
| Embodiment 7-6 | 40000 | Good |
| Embodiment 7-7 | 50000 | Difference |
By last table explanation, the molecular weight difference of mPEG, the yield of gained target product PEG-TCS is different, and is wherein better between 5000-40000.
According to all the other conditions identical with the condition of identical mPEG-ALD/TCS (w/w) match ratio of embodiment 7 under, the target product PEG-TCS yield (%) of different PH is as follows:
| Numbering | PH | Target product PEG-TCS yield (%) |
| Embodiment 7-8 | 2.5 | Difference |
| Embodiment 7-9 | 3 | Better |
| Embodiment 7-10 | 4.5 | Good |
| Embodiment 7-11 | 6.5 | Best |
| Embodiment 7-12 | 7.5 | Best |
| Embodiment 7-13 | 8 | Better |
| Embodiment 7-14 | 9 | Difference |
By last table explanation, pH value should be chosen between the 3-8, and is wherein better between 4-7.
According to all the other conditions identical with the condition of identical mPEC-ALD/TCS (w/w) match ratio of embodiment 7 under, target product PEG-TCS yield (%) is as follows under different time and the temperature conditions:
| Numbering | Hour | Temperature ℃ | Target product PEG-TCS yield (%) |
| Embodiment 7-8 | 3.5 | 40 | Difference |
| Embodiment 7-9 | 4 | 37 | Better |
| Embodiment 7-10 | 22 | 18 | Best |
| Embodiment 7-11 | 18 | 25 | Best |
| Embodiment 7-12 | 24 | 15 | Good |
| Embodiment 7-13 | 48 | 4.0 | Better |
| Embodiment 7-14 | 48 | 3.0 | Difference |
By the explanation of last table, response time and temperature are selected the face broad, but both economical time and temperature should be between 18-22 hours, 18-25 ℃.
Find also that by experiment the ionic strength of buffer system is between 10-500mmol/L, catalyst amount is between 5-100mM, and reaction can both take place.
Get the target product of embodiment 7 gained and do following performance test:
One, see Fig. 1 and Fig. 2, resulting PEG-TCS medicine is measured through SDS-PAGE and SE-HPLC, and purity is greater than 99%.
Two, trichosanthin and PEG-TCS molecular weight determination.
With SA is substrate, with the BIFLEX of BRUKER company
TMIII MALDI-TOF mass spectrograph is measured molecular weight.Vertical coordinate is an abundance of ions in the MALDI-TOF-MS collection of illustrative plates of trichosanthin and PEG-TCS (seeing Fig. 3, Fig. 4), and abscissa is ionic mass-to-charge ratio (m/z).
M/z 27141.071 peaks are TCS protonated molecular ion peak (single electric charge peak) among the trichosanthin MALDI-TOF-MS figure, i.e. [M+H]
+The peak.M/z 54331.016 peaks are the dimer peak [2M+H] of TCS
+M/z 13571.136 peaks are the double charge peak [M+2H] of TCS
++
M/z 49646.418 peaks are PEG-TCS protonated molecular ion peak (single electric charge peak) among the PEG-TCS MALDI-TOF-MS figure, i.e. [M+H]
+The peak.The m/z98016.250 peak is the dimer peak [2M+H] of PEG-TCS
+M/z 24894.295 peaks are the double charge peak [M+2H] of PEG-TCS
++
The relative molecular mass of PEG-TCS is 49646.418.After showing the trichosanthin molecule and peg molecule combining, make former relative molecular mass increase (peg molecule) more than 20,000, measurement result is not seen trichosanthin peak and other impurity peaks.Proof PEG-TCS pharmaceutical purity metallization processes is reasonable, and the PEG-TCS of purification is that a peg molecule connects a trichosanthin molecule.
Three, trichosanthin and the quality determination of PEG-TCS trypsin digestion peptide.
The trypsin digestion that trichosanthin and PEG-TCS were handled with TPCK respectively, the enzymatic hydrolysate lyophilization.With HCCA is substrate, with the BIFLEX of BRUKER company
TMIII MALDI-TOF mass spectrograph is measured trypsin digestion peptide quality.(seeing Fig. 5 and Fig. 6 and table 7).The N end that this shows trichosanthin is connecting Polyethylene Glycol.
Four, the external affinity test of PEG-TCS medicine and rabbit anti-serum
The antibody of preparation rabbit variola amyloid proteins is with the antibody binding capacity of conventional ELISA method mensuration trichosanthin and PEG-TCS and rabbit variola amyloid proteins.Fig. 7 result shows: compare with trichosanthin, the antibody binding capacity of PEG-TCS and rabbit Chinese People's Anti-Japanese Military and Political College pollen protein descends.
Five, the PEG-TCS medicine causes the test of Cavia porcellus systemic anaphylaxis
60 of the purebred Cavia porcelluss of white, body weight 250～300g, the male and female dual-purpose is divided into two groups.Trichosanthin and PEG-TCS medicine are pressed 5 times of (0.1mg/Kg Cavia porcellus) lumbar injections of human dosage, capacity 0.2ml/ only, the next day 1 time, continuous 3 times, make its sensitization.Trichosanthin and PEG-TCS medicine are pressed 10 times of human dosage (0.2mg/Kg Cavia porcellus), and capacity 0.2ml/ only respectively at the intravenous injection of shank shin in 14,21 after last sensitization.Observe in the 30min and the record result.
Table 1 Cavia porcellus systemic anaphylaxis progression criterion
| The order of | Reaction symptom | |
| 0 | No | |
| 1 | Have only and slightly grab nose, tremble or | |
| 2 | Have several times and cough, grab nose, tremble or | |
| 3 | Repeatedly or continuously cough, with dyspnea or spasm, tic etc. | |
| 4 | Spasm, tic, gatism, shock death |
Annotate: the order of reaction reaches 2 grades or when above, anaphylaxis is positive
Table 2 trichosanthin causes Cavia porcellus systemic anaphylaxis situation
| Group | Number of animals (only) | Each dosage (mg/kg) | The reaction rank | Number positive (only) | Positive rate (%) | |||||
| | Attack | 0 | 1 | 2 | 3 | 4 | ||||
| TCS | 15 | 0.1 | (0.2 14 days) | 0 | 0 | 0 | 15 | 15 | 100 | |
| TCS | 15 | 0.1 | (0.2 21 days) | 0 | 0 | 0 | 0 | 15 | 15 | 100 |
| PEG-TCS | 15 | 0.1 | (0.2 14 days) | 15 | 0 | 0 | 0 | 0 | 0 | 0 |
| PEG-TCS | 15 | 0.1 | (0.2 21 days) | 14 | 1 | 0 | 0 | 0 | 0 | 0 |
Annotate: " 0 " expression does not have this rank reaction
Table 3 PEG-TCS group number of times of attack and anaphylactoid relation
| Group | Number of animals (only) | 14 days | 21 days | 28 days | 35 days | 42 days | The total positives rate | |||||
| Number positive | Positive rate | Number positive | Positive rate | Number positive | Positive rate | Number positive | Positive rate | Number positive | Positive rate | |||
| PEG-TCS | 15 | 0 | 0 | 0 | 0 | 1 | 1/15 | 1 | 1/15 | 0 | 0 | 2/15 |
| PEG-TCS | 15 | - | - | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 1/15 | 1/15 |
Annotate: the PEG-TCS group refers to attack the Cavia porcellus that positive reaction do not occur in the table 2, attacks one weekly continuously
Inferior."-" expression is not attacked; " 0 " expression is attacked does not have positive reaction
The test of Cavia porcellus systemic anaphylaxis shows that trichosanthin has extremely strong systemic anaphylaxis; The PEG-TCS medicine has extremely low systemic anaphylaxis; After PEG-TCS sensitization, continuous four～five attacks, its anaphylaxis is still extremely low, and the PEG-TCS medicine can repeat repeatedly to use.
Six, the induced labor activity test of PEG-TCS medicine
With mid-term (12 days) pregnant Kunming mouse be model, intraperitoneal injection killed mice in 48 hours, dissected and calculated total young tire number and stillborn fetus number (comprising the fetus absorption point).The mice that fetal mortality surpasses total young tire several 50% is that induced labor is effective.The induced labor activity of TCS and PEG-TCS sees the following form:
The induced labor activity of table 4 PEG-TCS medicine
| PEG-TCS | |||||
| Dosage (ug/g mice) | Conceived number (only) | Young tire number (only) | Death toll (only) | Mortality rate | The induced |
| 2 | 10 | 106 | 106 | 100％ | 100％ |
| 1 | 9 | 85 | 85 | 100％ | 100％ |
| 0.4 | 10 | 100 | 99 | 99％ | 99％ |
| 0.2 | 7 | 67 | 41 | 61.2％ | 61.2％ |
The induced labor activity of table 5 trichosanthin medicine
| TCS | |||||
| Dosage (ug/g mice) | Conceived number (only) | Young tire number (only) | Death toll (only) | Mortality rate | The induced |
| 2 | 8 | 78 | 78 | 100％ | 100％ |
| 1 | 7 | 66 | 40 | 60.6％ | 60.6％ |
| 0.4 | 4 | 44 | 16 | 36.4％ | 36.4％ |
| 0.2 | 5 | 58 | 58 | 0 | 0 |
Induced labor activity test explanation, PEG-TCS can improve the induced labor activity in the mice body.
Seven, the external anti-HIV-1 activity test of PEG-TCS medicine
1, the titration of viral infection
HIV-1 presses Johnson ﹠amp; The described method improvement of Byington (1990) carries out titration, is summarized as follows: the HIV-1 stock solution is done 4 times of dilutions on 96 orifice plates, and 10 gradients, 6 repeating holes of every gradient, every hole adds C8166 cell 50 μ l (3 * 10
5/ ml), 37 ℃ of every hole final volume 200 μ l., 5%CO
2Cultivate.Added fresh RPMI-1640 culture medium 100 μ l on the 3rd day.(whether Cytopathic effect CPE), has the formation of syncytium (syncytium) to determine with every hole to observe HIV-1 inducing cell pathological changes effect in every hole on the 7th day under inverted microscope; Press Reed ﹠amp; The Muench method is calculated the TCID of virus
50(50% TissueCulture Infection Dose) causes that promptly the viral supernatant dilution factor of CPE appears in cell.
2, chemical compound detects the cytotoxicity of C8166
3 * 10
5/ mlC8166 cell suspension 100ul and different compound solution mixing, 37 ℃, 5%CO
2Cultivated three days, and adopted mtt assay to detect cytotoxicity.490nm ELx800ELISAReader measures the OD value.CC
50(50％ Cytotoxic Concentration)
3, the PEG-TCS medicine induces the C8166 cell to form plasmodial inhibitory action to HIV-1
With 6 * 10
5/ ml C8166 cell 50ul/ hole is inoculated on 96 orifice plates that contain 100 μ l/ hole doubling dilution chemical compounds, adds the HIV-1 dilution supernatant of 50 μ l then, and M.O.I. is 0.03TCID50.The control wells that does not contain chemical compound is set simultaneously.37 ℃, 5%CO
2Cultivated three days, (100 *) counting HIV-1 induces the plastidogenetic syncytium number of C8166 under the inverted microscope.EC
50(50% Effective Concentration) reaches 50% o'clock compound concentration for suppressing syncytium formation.CC
50Drug level when (50% Cytotoxic Concentration) is 50% host cell generation cytotoxicity, SI (Selectivity Index) is CC
50/ EC
50Ratio.
Table 6 PEG-TCS medicine suppresses HIV-1 and induces the C8166 cell to form syncytium and to the cytotoxic effect of C8166
| Chemical compound | CC 50(μg/ml ) | EC 50(μg/ml ) | SI(CC 50/EC 50) |
| AZT TCS PEG-TCS | 420.3 35 275 | 0.065 0.3 50 | 6466.2 116.6 5.5 |
Table 7 TCS and PEG-TCS trypsin digestion hybrid peptide MALDI-TOF-MS quality
Relatively (Matrix HCCA)
| TCS pancreatin peptide hydrolysis serial number | TCS pancreatin peptide hydrolysis sequence | The peptide theoretical molecular | TCS peptide hydrolysis measured value | PEG-TCS peptide hydrolysis measured value |
| T20 | NNM | 377.41 | 377.065 | |
| T21 | NNMA | 448.49 | ||
| T16 | RVDK | 516.59 | 516.596 | 517.259 |
| T10 | DAMRK | 619.73 | 619.34 | |
| T1 | DVSFR | 622.67 | 622.635 | |
| T3 | ALPNER | 698.77 | 698.754 | 699.198 |
| T6 | SSLPGSQR | 830.90 | 830.891 | 831.203 |
| T22 | GVFISNLR (non-specificity) | 905.01 | 905.024 | 905.288 |
| T12 | LQTAAGKIR | 957.15 | ||
| T15 | FIEQQIGK | 962.13 | 962.036 | 962.244 |
| T5 | LYDIPLLR | 1002.21 | 1002.166 | 1002.335 |
| T4 | KLYDIPLLR | 1130.39 | 1130.376 | 1130.390 |
| T9 | YVFKDAMRK | 1157.41 | ||
| T14 | YKFIEQQIGK | 1253.49 | ||
| T11 | VTLPYSGNYER | 1298.44 | 1298.35 | |
| T2 | LSGATSSSYGVFISNLR | 1758.98 | 1758.863 | |
| T8 | AGDTSYFFNEASATEAAK | 1879.99 | 1879.787 | 1879.973 |
| T17 | TFLPSLAIISLENSWSALSK | 2177.5 | 2177.312 | 2177.511 |
| T19 | VTITNVDAGVVTSNIALLLNR | 2183.59 | 2183.464 | 2183.504 |
| T18 | QIQIASTNNGQFESPVVLINAQN QR | 2770.15 | 2769.948 | |
| T7 | YALIHLTNYADETISVAIDVTNVY IMGYR | 3319.86 | 3319.487 | 3319.437 |
| T13 | ENIPLGLPALDSAITTLFYYNANS AASALMVLIQSTSEAAR | 4298.97 | 4299.098 | 4299.042 |
Claims (7)
1, a kind of PEG-TCS medicine, N-terminal by Polyethylene Glycol and trichosanthin is connected by the amine key, a TCS molecule is in conjunction with a peg molecule, the molecule that is connected with the N-terminal of trichosanthin is the mono methoxy polyethylene glycol anakmetomeres, it is characterized in that: the molecule that the N-terminal of trichosanthin is connected is a mono methoxy polyethylene glycol aldehyde.
2, a kind of preparation method of PEG-TCS medicine as claimed in claim 1, it is characterized in that: the part by weight (W/W) of Polyethylene Glycol and the reaction of trichosanthin generation standard reductive alkylation is between 0.5-20, the pH value of the buffer system of generation standard reductive alkylation reaction is between 3-8, the temperature of reaction is between 4-37 ℃, and the time of reaction is 4-48 hour.
3, the preparation method of PEG-TCS medicine according to claim 2 is characterized in that: described Polyethylene Glycol is molecular weight mono methoxy polyethylene glycol anakmetomeres between 10000-40000Da.
4, the preparation method of PEG-TCS medicine according to claim 2 is characterized in that: described Polyethylene Glycol is molecular weight mono methoxy polyethylene glycol aldehyde between 5000-40000Da.
5, according to the preparation method of claim 2 or 3 or 4 described PEG-TCS medicines, it is characterized in that: Polyethylene Glycol and trichosanthin generation reduction reaction alkylated reaction are: the ratio (W/W) of Polyethylene Glycol and trichosanthin generation reduction reaction alkylated reaction is between 4-10, pH value is between 4-7, temperature is between 8-25 ℃, and the time is between 16-24 hour.
6, according to the preparation method of claim 2 or 4 described PEG-TCS medicines, it is characterized in that: PEG-TCS generation standard reductive alkylation catalyst for reaction is Na CNB H
3
7, the preparation method of PEG-TCS medicine according to claim 4 is characterized in that:
(1), providing ionic strength is the buffer system of 10-500mmol/L, pH4.5-6.5;
(2), trichosanthin and free oxygen base peg molecule reacted 18-22 hour in the system that step (1) provides, temperature is at 18-25 ℃; Using consumption is the NaCNBH of 5～100mM
3Be catalyst;
(3), after the product process cationic exchange resin adsorption that step (2) reaction obtains, use eluting salt, collect and mainly contain the PEG-TCS eluting peak that a peg molecule connects a trichosanthin molecule, after concentration, go up the separation and purification of molecular sieve gel chromatographic column, collect the PEG-TCS that a peg molecule connects a trichosanthin molecule.
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