Antiviral Therapy 11:35 45

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1 A Phase I/II study evaluating escalating doses of recombinant human albumin interferon-α fusion protein in chronic hepatitis C patients who have failed previous interferon-α-based therapy Vijayan Balan 1 *, David R Nelson 2, Mark S Sulkowski 3, Gregory T Everson 4, Louis R Lambiase, Rusell H Wiesner 6, Rolland C Dickson 7, Anthony B Post 8, Robert R Redfield 9, Gary L Davis 1, Avidan U Neumann 11, Blaire L Osborn 12, William W Freimuth 12 and G Mani Subramanian 12 1 Mayo Clinic, Phoenix, AZ, USA 2 University of Florida, Gainesville, FL, USA 3 Johns Hopkins University, Baltimore, MD, USA 4 University of Colorado, Denver, CO, USA University of Florida at Jacksonville, Jacksonville, FL, USA 6 Mayo Clinic, Rochester, MN, USA 7 Mayo Clinic, Jacksonville, FL, USA 8 University Hospital of Cleveland, Cleveland, OH, USA 9 University of Maryland, MD, USA 1 Baylor University Medical Center, Dallas, TX, USA 11 Bar-Ilan University, Ramat-Gan, Israel 12 Human Genome Sciences, Rockville, MD, USA Antiviral Therapy 11:3 4 *Corresponding author: Tel: ; Fax: ; Balan.Vijayan@mayo.edu Albumin interferon-α (IFN-α) is a novel 8.7-kDa recombinant protein consisting of IFN-α that is genetically fused to human serum albumin. In this Phase I/II, multicentre, open-label study, we evaluated the safety and tolerability, pharmacokinetics and pharmacodynamics of albumin IFN-α in IFN-α-experienced patients with chronic hepatitis C. Albumin IFN-α was administered in 22 escalating doses (7 9 µg) in a single injection or in two injections 14 days apart. In the 119 patients studied, there were no discontinuations because of adverse events, and albumin IFN-α had a favourable safety profile at doses up to 9 µg. The most common adverse events were headache (6%), fatigue (2%), injection site erythema (38%), arthralgias (32%) and pyrexia (27%). Reduced clearance resulted in a mean elimination half-life of 19 h, which supports dosing at 2- to 4-week intervals. Induction of the IFN-specific gene OAS1 was maintained for 28 days following a single injection of albumin IFN-α at doses of 4 µg. Dosedependent antiviral activity was observed in this IFN-α-experienced study population. Antiviral activity of 1.-log reductions in HCV RNA was observed in 47% (37/78) of patients in the 12- to 9-µg cohorts and in 9% (16/27) in the 4- to 9-µg double-injection cohorts. These results support further clinical studies of albumin IFN-α for the treatment of patients with chronic hepatitis C. Introduction Hepatitis C virus (HCV) infection is a major global healthcare problem. Worldwide, approximately 17 million people have chronic hepatitis C (CHC) [1]. In the United States, approximately 3 4 million adults are chronically infected [2]. Important advances in the treatment of patients with CHC have included the development of interferon-α (IFN-α) plus ribavirin combination therapy, followed by pegylation of IFN-α, also administered with ribavirin [3,4]. Although pegylation has improved the pharmacokinetics resulting in improved convenience of IFN-α therapy and increased rates of sustained virological response (SVR), approximately 4% of treated patients still do not achieve an SVR. Albumin IFN-α (Albuferon TM, Human Genome Sciences, Rockville, MD, USA) is a novel recombinant 26 International Medical Press

2 V Balan et al. protein translated from a gene produced from the fusion of the human genes for IFN-α and human serum albumin. The resulting 8.7-kDa molecule is a single polypeptide that combines the antiviral properties of IFN-α with the long serum half-life of albumin []. In non-human primates, after a single subcutaneous injection of albumin IFN-α 3 µg/kg, the apparent clearance (clearance divided by bioavailability) was 1.4 ml/h/kg, the terminal half-life was 93 h, and bioavailability was 64% [6]. The rate of clearance of albumin IFN-α was approximately 14-fold slower and the half-life 18-fold longer than for IFN-α given subcutaneously [6]. Sera from monkeys treated with a single dose of albumin IFN-α demonstrated antiviral activity for 8 days based on an in vitro bioassay, compared with 1 day in standard IFN-treated animals [6]. Significant increases in 2, -oligoadenylate synthetase (OAS1) mrna relative to IFN-α-treated or vehicle-treated animals were maintained for 1 days after one subcutaneous dose [6]. Thus, the improved pharmacokinetics of albumin IFN-α correlated with extended pharmacodynamic activity. These findings taken together suggest that the improved pharmacokinetics of albumin IFN-α compared with standard IFN-α has the potential to offer less frequent dosing and possibly improved efficacy for the therapy of HCV. Therefore, the present Phase I/II study was undertaken to determine the safety, tolerability and pharmacokinetics of albumin IFN-α in humans with CHC who did not respond to other IFN-α-containing regimens. Materials and methods Patient selection Adult patients with CHC who had failed therapy with IFN-α or pegylated IFN-α monotherapy or combination therapy were included in this study. Patients were excluded if they had decompensated liver disease, other types of chronic liver disease, <7 platelets/mm 3, <2 neutrophils/mm 3, a history of severe psychiatric disease, immunologically mediated disease, uncontrolled thyroid disease, HIV infection, a thrombotic disorder, theophylline use, alcohol or drug dependence, or substantial coexisting medical conditions. Study agent Albumin IFN-α is a single polypeptide chain comprising recombinant human albumin fused to recombinant IFN-α. The fusion protein is secreted in fermentation from a host vector system using Saccharomyces cerevisiae. The yeast cells are separated and the product is purified using multiple chromatographic and ultrafiltration steps. Protein concentration was assessed with the bicinchoninic acid method [7]. Study design This was a Phase I/II, open-label, dose escalation, multicentre study. Each patient was assigned to one out of 22 cohorts to receive sequential subcutaneous doses of albumin IFN-α ranging from 7 9 µg. Within each dose cohort, five to six patients received a single subcutaneous injection [single injection (SI)] or two subcutaneous injections [double injection (DI)] of albumin IFN-α. Patients in the DI cohorts received two injections 14 days apart. Starting with cohort 9 (12 µg), the dose escalation proceeded with each subsequent set of cohorts receiving SIs of albumin IFN-α until either the maximum tolerated dose (MTD) or the highest dose level studied was achieved. Dose levels of 4 9 µg included SI and DI cohorts. The MTD was defined as one dose level below the dose that produced grade 3 or 4 adverse events in two or more patients in a cohort. As this was a sequential dose escalation study design, the protocol was amended several times to include higher dose cohorts based on a favourable safety profile. Hence the first cohorts were patients who received either one (SI) or two (DI) injections at a dose range of 7 8 µg. Subsequently, the protocol was amended to allow SI cohorts of doses up to 9 µg. Double injections at 4 9 µg were added to evaluate antiviral effects over a 28-day period and to assess whether there were additional adverse events with the administration of the second dose. After each dose of albumin IFN-α, blood was collected at specified times and drug concentrations, antibody titres, HCV RNA and alanine aminotransferase (ALT) levels were determined. For SI patients, samples were obtained at (predose, within 3 min of the first injection), 1, 3, 6, 12 and 24 h (day 1) and on days 2, 4, 7, 1, 14, 21, 28 and at day 6 in some patients. For DI patients, samples were obtained at the same intervals after the first injection and additional samples were obtained after the second injection at 1, 3, 6, 12 and 24 h (day 1), and on days 16, 18, 21, 24, 28, 3, and 42 and day 7 in some patients. The institutional review boards of participating centres approved the protocol and all amendments. All patients provided written informed consent. Human Genome Sciences, Rockville, MD, USA, sponsored the study. The sponsor designed the study in collaboration with the authors and other expert hepatologists. The study was conducted according to the guideline provisions of Good Clinical Practices, as defined in the US Code of Federal Regulations on the Protection of Human Patients for the United States. Human Genome Sciences was responsible for the collection and statistical analysis of the data. The lead investigator had unlimited access to the data and interpreted the results. No limitations on publications were imposed. The first author International Medical Press

3 Phase I/II study of albumin interferon-α in hepatitis C made final decisions regarding all aspects of the manuscript, and all named authors participated in the interpretation of results and the writing of the manuscript. Safety and tolerability Evaluation of safety and tolerability included adverse event monitoring, physical examination findings and clinical laboratory assessments (haematology, serum chemistry, urinalysis). Safety was assessed from baseline prior to dosing to up to 6 days post-dosing. The development of an antibody response (blocking antibodies to albumin IFN-α, human serum albumin and IFN-α neutralization) was also assessed prior to dosing and up to 6 days post-dosing via immunogenicity assays based on enzyme-linked immunosorbent assay (ELISA) methodology [6] or standardized neutralization assays [8]. Pharmacokinetics Blood samples for plasma albumin IFN-α concentration analyses were obtained prior to dosing and at 13 time points between day and 6 from patients in the SI cohorts. In the DI cohorts, samples were obtained prior to dosing and at 23 time points between days and 7 following dosing from patients. Plasma albumin IFN-α concentrations were measured using an IFN capture, IFN detection sandwich ELISA [6]. Concentration data were modelled using noncompartmental analyses on uniformly weighted data and the nonlinear regression program WinNonlin (Version 4.1, Pharsight Corp, Mountain View, CA, USA). Dose proportionality was evaluated by 1-way analysis of variance (ANOVA; Prism software, GraphPad, San Diego, CA, USA). Parameters expected to vary by dose maximum plasma concentration (C max ) and area under the plasma concentration time curve (AUC) were dose-normalized prior to evaluation. Oneway ANOVA was also used to evaluate the impact of increasing body mass index (BMI) on dose-normalized C max. A Spearman s correlation was used to evaluate the impact of body weight on dose-normalized AUC. HCV RNA levels and genotype HCV RNA analysis was performed with the Amplicor HCV Monitor Kit version 2 (Roche Diagnostic Systems, Branchburg, NJ, USA), and Quest Diagnostics (Teterboro, NJ, USA) performed the assays. Genotype was determined by Line Probe assay (Quest Diagnostics). OAS1 mrna levels were quantitated by a research-based TaqMan PCR assay [9]. A standard curve was generated to determine the pg of OAS1 mrna in each sample normalized to the amount of 18S RNA in the sample and represented as pg of OAS1 mrna in a standard amount of total RNA (2 ng). Statistical methods The safety analysis included all patients who received at least one dose of albumin IFN-α. For the safety and virological outcomes, the data were summarized using mean, median, standard error, standard deviation and number of patients in a group for continuous data, or as counts and percentages for categorical data. Differences in antiviral response and adverse events were analysed using the Fisher s exact test. The changes from baseline in HCV RNA and safety laboratory parameters were analysed using ANOVA. The dose-response trend in antiviral response and occurrence of adverse events were analysed using the Cochran Armitage trend test, as necessary. All statistical tests were 2-sided and performed at a significance level of. unless otherwise specified. Because of the exploratory nature of this study, no adjustments for multiple data comparisons were made. All statistical analyses were performed using SAS (SAS Institute), R statistical package (The R Foundation for Statistical Computing), and Prism and WinNonlin software. Results Patient demographics Of 147 patients screened, 119 were assigned to either the SI (n=71) or DI (n=48) treatment groups and received at least one dose of the study drug. A patient flow diagram by treatment group is shown in Figure 1. Patient demographics and baseline HCV characteristics of the 119 patients are summarized in Table 1. Of the 119 patients, % (6/119) were male and 82% (98/119) were white, not of Hispanic origin; the median age was 48 years; 78% (93/119) of patients were overweight (BMI 2 kg/m 2 ); and 43% were obese (BMI 3 kg/m 2 ). Baseline HCV characteristics were consistent with an IFN non-responder population. The majority of patients had HCV genotype 1 (92%), with a mean viral load of approximately 6.2 log IU/ml. Treatment groups were balanced in demographic parameters such as genotype distribution, baseline HCV RNA levels, hepatitis C duration, and duration of prior IFN-α therapy. There were significantly greater proportions of pegylated IFN-α treatment failures enrolled in the higher-dose cohorts (12 9 µg; 8% [66/78]) than in the lower-dose cohorts (7 8 µg; 6% [23/41]) (P=.1). Overall, study patients had a substantial prior treatment experience, with the median duration of prior IFN-α-based therapy of 48.2 weeks. Safety and tolerability Albumin IFN-α was well tolerated, and no patient was discontinued because of adverse events. Adverse events were transient and most were mild to moderate in severity. Six patients (%) had nine adverse events that were reported as grade 3 or greater. These included one Antiviral Therapy 11:1 37

4 V Balan et al. Figure 1. Patient flow diagram n=147 All n=119 Screening failures n=28 Did not meet entry criteria n=2 Subject request n=2 Other n=1* n=71 Single injection n=48 Double injection n=2 7 8 µg n= µg n=18 32 µg n= µg n= µg n= µg *Patient on pegylated interferon-α2a within 6 days of treatment. Table 1. Baseline characteristics* by treatment arm SI cohorts DI cohorts 7 8 µg µg 32 µg 6 9 µg 7 8 µg 4 9 µg n=2 n=16 n=18 n=17 n=21 n=27 P-value Male gender, n (%) 1 () 1 (63) 9 () 12 (71) 9 (43) 1 (6).617 Race, n (%).441 White (non-hispanic) 17 (8) 9 (6) 16 (89) 1 (88) 17 (81) 24 (89) Asian or Pacific Islander 1 (6) Black (non-hispanic) 2 (1) 4 (2) 1 (6) 2 (12) 3 (14) 2 (7) Hispanic 1 () 2 (13) 1 () 1 (4) Other 1 (6) Age, years, median (range) 47 (32 8) (38 66) 47 (31 64) (36 69) 49 (39 67) 49 (28 7).488 BMI, kg/m 2, median (range) 3 (22 2) 28 (17 41) 3 (2 43) 3 (23 4) 31 (23 3) 28 (19 7).3477 Distribution of BMI, n (%) <2 kg/m 2 3 (1) (31) (28) 2 (12) 3 (1) 7 (26) 2 3 kg/m 2 8 (4) 6 (38) 4 (22) 7 (41) 6 (3) 11 (41) >3 kg/m 2 9 (4) (31) 9 () 8 (47) 11 () 9 (33) Distribution of HCV genotypes, n (%) (8) 1 (94) 17 (94) 16 (94) 2 (9) 24 (89) 2 2 (1) 1 (6) 1 (6) 1 (6) 1 (4) 3 1 () 1 (4) 4 1 (4) 1 () Baseline* HCV RNA, log IU/ml, 6.1 (4.4 7.) 6. (.1 7.3) 6. ( ) 6. ( ) 6.4 (.6 6.9) 6. ( ).1812 median (range) Baseline* ALT level, IU/ml, 92 (3 166) 48 (3 99) 73 (23 17) 66 (2 248) 7(19 17) 49 (26 374).327 median (range) Duration of prior IFN-α therapy, 48 (19 12) 48 (13 233) 4 (24 187) 48 (12 8) 3 (18 33) 46 (14 21).34 week median (range) Underwent prior therapy with 9 (4) 13 (81) 14 (78) 14 (82) 14 (67) 2 (93).91 peg-ifn, n (%) *Baseline values measured at day or screening if day was missing. ALT, alanine aminotransferase; BMI, body mass index; DI, double injection; IFN, interferon; peg, pegylated; SI, single injection International Medical Press

5 Phase I/II study of albumin interferon-α in hepatitis C patient each with anxiety, photosensitivity reaction and neutropaenia (absolute neutrophil count [ANC] <7/µl at day 14) that were deemed as probably study-drug related. Two patients experienced sinusitis and back pain that were considered unrelated to the study medication. There was one serious adverse event in the 32-µg SI cohort, which was a transient hallucination (grade 3 in severity) that lasted for 2 days and resolved spontaneously. The MTD was not identified in this clinical study. The five most common adverse events were headache (6%), fatigue (2%), injection site erythema (38%), arthralgias (32%) and pyrexia (27%). There were no injection site severe inflammatory reactions noted. Table 2 provides a summary of all adverse events reported in the study and that occurred in at least three patients at any dose level or eight patients overall. A dose-response trend (P<.2) was observed for headache in the SI cohorts and headache, fatigue, and nausea in the DI cohorts. Laboratory abnormalities The most common laboratory abnormality was reduction in ANC. The percentage changes from baseline in ANC in the SI and DI cohorts at days 14 and 28 are shown in Figure 2. Maximum reductions occurred after the first injection and during the first week, with significant recovery by day 28 in the SI cohort. In the DI cohort, there were minimal subsequent reductions in ANC after the second dose at day 14. The mean reduction in ANC was 34.3% at day 14 and 22.8% at day 28. Neutropaenia (ANC <7/µl) occurred at any time during the study in 11.8% (14/119) of patients. ANC levels <7/µl at day 14 were observed only in one patient in the 9-µg DI cohort for whom the second dose of albumin IFN-α was delayed 1 week. When comparing the low- and high-dose DI cohorts, there were no dose-related increases in the incidence of reversible neutropaenia (1/21 versus /27; P=.21). Thrombocytopaenia or platelet counts <,/µl were observed in two patients during the study. The mean percentage changes in platelet count from baseline to day 14 and to day 28 were 8.1% and 6.2% in the SI cohorts and 11.7% and 13.3% in the DI cohorts, respectively (Figure 3). There were no shifts in platelet counts to grade 4 (<2,/µl), and there were no adverse events involving low platelet counts reported during the study. Table 2. Incidence of adverse events occurring in at least three patients and reported using preferred terms listed in the Medical Dictionary of Regulatory Activities SI cohorts DI cohorts Total 7-8 µg µg 32- µg 6-9 µg 7-8 µg 4-9 µg Event, n (%) n=2 n=16 n=18 n=17 n=21 n=27 n=119 P-value Headache 6 (3) 8 () 13 (72) 11 (6) 8 (38) 2 (74) 66 (6).132 Fatigue 9 (4) 6 (38) 6 (33) 11 (6) 9 (43) 21 (78) 62 (2).194 site erythema 7 (3) (31) 4 (22.2) 7 (41) 12 (7) 1 (37) 4 (38).3612 Arthralgia (2) 2 (13) 8 (44) 3 (18) 8 (38) 12 (44) 38 (32).1367 Myalgia 7 (3) 3 (19) 6 (33) 4 (24) 3 (14) 1 (37) 33 (28).467 Nausea 3 (1) 1 (6) (28) (29) 4 (19) 14 (2) 32 (27).18 Pyrexia 4 (2) 3 (19) (28) (29) (24) 1 (37) 32 (27).7929 Chills 2 (1) 2 (13) 3 (17) 6 (3) (24) 7 (26) 2 (21).447 Insomnia 4 (2) 4 (2) 2 (11) 1 (6) 6 (29) 4 (1) 21 (18).471 Diarrhoea 2 (1) 2 (13) 1 (6) 3 (18) 3 (14) 8 (3) 19 (16).3881 Dizziness 2 (1) 1 (6) 2 (11) 4 (24) 3 (14) 7 (26) 19 (16).63 Irritability (2) 1 (6) 1 (6) (24) 2 (7) 14 (12).1744 Pain 2 (1) 3 (19) 4 (22) 1 (6) 1 () 3 (11) 14 (12).31 Abdominal pain upper 1 () 2 (13) 3 (17) 2 (12) 2 (1) 3 (11) 13 (11).9191 Dry skin 3 (1) 1 (6) 2 (12) 3 (14) 3 (11) 12 (1).977 Back pain 3 (17) 2 (12) 4 (19) 2 (7) 11 (9).6378 Upper respiratory tract infection 1 () 2 (13) 2 (11) 2 (12) 3 (14) 1 (4) 11 (9).746 Asthenia 1 () 1 (6) 1 (6) 3 (14) 3 (11) 9 (8).8444 Palmar erythema 1 () 2 (13) 1 (6) 1 (6) 4 (19) 9 (8).948 Cough 1 () 2 (13) 1 (6) 1 () 3 (11) 8 (7).814 Depression 1 (6) 3 (18) 2 (1) 2 (7) 8 (7).6813 DI, double injection; SI, single injection. Antiviral Therapy 11:1 39

6 V Balan et al. Figure 2. Mean percentage change from baseline in ANC: SI and DI cohorts SI cohorts DI cohorts 7 8 µg µg 32 µg 6 9 µg 7 8 µg 4 9 µg 1-1 Mean percentage change in ANC Day 14 Day ANC, absolute neutrophil count; DI, double injection; SI, single injection. Overall, reductions in haemoglobin levels were modest. Four patients had decreases in haemoglobin >2. g/dl, and two patients had haemoglobin levels <1 g/dl. Mean reductions in haemoglobin at day 14 and day 28 were.24 and.32 g/dl in the lower-dose cohorts and.68 and.9 g/dl in the higher-dose cohorts, respectively. The differences in mean reductions between the lower-dose and higher-dose cohorts were statistically significant at day 14 (P=.7) and day 28 (P<.1). Transient hyperglycaemia ( 2 grade shift in serum glucose) was observed at any time during the study in 44% (18/41) of patients receiving 7 8 µg and 17% (13/78) of patients receiving 12 9 µg. Transient elevations in ALT (greater than threefold over baseline) were observed in one patient 7 days post injection; ALT levels returned to baseline values by the end of the study. A significant change in thyroid-stimulating hormone (TSH) level was observed in only one patient for whom TSH increased to 6.3 mu/l on day 42 from 1.41 mu/l at screening. This patient had no clinical signs or symptoms of thyroid dysfunction. Immunogenicity Among the 119 patients who participated in the study, 1 (12.6%) had pre-existing IFN antibodies. The observed incidence of emerging albumin IFN-α antibody-positive patients was 1.1%, with an observed incidence of emerging neutralizing antibodies of 2.% (3/119). Also, four patients (3.4%) had preexisting antibodies to albumin, while the observed incidence of emerging albumin antibodies in patients was 1.7% (two patients). The vast majority of patients with albumin IFN-α antibodies had low titres (<1 ng/ml). There was no correlation between the development of these antibodies and the occurrence of adverse events, laboratory abnormalities, or antiviral response. Pharmacokinetics Mean plasma concentrations of albumin IFN-α are presented in Figure 4A (SI cohorts, 12 9 µg) and Figure 4B (DI cohorts, 4 9 µg). The median time to reach maximum albumin IFN-α plasma concentrations (t max ) was 4 days following a single subcutaneous injection (Table 3). C max increased in a dose-proportional manner following single subcutaneous injections of 2 9 µg (Figure ). Mean C max ranged between 1.6 ng/ml following a dose of 2 µg to 34 ng/ml following a single subcutaneous dose of 9 µg. Albumin IFN-α was eliminated in a monophasic manner; mean elimination half-life (t fi ) was 19 hours (6.6 days) following the single dose and, thus, some drug accumulation was observed in patients in DI cohorts. In patients receiving DI doses of 4 9 µg, mean trough albumin IFN-α 4 26 International Medical Press

7 Phase I/II study of albumin interferon-α in hepatitis C Figure 3. Mean percentage change from baseline in platelet count: SI and DI cohorts SI cohorts DI cohorts µg µg 32 µg 6 9 µg µg µg Mean percentage change in platelet count Day 14 Day DI, double injection; SI, single injection. concentrations were % greater following the second dose at day 28 than on day 14 of the study. Albumin IFN-α concentrations of up to 3 ng/ml were detected 28 days after the second dose in the DI cohorts. There were no significant differences in dose-normalized C max or dose-normalized AUC between genders or between patients with normal body weights and those who were overweight (BMI between 2 and 3). A significant (P=.116) correlation was found between greater body weight and lower dose-normalized AUC. Obese patients (BMI >3) had significantly lower dosenormalized C max concentrations than non-obese patients (P=.24). Antiviral response Reductions from baseline in HCV RNA (log IU/ml) over 28 days in the SI (Figure 6) and DI (Figure 7) cohorts were greater among patients receiving the higher doses and were maintained for up to 28 days following a single injection. Antiviral activity of 1 log reduction in HCV RNA in the subjects that received 12 µg or more of albumin IFN-α, was observed in 47% (37/78). A dose response was observed. In the SI cohorts, the percentage of subjects with antiviral response increased from % in the 7- to 8-µg cohorts to 41% (21/1) in the 12- to 9-µg cohorts. In the DI cohorts, the percentage of subjects with antiviral response increased from 14% (3/21) in the 7- to 8-µg cohorts to 9% (16/27) in the 4- to 9-µg cohorts. Mathematical modelling of the HCV-RNA decline according to the viral dynamics model of Neumann et al. [1] revealed a biphasic response. Figure 8 illustrates the kinetics of viral load reductions in two patients receiving albumin IFN-α injections 7 µg at day and day 14. In one patient, there was no observable rebound 2 weeks after the injections and the kinetics are consistent with a biphasic decline due to an approximately constant antiviral effectiveness. In the other patient, there was a limited viral rebound starting 1 week after the first injection. Nevertheless, after the second injection the viral decline followed the biphasic pattern. Induction of IFN-specific genes The pharmacodynamics of albumin IFN-α was assessed by monitoring OAS1 mrna concentrations in peripheral blood; OAS1 is a well-characterized IFNspecific gene [9]. While the OAS1 mrna concentrations varied among patients at baseline, OAS1 mrna induction was observed in all dose cohorts. The durability of OAS1 mrna induction was assessed by comparing fold induction over baseline at day 28 following a single dose of albumin IFN-α among the different dose groups as shown in Figure 9. The median fold induction at day 28 days is greatest in the highest Antiviral Therapy 11:1 41

8 V Balan et al. Figure 4. Mean plasma albumin interferon-α concentrations A 6, Concentration, pg/ml, 4, 3, 2, 1, 18 µg 24 µg 12 µg 32 µg 4 µg 6 µg 7 µg 9 µg µg Time, days B Concentration, pg/ml 8, 7, 6,, 4, 3, 2, Dosing 4 µg x 2 µg x 2 6 µg x 2 7 µg x 2 9 µg x 2 1, Time, days (A) SI cohorts; (B) DI cohorts. Table 3. Mean pharmacokinetic parameters: SI cohorts SI cohorts 7 8 µg µg 32 µg 6 9 µg t 1/2, h, mean ± SD 12 ± ±7 136 ±34 76 ±62 t max, h, median (range) 98 (49 169) 9 (49 168) 96 (46 172) 96 (13 17) Vz/F, ml/kg, mean ± SD 142 ±42 19 ± ±69 28 ±13 CL/F, ml/h/kg, mean ± SD.67 ± ± ± ±.41 CL/F, apparent clearance; SI, single injection; t fi, elimination half-life; T max, time to maximum plasma concentrations; Vz/F, apparent volume of distribution International Medical Press

9 Phase I/II study of albumin interferon-α in hepatitis C Figure. C max versus dose following single subcutaneous injections of 2 9 µg C max, ng/ml Figure 6. Mean change from baseline in HCV RNA: SI cohort Mean change from baseline in HCV RNA, log IU/ml SI, single injection. SI cohorts Study day 7 8 µg µg 32 µg 6 9 µg Figure 7. Mean change from baseline in HCV RNA: DI cohort Mean change from baseline in HCV RNA, log IU/ml C max, maximum plasma concentration DI, double injection. r 2 = Dose, µg DI cohorts Study day 7 8 µg 4 9 µg dose group and this is consistent with the presence of significant detectable drug after a single dose of albumin IFN-α. Discussion Albumin IFN-α is a novel 8.7-kDa recombinant protein consisting of IFN-α2b genetically fused to human serum albumin. This fusion polypeptide was hypothesized to extend the circulating half-life of the IFN-α and thus prolong the drug s therapeutic activity. In this Phase I/II study conducted in an IFN-α-experienced predominantly genotype 1 CHC population, albumin IFN-α had a favourable safety and tolerability profile in the 119 patients treated with one or two injections. The common adverse events were similar to those previously observed with other IFNs and included headache, fatigue, arthralgias and pyrexia. Reductions in neutrophil and platelet concentrations occurred predominantly during the first week and stabilized after the second injection at day 14. There were no discontinuations and only one patient had a delay in receiving the second dose. Importantly, no dose-limiting toxicities were observed. Hence the MTD was not identified in this study. Pegylation of IFN alters the uptake and excretion of standard IFN and prolongs its t fi [11]. Albumin IFN-α has a t fi of ~7 days. This is approximately twofold longer than pegylated IFN-α2a, and fourfold greater than pegylated IFN-α2b, both of which are dosed once weekly [12,13]. Hence the pharmacokinetics of albumin IFN-α support further assessments of dosing in 2- to 4-week intervals. In the higher-dose cohorts, albumin IFN-α concentrations are consistently detected up to 28 days following a single dose. Patients in the higher DI cohorts (4 9 µg) showed the expected drug accumulation, with trough concentrations averaging % greater following the second dose of albumin IFN-α; however, this increase was not associated with increased adverse events or laboratory abnormalities. The increase in trough albumin IFN-α concentrations observed in this study are consistent with the approximately twofold increase in trough concentrations reported for pegylated IFN-α2a following multiple doses [12]. Albumin IFN-α would be expected to reach over 9% of its steady-state concentration by the fourth dose on the every 2-week schedule. Therefore, the reduced dosing intervals proposed with albumin IFN-α may provide convenience, improve compliance, and may translate into better clinical outcomes. The improved pharmacokinetics of albumin IFN-α correlated with prolonged pharmacodynamic activity. Induction of the IFN-specific gene OAS1 mrna was maintained for 28 days following a single injection of Antiviral Therapy 11:1 43

10 V Balan et al. Figure 8. Non-linear fitting of HCV RNA decline with biphasic viral decline for at least 28 days Patient 1 Patient 2 6 HCV RNA Fitted viral kinetics Biphasic viral decline 6 HCV RNA Fitted viral kinetics Biphasic viral decline HCV RNA, log IU/ml Study day Study day Individual patient examples receiving double injections of albumin IFN-α 7 µg. Biphasic model from Neumann et al. [1] VK, viral kinetics. Figure 9. Median OAS1 fold-induction at day 28: SI cohorts SI, single injection. 7 8 µg µg 32 µg P= µg albumin IFN-α. The lack of dose response in transcriptional activation (OAS1 gene induction) is not surprising as maximal gene expression was achieved with an albumin IFN-α dose 4 µg. Antiviral activity showed dose-dependent increases following a single dose and was maintained for up to 28 days in the higher-dose cohorts. A greater proportion of patients showed a 1 log reduction in HCV RNA in the 4 9 µg DI cohorts (9%) compared with the 7 8 µg DI cohorts (14%). The viral kinetics observed following the administration of albumin IFN-α in this highly IFN-α-experienced population is interesting. Neumann et al. previously described HCV RNA kinetics during treatment with standard IFN-α, wherein after initiation of therapy there was a rapid dose-dependent decline of viral load, termed the first-phase decline [1]. The viral decline then enters a second phase of relatively slow but continuous exponential decay during the following weeks of therapy, which is due to an approximately constant antiviral effectiveness [14]. In this report, we demonstrate in a subset of patients this biphasic response pattern with albumin IFN-α wherein a first-phase dose-dependent initial reduction in viral load is followed by subsequent second-phase decline in HCV RNA. The presence of antibodies to albumin IFN-α was evaluated in the current study population. The vast majority of albumin IFN-α antibody titres were low (<1 ng/ml) and generally did not show significant increases after treatment with albumin IFN-α. A total of 2 patients tested positive for antibodies to IFN at any time during the study period; of these patients, 1 were positive prior to the initiation of dosing. Twelve additional patients were antibody negative prior to dosing and antibody positive at one or more times following the initiation of dosing. Both incidences are in line with the published immunogenicity rates for pegylated IFNs (8 1%) in treatment-naive patients. All patients had previously been treated for a minimum of 12 weeks with an IFN-α-containing regimen and, hence, it is not surprising to detect in a subset of patients the presence of pre-existing antibodies or to detect low titres of antibodies on re-exposure to an IFN. Of six patients who tested positive for albumin antibodies, four were positive prior to the initiation of International Medical Press

11 Phase I/II study of albumin interferon-α in hepatitis C dosing. This is consistent with previous observations of increased incidence of albumin antibodies among patients with CHC [1,16]. In summary, the immunogenicity of albumin IFN-α does not appear to correlate with patient demographics, disease status (including prior treatment history) or the magnitude or duration of exposure to the study drug. In conclusion, this is the first report of a therapeutic recombinant protein that has been genetically fused to human serum albumin and has shown beneficial biological activity. In CHC patients, albumin IFN-α should be able to reduce dosing frequency and may improve efficacy through greater compliance. In addition, the less frequent dosing and improved pharmacokinetics may reduce the adverse events associated with IFN therapies. Thus, the information presented here supports further clinical studies of this novel protein in patients with CHC. Acknowledgements The authors would also like to acknowledge the contributions of the following individuals: Tina Bambury, RN, Rebecca Rush, RN, Marianne Rosati, CRNP Mayo Clinic, Phoenix, AZ, USA; Joy Peter, RN, Mitzi Tucker, University of Florida, Gainesville, USA; Heather Thomas, RN, Johns Hopkins University, Baltimore, MD, USA; Melissa Billington, University of Maryland, Baltimore, MD, USA; Jennifer DeSanto, RN, University of Colorado, Denver, CO, USA; Myrna Mattos, RN, University of Florida at Jacksonville, FL, USA; Linda Lairson, RN, Mayo Clinic, Rochester, MN, USA; Ellen Miceli, RN, Mayo Clinic, Jacksonville, FL, USA; Laura Veri, RN, University Hospitals of Cleveland, Cleveland, OH, USA; and Robert Abelson, John Zhong, Victor Roschke, Lu-Anne Novello and William W Freimuth, Human Genome Sciences, Rockville, MD, USA. References 1. World Health Organization [homepage on the Internet]. Hepatitis C fact sheet [updated 2 October; cited 24 Nov 2]. WHO Media Centre; [about 2 screens]. Available from: Received 6 September 2, accepted 7 November 2 2. Centers for Disease Control and Prevention [homepage on the Internet]. [updated 24; cited 2 Aug 3]. CDC Resource Center: Disease burden from viral hepatitis A, B, and C in the United States. [about 2 screens]. Available from: disease_burden24.pdf 3. Fried MW, Shiffman ML, Reddy KR, et al. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J Med 22; 347: Manns MP, McHutchison JG, Gordon SC, et al. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomised trial. Lancet 21; 38: Masci P, Bukowski RM, Patten PA, Osborn BL, Borden EC. New and modified interferon alfas: preclinical and clinical data. Current Oncol Rep 23; : Osborn BL, Olsen HS, Nardelli B, et al. Pharmacokinetic and pharmacodynamic studies of a human serum albumin-interferon-alpha fusion protein in cynomolgus monkeys. J Pharmacol Exp Ther 22; 33: Smith PK, Krohn RI, Hermanson GT, et al. Measurement of protein using bicinchoninic acid. Anal Biochem 198; 1: Grossberg SE, Kawade Y, Kohase M, Klein JP. The neutralization of interferons by antibody. II. Neutralizing antibody unitage and its relationship to bioassay sensitivity: the tenfold reduction unit. J Interferon Cytokine Res 21; 21: Rebouillat D, Hovanessian AG. The human 2, -oligoadenylate synthetase family: interferon-induced proteins with unique enzymatic properties. J Interferon Cytokine Res 1999; 19: Neumann AU, Lam NP, Dahari H, et al. Hepatitis C viral dynamics in vivo and the antiviral efficacy of interferonalpha therapy. Science 1998; 282: Zeuzem S, Welsch C, Herrmann E. Pharmacokinetics of peginterferons. Semin Liver Dis 23; 23 Suppl 1: Pegasys (peginterferon alfa-2a) Package insert. 24. Hoffmann-La Roche Inc, Nutley, NJ, USA 13. Glue P, Fang JW, Rouzier-Panis R, et al. Pegylated interferon-alpha2b: pharmacokinetics, pharmacodynamics, safety, and preliminary efficacy data. Hepatitis C Intervention Therapy Group. Clin Pharmacol Ther 2; 68: Herrmann E, Neumann AU, Schmidt JM, Zeuzem S. Hepatitis C virus kinetics. Antivir Ther 2; : Sansonno DE, DeTomaso P, Papanice MA, Manghisi OG. An enzyme-linked immunosorbent assay for the detection of autoantibodies to albumin. J Immunol Methods 1986; 9: Nardiello S, Pizzella T, Ronga C, Galanti B. Levels of antialbumin autoantibodies in acute and chronic hepatitis B virus infection. Hepatogastroenterology 199; 37 Suppl 2: Antiviral Therapy 11:1 4