WO2013029062A1

WO2013029062A1 - Peginterferon lambda 1 conjugates, processes for their preparation, pharmaceutical compositions containing these conjugates and processes for making the same

Info

Publication number: WO2013029062A1
Application number: PCT/VN2011/000009
Authority: WO
Inventors: Nhan HO
Original assignee: NANOGEN PHARMACEUTICAL BIOTECHNOLOGY CO Ltd
Current assignee: NANOGEN PHARMACEUTICAL BIOTECHNOLOGY CO Ltd
Priority date: 2011-08-25
Filing date: 2011-11-15
Publication date: 2013-02-28
Anticipated expiration: 2014-02-25

Abstract

This invention relates to new peginterferon lambda 1 conjugates (peg-ΓΡΝλΙ), processes for their preparation, pharmaceutical compositions containing these conjugates and processes for making the same. These conjugates have increased blood half-lives and persistence time compared to IFNλl and are effective in treatment of hepatitis В and hepatitis C.

Description

PEGINTERFERON LAMBDA 1 CONJUGATES, PROCESSES FOR THEIR PREPARATION, PHARMACEUTICAL COMPOSITIONS CONTAINING THESE CONJUGATES AND PROCESSES FOR MAKING THE SAME

Field of the invention

This invention relates to new pegylated derivatives of recombinant human interferon lambda 1 (peg-interferon lambda 1 conjugates, peg-IF i) having increased half-lives in circulation, processes for their preparation, pharmaceutical compositions containing these conjugates and processes for making the same.

Background of the invention

Hepatitis C virus (HCV) is a major health problem and the leading cause of chronic liver disease throughout the world. It is estimated that at least 180 million persons worldwide are chronically infected with HCV. In Vietnam, the proportion of infected HCV individuals in the population is 4-9%. Approximately 55-85% of acutely infected HCV individuals will convert to chronic infection, 5- 25% of these chronic carriers are at risk of developing cirrhosis after 25-30 years and of those with cirrhosis, 30% are at risk of liver decompensation over 10 years and 1-3% will develop liver cancer each year. According to epidemiological research, HCV is the cause of 40% of individuals in final stage cirrhosis and 60% in hepatoma.

Currently, a-interferons are the therapies of choice for the treatment of chronic HCV infection. a-Interferons can give a persistent response to HCV in approximately 70% of cases, however these interferons cause many side-effects, even in case of peg-interferon alpha. These side-effects can sometimes limit treatment, leaving treatment incomplete. Side-effects include influenza-like symptoms and hematologic effects such as thalassemia and anemia.

Interferons are proteins produced by immune cells (leukocytes) in response to viral infection. 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, AIDS. Interferons alpha are also effective in reducing malignant tumours and treating Kaposi's sarcoma, melanoma, and renal cell carcinoma. Moreover, alpha-interferons are applicable in prevention and treatment of diseases in cattle and other livestock. For example, alpha-interferons enhance the activity of vaccines used in prophylaxis and treatment of foot and mouth disease and porcine reproductive and respiratory syndrome. They are therapeutically effective in treatment of bacterial diseases such as respiratory infection, mastitis, bovine salmonellosis, and viral diseases such as blue-ear pig disease (also called porcine reproductive and respiratory syndrome-PRRS), pig diarrhea, influenza, Marek's disease, and Gumboro's disease in domestic birds.

Alpha-interferons 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.

With the development of recombinant DNA technology, we can now introduce interferon alpha genes into microorganisms that enable production of large amounts of interferons. However, these methods also present certain advantages and difficulties, mainly in the steps of expression and large-scale protein production.

IL-29 is a member of the helical cytokine family and is a type III interferon. It is also known as interferon lambda 1 (IFN l) and is highly similar in amino acid sequence to IL-28, the other type III interferon. IL-28 and IL-29 (ΙΡΝλΙ ) 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 . IFNs exhibit several common features with type I IFNs: antiviral activity, antiproliferative activity and in vivo antitumour activity. Importantly, however, IFNs bind to a distinct membrane receptor, composed of IFNLR1 and IL10R2. This specific receptor usage suggests that this cytokine family does not merely replicate the type I IFN system and justifies its designation as a separate class, the type III IFN by the nomenclature committee of the International Society of Interferon and Cytokine Research.

The major disadvantage with the therapeutic use of most biologicals is that they are administered parenterally, e.g. intravenously (i.v.), subcutaneously (s.c), intramuscularly (i.m.) etc. This means that delivery to the patient is associated with pain and discomfort. Furthermore, because of their usually very short half- lives, biologicals require frequent administration to the patient in order to maintain therapeutic blood levels of the drug. Injections that cannot be self- administered require frequent trips to the clinic and trained medical personnel, making such therapy inconvenient and expensive Many examples exist of biological drugs that require frequent administration. Interferon alpha-2a (Roferon, Roche) and 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 (McHutchison, et al., Engl. J. Med. 1998, 339, 1485-1492; Glue, et al., Clin. Pharmacol. Ther. 2000, 68, 556-567) and therefore require administration 3 times a week. Repeated injections with interferon beta- lb (Betaseron) are also required to treat the patients of multiple sclerosis (MS). The recommended dosing is subcutaneous injection given every other day. Likewise, filgrastim (granulocyte colony stimulating factor, or G-CSF) is injected every day for two weeks.

One very successful and well accepted method of overcoming the above requirement of frequent high dose injections to maintain threshold levels of the drug in the body is to increase the in vivo half-life of the therapeutic protein by conjugating it with a polymer, preferably polyethylene glycol (PEG). PEG molecules with their long chains not only create a protective shield around the pegylated drug molecule in aqueous solution, thereby reducing the immunogenicity of protein drugs while also protecting them from the action of proteases, but they further help increase circulation half-life of the drug by increasing its hydrodynamic volume which reduces its loss from the filtration mechanisms of the kidney glomeruli network. After their separation from the protein molecule, the PEG moieties are cleared without any structural changes and their clearance is proportional to their molecular weight. Conjugation of proteins to PEG has been reported since 1970s. Usually PEG moieties are attached to the protein by first activating the PEG moiety and then reacting it with the side chains of lysine residues 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-protein conjugates were formed by reacting a biologically active material with a molar excess concentration of a highly activated polymer having a terminal linking group without regard to where the polymer would attach to the protein, and leading to a physiologically active, non-immunogenic, water soluble polypeptide composition. Pegylation of interferons has been reported in U.S. Pat. Nos. 4,766,106 and 4,917,888 which describe inter alia beta interferon conjugated with activated polymers including mPEG-2,4,6-trichloro-S-triazine, mPEG-N- succinimidyl glutarate or mPEG-N-succinimidyl succinate. One such disclosure in U.S. Pat. No. 5,951,974 describes the conjugation of interferon to a substantially non-antigenic polymer at a histidine site. Another such disclosure in U.S. Pat. No. 5,981,709 describes an alpha interferon-polymer conjugate with a relatively long circulating half-life in vivo.

Peg-interferon lambda 1 (peg-ΙΡΝλΙ) is a pegylated derivative of human recombinant ΙΡΝλΙ (wherein polyethylene glycol is conjugated to the ΙΡΝλΙ molecule) useful in treatment of chronic hepatitis C in adult patients. It 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.

Summary of the invention

The present invention provides novel peg-IFN l conjugates. As presented hereafter, conjugates of the invention have a linear PEG chain structure. As compared to unmodified ΙΡΝλΙ (that is, ΙΡΝλΙ not conjugated with PEG), these conjugates have increased circulating half-life and persistence in plasma.

The present invention is directed to physiologically active peg-ΙΡΝλΙ conjugates having the formulae: O

II

CH₃0(CH₂CH₂0)— C— O-NH— Interferon lambda 1 wherein, n is a number of units of ethylene glycol in PEG structure and it is a positive integer selected from any numbers such that the molecular weight of PEG moiety is about 40kDa. The number n is selected in the range from 500 to 550. The PEG chain binds to the ΙΡΝλΙ via an amide bond on a primary amino group of, for example, lysine, or the N-terminal of ΙΡΝλΙ . ΙΡΝλΙ could be a natural or recombinant protein. In a preferred embodiment, ΙΡΝλΙ is a human protein derived from any source such as tissues, protein synthesis, or cell culture using natural cells or recombinant cells. In a still preferred embodiment, ΙΡΝλΙ is a human recombinant protein.

The conjugates of the invention have similar effects to those of ΙΡΝλΙ, for example, they may be used as anti-proliferative agents, antiviral agents, or antitumor agents. Specifically, the conjugates of invention are effective in treatment of hepatitis B and hepatitis C, but they have a longer persistence time in blood than IFN l . Therefore, the present invention also provides pharmaceutical compositions containing the above-mentioned human recombinant peg-IFN l conjugates and processes for making the same to use in treatment of hepatitis B and hepatits C.

In one embodiment, pharmaceutical compositions containing the conjugates of the invention are prepared as sterile lyophilized powders for injection or as solutions for injection in vials or pre-filled syringes. These pharmaceutical compositions can be formulated by way of mixing the conjugates with relevant pharmaceutically acceptable carriers and excipients.

In one embodiment, the invention provides processes for preparation of human recombinant peg-ΙΡΝλΙ conjugates. First, human recombinant IFN l is produced by recombinant DNA technology in E.coli, then reacted with a pegylating agent (a-methoxy-ro-(4-nitrophenoxy carbonyl)) polyoxyethylene (PEG-pNC) to produce the peg-ΙΡΝλΙ (linear chain PEG 40kDa conjugated to IFNll). This product bypasses the action of extracellular enzymes and kidney filtration when injected into the patient's body, therefore its blood half-life is extended.

The pegylation reaction between (a-methoxy-co-(4-nitrophenoxy carbonyl)) poly oxy ethylene (PEG-pNC) 40 kDa and ΙΡΝλΙ is as follows (wherein NH₂ group is a site of interferon lambda 1 molecule):

+ NH₂— Interferon lambda 1

CH₃0(CH₂CH₂O)_n—

The conjugates of the invention are generated by way of covalently binding Interferon lambda 1 with pre-activated PEG (PEG is activated by substituting the PEG hydroxyl with a link group to create the reaction agent that is (a-methoxy-G)-(4-nitrophenoxy carbonyl)) polyoxyethylene (PEG-pNC) ).

The invention will herein focus on description of manufacturing peg- IFN l conjugates from the step of producing ΙΡΝλΙ material to carrying out the pegylation reaction and processes for purifying and assaying the conjugated products.

Brief description of the drawings

Figure 1 shows the nucleic acid sequence used to produce human recombinant ΙΡΝλΙ in Nanogen Pharmaceutical Biotechnology Co., Ltd. after this sequence was synthesized and introduced into the expression vector pNanogen 1-IL29.

Figure 2 shows the amino acid sequence of human recombinant ΙΡΝλΙ produced by Nanogen Pharmaceutical Biotechnology Co., Ltd.

Figure 3 shows plasmid pNanogen 1-IL29 containing the gene encoding human ΙΕΝλΙ (interleukin-29). Figure 4 shows the result of analyzing plasmid pNanogen 1-IL29.

Figure 5 shows the result of electrophoresis process for examination of the ability of is. coli containing pNanogen 1-IL29 used to produce ΙΡΝλΙ .

Figure 6 shows the spectrum of the salt phase and SDS-PAGE electrophoresis after refolding protein.

Figure 7 shows the spectrum and SDS-PAGE electrophoresis after cation 1 phase.

Figure 8 shows the spectrum and SDS-PAGE electrophoresis after cation 2 phase.

Figure 9 shows the spectrum and SDS-PAGE electrophoresis after gel filtration phase.

Figure 10 shows the spectrum of the purification process and SDS-PAGE electrophoresis of peginterferon lambda 1.

Figure 11 shows the identification results of ΠΤνΓλΙ and peg-ΙΡΝλΙ .

Figure 12 shows the Maldi-Tof mass-spectrum of peg-ΙΡΝλΙ produced by Nanogen Pharmaceutical Co., Ltd.

Detailed description of the invention

The invention includes the following main contents: laboratory work to create a recombinant bacterial strain containing the gene encoding ΙΡΝλΙ, industrial manufacturing of ΙΡΝλΙ, pegylation reaction with ΕΡΝλΙ, purification of the produced peg-ΙΡΝλΙ , assay of the properties and characteristics of peg- IFN l .

We artificially synthesized the gene encoding ΙΡΝλΙ based on the published sequence available from the National Center for Biotechnology Information (said encoding gene was modified to conform to the industrial production process on E.coli), created the gene transfer vectors, introduced these vectors into the bacteria, and selected the bacterial strain that best produced IFN l. The industrial production process of IFN l includes the steps of: fermenting the initial material, collecting the solution of crude proteins, and purifying the ΙΡΝλΙ protein. The fermentation process was carried out in a 10 litre fermenting tank containing nutrient medium and production of ΙΡΝλΙ was induced by lactose. The biomass obtained was separated and purified. ΙΡΝλΙ was collected and refined through 4 steps including: refolding the protein, separating the protein by ion exchange chromatography (cation 1 and cation 2), and refining the protein on a gel.

The pegylation process is a reaction between linear chain (a-methoxy-ω- (4-nitrophenoxy carbonyl)) polyoxyethylene (PEG-pNC - molecular weigh 40kDa) and ΙΡΝλΙ, the product was purified on HPLC system and tested for quality.

The invention will be more fully appreciated by reference to the following examples, which are to be considered merely illustrative and not limiting to the scope of the invention as claimed.

Examples of invention

Example 1: Process for creation of E. coli strain containing the gene encoding human recombinant interferon lambda 1 ( IFN l)

The gene encoding IFN l was artificially synthesized based on the protein sequence data available from NCBI or other medicine databases. This is a novel method which reduces the time required to isolate the gene but still gives a result as accurate as the conventional method. The nucleic acid sequence used to produce ΙΡΝλΙ in Nanogen Pharmaceutical Biotechnology Co., Ltd. is presented in Figure 1 and the amino acid sequence of this protein is presented in Figure 2.

The expression vector pNanogen-IL29 (comprising the T7 transcription promoter region, the ΙΡΝλΙ 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 ΙΡΝλΙ. 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 hsdS_B (rB^'mB^' )gal dcm (DE3). The strain containing the IFNll gene was called E.Coli- pNanogenl-IL29. It has the ability to produce higher than lOOmg of IFN l 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 IFmi

The fermentation process was carried out in a 140 litres fermentation tank with nutrient medium at a temperature 37±0.5°C, air pressure 0.5m³/h, pH 7.0±0.2, stirring rate 300rpm and maintained the pH value between 6.8-7.2 by adding H₃PO4 or NH₄OH. After 8 hours (when E. coli grew in log phase is the time that cells develop most strongly), the temperature was cooled to 30±0.5°C and the stirring rate was reduced to 200rpm to start the process for generation of IFN l . The fermentation process was stopped after 4 hours, then centrifuged the cold product at 6000rpm to obtain biomass.

The biomass was disrupted in a cell lysis solution (12ml solution per lg 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 6000rpm for 30 minutes to give a pellet. The pellet was then washed with an inclusion body wash buffer (12ml 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. The pellet was dissolved in 2M urea solution and incubated ice-cold for 1 hour, the suspension was then centrifuged at 13,000rpm for 30 minutes to give the pellet. The pellet was dissolved in a wash solution and centrifuged at 13,000rpm 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, then centrifuged at 13,000rpm for 30 minutes. The solution containing protein was recovered and purified in next step.

The components of culture medium and solutions used to separate ΙΡΝλΙ are presented in Table 1. Table 1

Example 3: Process for purification of human recombinant IFN l

IFN l was refolded by dissolving the inclusion bodies in refolding solution (25mM Tris buffer, ImM EDTA, 1,2M Guanidine pH 8,2) such that the final concentration of the inclusion bodies were 500 μg/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 phosphate buffer (lOmM, pH 8.0). In the step "cation 1", said 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 IF ,1 with purity higher than 95% (see the spectrum and electrophoresis results in figures 5, 6, 7, 8, 9).

Example 4: Pegylation process

The solution of 5mg/ml human recombinant ΙΡΝλΙ (MW~20,lkDa) in 50mM sodium borate-phosphate pH 8.0 was added (a-methoxy-co-(4- nitrophenoxy carbonyl)) polyoxyethylene (PEG-pNC) (MW~40kDa) at a molar ratio of PEG-pNC ΉΝλΙ about 3: 1. The reaction mixture was kept at 2-4°C for 20 hours. The reaction was stopped by adjusting the pH to 4.0 using 30%w/w acetic acid solution. The resulting mixture was then diluted 5 -fold with water.

Example 5: Purification of peg-IFN l

The solution containing peg-ΙΡΝλΙ, quenched reagent, and unmodified ΙΡΝλΙ was purified on a cation column (this column was prefilled with Sepharose CM gel and equilibrated in lOmM sodium phosphate pH 6.0), eluted with a solution of lOmM sodium phosphate, 0.5M NaCl pH 6.0. The eluted fractions containing protein were transferred into preservative buffer using a solution of lOmM sodium phosphate pH 6.0. This product was then subjected to a sterile filtration process and stored at -20°C.

Figure 10 shows the spectrum of the purification process and SDS-PAGE electrophoresis of ΙΡΝλΙ. The resulting peg-ΙΡΝλΙ had a purity higher than 95% and antiviral EMC activity on Hep-2C cell with ED₅₀ about 10-50ng/ml (see example 6).

Example 6: Examination of antiviral activity of IFN l and peg- IFN l

The examination was based on the antiviral ability in EMC viral model and Hep-2C cell according to the study of Ank et al. (J Virol. 2006 May;80(9):4501-9 ). The experiment was carried out with 3 lots (IL2900101 1 1, IL290020311 and IL290030411), the obtained results indicated that the antiviral activity of Nanogen's interferon lambda 1 (ED₅₀ about l-5ng/ml) equivalent to the study results of Sheppard et al. (Nat Immunol. 2003 Jan;4(l):63-8.) (see Table 2).

The antiviral activity of peg-ΙΡΝλΙ was checked similarly to ΙΡΝλΙ . The experiment was carried out with 5 lots (PIL290010111, PIL290020211, PIL29003031 1, PIL290040411, PIL290050511). Similar results were obtained in all lots, with an ED₅₀ about 10-50ng/ml (see Table 3).

Table 2: Results of experiments to examine the antiviral activity of Nanogen's interferon lambda 1

Table 3: Results of experiments to examine the antiviral activity of

Nanogen's peg-ΙΡΝλΙ

Example 7: Identification of IFN l and peg-IFN l

Western blotting method was used to identify ΙΡΝλΙ and peg-ΙΡΝλΙ using anti-ΙΡΝλΙ antibody. The protein solution after being analyzed on an SDS-PAGE gel was transferred to a nitrocellulose membrane and probed with the anti-ΙΡΝλΙ antibody. Antibody was detected with peroxidase coupled protein A and TMB substrate (see Figure 1 1).

Example 8: Molecular weight of peg-IFN l The MALDI-TOF assay was applied to determine the molecular weight of peg-IFNXl. The result is showed in Figure 12, Nanogen's peg-ΙΡΝλΙ has a molecular weight of appropriate 62kDa.

Example 9: Purity of peg-IFN l

Five lots (PIL290010111, PIL290020211, PIL290030311, PIL29004041 1, PIL29005051 1) were used to determine the purity of peg-ΙΡΝλΙ by SDS-PAGE electrophoresis. The electrophoresis gel was stained with comassie blue, destained, then analyzed using Phoretix software (TotalLab, England). All tested lots showed purity higher than 95%.

Example 10: Toxicity of peg-IFN l

Acute toxicity of peg-IFNXl

The acute toxicity of peg-IFNXl was assessed in Swiss mice and rats. ICR mice and Sprague-Dawley rats were inspected for two weeks and healthy 5 week old animals were chosen for the study. Peg-IFN l was administered at three different dosages (high dose 3mg/kg, medium dose 0.3mg/kg, low dose 0.03mg/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 for 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.

Table 4

(*): ANOVA, single factor, compare to the vehicle treatment, (p>0.05)

All animals survived for the test period even at the highest dosage. The body weight did not significantly change in the treated animals compared to the control. There were no clinical signs or organ abnormalities observed in either group of the tested animals. From these data, it is concluded that the lethal dose (LD50) of Nanogen's peg-ΙΡΝλΙ in mouse and rat was greater than 3mg/kg.

Subacute toxicity of peg-ΙΡΝλΙ

Animals (5 weeks old rats) were administered peg-ΙΡΝλΙ at three different dosages (high dose 3mg/kg, medium dose 0.3mg/kg, low dose 0.03mg/kg) by subcutaneous or intraperitoneal injection once a day for 4 weeks. The rats were examined throughout the study for any clinical and behavioral adverse effects caused by Nanogen's peg-ΙΡΝλΙ administration. After the test period, the survived rats were sacrificed for autopsy and biochemical analyses. Blood samples were also collected from abdominal artery to conduct hematologic tests.

The test method and results are summarized in table 5.

Table 5

Urinalysis Normal Normal

Hematology Normal Normal

Serum biochemistry Normal Normal

Absolute and relative organ

Normal Normal weight

Autopsy result Normal Normal

Histopathological examination Normal Normal

There was no death in any groups during the entire study and no clinical signs were detected from the tested rats. The tested rats were normal in other examination categories and analyses even in the high dosage group. Therefore, the study shows that Nanogen's peg-ΙΡΝλΙ does not have toxic effects in rats when it is administered repeatedly at the dosage of 3mg/kg.

Immunological toxicity of peg-ΓΡΝλ!

A study was carried out to investigate immunologic potential of Nanogen's peg-ΙΡΝλΙ in guinea pigs. Healthy male Hartley guinea pigs with body weight of 300-500 gram were injected with peg-ΙΡΝλΙ twice a week for 3 weeks either at a high dose (3mg/kg) or low dose (0.03mg/kg) and ovalbumin as control. Fourteen days after the final sensitization, the anaphylaxis test was performed by intravenously injecting a high dose of peg-ΙΡΝλΙ. The study included peg-ΙΡΝλΙ incorporated in Freund's complete adjuvant (FCA). The sensitized guinea pigs were observed for active systemic anaphylaxis reactions after injection of a high dose peg-ΓΡΝλΙ . 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.

Table 6

1 - + + + - - - -

2 _ +-- +-- + _ _

Peg-IFN l

3 + + +

(3mg/kg) + FCA

4 - + + - + - - -

5 - + + - - -

' ' 1. Licking nose, rubbing nose; 2. Ruffling fur;

3. Labored breathing; 4. Sneezing, coughing;

5. Evacuation of feces, micturition; 6. Convulsion; 7. Prostration;

8. Death. -, negative ; +, positive

In the active systemic anaphylactic test, the guinea pigs slightly sensitized with high dose of peg-ΙΡΝλΙ (3mg/kg) incorporated in Freund's complete adjuvant (FCA) showed some indications of anaphylactic reaction. On the other hand, no guinea pigs sensitized with low dose and high dose of peg-IFN l (0.03 and 3mg/kg) alone showed any anaphylactic reaction. No guinea pigs were dead after administration with Nanogen's peg-IF^l and negative treatment (PBS), but 3 pigs were dead after administration with ovalbumin (positive treatment). Therefore, it can be concluded that Nanogen's peg-IFN l dose not induce systemic allergic reaction when administered alone in its clinical use.

Claims

1. A physiologically active Peg-IFN l conjugate has the following formulae:

O

II

CH₃0(CH₂CH₂0)— C— O-NH— Interferon lambda 1 wherein, the PEG moiety has a linear chain structure, n is a positive integer selected in the range from 500 to 550 such that the molecular weight of PEG moiety is about 40kDa.

2. The conjugate of claim 1, wherein its blood half-life and persistence time are prolonged compared to ΙΡΝλΙ .

3. A conjugate of claim 1 or claim 2, wherein said conjugate is effective in treatment of hepatitis B and hepatitis C.

4. A process for preparation of a human recombinant conjugate according to any one of claims 1 to 3 includes step of covalently binding (a-methoxy-G (4- nitrophenoxy carbonyl)) polyoxyethylene (PEG-pNC) 40kDa with ΙΡΝλΙ through pegylation reaction as follow: + NH₂— Interferon lambda 1

CH₃0(CH₂CH₂0)_n—

wherein, n is a positive integer selected in the range from 500 to 550 such that the molecular weight of PEG moiety is about 40kDa.

5. A pharmaceutical composition containing a conjugate according to any one of claims 1 to 3 and pharmaceutically acceptable carriers and excipients.

6. The pharmaceutical composition of claim 5, wherein said pharmaceutical composition is formulated as sterile lyophilized powder for injection.

7. The pharmaceutical composition of claim 5, wherein said pharmaceutical composition is formulated as solution for injection in vial or pre-filled syringe.

8. A pharmaceutical composition according to any one of claims 5 to 7, wherein said pharmaceutical composition is used in treatment of hepatitis B and hepatitis C.

9. A process for preparation of a pharmaceutical composition containing a conjugate according to any one of claims 1 to 3 comprising step of mixing the conjugate with pharmaceutically acceptable carriers and excipients.