Pegylated interferon alpha-2a (40 kDa) in the treatment of chronic hepatitis B

Chronic hepatitis B virus (HBV) is a serious and life-threatening disease afflicting 350 million of the world's population. So far, current monotherapy with conventional interferon-alpha, lamivudine, and adefovir dipivoxil remains unsatisfactory. In addition, the use of conventional interferon-alpha needs to be administered subcutaneously daily or thrice weekly and is associated with frequent adverse events. Although nucleoside-nucleotide analogs such as lamivudine and adefovir dipivoxil are well tolerated and can normalize serum alanine aminotransaminase rapidly, 1-year therapy with either lamivudine or adefovir dipivoxil results in low hepatitis B e antigen (HBeAg) seroconversion rates. In HBeAg negative patients, most of the patients would relapse after lamivudine has been discontinued. Pegylated interferon alpha-2a, an immunomodulatory agent, is a new drug that has just completed phase III clinical trials for the treatment of both HBeAg positive and HBeAg negative chronic HBV infection. The advantage of pegylated interferon alpha-2a in achieving sustained virological response over nucleoside-nucleotide analogs is particularly obvious in the HBeAg negative group. In both of these phase III studies, sustained off-treatment response is superior to the use of lamivudine. These recent data put pegylated interferon alpha-2a as the first choice of anti-HBV therapy, especially in young and motivated patients with chronic HBV infection.     

Pegylated liposomal doxorubicin in the treatment of AIDS-related Kaposi's sarcoma

Kaposi's sarcoma is a vascular tumor of skin and viscera first described in 1872. Prior to the 1980s, this disease was rarely seen in the Western world, but was quite prevalent in Sub-Saharan African countries. Since the onset of the HIV pandemic in the 1980s, the incidence of Kaposi's sarcoma has increased markedly in Africa and continues to be a significant problem in association with AIDS in Western countries. Many therapies have been demonstrated to be effective in the treatment of HIV-related Kaposi's sarcoma, including alitretinoin gel, interferon alpha, and various forms of cytotoxic chemotherapy. Antiretroviral therapy combined with cytotoxic agents has yielded significantly greater efficacy than chemotherapy alone. However, as reviewed in this report, pegylated liposomal doxorubicin has been established as the treatment of choice for patients with AIDS-associated Kaposi's sarcoma in Western countries. Compelling preclinical and clinical evidence, reviewed herein, has demonstrated that the nanoparticle (pegylated liposome) delivery system of this formulation leads to greater tumor localization of doxorubicin and consequent improved efficacy, as well as reduced toxicity.     

Rational design of a potent, long-lasting form of interferon: a 40 kDa branched polyethylene glycol-conjugated interferon alpha-2a for the treatment of hepatitis C

A potent, long-lasting form of interferon alpha-2a mono-pegylated with a 40 kilodalton branched poly(ethylene glycol) was designed, synthesized, and characterized. Mono-pegylated interferon alpha-2a was comprised of four major positional isomers involving Lys31, Lys121, Lys131, and Lys134 of interferon. The in vitro anti-viral activity of pegylated interferon alpha-2a was found to be only 7% of the original activity. In contrast, the in vivo antitumor activity was severalfold enhanced compared to interferon alpha-2a. Pegylated interferon alpha-2a showed no immunogenicity in mice. After subcutaneous injection of pegylated interferon alpha-2a, a 70-fold increase in serum half-life and a 50-fold increase in mean plasma residence time concomitant with sustained serum concentrations were observed relative to interferon alpha-2a. These preclinical results suggest a significantly enhanced human pharmacological profile for pegylated interferon alpha-2a. Results of Phase II/III hepatitis C clinical trials in humans confirmed the superior efficacy of pegylated interferon alpha-2a compared to unmodified interferon alpha-2a.     

Advances in therapy for hepatitis C infection

The first approved therapy for chronic hepatitis C virus (HCV) infection was recombinant interferon. Subsequently, controlled studies demonstrated that the combination of interferon-alpha and ribavirin leads to significantly higher virologic sustained responses in patients with chronic hepatitis C. A novel modification of the interferon molecule resulted in the formulation of pegylated interferons, which have a longer half-life than standard interferon. Two recent trials have established the superiority of pegylated interferons compared with interferon-alpha in inducing sustained virologic responses in patients with chronic HCV infection, with or without cirrhosis. Presumably, pegylated interferons will replace standard interferon in treating HCV infection. Phase 3 trials of pegylated interferons in combination with ribavirin are currently under way. Noninterferon-based therapies for the treatment of HCV infection are also in the developmental and experimental phases. Our aims in this review are to present the currently available therapeutic options for HCV infection and the evidence supporting their use in typical patients with chronic hepatitis C or in patients with special circumstances. We also briefly review novel therapeutic approaches, including noninterferon-based therapies.     

Ultrafiltration characteristics of pegylated proteins

There is growing clinical interest in the use of pegylated recombinant proteins with enhanced stability, half-life, and bioavailability. The objective of this study was to develop a quantitative understanding of the ultrafiltration characteristics of a series of pegylated proteins with different degrees of pegylation. Sieving data were compared with available theoretical models and with corresponding results for the partition coefficient in size exclusion chromatography (SEC). The sieving coefficients of the pegylated proteins depended not only on the protein size and the total molecular weight of the polyethylene glycol (PEG) but also on the number of PEG chains. This is in sharp contrast to the partition coefficient in SEC, which was uniquely determined by the total molecular weight of the PEG and protein. This difference is due to the deformation and/or elongation of the PEG chains caused by the convective flow into the membrane pores, an effect that is not present in SEC. These results provide important insights into the transport and separation characteristics of pegylated proteins.     

The errors in assembly of MuLV in interferon treated cells

Interferon treatment of JLSV-6 cells chronically infected with Rauscher MuLV leads to the formation of noninfectious particles (interferon virions) containing the structural proteins of env and gag genes as well as additional viral polypeptides. In the control virions the major glycoprotein detected is gp71, interferon virions contain in addition to gp71 and 85k dalton (gp85) glucosamine-containing, fucose-deficient glycoprotein which is recognized by antiserum to MuLV but not by the gp71 antiserum. The surface iodination of the intact virions indicates that both gp71 and gp85 are the major components of the external virions envelope. However, unlike the control virions in which gp71 associates with p15E (gp90), the gp71-p15E complex was not detected in interferon virions. The analysis of the iodinated proteins of the disrupted interferon virions revealed the presence of 85k and 65k dalton polypeptides preciptable with antiserum against MuLV, which are not present in the control virions. The difference in the polypeptide pattern of virions produced in the presence of interferon does not seem to be a consequence of the slowdown in the synthesis of viral proteins or their processing in the interferon-treated cells. Both the structural proteins of env and gag genes seem to be synthesized and processed at a comparable rate in the interferon-treated and -untreated cells. These results indicate an alteration of virus assembly in the presence of interferon.     

干扰素研究及应用进展

1957年,英国科学家Isaacs和Lindenmann利用鸡胚绒毛尿囊研究流感干扰现象时,发现一种能够干扰病毒繁殖的物质,称之为干扰素（IFN）,由于干扰素对许多疾病有很好的治疗作用,一直是医学领域研究的重要物质之一。现在干扰素已成功地用于肝炎、肿瘤治疗等方面。为了充分发挥干扰素的作用,人们稍用基因工程的方法来对干扰素的产量及功效进行改进,已达到更好的应用,因此基因工程干扰素越来越多地成了研究的重点。     

Prediction of the secondary and tertiary structures of interferon from four homologous amino acid sequences

The secondary and tertiary structures of interferon were predicted from four homologous amino acid sequences. Three methods of secondary structure prediction gave differing results that were interpreted to suggest that there might be four α-helices that are important in the tertiary fold. The validity of this interpretation was assessed by the application of the methods to predict the secondary structures of two proteins known to consist of four α-helices. A possible tertiary model for interferon is then proposed in which the four α-helices pack into a right-handed bundle similar to that observed in several known protein structures. This model was shown to be stereochemically feasible by an α-helix docking algorithm. One of the resultant structures is shown to be compatible with the known disulphide linkages in interferon. Certain residues that are conserved between the different sequences lie near each other in our model and these residues might form a functional site. In the absence of a crystal structure for interferon, a predicted tertiary model will help further structural and functional studies.     

Acute inflammatory demyelinating polyneuropathy after treatment with pegylated interferon alfa-2a in a patient with chronic hepatitis C virus infection: a case report

ABSTRACT: INTRODUCTION: The combination of polyethylene glycol (PEG)ylated interferon (pegylated interferon) and ribavirin has been shown to be an effective treatment for chronic hepatitis C virus. In general, common side effects related to this combination therapy are mild and are well tolerated. However, peripheral neuropathy including demyelinating polyneuropathy related to PEG-interferon alpha2a (pegylated interferon alfa-2a) is extremely rare. In the literature, only one case of acute inflammatory demyelinating polyneuropathy related to PEG-interferon alpha2a has been published previously. CASE PRESENTATION: To the best of our knowledge we present only the second case of acute inflammatory demyelinating polyneuropathy related to PEG-interferon alpha2a, occurring in a 63-year-old Caucasian man. He developed tingling, numbness, and weakness of his upper and lower extremities with acute neurological deficits after five weeks of a combination therapy with PEG-interferon alpha2a and ribavirin for chronic hepatitis C virus infection. His clinical course, neurological findings, and his electromyogram results were all consistent with acute inflammatory demyelinating polyneuropathy. Our patient recovered completely after interferon was stopped and symptomatic treatment and a further electromyogram showed a disappearance of neuropathy. Four weeks later, PEG-interferon alpha2a was reintroduced with a gradually increasing dose without any reappearance of neurological symptoms allowing hepatitis C seroconversion. CONCLUSIONS: Recognition of this rare yet possible presentation is important for early and accurate diagnosis and treatment. This case report also suggests that the reintroduction of PEGylated interferon in patients who had presented with acute inflammatory demyelinating polyneuropathy related to interferon alpha may be safe, but this must be confirmed by further studies.     

Pegylated liposomal doxorubicin in ovarian cancer

Pegylated liposomal doxorubicin is a formulation of doxorubicin in which the molecule itself is packaged in a liposome made of various lipids with an outer coating of polyethylene glycol. Liposomal technology is being used in increasing amounts in the therapy of a variety of cancers, including ovarian cancers. This article reviews the mechanistic actions of this formulation, the Phase II and Phase III data that helped define the role of pegylated liposomal doxorubicin in recurrent ovarian cancer, as well as a discussion of some of the side-effects and their management.     

聚乙二醇修饰重组人干扰素α-2b的碘染色法

目的：建立检测聚乙二醇位点特异性修饰重组人干扰素α-2b反应的方法。方法：采用分子量20000的甲氧基聚乙二醇马来酰亚胺修饰重组人干扰素α-2b,反应混合物样品经十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）后,碘染色法判断反应产物组成。结果：该修饰反应产物除含有单PEG化的干扰素α-2b外,还有不同修饰程度的多PEG化干扰素。结论：本方法方便快捷、分辨率高、特异性强,同时可用于其它聚乙二醇修饰蛋白质的分析研究。     

α-2-HS-glycoprotein is a potential marker predicting hepatitis B e antigen seroconversion in patients with chronic hepatitis B during treatment with pegylated interferon alfa-2b

The efficacy of interferon (IFN) is limited in about 1/3 of patients with chronic hepatitis B (CHB). We used two-dimensional electrophoresis (2-DE)-based proteomic strategies to identify potential serum markers predicting hepatitis B e antigen (HBeAg) seroconversion in these patients during IFN therapy. Two groups of patients were enrolled: training and validation. In the training group, 2-DE experiments and subsequent identification of altered levels of proteins showed that α-2-HS-glycoprotein, leucine-rich α-2-glycoprotein, and haptoglobin were significantly upregulated as compared with baseline levels in the HBeAg seroconversion group, whereas apolipoprotein C-III precursor, leucine-rich α-2-glycoprotein, and α-albumin were downregulated in the non-seroconversion group. For patients with HBeAg seroconversion in the training group, Western blot analyses showed that α-2-HS-glycoprotein levels in 75% of patients were significantly upregulated at the end of the treatment as compared with baseline levels. Subsequent experiments in the validation group showed that α-2-HS-glycoprotein levels were significantly increased at week 4 in 83.33% of patients in the HBeAg seroconversion group. Dynamic changes in the serum level of α-2-HS-glycoprotein may be a potential early marker for predicting HBeAg seroconversion during IFN treatment for CHB.     

Agranulocytosis during treatment of chronic hepatitis C complicated by hyperthyreosis. Case reports

Agranulocytosis is a life-threatening disorder characterised by a greatly decreased number of circulating neutrophils below 500/μL. This article presents two cases of agranulocytosis in patients treated with pegylated interferon and ribavirin due to chronic hepatitis C. Interferon induced hyperthyroidism, which required the use of a tyreostatic. Anti-thyroid drugs (ATD) used to treat hyperthyroidism can cause agranulocytosis. The synergistic reaction of ATD and interferon on bone marrow cannot be excluded.     

Interferon-based treatment of chronic hepatitis C

The treatment of patients with chronic hepatitis C has rapidly evolved in the past 10 years centered on the use of interferon alpha 2 as an antiviral and immunomodulatory agent against hepatitis C virus. Firstly used as a monotherapy associated with a deceiving long-term efficacy, interferon alpha was then combined with ribavirin, a nucleoside analog with large antiviral properties. Combination of both drugs dramatically improved the efficacy of treatment with 50% of patients reaching a sustained viral response, characterized by the final eradication of the virus from the infected individual. Surprisingly, this synergistic effect remains greatly unexplained. The third step consisted in the use of pegylated interferon in order to adapt its pharmacokinetics and to allow a better efficacy with a more tolerable dosing schedule: once weekly subcutaneous injection instead of thrice weekly. Pegylated interferon combined with ribavirin during 24-48 weeks of treatment is the current standard of care with nearly 60% of sustained virologic response, overall. Development of new forms of interferon alpha are on the way with promising preliminary results.     

Detection and molecular weight determination of polyethylene glycol-modified hirudin by staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

This article describes a general method for detecting pegylated proteins directly after SDS-PAGE. The proteins to which polyethylene glycol (PEG) molecules are attached are stained with a barium iodide solution. The staining is based on the formation of a barium iodide complex with PEG. The described method combines a specific staining of PEG molecules with the high resolution of the SDS-PAGE method. It is shown that pegylated protein is detectable on SDS-PAGE as well as on IEF at concentrations that are not detectable by Coomassie protein staining. This paper also describes the determination of the molecular weight of pegylated hirudin by calibrating SDS-PAGE with polyethylene glycol of different molecular weight. Under the conditions used, PEG showed linear mobility during electrophoresis. However, the use of nonpegylated proteins as standards resulted in incorrect molecular weight values due to the lower mobility of the pegylated protein during electrophoresis. The method described might reflect a general method for determining molecular weight of pegylated proteins.     

The structural and accessory proteins M,ORF 4a,ORF 4b,and ORF 5 of Middle East respiratory syndrome coronavirus (MERS-CoV) are potent interferon antagonists

The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic respiratory virus with pathogenic mechanisms that may be driven by innate immune pathways. The goal of this study is to characterize the expression of the structural (S, E, M, N) and accessory (ORF 3, ORF 4a, ORF 4b, ORF 5) proteins of MERS-CoV and to determine whether any of these proteins acts as an interferon antagonist. Individual structural and accessory protein-coding plasmids with an N-terminal HA tag were constructed and transiently transfected into cells, and their native expression and subcellular localization were assessed using Wes tern blotting and indirect immunofluorescence. While ORF 4b demonstrated majorly nuclear localization, all of the other proteins demonstrated cytoplasmic localization. In addition, for the first time, our experiments revealed that the M, ORF 4a, ORF 4b, and ORF 5 proteins are potent interferon antagonists. Further examination revealed that the ORF 4a protein of MERS-CoV has the most potential to counteract the antiviral effects of IFN via the inhibition of both the interferon production (IFN-β promoter activity, IRF-3/7 and NF-κB activation) and ISRE promoter element signaling pathways. Together, our results provide new insights into the function and pathogenic role of the structural and accessory proteins of MERS-CoV.     

Molecularly imprinted cryogels for human interferon‐alpha purification from human gingival fibroblast culture

Interferons are important proteins for the immune system because of their antiviral, anti‐proliferating and immunomodulatory activities. Therapeutic value of these proteins against certain types of tumors caused interest and investigations aimed to obtain highly purified interferons. Molecular imprinting is an efficient method for purification with high selectivity, specificity and good reproducibility. In this study, we utilized advantages of molecular imprinting technique for the purification of interferon from human gingival fibroblast culture. For this purpose, interferon α‐2b imprinted poly(hydroxyethyl methacrylate) cryogel (hIFN‐α‐MIP) was prepared. Optimum adsorption conditions were determined, and maximum adsorption capacity of hIFN‐α‐MIP cryogel was found as 254.8 × 10⁴ IU/g from aqueous solution. All interferon measurements are expressed as International Unit (IU), which is a unit measurement used to quantify biologically active substances like interferon based on their biological activity or effect. Selectivity experiments were performed using competitive proteins and repeated adsorption–desorption studies showed that the adsorption capacity maintained almost at a constant value after ten cycles. For the purification of interferon from human gingival fibroblast culture, fast protein liquid chromatography was used and the specific activity of the purified interferon α‐2b on HeLa cell line was found between the values 3.45 × 10⁸ IU/mg and 3.75 × 10⁸ IU/mg. The results are promising, and the molecular imprinting technique is effective for the purification of interferon α‐2b. Copyright © 2013 John Wiley & Sons, Ltd.     

Identification of the major positional isomer of pegylated interferon alpha-2b

Interferons display a wide range of antiviral, antiproliferative, and immunomodulatory activities on a variety of cell types and have been used to treat many diseases including hairy-cell leukemia and hepatitis B and C and have also been applied to other therapeutic areas. To improve the pharmacological properties of interferon (IFN) alpha-2b, a long-acting pegylated form (PEG-IFN) has been developed [PEG, monomethoxy poly(ethylene glycol) with average molecular mass of 12 000 Da]. PEG-IFN is a mixture of pegylated proteins with differing sites of PEG attachment. To identify the major positional isomer in the pegylated material [PEG-IFN(His-34)], NMR studies were conducted on a subtilisin-digested N-acetylated peptide of the major positional isomer [PEG-IFN(His-34)dig], synthetic peptide analogues containing His-34, as well as unmodified IFN and PEG-IFN(His-34). Our studies reveal a novel interferon-polymer attachment site as a histidine-linked interferon conjugate. We show that the major component of PEG-IFN is pegylated in the imidazole side chain of histidine-34. Chemical shift data suggest that pegylation occurs mainly at the N(delta)(1) position in the imidazole side chain of this residue. This positional isomer, PEG-IFN(His-34), comprises approximately 47% of the total pegylated species when PEG-IFN is synthesized under the current experimental conditions at pH 6.5 with an electrophilic derivative of PEG, succinimidyl carbonate PEG. The reversibility of the histidine modification was examined. The PEG-imidazole adduct in the intact protein, PEG-IFN(His-34), is labile but much more stable than in the peptide, PEG-IFN(His-34)dig. Apparently, the tertiary structure of the intact protein protects the His(34)-imidazole ring from depegylation.