TWI555758B - An interferon conjugate modified by peg - Google Patents

An interferon conjugate modified by peg Download PDF

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TWI555758B
TWI555758B TW101118668A TW101118668A TWI555758B TW I555758 B TWI555758 B TW I555758B TW 101118668 A TW101118668 A TW 101118668A TW 101118668 A TW101118668 A TW 101118668A TW I555758 B TWI555758 B TW I555758B
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interferon
conjugate
integer
formula
activated
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王亞里
呂愛鋒
孫長安
王瑞軍
李蘊波
房雷
徐剛
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江蘇恆瑞醫藥股份有限公司
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Description

一種聚乙二醇干擾素偶聯物 Polyethylene glycol interferon conjugate

本發明涉及一種聚乙二醇干擾素偶聯物，該偶聯物藉由一種新穎的生物蛋白分子的聚乙二醇修飾技術合成，具有抗病毒感染、抗腫瘤、免疫調節等生物學活性，本發明的應用領域涉及生物化學、藥物化學以及人類疾病的治療。 The invention relates to a pegylated interferon conjugate synthesized by a novel bio-protein molecule polyethylene glycol modification technology, which has anti-viral infection, anti-tumor, immunomodulation and other biological activities. The field of application of the invention relates to the treatment of biochemistry, medicinal chemistry and human diseases.

干擾素(Interferon)是一組具有多種功能的活性蛋白質，是一類重要的家族性細胞因數，具有廣譜的抗病毒、抗細胞增殖、和免疫調節作用。哺乳動物的干擾素可以分為α、β、γ、ω等幾種類型，其中α干擾素又可分十餘種亞型，經大量臨床研究證明α型干擾素是一種重要的抗病毒和抗腫瘤治療藥物。目前我國在臨床使用最廣泛的主要是重組人干擾素α 1b、α 2b和α 2a。另外，美國Amgen公司根據13種α型干擾素的基因序列同源性，設計出的一種全新的蛋白質工程藥物幹複津(INFERGEN®，IFN-Con-1)於1997年經FDA批准上市用於治療丙型肝炎，其病毒活性是α 2b干擾素的5至10倍。 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, such as α, β, γ, and ω. Among them, interferon alpha can be divided into more than ten subtypes. A large number of clinical studies have proved that alpha interferon is an important antiviral and antibiotic. Tumor treatment drugs. At present, the most widely used in China is recombinant human interferon α 1b, α 2b and α 2a. Further, according to US Amgen, 13 kinds of gene sequence homology of α interferon, a new design of protein engineering pharmaceutical INFERGEN (INFERGEN ®, IFN-Con- 1) in 1997 and approved for marketing by the FDA for For the treatment of hepatitis C, its viral activity is 5 to 10 times that of α 2b interferon.

但是，一般來說，許多有生物技術(如基因重組)表達所得的產品，包括干擾素，都是藉由非腸道給藥途徑而進入生物體內發揮其藥效作用。此類由非腸道給藥的生物大分子藥物產品通常存在下面的一些問題：(1)生物大分子藥物往往具有致敏性反應，生物體內產生的抗體會對生物體造成嚴重的傷害並影響治療的進行；(2)生物大分子藥物本 身因受到抗體的影響或因蛋白分解酶而引起的代謝作用，致使其生物半衰期大為縮短；(3)生物大分子的穩定性差，保存困難。所以，無論是干擾素α 1b、α 2b和α 2a還是重組集成干擾素(infergen)，作為蛋白質藥物，由於穩定性差，血漿廓清率高，體內半衰期短，易產生抗原抗體反應等，在臨床治療中受到很大的限制，這些缺點造成的結果是：需頻繁注射干擾素才能達到有效的血漿治療濃度；而且，每次注射後均會導致血液濃度的較大波動，形成藥物濃度的峰值與穀值。這樣就可能增加了治療費用以及給藥不便和不良反應的風險。因此，人們試圖採用各種藥物傳遞技術(Drug Delivery Technology)，來提高蛋白質藥物的療效。而目前在藥物傳遞技術中研究最為廣泛的是聚乙二醇修飾技術(PEGNOLOGY)。 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 body can cause serious damage to the organism and affect it. Treatment progress; (2) biomacromolecules The biological half-life is greatly shortened due to the influence of antibodies or the metabolism caused by proteolytic enzymes; (3) the stability of biomacromolecules is poor and storage is difficult. Therefore, whether interferon α 1b, α 2b and α 2a are recombinantly integrated 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 There are great limitations in these, and the result of these shortcomings is that frequent injection of interferon is required to achieve an effective plasma treatment concentration; moreover, each injection will cause a large fluctuation in blood concentration, forming a peak and valley of drug concentration. value. This may increase the cost of treatment as well as the risk of inconvenience and adverse reactions. Therefore, people try to use various drug delivery technologies (Drug Delivery Technology) to improve the efficacy of protein drugs. At present, the most widely studied in drug delivery technology is polyethylene glycol modification technology (PEGNOLOGY).

1980年，Davis等人在美國專利4,179,337中公開了利用單一分子不同聚合度的聚乙二醇對蛋白藥物進行的化學修飾，在保持藥物的生物活性同時，藥物的抗原性降低，Verones等人在Applied Biochem and Biotech 11,142(1985)上公開發表了用氯甲酸苯酯活化的聚乙二醇修飾核糖核酸酶和超氧化物歧化酶，增加了蛋白的生物半衰期。上述現有技術文獻報導已表明聚乙二醇修飾技術的確可以解決非腸道給藥生物大分子藥物存在的一些問題。 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, Verones et al. Applied Biochem and Biotech 11, 142 (1985) disclose polyethylene glycol-modified ribonucleases and superoxide dismutases activated with phenyl chloroformate, which increase the biological half-life of the protein. The above prior art reports have shown that polyethylene glycol modification techniques can indeed solve some of the problems associated with parenteral administration of biomacromolecules.

目前，市場上已經有兩種聚乙二醇化干擾素產品，分別是Schering Plough公司的PEG-IFN α 2b(PEG-INTRON)與Hoffman-1a Roche公司的PEG-IFN α 2a(PEGASYS)，提 高了干擾素在體內的半衰期，達到每週注射一次，使得干擾素的生物利用度得到了一定程度的提升。這兩個產品已佔有國外干擾素市場60%，獲得了巨大的經濟效益。 Currently, there are two kinds of pegylated interferon products on the market, namely PEG-IFN α 2b (PEG-INTRON) from Schering Plough and PEG-IFN α 2a (PEGASYS) from Hoffman-1a Roche. The half-life of interferon in the body is increased, and the weekly injection is achieved, so that the bioavailability of interferon is improved to some extent. These two products have accounted for 60% of the foreign interferon market and have achieved huge economic benefits.

本發明的目的在於提供一種生物學活性更好、生物利用度更高的的聚乙二醇修飾的干擾素偶聯物，其結構如式(I)所示：P-NH-CH2-X-S-Y-Q(I) 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-CH 2 -XSYQ (I)

其中，P是干擾素，包括所有類型的干擾素，以及這些類型所有的亞類，以及不同類型和/或亞類干擾素組成的複合干擾素；P可以是任何來源的，包括天然來源、組織培養或由基因重組技術得到的干擾素；P較佳為天然的或人工重組的干擾素蛋白，更佳為重組人干擾素，更較佳為重組人干擾素α、β、γ或ω，最佳為α 2b；P既可藉由本領域公開的技術制得，也可在市場上購得；X是-(CH2)k-或-CH2(OCH2CH2)k-，k選自2至10的整數，較佳為1制4，進一步較佳為為2；Q為甲氧基聚乙二醇，其平均分子量較佳為5,000至40,000道爾頓，更較佳為20,000道爾頓；Y選自Y1至Y12，其中，m、n各自獨立地選自2至10的整數，較佳為為2， 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; P can be of any origin, including natural sources, tissues An interferon cultured or obtained by genetic recombination technology; P is preferably a natural or artificially recombinant interferon protein, more preferably recombinant human interferon, more preferably recombinant human interferon alpha, beta, gamma or ω, most Preferably, α 2b; P can be obtained by a technique disclosed in the art or commercially available; X is -(CH 2 ) k - or -CH 2 (OCH 2 CH 2 ) k -, k is selected from An integer of 2 to 10, preferably 1 to 4, further preferably 2; Q is a methoxypolyethylene glycol having an average molecular weight of preferably 5,000 to 40,000 Daltons, more preferably 20,000 Dao. Y is selected from Y1 to Y12, wherein m and n are each independently selected from an integer of 2 to 10, preferably 2,

具體而言，本發明提供一種結構通式為(II)的偶聯物： In particular, the invention provides a conjugate of the formula (II):

其中P是指干擾素，X是-(CH2)k-或-CH2(OCH2CH2)k-，k的數目選自1至10，m、n的數目選自2至10，m1選自100至2000之間的整數。 Wherein P is a-interferon, X is - (CH 2) k - or -CH 2 (OCH 2 CH 2) k -, k is the number selected from 1 to 10, m, n is a number selected from 2 to 10, m 1 is selected from an integer between 100 and 2000.

其中X較佳為為-(CH2)k-，k的數目可進一步較佳為自1至4，最佳為的是2。 Wherein X is preferably -(CH 2 ) k -, and the number of k may further preferably be from 1 to 4, most preferably 2.

對上述較佳實施方案進一步較佳為，本發明還提供了最佳的一個實施方案，對應的結構式為： It is further preferred for the above preferred embodiment that the present invention also provides a preferred embodiment, the corresponding structural formula is:

m1選自450至600的整數，其中P是指干擾素，可進一步較佳為重組人干擾素，再較佳為重組人干擾素α，最佳為重組人干擾素α 2b。 m 1 is selected from an integer of 450 to 600, wherein P means interferon, and further preferably recombinant human interferon, more preferably recombinant human interferon alpha, most preferably recombinant human interferon alpha 2b.

本發明還提供一種結構通式為(III)的偶聯物： The invention also provides a conjugate of the formula (III):

其中P是指重組人干擾素，X是-(CH2)k-或-CH2(OCH2CH2)k-，k的數目選自1至10，m的數目選自2至10，m1選自100至2000之間的整數。 Wherein P refers to recombinant human interferon, X is -(CH 2 ) k - or -CH 2 (OCH 2 CH 2 ) k -, the number of k is selected from 1 to 10, and the number of m is selected from 2 to 10, m 1 is selected from an integer between 100 and 2000.

對結構式(III)進一步較佳為，所對應的最佳為實施方案需要同時滿足以下條件：P是指重組人干擾素α；X是-(CH2)k-，其中k是2；m的數目選自2；m1選自450至600的整數。 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 alpha; X is -(CH 2 ) k -, wherein k is 2; The number is selected from 2; m 1 is selected from an integer from 450 to 600.

本發明還提供一種結構通式為(IV)的偶聯物： The invention also provides a conjugate of the formula (IV):

其中P是指重組人干擾素，X是-(CH2)k-或-CH2(OCH2CH2)k-，k的數目選自1至10，m的數目選自2至10，m1選自100至2000之間的整數。 Wherein P refers to recombinant human interferon, X is -(CH 2 ) k - or -CH 2 (OCH 2 CH 2 ) k -, the number of k is selected from 1 to 10, and the number of m is selected from 2 to 10, m 1 is selected from an integer between 100 and 2000.

對結構式(IV)進一步較佳為，所對應的最佳為實施方案需要同時滿足以下條件：P是指重組人干擾素α；X是-(CH2)k-，其中k是2；m的數目選自2；m1選自450至600的整數。 It is further preferred for the structural formula (IV) that the corresponding optimal embodiment needs to satisfy the following conditions simultaneously: P means recombinant human interferon alpha; X is -(CH 2 ) k -, wherein k is 2; The number is selected from 2; m 1 is selected from an integer from 450 to 600.

本發明還提供一種結構通式為(V)的偶聯物： The invention also provides a conjugate of the formula (V):

其中P是指重組人干擾素，X是-(CH2)k-或-CH2(OCH2CH2)k-，k的數目選自1至10，m1選自100至2000之間的整數。 Wherein P refers to recombinant human interferon, X is -(CH 2 ) k - or -CH 2 (OCH 2 CH 2 ) k -, the number of k is selected from 1 to 10, and m 1 is selected from 100 to 2000. Integer.

對結構通式為(V)的偶聯物進一步較佳為，所對應的最佳為實施方案需要同時滿足以下條件：P是指重組人干擾素α；X是-(CH2)k-，其中k是2；m1選自450至600的整數。 Further preferred for the conjugate of the formula (V), the corresponding optimal embodiment needs to satisfy the following conditions: P means recombinant human interferon alpha; X is -(CH 2 ) k -, Wherein k is 2; m 1 is selected from an integer of 450 to 600.

本發明還提供一種結構通式為(VI)的偶聯物： The invention also provides a conjugate of the formula (VI):

其中P是指重組人干擾素，X是-(CH2)k-或-CH2(OCH2CH2)k-，k的數目選自1至10，m的數目選自2至10，m1選自100至2000之間的整數。 Wherein P refers to recombinant human interferon, X is -(CH 2 ) k - or -CH 2 (OCH 2 CH 2 ) k -, the number of k is selected from 1 to 10, and the number of m is selected from 2 to 10, m 1 is selected from an integer between 100 and 2000.

對結構式(VI)進一步較佳為，所對應的最佳為實施方案需要同時滿足以下條件：P是指重組人干擾素α；X是-(CH2)k-，其中k是2；m的數目選自2，m1選自450至600的整數。 The structure of formula (VI) is further preferred, as the corresponding preferred embodiment of the need to meet the following criteria: P refers to a recombinant human interferon α; X is - (CH 2) k -, wherein k is 2; m The number is selected from 2, and m 1 is selected from an integer of 450 to 600.

本發明還提供一種結構通式為(VII)的偶聯物： The invention also provides a conjugate of the formula (VII):

其中P是指重組人干擾素，X是-(CH2)k-或-CH2(OCH2CH2)k-，k的數目選自1至10，m、n的數目各自獨立地選自2至10的整數，m1選自100至2000之間的整數。 Wherein P refers to recombinant human interferon, X is -(CH 2 ) k - or -CH 2 (OCH 2 CH 2 ) k -, the number of k is selected from 1 to 10, and the number of m and n are each independently selected from An integer from 2 to 10, m 1 is selected from an integer between 100 and 2000.

對結構通式為(VII)的偶聯物進一步較佳為，所對應的最佳為實施方案需要同時滿足以下條件：P是指重組人干擾素α；X是-(CH2)k-，其中k是2；m、n的數目選自2；m1選自450至600的整數。 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 -(CH 2 ) k -, Wherein k is 2; the number of m, n is selected from 2; and m 1 is selected from an integer of 450 to 600.

本發明還提供一種結構通式為(VIII)的偶聯物： The invention also provides a conjugate of the formula (VIII):

其中P是指重組人干擾素，X是-(CH2)k-或-CH2(OCH2CH2)k-，k的數目選自1至10，m的數目選自2至10，m1選自100至2000之間的整數。 Wherein P refers to recombinant human interferon, X is -(CH 2 ) k - or -CH 2 (OCH 2 CH 2 ) k -, the number of k is selected from 1 to 10, and the number of m is selected from 2 to 10, m 1 is selected from an integer between 100 and 2000.

對結構式(VIII)進一步較佳為，所對應的最佳為實施方案需要同時滿足以下條件：P是指重組人干擾素α；X是-(CH2)k-，其中k是2；m的數目選自2；m1選自450至600的整數。 It is further preferred for the structural formula (VIII) that the corresponding optimal embodiment needs to satisfy the following conditions simultaneously: P means recombinant human interferon alpha; X is -(CH 2 ) k -, wherein k is 2; The number is selected from 2; m 1 is selected from an integer from 450 to 600.

本發明還提供一種結構通式為(IX)的偶聯物： The invention also provides a conjugate of the formula (IX):

其中P是指重組人干擾素，X是-(CH2)k-或-CH2(OCH2CH2)k-，k的數目選自1至10，m，n的數目選自2至10，m1選自100至2000之間的整數。 Wherein P refers to recombinant human interferon, X is -(CH 2 )k- or -CH 2 (OCH 2 CH 2 ) k -, the number of k is selected from 1 to 10, and the number of n is selected from 2 to 10 m 1 is selected from an integer between 100 and 2000.

對結構式(IX)進一步較佳為，所對應的最佳為實施方案需要同時滿足以下條件：P是指重組人干擾素α；X是-(CH2)k-，其中k是2；m，n的數目選自2；m1選自450至600的整數。 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 -(CH 2 ) k -, wherein k is 2; The number of n is selected from 2; m 1 is selected from an integer from 450 to 600.

本發明還提供一種結構通式為(X)的偶聯物： The invention also provides a conjugate of the formula (X):

其中P是指重組人干擾素，X是-(CH2)k-或-CH2(OCH2CH2)k-，k的數目選自1至10，m的數目選自2至10，m1選自100至2000之間的整數。 Wherein P refers to recombinant human interferon, X is -(CH 2 ) k - or -CH 2 (OCH 2 CH 2 ) k -, the number of k is selected from 1 to 10, and the number of m is selected from 2 to 10, m 1 is selected from an integer between 100 and 2000.

對結構通式為(X)的偶聯物進一步較佳為，所對應的最佳為實施方案需要同時滿足以下條件：P是指重組人干擾素α；X是-(CH2)k-，其中k是2；m的數目選自2，m1選自450至600的整數。 It is further preferred that the conjugate of the formula (X) is further preferred, and the corresponding embodiment is required to simultaneously satisfy the following conditions: P means recombinant human interferon alpha; X is -(CH 2 ) k -, Wherein k is 2; the number of m is selected from 2, and m 1 is selected from an integer of 450 to 600.

本發明還提供一種結構通式為(XI)的偶聯物： The invention also provides a conjugate of the formula (XI):

其中P是指重組人干擾素，X是-(CH2)k-或-CH2(OCH2CH2)k-，k的數目選自1至10，m的數目選自2至10，m1選自100至2000之間的整數。 Wherein P refers to recombinant human interferon, X is -(CH 2 ) k - or -CH 2 (OCH 2 CH 2 ) k -, the number of k is selected from 1 to 10, and the number of m is selected from 2 to 10, m 1 is selected from an integer between 100 and 2000.

對結構式(XI)進一步較佳為，所對應的最佳為實施方案需要同時滿足以下條件：P是指重組人干擾素α；X是-(CH2)k-，其中k是2；m的數目選自2，m1選自450至600的整數。 It is further preferred for the structural formula (XI) that the corresponding optimal embodiment needs to satisfy the following conditions simultaneously: P means recombinant human interferon alpha; X is -(CH 2 ) k -, wherein k is 2; m The number is selected from 2, and m 1 is selected from an integer of 450 to 600.

本發明還提供一種結構通式為(XII)的偶聯物： The invention also provides a conjugate of the formula (XII):

其中P是指重組人干擾素，X是-(CH2)k-或-CH2(OCH2CH2)k-，k的數目選自1至10，m、n的數目各自獨立地選自2至10的整數，m1選自100至2000之間的整數。 Wherein P refers to recombinant human interferon, X is -(CH 2 ) k - or -CH 2 (OCH 2 CH 2 ) k -, the number of k is selected from 1 to 10, and the number of m and n are each independently selected from An integer from 2 to 10, m 1 is selected from an integer between 100 and 2000.

對結構通式為(XII)的偶聯物進一步較佳為，所對應的最佳為實施方案需要同時滿足以下條件：P是指重組人干擾素α；X是-(CH2)k-，其中k是2；m、n的數目選自2；m1選自450至600的整數。 Further preferred for the conjugate of the formula (XII), the corresponding optimal embodiment needs to satisfy the following conditions: P means recombinant human interferon alpha; X is -(CH 2 ) k -, Wherein k is 2; the number of m, n is selected from 2; and m 1 is selected from an integer of 450 to 600.

發明還提供一種結構通式為(XIII)的偶聯物： The invention also provides a conjugate of the formula (XIII):

其中P是指重組人干擾素，X是-(CH2)k-或-CH2(OCH2CH2)k-，k的數目選自1至10，m、n的數目各自獨立地選自2至10的整數，m1選自100至2000之間的整數。 Wherein P refers to recombinant human interferon, X is -(CH 2 ) k - or -CH 2 (OCH 2 CH 2 ) k -, the number of k is selected from 1 to 10, and the number of m and n are each independently selected from An integer from 2 to 10, m 1 is selected from an integer between 100 and 2000.

對結構通式為(XIII)的偶聯物進一步較佳為，所對應的最佳為實施方案需要同時滿足以下條件：P是指重組人干擾素α；X是-(CH2)k-，其中k是2；m、n的數目選自2；m1選自450至600的整數。 Further preferred for the conjugate of the formula (XIII), the corresponding preferred embodiment is required to simultaneously satisfy the following conditions: P means recombinant human interferon alpha; X is -(CH 2 ) k -, Wherein k is 2; the number of m, n is selected from 2; and m 1 is selected from an integer of 450 to 600.

本發明的又一目的在於提供一種製備所述干擾素偶聯物的方法，該方法主要包括兩個階段：首先是干擾素和含有已保護巰基的醛類物質反應，形成藉由-NH-CH2-鍵連接的活化干擾素蛋白；然後，所述活化的干擾素脫保護，與活性甲氧基聚乙二醇衍生物偶聯，藉由分離而得到。具體而言，其步驟包括：(1)製備結構通式為(XIV)的小分子醛化合物： 其中k的數目選自2至10，較佳為2；(2)將結構通式為(XIV)的小分子醛化合物與干擾素在緩衝液裏反應，並加入還原劑得到結構通式為(XV)的活化 干擾素； 其中P是指干擾素，較佳為重組人干擾素α 2b，緩衝液的pH選自4.0至7.0，較佳為4.5，還原劑可以是硼氰化鈉、氰基硼氰化鈉，較佳為氰基硼氰化鈉；(3)利用本領域公知的技術在含有活化干擾素的緩衝液裏加入脫保護劑脫去結構通式為(XV)的活化干擾素的乙醯基保護基，隨後加入結構通式為(XVI)的活化甲氧基聚乙二醇進行聚乙二醇化反應， 其中加入的脫保護劑較佳為羥胺；緩衝體系的pH選自5.0至7.0，較佳為pH6.2；AG選自： 其中m、n的數目選自2至10，較佳為2；(4)聚乙二醇干擾素偶聯物的純化方法採用本領域技術人員公知的技術，如離子交換層析、凝膠層析。 It is still another object of the present invention 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 by -NH-CH. The 2 -interlinked activated interferon protein; then, the activated interferon is deprotected, coupled to a reactive methoxypolyethylene glycol derivative, and isolated. Specifically, the steps thereof include: (1) preparing a small molecule aldehyde compound of the formula (XIV): Wherein the number of k is selected from 2 to 10, preferably 2; (2) the small molecule aldehyde compound of the formula (XIV) is reacted with an interferon in a buffer, and a reducing agent is added to obtain a structural formula ( XV) activated interferon; Wherein P means interferon, preferably recombinant human interferon alpha 2b, the pH of the buffer is selected from 4.0 to 7.0, preferably 4.5, and the reducing agent may be sodium borohydride or sodium cyanoborohydride, preferably. Is sodium cyanoborohydride; (3) a deprotecting agent is added to the buffer containing the activated interferon to remove the acetamino protecting group of the activated interferon of the formula (XV) by a technique known in the art. Subsequently, an activated methoxypolyethylene glycol having the general formula (XVI) is added for pegylation. The deprotecting agent to be added is preferably hydroxylamine; the pH of the buffer system is selected from 5.0 to 7.0, preferably pH 6.2; and the AG is selected from the group consisting of: Wherein the number of m, n is selected from 2 to 10, preferably 2; (4) the purification method of the pegylated interferon conjugate is carried out by techniques well known to those skilled in the art, such as ion exchange chromatography, gel layer Analysis.

本發明製備所述干擾素偶聯物的方法中，結構通式為(XIV)的小分子醛化合物可按文獻公開的已知方法製備，如US4338435。 In the process for preparing the interferon conjugate of the present invention, a small molecule aldehyde compound of the formula (XIV) can be prepared according to a known method disclosed in the literature, such as U.S. Patent 4,338,435.

本發明的又一目的在於提供一種含有該被修飾的干擾 素偶聯物的醫藥組成物，其包含：(1)本發明所提供的治療量的聚乙二醇化干擾素偶聯物；(2)藥學可接受的藥物載體。本發明提供的干擾素偶聯物可以藉由本領域公知的方法用藥學可接受載體或賦形劑製成適合注射的醫藥組成物，欲使用的醫藥組成物可製成製劑，並按與合適的醫療慣例一致的方式給藥。本發明的化合物可以在含有132 mM氯化鈉的10 mM磷酸鈉/鉀緩衝液pH7中配製。任選地，醫藥組成物可以含有防腐劑。醫藥組成物可以含有不同量的干擾素，例如10-1000微克/毫升，例如50微克或400微克。 It is still another object of the present invention to provide an interference containing the modification A pharmaceutical composition of a conjugate comprising: (1) a therapeutic amount of a pegylated interferon conjugate provided by the present invention; (2) a pharmaceutically acceptable pharmaceutical 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 chloride. Optionally, the pharmaceutical composition may contain a preservative. The pharmaceutical composition may contain varying amounts of interferon, for example 10-1000 micrograms per milliliter, such as 50 micrograms or 400 micrograms.

進一步，本發明的目的在於提供所述干擾素偶聯物以及含有它們的醫藥組成物在製備抗病毒感染、抗腫瘤、免疫調節藥物中的用途。上述疾病具體包括：慢性乙型肝炎、丙型肝炎和戊型肝炎等；病毒性疾病如帶狀皰疹、扁平和尖銳濕疣、乳頭瘤病毒感染、流行性出血熱和小兒呼吸道合胞病毒肺炎等；惡性腫瘤如毛細胞性白血病、慢性粒細胞白血病、黑色素瘤、淋巴瘤、多發性骨髓瘤、腎細胞癌、卵巢癌、直腸癌、肝癌、肺癌等。 Further, it is an object of the present invention to provide use of the interferon conjugate and a pharmaceutical composition containing the same for the preparation of an antiviral infection, antitumor, immunomodulatory drug. The above diseases specifically include: chronic hepatitis B, hepatitis C and hepatitis E; viral diseases such as herpes zoster, flat and condyloma acuminata, papillomavirus infection, epidemic hemorrhagic fever and pediatric respiratory syncytial virus pneumonia 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.

干擾素或本發明提供的聚乙二醇干擾素偶聯物的生物活性可由宿主細胞致病效應測試法的顏色反應而確定。宿主細胞致病效應(CytoPathic Effect,or CPE)測試法系由Foti提出(Methods in Enzymology,119,533,198)。本法利用人類宿主細胞經干擾素處理後產生抵抗病毒感染的活性而能防止細胞病變死亡的原理設計而成的一種簡易快捷 的顏色吸收反應測試方法。簡單而言，宿主細胞在經干擾素處理後可抵抗病毒入侵因而可防止宿主細胞死亡。干擾素的抗病毒生物活性即可經由存活細胞所吸收的染料而直接定量測量。由此而來的採用細胞病變抑制法藉由WISH-VSV系統測定干擾素的體外抗病毒活性，是本技術領域公知的方法，具體參照2005版《中華人民共和國藥典》三部附錄XC“干擾素的生物活性測定”。 The biological activity of the interferon or the peginterferon 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 Foti (Methods in Enzymology, 119, 533, 198). This method is simple and quick to design a principle that the human host cell is treated with interferon to produce anti-viral infection and can prevent cytopathic death. The color absorption reaction test method. Briefly, host cells are resistant to viral invasion after interferon treatment and thus prevent host cell death. The antiviral biological activity of the interferon can be directly quantified by the dye absorbed by the viable cells. Thus, the in vitro antiviral activity of interferon by the WISH-VSV system using the cytopathic inhibition method is a well-known method in the art, and specifically refers to the 2005 edition of the Pharmacopoeia of the People's Republic of China, the three appendix XC "interferon. Biological activity assay".

藉由對本發明提供的聚乙二醇干擾素偶聯物的測試資料表明：本發明提供的聚乙二醇干擾素偶聯物可以達到修飾干擾素α 2b分子表面胺基酸的目的且保留較多的干擾素α 2b的生物活性並提供良好的藥物代謝特性。 The test data of the pegylated interferon conjugate provided by the present invention indicates that the pegylated interferon conjugate provided by the present invention can achieve the purpose of modifying the surface amino acid of the interferon α 2b molecule and retaining The interferon alpha 2b is biologically active and provides good drug metabolism properties.

在本發明所提供的化合物中，干擾素與連接體以-NH-CH2-鍵形式連接，由此增強了PEG修飾反應的專一性。 In the compounds provided herein, the interferon linker to -NH-CH 2 - linkages form, thereby enhancing the specificity of PEG-modified reaction.

實施例1：乙醯基巰基丙醛的製備 Example 1: Preparation of acetyl hydrazinopropanal

將11.2 g(0.2 mmol)丙烯醛與乾燥的100 ml THF加入到反應瓶中，冷卻至0℃，然後緩慢滴加1.52 g(0.02 mol)硫代乙酸/20 ml THF的混合溶液。滴加完畢保溫反應2小時後。35℃減壓濃縮除去過量的丙烯醛。然後快速上柱(洗脫液純正己烷→正己烷/乙酸乙酯=50/1)，合併收集產物，減壓濃縮至乾得油狀液體0.6 g。 11.2 g (0.2 mmol) of acrolein and dry 100 ml of THF were added to the reaction flask, cooled to 0 ° C, and then a mixed solution of 1.52 g (0.02 mol) of thioacetic acid / 20 ml of THF was slowly added dropwise. After the dropwise addition, the reaction was allowed to stand for 2 hours. The excess acrolein was removed by concentration under reduced pressure at 35 °C. Then, the column was quickly applied (eluent pure n-hexane → n-hexane / ethyl acetate = 50/1), and the product was collected, and concentrated to dryness to give an oily liquid (0.6 g).

實施例2 活化干擾素的製備 Example 2 Preparation of activated interferon

取600 mg干擾素α 2b(來自上海恒瑞醫藥有限公司)，蛋白濃度為1.0 mg/ml，共600 ml，體系為0.1 M乙酸緩衝液，pH 4.5；取39.6 mg乙醯基巰基丙醛溶於400 ul乙腈後加入以上蛋白溶液；再稱取756 mg氰基硼氫化鈉加入上述反應液，並慢速攪拌反應，冰浴調控反應溫度在10℃，反應4小時；然後將反應液轉入透析袋(截留分子量3500)，對0.1 M磷酸鈉鹽緩衝液，含2 mM EDTA，pH 6.25進行透析，以去除過量的小分子醛，然後再加入羥胺脫去乙醯基，得活化干擾素。 Take 600 mg of interferon α 2b (from Shanghai Hengrui Pharmaceutical Co., Ltd.), the protein concentration is 1.0 mg / ml, a total of 600 ml, the system is 0.1 M acetic acid buffer, pH 4.5; take 39.6 mg acetamidopropionaldehyde Add the above protein solution after 400 ul of acetonitrile; add 756 mg of sodium cyanoborohydride to the above reaction solution, and stir the reaction slowly, adjust the reaction temperature at 10 ° C for 4 hours in an ice bath; then transfer the reaction solution The dialysis bag (molecular weight cutoff 3500) was dialyzed against 0.1 M sodium phosphate buffer, containing 2 mM EDTA, pH 6.25 to remove excess small molecule aldehyde, and then hydroxylamine was added to remove the ethyl thiol to activate the interferon.

說明：以下實施例(3至8)為將實施例2得到的活化干擾素用活性聚乙二醇修飾，並得到聚乙二醇干擾素的過程。目標物的代號及結構示意圖見下表，其中IFN是活化干擾素α 2b： 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 alpha 2b:

實施例3：HH-IFN-001的製備 Example 3: Preparation of HH-IFN-001

藉由實施例2得到的100 mg活化干擾素α 2b(1.0 mg/ml，0.1 M磷酸鈉鹽緩衝液，含2 mM EDTA，pH 6.25)，加入0.25 g mPEG-馬來醯胺(5kD，來自SUNBIO)，攪拌反應60分鐘；再加入N-甲基馬來醯亞胺至濃度為5 mM，室溫反應30分鐘，將蛋白上剩餘的巰基予以反應；然後將反應液透析至20 mM乙酸緩衝液體系。 100 mg of activated interferon alpha 2b (1.0 mg/ml, 0.1 M sodium phosphate buffer, containing 2 mM EDTA, pH 6.25) obtained in Example 2, was added 0.25 g mPEG-maleamide (5 kD from SUNBIO), the reaction was stirred for 60 minutes; N-methyl maleimide was added to a concentration of 5 mM, and reacted at room temperature for 30 minutes to react the remaining thiol groups on the protein; then the reaction solution was dialyzed to 20 mM acetate buffer. Liquid system.

反應液使用離子交換層析(SP Sepharose H.P)純化， 得到聚乙二醇化干擾素(HH-IFN-001)，約17.5mg，經MALDI-TOF-MS檢測分子量為24.6KD。 The reaction solution was purified by ion exchange chromatography (SP Sepharose H.P.). The pegylated interferon (HH-IFN-001) was obtained, which was about 17.5 mg, and the molecular weight was 24.6 KD by MALDI-TOF-MS.

實施例4：HH-IFN-002的製備 Example 4: Preparation of HH-IFN-002

向實施例2得到的含有游離巰基的100 mg活化干擾素α 2b(1.0 mg/ml，0.1M磷酸鈉鹽緩衝液，含2 mM EDTA，pH 6.25)，加入0.5 g mPEG-馬來醯胺(10kD，來自SUNBIO)，攪拌反應60分鐘；再加入N-甲基馬來醯亞胺至濃度為5 mM，室溫反應30分鐘，將蛋白上剩餘的巰基予以反應；然後將反應液透析至20 mM乙酸緩衝液體系。 To 100 mg of activated interferon alpha 2b (1.0 mg/ml, 0.1 M sodium phosphate buffer, containing 2 mM EDTA, pH 6.25) containing free thiol obtained in Example 2, 0.5 g of mPEG-maleamide was added ( 10kD, from SUNBIO), stir the reaction for 60 minutes; add N-methyl maleimide to a concentration of 5 mM, react at room temperature for 30 minutes, react the remaining sulfhydryl groups on the protein; then dialyze the reaction solution to 20 mM acetate buffer system.

反應液使用離子交換層析(SP Sepharose H.P)純化，得到聚乙二醇化干擾素(HH-IFN-002)，約15.3 mg，經MALDI-TOF-MS檢測分子量為29.9KD。 The reaction solution was purified by ion exchange chromatography (SP Sepharose H.P.) to obtain pegylated interferon (HH-IFN-002), about 15.3 mg, and the molecular weight was 29.9 KD by MALDI-TOF-MS.

實施例5：HH-IFN-003的製備 Example 5: Preparation of HH-IFN-003

向實施例2得到的含有游離巰基的100 mg活化干擾素α 2b(1.0 mg/ml，0.1M磷酸鈉鹽緩衝液，含2 mM EDTA，pH6.25)，加入1.0 g mPEG-馬來醯胺(20kD，來自SUNBIO)，攪拌反應60分鐘；再加入N-甲基馬來醯亞胺至濃度為5 mM，室溫反應30分鐘，將蛋白上剩餘的巰基予以反應；然後將反應液透析至20 mM乙酸緩衝液體系。 100 mg of activated interferon alpha 2b (1.0 mg/ml, 0.1 M sodium phosphate buffer, containing 2 mM EDTA, pH 6.25) containing free thiol obtained in Example 2, and 1.0 g of mPEG-maleamide was added. (20kD, from SUNBIO), stir the reaction for 60 minutes; add N-methyl maleimide to a concentration of 5 mM, react at room temperature for 30 minutes, react the remaining sulfhydryl groups on the protein; then dialyze the reaction solution to 20 mM acetate buffer system.

反應液使用離子交換層析(SP Sepharose H.P)純化，得到聚乙二醇化干擾素(HH-IFN-003)，約16.0 mg，經MALDI-TOF-MS檢測分子量為40.3KD。 The reaction solution was purified by ion exchange chromatography (SP Sepharose H.P.) to obtain pegylated interferon (HH-IFN-003), about 16.0 mg, and the molecular weight was 40.3 KD by MALDI-TOF-MS.

實施例6：HH-IFN-004的製備 Example 6: Preparation of HH-IFN-004

向實施例2得到的含有游離巰基的100 mg活化干擾素 α 2b(1.0 mg/ml，0.1 M磷酸鈉鹽緩衝液，含2 mM EDTA，pH 6.25)，加入2.0g mPEG-馬來醯胺(40kD，來自SUNBIO)，拌反應60分鐘，再加入N-甲基馬來醯亞胺至濃度為5 mM，室溫反應30分鐘，將蛋白上剩餘的巰基予以反應；然後將反應液透析至20 mM乙酸緩衝液體系。 100 mg of activated interferon containing free thiol obtained in Example 2 α 2b (1.0 mg / ml, 0.1 M sodium phosphate buffer, containing 2 mM EDTA, pH 6.25), adding 2.0 g of mPEG-maleamide (40 kD from SUNBIO), mixing for 60 minutes, then adding N- Methyl maleimide was added to a concentration of 5 mM at room temperature for 30 minutes to react the remaining thiol groups on the protein; the reaction solution was then dialyzed into a 20 mM acetate buffer system.

反應液使用離子交換層析(SP Sepharose H.P)純化，得到聚乙二醇化干擾素(HH-IFN-004)，約14.5 mg，經MALDI-TOF-MS檢測分子量為60.1KD。 The reaction solution was purified by ion exchange chromatography (SP Sepharose H.P.) to obtain pegylated interferon (HH-IFN-004), about 14.5 mg, and the molecular weight was 60.1 KD by MALDI-TOF-MS.

實施例7：HH-IFN-005的製備 Example 7: Preparation of HH-IFN-005

向實施例2得到的含有游離巰基的100 mg活化干擾素α 2b(1.0 mg/ml，0.1M磷酸鈉鹽緩衝液，含2mM EDTA，pH 6.25)，加入1.0g mPEG-鄰吡啶基-二硫化物(20KD，來自SUNBIO)，攪拌反應60分鐘，再加入N-甲基馬來醯亞胺至濃度為5 mM，室溫反應30分鐘，將蛋白上剩餘的巰基予以反應；然後將反應液透析至20 mM乙酸緩衝液體系。反應液使用離子交換層析(SP Sepharose H.P)純化，得到聚乙二醇化干擾素(HH-IFN-005)，約16.0 mg，經MALDI-TOF-MS檢測分子量為41.0KD。 To 100 mg of activated interferon α 2b (1.0 mg/ml, 0.1 M sodium phosphate buffer containing 2 mM EDTA, pH 6.25) containing free thiol obtained in Example 2, 1.0 g of mPEG-o-pyridyl-disulfide was added. (20KD from SUNBIO), stir the reaction for 60 minutes, then add N-methyl maleimide to a concentration of 5 mM, react at room temperature for 30 minutes, react the remaining sulfhydryl groups on the protein; then dialyze the reaction solution Up to 20 mM acetate buffer system. The reaction solution was purified by ion exchange chromatography (SP Sepharose H.P.) to obtain pegylated interferon (HH-IFN-005), about 16.0 mg, and the molecular weight was 41.0 KD by MALDI-TOF-MS.

實施例8：HH-IFN-006的製備 Example 8: Preparation of HH-IFN-006

向實施例2得到的含有游離巰基的100 mg活化干擾素α 2b(1.0 mg/ml，0.1 M磷酸鈉鹽緩衝液，含2 mM EDTA，pH 6.25)，加入1.0g碘乙醯胺-mPEG(20KD，來自NOF CORPORATION)，攪拌反應60分鐘，再加入N-甲基馬來醯亞胺至濃度為5 mM，室溫反應30分鐘，將蛋白上剩餘的 巰基予以反應；然後將反應液透析至20 mM乙酸緩衝液體系。 To 100 mg of activated interferon α 2b (1.0 mg/ml, 0.1 M sodium phosphate buffer, containing 2 mM EDTA, pH 6.25) containing free thiol obtained in Example 2, 1.0 g of iodoacetamide-mPEG was added ( 20KD from NOF CORPORATION), stir the reaction for 60 minutes, then add N-methyl maleimide to a concentration of 5 mM, react at room temperature for 30 minutes, and leave the remaining protein. The thiol was reacted; the reaction was then dialyzed into a 20 mM acetate buffer system.

反應液使用離子交換層析(SP Sepharose H.P)純化，得到聚乙二醇化干擾素(HH-IFN-006)，約16.5 mg，經MALDI-TOF-MS檢測分子量為39.8KD。 The reaction solution was purified by ion exchange chromatography (SP Sepharose H.P.) to obtain pegylated interferon (HH-IFN-006), about 16.5 mg, and the molecular weight was 39.8 KD by MALDI-TOF-MS.

實施例9：干擾素α 2b及6種聚乙二醇干擾素偶聯物的體外抗病毒活性測試 Example 9: In vitro antiviral activity test of interferon alpha 2b and six pegylated interferon conjugates

採用細胞病變抑制法藉由WISH-VSV系統測定干擾素體外抗病毒活性。 The in vitro antiviral activity of interferon was determined by the WISH-VSV system using cytopathic inhibition.

本實施例測定了7種干擾素(或衍生物)的體外抗病毒活性，具體包括：IFN α 2b(上海恒瑞醫藥股份有限公司提供)、HH-IFN-001(本發明實施例3製備)、HH-IFN-002(本發明實施例4製備)、HH-IFN-003(本發明實施例5製備)、HH-IFN-004(本發明實施例6製備)、HH-IFN-005(本發明實施例7製備)、HH-IFN-006(本發明實施例8製備)。體外抗病毒活性測定結果見表1： This example measures the in vitro antiviral activity of seven interferons (or derivatives), specifically including: IFNα 2b (provided by Shanghai Hengrui Pharmaceutical Co., Ltd.), HH-IFN-001 (prepared by Example 3 of the present invention) , HH-IFN-002 (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-005 (this) Inventive Example 7 Preparation), HH-IFN-006 (Prepared in Example 8 of the present invention). The results of in vitro antiviral activity assay are shown in Table 1:

表1中資料顯示，本發明所提供的聚乙二醇干擾素α 2b偶聯物與修飾前的干擾素α 2b相比，仍保留有約7至15%的抗病毒活性。 The data in Table 1 shows that the peginterferon alfa 2b conjugate provided by the present invention retains about 7 to 15% of antiviral activity compared to the pre-modified interferon alpha 2b.

實施例10：食蟹猴單次皮下給藥受試品HH-IFN-003和參考比較品PegIntron後的血清抗原濃度和藥物代謝動力學比較實驗 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 PegIntron in cynomolgus monkeys

用Hu-IFN-α ELISA藥盒可以測定血清中受試品HH-IFN-003和參考比較品PegIntron的濃度。Hu-IFN-α Immunoassay是一種放大的雙夾心抗體免疫分析法，試驗操作步驟遵照藥盒說明書，96孔板預先用IFN-α的單抗包被，分別加入受試品HH-IFN-003和參比品PegIntron，孵育一定時間，洗板，加抗體稀釋液，封板，室溫孵育，再洗板，另加入攜有HRP(辣根過氧化物酶)耦合物，封板，室溫孵育，徹底洗板，最後加入顯色底物室溫避光孵育直 到出現顏色；加入終止液終止反應，用酶標儀測定450 nm波長處的光吸收值。光吸收值的對數值與樣品中HH-IFN-003和PegIntron的濃度對數值呈正相關，對濃度對數和光吸收值對數進行雙對數線性回歸，繪製標準曲線，計算未知樣品濃度。 The concentration of the test article HH-IFN-003 and the reference comparator PegIntron in serum can be determined using a Hu-IFN-α ELISA kit. Hu-IFN-α Immunoassay is an amplified double-sandwich antibody immunoassay. The test procedure is in accordance with the instructions of the kit. 96-well plates are pre-coated with IFN-α monoclonal antibody and added to the test substance HH-IFN-003 and Reference product PegIntron, incubate for a certain period of time, wash the plate, add antibody dilution, seal the plate, incubate at room temperature, wash the plate, add HRP (horseradish peroxidase) coupling, seal the plate, incubate at room temperature Wash the plate thoroughly, and finally add the chromogenic substrate at room temperature and incubate in the dark. The color appears; the stop solution is added to terminate the reaction, and the absorbance at 450 nm is measured by a microplate reader. The logarithm of the light absorption value is positively correlated with the logarithm of the concentration of HH-IFN-003 and PegIntron in the sample. The logarithm of the logarithm of the concentration and the logarithm of the light absorption value are subjected to a double logarithmic linear regression, and a standard curve is drawn to calculate the unknown sample concentration.

實驗結果：與單次皮下給藥先靈葆雅公司參比品組PegIntron，10μg．kg-1相比，HH-IFN-003受試品組各時間點血清抗原濃度在數值上均大於PegIntron組(見圖1)，AUC(0-96h)，和AUC(0-inf)均高於PegIntron組，藥物代謝動力學參數見表2。表面單次皮下給藥後，HH-IFN-003的體內暴露量要高於PegIntron。 Experimental results: PegIntron, 10 μg, with a single subcutaneous administration of Schering-Plough's reference product group. Compared with kg-1, the serum antigen concentration of the HH-IFN-003 test group was higher than that of the PegIntron group (see Figure 1), AUC (0-96h), and AUC (0-inf). For the PegIntron group, the pharmacokinetic parameters are shown in Table 2. HH-IFN-003 was exposed in vivo to PegIntron after a single subcutaneous administration of the surface.

實施例11：食蟹猴單次皮下給藥10μg．kg-1 HH-IFN-003及PegIntron後血清抗病毒活性比較實驗 Example 11: Single dose of cynomolgus monkey subcutaneously administered 10 μg. Comparative experiment of serum antiviral activity after kg -1 HH-IFN-003 and PegIntron

PEG-IFN α-2b與IFN α-2b活性測定方法採用細胞病變抑制試法(CPE法)，以WISH細胞作為病毒的宿主細胞(易感細胞)，細胞受到病毒感染攻擊後會產生細胞病變效應 (CPE)，細胞發生病變，死亡，並從培養皿底部脫落。在抗病毒活性藥物的干預下，未發生病變的細胞附著在培養皿上，利用結晶紫染料，可使活細胞染色，再用脫色液將染料脫出，藉由測定其OD值來反應細胞附著在培養皿上的存活狀態。 PEG-IFN α-2b and IFN α-2b activity assay using cytopathic inhibition assay (CPE method), WISH cells as viral host cells (susceptible cells), cells will be cytopathic effect after viral infection attack (CPE), the cells develop lesions, die, and fall off the bottom of the dish. Under the intervention of antiviral active drugs, the cells without lesions are attached to the culture dish, and the living cells are stained by the crystal violet dye, and the dye is removed by the decolorizing solution, and the cell adhesion is determined by measuring the OD value. The state of survival on the culture dish.

將處於對數生長期的WISH細胞消化，用含10%FBS的MEM培養液調整細胞濃度，接種於96孔培養板中，37℃，5%CO2，孵育24小時。以PBS清洗後，分別加入以2%FBS的MEM培養液稀釋調配的含有受試品HH-IFN-003或參比品PegIntron的血清樣本，每個濃度設三複孔，並設無藥物對照孔，繼續培養24小時。加入以2%FBS的MEM培養液稀釋調配的100倍TCID50(50%細胞感染量)的病毒，並設無病毒感染正常細胞孔和病毒感染對照組，繼續培養24小時，中性紅染色後甲醛固定，洗滌後用脫色液溶解，在540nm處得到吸光度(OD)值。根據不同稀釋度的待測樣品抵抗病毒感染發生的細胞病變(CPE)的保護能力，計算出干擾素抵抗病毒感染抑制率(%)，並得到半數有效保護濃度(EC50)。 The WISH cells in the logarithmic growth phase were digested, and the cell concentration was adjusted with a MEM medium containing 10% FBS, seeded in a 96-well culture plate, and incubated at 37 ° C, 5% CO 2 for 24 hours. After washing with PBS, serum samples containing the test substance HH-IFN-003 or the reference PegIntron diluted in 2% FBS MEM medium were added, and each concentration was set to three replicate wells, and no drug control wells were set. Continue to train for 24 hours. The virus was diluted with 100% TCID50 (50% cell infection) in 2% FBS MEM medium, and the virus-infected normal cell well and virus-infected control group were added, and the culture was continued for 24 hours. After neutral red staining, formaldehyde was added. After fixation, it was dissolved with a decolorizing solution after washing, and an absorbance (OD) value was obtained at 540 nm. According to the protection ability of the sample to be tested with different dilutions against cytopathic effect (CPE) of viral infection, the inhibition rate (%) of interferon resistance virus infection was calculated, and half effective protection concentration (EC50) was obtained.

實驗結果：食蟹猴單次皮下給藥10μg．kg-1 HH-IFN-003後各時間點血清抗病毒活性的絕對值高於同劑量的PegIntron。見表3、第2圖。這個結果表明，由於HH-IFN-003血藥濃度要高於同劑量的PegIntron，因此其體內的抗病毒藥效也優於PegIntron。 Experimental results: cynomolgus monkeys were given a single subcutaneous dose of 10 μg. The absolute value of serum antiviral activity at each time point after kg -1 HH-IFN-003 was higher than that of the same dose of PegIntron. 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 PegIntron.

第1圖是食蟹猴單次皮下給藥相同劑量HH-IFN-003與PEG-IFN α-2b(PegIntron，先靈葆雅)後的血清抗原濃度-時間變化比較圖。 Figure 1 is a comparison of serum antigen concentration-time changes after a single subcutaneous administration of the same dose of HH-IFN-003 and PEG-IFN α-2b (PegIntron, Schering-Plough) in cynomolgus monkeys.

第2圖是食蟹猴單次皮下給藥10μg．kg-1 HH-IFN-003及PegIntron後血清抗病毒活性-時間變化的比較圖。 Figure 2 is a single subcutaneous administration of cynomolgus monkey 10μg. Comparison of serum antiviral activity-time changes after kg-1 HH-IFN-003 and PegIntron.

由於本案的圖為試驗的結果數據，並非本案的代表圖。故本案無指定代表圖。 Since the picture in this case is the result data of the test, it is not a representative figure of the case. Therefore, there is no designated representative map in this case.

Claims (25)

一種結構如式(I)所示的干擾素偶聯物，P-NH-CH2-X-S-Y-Q(I)其中，P是干擾素；X是-(CH2)k-或-CH2(OCH2CH2)k-，k選自1至10的整數；Q是甲氧基聚乙二醇，其中，每個甲氧基聚乙二醇基團的平均分子量為5,000至40,000道爾頓；Y選自Y1至Y12： 其中m、n各自獨立地選自2至10之間的整數。 An interferon conjugate of the formula (I), P-NH-CH 2 -XSYQ (I) wherein P is an interferon; X is -(CH 2 ) k - or -CH 2 (OCH 2 CH 2 ) k -,k is selected from an integer from 1 to 10; Q is a methoxypolyethylene glycol wherein each methoxypolyethylene glycol group has an average molecular weight of 5,000 to 40,000 Daltons; From Y1 to Y12: Wherein m and n are each independently selected from an integer between 2 and 10. 如申請專利範圍第1項所述的干擾素偶聯物，其中，該偶聯物選自式(II)至(XIII)所示化合物： 其中，P是干擾素，X是-(CH2)k-或-CH2(OCH2CH2)k-，k選自1至10，m、n各自獨立地選自2至10之間的整數，m1選自100至2000之間的整數。 The interferon conjugate of claim 1, wherein the conjugate is selected from the group consisting of compounds of formula (II) to (XIII): Wherein P is an interferon, X is -(CH 2 ) k - or -CH 2 (OCH 2 CH 2 ) k -, k is selected from 1 to 10, and m and n are each independently selected from 2 to 10 An integer, m 1 is selected from an integer between 100 and 2000. 如申請專利範圍第1或2項所述的干擾素偶聯物，其中，該干擾素選自所有類型的干擾素及其亞類中的任意一種，或者不同類型和/或亞類干擾素組成的複合干擾素。 The interferon conjugate according to claim 1 or 2, wherein the interferon is selected from any one of all types of interferons and subclasses thereof, or a different type and/or subclass of interferon composition Composite interferon. 如申請專利範圍第3項所述的干擾素偶聯物，其中，該干擾素是重組人干擾素α、β、γ或ω。 The interferon conjugate of claim 3, wherein the interferon is recombinant human interferon alpha, beta, gamma or ω. 如申請專利範圍第4項所述的干擾素偶聯物，其中，該干擾素是重組人干擾素α。 The interferon conjugate of claim 4, wherein the interferon is recombinant human interferon alpha. 如申請專利範圍第4項所述的干擾素偶聯物，其中，該干擾素是重組人干擾素α 2b。 The interferon conjugate of claim 4, wherein the interferon is recombinant human interferon alpha 2b. 如申請專利範圍第4至6項中任一項所述的偶聯物，其中，m=2，n=2。 The conjugate according to any one of claims 4 to 6, wherein m=2, n=2. 如申請專利範圍第7項所述的偶聯物，其中，X是-(CH2)k-，k為1至4之間的整數。 The conjugate of claim 7, wherein X is -(CH 2 ) k -, and k is an integer between 1 and 4. 如申請專利範圍第8項所述的偶聯物，其中，k=2。 The conjugate of claim 8, wherein k=2. 如申請專利範圍第8或9項所述的干擾素偶聯物，其中，m1選自450至600之間的整數。 The interferon conjugate of claim 8 or 9, wherein m 1 is selected from an integer between 450 and 600. 如申請專利範圍第1項所述的干擾素偶聯物，其中，每個甲氧基聚乙二醇基團的平均分子量為20,000道爾頓。 The interferon conjugate of claim 1, wherein each methoxypolyethylene glycol group has an average molecular weight of 20,000 Daltons. 一種製備申請專利範圍第1至11項中任一項所述干擾素偶聯物的方法，其包括以下步驟：1)干擾素和含有已保護巰基的醛類物質反應，形成藉由-NH-CH2-鍵連接的活化干擾素蛋白；2)該活化的干擾素蛋白脫保護，與活性甲氧基聚乙二醇衍生物偶聯。 A method of preparing an interferon conjugate according to any one of claims 1 to 11, which comprises the steps of: 1) reacting an interferon with an aldehyde having a protected sulfhydryl group to form by -NH- CH 2 - linkages interferon protein activation; 2) the interferon protein activated deprotection, coupling with the active methoxy polyethylene glycol derivative. 如申請專利範圍第12項所述的方法，其中，該醛類物質是式(XIV)的小分子醛化合物， 其中k的數目選自2至10之間的整數。 The method of claim 12, wherein the aldehyde substance is a small molecule aldehyde compound of the formula (XIV), Wherein the number of k is selected from an integer between 2 and 10. 申請專利範圍第13項所述的方法，其中，k的數目為2。 The method of claim 13, wherein the number of k is 2. 如申請專利範圍第12至14項中任一項所述的方法，其中，該活性甲氧基聚乙二醇衍生物是通式為(XVI)的活化甲氧基聚乙二醇， AG選自： 其中m、n的數目選自2至10之間的整數。 The method of any one of claims 12 to 14, wherein the reactive methoxypolyethylene glycol derivative is an activated methoxypolyethylene glycol of the formula (XVI). AG is selected from: Wherein the number of m, n is selected from an integer between 2 and 10. 如申請專利範圍第15項所述的方法，其中，m、n的數目為2。 The method of claim 15, wherein the number of m and n is 2. 如申請專利範圍第12項所述的方法，包括以下步驟：(1)製備結構通式為(XIV)的小分子醛化合物， 其中k的數目選自2至10之間的整數；(2)將結構通式為(XIV)的小分子醛化合物與干擾素在緩衝液裏反應，並加入還原劑得到結構通式為(XV)的活化干擾素， 其中P是指干擾素；(3)在含有活化干擾素的緩衝液裏加入脫保護劑脫去結構通式為(XV)的活化干擾素的乙醯基保護基，隨後加入結構通式為(XVI)的活化甲氧基聚乙二醇進行聚乙二醇化反應，得到聚乙二醇干擾素偶聯物， AG選自： 其中m、n的數目選自2至10之間的整數；(4)純化聚乙二醇干擾素偶聯物。 The method of claim 12, comprising the steps of: (1) preparing a small molecule aldehyde compound of the formula (XIV), Wherein the number of k is selected from an integer between 2 and 10; (2) reacting a small molecule aldehyde compound of the formula (XIV) with an interferon in a buffer, and adding a reducing agent to obtain a structural formula (XV) Activated interferon, Wherein P refers to interferon; (3) a deprotecting agent is added to the buffer containing activated interferon to remove the ethylthio protecting group of the activated interferon of the formula (XV), and then the structural formula is added ( XVI) activated methoxy polyethylene glycol is PEGylated to obtain a pegylated interferon conjugate, AG is selected from: Wherein the number of m, n is selected from an integer between 2 and 10; (4) the purified peginterferon conjugate is purified. 如申請專利範圍第17項所述的方法，其中，k的數目為2。 The method of claim 17, wherein the number of k is two. 如申請專利範圍第17項所述的方法，其中，P是重組人干擾素α 2b。 The method of claim 17, wherein P is recombinant human interferon alpha 2b. 如申請專利範圍第17項所述的方法，其中，m、n的數目為2。 The method of claim 17, wherein the number of m and n is 2. 一種醫藥組成物，其包含：1)如申請專利範圍第1至11項中任一項所述的治療有效量的聚乙二醇化干擾素偶聯物；2)藥學可接受的藥物載體。 A pharmaceutical composition comprising: 1) a therapeutically effective amount of a pegylated interferon conjugate as described in any one of claims 1 to 11; 2) a pharmaceutically acceptable pharmaceutical carrier. 一種申請專利範圍第1至11項中任一項所述的聚乙二醇化干擾素偶聯物的用途，其係用在製備抗病毒、抗腫瘤、免疫調節藥物。 The use of the pegylated interferon conjugate of any one of claims 1 to 11 for the preparation of an antiviral, antitumor, immunomodulatory drug. 如申請專利範圍第22項所述的聚乙二醇化干擾素偶聯物的用途，其係用在製備治療慢性乙型肝炎、丙型肝炎和戊型肝炎、帶狀皰疹、扁平和尖銳濕疣、乳頭瘤病毒 感染、流行性出血熱、小兒呼吸道合胞病毒肺炎、毛細胞性白血病、慢性粒細胞白血病、黑色素瘤、淋巴瘤、多發性骨髓瘤、腎細胞癌、卵巢癌、直腸癌、肝癌、肺癌的藥物。 The use of a pegylated interferon conjugate as described in claim 22, 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 myeloma, renal cell carcinoma, ovarian cancer, rectal cancer, liver cancer, lung cancer . 一種申請專利範圍第21項所述的醫藥組成物的用途，其係用在製備抗病毒、抗腫瘤、免疫調節藥物。 A use of the pharmaceutical composition according to claim 21, which is for the preparation of an antiviral, antitumor, and immunomodulatory drug. 如申請專利範圍第24項所述醫藥組成物的用途，其係用在製備治療慢性乙型肝炎、丙型肝炎和戊型肝炎、帶狀皰疹、扁平和尖銳濕疣、乳頭瘤病毒感染、流行性出血熱、小兒呼吸道合胞病毒肺炎、毛細胞性白血病、慢性粒細胞白血病、黑色素瘤、淋巴瘤、多發性骨髓瘤、腎細胞癌、卵巢癌、直腸癌、肝癌、肺癌的藥物。 The use of the pharmaceutical composition as described in claim 24, for the treatment of chronic hepatitis B, hepatitis C and hepatitis E, herpes zoster, flat and condyloma acuminata, papillomavirus infection, prevalence Hemorrhagic fever, pediatric respiratory syncytial virus pneumonia, hairy cell leukemia, chronic myeloid leukemia, melanoma, lymphoma, multiple myeloma, renal cell carcinoma, ovarian cancer, rectal cancer, liver cancer, lung cancer.
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