WO2013028233A1 - Peg-interferon lambda 1 conjugates - Google Patents

Peg-interferon lambda 1 conjugates Download PDF

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Publication number
WO2013028233A1
WO2013028233A1 PCT/US2012/027317 US2012027317W WO2013028233A1 WO 2013028233 A1 WO2013028233 A1 WO 2013028233A1 US 2012027317 W US2012027317 W US 2012027317W WO 2013028233 A1 WO2013028233 A1 WO 2013028233A1
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Prior art keywords
ιρνλι
peg
conjugate
alkyl
ιενλι
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PCT/US2012/027317
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English (en)
French (fr)
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Nhan HO
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Nanogen Pharmaceutical Biotechnology
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Application filed by Nanogen Pharmaceutical Biotechnology filed Critical Nanogen Pharmaceutical Biotechnology
Priority to CA2846092A priority Critical patent/CA2846092A1/en
Priority to RU2014111179/10A priority patent/RU2014111179A/ru
Priority to BR112014004302A priority patent/BR112014004302A2/pt
Priority to EP12825651.8A priority patent/EP2748328A4/en
Priority to AU2012299423A priority patent/AU2012299423A1/en
Priority to CN2012800033678A priority patent/CN103228792A/zh
Priority to JP2014527142A priority patent/JP2014525939A/ja
Publication of WO2013028233A1 publication Critical patent/WO2013028233A1/en
Priority to ZA2014/01159A priority patent/ZA201401159B/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]

Definitions

  • the present application discloses pegylated derivatives of recombinant human interferon lambda 1 (PEG-interferon lambda 1 conjugates or PEG- ⁇ ), processes for their preparation, pharmaceutical compositions containing these conjugates and processes for making the same.
  • PEG-interferon lambda 1 conjugates or PEG- ⁇ pegylated derivatives of recombinant human interferon lambda 1 conjugates or PEG- ⁇
  • HCV Hepatitis C virus
  • AI a-interferons
  • Interferons are currently used for the treatment of many viral diseases such as hepatitis B, hepatitis C, hepatitis D, condyloma acuminata, lepromatous leprosy, chronic leukaemia and AIDS.
  • AI are also effective in reducing malignant tumors and treating Kaposi's sarcoma, melanoma, and renal cell carcinoma.
  • AI are applicable in the prevention and treatment of diseases in cattle and other livestock.
  • AI enhance the activity of vaccines used in prophylaxis and treatment of foot and mouth disease and porcine reproductive and respiratory syndrome.
  • AI have been produced from human cell lines incubated in tissue culture media or leukocytes derived from donors. However, these methods are time consuming, labor intensive, expensive, and not amenable to large scale manufacturing. Furthermore, there is the risk of septicaemia caused by infectious agents from the cell lines.
  • IL-29 is a member of the helical cytokine family and is a type III interferon. It is also known as interferon lambda 1 ( ⁇ ) and is highly similar in amino acid sequence to IL-28, the other type III interferon.
  • IL-28 and IL-29 ( ⁇ ) were recently described as members of a new cytokine family that shares with type I interferon (IFN), the same Jak/Stat signaling pathway driving expression of a common set of genes. Accordingly, they have been named ⁇ .
  • IFN type I interferon
  • ⁇ exhibit several common features with type I IFNs: antiviral activity, antiproliferative activity and in vivo antitumor activity. Importantly, however, ⁇ bind to a distinct membrane receptor, composed of IFNLR1 and IL10R2.
  • Interferon alpha-2a Roferon, Roche
  • Interferon alpha-2b Intron A, Schering A G
  • the two recombinant forms of human interferon alpha used in the treatment of chronic hepatitis B and C have a serum half-life of less than 12h
  • PEG polyethylene glycol
  • PEG moieties are attached to the protein by first activating the PEG moiety and then reacting the activated PEG agent with the side chains of an amino acid of a protein, such as the lysine residue and/or the N-terminal amino group on the protein.
  • the most frequently used PEG is monofunctional PEG because this moiety resists cross-linking and aggregation.
  • One such example has been disclosed by Davis et al. in U.S. Pat. No. 4,179,337.
  • PEG-interferon lambda 1 is a pegylated derivative of human recombinant ⁇ (wherein polyethylene glycol is conjugated to ⁇ , also referred to as the "conjugate") that is useful in the treatment of chronic hepatitis C in adult patients.
  • PEG- ⁇ bypasses the action of extracellular enzymes and resists filtration in the kidney after injection into the patient's body; therefore its half-life in circulation is extended. That is, the conjugate has significantly improved stability, better solubility, and enhanced circulating half-life and plasma residence times when compared to the corresponding non-PEG-conjugated ⁇ .
  • Interferon lambda 1 (IFN ⁇ , Zcyto21 or IL-28A) is known in the art, for example, from U.S. Pat. Nos. 7,038,032, 6,927,040, 7,135,170, 7,157,559 and 7,351,689; and PCT publication Nos. WO 05/097165, WO 07/012,033, WO 07/013,944 and WO 07/041,713; all of which are herein incorporated by reference in their entirety.
  • the present application discloses novel PEG- ⁇ conjugates.
  • conjugates of the present application have a linear PEG chain structure.
  • these conjugates have increased circulating half-life and persistence in plasma.
  • Water soluble PEGs include polyethylene glycol (PEG), monomethoxy-PEG (mPEG), mono-Ci-io alkoxy-PEG and mono-Ci-3 alkoxy-PEG.
  • PEGs that may be employed may have a molecular weigth of about 600 to 60,000 and include those, for example with about 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa and 60 kDa.
  • the PEG employed in the present conjugates are mPEG with a molecular weight of 40 kDa.
  • R OCCHzCHzO) 'n, L— X— nsra i I
  • R is H or Ci_ 3 alkyl
  • m is 1, 2, 3 or 4
  • n is a positive integer selected in the range from 400 to 550
  • P is a C 1-10 alkyl or heteroalkyl linker
  • X is -0-, -NH- or -S-
  • ⁇ is interferon lambda 1 ; or a pharmaceutically acceptable salt thereof.
  • interferon lambda 1 is a human recombinant interferon.
  • the ⁇ may be a natural or recombinant protein.
  • the ⁇ is a human protein derived from a source such as tissues, protein synthesis, or cell culture using natural cells or recombinant cells.
  • ⁇ is a human recombinant protein.
  • the conjugate interferon lambda 1 of the formula I is the SPQ ID 2.
  • the PPG chain is coupled to the ⁇ via an amide bond on a primary amino group of, for example, lysine, or the N-terminal of ⁇ .
  • n is about 500 to 550.
  • n is about 420, 520 or 455.
  • the molecular weight of the Peg group is about 35 kDa to 45 kDa, or about 40 kDa.
  • m is 1 or 2.
  • Ci-io alkyl or heteroalkyl may be a linear or a branched alkyl or heteroalkyl group.
  • the C 1-10 alkyl group is a -C(O)- group.
  • m is 1 and P-X- is selected from the group consisting of the formulae:
  • the two PEG groups, PEG, alkyl-PEG or m- PEGs are attached to the two formarnide groups (i.e. -C(O)NH-) of the above formula.
  • X is -NH- or -O- and m is 2.
  • the linker is attached to two PEG groups.
  • X is -NH-, and the group attached to the linker is the residue of a lysine on ⁇ .
  • the -NH- group (i.e., the amino group) attached to the linker is the residue of a histidine.
  • X is -0-, and the group attached to the linker may be derived from the residue of a serine on ⁇ .
  • R is -CH 3 .
  • the linker is attached to the residue of a lysine, a serine, a histidine or mixtures thereof on the ⁇ .
  • the linker is attached to a positional isomer of the residue of a lysine, a serine, a histidine or mixtures thereof on the ⁇ .
  • R is H or -CH 3
  • m is 1
  • L is -C(O)-
  • X is -NH-.
  • n is 500 to 550.
  • conjugate comprises the formula II:
  • ⁇ is interferon lambda 1; and n is 500 to 550.
  • n is a number of units of ethylene glycol in the PEG structure and it is a positive integer selected from any numbers such that the molecular weight of PEG moiety is about 40 kDa, and ⁇ is interferon lambda 1.
  • the conjugate has a blood serum half-life and persistence time that are prolonged or extended when compared to ⁇ .
  • the PEG is attached to a methionine at the N-terminal of the ⁇ .
  • the conjugate is effective in the treatment of hepatitis B and hepatitis C.
  • a process for the preparation of a human recombinant conjugate as disclosed above comprises the step of covalently binding (a-methoxy-co-(4-nitrophenoxy carbonyl)) polyoxyethylene (PEG-pNC) 40 kDa with ⁇ through a conjugation reaction as follows:
  • n is a positive integer selected such that the molecular weight of PEG moiety is about 40 kDa; and isolating the conjugate. In one aspect, n is from about 500 to 550.
  • composition containing a conjugate as disclosed above and pharmaceutically acceptable carriers and excipients.
  • compositions comprising the conjugate of the present application can be also prepared using the compositions comprising the conjugate of the present application.
  • the formulations may comprise a therapeutically effective amount of the composition comprising the conjugate together with pharmaceutically acceptable carriers as known in the art.
  • pharmaceutically acceptable carriers for example, adjuvants, diluents, preservatives and/or solubilizers, if needed, may be used.
  • compositions comprising the conjugate may include diluents of various buffers (e.g., Tris-HCl, acetate, phosphate) having a range of pH and ionic strength, carriers (e.g., human serum albumin), solubilizers (e.g., polyoxyethylene sorbitan or TWEEN®, polysorbate), and preservatives (e.g., thimerosol, benzyl alcohol), as disclosed, for example, in U.S. Patent No. 4,496,537.
  • the pharmaceutical composition is formulated as a sterile lyophilized powder for injection.
  • the composition comprises a combination of pharmaceutically acceptable vehicles, including saline, buffered saline and 5% dextrose in water.
  • the pharmaceutical composition is formulated as a solution for injection in vials or pre-filled syringes.
  • the pharmaceutical composition is used in the treatment of hepatitis B and hepatitis C.
  • Pharmaceutical formulations and methods for preparing such formulations are well known in the art and are disclosed, for example, in Remington, The Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co., Easton, Pa. 19 th ed. 1995.
  • R is H or C 1-3 alkyl; m is 1, 2, 3 or 4; n is a positive integer selected in the range from 400 to 550; L is a C 1-10 alkyl or heteroalkyl linker; X is -0-, -NH- or -S-; and ⁇ is interferon lambda 1; or a pharmaceutically acceptable salt thereof; the process comprising: contacting the ⁇ with a pre-activated Peg under conditions that are sufficient to facilitate covalent conjugation with an amino acid residue of the ⁇ .
  • a PPG- ⁇ conjugate prepared by the process as described herein.
  • a method of preparing the above conjugate comprising contacting the ⁇ with a sufficient amount of an activated PPG or mPPG under conditions that are sufficient to facilitate covalent attachment of the PPG or mPPG on the ⁇ .
  • the activated mPPG is mPPG-pNC.
  • the attachment of the activated mPPG is on a methionine at the N- terminal of the ⁇ .
  • the mPPG has a molecular weight of about 40 kDa.
  • the activated oxycarbonyl agent is a mono- or di-activated agent.
  • a method for inhibiting the proliferation of a cancer cell in a patient comprising contacting the cancer cell with the conjugate described above, wherein the conjugate has a blood serum half-life and persistence time that are prolonged or extended when compared to ⁇ .
  • the conjugate of the present application has a blood serum half-life that is extended by more than twice, three times, five times, eight times or more than 10 times the serum half life of the corresponding unconjugated ⁇ .
  • a method for treating a proliferative disorder in a mammal comprising administering to the mammal a therapeutically effective amount of the above conjugate.
  • the conjugate may be used for treating interferon-susceptible conditions or conditions which would respond positively or favorably to interferon based therapy.
  • the treatment using the conjugate results in substantially reduced or elimination of side effects when compared to conventional treatment with interferons.
  • exemplary conditions which can be treated with the conjugates of the present application include, but are not limited to, cell proliferation disorders, in particular cancer (e.g., hairy cell leukemia, Kaposi's sarcoma, chronic myelogenous leukemia, multiple myeloma, basal cell carcinoma and malignant melanoma, ovarian cancer and cutaneous T cell lymphoma), and viral infections.
  • cancer e.g., hairy cell leukemia, Kaposi's sarcoma, chronic myelogenous leukemia, multiple myeloma, basal cell carcinoma and malignant melanoma, ovarian cancer and cutaneous T cell lymphoma
  • the conjugates may be used to treat conditions which would benefit from inhibiting the replication of interferon-sensitive viruses.
  • Viral infections which may be treated with the conjugate of the present application include hepatitis A, hepatitis B, hepatitis C, other non-A/non-B hepatitis, herpes virus, Ppstein-Barr virus (PBV), cytomegalovirus (CMV), herpes simplex, human herpes virus type 6 (HHV-6)), papilloma, poxvirus, picomavirus, adenovirus, rhinovirus, human T lymphotropic virus-type 1 and 2 (HTLV-1/-2), human rotavirus, rabies, retroviruses including human immunodeficiency virus (HIV), encephalitis and respiratory viral infections.
  • PBV Ppstein-Barr virus
  • CMV cytomegalovirus
  • HHV-6 herpes simplex
  • HHV-6 herpes simplex
  • HHV-6 herpes simplex
  • HHV-6 herpes simplex
  • HHV-6 herpes simplex
  • a method of treating a patient infected or at risk of infection with a viral infection comprising administering to a patient in need thereof, a therapeutically effective amount of a conjugate of the formula I:
  • R OCCHzCHzO) 'n, L— X— nsra i I
  • R is H or C 1-3 alkyl
  • m is 1, 2, 3 or 4
  • n is a positive integer selected in the range from 500 to 550
  • L is a C 1-10 alkyl or heteroalkyl linker
  • X is -0-, -NH- or -S-
  • ⁇ is interferon lambda 1; or a pharmaceutically acceptable salt thereof; or a pharmaceutical formulation comprising the conjugate of the formula I.
  • the conjugate interferon lambda 1 of the formula I is the SEQ ID 2.
  • the mammal is a human.
  • the viral infection is caused by a hepatitis C virus, or the viral infection results in advance liver cirrhosis.
  • the patient is an HCV resistant or refractory patient.
  • the PEG- ⁇ is administered in a dose of about 0.5 g kg to 10.0 g kg weekly. In one aspect of the method, the PEG- ⁇ is administered in a dose of about 2.5 g kg weekly. In another aspect, the PEG- ⁇ is administered for about 8 weeks to about 52 weeks. In another aspect, the PEG- ⁇ is administered for about 12 weeks, about 16 weeks, about 20 weeks or about 24 weeks.
  • the PEG- ⁇ is administered until the patient is determined to be free of HCV RNA in blood serum.
  • the administration of the conjugate provides significant improvement over the standard PEG-INF-a therapy because the method does not result in the significant reductions in neutrophil counts, platelet counts or hemoglobin levels.
  • the method further comprises the administration of a nucleoside analogue selected from ribavirin and viramidine.
  • the ribavirin is administered orally in a dose of 5 mg/kg to 25 mg/kg daily; or 15 mg/kg to 25 mg/kg daily.
  • the ribavirin is administered in a dose of about 10 mg/kg to 30 mg/kg once or twice daily, or about 15 mg/kg daily once or twice daily.
  • the conjugate is administered parenterally.
  • HBV may be treated using a dose of about 200 ,ug of the PEG-IfWJ conjugate per week, combined with tenofovir (tenofovir disoproxil fumarate) at about 300 mg per day. Under this treatment, it is determined that most patients are clear of HBV after about four injections over about 30 days.
  • the HBV is found to be suppressed, and the HBsAg (virus surface antigen) is released, which triggers the immune system to make the antibody against this antigen, resulting in the optimal endpoint in the particular treatment.
  • the treatment may be continued as disclosed herein, for about 12 weeks to 24 weeks, depending on the patient's initial viral load.
  • Alkyl means a straight or branched, saturated or unsaturated, aliphatic radical having a chain of carbon atoms, optionally substituted with oxygen (e.g., a Ci alkyl may be -C(O)-), nitrogen atoms (e.g., a Ci alkyl may be -C(NH)-) or sulfur atoms (e.g., a C 2 alkyl may be
  • Ci- 6 alkyl includes alkyls having between 1 and 6 carbons (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, vinyl, isopropenyl, 1-butenyl, ethynyl, 1-propynyl and the like).
  • a “heteroalkyl” or “heteroalkylene” is an alkyl that may have an oxygen, nitrogen or sulfur between the carbon atoms.
  • heteroalkyl groups include -C(0)NH-, -OC(O)-, -CH 2 CH 2 C(0)-NH-, -CH 2 -0-CH 2 -CH 2 -, -CH 2 -NH-CH 2 -CH 2 -, -CH 2 -S-CH 2 -CH 2 - and -CH 2 0- CH 2 -CH 3 and the like.
  • PEG polyethylene glycol as used in the art, and generally includes both alkyl-PEG such as mPEG (methoxy-polyethylene glycol) and PEG, unless specified otherwise.
  • a “therapeutically effective amount” is an amount of the PEG- ⁇ ⁇ conjugate that is sufficient to produce a clinically significant change in the treated condition, such as a clinically significant change in the viral load or immune function, a significant reduction in morbidity or a significantly increased histological score, or combinations thereof.
  • treatment refers to a therapeutic treatment and prophylactic or preventive measures.
  • Patients who are in need of treatment include patients already infected with hepatitis C virus as well as those in which the hepatitis C disease is to be prevented.
  • the conjugate is administered by injection or infusion.
  • the conjugate is administered intravenously, intramuscularly, subcutaneousiy, inJxader ally or intraperitoneally.
  • the conjugate is administered to the patient in a dose amount selected from less than 0.5 p.g kg, 0.5 to 1.0 ug/kg, 1 .0 to 1.5 ug/kg, 1.5 to 2.0 .ug/kg, 2,0 to 2.5 Lig/kg, 2.5 to 3.0 p.g/kg, 3.0 to 3.5 ⁇ g/k:g, 3.5 to 4.0 ,ug/kg, 4.0 to 4.5 Mg/ ' kg, 4.5 to 5.0 Lig/kg, 5.0 to 5.5 ⁇ -g/kg, 5.5 to 6.0 ⁇ g/k:g, 6.0 to 6.5 ,ug/kg, 6.5 to 7.0 , ug/kg, 7.0 to 7.5 ⁇ /kg, 7.5 to 8.0 , ug/kg, 8.0 to 8.5 ⁇ ig/kg.
  • the conjugate is administered in a fixed dose of about 60-80 ⁇ » 80-100 ⁇ ig, 100-120 120-140 g, 140- 160 ug, 160- 180 ⁇ g, 180 -200 ⁇ ig, 200-220 ⁇ g, 220- 240 ⁇ , ⁇ , 240-260 .g, 260-280 ug, or about 280-300 ⁇ . ⁇ .
  • the conjugate is administered subcutaneously at 200 ⁇ g for 12 consecutive weeks.
  • a pharmaceutical composition containing the above conjugate and pharmaceutically acceptable carriers and excipients In another embodiment, the pharmaceutical composition is used in treatment of hepatitis B and hepatitis C. In another embodiment, there is provided a process for the preparation of a pharmaceutical composition containing the above conjugate comprising mixing the conjugate with pharmaceutically acceptable carriers and excipients.
  • the conjugates of the application have similar effects or activities as those of ⁇ .
  • the conjugates may be used as anti-proliferative agents, antiviral agents, or antitumor agents.
  • the conjugates of the present application are effective in treatment of hepatitis B and hepatitis C, and they have a longer persistence time in blood than ⁇ .
  • pharmaceutical compositions containing the conjugates of the present application are prepared as sterile lyophilized powders for injection, or as solutions for injection in vials or pre- filled syringes. These pharmaceutical compositions may be formulated by mixing the conjugates with relevant pharmaceutically acceptable carriers and excipients.
  • the present application provides processes for the preparation of human recombinant PEG- ⁇ conjugates.
  • human recombinant ⁇ is produced by recombinant DNA technology in E.coli, then reacted with a pegylating agent (such as a-methoxy- co-(4-nitrophenoxy carbonyl)) polyoxyethylene (PEG-pNC) to produce the PEG- ⁇ .
  • a pegylating agent such as a-methoxy- co-(4-nitrophenoxy carbonyl) polyoxyethylene (PEG-pNC)
  • PEG-pNC polyoxyethylene
  • the PEG- ⁇ is a linear chain PEG 40 kDa that is conjugated to ⁇ . This product bypasses the action of extracellular enzymes and kidney filtration when injected into the patient's body, therefore its blood serum half-life is extended.
  • the -NH 2 group is a methionine residue at the N-terminal on a site of the interferon lambda 1 molecule.
  • the -NH 2 group is the amine of a lysine residue on a site of the interferon lambda 1 molecule.
  • the conjugates of the present application may be prepared by covalently binding Interferon lambda 1 with pre-activated PEG.
  • PEG may be activated by substituting the PEG hydroxyl group with a linking group to form the coupling agent, or an activated PEG agent that is (a-methoxy-co-(4-nitrophenoxy carbonyl)) polyoxyethylene (PEG-pNC).
  • PEG-pNC an activated PEG agent that is (a-methoxy-co-(4-nitrophenoxy carbonyl)) polyoxyethylene
  • the PEG- ⁇ conjugates may be prepared by the preparation of ⁇ and the pegylation of the ⁇ . Also disclosed are processes for purifying and assaying the conjugated products.
  • Figure 1 exemplifies a nucleic acid sequence (SEQ ID 1) used to produce human recombinant ⁇ after the sequence was synthesized and introduced into the expression vector pNanogen 1-IL29.
  • Figure 2 is a representative amino acid sequence (SEQ ID 2) of human recombinant ⁇ produced by Nanogen Pharmaceutical Biotechnology Co., Ltd.
  • Figure 3 exemplifies a plasmid pNanogen 1-IL29 containing the gene encoding human ⁇ (interleukin-29).
  • Figure 4 depicts a result of analyzing plasmid pNanogen 1-IL29.
  • Figure 5 exemplifies a result of an electrophoresis process for examining the ability of E. coli containing pNanogen 1-IL29 used to produce ⁇ .
  • Figure 6 is a representative spectrum of the salt phase and SDS-PAGE electrophoresis after refolding protein.
  • the spectrum of Figures 6, 7, 8 and 9 are all coomassi blue stained.
  • Figure 7 exemplifies a spectrum and SDS-PAGE electrophoresis after cation 1 phase.
  • Figure 8 exemplifies a spectrum and SDS-PAGE electrophoresis after cation 2 phase.
  • Figure 9 exemplifies a spectrum and SDS-PAGE electrophoresis after a gel filtration phase.
  • Figure 10 exemplifies a spectrum of the purification process and SDS-PAGE
  • Figure 11 exemplifies an identification results of ⁇ and PEG- ⁇ .
  • Figure 12 exemplifies a Maldi-Tof mass-spectrum of PEG- ⁇ produced by Nanogen
  • the present application discloses processes for preparing a
  • the present application discloses an artificial synthesis of the gene encoding ⁇ based on the published sequence available from the National Center for
  • Biotechnology Information (the encoding gene was modified to conform to the industrial production process on E.coli), creating of the gene transfer vectors, introducing these vectors into the bacteria, and selecting the bacterial strain that best produced ⁇ .
  • the industrial manufacturing process for ⁇ includes the steps of: fermenting the initial material, collecting the solution of crude proteins and purifying the ⁇ protein.
  • the fermentation process may be carried out in a 10 liter fermenting tank containing a nutrient medium and production of ⁇ was induced by lactose. The biomass obtained was separated and purified.
  • ⁇ was collected and refined through a number of steps including: refolding the protein, separating the protein, for example by ion exchange chromatography (cation 1 and cation 2), and refining the protein on a gel.
  • the pegylation process comprises a reaction between the linear chain (a-methoxy-co-(4-nitrophenoxy carbonyl)) polyoxyethylene (PEG-pNC- with a molecular weight of 40 kDa) and ⁇ .
  • the resulting conjugate product may be purified by chromatography, such as using an HPLC system, and tested for quality and purity.
  • Example 1 Process for preparing E. coli strain containing the gene encoding human recombinant interferon lambda 1 ( ⁇ )
  • the gene encoding ⁇ was artificially synthesized based on the protein sequence data available from NCBI or other databases.
  • the novel method provided herein reduces the time required to isolate the gene but still provides a result as accurate as the conventional method.
  • the nucleic acid sequence used to produce ⁇ in Nanogen Pharmaceutical Biotechnology Co., Ptd. is shown in Figure 1 and the amino acid sequence of this protein is shown in Figure 2.
  • the expression vector pNanogen-IL29 (comprising the T7 transcription promoter region, the ⁇ transgene, the T7 reverse priming site, the T7 transcription terminator, the fl origin, the kanamycin resistance gene, and the pUC origin of replication) was specifically designed to enable high expression of the protein and facilitate fermentation for industrial production of a large quantity of ⁇ .
  • Figures 3, 4 show the process for creation of vector pNanogen 1-IL29.
  • Vector pNanogen 1-IL29 was then transferred into an E.coli strain suitable for expression of promoter T7.
  • This strain has a genotype F ompT hsdSs (rB ' mB ' )gal dcm (DE3).
  • the strain containing the ⁇ gene is termed E.Co/z ' -pNanogenl-IL29. It has the ability to produce higher than 100 mg of ⁇ per litre by fermentation (see Figure 5) and was introduced into the original strain bank.
  • Example 2 Process for fermentation of E. coli to produce human recombinant ⁇
  • the fermentation process was carried out in a 140 liter fermentation tank with nutrient medium at a temperature of 37+0.5 °C, air pressure 0.5 m 3 /h, pH 7.0+0.2, stirring rate of 300 rpm and the pH was maintained at between 6.8-7.2 by adding H 3 PO 4 or NH 4 OH.
  • the temperature was cooled to 30+0.5 °C and the stirring rate was reduced to 200 rpm to start the process for the generation of ⁇ .
  • the fermentation process was stopped after 4 hours and the cold product was centrifuged at 6000 rpm to obtain biomass.
  • the biomass was disrupted in a cell lysis solution (12 ml solution per 1 g wet biomass) by homogenizing in a homogenizing device. The temperature was maintained at 4 °C for 1 hour, then the cells were disrupted 2 times by an ultrasonic device. The resulting suspension was centrifuged at 6000 rpm for 30 minutes to give a pellet. The pellet was then washed with an inclusion body wash buffer (12 ml buffer per lg wet biomass), the resulting suspension was kept at 4 °C for 1 hour, then centrifuged twice at 13,000 rpm for 30 minutes to obtain a pellet.
  • an inclusion body wash buffer (12 ml buffer per lg wet biomass
  • the pellet was dissolved in 2M urea solution and incubated ice-cold for 1 hour, the suspension was then centrifuged at 13,000 rpm for 30 minutes to give the pellet. The pellet was dissolved in a wash solution and centrifuged at 13,000 rpm for 30 minutes to give a resulting pellet. The pellet was then dissolved in 6M guanidine solution, the suspension was kept ice-cold for 12-16 hours, and centrifuged at 13,000 rpm for 30 minutes. The solution containing protein was recovered and purified in next step.
  • ⁇ was refolded by dissolving the inclusion bodies in refolding solution (25mM Tris buffer, ImM PDTA, 1.2M guanidine, pH 8.2) such that the final concentration of the inclusion bodies were 500 ⁇ g/ml.
  • the mixture was then kept at 2-8 °C for 16-24 hours.
  • the resulting mixture was desalted before being subjected to a purification step on a Sephadex G25 column.
  • the salt exchange buffer was a phosphate buffer (lOmM, pH 8.0).
  • step “cation 1” the desalted mixture was loaded onto a Sephadex G25 column (this column was prefilled with CM- Sepharose FF gel and equilibrated in lOmM phosphate buffer pH 8.0), the product was eluted using lOmM sodium phosphate + 0.5M NaCl pH 8.0.
  • the resulting protein solution was desalted and chromatographed as above (step “cation 2"). The protein solution was then filtered through a gel column to give the product human recombinant ⁇ with purity greater than 95% (see the spectrum and electrophoresis results in Figures 5, 6, 7, 8 and 9).
  • the reaction conditions for the conjugation reaction of the activated PEG or m-PEG reagent to the ⁇ further include conducting the reaction using about equi-molar to a relatively small molar excess of the activated PEG or m-PEG with respect to ⁇ .
  • the conjugation may be carried out with about 1-10 fold molar excess; or about 1.5 to 7 fold molar excess; or about 1.75 to 5 fold molar excesses.
  • the conjugation reaction can be carried out at about room temperature, or about 20-25 °C.
  • the conjugation reaction may be allowed to proceed for about 1 to 10 hrs, 1 to 5 hrs, 1 to 3 hrs or about 1 to 2 hrs, before the reaction is terminated by quenching.
  • the reaction conditions provide a mixture of the PEG- ⁇ positional isomers.
  • each isomer contains a single PEG-linker unit attached to the ⁇ via an amino acid residue as disclosed herein.
  • the resulting composition containing these conjugates may be used or may be separated by chromatography using standard purification methods, including ultrafiltration, ion exchange chromatography, affinity chromatography and size exclusion chromatography.
  • the purification method used for the separation and purification of the conjugates is cation exchange
  • the site of conjugation on the ⁇ may be influenced by the pH of the reaction medium. Modification of the particular pH of the conjugation process will result in certain preferred sites of conjugation. For example, under certain conditions, the conjugation at basic pH values, such as pH of 7.5 or higher, 8.0 or higher, 8.5 or higher or 9.0 or higher, favors the conjugation to a lysine group of the ⁇ .
  • the pegylation reagent suh as PEG-pNC, forms a carbamate linker between the PEG and ⁇ .
  • Additional pegylation reagents that may be employed in the above process include oxycarbonyl-oxy-N-dicarboximide (such as succinimidyl carbonate, succinimidyl succinate), para-nitroaryl carbonates, para-nitrophenyl carbonates, carbonyl di-imidazole, benzotriazole carbonates, pyridyl carbonates, N-succinimide, N-phthalimide, N-glutarimide, and N-tetrahydrophthalimide as disclosed in U.S. Patent No. 5,122,614.
  • oxycarbonyl-oxy-N-dicarboximide such as succinimidyl carbonate, succinimidyl succinate
  • para-nitroaryl carbonates para-nitrophenyl carbonates
  • carbonyl di-imidazole benzotriazole carbonates
  • Representative activated PEG or mPEG compounds 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.
  • Antiviral activities of the conjugates of the formula la in the above table at ED 50 are about 25.00 to 28.00; with a Mean (ng/ml) of about 1.0 to about 30.0; SD of about 0.1 to about 1.0 and RSD of about 3.0 to 7.0.
  • the conjugates of the formula la in the above table are administered to patienst at 200 ⁇ g (weekly subcutaneous injection) + ribavirin 15 mg/kg (daily). In the first 4 weeks, all patients are determined to be free of HCV RNA (free virus in serum). The treatment protoccols are continued for 12 weeks. All patients achieve primary endpoint of total viral surpression after 12 weeks treatment and 12 weeks follow up.
  • Figure 10 shows the spectrum of the purification process and SDS-PAGE electrophoresis of ⁇ .
  • the resulting PEG- ⁇ had a purity that is higher than 95% and antiviral EMC activity on Hep-2C cell with ED 50 about 10-50 ng/ml (see example 6).
  • Example 6 Examination of antiviral activity of IF l and PEG- ⁇
  • Peglamda PEG- ⁇ 200 ⁇ g (weekly subcutaneous injection) + ribavirin 15 mg/kg (daily).
  • ribavirin 15 mg/kg (daily).
  • All patients were determined to be free of HCV RNA (free virus in serum). The treatment was continued for 12 weeks.
  • the presently disclosed treatment protocol was found to be effective for greater than 80%, 85%, 90 % or greater than 95% of the HCV resistant patient population. Accordingly, the treatment methods using the PEG- ⁇ demonstrate efficacy in HCV including cases of resistance to current standard therapy of peginterferon alfa-2a with ribavirin. No significant side effects that are typically associated with the combination therapy of PEGASYS® with ribavirin were observed.
  • Example 7 Identification of ⁇ and PEG- ⁇
  • Example 8 Molecular weight of PEG- ⁇ [0093] The MALDI-TOF assay was applied to determine the molecular weight of PEG-IFWJ. The result is provided in Figure 12. In the present example, the Nanogen's PEG- ⁇ has a molecular weight of approximately 62 kDa.
  • electrophoresis gel was stained with coomassie blue, destained and then analyzed using Phoretix software (TotalLab, England). All tested lots showed purity higher than 95%.
  • Acute toxicity of PEG- ⁇ The acute toxicity of PEG- ⁇ was assessed in Swiss mice and rats. Healthy ICR mice and Sprague-Dawley rats, at 5 week old, were chosen for the study. The animals were inspected for two weeks. PEG- ⁇ was administered at three different dosages (high dose 3 mg/kg, medium dose 0.3mg/kg, low dose 0.03 mg/kg and the vehicle treatment (phosphate buffer saline, pH 7.2)) by subcutaneous or intraperitoneal injection. Animals were observed for clinical signs, body weight changes, and mortality 14 days after treatment. At the end of the study, all animals were sacrificed, and their tissues and organs were examined for abnormalities. The results are summarized in table 4.
  • the lethal dose (LD50) of Nanogen's PEG-IFTSai in mouse and rat was greater than 3 mg/kg.
  • Subacute toxicity of PEG- ⁇ Animals (5 weeks old rats) were administered PEG- ⁇ at three different dosages (high dose 3 mg/kg, medium dose 0.3 mg/kg, low dose 0.03 mg/kg) by subcutaneous or intraperitoneal injection once a day for 4 weeks.
  • the sensitized guinea pigs were observed for active systemic anaphylaxis reactions after injection of a high dose PEG-IFWJ. A list of indications was used as a sign of anaphylactic reaction and their occurrence was monitored in each tested animal.
  • Table 6 shows the study method and results.

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CA2846092A CA2846092A1 (en) 2011-08-25 2012-03-01 Peg-interferon lambda 1 conjugates
RU2014111179/10A RU2014111179A (ru) 2011-08-25 2012-03-01 КОНЪЮГАТЫ ИНТЕРФЕРОНА λ1 И ПОЛИЭТИЛЕНГЛИКОЛЯ
BR112014004302A BR112014004302A2 (pt) 2011-08-25 2012-03-01 conjugado, composição farmacêutica, processo para preparar um conjugado, métodos para inibir a proliferação de uma célula cancerosa, para tratar um distúrbio, e para tratar um paciente
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EP3375452A1 (en) 2017-03-16 2018-09-19 Evangelos Andreakos Use of lambda interferons in the treatment of disorders and related diseases
WO2021013204A1 (zh) 2019-07-22 2021-01-28 厦门特宝生物工程股份有限公司 一种基于干扰素的疾病治疗方法
WO2022156735A1 (zh) 2021-01-21 2022-07-28 厦门特宝生物工程股份有限公司 一种基于干扰素的癌症治疗方法和药物组合
WO2022156733A1 (zh) 2021-01-21 2022-07-28 厦门特宝生物工程股份有限公司 一种预防癌症复发的方法和药物组合
WO2024121424A1 (en) 2022-12-09 2024-06-13 Daniel Zagury Composite aids vaccine generating anti-hiv specific neutralizing antibodies and/or anti-hiv cytotoxic t cells

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PL3416675T3 (pl) * 2016-02-19 2021-10-11 Eiger Biopharmaceuticals, Inc. Leczenie zakażenia wirusem zapalenia wątroby typu delta za pomocą interferonu lambda
RU2678332C1 (ru) * 2017-09-08 2019-01-28 Общество с ограниченной ответственностью "Саентифик Фьючер Менеджмент" (ООО "СФМ") Пегилированный интерферон лямбда, обладающий высокой биодоступностью при пероральном применении, и способ его получения

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3375452A1 (en) 2017-03-16 2018-09-19 Evangelos Andreakos Use of lambda interferons in the treatment of disorders and related diseases
WO2018167287A1 (en) 2017-03-16 2018-09-20 Biomedical Research Foundation Of The Academy Of Athens Use of lambda interferons in the treatment of obesity-related disorders and related diseases
DE112018001364T5 (de) 2017-03-16 2019-12-05 Evangelos Andreakos Verwendung von Lambda-Interferonen bei der Behandlung von mit Adipositas zusammenhängenden Störungen und verwandten Erkrankungen
WO2021013204A1 (zh) 2019-07-22 2021-01-28 厦门特宝生物工程股份有限公司 一种基于干扰素的疾病治疗方法
WO2022016844A1 (zh) 2019-07-22 2022-01-27 厦门特宝生物工程股份有限公司 一种基于干扰素的癌症治疗方法和药物组合
WO2022156735A1 (zh) 2021-01-21 2022-07-28 厦门特宝生物工程股份有限公司 一种基于干扰素的癌症治疗方法和药物组合
WO2022156733A1 (zh) 2021-01-21 2022-07-28 厦门特宝生物工程股份有限公司 一种预防癌症复发的方法和药物组合
WO2024121424A1 (en) 2022-12-09 2024-06-13 Daniel Zagury Composite aids vaccine generating anti-hiv specific neutralizing antibodies and/or anti-hiv cytotoxic t cells

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