CN1268517A - Liver target high-carbohydrate density galastosyl interferon conjugate - Google Patents

Abstract

The present invention provides a high-sugar density galactosyl interfereon coupling substance which is formed by using branching bridge reagent to connect 2-5 galactosyls on each coupling point of interferon molecular. Said coupling substance may be used to develop medicine for treating viral hapatitis. Its chemical structure is also disclosed.

Description

The liver target high-carbohydrate density galastosyl interferon conjugate

What the present invention relates to is that a class is by branch bridge reagent, make Interferon, rabbit (a kind of medical protein) but 2-5 galactosyl in the connection of each link coupled site (normally free amino) in the molecule, thereby form the galactosyl interferon conjugate of high-carbohydrate density.In body, this conjugate demonstrates significant liver target feature.

By the literature search and the Cha Xin of system, it is present not look into the galactosyl interferon conjugate of seeing chemical structure type of the present invention as yet, looks into and sees that relevant document and patent report are as follows:

[1] the galactosyl interferon conjugate of liver target such as Zhong Yuguo

Chinese invention patent ublic specification of application application number: 9311196

Open day of publication number: CN1087093A: on May 25th, 1994

Glycosylation cytokine such as [2] Bin Kou is straight

Chinese invention patent ublic specification of application application number: 93117879.7

Open day of the number of opening: CN1087916A: on June 15th, 1994

[3] the synthetic and liver target research of galactosyl Interferon, rabbit such as Guan Changtian

China's Journal of Nuclear Medicine 1996,11 (2): 234

[4] the synthetic and biological property research of liver target galactosyl Interferon, rabbit such as Zhong Yuguo

China's pharmaceutical chemistry magazine 1995,5 (3): 164-8

[5] the synthetic and liver target research of liver target galactosyl cytochrome C such as Zhong Yuguo

Journal of West China University of Medical Sciences 1996,27 (2): 130-3

[6] Guan Changtian etc. is with the feasibility study of NGA as the hepatic targeting drug carrier

China Journal of Nuclear Medicine 1992,12 (2): 166-8

Document [1], [2] but the structure of two its galactosyl interferon conjugates of patent is that the coupling site connects 1 galactosyl in the interferon molecule, with the present invention significant difference is arranged all on 26S Proteasome Structure and Function; Document [3] [4] [5] [6] is fundamental research and the methodological study that the invention people carries out for the present invention, the unexposed content of delivering patent of the present invention.

The eighties foreign scholar confirms that the HBP (HBP) that exists only on the hepatocyte of mammal film is the acceptor (ASGPR) of asialoglycoprotein.Human albumin is modified the galactosyl albumin (NGA) of back gained with galactosyl, it is the part of the ASGPR of a synthetic, be typical part---acceptor identification, and have part---all biological of receptors bind is learned feature, and abroad NGA being used is the carrier of hepatic targeting drug.

Interferon, rabbit is a protogonocyte element in the human body, for a part amount is about 1.6 ten thousand activated protein, has multiple biological activitys such as antiviral and antitumor, for current generally acknowledged to choice drug B-mode, that hepatitis C carries out causal treatment.Since behind the drug administration by injection with the circulation of blood NATURAL DISTRIBUTION, be transported to the 5-7% that implantation dosage is only arranged of liver, will be to the hepatitis virus generation drug effect in the liver cell, require the pharmacological agent of heavy dose of (3,000,000 unit/sky) long-time (three months), thereby medical expense is high, side reaction increases, its curative effect not ideal enough (viral negative conversion rate only has about 30%).

The present inventor has carried out the research of liver targeted interferon at home first: use chemical process, galactosyl is connected human leukocyte interferon (INF-α), on genetic engineering interferon (rINF α) protein molecular, make it to have the ASGPR part and keep the active double biological characteristics of INF.Result of study shows, prepared galactosyl interferon conjugate (Gal-INF-α, Gal-rINF-α ₁) its liver distributive law is the 20-25% of implantation dosage, antiviral activity does not carry out the raw material Interferon, rabbit that semi-lactosi modifies and has improved 2-3 doubly.This carbohydrate density is the conjugate of 2-3, although liver target has improved 2-3 doubly than the raw material Interferon, rabbit, but still not ideal enough, know that from contriver's fundamental research as carbohydrate density is brought up to 8-12, the liver distributive law is expected to reach 30-50%.

In interferon molecule, but the binding site (free amine group) that semi-lactosi is modified is limited, and in order to improve carbohydrate density, the inventor has designed and synthesized the branch bridge reagent (M that can connect a plurality of semi-lactosis ₁, M ₂, M ₃, M ₄), and with Interferon, rabbit (rINF α ₁, rINF-α ₂B) carry out coupling, by purifies and separates and biological characteristic research experiment, prepared digalactosyl Interferon, rabbit (2Gal-rINF _{α 2}B, T ₁) protein electrophoresis list band, carbohydrate density=10, liver distributive law 30-35%.

The object of the present invention is to provide class connection to go up the branch bridge reagent of 2-5 semi-lactosi and the galactosylation interferon conjugate that the Interferon, rabbit coupling forms high-carbohydrate density, this conjugate has significant liver target.Be developed further into medicine for viral hepatitis, will reduce Interferon, rabbit dosage, heighten the effect of a treatment, reduce untoward reaction, save medical expense, this is expected making a breakthrough property effect to clinical treatment B-mode, hepatitis C.

The present invention is a kind of two ends at aromatic ring branch bridge and fat chain branch bridge, with the semi-lactosi of glucoside key, peptide bond, the coupled differing molecular number of imido ester bond and Interferon, rabbit and the high-carbohydrate density galactosyl interferon conjugate for preparing, the chemical structure of general formula of this conjugate is as follows respectively.

G=M ₁, M ₂, M ₃, M ₄X=O, NH n=2-6 INF=rINF α ₂B, rINF α ₁G is semi-lactosi branch bridge reagent, wherein M ₁Be disaccharide aromatic ring bridge reagent, M ₂Be disaccharide fat chain bridge reagent, M ₃Be three glycolipid chain bridge reagent, M ₄Be aromatic ring aromatic ring bridge reagent, its chemical structural formula is as follows:

Mentioned reagent M ₁, M ₂, M ₃, M ₄Respectively with r-INF α ₁, r-INF α ₂After the b coupling is purified, get high-carbohydrate density lactose base interferon conjugate T respectively ₁T ₂T ₃T ₄, its chemical structure is as follows:

The preparation method of target compound of the present invention is as follows:

Aromatic ring branch bridge type: the synthetic branched sugar reagent M of choosing ₁, the coupling Interferon, rabbit prepares target compound T ₁Be example.

Two semi-lactosi aromatic ring branch bridge reagent M ₁Synthetic: with 3, the 5-resorcylic acid is starting raw material and the condensation of 6-aminocaprolc acid methyl esters, 3,5-dihydroxy-benzene formyl methoxycarbonyl hexylamine combines with sulfo-semi-lactosi reagent behind the usefulness ethylene dibromide hydrocarbonylation hydroxyl and gets M ₁

Two semi-lactosi aromatic ring bridge interferon conjugate T ₁Preparation: with M ₁Use methyl alcohol---after the sodium methoxide solution hydrolysis, boil off methyl alcohol, residue is dissolved in the phosphate buffer solution of pH=8, adds rINF α ₂B and condensing agent EDC, treat that condensation reaction is finished after, reaction solution dialysed to the extracellular fluid dialysis sugar-free to be detected, sterile filtration, lyophilize gets T ₁Embodiment 1 is seen in concrete processing condition and operating process.

Fat chain branch bridge type: the synthetic branched sugar reagent M of choosing ₂, coupled Interferon, rabbit prepares target compound T ₂Be example.

Two galactolipid chain bridge reagent M ₂Synthetic: with diethyl malonate is starting raw material, through condensation, cyclisation, decarboxylation reduction, addition, open loop, iodo etc. seven go on foot the diiodo-thing, get M with the condensation of sulfo-semi-lactosi reagent again ₂

Two galactolipid chain bridge interferon conjugate T ₂Preparation: with M ₂Placing methyl alcohol---sodium methoxide solution alcohol solves imino esters, flings to methyl alcohol with nitrogen, and residue is dissolved in the phosphoric acid buffer, adds rINF α ₁After treating that condensation reaction is finished, reaction solution dialysed to the extracellular fluid dialysis sugar-free to be detected, sterile filtration, and lyophilize gets T ₂

The purity of target compound of the present invention, carbohydrate density and liver target are surveyed inspection and (are used target compound T ₁Be example)

Purity and carbohydrate density detect: adopt the sds polyacrylamide gel electrophoresis method to digalactosyl interferon conjugate (2Gal-rINF α ₂B is T ₁) (Fig. 1 indicates A) and raw material rINF α ₂B (Fig. 1 indicates B) and standard molecular weight albumen (Fig. 1 indicates C) carry out electrophoresis.The results are shown in electrophorogram (Fig. 1), the result shows, T ₁With rINF α ₁Protein electrophoresis be both electrophoretic mobilities of single band (seeing A.B) there were significant differences, reach the purifying requirement.

The measuring and calculating of carbohydrate density: make typical curve with the standard molecular weight PROTEIN C, record T ₁Molecular weight is 18700Da, rINF α ₂B is 15200Da, and M ₁Molecular weight be 694Da.Per molecule T then ₁Contain 5 molecule M approximately ₁(18700-15200/694=5.04), and each M ₁Molecule contains two semi-lactosis, then T ₁Carbohydrate density be 10.

Liver target detects: with T ₁With raw material rINF α ₂B uses radio isotope respectively ¹³¹I (chloramine-t method) carries out isotope labeling and gets ¹³¹I-T ₁With ¹³¹I-rINF α ₂B is injected into respectively in the rabbit vein, makes rabbit whole body radiography with Siermens Basicar r photographic camera, take to collect separately 5-30 minute stack photo (Fig. 2, Fig. 3).Fig. 2 be raw material ( ¹³¹I-rINF α ₂B) photo is the blood pond developing of NATURAL DISTRIBUTION, and wherein the radiation video picture of liver, kidney, bladder is high-visible.Accompanying drawing 3 is ¹³¹I-T ₁Resemble, can see significantly that nucleic concentrates in the hepatic region, kidney and bladder developing are faint.Both contrasts can be seen T intuitively ₁Liver target.

Experiment distributes in the mouse body: with T ₁With rINF α ₂B uses respectively ¹²⁵I mark (chloramine-t method), gained ¹²⁵I-T ₁With ¹²⁵I-rINF α ₂B injects respectively and respectively organizes mouse tail vein, regularly puts to death mouse, dissects and complete each internal organs of collection mouse, measures the exit dose among each internal organs official, calculates it and accounts for the percentage that injects total exit dose, the results are shown in Table 1. tables 2.Detected result shows ¹²⁵I-T ₁Liver distribution peak value was 36.50 ± 2.54 in 5 minutes, ¹²⁵I-rINF α ₂B is 11.97 ± 0.25, T ₁The liver uptake ratio be raw material rINF α ₂3.1 times of b.

The distribution result of experiment has fully shown genetic engineering interferon alpha in the above mouse body ₂After b adopts this coupling method to connect galactosyl, formed digalactosyl genetic engineering interferon alpha ₂B conjugate (2Gal-IFN α ₂B) with the raw material genetic engineering interferon alpha ₂B (IFN α ₂B) compare, have tangible liver target.1 pair of semi-lactosi aromatic ring of embodiment bridge Interferon, rabbit (rINF α ₂B) conjugate (T ₁) preparation Two semi-lactosi aromatic ring bridge reagent M ₁Synthetic: with the 6-Aminocaproic Acid methyl esters under HOBT catalysis, with DCC and 3.5-resorcylic acid (1) condensation, generation intermediate (2).Under alkaline condition,, get intermediate (3) with glycol dibromide generation alkylation reaction.After being dissolved in two bromo-derivative 200mg (0.41mmol) in 15ml acetone and the 6ml distilled water subsequently, add 2-S-(2,3,4,6-four-O-acetyl-β-1)-and galactopyranose base-2-isothiourea hydrobromate 800mg (1.67mmol), salt of wormwood 274mg (1.99mmol), stirring at room is after 10 minutes, add sodium bisulfite 172mg (1.65mmol), stirred after 24 hours this reaction mixture lucifuge chamber, and concentrating under reduced pressure adds the 30ml chloroform in resistates, the 15ml washing once, concentrating under reduced pressure behind the anhydrous sodium sulfate drying, gained resistates be through silica gel column layer, petroleum ether-ethyl acetate (1: 1) wash-out, elutriant concentrates, crystallization gets white powder solid M ₁150mg, mp57-58 ℃, yield 29%.IR：3414，2940，1750，1647cm ¹；HNMR(200HMZ；CDCL ₃)：6.91(2H，d，ArH)，6.54(1H，S，ArH)，5.43(2H，d，HC-S)5.05(2H，dd，2X-CHOAc)4.60(4H，dm，2X，-CHOAC)，4.19(6H，m，2XCH ₂OAc，2X-CHO)4.00(4H，dd，2XCH ₂OAr)，3.65(3H，S，CH ₃OCO ^-)，3.40(2H，m，NCH ₂)，3.16(2H，m，CH ₂S)2.95(2H，m，CH ₂S)，2.32(2H，t，CH ₂CO ₂)2.14(6H，s，2XCH ₃CO)，2.10(6H，s，2XCH ₃CO)，2.05(6H，s，CH ₃CO)，1.98(6H，s，CH ₃CO)，1.6l(4H，m，-CH ₂-，CH ₂ ^-)，1.42(2H，m，CH ₂)ppm。

Digalactosyl virtue bridge Interferon, rabbit (rINF α ₂B) conjugate (T ₁) preparation:

With branched sugar reagent M ₁200mg, 0.16mmol be dissolved in the 20ml anhydrous methanol, add the methanol solution 4.0mol (0.4mmol) of the sodium methylate of 0.1N subsequently, stirring at room is after 6 hours, concentrating under reduced pressure, add 4.0ml water in the resistates, continued stirring at room 10 hours, add the phosphoric acid buffer (4ml) of pH=8.0, behind the hydrochloric acid accent pH=8.0 with 1N, add EDC 36mg (0.16mmol), behind the stirring and evenly mixing, add 4mg genetic engineering interferon a ₂B in 4 ℃ stir 24 hours after, reaction solution is added in the pretreated dialysis tubing, the phosphoric acid buffer dialysis that contains 0.73% sodium-chlor with 0.1M, changed an extracellular fluid dialysis in per six hours, until using phenol---dialyzate sterile filtration till the sulfuric acid process detection extracellular fluid dialysis sugar-free, lyophilize gets T ₁

Adopt SDS-polyacrylamide gel electrophoresis (Fig. 1), can measure conjugate T respectively ₁(2Gal-IFNa ₂B) and raw materials used Interferon, rabbit (IFNa ₂B) molecular weight be respectively 18700Da and 15200Da, and modification group is 694Da, so calculate conjugate T thus ₁In the branch semi-lactosi be 5, and each branch semi-lactosi reagent contains two galactose residues, so conjugate T ₁(2Gal-IFN α ₂B) carbohydrate density should be 10.

Use method for preparing, purifying has also been confirmed structure and carbohydrate density is 10 conjugate T ₁(2Gal-IFN α ₂B) carry out its liver target research in animal body as test with sample.

With this conjugate T ₁(2Gal-IFN α ₂B) carry out with chloramine-t method ¹²⁵The I mark, and, carry out purification process with the PBS wash-out with Sephadex G50 chromatography notes, the Tricholroacetic Acid precipitator method are measured its radiochemicsl purity and are reached more than 99%, and with same ¹²⁵The raw material Interferon, rabbit of I mark ( ¹²⁵I-IFN α ₂B) being contrast, is the mouse tail vein injection of 15-20g from body weight respectively, does the experiment that distributes in the mouse body.Every mouse of test group is injected the conjugate T that mark is crossed ₁2ug is after every mouse of control group is injected the contrast 2ug that mark crosses, respectively at phase sacrificed by decapitation simultaneously not such as 5 ', 10 ', 15 ', 30 ', 60 ', 120 '.Dissect to take out each internal organs, measure the radioactivity of each internal organs, the result represents (see Table 1, table 2) with the per-cent that the radioactivity accumulation in each internal organs accounts for injection rate.

Can see by table 1, ¹²⁵I-2Gal-IFN α ₂B mainly is distributed in liver, kidney, enteron aisle and the blood, and the liver intake peaked at 5 minutes, accounted for 36.5% of gross activity.Enteron aisle radioactivity from 5 ' to 30 ' rises gradually, and peaks 30 ' time, and radioactivity begins to descend subsequently.The radioactivity that can infer enteron aisle thus mainly comes from liver.The radioactivity of kidney reached maximum ingestion 10.2% in 5 minutes, reduce explanation subsequently gradually ¹²⁵I-2Gal-IFN α ₂B can be from renal excretion.In addition, at control group ¹²⁵I-IFN α ₂(see Table 2) among the b, the kidney increased radioactivity is higher, reached in 5 minutes peak value 10.49% with ¹²⁵I-2Gal-IFN α ₂B master's peak value is suitable, shows that it equally also can be from renal excretion, but the liver intake equally also during peaking, only was 11.97% at 5 minutes.Descend gradually subsequently. ¹²⁵I-2Gal-IFN α ₂B with ¹²⁵I-IFN α ₂The ratio of the liver of b picked-up peak value is 3.05: 1, and both are after 120 minutes, ¹²⁵I-2Gal-IFN α ₂The uptake ratio of the liver of b still is higher than ¹²⁵I-IFN α ₂B.Table 1: ¹²⁵I-2Gal-Interferon α ₂Distribution experimental result in the mouse body of b

Time (mins)

5 10 15 30 60 120 blood 12.00 ± 0.93 9.17 ± 2.74 8.93 ± 0.12 8.95 ± 1.12 6.56 ± 0.45 6.56 ± 1.94 heart 0.50 ± 0.13 0.48 ± 0.16 0.46 ± 0.13 0.32 ± 0.03 0.26 ± 0.02 0.33 ± 0.12 liver 36.50 ± 2.54 31.2 ± 2.37 23.87 ± 1.93 9.99 ± 1.87 6.53 ± 0.18 4.37 ± 0.36 spleens 0.75 ± 0.27 0.87 ± 0.16 0.53 ± 0.13 1.13 ± 0.02 0.63 ± 0.07 0.37 ± 0.01 lung 1.48 ± 0.28 1.37 ± 0.39 1.33 ± 0.21 1.48 ± 0.46 1.11 ± 0.17 0.87 ± 0.29 kidney 10.20 ± 1.18 8.42 ± 0.48 6.63 ± 0.32 5.15 ± 0.62 3.78 ± 0.12 2.79 ± 0.32 intestines 4.06 ± 2.11 4.51 ± 1.18 4.02 ± 0.09 6.17 ± 0.51 4.67 ± 1.23 1.49 ± 1.79 tables 2:¹²⁵I-2Gal-Interferon α ₂Distribution experimental result in the mouse body of b

Time (mins)

5 10 15 30 60 120 blood 15.94 ± 0.37 11.96 ± 0.75 10.06 ± 0.49 8.05 ± 0.52 6.53 ± 1.09 3.82 ± 0.59 heart 0.85 ± 0.08 0.51 ± 0.01 0.50 ± 0.07 0.34 ± 0.03 0.28 ± 0.03 0.20 ± 0.01 liver 11.97 ± 0.26 10.97 ± 1.87 7.03 ± 1.37 5.75 ± 1.67 3.53 ± 0.50 2.42 ± 0.12 spleen 1.12 ± 0.25 0.93 ± 0.12 0.81 ± 0.29 0.56 ± 0.31 0.43 ± 0.12 0.37 ± 0.03 lung 3.62 ± 1.18 2.00 ± 0.09 2.21 ± 0.24 1.72 ± 0.56 1.71 ± 0.28 0.85 ± 0.18 kidneys 10.49 ± 1.28 7.42 ± 1.44 6.16 ± 0.69 3.65 ± 0.76 2.01 ± 0.03 1.39 ± 0.14 intestines 7.99 ± 4.20 8.28 ± 0.61 9.49 ± 3.73 6.57 ± 0.77 6.59 ± 0.77 4.36 ± 0.97 embodiment, 2 digalactosyl amine chain bridge interferon (rIFN α₁) conjugate (T ₂) preparation:

Digalactosyl amine chain bridge reagent M ₂Synthetic: with diethyl malonate is starting raw material; through formaldehyde condensation; the decarboxylation of acetone cyclisation post-heating gets (7); get (9) with back combination of Li-Al hydrogen reduction with propylene; sulfonylation gets disulfonic acid diol ester (11) after the deprotection open loop; with sodium iodide handle (11) diiodide (12), be dissolved in diiodide (12) 379mg (1mmol) in 20ml acetone and the 12ml distilled water after, add 2-S-(2; 3; 4,6-four-O-acetyl-O-galactopyranose base)-and the different sulphur arteries and veins of 2-hydrobromate 920mg (4mmol) adds salt of wormwood 579mg (4.2mmol) subsequently, and the room temperature lucifuge stirred after 20 hours; the concentrating under reduced pressure reaction solution; add the 40ml chloroform in resistates, 20ml washes once, anhydrous sodium sulfate drying; concentrating under reduced pressure; resistates is through silica gel column chromatography, and sherwood oil-acetone (2: 1) wash-out gets colorless oil M ₂450mg, yield 53%.IR:2936,2334,1752cm ^-1HNMR (200MH _zInCDCl ₃): 5.42 (2H, d, 2XCHS), 5.15 (2H, t, 2XCHOAC), 5.03 (2H, dd, 2XCHOAC), 4.50 (2H, d, 2XCHOAC), 4.13 (4H, m, 2XCH ₂OAC), 3.96 (2H, m, 2XCH..0), 3.59 (4H, m, 2XOCH ₂), 2.85 (4H, m2XSCH ₂), 2.61 (2H, t, CH ₂CN), 2.15 (6H, s, 2XCH ₃CO), 2.07 (6H, s, 2XCH ₃CO), 2.06 (6H, S, 2XCH ₃CO), 2.04 (6H, s, 2XCH ₃CO), 1.97 (6H, s, 2XCH ₃CO), 1.92 (1H, m, CH) ppm digalactosyl fat chain bridge Interferon, rabbit (rINF α ₁) conjugate (T ₂) preparation: with 9.2mg (0.1mmol) M ₂Be dissolved in the 40ml anhydrous methanol, under the stirring at room, add the 8mg sodium methylate, after the stirring at room 48 hours, volatilize methyl alcohol, 20ml phosphoric acid buffer (pH8) is added in the residue with argon nitrogen stream, the dissolving back transfers to pH8 with 1NHCl, under 4 ℃, adds Interferon, rabbit (rINF α ₁) 4mg, 4 ℃ were stirred 24 hours, and free sugar and inorganic salt are removed in the reaction solution dialysis, sterile filtration, frost drying gets T ₂Embodiment 3 three galactosyl fat chain bridge Interferon, rabbit (rINF α ₂B) conjugate (T ₃) preparation

Three galactosyl fat chain bridge reagent (M ₃) synthetic:

With the dendroid tetramethylolmethane is starting raw material, with after the vinyl cyanide addition four nitrile things (14), alcohol solves four esters (15) subsequently, get alcohol ester (16) with the borine partial reduction, the sulfonic acid esterification gets (17), with (17) and 2-s-(2,3,4,6-four-O acetyl-β-O-galactopyranose base)-condensation of 2-isothiourea hydrobromate, product gets (M through purification by silica gel column chromatography ₃) its structure identifies that spectroscopic data is as follows: IR:2932,1752,1748cm ^-1HNMR (200MH _zN CDCl ₃), 5.43 (3H, d, 3XCHS), 5.17 (3H, t, 3XCHOAC), 5.04 (3H, dd, 3XCHOAC), 4.32 (3H, d, 3XCHOAC), 4.05 (6H, m, 3XCH ₂OAC), 3.92 (3H, m, 3XCHO), 3.68 (3H, s, CH ₃OCO), 3.67 (2H, t, OCH ₂) 3.32..3.4Z (14H, m, 7XOCH ₂), 2.76 (6H, m, 3XSCH ₂), 2.55 (2H, t, CH ₂), 2.16 (9H, s, 3XCH ₃CO), 2.11 (9H, s, 3XCH ₃CO), 2.09 (9H, s, 3XCH ₃CO), 2.00 (9H, s, 3XCH ₃CO) ppm, three galactosyl Interferon, rabbit (rINF α ₂B) conjugate (T ₃) preparation: with M ₃200mg is dissolved in the 20ml anhydrous methanol, methanol solution of sodium methylate 4.0ml (0.2mmol) stirring at room of adding 0.1N 8 hours, concentrating under reduced pressure, add 4.0ml water in the resistates and continued stirring at room 10 hours, the phosphoric acid buffer 10.0ml that adds pH=8.0, after transferring to pH8 with the hydrochloric acid of 1N is meticulous, add the EDC 40mg back that stirs and add 4mg genetic engineering interferon (rINF α ₂B), in 4 ℃ of stirring reactions 24 hours, reaction solution is added in the pretreated dialysis tubing, dialyse with the phosphoric acid buffer that contains 0.73% sodium-chlor of 0.1M, changed an extracellular fluid dialysis in per six hours, till detecting the reaction of extracellular fluid dialysis sugar-free with phenol one sulfuric acid process.Reaction solution is after sterile filtration, and lyophilize gets (T ₃).

Description of drawings: Fig. 1 is the two semi-lactosi-interferon alphas of the present invention ₂B (A), interferon alpha ₂The electrophorogram of b (B) and standard molecular weight albumen (C).Fig. 2 is the present invention ¹³¹The iodo-interferon alpha ₂B rabbit whole body radiation video picture figure (5-30 branch).Fig. 3 is the present invention ¹³¹Iodine T ₁(digalactosyl interferon alpha ₂B) rabbit whole body radiation video picture figure (5-30 branch).

Claims (3)

1, a kind of liver target high-carbohydrate density galastosyl interferon conjugate, the two ends that it is characterized in that aromatic ring branch bridge and fat chain branch bridge, generate high-carbohydrate density branch bridge interferon conjugate with glucoside key, peptide bond, the coupled 2-5 of an imido ester bond semi-lactosi and Interferon, rabbit respectively, its chemical structure of general formula as after.

2,, it is characterized in that link coupled semi-lactosi shared molecular ratio in the conjugate molecule is that carbohydrate density should be 4-20: 1 according to the described conjugate of claim 1.

3,, it is characterized in that conjugate should be greater than 30% of dosage at mammiferous liver uptake ratio according to the described conjugate of claim 1. The two semi-lactosi aromatic ring bridge reagent (M of G= ₁) two galactolipid chain bridge reagent (M ₂) X=O, NH three semi-lactosi aromatic ring bridge reagent (M ₃) n=2-6 three galactolipid chain bridge reagent (M ₄)

INF=human leukocyte interferon (INF _α)

Genetic engineering interferon _{α 1}(rINF _{α 1})

Genetic engineering interferon _{α 2}B (rINF _{α 2}B)

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