JP2014525939A - Pegylated interferon λ1 complex - Google Patents

Abstract

The present application discloses novel pegylated interferon λ1 conjugates (PEG-IFNλ1), methods for producing them, pharmaceutical compositions containing these conjugates, and methods for producing the same. These complexes have an increased blood half-life and duration compared to IFNλ1, and are effective in treating hepatitis B and hepatitis C.

Description

Related applications

  This application claims the benefit of Vietnamese Patent Application No. VN1-2011-02222 filed on August 25, 2011, the entirety of which is incorporated herein by reference.

  In one embodiment, the present application discloses pegylated derivatives of recombinant human interferon λ1 (peginterferon λ1 complex or PEG-IFNλ1), methods for their production, pharmaceutical compositions containing these complexes, and methods for their production.

  Hepatitis C virus (HCV) is a major health problem worldwide and the leading cause of chronic liver disease. It is estimated that at least 180 million people worldwide are chronically infected with HCV. In Vietnam, the proportion of HCV infected people in the population is 4-9%. About 55 to 85% of those who are acutely infected with HCV transition to chronic infection, 5 to 25% of these chronic carriers are at risk of developing cirrhosis after 25 to 30 years, and 30% of those with cirrhosis There is a risk of liver decompensation for over 10 years, and 1-3% each year can develop liver cancer. Epidemiological studies show that HCV is responsible for 40% of patients with final stage cirrhosis and 60% of hepatocellular carcinomas.

Currently, the selected therapy for the treatment of chronic hepatitis C is α-interferon (AI). AI can persistently respond to HCV in about 70% of cases, but these interferons cause many side effects (even with pegylated interferon alpha). These side effects often limit treatment and patient treatment can remain incomplete. Side effects include influenza-like syndromes and hematological effects (eg thalassemia and anemia).
Interferons are currently used for the treatment of many viral diseases such as hepatitis B, hepatitis C, hepatitis D, warts, lepromatous leprosy, chronic leukemia and AIDS. AI is also effective in reducing malignant tumors and treating Kaposi's sarcoma, melanoma, and renal cell carcinoma. In addition, AI can be applied in the prevention and treatment of diseases in cattle and other livestock. For example, AI enhances the activity of vaccines used in the prevention and treatment of foot-and-mouth disease and porcine reproductive and respiratory syndrome.

AI is produced from human cell lines or donor-derived leukocytes cultured in tissue culture medium. However, these methods are time consuming, labor intensive, expensive and not suitable for large scale manufacturing. In addition, there is a risk of sepsis caused by infectious agents from cell lines.
With the development of recombinant DNA technology, we can now produce large quantities of interferon by introducing AI genes into microorganisms. However, these methods also present some advantages and difficulties primarily in the expression process and the large-scale protein production process.

  IL-29 is a member of the helical cytokine family and is a type III interferon. It is also known as interferon λ1 (IFNλ1) and has a high similarity in amino acid sequence with IL-28 (another type III interferon). IL-28 and IL-29 (IFNλ1) have recently been members of a new cytokine family (type I interferon (IFN), the same Jak / Stat signal pathway that drives expression of a common set of genes)) It was described. Accordingly, it is named IFNλ. IFNλ exhibits several common characteristics with type I IFN: antiviral activity, antiproliferative activity and in vivo antitumor activity. Importantly, however, IFNλ binds to separate membrane receptors (consisting of IFNLR1 and IL10R2).

  The main disadvantage of most biological therapeutic uses is that they are administered parenterally (eg intravenous (i.v.), subcutaneous (s.c.), intramuscular (i.m.), etc.). This means that delivery to the patient is accompanied by pain and discomfort. Moreover, because they usually have a very short half-life, biologics need to be administered to patients frequently to maintain therapeutic serum or plasma concentrations of the drug. Injections that cannot be self-administered require frequent visits to clinics and skilled health care workers, making the therapy inconvenient and expensive. Two recombinant forms of human interferon alpha, interferon alpha-2a (Roferon, Roche) and interferon alpha-2b (Intron A, Schering AG), used in the treatment of chronic hepatitis B and C, have a serum half-life. Since it is less than 12 hours (Non-patent Document 1), it is necessary to administer 3 times / week. Also, multiple injections of interferon β-1b (Betaseron) are required to treat patients with multiple sclerosis (MS).

  One method for maintaining a threshold level of a drug in the body, overcoming the need for frequent and high dose injections as described above, is a very successful and widely accepted method. It is to increase its in vivo half-life by conjugating a therapeutic protein with a polymer (eg, polyethylene glycol (PEG or Peg)). Long-chain PEG molecules not only protect them from protease action while reducing the immunogenicity of protein drugs by creating a protective shield around pegylated drug molecules in aqueous solution, They further facilitate increasing the circulating half-life of the drug by increasing its hydrodynamic volume (thus reducing its loss from the renal glomerular network filtration mechanism). After their separation from the protein molecules, the PEG moieties are removed without any structural changes, and their clearance is proportional to their molecular weight.

  Usually, PEG is activated by first activating the PEG moiety and then reacting the activated PEG substance with the amino acid side chain of the protein (eg, a lysine residue and / or N-terminal amino group on the protein). Bind the moiety to the protein. The most frequently used PEG is the monofunctional PEG. This is because this part is resistant to cross-linking and aggregation. One example is disclosed in Patent Document 1 by Davis et al.

U.S. Pat.No. 4,179,337

McHutchison, et al., Engl. J. Med. 1998, 339, 1485-1492; Glue, et al., Clin. Pharmacol. Ther. 2000, 68, 556-567

Summary of invention:
Pegylated interferon λ1 (PEG-IFNλ1) is a pegylated derivative of human recombinant IFNλ1 (polyethylene glycol is bound to IFNλ1 and is also referred to as a “complex”), which is used to treat chronic hepatitis C Useful in treatment. PEG-IFNλ1 extends its half-life in the circulation after infusion into the patient's body by avoiding the action of extracellular enzymes and resisting filtration in the kidney. That is, the conjugate has significantly improved stability, better solubility, and increased circulation half-life and plasma residence time compared to the corresponding non-PEG conjugate IFNλ1.

  Interferon λ1 (IFNλ1, Zcyto21 or IL-28A) is known in the art (eg, US Pat. Nos. 7,038,032, 6,927,040, 7,135,170, 7,157,559 and 7,351,689; and International Publication WO 05/097165, WO 07 / From 012,033, WO 07 / 013,944 and WO 07 / 041,713; all of which are hereby incorporated by reference in their entirety).

In one embodiment, the present application discloses a novel PEG-IFNλ1 conjugate. In one embodiment, the conjugate of the present application has a linear PEG chain structure. Compared to unmodified IFNλ1 (ie, IFNλ1 not conjugated to PEG or mPEG), these complexes have increased circulating half-life and persistence in plasma. Water-soluble PEGs include polyethylene glycol (PEG), monomethoxy-PEG (mPEG), mono-C _1-10 alkoxy-PEG and mono-C _1-3 alkoxy-PEG. These PEGs that may be used may have a molecular weight of about 600-60,000, including, for example, those that are about 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa and 60 kDa. In one embodiment, the PEG used in the complex is mPEG with a molecular weight of 40 kDa.

（式中、RはHまたはC_1-3アルキルであり;mは1、2、3または4であり;nは400〜550の範囲で選択された正の整数であり;LはC_1-10アルキルまたはヘテロアルキルリンカーであり;Xは-O-、-NH-または-S-であり;そして、IFNλ1はインターフェロンλ1である）で示される化合物またはその医薬的に許容される塩を含有する、生理的に活性なPEG-IFNλ1複合体を提供する。一態様において、インターフェロンλ1はヒト組換えインターフェロンである。別の態様において、該IFNλ1は天然または組換えタンパク質であってよい。別の態様において、該IFNλ1は、組織、タンパク質合成、または天然の細胞もしくは組換え細胞を用いた細胞培養といった供給源由来のヒトタンパク質である。さらに別の態様において、IFNλ1はヒト組換えタンパク質である。別の態様において、式Iの複合体インターフェロンλ1はSEQ ID 2である。一実施態様において、PEG鎖は、IFNλ1の第1級アミノ基（例えば、リジン）またはN末端上でアミド結合を介してIFNλ1に結合している。 In one embodiment, Formula I:

Wherein R is H or C _1-3 alkyl; m is 1, 2, 3 or 4; n is a positive integer selected in the range of 400-550; L is C _{1- 10 is} an alkyl or heteroalkyl linker; X is —O—, —NH— or —S—, and IFNλ1 is interferon λ1) or a pharmaceutically acceptable salt thereof Physiologically active PEG-IFNλ1 conjugates are provided. In one embodiment, interferon λ1 is human recombinant interferon. In another embodiment, the IFNλ1 may be a natural or recombinant protein. In another embodiment, the IFNλ1 is a human protein from a source such as tissue, protein synthesis, or cell culture using natural or recombinant cells. In yet another embodiment, IFNλ1 is a human recombinant protein. In another embodiment, the complex interferon λ1 of formula I is SEQ ID 2. In one embodiment, the PEG chain is attached to IFNλ1 via an amide bond on the primary amino group (eg, lysine) or N-terminus of IFNλ1.

からなる群から選択される。 Heteroalkyl is defined as C _1-10 alkyl in which at least one of the carbons of the C _1-10 alkyl is substituted with —O—, —C (═O) —, —NH—, or a combination thereof. Is done. Examples of the combination include —OC (═O) —, —C (═O) O—, —NHC (═O) —, —C (═O) NH— and the like. In one embodiment, n is about 500-550. In another embodiment, n is about 420, 520 or 455. In another embodiment, the molecular weight of the Peg group is about 35 kDa to 45 kDa or about 40 kDa. In another embodiment of the above, m is 1 or 2. C _1-10 alkyl or heteroalkyl may be a linear or branched alkyl or heteroalkyl group. In one embodiment, the C _1-10 alkyl group is a —C (O) — group. In one embodiment, m is 1 and LX- is of the formula:

Selected from the group consisting of

である。 In one embodiment of the above complex, m is 2 and LX- is of the formula:

It is.

In another embodiment of the conjugate, two PEG groups (PEG, alkyl-PEG or m-PEG) are attached to two formamide groups of the above formula (ie, —C (O) NH—). Yes. In another embodiment, X is —NH— or —O— and m is 2. In another embodiment, the linker is attached to two PEG groups. In another embodiment, X is —NH— and the group attached to the linker is a lysine residue on IFNλ1. In one embodiment, the —NH— group (ie, amino group) attached to the linker is a histidine residue. In another embodiment, X is —O— and the group attached to the linker may be derived from a serine residue on IFNλ1. In one variation of the above formula, R is —CH ₃ . In another embodiment, the linker is attached to lysine, serine, histidine or mixtures thereof on IFNλ1. In another embodiment, the linker is attached to a regioisomer of a residue of lysine, serine, histidine or mixtures thereof on IFNλ1. In another embodiment of the complex, R is H or —CH ₃ , m is 1, L is —C (O) —, and X is —NH—. In another embodiment, n is 500-550.

（式中、INFλ1はインターフェロンλ1であり;そして、nは500〜550である）の化合物を含有する。一態様において、nはPEG構造中の多数のエチレングリコールユニットであり、PEG部分の分子量が約40 kDaであるようにいくつかの数から選択される正の整数であり、そして、INFλ1はインターフェロンλ1である。上記の別の実施態様において、該複合体は、IFNλ1と比較して長期化または延長された血清半減期および持続時間を有する。該複合体の別の態様において、該PEGはIFNλ1のN末端にてメチオニンに結合している。さらに別の態様において、該複合体はB型肝炎およびC型肝炎の処置において有効である。 In another embodiment, the conjugate (Nanogen PEG-IFNλ1) has the formula II:

In which INFλ1 is interferon λ1; and n is 500-550. In one embodiment, n is a number of ethylene glycol units in the PEG structure, a positive integer selected from several numbers such that the molecular weight of the PEG moiety is about 40 kDa, and INFλ1 is interferon λ1 It is. In another embodiment of the above, the complex has a prolonged or extended serum half-life and duration compared to IFNλ1. In another embodiment of the conjugate, the PEG is attached to methionine at the N-terminus of IFNλ1. In yet another embodiment, the complex is effective in the treatment of hepatitis B and hepatitis C.

（式中、nはPEG部分の分子量が約40 kDaであるように選択される正の整数である）の通りの結合反応によって(α-メトキシ-ω-(4-ニトロフェノキシカルボニル))ポリオキシエチレン(PEG-pNC)（40 kDa）をIFNλ1と共有結合させる工程;ならびに、複合体を単離する工程を含む。一態様において、nは約500〜550である。 In another embodiment, a method for producing the human recombinant complex disclosed above is provided, the method comprising:

(Wherein n is a positive integer selected such that the molecular weight of the PEG moiety is about 40 kDa) (α-methoxy-ω- (4-nitrophenoxycarbonyl)) polyoxy Covalently coupling ethylene (PEG-pNC) (40 kDa) to IFNλ1; and isolating the conjugate. In one embodiment, n is about 500-550.

In another embodiment, a pharmaceutical composition is provided comprising the above complex and a pharmaceutically acceptable carrier and excipient. Moreover, a normal pharmaceutical formulation can be manufactured using the composition containing the composite_body | complex of this application. The formulation may comprise a composition containing a therapeutically effective amount of the complex together with a pharmaceutically acceptable carrier known in the art. For example, adjuvants, diluents, preservatives and / or solubilizers may be used as necessary. The pharmaceutical composition containing the complex has various buffer (eg Tris-HCl, acetate, phosphate) diluents (with a wide range of pH and ionic strength, eg, as described in US Pat. No. 4,496,537 ), Carriers (eg, human serum albumin), solubilizers (eg, polyoxyethylene sorbitan or TWEEN®, polysorbate), and preservatives (eg, thimerosol, benzyl alcohol). In one embodiment, the pharmaceutical composition is formulated as a sterile lyophilized powder for injection. In another embodiment, the composition contains a combination of pharmaceutically acceptable vehicles (including saline, buffered saline and 5% dextrose). In another embodiment, the pharmaceutical composition is formulated as an infusion solution in a vial or pre-filled syringe. In one embodiment described above, the pharmaceutical composition is used for the treatment of hepatitis B and hepatitis C. Pharmaceutical formulations and methods for making the formulations are well known in the art, for example, “Remington, The Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co., Easton, Pa. 19 ^th ed. 1995. Is disclosed in.

（式中、RはHまたはC_1-3アルキルであり;mは1、2、3または4であり;nは400〜550の範囲で選択される正の整数であり;LはC_1-10アルキルまたはヘテロアルキルリンカーであり;Xは-O-、-NH-または-S-であり;そして、IFNλ1はインターフェロンλ1である）で示される化合物またはその医薬的に許容される塩を含有するPeg-IFNλ1複合体を製造するための方法であって、IFNλ1のアミノ酸残基との共有結合を促進するのに十分な条件下においてIFNλ1を予め活性化させたPegと接触させることを含む方法を提供する。一実施態様において、本明細書に記載の方法により製造されたPEG-IFNλ1複合体を提供する。一態様において、IFNλ1上でのPEGもしくはmPEGの共有結合を促進させるのに十分な条件下においてIFNλ1を十分な量の活性化されたPEGもしくはmPEGと接触させることを含む、上記の複合体を製造する方法を開示する。別の態様において、活性化されたmPEGはmPEG-pNCである。別の態様において、活性化されたmPEGはIFNλ1のN末端のメチオニン上で結合する。別の態様において、該mPEGは約40 kDaの分子量を有する。別の態様において、活性化されたオキシカルボニル剤はモノ-またはジ-活性化剤である。 In another embodiment, Formula I:

Wherein R is H or C _1-3 alkyl; m is 1, 2, 3 or 4; n is a positive integer selected in the range of 400-550; L is C _{1- 10 is} an alkyl or heteroalkyl linker; X is —O—, —NH— or —S—, and IFNλ1 is interferon λ1) or a pharmaceutically acceptable salt thereof A method for producing a Peg-IFNλ1 complex comprising contacting IFNλ1 with a preactivated Peg under conditions sufficient to promote covalent binding to an amino acid residue of IFNλ1 provide. In one embodiment, PEG-IFNλ1 conjugates produced by the methods described herein are provided. In one embodiment, producing the above conjugate comprising contacting IFNλ1 with a sufficient amount of activated PEG or mPEG under conditions sufficient to promote covalent attachment of PEG or mPEG on IFNλ1 A method is disclosed. In another embodiment, the activated mPEG is mPEG-pNC. In another embodiment, the activated mPEG binds on the N-terminal methionine of IFNλ1. In another embodiment, the mPEG has a molecular weight of about 40 kDa. In another embodiment, the activated oxycarbonyl agent is a mono- or di-activator.

  In another embodiment, there is provided a method of inhibiting cancer cell growth in a patient comprising contacting cancer cells with the complex as described above, wherein the complex is prolonged or prolonged compared to IFNλ1. Serum half-life and duration. In one embodiment, the complex of the present application has a serum half-life that is extended by two, three, five, eight, or ten or more times the serum half-life of the corresponding non-complexed IFNλ1.

  In another embodiment, a method of treating a proliferative disorder in a mammal comprising administering to the mammal a therapeutically effective amount of the complex described above is provided. In one embodiment, the complex may be used to treat an interferon sensitive condition or a condition that responds positively or favorably to an interferon-based therapy. In one embodiment, treatment with the complex results in a substantial reduction or elimination of side effects compared to normal treatment with interferon.

  In one aspect, examples of conditions that can be treated using the complexes of the present application include, but are not limited to, cell proliferative disorders, specifically cancer (eg, hairy cell leukemia, Kaposi's sarcoma, chronic myelogenous leukemia). , Multiple myeloma, basal cell and malignant melanoma, ovarian cancer, and cutaneous T-cell lymphoma), and various infections. In another embodiment, the complex may be used to treat conditions that benefit from inhibiting replication of interferon sensitive viruses. Viral infections that may be treated using the complex of the present application include hepatitis A, hepatitis B, hepatitis C, other non-A / non-B hepatitis, herpes virus, Epstein-Barr virus (EBV), Cytomegalovirus (CMV), herpes simplex, human herpesvirus type 6 (HHV-6), papilloma, poxvirus, picornavirus, adenovirus, rhinovirus, human T cell leukemia virus type 1 and 2 (HTLV-1 / -2), human rotavirus, rabies, retrovirus (including human immunodeficiency virus (HIV)), encephalitis, and respiratory virus infection.

（式中、RはHまたはC_1-3アルキルであり;mは1、2、3または4であり;nは500〜550の範囲で選択される正の整数であり;LはC_1-10アルキルまたはヘテロアルキルリンカーであり;Xは-O-、-NH-または-S-であり;そして、IFNλ1はインターフェロンλ1である）の複合体またはその医薬的に許容される塩、あるいは該式Iの複合体を含有する医薬製剤を投与することを含む該方法を提供する。一態様において、該式Iの複合体インターフェロンλ1はSEQ ID 2である。一態様において、哺乳動物はヒトである。該方法の別の態様において、該ウイルス感染症はC型肝炎ウイルスによって引き起こされるか、あるいは該ウイルス感染症によって進行性（advance）肝硬変が引き起こされる。特定の態様において、該患者はHCV耐性もしくは抵抗性患者である。上記方法の別の態様において、該PEG-IFNλ1は毎週約0.5 μg/kg〜10.0 μg/kgの用量で投与される。該方法の一態様において、該PEG-IFNλ1は毎週約2.5 μg/kgの用量で投与される。別の態様において、該PEG-IFNλ1は約8週間〜約52週間投与される。別の態様において、該PEG-IFNλ1は約12週間、約16週間、約20週間、または約24週間投与される。別の態様において、該PEG-IFNλ1は患者が血清中にHCV RNAを有しないと判断されるまで投与される。別の態様において、該方法は好中球数、血小板数またはヘモグロビン濃度の顕著な低下をもたらさないので、該複合体を投与することによって、標準的なPEG-INF-α療法以上に顕著に改善される。上記方法のさらに別の態様において、該方法はさらに、リバビリンまたはビラミジンから選択されるヌクレオシドアナログの投与を含む。該方法の別の態様において、該リバビリンは毎日5 mg/kg〜25 mg/kg;または毎日15 mg/kg〜25 mg/kgの用量で経口投与される。一態様において、該リバビリンは約10 mg/kg〜30 mg/kg（1日1回もしくは2回）、または約15 mg/kg（1日1回もしくは2回）の用量で投与される。上記の一態様において、該複合体は非経口投与される。 In another embodiment, a method of treating a patient infected with or at risk of being infected with a viral infection, wherein the patient has a therapeutically effective amount of Formula I:

Wherein R is H or C _1-3 alkyl; m is 1, 2, 3 or 4; n is a positive integer selected in the range of 500-550; L is C _{1- 10} alkyl or heteroalkyl linker; X is —O—, —NH— or —S—; and IFNλ1 is interferon λ1), or a pharmaceutically acceptable salt thereof, or the formula The method comprises administering a pharmaceutical formulation containing the complex of I. In one embodiment, the complex interferon λ1 of formula I is SEQ ID 2. In one embodiment, the mammal is a human. In another embodiment of the method, the viral infection is caused by hepatitis C virus, or the viral infection causes advanced cirrhosis. In certain embodiments, the patient is an HCV resistant or resistant patient. In another embodiment of the above method, the PEG-IFNλ1 is administered at a dose of about 0.5 μg / kg to 10.0 μg / kg weekly. In one embodiment of the method, the PEG-IFNλ1 is administered at a dose of about 2.5 μg / kg weekly. In another embodiment, the PEG-IFNλ1 is administered for about 8 weeks to about 52 weeks. In another embodiment, the PEG-IFNλ1 is administered for about 12 weeks, about 16 weeks, about 20 weeks, or about 24 weeks. In another embodiment, the PEG-IFNλ1 is administered until it is determined that the patient does not have HCV RNA in the serum. In another embodiment, the method does not result in a significant decrease in neutrophil count, platelet count or hemoglobin concentration, so administration of the conjugate significantly improves over standard PEG-INF-α therapy. Is done. In yet another embodiment of the above method, the method further comprises administration of a nucleoside analog selected from ribavirin or viramidine. In another embodiment of the method, the ribavirin is administered orally at a dose of 5 mg / kg to 25 mg / kg daily; or 15 mg / kg to 25 mg / kg daily. In one embodiment, the ribavirin is administered at a dose of about 10 mg / kg to 30 mg / kg (once or twice daily), or about 15 mg / kg (once or twice daily). In one embodiment of the above, the complex is administered parenterally.

  In one embodiment, a dose of about 200 μg per week of PEG-IFNλ1 conjugate may be used in combination with about 300 mg of tenofovir (tenofovir disoproxil fumarate) per day to treat HBV. In this treatment, most patients are judged to be free of HBV after about 4 injections over about 30 days. Under these conditions, the HBV is known to be suppressed and HBsAg (viral surface antigen) is released, which triggers the immune system to produce antibodies against this antigen, resulting in the specific The optimal end point of treatment is provided. The treatment can be continued for about 12 to 24 weeks, depending on the patient's initial viral load, as described herein.

Definition:
`` Alkyl '' is a straight or branched, saturated or unsaturated, aliphatic radical having a chain of carbon atoms, optionally substituted with an oxygen atom (e.g., C ₁ alkyl is —C (O) - and may be) or is substituted with a nitrogen atom (eg, C ₁ alkyl is -C (NH) - a may be), or a sulfur atom (e.g., C ₂ alkyl is -CH Means an aliphatic radical substituted with ₂ C (S)-). C _X alkyl and C _XY alkyl (eg, C _1-10 alkyl or C _1-6 alkyl) are typically used where X and Y indicate the number of carbon atoms in the chain. For example, C _1-6 alkyl includes alkyl having 1 to 6 carbons (for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, vinyl, isopropenyl, 1-butenyl, ethynyl, 1-propynyl, etc.) Is included.

“Heteroalkyl” or “heteroalkylene” is an alkyl that may have an oxygen, nitrogen or sulfur atom between the carbon atoms. Examples of the heteroalkyl group include -C (O) NH-, -OC (O)-, -CH ₂ CH ₂ C (O) -NH-, -CH ₂ -O-CH ₂ -CH _2- , _{-CH 2 -NH-CH 2 -CH 2} -, - CH 2 -S-CH 2 -CH 2 - and the like, and -CH ₂ O-CH ₂ -CH ₃ and the like.

  The term “PEG” as in “PEG-IFNλ1” means polyethylene glycol as used in the art, and unless otherwise specified generally alkyl-PEG (eg mPEG (methoxy-polyethylene glycol) ) And PEG.

  “Therapeutically effective amount” means a clinically significant change in the treated condition (eg, a clinically significant change in viral load or immune function, a significant decrease in morbidity, or a significant histological score) The amount of PEG-IFNλ1 conjugate sufficient to produce a large increase, or a combination thereof).

  “Treatment” or “treating” as used herein refers to therapeutic treatment and prophylactic measures. Patients in need of treatment include those already infected with hepatitis C virus as well as those who should be prevented from hepatitis C.

  In one embodiment of the above method, the complex is administered by injection or infusion. In another embodiment, the complex is administered intravenously, intramuscularly, subcutaneously, intradermally or intraperitoneally. In another embodiment, the complex is less than 0.5 μg / kg, 0.5-1.0 μg / kg, 1.0-1.5 μg / kg, 1.5-2.0 μg / kg, 2.0-2.5 μg / kg, 2.5-3.0 μg / kg. 3.0-3.5 μg / kg, 3.5-4.0 μg / kg, 4.0-4.5 μg / kg, 4.5-5.0 μg / kg, 5.0-5.5 μg / kg, 5.5-6.0 μg / kg, 6.0-6.5 μg / kg, 6.5-7.0 μg / kg, 7.0-7.5 μg / kg, 7.5-8.0 μg / kg, 8.0-8.5 μg / kg, 8.5-9.0 μg / kg, 9.0-9.5 μg / kg, 9.5-10.0 μg / kg, or The patient is administered at a dose selected from 10.0 μg / kg or more. In another embodiment, the complex is about 60-80 μg, 80-100 μg, 100-120 μg, 120-140 μg, 140-160 μg, 160-180 μg, 180-200 μg, 200-220 μg. , 220-240 μg, 240-260 μg, 260-280 μg, or about 280-300 μg. In one embodiment, the complex is administered subcutaneously at 200 μg for 12 consecutive weeks.

  In another embodiment, there is provided a pharmaceutical composition comprising the complex and a pharmaceutically acceptable carrier and excipient. In another embodiment, the pharmaceutical composition is used for the treatment of hepatitis B and hepatitis C. In another embodiment, there is provided a method for producing a pharmaceutical composition containing the complex comprising mixing the complex with a pharmaceutically acceptable carrier and excipient.

  The complex of the present application has the same effect or activity as IFNλ1. For example, the complex can be used as an antiproliferative, antiviral, or antitumor agent. Specifically, the complexes of the present application are effective in the treatment of hepatitis B and hepatitis C, and they have a longer blood duration than IFNλ1. In one embodiment, the pharmaceutical composition containing the complex of the present application is manufactured as a sterile lyophilized powder for injection or as an infusion solution in a vial or pre-filled syringe. These pharmaceutical compositions can be formulated by mixing the complex with suitable pharmaceutically acceptable carriers and excipients.

  In another embodiment, the present application provides a method for producing a human recombinant PEG-IFNλ1 conjugate. First, human recombinant IFNλ1 is produced by recombinant DNA technology in E. coli and then reacted with a PEGylating agent (eg, α-methoxy-ω- (4-nitrophenoxycarbonyl)) polyoxyethylene (PEG-pNC)) To produce PEG-IFNλ1. In one embodiment, PEG-IFNλ1 is a linear chain PEG 40 kDa linked to IFNλ1. This product avoids the action of extracellular enzymes and filtration in the kidney after injection into the patient's body, thereby extending its serum half-life.

The pegylation reaction between (α-methoxy-ω- (4-nitrophenoxycarbonyl)) polyoxyethylene (PEG-pNC) 40 kDa and IFNλ1 to form the complex is shown below. In one embodiment, the —NH ₂ group is the N-terminal methionine residue on the interferon λ1 molecule. In another embodiment, the —NH ₂ group is an amine of a lysine residue on the interferon λ1 molecular site.

  In one embodiment, the conjugates of the present application can be made by covalently coupling interferon λ1 and pre-activated PEG. In one embodiment, PEG is activated by replacing the PEG hydroxyl group with a linking group to produce a coupling agent, or an activated PEG agent (ie, (α-methoxy-ω- (4-nitrophenoxycarbonyl) ) Polyoxyethylene (PEG-pNC)) may be obtained. In one embodiment, the PEG-IFNλ1 conjugate can be produced by production of IFNλ1 and pegylation of the IFNλ1. A method for purifying and assaying the complex product is also disclosed.

FIG. 1 illustrates the nucleic acid sequence (SEQ ID 1) used to produce human recombinant IFNλ1, after the sequence was synthesized and introduced into the expression vector pNanogen 1-IL29.

FIG. 2 is a representative amino acid sequence (SEQ ID 2) of human recombinant IFNλ1 from Nanogen Pharmaceutical Biotechnology Co., Ltd.

FIG. 3 illustrates plasmid pNanogen 1-IL29 containing the gene encoding human IFNλ1 (interleukin-29).

FIG. 4 shows the analysis result of plasmid pNanogen 1-IL29.

FIG. 5 illustrates the results of an electrophoresis process to examine the ability of E. coli with pNanogen 1-IL29 used to produce IFNλ1.

FIG. 6 is a representative spectrum of salt phase and SDS-PAGE electrophoresis after protein refolding. The spectra in FIGS. 6, 7, 8 and 9 are all Coomassie blue stained.

FIG. 7 illustrates the spectrum and SDS-PAGE electrophoresis after the cation 1 phase.

FIG. 8 illustrates the spectrum and SDS-PAGE electrophoresis after the cation 2 phase.

FIG. 9 illustrates the spectrum and SDS-PAGE electrophoresis after the gel filtration phase.

FIG. 10 illustrates the spectrum of the purification process of pegylated interferon λ1 and SDS-PAGE electrophoresis.

FIG. 11 illustrates the identification results of IFNλ1 and PEG-IFNλ1.

FIG. 12 illustrates the Maldi-Tof mass spectrum of PEG-IFNλ1 from Nanogen Pharmaceutical Co., Ltd.

(Detailed description of the invention)
In one embodiment, the present application provides for the production of recombinant strains comprising a gene encoding IFNλ1, large-scale or industrial production of IFNλ1, PEGylation of IFNλ1, purification of the produced PEG-IFNλ1, and assay of PEG-IFNλ1. Providing a method for

  In one embodiment, the present application provides an artificial synthesis of a gene encoding IFNλ1 based on published sequences available from the National Center for Biotechnology Information (the encoded gene has been modified to be compatible with industrial production processes in E. coli. ), Preparation of gene transfer vectors, introduction of these vectors into fungi, and selection of strains that most produced IFNλ1.

  In one embodiment, the industrial production process of IFNλ1 includes the following steps: fermentation of initial material, collection of crude protein solution, and purification of IFNλ1 protein. In a typical process, the fermentation process may be performed in a 10 liter fermentation tank containing a nutrient medium and induced production of IFNλ1 by lactose. The obtained biomass was separated and purified. IFNλ1 was collected and purified by a number of steps including: protein refolding, protein separation (eg, by ion exchange chromatography (cation 1 and cation 2)), and purification of the protein on a gel.

  In one embodiment, the pegylation process comprises a reaction between a linear chain (α-methoxy-ω- (4-nitrophenoxycarbonyl)) polyoxyethylene (PEG-pNC-, molecular weight 40 kDa) and IFNλ1 . The resulting complex product may be purified by chromatography (eg, using an HPLC system) and tested for quality and purity.

  The present application will be fully understood by reference to the following examples, which are merely illustrative and are not to be construed as limiting the claims.

Example:
Example 1: Method for producing E. coli strain having gene encoding human recombinant interferon λ1 (IFNλ1)

  The gene encoding IFNλ1 was artificially synthesized based on protein sequence data available from NCBI or other databases. The novel methods described herein reduce the time required to isolate genes while remaining as accurate as conventional methods. The nucleic acid sequence used to produce IFNλ1 at Nanogen Pharmaceutical Biotechnology Co., Ltd. is shown in FIG. 1, and the amino acid sequence of the protein is shown in FIG.

  Expression vector pNanogen-IL29 (containing T7 transcription promoter region, IFNλ1 transgene, T7 reverse priming site, T7 transcription terminator, f1 replication origin, kanamycin resistance gene, and pUC replication origin) enables high expression of the protein Specially designed to promote fermentation and for industrial production of large amounts of IFNλ1. 3 and 4 show a method for producing the vector pNanogen 1-IL29.

The vector pNanogen 1-IL29 was then transferred to an E. coli strain suitable for the expression of promoter T7. This strain, genotype F ^- having _{gal dcm (DE3) ompT hsdS B} (rB - - mB). The strain having the IFNλ1 gene is referred to as E. Coli-pNanogen1-IL29. It has the ability to produce more than 100 mg of IFNλ1 per liter by fermentation (see FIG. 5), which was introduced into the original strain bank.

  Example 2: E. coli fermentation process to produce human recombinant IFNλ1

The fermentation process was carried out in a 140 L fermentation tank containing nutrient medium at 37 ± 0.5 ° C, air pressure 0.5 m ³ / h, pH 7.0 ± 0.2, stirring speed 300 rpm (H ₃ PO ₄ or NH ₄ OH The pH was maintained between 6.8 and 7.2 by adding). After 8 hours (E. coli grows in log phase, when cells grow most efficiently), the temperature is cooled to 30 ± 0.5 ° C., the agitation speed is reduced to 200 rpm, and IFNλ1 The process for the production of was started. The fermentation process was stopped after 4 hours and the cold product was centrifuged at 6000 rpm to obtain biomass.

  The biomass was disrupted by homogenization in a homogenizer in cell lysis solution (12 ml solution per g wet biomass). After maintaining the temperature at 4 ° C. for 1 hour, the cells were broken twice by an ultrasonic device. The resulting suspension was centrifuged at 6000 rpm for 30 minutes to obtain a pellet. The pellet is then washed with inclusion body wash buffer (12 ml buffer per gram of wet biomass) and the resulting suspension is maintained at 4 ° C. for 1 hour and then centrifuged twice at 13,000 rpm for 30 minutes. To obtain a pellet. The pellet was dissolved in 2M urea solution and incubated on ice for 1 hour, and then the suspension was centrifuged at 13,000 rpm for 30 minutes to obtain a pellet. The pellet was dissolved in a washing solution and centrifuged at 13,000 rpm for 30 minutes to obtain a pellet. The pellet was then dissolved in 6M guanidine solution and the suspension was kept on ice for 12-16 hours and centrifuged at 13,000 rpm for 30 minutes. The solution containing the protein was collected and purified in the next step.

The culture medium and solution components used to separate IFNλ1 are shown in Table 1.
table 1

  Example 3: Purification process of human recombinant IFNλ1

  IFNλ1 is refolded by dissolving the inclusion bodies in refolding solution (25 mM Tris buffer, 1 mM EDTA, 1.2 M guanidine, pH 8.2) (so that the final concentration of the inclusion bodies is 500 μg / ml). I let you. The mixture was then maintained at 2-8 ° C. for 16-24 hours. The resulting mixture was desalted and then processed in a purification step on a Sephadex G25 column. The salt exchange buffer was a phosphate buffer (10 mM, pH 8.0). In step “cation 1”, the desalted mixture is applied to a Sephadex G25 column (this column is pre-packed with CM-Sepharose FF gel and equilibrated in 10 mM phosphate buffer pH 8.0). The product was eluted using 10 mM sodium phosphate + 0.5 M NaCl pH 8.0. The resulting protein solution was desalted and chromatographed as described above (step “cation 2”). The protein solution was then filtered through a gel column to obtain human recombinant IFNλ1 as a product with a purity of 95% or more (see spectra and electrophoresis results in FIGS. 5, 6, 7, 8 and 9).

  Example 4: PEGylation process for composite production

  To a solution of 5 mg / ml human recombinant IFNλ1 (MW-20.1 kDa) in 50 mM sodium borate-phosphate pH 8.0, add (α-methoxy-ω- (4-nitrophenoxycarbonyl)) polyoxyethylene (PEG-pNC ) (MW-40 kDa) was added at a molar ratio of about 3: 1 PEG-pNC: IFNλ1. The reaction mixture was maintained at 2-4 ° C. for 20 hours. The reaction was stopped by adjusting the pH to 4.0 using a 30% w / w acetic acid solution. Thereafter, the obtained mixed solution was diluted 5 times with water.

  In a typical exemplary process, the reaction conditions for the coupling reaction of activated PEG or m-PEG reagent to IFNλ1 further include an approximately equimolar to relatively small molar excess of activated PEG or m relative to IFNλ1. -Performing the reaction with PEG. In one variation, the conjugation is performed at a molar excess of about 1 to 10 times; a molar excess of about 1.5 to 7 times; or a molar excess of about 1.75 to 5 times. In one variation, the binding reaction can be performed at about room temperature or about 20-25 ° C. After the binding reaction is allowed to proceed for about 1-10 hours, 1-5 hours, 1-3 hours, or about 1-2 hours, the reaction may be terminated by quenching. In some cases, the reaction conditions result in a PEG-IFNλ1 regioisomer. In one embodiment, each isomer comprises a single PEG-linker unit attached to IFNλ1 via an amino acid residue described herein. In certain cases where one or more PEG-linker units are attached to IFNλ1, the resulting composition (including these conjugates) may be used, if desired, or standard May be separated by chromatography using various purification methods, including ultrafiltration, ion exchange chromatography, affinity chromatography and size exclusion chromatography. In one embodiment, the purification method used for separation and purification of the complex is cation exchange chromatography as described herein.

  Under certain conditions, the binding site on the IFNλ1 can be influenced by the pH of the reaction medium. Modification of the specific pH of the binding process results in certain preferred binding sites. For example, under certain conditions, binding at a basic pH value (eg, pH 7.5 or higher, 8.0 or higher, 8.5 or higher, or 9.0 or higher) favors the binding of IFNλ1 to the lysine group.

  In the above method, the PEGylation reagent (eg PEG-pNC) forms a carbamate linker between PEG and IFNλ1. Additional pegylation reagents that may be used in the above process include oxycarbonyl-oxy-N-dicarboximide (eg, succinimidyl carbonate, succinimidyls, as disclosed in US Pat. No. 5,122,614). Succinate), para-nitroaryl carbonate, para-nitrophenyl carbonate, carbonyl di-imidazole, benzotriazole carbonate, pyridyl carbonate, N-succinimide, N-phthalimide, N-glutarimide, and N-tetrahydrophthalimide. Exemplary activated PEG or mPEG components that may be used to form the conjugate include PEG-2,4,6-trichloro-S-triazine, mPEG-2,4,6-trichloro-S-triazine PEG-N-succinimidyl glutarate, mPEG-N-succinimidyl glutarate, PEG-N-succinimidyl succinate and mPEG-N-succinimidyl succinate.

  Representative compounds of the examples:

The table below provides a summary of selected example compounds described herein:

The process for purification of the complex of formula Ia in the table above is performed using the methods described herein. The obtained PEG-IFNλ1 has a purity of about 95% or more. The spectrum and SDS-PAGE electrophoresis of the complex after the gel filtration phase are illustrated in FIG. The spectrum of the purification process of the complex and SDS-PAGE electrophoresis are illustrated in FIG. The complex has antiviral EMC activity (with an ED ₅₀ in the range of about 10-50 ng / ml) on Hep-2C cells. The antiviral activity of the complex of Formula Ia in the above table at ED ₅₀ (ng / ml) is about 25.00-28.00; average (ng / ml) about 1.0 to about 30.0; SD about 0.1 to about 1.0 and RSD about 3.0-7.0.

  The complex of formula Ia in the above table is administered to the patient at 200 μg (weekly subcutaneous injection) + ribavirin 15 mg / kg (daily). In the first 4 weeks, all patients are determined to have no HCV RNA (no virus in serum). The treatment protocol is continued for 12 weeks. After 12 weeks of treatment and 12 weeks of follow-up, all patients reach the primary endpoint of complete virus suppression.

  Example 5: Purification of PEG-IFNλ1

  A solution containing PEG-IFNλ1, quenched reagent and unmodified IFNλ1 was purified on a cation column (the column was pre-packed with Sepharose CM gel and equilibrated in 10 mM sodium phosphate pH 6.0). Elution was performed with a sodium acid, 0.5M NaCl (pH 6.0) solution. The eluted fraction containing protein was transferred to a storage buffer using 10 mM sodium phosphate (pH 6.0) solution. The product was then processed through a sterile filtration process and stored at -20 ° C.

FIG. 10 shows the spectrum of the purification process of IFNλ1 and SDS-PAGE electrophoresis. The obtained PEG-IFNλ1 had a purity of 95% or more, and the antiviral EMC activity in Hep-2C cells was ED _{50 of} about 10-50 ng / ml (see Example 6).

  Example 6: Examination of antiviral activity of IFNλ1 and PEG-IFNλ1

The study was based on the antiviral activity in EMC virus models and Hep-2C cells according to the work of Ank et al. (J Virol. 2006 May; 80 (9): 4501-9). The experiment was performed using 3 lots (IL290010111, IL290020311 and IL290030411). From the results, the antiviral activity (ED ₅₀ about 1-5 ng / ml) of interferon λ1 manufactured by Nanogen is equivalent to the research result of Sheppard et al. (Nat Immunol. 2003 Jan; 4 (1): 63-8). (See Table 2).

The antiviral activity of PEG-IFNλ1 was compared with IFNλ1. The experiment was performed using 5 lots (PIL290010111, PIL290020211, PIL290030311, PIL290040411, PIL290050511). Similar results were obtained in all lots with an ED _{50 of} about 10-50 ng / ml (see Table 3).

  Clinical information of patients before treatment:

  All patients in this treatment group were diagnosed with chronic HCV and had been previously treated with pegasys (peginterferon α2a) and pegintron (peginterferon α2b) for more than 6 months in combination with ribavirin (15 mg / kg). (There was no more than 1 log decrease in HCV RNA). HCV RNA> 500,000 IU / ml serum; HCV genotype 1-6; quantity 150; age 26-78 years; average age 52 years; some patients have high ferritin, low platelets (<50,000 / ml) Low Hb. Most patients have high fibrosis with a fibroscale F4 (high AST / ALT ratio of 1 or greater (liver) due to chronic infection with HCV. It has an index of fibrosis)). Some patients were on insulin treatment for diabetes. All patients over the age of 50 in the treatment group have hypertension.

  Treatment regimen:

  Peglamda (PEG-IFNλ1) 200 μg (weekly subcutaneous injection) + ribavirin 15 mg / kg (daily). In the first 4 weeks, all patients were determined to have no HCV RNA (no virus in serum) It was. The treatment was continued for 12 weeks.

表3:Nanogen製PEG-IFNλ1の抗ウイルス活性

Results: All patients reached the primary endpoint of complete virus suppression after 12 weeks of treatment and 12 weeks of follow-up.

Table 2: Antiviral activity of Nanogen interferon λ1 (PEG-IFNλ1)

Table 3: Antiviral activity of PEG-IFNλ1 from Nanogen

  Therapy for HCV resistant patients:

  It has been found that over 50 patients previously treated with standard therapies (eg, Pegasys® (a combination of pegylated interferon α-2a) and ribavirin) have no effect. Non-responder patients (defined as those who do not show clearance of HCV RNA from serum after 24 weeks of treatment according to HCV treatment guidelines provided by AASLD) and null responder patients (2 log of HCV RNA at 12 weeks) Defined as patients who do not decrease to less than) were assigned to treatment groups with PEG-IFNλ1 of the present application.

  After treatment with PEG-IFNλ1 for 4-12 weeks or 4-24 weeks using the treatment protocol described, virtually all patients were tested and determined to be HCV RNA negative; or all patients Had a sustained virological response (SVR) (defined as no virus detected 24 weeks after the final treatment dose).

  In other studies with HCV resistant patients, the treatment protocol described herein has been found to be effective in over 80%, over 85%, over 90%, or over 95% of the HCV resistant patient population. Therefore, the treatment method using the PEG-IFNλ1 demonstrates that it is effective in HCV (including the case where the current standard therapy is resistant to the combination of pegylated interferon α-2a and ribavirin). There were no significant side effects typically associated with combination therapy with Pegasys® and ribavirin.

  Example 7: Identification of IFNλ1 and PEG-IFNλ1

  Western blotting using anti-IFNλ1 antibody was used to identify IFNλ1 and PEG-IFNλ1. The protein solution after analysis by SDS-PAGE gel was transferred to a nitrocellulose membrane and probed with an anti-IFNλ1 antibody. The antibody was detected using peroxidase binding protein A and TMB substrate (see FIG. 11).

  Example 8: Molecular weight of PEG-IFNλ1

  MALDI-TOF assay was applied to determine the molecular weight of PEG-IFNλ1. The results are shown in FIG. In this example, PEG-IFNλ1 from Nanogen has a molecular weight of about 62 kDa.

  Example 9: Purity of PEG-IFNλ1

  The purity of PEG-IFNλ1 was determined by SDS-PAGE electrophoresis using 5 lots (PIL290010111, PIL290020211, PIL290030311, PIL290040411, PIL290050511). The electrophoresis gel was stained with Coomassie blue, destained and then analyzed using Phoretix software (TotalLab, England). All test lots showed a purity of over 95%.

  The following test uses the PEG-IFNλ1 produced above.

  Example 10: Toxicity of PEG-IFNλ1

(^*): ANOVA, 1要因（single factor）, ビヒクル処置と比較, (p＞0.05)
¹ 異常なしを意味する Acute toxicity of PEG-IFNλ1: The acute toxicity of PEG-IFNλ1 was evaluated in Swiss mice and rats. Five week old healthy ICR mice and Sprague Dawley rats were chosen for the study. The animals were inspected for 2 weeks. PEG-IFNλ1 subcutaneously or intraperitoneally at three different doses (high dose 3 mg / kg, medium dose 0.3 mg / kg, low dose 0.03 mg / kg and vehicle treatment (phosphate buffered saline, pH 7.2)) Administered by injection. Animals were observed for clinical signs, weight changes, and deaths on day 14 after treatment. At the end of the study, all animals were sacrificed and examined for abnormalities in their tissues and organs. The results are summarized in Table 4.

Table 4

( ^* ): ANOVA, single factor, compared to vehicle treatment, (p> 0.05)
¹ means no abnormality

  All animals survived during the study, even at high doses. There was no significant change in body weight in treated animals compared to controls. No clinical signs or organ abnormalities were observed in any group of test animals.

  Based on these results, the lethal dose (LD50) of PEG-IFNλ1 produced by Nanogen in mice and rats was 3 mg / kg or more. Subacute toxicity of PEG-IFNλ1: Animals (5-week-old rats) were administered PEG-IFNλ1 at three different doses (high dose 3 mg / kg, medium dose 0.3 mg / kg, low dose 0.03 mg / kg) It was administered once a day for 4 weeks by subcutaneous or intraperitoneal injection.

  Throughout the study, the rats were examined for any clinical and behavioral side effects caused by administration of Nanogen PEG-IFNλ1. After the test period, surviving rats were sacrificed for necropsy and biochemical analysis. A blood sample was taken from the abdominal artery and a blood test was performed.

The test methods and results are summarized in Table 5.
Table 5

  There were no deaths in any group during the entire study period, and there were no clinical signs from the test rats. Test rats were normal in other test categories and analyses, even in the high dose group. The study thus shows that Nanogen PEG-IFNλ1 has no toxic effects in rats when repeatedly administered at a dose of 3 mg / kg.

  Immunological toxicity of PEG-IFNλ1: A study was conducted in guinea pigs to investigate the immunological potential of PEG-IFNλ1 from Nanogen. Healthy male Hartley guinea pigs (weighing 300-500 g), PEG-IFNλ1 either at high dose (3 mg / kg) or low dose (0.03 mg / kg) twice a week for 3 weeks, and controls Was injected with ovalbumin. On day 14 after the last sensitization, an anaphylaxis study was performed by intravenous injection of a high dose of PEG-IFNλ1. The study included PEG-IFNλ1 incorporated into Freund's complete adjuvant (FCA). Sensitized guinea pigs were observed for active systemic anaphylactic reaction after injection of high doses of PEG-IFNλ1. The list of indications was used as a sign of anaphylactic reaction and their appearance was monitored in each test animal.

⁽¹⁾ 1. 鼻を舐める, 鼻をこする; 2. 毛を逆立てる;
3. 努力性呼吸; 4. くしゃみ, 咳;
5. 便の排出, 排尿; 6. 痙攣; 7. 衰弱;
8. 死亡. -, ネガティブ ; +, ポジティブ Table 6 shows the study methods and results.
Table 6

⁽¹⁾ 1. Lick the nose, rub the nose; 2. Turn the hair upside down;
3. forced breathing; 4. sneezing, coughing;
5. stool discharge, urination; 6. spasm; 7. weakness;
8. Death.-, Negative; +, positive

  In the active systemic anaphylaxis study, guinea pigs slightly sensitized with high dose PEG-IFNλ1 (3 mg / kg) incorporated in Freund's complete adjuvant (FCA) showed some signs of anaphylactic reaction . On the other hand, guinea pigs sensitized with only low and high doses of PEG-IFNλ1 (0.03 and 3 mg / kg) did not show any anaphylactic reaction. Guinea pigs did not die after administration of Nanogen PEG-IFNλ1 and negative treatment (PBS), but three guinea pigs died after administration of ovalbumin (positive treatment). Therefore, it can be concluded that Nanogen PEG-IFNλ1 does not induce systemic allergic reactions when administered alone in its clinical application.

  It will be apparent to those skilled in the art that various modifications and variations can be made to the compounds, compositions and methods of the present invention without departing from the spirit or scope of the invention. Accordingly, the present invention is intended to embrace all such modifications and variations as fall within the scope of the appended claims and their equivalents.

Claims (20)

Formula I:

[Where
R is H or C _1-3 alkyl;
m is 1, 2, 3 or 4;
n is a positive integer selected in the range of 400 to 550;
L is a C _1-10 alkyl or heteroalkyl linker;
X is -O-, -NH- or -S-; and
IFNλ1 is interferon λ1]
A physiologically active PEG-IFNλ1 complex comprising a compound represented by the formula (I) or a pharmaceutically acceptable salt thereof: The complex of claim 1, wherein R is H or —CH ₃ , m is 1, L is —C (O) —, and X is —NH—.   The composite according to claim 1 or 2, wherein n is 500 to 550. formula:

(Wherein INFλ1 is the interferon λ1 of SEQ ID 2;
n is 500 to 550)
The composite_body | complex of Claim 1 containing the compound shown by these.   5. The complex according to any one of claims 1 to 4, wherein the complex has a prolonged or prolonged serum half-life and duration compared to IFNλ1.   6. The complex according to any one of claims 1 to 5, wherein the PEG is bound to methionine at the N-terminus of IFNλ1.   A pharmaceutical composition comprising the complex according to any one of claims 1 to 6, and a pharmaceutically acceptable carrier and excipient.   The pharmaceutical composition according to claim 7, wherein the pharmaceutical composition is used for treatment of hepatitis B and hepatitis C. Binding reaction:

(Wherein n is a positive integer selected in the range of 500 to 550 so that the molecular weight of the PEG moiety is about 40 kDa)
And (α-methoxy-ω- (4-nitrophenoxycarbonyl)) polyoxyethylene (PEG-pNC) 40 kDa covalently linked to IFNλ1; and isolating the complex. A method for producing the human recombinant complex according to 1. Formula I:

[Where
R is H or C _1-3 alkyl;
m is 1, 2, 3 or 4;
n is a positive integer selected in the range of 400 to 550;
L is a C _1-10 alkyl or heteroalkyl linker;
X is -O-, -NH- or -S-; and
IFNλ1 is interferon λ1]
A method for producing a Peg-IFNλ1 complex containing a compound represented by the formula (I) or a pharmaceutically acceptable salt thereof:
The production method comprising contacting IFNλ1 with a preactivated Peg under conditions sufficient to promote a covalent bond with an amino acid residue of IFNλ1.   A PEG-IFNλ1 complex produced by the production method according to claim 10.   A method for inhibiting the growth of cancer cells in a patient, comprising contacting a cancer cell with the complex according to any one of claims 1 to 4, wherein the complex is prolonged or compared to IFNλ1. The method has an extended serum half-life and duration.   A method of treating a proliferative disease in a mammal comprising administering to the mammal a therapeutically effective amount of the complex of claim 1. A method of treating a patient infected with or at risk of being infected with a virus, wherein a therapeutically effective amount of Formula I:

[Where
R is H or C _1-3 alkyl;
m is 1, 2, 3 or 4;
n is a positive integer selected in the range of 500 to 550;
L is a C _1-10 alkyl or heteroalkyl linker;
X is -O-, -NH- or -S-; and
IFNλ1 is interferon λ1]
Or a pharmaceutically acceptable salt thereof; or administering a pharmaceutical formulation containing the complex of formula I. R is -CH _3, m is 1, L is -C (O) -, X is -NH-, and, IFNramuda1 is SEQ ID 2, The method of claim 14 .   The method according to claim 14 or 15, wherein the viral infection is caused by hepatitis C virus or progressive cirrhosis is caused by the viral infection.   17. The method of any one of claims 14 to 16, wherein the PEG-IFNλ1 is administered weekly at a dose of about 0.5 μg / kg to 10.0 μg / kg.   18. The method according to any one of claims 14 to 17, further comprising administering a nucleoside analog selected from ribavirin or viramidine.   19. The method of claim 18, wherein the ribavirin is administered daily at a dose of 5 mg / kg to 25 mg / kg.   The method according to any one of claims 14 to 17, wherein the patient is an HCV resistant or refractory patient.

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