Recombinant Human Interferon-alpha Research Articles

Effect of human recombinant interferon alpha 2b (Viferon) in treatment of severe bacterial infections in neonates. (B32)

Abstract Neonates have decreased cytotoxic mechanisms and low levels of endogenous interferon production. Aim: Clinical efficacy and mechanisms of adjunctive immunotherapy with human recombinant interferon alpha 2b (Viferon) were studied in neonates suffering from severe bacterial infections. Design. 48 neonates with bacterial pneumonia were observed in NICU. 23 infants (group 1) were treated with Viferon-1, 150,000 IU per suppositorium q8h per rectum during 7 days in addition to combined antibacterial and supportive therapy. 25 babies (group 2) received the same basic therapy plus placebo instead of Viferon in similar way. Results. Clinical course of pneumonia was more favourable in neonates treated with Viferon. The duration of central venous line usage and total parenteral nutrition were significantly less in group 1 than in group 2. Inflammatory signs in blood analyses were significantly decreased after the treatment with Viferon in comparison with placebo-group. Infants treated with Viferon had significantly higher levels of CD3+, CD4+ and CD25+-lymphocytes, higher serum levels of IFN, elevation of mitogen-induced IFN-gamma production after Viferon than infants from group 2. Conclusion. Immunotherapy with Viferon improves the clinical course of severe bacterial infections and restores the balance in immune system of infected newborn infants.

The inhibition of aggregation of recombinant human consensus interferon-α mutant during Pichia pastoris fermentation

Lower induction temperature and polyoxyethylene sorbitan monolaurate (Tween-20) were successfully used to inhibit the aggregation of recombinant human consensus interferon-alpha mutant (cIFN) during Pichia pastoris fermentation. When the induction temperature was decreased from 30 to 20 degrees C, the cIFN secreted into the medium was in the form of monomers instead of aggregates. The maximum specific activity at 20 degrees C was 4.04 times as high as that at 30 degrees C. There was no obvious effect on the cell growth at 20 degrees C, but the total protein level was decreased. Similar inhibition effect on cIFN aggregation was observed when 0.2 g l(-1) Tween-20 was added during induction. Furthermore, there was a synergistic effect found between induction temperature and Tween-20 on the inhibition of cIFN aggregation. The maximum specific activity with Tween-20 at 20 degrees C was 19.9-fold higher than that without Tween-20 at 30 degrees C.

Complete remission of multiple myeloma after autoimmune hemolytic anemia: Possible association with interferon‐α

A patient with multiple myeloma (MM) was being maintained on human recombinant interferon-alpha (INF-alpha) after VAD and autologous bone marrow transplantation (pretreated with melphalan). An episode of immune thrombocytopenia and (Coombs positive) autoimmune hemolytic anemia (AIHA) was noted while on maintenance INF-alpha, which remitted when it was withdrawn. Following this event, he achieved a state of stable disease that persists (more than 3 years) with no specific myeloma treatment. This sequence of events suggests a relationship between an immunological reaction induced by INF-alpha and the prolonged phase of stable disease.

A strategy for high-level expression of soluble and functional human interferon α as a GST-fusion protein in E.coli

Escherichia coli is the most extensively used host for the production of recombinant proteins. However, most of the eukaryotic proteins are typically obtained as insoluble, misfolded inclusion bodies that need solubilization and refolding. To achieve high-level expression of soluble recombinant human interferon alpha (rhIFNalpha) in E. coli, we have first constructed a recombinant expression plasmid (pGEX-hIFNalpha2b), in which we merged the hIFNalpha2b cDNA with the glutathione S-transferase (GST) coding sequence downstream of the tac-inducible promoter. Using this plasmid, we have achieved 70% expression of soluble rhIFNalpha2b as a GST fusion protein using E. coli BL21 strain, under optimized environmental factors such as culture growth temperature and inducer (IPTG) concentration. However, release of the IFN moiety from the fusion protein by thrombin digestion was not optimal. Therefore, we have engineered the expression cassette to optimize the amino acid sequence at the GST-IFN junction and to introduce E. coli preferred codon within the thrombin cleavage site. We have used the engineered plasmid (pGEX-Delta-hIFNalpha2b) and the modified E. coli trxB(-)/gor(-) (Origami) strain to overcome the problem of removing the GST moiety while expressing soluble rhIFNalpha2b. Our results show the production of soluble and functional rhIFNalpha2b at a yield of 100 mg/l, without optimization of any step of the process. The specific biological activity of the purified soluble rhIFNalpha2b was equal to 2.0 x 10(8) IU/mg when compared with the WHO IFNalpha standard. Our data are the first to show that high yield production of soluble and functional rhIFNalpha2b tagged with GST can be achieved in E. coli.

Aggregation of recombinant human interferon alpha 2b in solution: Technical note

Sodium phosphate buffer increased the aggregation of rhIFN-α2b in the range of 1.55 to 1.8103 day−1, as determined by SDS/PAGE under reduced and nonreduced conditions. In contrast, sodium citrate buffer decreased the aggregation rate of this cytokine, as compared with those samples in sodium phosphate buffer. Results from sodium citrate-phosphate buffer were very similar to those obtained with sodium citrate solutions. On the other hand, EDTA Na2×2H2O reduced the aggregation rate of rhIFN-α2b, showing an aggregation kinetic constant in the range of 0.52 to 0.75×103 day−1. Polysorbates 20 and 80 were less effective than the chelating agent in preventing this degradation pathway. Additionally, metal ions (Zn2+ and Cu2+) increased the aggregation kinetic constant of rhIFN-α2b, probably through undetermined metal-catalyzing reactions. Taken together, these data can be useful for the development of new formulations containing rhIFN-α2b as an active ingredient.

PEG-Intron Therapy in Patients with Philadelphia Chromosome-Negative Myeloproliferative Disorders (MPD): Final Result of a Phase II Study.

The conventional management of patients (pts) with high-risk Philadelphia chromosome-negative (Ph-negative) MPD revolves around the administration of cytoreductive agents such as hydroxyurea, anagrelide, and recombinant human interferon alpha (IFN-a). IFN-a has shown significant activity in the treatment of Ph-negative MPD, particularly essential thrombocythemia (ET) and polycythemia vera (PV). PEG-Intron is a pegylated IFN-a that has a significant advantage over regular IFN-a in that it is administered once a week. We are reporting the final results of a phase II study evaluating the efficacy and tolerability of PEG-Intron in pts with Ph-negative MPD treated at M. D. Anderson Cancer Center, Houston, Texas. PEG-Intron was given subcutaneously weekly with intent to treat pts as long as they benefit from the therapy. Forty pts were enrolled, 26 males (65%), with a median age of 54 years (range 28 to 81). Pts diagnoses were: 14 ET (35%); 11 myelofibrosis (MF; 27%); 6 Ph-negative chronic myeloid leukemia (CML; 15%); 4 hypereosinophilic syndrome (HES; 10%); 4 PV (10%); and 1 myelodysplastic syndrome/myeloproliferative disease (MDS/MPD; 3%). Thirty-four pts are evaluable for response evaluation, as they have received minimum of 3 months of therapy, and have received more than 50% of the medication during the time on study: complete remission (CR) was achieved in 13 pts (38%) while partial response (PR) was noted in 4 pts (12%); 17 pts (50%) had no response. Six pts were not evaluable for response: 2 refused therapy before starting; 3 stopped therapy early due to toxicity, and 1 had progression of the disease before 3 month response evaluation. Of the 17 responders 12 (71%) achieved their best response by 3 months on the therapy. Overall response among all the patients enrolled varied by the type of diagnosis: of the 14 ET pts 7 achieved CR and 2 achieved PR (64% overall response rate); of the 11 MF pts one achieved CR; of the 6 Ph-negative CML 2 achieved CR; of the 4 pts with HES one achieved CR; of 4 PV pts 2 achieved CR and 2 PR; and 1 MDS/MPD pt was not evaluable for response. Initially, starting dose of PEG-Intron was 3 mcg/kg per week. However, of the first 14 pts on the study all but 1 had to reduce the dose at least once within the first 3 months, and therefore the starting dose of PEG-Intron was reduced to 2 mcg/kg per week. Most of the pts maintained the 2 mcg/kg per week throughout their time on the therapy. Recorded grade 3 toxicities were related to myelosuppression, disturbance of GI tract, and musculo-skeletal system (22 pts). Grade 4 toxicities were recorded in 4 pts: 1 developed anemia and the other 3 developed thrombocytopenia. Of the 38 pts that have received any therapy 5 (4 in CR and 1 in PR) are still receiving therapy for a median duration of 28+ months (range, 21+ to 41+). Of the 33 pts that stopped the therapy 17 stopped because of no response, 6 due to unrelated medical reasons, 5 due to toxicity, 2 due to other reasons, 1 was lost to follow-up, 1 lost response, and 1 progressed to acute myeloid leukemia. After a median follow-up of 24 months (range, 1 to 42) 26 pts are alive. In conclusion, PEG-Intron, with proper dose modifications, is effective in controlling disease in a significant proportion of Ph-negative MPD pts, particularly patients with ET and PV. However, toxicities encountered with PEG-Intron therapy are still significant.

Treatment with Pegylated Interferon-Alfa-2A (PEG-IFN-α-2A; PEGASYS™) for Patients with Essential Thrombocythemia (ET) and Polycythemia Vera (PV).

Patients (pts) with high-risk Philadelphia chromosome-negative myeloproliferative disorders (MPDs) are customarily treated with cytoreductive agents such as recombinant human interferon-alpha (IFN-α), hydroxyurea (HU), and anagrelide (AG). Despite the significant activity of IFN-α in MPDs, responses have been frequently limited by poor tolerance and inconvenient dosing schedules. PEG-IFN-α is formulated by covalently attaching polymers of ethylene glycol to the native IFN-α molecule, which results in increased serum half-life, decreased renal excretion, thus allowing for weekly administration while maintaining acceptable toxicity profile. Based on the superior pharmacokinetic and pharmacodynamic profile of PEG-IFN-α relative to IFN-α we designed a phase II study of subcutaneous (sc) PEG-IFN-α-2a (Pegasys™) for pts with ET and PV. A total of 23 pts have been enrolled so far (13 ET, 10 PV). First 3 pts received PEG-IFN-α-2a at 450mcg weekly but experienced toxicity and had to reduce the dose; next 3 pts started at 360mcg weekly and again had to reduce the dose due to toxicity; last 17 pts started at 270mcg weekly and tolerated therapy well. Dose modifications of 90 mcg are allowed according to response or toxicity. Median age is 54 years (range, 23 to 73), time from diagnosis to PEG-IFN-α-2a therapy 64 months (range, 1 to 282), Hb 13.5 g/dL (range, 8.9 to 18.5), WBC 5×109/L, (range, 4.3 to 49), platelets 592×109/L (range, 298 to 1000). All pts had prior therapies (median 2, range 1–6), including HU (n=17), AG (n=13), IFN-α (n=6: 4 oral and 2 sc), imatinib mesylate (n=2) and others (n=3). In addition, 8 PV pts had received phlebotomies. The JAK2 V617F mutation was detected in 6 (46%) of 13 ET pts and in 9 (90%) of 10 PV pts. All pts had diploid cytogenetics. After a median follow-up of 6 months (range, 1 to 15), 11 of 13 (85%) ET pts have achieved a complete response (platelets <440×109/L, off HU and AG, in the absence of thromboembolic events); 2 pts without response received less than 2 months of therapy so far. Six of 10 (60%) PV pts achieved complete remission (Hb <15 g/dL, off HU and AG, no phlebotomy, with disappearance of splenomegaly), 3 (30%) achieved partial response (no phlebotomy, off HU and AG, but still palpable spleen), and 1 have not responded so far (less than 3 months on the therapy), for overall response of 90%. The median time to response was 1.5 months (range, 0.5 to 5.6). While the first 6 pts on the study, that received high PEG-IFN-α-2a dose (450 or 360mcg weekly), experienced grade 3 toxicity, among last 17 pts that received 270mcg weekly, there were only 5 cases of grade 3 toxicity (anemia, increased liver function tests, and neutropenia x3) requiring dose reduction. However, to preserve pts quality of life, the dose of PEG-IFN-α-2a was also reduced for the persistent grade 2 toxicity (> 2 weeks). Overall, 17 pts (74%) reduced the dose so far, while 6 pts (all on 270mcg weekly) remain at the same initial dose. Only 2 pts (1 ET, 1 PV) discontinued therapy, due to retinal infiltrates and depression after 8 and 6 months on therapy, respectively. In summary, therapy with PEG-IFN-α-2a has an acceptable toxicity profile and significant activity in pts with ET and PV. Updated clinical and molecular results will be presented.

Pegylated Interferon Therapy for Patients with Philadelphia Chromosome-Negative Myeloproliferative Disorders

The conventional management of patients with high-risk Philadelphia chromosome-negative (Ph-negative) myeloproliferative disorders (MPDs) revolves around the administration of cytoreductive agents such as hydroxyurea, anagrelide, and recombinant human interferon alpha (IFN-alpha). IFN-alpha has shown significant activity in the treatment of chronic myelogenous leukemia (CML) and Ph-negative MDPs. However, the response rates of IFN-alpha therapy frequently have been hampered by high dropout rates due to side effects and inconvenient dosing schedules. Pegylated (PEG) IFN-alpha is formulated by covalently attaching polymers of ethylene glycol of large molecular weight to the native IFN-alpha molecule. Such chemical modification increases serum half-life, decreases renal excretion, and results in prolonged patient exposure to PEG-IFN-alpha, thus allowing for weekly administration while maintaining acceptable toxicity, tolerability, and activity profiles. The lack of adequate therapies for patients with MPDs and the superior pharmacokinetic and pharmacodynamic profile of PEG-IFN-alpha relative to standard IFN-alpha has prompted the investigation of the activity and safety of PEG-IFN-alpha in patients with essential thrombocythemia, polycythemia vera, and idiopathic myelofibrosis. We summarize the available data on the use of PEG-IFN-alpha in patients with Ph-negative MPDs.

Expression and Aggregation of Recombinant Human Consensus Interferon-α Mutant by Pichia pastoris

A recombinant human consensus interferon-alpha mutant (cIFN) was expressed in Pichia pastoris. The maximum dry cell weight, cIFN concentration and antiviral activity were 160 g l(-1), 1.24 g l(-1) and 4.1 x 10(7) IU ml(-1), respec tively. The cIFN secreted into the medium was in the form of aggregates dominantly by non-covalent interaction and partially by disulphide bond. When the fermentation supernatant was disaggregated with 6 M guanidine hydrochloride, the antiviral activity of cIFN achieved 2.2 x 10(8) IU ml(-1).

Randomized, placebo-controlled trial of nonpegylated and pegylated forms of recombinant human alpha interferon 2a for suppression of dengue virus viremia in rhesus monkeys.

Dengue fever and dengue hemorrhagic fever are caused by infection with any one of the four dengue viruses (DVs) and are significant public health burdens throughout the tropics. Higher viremia levels are associated with greater dengue disease severity. A therapeutic intervention to suppress viremia early in DV infection could potentially ameliorate severe disease. Recombinant alpha interferon 2a (rIFN-alpha-2a, Roferon-A) suppressed DV replication in human peripheral blood mononuclear cells in vitro. We therefore examined the effects of rIFN-alpha-2a and pegylated recombinant IFN-alpha-2a (PEG-rIFN-alpha-2a, PEGASYS) on DV serotype 2 (DV-2) viremia in rhesus monkeys. Flavivirus-naïve monkeys were inoculated with DV-2 and randomized to receive a single dose of rIFN-alpha-2a (10 million international units/m2) versus placebo or PEG-rIFN-alpha-2a (6 microg/kg) versus placebo 1 day after the onset of viremia. Serial daily viremia levels were measured, and convalescent-phase DV-2 neutralizing antibody titers were determined. Compared to placebo, a single injection of rIFN-alpha-2a temporarily suppressed DV-2 replication and delayed the time to peak viremia by a median of 3 days. However, measures of total viral burden were not different between the two groups. A single injection of PEG-rIFN-alpha-2a significantly lowered daily viremia levels and improved virus clearance, starting 48 h after administration. There were no significant differences in DV-2 neutralizing antibody titers between the treatment and placebo groups at 30 and 90 days postinfection. Based on their individual effects, future studies should investigate a combination of rIFN-alpha-2a and PEG-rIFN-alpha-2a for suppression of dengue virus viremia and as a potential therapeutic intervention.

Some factors affecting the stability of interferon alpha 2b in solution

In this paper we evaluated the influence of the protein concentration and a formulation vehicle on the stability of recombinant human Interferon alpha 2b (rhIFN-α2b) in solution. The effect of the protein content (from 1 to 100 MIU/ml) at 37 °C, showed that higher concentration of this cytokine protected against the loss of bioactivity (antiviral titration) better than the lower concentrations. In contrast, rhIFN-α2b at 50 and 100 MIU/ml decreased the SDS/PAGE- and RP-HPLC-determined purity faster than samples at 1 or 10 MIU/ml. According to these results, 10 MIU/ml rhIFN-α2b was the best choice to evaluate the influence of a formulation on the stability of this cytokine. Taking this into consideration, we studied the stability of a liquid and albumin-free formulation of this protein at the recommended storage temperature (5 ± 3 °C) and under accelerated conditions (28 ± 2 °C). Accelerated storage results showed an acceptable biochemical stability of the active ingredient throughout 2 months. Real-time storage data confirmed the good biochemical stability of this formulation for 30 months.

Kinetic studies of recombinant human interferon-alpha (rhIFN-α) expression in transient state continuous cultures

Kinetic modeling of rhIFN-α expression was done in continuous cultures using complex media at dilution rates varying from 0.2 to 0.5 h −1. The human IFN-α gene was inserted under the T7 promoter in BL21 (DE3) codon plus (RIL) cells and induced with 1 mM IPTG. Post-induction growth and production profiles were monitored over time. Wash out of the cells was observed only at a dilution rate of 0.5 h −1, which gave a post-induction specific growth rate ( μ) of 0.37 h −1. The rate of accumulation of rhIFN-α inside the cells as inclusion bodies was used to calculate the specific product formation rate ( q p). q p was found to be strongly correlated with the dilution rates used with the maximum q p being 0.17 g g −1 h −1 at the first hour of induction at a dilution rate of 0.5 h −1.The decline in q p with time could be related to the build up of inclusion bodies inside the cells which reached a maximum value of 50% of the total protein in the fourth hour post-induction.

Structural, Kinetic, and Thermodynamic Analysis of the Binding of the 40 kDa PEG−Interferon-α2a and Its Individual Positional Isomers to the Extracellular Domain of the Receptor IFNAR2

Type-I Interferons exert antiviral and antiproliferative activities through the binding to a common cell surface receptor comprising two subunits, IFNAR1 and IFNAR2. Human recombinant Interferon-alpha(2a) (IFNalpha(2a)) is a potent drug (Roferon-A) used to treat various cancers and viral diseases including Hepatitis B/C infections. To significantly improve the pharmacological properties of the drug, a pegylated form of IFNalpha(2a) was developed (PEGASYS). This 40 kDa PEG-conjugated IFNalpha(2a) ((40)PEG-IFNalpha(2a)) is obtained by the covalent binding of one 40 kDa branched PEG-polymer to a lysine side-chain of IFNalpha(2a). Here, we report the detailed structural, kinetic, and thermodynamic analysis of the binding to the extracellular domain of the receptor IFNAR2 of (40)PEG-IFNalpha(2a) and its isolated positional isomers modified at K31, K134, K131, K121, K164, and K70, respectively, in comparison with unmodified IFNalpha(2a). Our binding studies, using the surface plasmon resonance technique, show that the pegylation does not abolish the binding to the receptor, but significantly reduces the affinity mainly due to a change of the association rate. The results are supported by modeling and simulation of the binding, using Self-Avoiding-Walk calculations for the polymer conformations. A correlation between the structural parameters and the kinetic and thermodynamic parameters of the binding of the positional isomers could be established. For the Isomer-K31 and -K164, the PEG-polymer attachment point is located in proximity to the binding interface, and the isomers display affinity in the range 150-520 nM in an enthalpy-driven binding process. In contrast for the Isomer-K134, -K131, -K121, and -K70, the PEG-polymer is attached remotely from the binding interface, and the isomers exhibit a higher affinity (32-76 nM) in an entropy-driven binding process. This study constitutes an essential collection of knowledge on which the interaction of (40)PEG-IFNalpha(2a) and its positional isomers with its cellular receptors can be better understood.

Reproductive Effects Of human Interferon-Alpha-2b Administration on Male Albino Mice Testes.An Experimental Study.

Background: Recombinant human interferon alpha (rh-IFN-α) is used therapeutically in malignant disorders and chronic hepatitis. The phenotypic effects of this drug at the structural levels on testicular tissue were hardly ever addressed. Hence, this work was designed in adult male albino mice to study the phenotypic effects of rh-INF-α-2b on testicular tissue as well as assessing its effects on serum testosterone and gonadotropins levels. Objective: This research was planned to through light on the effects of interferon-alpha- 2b (IFN-alpha-2b) on the hypothalamic-pituitary-testicular (HPT) axis of the adult male albino mice. Design: Experimental study. Setting: National Hepatology and Tropical Medicine Research Institute (NHTMRI). The study was conducted from November (2004) to February (2005). Materials and methods: Thirty sexually mature male mice were divided into three groups (10 mice in each group), namely: the control, the experimental and the recovery groups. Mice in the experimental and recovery groups were administered recombinant human interferon alpha intraperitoneally at a dose of 3000 U / mouse weekly for 12 weeks in a volume of 1.0-microliter isotonic normal saline, then animals in the recovery group were left to recover for a further period of two months. At the end of the experiment, serum concentrations of gonadotropins and testosterone were measured and then all animals were then sacrificed to study histopathologically the possible effects of interferon on the testicular tissue. Results: rh-IFN-α-2b induced remarkable decline in the serum levels of both follicle stimulating hormone (FSH) and luteinizing hormone (LH) in mice of the experimental group compared to the corresponding control and mice of recovery group. At the same time, testosterone was moderately increased in the experimental group, and then returned to its normal levels within 2 months after cessation of treatment. Histopathologically, in the experimental group, there were focal thickening of the basement membrane, degenerative changes and clumping of the germinal epithelial cells in the center of seminiferous tubules, partial desquamation of the germinal epithelium from basement membrane, reduction in the germ cell height, partial arrest of maturation and increased number of Sertoli cells. Increased number of Leydig’s cells and hypervascularity were detected in the interstitial spaces. In the recovery group, there was lessening of the germ cell hypoplasia manifested by restoration of spermatogenic cells and accidental disruption in the basement membrane. Most of the spermatogenic and Sertoli cells restored their polarity, height and maturation. Conclusion: our results suggest that rh-INF-α-2b temporally affects the hypothalamic- pituitary-testicular axis (HPT), both centrally and peripherally (at the testicular level), through the lessening of FSH, LH, raise of testosterone serum levels and direct phenotypic effect on the testicular tissue. 67

OPPOSITE EFFECTS OF SEROTONIN AND INTERFERON-α ON THE MEMBRANE POTENTIAL AND FUNCTION OF HUMAN NATURAL KILLER CELLS

In an earlier article, we reported that serotonin (5-hydroxytryptamine, 5-HT) inhibits the natural killer cell (NK) cytotoxicity of human whole blood in a dose-dependent manner and that natural human interferon-alpha (IFN-alpha) partially eliminates this effect. Because natural IFN-alpha might contain factors other than IFN, we repeated these experiments with recombinant human interferon-alpha (rhIFN-alpha) and separated blood lymphocytes enriched with NK cells and then demonstrated that IFN really is responsible for this effect. Furthermore, this investigation was carried out to clarify the mechanisms of the action of 5-HT and of rhIFN-alpha on NK cells. The inhibition of the cytotoxicity was pronounced when 5-HT was added at the onset of the cytotoxic assay, whereas the pretreatment of lymphocytes for 18 h only led to a slight inhibition. Moreover, rhIFN-alpha applied 1 h before or 1 h after the addition of 5-HT decreased the inhibitory effect of 5-HT. Flow cytometric analysis involving the use of a voltage-sensitive dye, oxonol, revealed that 5-HT depolarized, whereas rhIFN-alpha hyperpolarized the plasma membrane of the lymphocytes. Thus, it seems likely that the inhibitory effect of 5-HT on the cytotoxicity of peripheral human lymphocytes is due to the depolarization on the plasma membrane of the effector cells and that rhIFN-alpha antagonizes this ability via its hyperpolarizing activity.

Fever of recombinant human interferon-alpha is mediated by opioid domain interaction with opioid receptor inducing prostaglandin E 2

We have reported that there are distinct domains in Interferon-alpha (IFNα) molecule mediating immune and opioid-like effects respectively. And the opioid effect of IFNα is mediated by μ opioid receptor. We report here the structural basis of fever induced by recombinant human IFNα. Two kinds of IFNα mutants were obtained and used to investigate the structural basis of fever of IFNα, which are 129Ser-IFNα and 38Leu-IFNα. The antiviral activity of 129Ser-IFNα almost disappeared, but there still retained the strong analgesic activity. The antiviral activity of 38Leu-IFNα remained, but the analgesic activity disappeared completely. It showed that IFNα and 129Ser-IFNα decreased cAMP production, induced the fever, and stimulated PGE 2 to release from the hypothalamus slices, which could be blocked by naloxone, but 38Leu-IFNα failed. It is the first demonstration that fever induced by IFNα is mediated by opioid domain of IFNα interacting with opioid receptor. It is inferred that high-activity and low side-effect IFNα or other cytokines could be obtained after being changed the motifs in the tertiary structure.

Feline epitheliotrophic T‐cell lymphoma with paraneoplastic eosinophilia – immunochemotherapy with vinblastine and human recombinant interferon α2b

A cat with epitheliotrophic T-cell lymphoma with paraneoplastic eosinophilia is described. Initial attempts to control the disease with conventional therapies failed. The addition of recombinant human interferon alpha(2b) (rhINFalpha(2b)) resulted in a clinical, haematogenous and sonographic improvement for 49 days. The overall survival time from initial diagnosis was 100 days. Relapse was correlated with the development of serum antibodies directed against rhINFalpha(2b). To our knowledge, this is the first report describing the clinical use of IFNalpha in the treatment of neoplasia in the cat.

Fourier transform infared spectroscopy investigation of protein conformation in spray‐dried protein/trehalose powders

Spray drying is a way to generate protein solids (powders), which is also true for lyophilization. Sugars are used to protect proteins from conformational changes and chemical degradations arising from drying processes and storage conditions such as the humidity. The influence of trehalose and humidity on the conformation and hydration of spray-dried recombinant human granolucyte colony stimulating factor (rhG-CSF) and recombinant consensus interferon-alpha (rConIFN) was investigated using Fourier transform IR spectroscopy. The spectral analysis of spray-dried powders in the amide I region demonstrated that trehalose stabilized the alpha-helical conformation of both rhG-CSF and rConIFN proteins. Exposure of the pure protein powders to 33% relative humidity (RH) resulted in the formation of beta sheets and loss of turns but no change in alpha-helical structure. Trehalose reduced the magnitude of the changes in beta sheets and turns. Exposure of the pure protein powders to 75% RH resulted in the loss of alpha-helical conformation with a corresponding increase in beta structures (beta sheets and turns). Trehalose did not protect proteins from the loss of alpha-helical structures, but it reduced the formation of antiparallel beta sheets. Hydrogen-deuterium exchange (H-D exchange) was used to further characterize these hydration-induced conformational changes. At 33% RH the percent exchange of the protein decreased with increasing trehalose content, indicating a greater protection of the protein from H-D exchange by a higher concentration of trehalose. Such protection correlates with decreased conformational changes of the protein by trehalose at this humidity. At 75% RH the degree of H-D exchange of the protein was insensitive to the powder composition in all powders. Surprisingly, the H-D exchange of trehalose was low at about 20-25%, which was nearly independent of the protein/trehalose ratio and humidity, indicating that the exchangeable protons on trehalose molecules are highly protected in protein-containing powders. The observed protein hydration is related to the effect of trehalose on the conformational changes of the protein under humidity.

Current and Future Therapy for Chronic Hepatitis C Virus Liver Disease

Therapy with recombinant human interferon alpha remains pivotal to the treatment for chronic hepatitis C virus liver disease. Semi-synthetic protein-polymer conjugates of interferon with polyethylene glycol have also been recently developed. These conjugates protect the protein from degradation; reduce the immunogenicity; and prolong exposure to drug by a sustained absorption, restricted volume of distribution and sustained high serum concentration. Therapy with pegylated interferons is associated with significantly greater sustained virological response rates (SVR) compared to the non-pegylated formulation. Ribavirin is a guanosine analog with minimal antiviral activity against HCV. It demonstrates significant clinical synergism when administered in combination with interferon. Amantadine blocks entry of influenza A virus into cells. Used in combination with ribavirin and interferon as triple therapy, it may have some benefit compared to dual or monotherapy. Current treatment with pegylated interferons combined with weight-based ribavirin, provides the highest sustained virological response rates. In the absence of suitable animal models, HCV dynamic studies in man have been helpful in defining the mechanisms of action of interferon in chronic HCV liver disease. Novel therapeutic agents are being developed as the replication cycle of HCV is being understood. However, their safety and efficacy remain to be established and availability for clinical use is unlikely within the next 3 to 5 years. This review describes current antiviral therapy in chronic HCV liver disease, addresses the potential role of viral dynamics in elucidating the mechanisms of action of the drugs and discusses future potential therapeutics agents.

Recombinant human interferon alpha-2a therapy in children with chronic immune thrombocytopenic purpura.

In a prospective study, 11 children with chronic immune thrombocytopenic purpura between ages 3 and 18 years were treated with recombinant human interferon alpha 2a (rhIFN alpha-2a). A dose of 3 x 10(6) U/m2 three times weekly for 4 to 5 weeks (one cycle) was administered. Patients were treated with one to four cycles of rhIFN alpha-2a, and the outcomes were measured initially and 18 to 30 months after the last cycle. Good therapeutic responses (defined as platelet count >100 x 10(9)/L) lasting for 18 to 30 months from the last interferon cycle were achieved in 6 of the 11 (55%) patients, including one with a probable spontaneous remission. Fair responses (platelet count 31-60 x 10(9)/L) for 18 months were achieved in 3 of the 11 (27%) patients. Only two patients, each treated only with one interferon cycle, exhibited no response. Side effects of treatment included fever and a flu-like syndrome, which were usually present during the first 14 days of therapy only. Interferon-alpha appears to be an effective therapeutic approach to children with chronic immune thrombocytopenic purpura, with the potential of sustained long-term remission. A randomized, placebo-controlled study is needed to confirm its role in this population.