TW201249874A - An interferon conjugate modified by PEG - Google Patents

Abstract

Herein provided is the interferon conjugates modified by PEG and their preparation, a composition comprising of a therapeutically effective amount of the interferon conjugates modified by PEG and the uses of the interferon conjugates modified by PEG, as well as the uses of the composition described above in the preparation of a medicament for the treatment of Virus affections and cancers, or as an immune regulators.

Description

201249874 VI. Description of the Invention: [Technical Field] The present invention relates to a pegylated interferon conjugate synthesized by a novel bio-protein molecule polyethylene glycol modification technique and having resistance Biological activities such as viral infection, anti-tumor, immunomodulation, and the field of application of the present invention relate to the treatment of biochemistry, medicinal chemistry, and human diseases. [Prior Art] Interferon is a group of active proteins with multiple functions. It is an important family of cytokines with broad-spectrum antiviral, anti-cell proliferation, and immunomodulatory effects. Mammalian interferons can be divided into several types: α, cold, τ, ω, etc. Among them, α interferon can be divided into more than ten subtypes. A large number of clinical studies have proved that α-interferon is an important antiviral and anti- Tumor treatment drugs. At present, the most widely used in China is recombinant human interferon a lb, a 2b and a 2a. In addition, Amgen, a new protein engineering drug designed by Amgen, based on the homology of 13 alpha interferons, was approved by the FDA for marketing in 1997. For the treatment of hepatitis C, its viral activity is 5 to 10 times that of a 2b interferon. However, in general, many products obtained by biotechnology (such as genetic recombination), including interferons, enter the organism through parenteral administration to exert their pharmacological effects. Such biomacromolecular drug products for parenteral administration usually have the following problems: (1) Biomacromolecules often have a sensitizing reaction, and antibodies produced in the living body cause serious damage to the organism and Influencing the progress of treatment; (2) Biomacromolecules drug 95622 3 Cao 201249874 The effect of antibodies or the metabolism caused by proteolytic enzymes, resulting in a shortened biological half-coating period; (3) biomacromolecules Poor stability and difficulty in storage. Therefore, whether interferon alb, a 2b and a2a or recombinant interferon (infergen), as a protein drug, due to poor stability, high plasma clearance rate, short half-life in vivo, easy to produce antigen-antibody reaction, etc. in clinical treatment Subject to great limitations, these shortcomings result in the need to frequently inject interferon to achieve an effective plasma treatment concentration; moreover, each injection will cause a large fluctuation in blood concentration 'the peak and valley of drug formation value. This may increase the cost of treatment and the risk of inconvenience and adverse reactions. Therefore, people have tried various drug delivery technologies (Drug Delivery Technology) to improve the efficacy of protein drugs. Currently, the most widely studied drug delivery technology is polyethylene glycol modification technology (PEGN0L0GY). In 1980, Davis et al., U.S. Patent No. 4,179,337, discloses the use of a single molecule of polyethylene glycol having a different degree of polymerization to chemically modify a protein drug while maintaining the biological activity of the drug while reducing the antigenicity of the drug, r〇 Nes et al., published in Applied Biochem and Biotech 11, 142 (1985), disclose polyethylene glycol-modified ribonuclease and superoxide dismutase activated by phenyl formate, increasing the biological half-life of the protein. The above prior art literature reports have shown that polyethylene glycol modification techniques can indeed solve some of the problems associated with parenteral administration of biomacromolecules. There are currently two types of pegylated interferon products on the market, namely PEG-IFNa2b (PEG-INTR0N) from Schering Plough and pEG-IFNa2a (PEGASYS) from H〇ffman-la R0che, 95622

4 201249874 High interferon half-life in the body, reaching weekly injections, so that the bioavailability of interferon has been improved to some extent. These two products have accounted for 60% of the foreign interferon market and have achieved huge economic benefits. SUMMARY OF THE INVENTION It is an object of the present invention to provide a polyethylene glycol modified interferon conjugate having better biological activity and higher bioavailability, the structure of which is as shown in formula (I):

P-NH-CH2-XSYQ (I) wherein P is an interferon, including all types of interferons, and all subclasses of these types, as well as interferon composed of different types and/or subclasses of interferon; Is of any origin, including natural sources, tissue culture or interferon obtained by genetic recombination techniques; P is preferably a natural or artificially recombinant interferon protein, more preferably recombinant human interferon, more preferably recombinant human interference Preferably, a, /3, 7 or ω, a2b; P can be obtained either by techniques disclosed in the art or commercially available; X is -(CH2)k- or -CH2(0CH2CH2)k And k is selected from an integer of 2 to 10, preferably 1 to 4, more preferably 2; Q is an epoxypolyethylene glycol having an average molecular weight of preferably 5,000 to 40,000 doers. More preferably, it is 20,000 Daltons; Y is selected from Y1 to Y12, wherein m and η are each independently selected from an integer of 2 to 10, preferably 2, 95622 5 201249874

Υ3 s-ch2ch2 Υ4 ο S CH2 J—^NH -CH2C Hz 7m Y5 95622 6 201249874 Ο _ S ten ratio —CH2 /m n Y6

O _CH2CH2——S-( CH2 o Y7 〇 ch2ch2——s-{- ch5 /m •NH_ Y8 〇

-NH ch2ch2——s-f- CH2 Y9 *CH〇 •NH— CH2 Y10 o CH2 -N-

CH 2 •NH- m

O

Yll 95622 201249874 or

CH2—π—NH—^ CH2 O ο

NH——CHZ—CH2——Y12. In particular, the invention provides a conjugate of the formula (II): p-nh-ch2-x

(II) wherein P is an interferon, X is -(CH2)k- or -CH2(OCH2CH2)k-, the number of k is selected from 1 to 10, and the number of m, η is selected from 2 to 10, and im is selected from An integer between 100 and 2000. Wherein X is preferably -(CH2)k-, and the number of k may further preferably be from 1 to 4, most preferably 2. It is further preferred for the above preferred embodiment that the present invention also provides a preferred embodiment, the corresponding structural formula is:

f^N^CH2j-C-NH-^CH2^-~NH—j|-^0CH2CH^-0CH3 Ο im is selected from an integer of 450 to 600, wherein Ρ refers to interferon, and further preferably recombination interference Further preferred is recombinant human interferon alpha, preferably recombinant human interferon a 2b. The invention also provides a conjugate of the formula (III): 95622 8 201249874

(III) wherein P is a recombinant human interferon, X is -(CH〇k- or -CH2(0CH2CH2)k-, the number of k is selected from 1 to 10, and the number of m is selected from 2 to 10'mi An integer from 1〇〇 to 2〇〇〇. It is further preferred for the structural formula (III) that the corresponding optimal embodiment needs to satisfy the following conditions simultaneously: P means recombinant human interferon α; _(CH2)k- 'where k is 2; the number of m is selected from 2; m丨 is selected from an integer of 450 to 6〇〇^

The invention also provides a conjugate of the formula (IV): P~NH-CH2-X-S

-〇CH2CH2f〇CH, UV; wherein P means recombinant human interferon, X is _(CH〇k- or _CH2(〇CH2CH2)k-, the number of k is selected from 1 to 10, and the number of m is selected from 2 To l〇, m丨 is selected from an integer between 100 and 2〇〇〇. For the L configuration (IV), it is preferred that the corresponding optimal condition is the recombinant human interferon alpha. ; Χ is the integer k. 'where 1^2; "1 is selected from 2; mi is selected from 450 to 600 months. Also provides a conjugate of structural formula (V): 95622 201249874 p— —N——c2—X—s——s——ch2ch2-(och2ch2^-och3 \ /ml (V) where P is the recombinant human interferon and X is _(CH2)k- or -CH2(0CH2CH2 k), the number of k is selected from 1 to 10, and m is selected from an integer between 100 and 2000. It is further preferred that the conjugate of the formula (V) is a preferred embodiment. It is desirable to simultaneously satisfy the following conditions: P means recombinant human interferon alpha; X is -(CH2)k-, wherein k is 2; mi is selected from an integer of 450 to 600. The present invention also provides a structural formula (VI) Conjugates:

(VI) wherein P is a recombinant human interferon, X is -(CH2)k- or -CH2(OCH2CH2)k-, the number of k is selected from 1 to 10, and the number of m is selected from 2 to 10, and im is selected from An integer between 100 and 2000. It is further preferred for the structural formula (VI) that the corresponding optimal embodiment needs to satisfy the following conditions simultaneously: P means recombinant human interferon α; X is -(CH2)k_, where k is 2; It is selected from 2, m丨 selected from an integer of 450 to 600. The invention also provides a conjugate of the formula (VII): 95622 10 201249874

H2 P-NH——C ——X-S-S

OCH3 (VII) wherein P means that the recombinant human interferon 'X is -(CH2)k- or -CH2(0CH2CH2)k-, the number of k is selected from 1 to 10', and the number of n is each independently selected from An integer from 2 to 10, im is selected from an integer between 1 and 2000. Further preferred for the conjugate of the formula (VII), the corresponding preferred embodiment is required to simultaneously satisfy the following conditions: P means recombinant human interferon alpha; X is -(CH2)k-, wherein k is 2; the number of m, n is selected from 2; mi is selected from an integer of 450 to 600. The present invention also provides a conjugate of the formula (VIII): 0 P-NH —CH 2 —X—s—CH 2 CH 2 — — S — — CH 2 j—^OCH^^j-OCHa (VIII) P refers to recombinant human interferon, X is _(CH2)k_ or 'CH2(0CH2CH2)k-, the number of k is selected from 1 to 10, the number of m is selected from 2 1 1〇, and the vessel is selected from 1〇〇 An integer between 2 and 〇〇〇. It is further preferred for the structural formula (VIII) that the corresponding optimum is carried out: the case needs to satisfy the following conditions simultaneously: p means recombinant human interferon. ;χ 疋—CUi2)k-, the integer of 9 i _ in k county. The number of m is selected from 2; mi is selected from 450 to the present invention. A conjugate of the formula (IX) is further provided: 95622 11 201249874 P-NH-CH2-XS-CH2CH2 ch2 ch2 foCH2CH24-〇CH3 (IX Wherein P refers to recombinant human interferon, X is _(CH2)k_ or -CH2(OCH2CH2)k-, the number of k is selected from 1 to 10, m, the number of n is selected from 2 to 10, and mi is selected from An integer between 100 and 2000. It is further preferred for the structural formula (IX) that the corresponding optimal embodiment needs to satisfy the following conditions simultaneously: P means recombinant human interferon alpha; X is -(CH2)k-, wherein k is 2; m, The number of η is selected from 2; m丨 is selected from an integer of 450 to 600. The present invention also provides a conjugate of the formula (X): Ο ο 丨丨Μ ί — Λ-- Ρ NH CH2 X S'CH2CH2· S CH2 ^ NH CH2CH2—^OCH2〇H2·^~OCH3 〇 (X) wherein P is a recombinant human interferon, X is -(CH2)k- or -CH2(0CH2CH2)k-, the number of k is selected from 1 to 10, and the number of m is selected from 2 to 10, and mi is selected from An integer between 100 and 2000. It is further preferred that the conjugate of the formula (X) is further preferred, and the corresponding embodiment needs to satisfy the following conditions simultaneously: P means recombinant human interferon 0!; 乂 is -((:}12) 1?-, wherein 1^ is 2; the number of 111 is selected from 2, 1111 is selected from an integer from 450 to 600. The present invention also provides a conjugate of the formula (XI): 95622 12 S) 201249874 F NH —*-CH5—XH / n / \ b, CH2—|—N—(CH2 ^OCH2CH2^〇ch3 o (XI) where p is a recombinant human interferon and X is _(CH2)k_ or -C^ The number of OCHOA-'k is selected from u10, the number of m is selected from 2 to 10, and m丨 is selected from an integer between 1 〇〇 and 2 。. Further preferred for structural formula (XI) is corresponding Preferably, the embodiment requires that the following conditions are met: p means that the recombinant human interferon alpha χ is -(CH2)k-, wherein k is 2; the number of m is selected from 2, and m is selected from an integer from 450 to 600. The present invention also provides a conjugate having the general formula (χπ): p_nh_CH2_x_s__CH2_^nh^ch2) JLnh^CH2^_nh_b. CH3 〇ml ο (XII) wherein Ρ refers to recombinant human interferon, X is _(CH〇k_ or -CH2(0CH2CH2)k-, and the number of k is selected from ^...^ each independently selected from 2 to An integer of 10, mi is selected from an integer between 1 〇〇 and 2 。. The conjugate of the formula (ΧΠ) is further preferably, and the corresponding optimum embodiment needs to satisfy the following conditions simultaneously. :P means recombinant human interferon ¢2 ·' X is -(CH2)k-, where k is 2; m, n is selected from 2; im is selected from an integer from 450 to 600. The invention also provides a structure Condensation of the formula ( 95): 95622 13 201249874 ρ_ΝΗ—CH2—S—CH2, ^略Ο ο

ΝΗ一CH2CH 2—^och2ch

Och3 (XIII) wherein P is a recombinant human interferon, X is -(CH2)k- or -CH2(0CH2CH2)k-'k is selected from i to 1〇, and the number of m and n are each independently selected from An integer from 2 to 10, mi is selected from an integer between 1 〇〇 and 2 。. Further preferred for the conjugate having the structural formula of «丨丨^, the corresponding optimal embodiment needs to satisfy the following conditions simultaneously: p refers to recombinant human interferon α 'X疋-(CH2), Wherein k is 2; the number of m, n is selected from 2; mi is selected from an integer from 450 to 600. Still another object of the present invention is to provide a method for preparing the interferon conjugate, which comprises two stages: first, an interferon and an aldehyde containing a protected sulfhydryl group are reacted to form _NH_CH2_ The linked interfering activated interferon protein; then, the activated interferon is deprotected, coupled to a reactive methoxypolyethylene glycol street bios, and isolated. The steps include: (1) preparing a small molecule aldehyde compound of the formula (XIV): (XIV) wherein the number of k is selected from 2 to 10, preferably 2; (2) structural formula The small molecular aldehyde compound of (XIV) is reacted with interferon in a buffer, and a reducing agent is added to obtain an activation of the structural formula (XV).

S 201249874 Interferon; 0 P——N——c^—^-c^-s---CH3 (XV) wherein P refers to interferon, preferably recombinant human interferon a 2b, pH selection of buffer From 4.0 to 7.0, preferably 4.5, the reducing agent may be sodium borohydride, sodium cyanoborohydride, preferably sodium cyanoborohydride; (3) activated by means of techniques well known in the art. Buffering of interferon. The deprotecting agent is added to the liquid to remove the ethyl sulfhydryl protecting group of the activated interferon of the formula (XV), followed by the activated methoxypoly polyethylene glycol of the formula (XVI). The PEGylation reaction, AG- (XVI) wherein the deprotecting agent is preferably hydroxylamine; the pH of the buffer system is selected from 5.0 to 7.0, preferably pH 6. 2;

S_s— ο

95622 15 201249874 Ο Ο o H2c=c——S- CH2 m H2C=C- s——h CH, H2C=S- s——l· CH, .NH-f~ CH2 -NH- o 〇• NH- CH2CH2_ CH2—η—NH—(CH2

CH2-η NH-(CH2 ju-NH-CH2——CH2- wherein the number of m, n is selected from 2 to 10, preferably 2; (4) purification method of pegylated interferon conjugate Techniques well known to those skilled in the art, such as ion exchange chromatography, gel chromatography. In the method for preparing the interferon conjugate of the present invention, a small molecule aldehyde compound of the formula (XIV) can be disclosed as disclosed in the literature. A method is known, for example, from US 4,338,435. It is yet another object of the present invention to provide an interference containing 95622 16 that is modified.

A pharmaceutical composition of a conjugate of 201249874 comprising: (1) a therapeutic amount of a pegylated interferon conjugate provided by the present invention; (2) a pharmaceutically acceptable drug carrier. The interferon conjugate provided by the present invention can be prepared into a pharmaceutical composition suitable for injection by a method known in the art using a pharmaceutically acceptable carrier or excipient, and the pharmaceutical composition to be used can be formulated into a preparation and, as appropriate, Medical practices are administered in a consistent manner. The compounds of the invention may be formulated in 10 mM sodium phosphate/potassium buffer pH 7 containing 132 mM sodium hydride. Optionally, the pharmaceutical composition may contain a preservative. The pharmaceutical composition may contain varying amounts of interferon, e.g., 10-1000 micrograms per milliliter, such as 50 micrograms or 4 micrograms. Further, it is an object of the present invention to provide the use of the interferon conjugates and pharmaceutical compositions containing them for the preparation of antiviral infection, antitumor, immunomodulatory drugs. The above diseases specifically include: chronic hepatitis B, hepatitis B, hepatitis and hepatitis E; viral diseases such as vesicular treatment, flat and sharp wet fatigue, papillomavirus infection, epidemic hemorrhagic fever and pediatric respiratory syncytial virus Pneumonia, etc.; malignant tumors such as hairy cell leukemia, chronic myeloid leukemia, melanoma, lymphoma, multiple myeloma, renal cell carcinoma, ovarian cancer, rectal cancer, liver cancer, lung cancer, and the like. The biological's tongue of the interferon or the pegylated interferon conjugate provided by the present invention can be determined by the color reaction of the host cell pathogenic effect test. The CytoPathic Effect (or CPE) test method was proposed by F〇ti (Methods in Enzymology, 119, 533, 198). This method utilizes the principle that the human host cell is treated with interferon to produce a virus-resistant lacquer and thus prevents cytopathic death. This method is simple and quick. The color absorption reaction method of the method is as follows: After treatment with interferon, it can resist viral invasion and thus prevent host cell death. The biological activity of the interferon 6^/L virus can be directly measured by the dye absorbed by the viable cells. 1. The resulting cytopathic inhibition method is performed by the foot-VSV secret ingredient. , is a method well known in the art, specifically refer to the _ version of the "Pharmacopoeia of the People's Republic of China" three appendix xc "the biological activity of interferon determination,. Μ矣 = 22 provided by pegylated interferon conjugate test The conjugated product of the smuggling + operation I 彳明 can achieve the purpose of repairing a 2b έίιΐΓ, the surface amino acid of the knife and retaining more interference, biological activity and providing good drug metabolism characteristics. In the compound provided by Lutiti, the interferon and the linker are cautiously linked by r', thereby enhancing the specificity of the PEG modification reaction. [Embodiment] Example of preparation of acetamidine-based propyl group γ Add 11:2 g (0.2 mmol) of chlorpyrifos and dried i〇〇mi Shan to the reaction flask to 'cold part to Q〇c, then slowly add Us(n〇1) guadai B I/, ml Mixed solution of THF. After the addition of the reaction, the reaction was carried out 2 The amount of the secret is then quickly applied to the column (the eluent is purely calcined - the calcined acid is used, Μ), and the product is collected and concentrated under reduced pressure to dryness to give an oily liquid, 0.6 g. Preparation 95622

S 18 201249874 Take 600 mg of interferon a 2b (from Shanghai Hengrui Pharmaceutical Co., Ltd.), the protein concentration is 1.0 mg / m 1, a total of 600 m 1, the system is 0.1 Μ acetic acid buffer, pH 4 5; Take 39.6 mg of ethionyl-propyl-propionic acid dissolved in 400 ul of acetonitrile and add the above protein solution; then weigh 756 mg of cyano-sodium hydride into the above reaction solution, and stir the reaction slowly, and adjust the ice bath. The reaction temperature was 10 ° C, and the reaction was carried out for 4 hours; then the reaction solution was transferred to a dialysis bag (molecular weight cutoff 3500), and dialyzed against 0.1 Μ sodium phosphate buffer solution containing 2 mM EDTA, pH 6. 25 to remove An excess of small molecule aldehyde is then added to the hydroxylamine to remove the ethyl thiol to activate the interferon. Description: The following Examples (3 to 8) are the procedures in which the activated interferon obtained in Example 2 was modified with a living polyethylene glycol to obtain pegylated interferon. The code and structure of the target are shown in the table below, where IFN is activated interferon a2b : 95622 19 201249874

Code structure remarks HH-IFN-001, ΡΝ ~^~ΗΥ^0 Η Η '〇PEG molecular weight is 5kD HH-IFN-002 IFN J~^~七Η O PEG molecular weight lOkD HH-IFN-003 IFN Jn ~^ ~7Η '6 Η Ο PEG molecular weight 20kD HH-IFN-004 Η 〇 PEG molecular weight 40kD HH-IFN-005 IFN, 八^s, s+/^o 七* Η PEG molecular weight 20kD HH-IFN-006 H 5 PEG molecular weight is 20 kD Example 3: Preparation of HH-IFN-001 100 mg of activated interferon a 2b obtained by Example 2 (1.0 mg/ml, 0.1 Μ sodium nitrate buffer, containing 2 mM EDTA, pH 6. 25), adding 0.25 g of mPEG-maleamide (5 kD from SUNBIO), stirring for 60 minutes; adding N-mercaptomaleimide to a concentration of 5 mM, room temperature The reaction was carried out for 30 minutes, and the remaining thiol group on the protein was reacted; then, the reaction solution was dialyzed into a 20 mM acetate buffer system. The reaction solution was purified by ion exchange chromatography (SP Sepharose Η. P), 95622 20

S 201249874 gave pegylated interferon (HH-IFN-001), about 17.5 mg, and the molecular weight was 24.6 KD by MALDI-T0F-MS. Example 4: Preparation of HH-IFN-002 1 〇〇mg activating interferon a2b containing free thiol obtained in Example 2 (1.0 mg/ml, 0.1 M sodium phosphate buffer, containing 2 mM EDTA, pH 6 25), adding g. 5gmPEG-maleamide (l〇kD from SUNBIO), stirring the reaction for 60 minutes; adding N-methyl maleimine to a concentration of 5 mM, reacting at room temperature for 30 minutes' The remaining sulfhydryl groups on the protein were reacted; the reaction solution was then dialyzed into a 20 mM acetate buffer system. The reaction mixture was purified by ion exchange chromatography (SP Sepharose Η. P) to give pegylated interferon (HH-IFN-002), about 15.3 mg, and the molecular weight was 29.9 KD by MALDI-TOF-MS. Example 5: Preparation of HH-IFN-003 1 〇〇mg activating interferon a2b containing free weigen obtained in Example 2 (1.0 mg/ml, 0.1 M sodium phosphate buffer, containing 2 mM EDTA, pH 6.25), add 1·〇gmPEG-maleamide (2〇kD from SUNBIO), stir the reaction for 60 minutes; then add N-mercaptopurine to the concentration of 5 mM, room temperature The reaction was carried out for 30 minutes, and the remaining thiol group on the protein was reacted; then, the reaction solution was dialyzed into a 20 mM acetate buffer system. The MWDI-TOF-MS has a molecular weight of 40. 3KD, which is obtained by MALDI-TOF-MS. . Example 6: Preparation of HH-IFN-004 1 mg of activated interferon containing free thiol group obtained from Example 2 95622 21 201249874 a2b (1.0 mg/ml '0.11 Μ sodium phosphate buffer, containing 2 mM EDTA 'pH 6. 25), add 2·OgmPEG-maleamide (40kD from SUNBIO) 'mix reaction for 60 minutes' and then add N-mercaptomarine to a concentration of 5 mM ' room temperature reaction The remaining thiol group on the protein was reacted for 30 minutes; then the reaction solution was dialyzed into a 20 mM acetate buffer system. The reaction solution was purified by ion exchange chromatography (SP Sepharose HP) to obtain pegylated interferon (HH- The MW-60 was measured by MALDI-TOF-MS. The molecular weight was 60. 1KD. Example 7: Preparation of HH-IFN-005 To 100 mg of activated interferon a 2b (1.0 mg/ml, 0.1 M sodium phosphate buffer) containing free thiol obtained in Example 2, containing 2 mM EDTA, pH 6. 25), add 1. 〇g mPEG-o-pyridyl-disulfide (20KD from SUNBIO), stir the reaction for 60 minutes' and then add N-methyl maleimide to a concentration of 5 mM, room The remaining sulfhydryl groups on the protein were reacted by warm reaction for 30 minutes; the reaction solution was then dialyzed into a 20 mM acetate buffer system. The reaction mixture was purified by ion exchange chromatography (SP Sepharose H.P.) to obtain pegylated interferon (HH-IFN-005), which was about 16. 〇 mg, and the molecular weight was 41. 0 KD by MALDI-TOF-MS. Example 8: Preparation of HH-IFN-006 100 mg of activated interferon a 2b containing free sulfhydryl groups obtained in Example 2 (1.0 mg/ml, 0.1 Μ sodium phosphate buffer, containing 2 mM EDTA, pH 6. 25), add 1. 〇g moth amine ~ mPEG (20KD, from NOj? CORPORATION), stir the reaction for 60 minutes, then add N-mercaptoma uglyimine to a concentration of 5 mM, The reaction was carried out for 30 minutes at room temperature, and the remaining 95622 22 201249874 thiol was reacted on the protein; then the reaction solution was dialyzed into a 20 mM acetate buffer system. The reaction mixture was purified by ion exchange chromatography (SP Sepharose Η. P) to obtain pegylated interferon (HH-IFN-006), which was about 16.5 mg, and the molecular weight was 39. 8 KD by MALDI-T0F-MS. Example 9: In vitro antiviral activity assay of interferon a2b and 6 pegylated interferon conjugates The in vitro antiviral activity of interferon was determined by the WISH-VSV system using the cytopathic inhibition method. This example measures the in vitro antiviral activity of seven interferons (or derivatives), specifically including: IFNa2b (provided by Shanghai Hengrui Pharmaceutical Co., Ltd.), HH-IFN-001 (prepared by Example 3 of the present invention), HH -IFN-〇〇2 (prepared in Example 4 of the present invention), HH-IFN-003 (prepared in Example 5 of the present invention), HH-IFN-004 (prepared in Example 6 of the present invention), hh_iFN_〇〇5 (this) Inventive Example 7 Preparation), HH-IFN-006C (Prepared in Example 8 of the invention). The results of in vitro antiviral activity assay are shown in Table 1: 95622 23 201249874 Table 1 In vitro antiviral activity assay results Name specific activity (xl08IU/mg) Activity retention (%) IFNa2b 1. 18 HH-IFN-001 0. 18 15% HH- IFN-002 0. 15 13% HH-IFN-003 0. 13 11% HH-IFN-004 0.08 7% HH-IFN-005 0. 12 — 10% HH-IFN-006 0. 11 9°/〇· — The data in Table 1 shows that the pegylated interferon a 2b conjugate provided by the present invention retains about 7 to 15% of antiviral activity compared to the pre-modified interferon α 2b. Example 10. Comparison of serum antigen concentration and pharmacokinetics of a single subcutaneous administration of a test article HH_ifn-003 and a reference product Peglntron in a cynomolgus monkey. The serum-test can be determined using a Hu-IFN-α ELISA kit. The concentration of the product HH-IFN-003 and the reference comparator Peglntron. Hu_IFN_a

Immunoassay is an amplified double-sandwich antibody immunoassay test procedure in accordance with the kit instructions '96-well plate pre-coated with iFN-α monoclonal antibody' separately added to the test article HH-IFN-003 and reference product pegIntr〇n For a certain period of time, wash the plate, add antibody dilution, seal the plate, incubate at room temperature, wash the plate, add the HRP (horseradish peroxidase) coupling, seal the plate, incubate at room temperature, wash thoroughly Plate, finally add chromogenic substrate at room temperature in the dark to incubate straight 95622 24

S 201249874 The color appears; the stop solution is added to terminate the reaction, and the absorbance at 45 〇 nm is measured by a microplate reader. The logarithm of the absorbance value is positively correlated with the logarithm of the concentration of HH_IFN_003 *PegInt· in the sample. The logarithm of the logarithm of the concentration and the logarithm of the absorbance are linearly regressed, and a standard curve is drawn to calculate the unknown sample concentration. Experimental results·Compared with the single subcutaneous administration of Peglntron, 10#g · kg-Ι, the reference concentration of Hh ifn 〇〇3 in the test group was greater than the serum antigen concentration at each time point. (4) Add group (see Figure υ, (〇-_, and AUC (0~inf) are higher than peglntr〇n group, pharmacokinetic parameters see I2. After single subcutaneous administration of surface, HH-IFN-003 The amount of exposure in vivo was higher than that of pegIntr〇n. Table 2 Single subcutaneous injection of cynomolgus monkey HH_IFN 〇〇3 and -PegIntroni%i^ mechanical parameters comparison parameters unit test ασ HH~IFN-003 Peglntron _ · kg&quot ;1 10 " g · kg'1 AUC(〇-96h) ng-h-mL 1 1167.0 ±143.5 782. 9 ±304. 2 AUC(o-inf) ng.h.mL1 1187.3 ±146.1 803. 2 ± 304. 4 Example 11: Comparison of serum antiviral activity of cynomolgus monkeys in a single subcutaneous administration of 10 g·kg-1 HH-IFN-003 and Peglntron. PEG-IFNa-2b and IFNa-2b activity assay using cells The lesion inhibition test method (CPE method) uses WISH cells as the host cells of the virus (susceptible cells), and the cells will have a cytopathic effect after being attacked by the virus. 95622 25 201249874 (CPE) 'The cells develop lesions, die, and fall off from the bottom of the culture dish. Under the intervention of antiviral active drugs, the cells without lesions are attached to the culture dish. 'Using crystal violet dye' can stain the living cells, and then use the decolorizing solution. Dye extraction 'Response to the viable state of cell attachment on the culture dish by measuring its 0D value. WISH cells in logarithmic growth phase were digested, and the cell concentration was adjusted with MEM medium containing 10% FBS, and inoculated into 96 wells. In the culture plate, 3rc, 5% C (h, incubate for 24 hours. After washing with pBS, add serum containing the test substance HH_IFN_〇〇3 or reference Peglntron diluted in 2% FBS in MEM medium. Samples, three wells per concentration, and no drug control wells' continued to culture for 24 hours. Add 100% TCID50 (50% cell infection) virus diluted in 2% FBS MEM medium, and set no The virus infects normal cell wells and virus-infected control group, and continues to culture for 24 hours. After neutral red staining, the nail is fixed. After washing, the strip is dissolved with decolorizing solution, and the absorbance (0D) value is obtained at 54 〇 nm. Sample to be tested Against the cytopathic effect (CPE) of the virus infection of protection is calculated interferon contact against viral infection inhibition rate (%), and concentration to give half the effective protection (EC50). Experimental results: The absolute value of serum antiviral activity at each time point after cynomolgus monkey single subcutaneous administration of 1 〇 V g · kg-1 Η Η -1F N - 0 0 3 was higher than that of the same dose of Peglntron. See Table 3 and Figure 2. This result indicates that since the blood concentration of HH-IFN-003 is higher than that of the same dose of pegintron, the antiviral efficacy in vivo is also superior to that of Peglntron.

95622 26 S 201249874 Table 3 Comparison of serum antiviral activity-time change in cynomolgus monkeys after single-dose Peglntron and HH-IFN-003 - Antiviral activity (IU · mL_1) Time point 10 " g · kg-丨10&quot g · kg_1 HH-IFN-003 Peglntron 0 ND ND 4 184. 7±89. 7 164. 7±36. 9 12 223.0+132.4 210.7±30. 9 24 180.7±113. 4 122. 7±17. 48 24.0+12.0 19. 7±7.4 96 5.0+1.0 3.3+3.2 [Simple description of the diagram] Figure 1 shows the same dose of HH-IFN-003 and PEG-IFNa -2b (Peglntron, a single subcutaneous administration of the crab. Serum antigen concentration _ time change comparison chart after Schering. Figure 2 is a comparison of serum antiviral activity-time changes after a single subcutaneous administration of 10/z g*kg-l HH-IFN-003 and Peglntron. [Main component symbol description] None 0 95622 27

Claims (1)

201249874 VII. Patent application scope: 1. An interferon conjugate of the formula (I), P-NH-CH2-XSYQ (I) wherein P is an interferon; X is -(CH2)k- Or -CH2(0CH2CH〇k-, k is selected from an integer from 1 to 10; Q is methoxypolyethylene glycol; Y is selected from Y1 to Y12: ^J^n^ch2^nh-(ch2)-W-j-- S-CH2CH2 Y4 95622 1 201249874 Ο S Y5 0 S -^CH2 ^-NH —(cH2 |-NH /mn Y6 O •CH2CH2——Sf CH2 o Y7 _ch2ch2——s ch2 )——u——nh- Y8 0 ch2ch2——sf-ch2 /m -NH- Y9 •CH〇•NH— CH2 Y10 95622 2 201249874 Ο "ch2- 0 R—( CH〇τη •NH—fCH2H-NH· 01 Yll 'NH— CH2 or 〇O m 0 -ch2—ch2—NH Y12 wherein m and n are each independently selected from an integer between 2 and 10 。. The interferon conjugate of claim 1, wherein the coupling The compound is selected from the compounds of formula (II) to (XIII): (II) Η 〇(III) 'I'icHaVl P-NH-CH2-X-S-U 'C~NH fm (士~(ocl h2ch才och3 mi (IV) 95622 3 201249874 P——NC -XSS-ΟΗ2〇Η2- ^〇〇Η2〇Η2^_ΟΟΗ3 (V) X-S—-f-CH2j—{-〇CH2CH2j—OCH3 2, m. : a SS~fCH2)-C-NH+-CH2|—NH—rrf〇CH2CH2—OCH; m, (VII) o P- NH-CH 2- (-S-CH2CH2〆*^-y CH2 j^^CH2CH2J-OCH3 O (VIII) P-NH-CHz-X——S- •CH2〇H2〆》~f CH2 -NH-一·f* ch2 rNH_ ^OCHzCHzJ-OCHa (IX) o "S, CH2CH2 - NHCH2CH2-^f OCH2CH2 十OCH3 (X) 95622 P——NH——CH2—X——s, 〇V o 4 i2Vc /mi N-1 ch2 )——f och2ch2 十-och3 m (XI) 201249874 (XIII) wherein, P is an interferon, X is -(CH2)k- or -CH2(0CH2CH2)k-, k is selected from 1 to 10, and m and η are each independently selected from an integer between 2 and 10' Im is selected from an integer between 100 and 2000. 3. The interferon conjugate of claim 1 or 2, wherein the interferon is selected from any of a variety of interferons and subclasses thereof, or interferes with different types and/or subclasses a composite interferon composed of primes. 4. The interferon conjugate of claim 3, wherein the interferon is recombinant human interferon alpha, point, 7 or ω. 5. The interferon conjugate of claim 4, wherein the interferon is recombinant human interferon alpha. 6. The interferon conjugate of claim 4, wherein the interferon is recombinant human interferon a 2b. 7. The conjugate according to any one of claims 4 to 6, wherein m = 2 and η = 2. 8. The conjugate of claim 7, wherein χ is - and k is an integer between 1 and 4. 9. The conjugate of claim 8, wherein k=2. The interferon conjugate of claim 8 or 9, wherein mi is selected from an integer between 450 and 600. 11. The interferon conjugate of claim 10, wherein each methoxypolyethylene glycol group has an average molecular weight of from 5,000 to 40,000 Daltons. 12. The interferon conjugate of claim 11, wherein each of the decyloxy polyethylene glycol groups has an average molecular weight of 20,000 Daltons. A method for producing an interferon conjugate according to any one of claims 1 to 12, which comprises the steps of: 1) reacting an interferon with an aldehyde having a protected sulfhydryl group to form a lend An activated interferon protein linked by a -NH-CH2- linkage; 2) the activated interferon protein is deprotected and coupled to a reactive methoxypolyethylene glycol derivative. 14. The method of claim 13, wherein the aldehyde substance is a small molecule aldehyde compound of the formula (XIV), (XIV) wherein the number of k is selected from an integer between 2 and 10. 15. The method of claim 14, wherein the number of k is two. 16. The method of any one of claims 13 to 15, wherein the active oxirane polyethylene glycol derivative is an activated oxirane polyethylene glycol of the formula (XVI), 95622 6 (XVI)201249874 mi AG selected from: CN4'2tr 〇〇0 』H2 fl H h2 h2 [I N-fc t-11-N-C -C - 0 -^1· S- O -s-s N o -s—so H2C=C——S -( CH2 m H o 95622 H2C=C- -S——L CH, H2C===h" s——l· CH, 7 -NH-f- CH2 ) 7 • NH· o _NH- CH2CH2_ 201249874 CH2—π—NH——^ CH2 O o CH2-π NH-(CH2 j--NH-CH2-CH2- wherein the number of m, n is selected from an integer between 2 and 10. 17. The method of claim 16, wherein m, The number of η is 2. 18. The method of claim 13, comprising the steps of: (1) preparing a small molecule aldehyde compound of the formula (XIV), if Η2, Π H—Jl V c -h-S—^-C^ k (XIV) wherein the number of k is selected from an integer between 2 and 10; (2) the small molecule aldehyde compound of the formula (XIV) and the interferon are in a buffer Reaction, and adding a reducing agent to obtain activating interferon of the formula (XV), 0 P - N - c ^ - ^ - C ^ s - - CH3 (XV) wherein P means interferon; Adding a deprotecting agent to the buffer containing activated interferon to remove the acetamino protecting group of the activated interferon of the formula (XV), followed by the addition of the activated oxime of the formula (XVI) to 95622 8 201249874 The polyethylene glycol is PEGylated to obtain a polyethylene glycol interferon conjugate, and AG-mi (XVI) AG is selected from the group consisting of: 〇i4h2^h_h2_h2 m _s_s_c2-0 -S——S 0 s—s-fS2iLa_^2^R ο 95622 H2C==h—so 〇H2C=Cs——f- ch2 9 CH2 m −NH-f- CH2 )- •NH_ o 201249874 HzC=c- s——(-CH, •CH〇O • NH- CH0CH〇-NH— CH2 O •CH〇-NH- CH2 -NH—fCH2^-NH- OOO •CH 〇-NH——CH2-NH—CH2——CH2 O wherein the number of m and n is selected from an integer between 2 and 10; (4) Purification of the pegylated interferon conjugate. The method of claim 18, wherein the number of k is 20. The method of claim 18, wherein P is recombinant human interferon a 2b. 21. as described in claim 18 The method of the present invention, wherein the number of m and η is 2. 22. A pharmaceutical composition comprising: 1) a therapeutically effective amount of PEGylation according to any one of claims 1 to 12. Interferon conjugate; 2) Pharmaceutically acceptable pharmaceutical carrier. Use of the polyethylene glycolated interferon conjugate of any one of claims 1 to 12 for the preparation of an antiviral, antitumor, immunomodulatory drug. 95622 10 201249874 24. The use of a pegylated interferon conjugate as described in claim 23, for the treatment of chronic hepatitis B, hepatitis C and hepatitis E, herpes zoster , flat and condyloma acuminata, papillomavirus infection, epidemic hemorrhagic fever, pediatric respiratory syncytial virus pneumonia, hairy cell leukemia, chronic myeloid leukemia, melanoma, lymphoma, multiple bone hammer tumor, renal cell carcinoma, ovary Drugs for cancer, rectal cancer, liver cancer, and lung cancer. 25. Use of the pharmaceutical composition of claim 22 for the preparation of an antiviral, antitumor, immunomodulatory drug. 26. The use of a pharmaceutical composition according to claim 24, for the treatment of chronic hepatitis B, hepatitis C and hepatitis E, herpes zoster, flat and condyloma acuminata, papilloma Viral infection, epidemic hemorrhagic fever, pediatric respiratory syncytial virus pneumonia, hairy white jk disease, chronic myeloid leukemia, melanoma, lymphoma, multiple myeloma, renal cell carcinoma, ovarian cancer, rectal cancer, liver cancer, Drugs for lung cancer. 95622 11 S