JOURNAL OF INTERFERON & CYTOKINE RESEARCH 28:113–122 (2008)© Mary Ann Liebert, Inc.DOI: 10.1089/jir.2007.0073

Novel Controlled-Release Lemna-Derived IFN-�2b (Locteron):Pharmacokinetics, Pharmacodynamics, and Tolerability in

a Phase I Clinical Trial

LEO G.J. DE LEEDE,1 JOHN E. HUMPHRIES,2 ANNE C. BECHET,1 EWOUD J. VAN HOOGDALEM,1

RUUD VERRIJK,1 and DAVID G. SPENCER2

ABSTRACT

Locteron™, a newly developed controlled-release formulation of Lemna-derived free (unpegylated) recombi-nant interferon-�2b (IFN-�2b, Biolex Therapeutics, Pittsboro, NC) in poly(ether-ester) microspheres (Poly-Active, OctoPlus N.V., Leiden, the Netherlands), was evaluated in 27 volunteers injected with either 20, 80,or 320 �g Locteron (equivalent to 6.25, 25, or 100 � 106 IU, respectively), 80 �g pegylated IFN-�2b (PEG-IFN-�2b), microspheres not containing IFN-�2b, or placebo. Serum free or PEG-IFN-�2b and two biomarkersof IFN activity, neopterin and 2�,5�-oligoadenylate synthetase (2�,5�-OAS), were measured. After injection of320 �g Locteron, serum IFN-�2b remained elevated through 14 days. The elimination half-life of Locteronwas more than 2-fold that of PEG-IFN-�2b. The effects of 80 �g Locteron and 80 �g PEG-IFN-�2b on bothneopterin and 2�,5�-OAS were in a comparable range. Serum persistence of both these biomarkers was sim-ilar at 14 days after 320 �g Locteron compared with 7 days after 80 �g PEG-IFN-�2b. Mild, moderate, orsevere influenza-like symptoms developed in all 6 subjects receiving 80 �g PEG-IFN-�2b. No such symptomsoccurred after 20 or 80 �g Locteron doses. Among the 4 recipients of 320 �g Locteron, 1 experienced mildand 2 experienced moderate influenza-like symptoms. Locteron merits further clinical investigation as a he-patitis C therapy suitable for dosing once per 2 weeks.

113

INTRODUCTION

INTERFERON-� (IFN-�) COMBINED WITH oral ribavirin has be-come a mainstay of treatment for hepatitis C, a chronic vi-

ral infection of the liver that places a significant burden on bothindividuals and healthcare resources. According to the WorldHealth Organization (WHO), an estimated 170 million personsworldwide are chronically infected with the hepatitis C virus(HCV), and 3–4 million persons are newly infected each year(www.who.int/mediacentre/factsheets/fs164/en/index.html; ac-cessed February 7, 2007). Approximately 20% of patients af-fected by chronic HCV infections progress to cirrhosis after anaverage of 20 years, and 5% will develop hepatocellular carci-noma (Hughes and Shafran 2006).

The primary goal of therapy for chronic HCV infection isviral eradication, that is, a sustained virologic response (SVR),and treatment must continue up to 48 weeks. Initially, subcu-taneous injection of unmodified recombinant IFN-� was com-monly employed, and because of the short circulating half-life

of the molecule (2–3 h) (Radwanski and others 1987), dosingthree times per week was required. In studies of combinationtherapy with IFN-� and ribavirin, SVRs of approximately 40%have been documented (McHutchison and others 1998; Poy-nard and others 1998; Reichard and others 1998). In an attemptto improve the SVR and side effect profile and to enhance ad-herence, pegylated forms of IFN-� (PEG-IFN) were developed.Both PEG-IFN-�2a and PEG-IFN-�2b have been approved forHCV treatment by once weekly dosing, and PEG-IFN emergedas the standard agent for IFN-based therapy. Combination reg-imens with PEG-IFN and ribavirin have resulted in higher re-sponse rates, with eradication of the virus in more than half oftreated patients (Lindsay and others 2001; Manns and others2001; Fried and others 2002; McHutchison and Fried 2003;Meyer-Wyss and others 2006). This advantage may arise at leastpartly from reduced exposure of the patient to circulating IFN-� nadirs. Improved adherence may be another contributor.

Pegylation, which entails the covalent attachment of an in-ert, water-soluble polymer of polyethylene glycol (PEG) to the

1OctoPlus N.V., Leiden, the Netherlands.2Biolex Therapeutics, Pittsboro, NC 27312.

IFN molecule in either a linear chain (PEG-IFN-�2b) or abranched-chain configuration (PEG-IFN-�2a), causes sterichindrance and reduces the specific activity of the IFN mole-cule. Thus, PEG-IFN-�2b retains only 28% of specific activitycompared with unmodified IFN-�2b, and PEG-IFN-�2a retains�7% vs. unmodified IFN-�2a (Caliceti 2004). Another limita-tion is the observed decline of serum PEG-IFN-�2b concen-tration to pretreatment levels within 7 days (Glue and others2000b). Thus, any delay in dosing might permit viral rebound(Formann and others 2003).

IFN therapy is associated with well-described adverse events,such as fatigue, influenza-like illness, neutropenia, thrombocy-topenia, and psychiatric symptoms, that can necessitate dis-continuation of therapy or dose modification (Fried 2002;Bagheri and others 2004). Adherence to combination therapyis important in achieving SVR (McHutchison and others 2002).In an analysis of results from three randomized trials, SVR wasincreased among patients receiving �80% of the prescribeddosage throughout the duration of the regimen (McHutchisonand others 2002).

Although PEG-IFN represents a definite advance and islikely to continue as first-line therapy in the near term, furtherimprovements might be achieved through even more conve-nient dosing regimens, prolonged maintenance of circulatingIFN concentration, increased bioavailability, and reduced ad-verse events (Mohl and Winter 2004; Peleg-Shulman and oth-ers 2004). One promising approach is through controlled re-lease of unmodified IFN. Locteron is a newly developedformulation of recombinant IFN-�2b (Biolex Therapeutics,Pittsboro, NC) in poly(ether-ester) microspheres (PolyActive,OctoPlus N.V., Leiden, the Netherlands) (van Dijkhuizen-Radersma and others 2002, 2003, 2005; Gasdaska and others2003). The recombinant IFN-�2b component of Locteron(BLX-883) is synthesized in a Lemna aquatic plant expressionsystem, and posttranslational cleavage yields a mixture of mol-ecules with either seven or eight amino acids removed from theC-terminus. IFN-�2b molecules produced by the Lemna sys-tem are nonglycosylated. The antiviral and antiproliferative ac-

tivities of BLX-883 in vitro are indistinguishable from those ofstandard Escherichia coli-derived IFN-�2b (Table 1) (Gasdaskaand others 2003). Locteron has been engineered to release freeIFN continuously over a period of 2 weeks. The present phaseI clinical trial has evaluated Locteron. The Locteron trial waspreceded by a trial examining the BLX-883 recombinant IFN-�2b component of Locteron directly injected with nopoly(ether-ester) microspheres.

MATERIALS AND METHODS

Trial design

This phase I clinical trial, carried out from September to De-cember 2005, was a randomized, double-blind, active-con-trolled and placebo-controlled, dose-escalation study evaluat-ing the pharmacokinetics, pharmacodynamics, and tolerabilityof Locteron after a single subcutaneous dose. Subjects eligiblefor study entry were healthy male volunteers 21–50 years ofage with a body mass index (BMI) of 20–28 kg/m2. Exclusioncriteria consisted of hypersensitivity to study medication, alco-hol or drug abuse, participation in another investigational drugtrial or blood donation within the preceding 90 days, cigarettesmoking, impaired renal or hepatic function, history of pul-monary disease, diabetes mellitus, coagulation disorders, pso-riasis or sarcoidosis, prescription medication or chronic non-prescription medication use, heart rhythm disorders, priortreatment with IFN or other therapeutic cytokine, herpes zosterinfection resolving �2 months before study entry, exposure tolive vaccine within the previous 4 weeks, and viral or bacterialinfection during the preceding 3 months. Regular use of con-comitant medication other than paracetamol was not permittedduring the study.

In addition to the primary phase I clinical trial of Locteron,a second phase I clinical trial was conducted earlier (Januaryto June 2005) to assess the pharmacokinetics, pharmacody-namics, and tolerability of BLX-883, the Lemna-derived IFN-

DE LEEDE ET AL.114

TABLE 1. IN VITRO COMPARISON OF LEMNA-DERIVED AND E. COLI-DERIVED IFN-�2ba

IFN-�2b sourceb

Lemna E. coliParameter (BLX-883) (Intron A)

% Daudi cell growth inhibition,mean (SD), n � 3c

46 h 16.5 (4.0) 14.7 (2.4)70 h 24.0 (2.9) 22.7 (3.6)93 h 25.4 (3.1) 25.3 (3.6)

Viral titer log reduction, n � 1d

Encephalomyocarditis virus 1.2 1.2Sindbis virus 0.5 0.5Vesicular stomatitis virus 0.4 0.4

aAdapted from data of Gasdaska and others 2003.bBLX-883 and Intron A concentrations were 103 IU/mL (equivalent to 3.85 ng/ml)

in all experiments. All results are expressed per ng protein.cProliferation of IFN-sensitive Daudi cells was assayed by trypan blue exclusion, and

there was no evidence of cell lysis.dViral titer log reductions were determined by incubation of HuH7 cells with BLX-

883 or Intron A, followed by viral challenge at a 1.0 multiplicity of infection.

�2b constituent of Locteron. The in vitro properties of BLX-883 have been described previously (Gasdaska and others2003). The second phase I clinical trial was undertaken to char-acterize BLX-883 in vivo. Eligible subjects were healthy malenonsmokers 18–65 years of age with a BMI of 18–28 kg/m2

and no clinically important physical, laboratory, electrocardio-gram (ECG), blood pressure, or heart rate abnormalities.

Subjects in both the Locteron and BLX-883 phase I trialsgranted their written informed consent prior to enrollment. Thesites of the two trials were Xendo Clinics, Groningen, TheNetherlands, and Charles River Laboratories, Edinburgh, Scot-land, respectively. Both trials were performed in conformitywith Good Clinical Practice.

Endpoints

In the Locteron trial, pharmacokinetic parameters were esti-mated from repeated determinations of circulating free or pe-gylated IFN-�2b. These parameters consisted of the area underthe time-concentration curve (AUC), maximum attained con-centration (Cmax), and elimination half-life (t1/2el). Pharmaco-dynamic end points were the posttreatment serum levels of twobiomarkers indicating IFN activity, namely, neopterin and 2�,5�-

oligoadenylate synthetase (2�,5�-OAS). Repeated neopterin and2�,5�-OAS measurements were also subjected to pharmacoki-netic analysis. Tolerability was judged on the basis of post-treatment adverse events and changes in hematologic variables,primarily serum leukocytes, neutrophils, hemoglobin, andplatelets.

In the BLX-883 trial, the temporal patterns of change inserum IFN-�2b concentration after injection of BLX-883 or E.coli-derived IFN-�2b were determined. The pharmacodynamicend point was circulating neopterin concentration. In the as-sessment of tolerability, adverse events were documented, andhematologic variables were evaluated.

Treatment

Subjects in the Locteron trial were admitted to the study cen-ter on the afternoon of the day before treatment, at which timethe following baseline assessments were performed: vital signsand body temperature, physical examination, ECG, biochem-istry and hematology, urinalysis, drug and alcohol screeningand serology, serum free or PEG-IFN-�2b, biomarkers, and im-munogenicity. At 0800–1000 h the next day, the subjects re-ceived one of the following subcutaneous injections: 20, 80, or

CONTROLLED-RELEASE LEMNA-DERIVED IFN-�2b 115

FIG. 1. Mean serum IFN-�2b concentration after adminis-tration of 0.5 � 106 IU BLX-883, 1.0 � 106 IU BLX-883,3.0 � 106 IU BLX-883, or 3.0 � 106 IU Intron A in groups of6 healthy human male volunteers each. Error bars show SD.

Neo

pter

in (

nmol

·L)

-1

Time (days)0 1 2 6 8 10 12

0

5

10

15

20

25 0.5 × 10 IU BLX-8836

1.0 × 10 IU BLX-8836

3.0 × 10 IU BLX-8836

3.0 × 10 IU Intron A6

FIG. 2. Mean circulating concentrations of neopterin after ad-ministration of 0.5 � 106 IU BLX-883, 1.0 � 106 IU BLX-883,3.0 � 106 IU BLX-883, or 3.0 � 106 IU Intron A in groups of6 healthy human male volunteers each. Error bars show SD.

320 �g Locteron (equivalent to 6.25, 25, or 100 � 106 IU, re-spectively), 80 �g PEG-IFN-�2b (PEG-Intron, Schering Corp.,Kenilworth, NJ), 2, 8, or 32 mg PolyActive microspheres notcontaining IFN-�2b, or 10% hydroxyethyl starch placebo (He-mohes, B. Braun, Melsungen, Germany). Locteron is composedof PolyActive microspheres containing 1% (w/w) IFN-�2b, andLocteron doses are expressed in terms of IFN-�2b contentrather than microsphere mass. Thus, the 2-mg, 8-mg, and 32-mg doses of control PolyActive microspheres provided the samemicrosphere quantities as the corresponding 20-�g, 80-�g, and320-�g Locteron doses.

The 20-�g Locteron and 2-mg PolyActive microsphere doseswere administered in 0.2 mL volumes per injection site, whereasthe higher doses were injected in 0.4 mL per site. The 320-�gLocteron and 32-mg PolyActive microsphere doses were di-vided among four injection sites, whereas lower doses were in-jected at a single site.

The volunteers in the BLX-883 trial were randomly allocatedto receive a single subcutaneous injection of 0.5, 1.0, or 3.0 �106 IU BLX-883 or 3.0 � 106 IU E. coli-derived IFN-�2b (In-tron A, Schering Corp.). Six volunteers were assigned to eachgroup. The two groups receiving 3.0 � 106 IU BLX-883 (equiv-alent to 9.7 �g) or 3.0 � 106 IU Intron A (equivalent to 11.5�g) crossed over after 4 weeks to a single 3.0 � 106 IU sub-cutaneous injection of Intron A or BLX-883, respectively.Those two groups were investigated on a single-blind basis,whereas an open-label design was adopted for the other twogroups.

Posttreatment assessments

In the Locteron trial, blood samples (5 mL) were drawn forpharmacokinetic evaluation of serum-free or pegylated IFN-�2b at 1, 3, 6, 12, and 24 h and 2, 3, 7, 10, 14, 17, and 28 daysafter injection. Adverse events were documented at all thesetime points except 17 days. Measurements of biomarkers were

made at 12 and 24 h and 2, 3, 7, 10, 14, and 28 days. Vitalsigns and body temperature, ECG, physical examination, andurinalysis were performed at frequent intervals throughout the28-day course of postinjection observation.

In the BLX-883 trial, 5-mL blood samples for measurementof serum IFN-�2b were collected at baseline and 1, 2, 3, 4, 6,8, 10, 12, 16, 20, 24, 28, 32 36, and 48 h postinjection. Circu-lating neopterin determinations were performed at baseline, 12,24, and 36 h and 2, 5, 7, 9, and 11 days. Hematologic variables,including leukocytes, neutrophils, hemoglobin, and platelets,were measured at baseline and 1, 2, 5, 7, 9, 15, 22, and 43 days.Adverse events occurring at any time up to 43 days wererecorded.

Laboratory methods

Enzyme-linked immunosorbent assay (ELISA) with com-mercial reagents was used for determination of serum-free IFN-

DE LEEDE ET AL.116

TABLE 2. ADVERSE EVENTS AFTER BLX-883 OR INTRON Aa

Number (%) of subjectswith adverse event

(BLX-883) (Intron A)Adverse event (n � 11) (n � 10)

Pyrexia 8 (72.7) 9 (90.0)Headache 8 (72.7) 6 (60.0)Myalgia 4 (36.4) 4 (40.0)Rigors 3 (27.3) 2 (20.0)Nausea 3 (27.3) 1 (10.0)Vomiting 1 (9.1) 1 (10.0)Nasal congestion 2 (18.2) 0 (0.0)Back pain 1 (9.1) 1 (10.0)Abdominal pain 0 (0.0) 1 (10.0)Pharyngolaryngeal pain 1 (9.1) 0 (0.0)Bowel sounds abnormal 0 (0.0) 1 (10.0)Herpes simplex 0 (0.0) 1 (10.0)Influenza 0 (0.0) 1 (10.0)

aDose of BLX-883 and Intron A � 3 � 106 IU.

FIG. 3. Mean circulating concentrations of leukocytes, neu-trophils, hemoglobin, and platelets after administration of 3 �106 IU BLX-883 or Intron A in groups of 6 healthy human malevolunteers each. Error bars show SD.

A

B

C

D

�2b (IFN-� ELISA kit, GE Healthcare, Buckinghamshire,U.K.) and PEG-IFN-�2b (IFN-� ELISA kit, PBL BiomedicalLaboratories, Piscataway, NJ). Commercial radioimmunoas-says were employed to measure serum neopterin (BRAHMSNeopterin RIA kit, BRAHMS AG, Hennigsdorf, Germany) and2�,5�-OAS (2-5A Kit “Eiken,” Eiken Chemicals Co., Ltd.,Tokyo, Japan).

Statistical analysis

Descriptive statistics consisted of the mean, standard devia-tion (SD), coefficient of variation (CV), median, and range. Be-tween-group difference in mean values was determined by cal-culating the 95% confidence interval (CI) for the difference.Absence of zero from the CI implies a statistically significantdifference (p � 0.05). The significance of between-group dif-ference in variability was assessed by the Levene test. The dose-response relationship between Locteron and biomarkers wascharacterized by linear regression. Estimation of pharmacoki-netic parameters was carried out using WinNonLin Enterprise4.1 pharmacokinetic modeling software (Pharsight Corp.,Mountain View, CA). Otherwise, data were analyzed with Rversion 2.4.1 statistical software (R Foundation for StatisticalComputing, Vienna, Austria).

RESULTS

Characterization of BLX-883

In a phase I clinical trial of 24 healthy men, a single 3.0 �106 IU dose of BLX-883 increased serum IFN-�2b (Fig. 1) andneopterin (Fig. 2) approximately as much as did the same doseof Intron A. BLX-883 doses of 0.5, 1.0, and 3.0 � 106 IU ledto progressively higher peak serum IFN-�2b (Fig. 1) andneopterin (Fig. 2) concentrations. Between the subjects receiv-ing 3.0 � 106 IU BLX-883 and Intron A, the incidence andtypes of adverse events differed little (Table 2), and the pat-terns of change in hematologic variables coincided very closely(Fig. 3).

Disposition of subjects

Of 51 candidate subjects screened for the Locteron phase Itrial, 27 were randomized and treated. Their mean (SD) age was31.0 years (9.2 years), weight 82.0 kg (7.0 kg), and BMI 24.3kg/m2 (1.5 kg/m2). Four subjects each received 20, 80, and 320�g Locteron, 6 subjects received 80 �g PEG-Intron, 2 each re-ceived 2, 8, and 32 mg PolyActive microspheres, and 3 receivedplacebo. All 27 subjects completed the study.

CONTROLLED-RELEASE LEMNA-DERIVED IFN-�2b 117

FIG. 4. Mean serum concentration of IFN-�2b up to 28 days after administration of 320 �g Locteron (n � 4) or PEG-Intronafter an 80-�g dose (n � 6). Data from 1 subject in the PEG-Intron group were excluded due to marked baseline elevation ofindeterminate origin. Error bars show SD.

Pharmacokinetics

Serum concentrations of IFN-�2b after administration of 320�g Locteron are shown in Figure 4. Most circulating IFN-�2bconcentration measurements after doses of either 20 or 80 �gwere below the assay limit of quantification. After the 320-�gdose, serum IFN-�2b increased continuously over a period of12 h. Thereafter, a comparatively broad plateau was reachedwith estimated mean peak concentration (Cmax) of 14.6 pg/mLat approximately 24 h. Plateau levels persisted until approxi-mately 3 days, after which serum IFN-�2b began to declinegradually. Nevertheless, the mean concentration at 7 days re-mained relatively high, equaling 58% of Cmax (Fig. 4). IFN-�2b levels remained measurable at 14 days.

Following an 80-�g PEG-Intron dose, maximal PEG-Intronlevels were reached at approximately 24 h (Fig. 4); however,the peak was relatively narrow, and serum PEG-Intron de-

creased substantially as early as 2 days and could no longer bedetected at 7 days. Variability in AUC (Fig. 4), as measured byCV, was lower by 28.4% for 320 �g Locteron than 80 �g PEG-Intron (p � 0.024).

Pharmacodynamics

Circulating concentrations of both neopterin and 2�,5�-OASincreased in response to administration of 20, 80, and 320 �gLocteron and 80 �g PEG-Intron (Fig. 5). Neopterin and 2�,5�-OAS AUC and Cmax data are presented in Table 3. The per-sistence of the posttreatment serum increments in neopterin and2�,5�-OAS, expressed as a percentage of Cmax with baseline ad-justment, is summarized in Table 4. At 7 days after 320 �gLocteron, the serum neopterin concentration was higher by 43%than after 80 �g PEG-Intron. At 14 days, 14% of the post-treatment neopterin increase persisted in the 320-�g Locterongroup, whereas neopterin had returned to baseline in the 80-�gPEG-Intron group. The residual increment in 2�,5�-OAS at 7days was also higher in recipients of 320 �g Locteron, althoughthe difference was not statistically significant. At 14 days, how-ever, the proportion of the posttreatment rise in 2�,5�-OAS wassignificantly higher by 27% among subjects receiving 320 �gLocteron than 80 �g PEG-Intron. In comparisons at 14 daysafter 320 �g Locteron vs. 7 days after 80 �g PEG-Intron (Table4), no significant differences were observed in either serumneopterin (p � 0.38) or serum 2�,5�-OAS (p � 0.16).

Linear regression analysis of neopterin and 2�,5�-OAS AUCas a function of Locteron dose is displayed in Figure 6. Overthe tested dose range of 20–320 �g, there was evidence of alinear relationship between administered Locteron and AUC ofboth neopterin and 2�,5�-OAS (p � 0.001 and p � 0.02, re-spectively, for the hypothesis of zero slope). From the regres-sion, a predicted mean Locteron dose of 118 �g would be re-quired to produce a neopterin elevation equaling that by 80 �gPEG-Intron, whereas a dose of 87 �g Locteron would matchthe 2�,5�-OAS response to 80 �g PEG-Intron. Thus, at an 80-�g dose, the effects of Locteron and PEG-Intron on the bio-markers were in a comparable range.

Tolerability

All doses of control PolyActive microspheres not containingIFN-�2b were well tolerated. The only treatment-emergent ad-verse event after PolyActive injection was headache in 1 sub-ject.

The most common adverse events of Locteron at the high-est dose (320 �g) were influenza-like illness, injection site re-

DE LEEDE ET AL.118

TABLE 3. AUC AND CMAX OF LOCTERON AND PEG-INTRON

AUC (mM � h), mean (SD) Cmax (nM), mean (SD)

Neopterin 2�,5�-OAS� Neopterin 2�,5�-OAS�

Locteron20 �g 4.09 (0.68) 0.60 (0.24) 11.7 (1.7) 1.66 (0.75)80 �g 4.36 (0.19) 1.00 (0.16) 15.0 (2.4) 2.84 (0.69)

320 �g 6.61 (0.74) 1.18 (0.32) 24.9 (3.0) 3.92 (1.59)80 �g PEG-Intron 4.83 (0.61) 0.84 (0.24) 26.3 (2.4) 3.25 (1.00)

FIG. 5. Serum concentration of 2�,5�-OAS and neopterin upto 28 days after administration of 20 �g Locteron, 80 �g Loc-teron, 320 �g Locteron, or 80 �g PEG-Intron. Group sizes were4 subjects at each Locteron dose and 6 for 80 �g PEG-Intron.Error bars show SD.

A

B

action, and headache. In no case did an adverse event promptstudy withdrawal. Episodes of influenza-like illness, which oc-curred exclusively within the first 120 h after treatment, aresummarized in Figure 7. No subject treated with 20 or 80 �gLocteron developed influenza-like illness. Three of the 4 sub-jects receiving 320 �g Locteron experienced influenza-like ill-ness. Symptoms were mild in 1 of these 3 subjects and mod-erate in the other 2. All subjects in the PEG-Intron groupdeveloped influenza-like illness, which was severe in 2 cases.In 1 of these 2 severe cases, discharge had to be delayed by 1day as a result of the influenza-like illness. The median totalduration of influenza-like illness without regard to severity wasshorter in the recipients of 320 �g Locteron (16.5 h, range0.0–42.7 h) than the recipients of 80 �g PEG-Intron (40.5 h,range 14.0–75.8 h), and the median total dose of paracetamolto manage these adverse events was smaller after 320 �g Loc-

teron (1000 mg, range 0–2000 mg) than after 80 �g PEG-In-tron (1750 mg, range 0–5500 mg), although these differenceswere not statistically significant (p � 0.17 and p � 0.42, re-spectively).

Mild injection site reactions were recorded in 1 of 4, 2 of 4,and 1 of 6 subjects receiving 80 and 320 �g Locteron and 80�g PEG-Intron, respectively. Erectile dysfunction in 1 subjecttreated with 320 �g Locteron was the only adverse event clas-sified as severe among all Locteron recipients. This adverseevent resolved within 5 days and may have been precipitatedby sleep deprivation and stress.

Circulating concentrations of leukocytes, neutrophils, hemo-globin, and platelets after administration of 320 �g Locteronand 80 �g PEG-Intron are shown in Figure 8. In the PEG-In-tron group, mean neutrophil concentration fell 63% after treat-ment to a nadir at 3 days, thereafter slowly rebounding over therest of the 28-day observation period toward the baseline level.At the 3 day nadir, neutrophil level had fallen below 1000/�Lin 2 of 5 subjects (40%), with available data for that time point.Following treatment with 320 �g Locteron, serum neutrophilsdeclined gradually by 59% to a minimum at 14 days and sub-sequently increased. At 14 days, the neutrophil level exceeded1000/�L in all 3 subjects of the 320-�g Locteron group, withavailable data.

Patterns of fluctuation in serum leukocytes were compara-ble to those in neutrophils. Temporal changes in hemoglobinand platelets were relatively small and generally similar be-tween the two groups.

No subject receiving Locteron displayed a positive antibodyresponse against IFN or Lemna proteins by the criterion of asustained 4-fold antibody level increase from baseline. In 1 re-cipient of 320 �g Locteron, a single antibody value was ele-vated 4-fold by screening anti-IFN-binding antibody assay;however, neutralizing antibody titers determined on samplesfrom this subject indicated the absence of neutralizing anti-bodies.

DISCUSSION

Chronic hepatitis C infection requires a complicated andlengthy treatment course. Currently, PEG-IFN-� combinedwith ribavirin is the most effective available treatment. Adher-ence to therapy, in both duration of treatment and maintenanceof the prescribed dose, is key to achieving SVR (Manns 2004).Adherence is of particular importance among patients infectedwith HCV genotype 1, the most common type and most diffi-cult to treat (McHutchison and others 2002). A more conve-

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TABLE 4. PERSISTENCE OF LOCTERON AND PEG-INTRON EFFECTS

Mean % of Cmax, baseline-adjusted

Time 320 �g Locteron 80 �g PEG-Intron DifferenceBiomarker (days) (SD) (SD) (CI)

Neopterin 7 62 (10) 20 (10) 43 (27–58)14 14 (5) 0 (4) 14 (8–21)

2�,5�-OAS� 7 87 (22) 65 (20) 22 (�9–53)14 47 (14) 19 (12) 27 (8–46)

FIG. 6. Dose proportionality of Locteron in relation to AUCof neopterin and 2�,5�-OAS based on linear regression. Datapoints depict individual subject AUC estimates from pharma-cokinetic analysis. Dashed lines indicate 95% CIs of the re-gressions, and dotted lines indicate 95% prediction intervals fornew observations. Shaded rectangles display means for 80 �gPEG-Intron, and error bars indicate SD. Group sizes were 4subjects at each Locteron dose and 6 for 80 �g PEG-Intron.

A

B

nient and better-tolerated dosage regimen, such as one requir-ing subcutaneous administration only once every 2 weeks in-stead of each week, could promote adherence over the manymonths of required therapy.

Controlled release of free IFN differs from the approachesof pegylation or fusion protein engineering that modify the IFNmolecule and work primarily by slowing clearance (Harris andothers 2001; Bain and others 2006). A possible advantage ofthis controlled-release strategy is that the delivered agent,namely, free IFN, is an endogenous molecule with already well-characterized effects. Continuously released free IFN might alsooffer tolerability advantages. For instance, dose modificationprompted by adverse events has been reported in a higher pro-portion of patients receiving PEG-Intron (42%) than unmodi-fied IFN (34%) (Fried 2002). The present study evaluated abiodegradable and biocompatible polymeric drug delivery sys-tem allowing tight control of release rate (van Dijkhuizen-

Radersma and others 2002, 2003, 2005). Other technologieshave also been investigated for sustained release of IFN-�, in-cluding lipid encapsulation (Bonetti and Kim 1993), polyglyc-erol esters of fatty acids (Yamagata and others 2000), poly-DL-lactide-poly(ethylene glycol) microspheres (Zhou and others2002), and implantation of triglyceride matrices (Mohl andWinter 2004).

The present phase I clinical trial results provide encourage-ment that Locteron can satisfy the performance criteria for freeIFN-�2b therapy with reduced dosing frequency. Elevation ofcirculating IFN-�2b was maintained far longer after a singlesubcutaneous dose of 320 �g Locteron than 80 �g PEG-Intron(Fig. 4). After 7 days, 58% of peak Locteron concentration per-sisted, whereas PEG-Intron was no longer detectable. The es-timated t1/2el of Locteron (85.1 h), signifying the net disap-pearance of IFN-�2b from the combined contributions ofsystemic clearance and replenishment by additional molecules

DE LEEDE ET AL.120

FIG. 7. Influenza-like illness in individual subjects duringthe first 120 h after administration of Locteron (n � 4 at eachdose) or PEG-Intron (n � 6). No subject experienced in-fluenza-like illness subsequent to 120 h. At no time did in-fluenza-like illness develop in any recipient of either 20 or 80�g Locteron.

FIG. 8. Mean circulating concentrations of leukocytes, neu-trophils, hemoglobin, and platelets after administration of 320�g Locteron or 80 �g PEG-Intron. Group sizes were 4 subjectstaking the 320-�g Locteron dose and 6 subjects taking 80 �gPEG-Intron. Error bars show SD.

A

B

C

D

entering the circulation from the microspheres, was more thantwice as long as the t1/2el of PEG-Intron (37.8 h), reflectingclearance only. The estimated t1/2el of PEG-Intron in the pres-ent trial of healthy subjects was similar to that reported in pa-tients with chronic hepatitis C infection receiving PEG-Intronmonotherapy (Glue and others 2000a). After Locteron admin-istration, IFN-�2b remained measurable through 14 days (Fig.4). This observation clearly suggests that Locteron may provesuitable for dosing once every 2 weeks, thereby promoting ad-herence. There is also the possibility of a further benefit relatedto antiviral effectiveness. As demonstrated in Figure 4, recipi-ents of once weekly PEG-Intron experience a serum IFN nadirevery 7 days, compared with every 14 days after Locteron treat-ment. There is empirical evidence indicating that reducing theoccurrence of IFN nadirs may improve the likelihood of SVR,which a sizeable percentage of patients fail to achieve undercurrent IFN therapy regimens. In a study of patients withchronic hepatitis C genotype 1, initial viral clearance was in-creased by twice vs. once weekly dosing of PEG-IFN (Formannand others 2003).

Locteron also exhibited more predictable pharmacokineticsthan PEG-Intron in this trial. The observed CVAUC of PEG-In-tron was 39% (Fig. 4), a value in close agreement with that inanother recent study (37%) (Bruno and others 2005). In con-trast, the CVAUC of Locteron was 10%.

In pharmacodynamic assessments using the IFN-inducibleenzymes neopterin and 2�,5�-OAS as biomarkers for IFN ac-tivity, Locteron produced a linear dose-response in both bio-markers over the tested range of 20–320 �g (Fig. 6). Impor-tantly, after a 320-�g Locteron dose, 14% of peak neopterin inserum and 47% of peak 2�,5�-OAS persisted after 14 days, fur-ther substantiating the potential feasibility of dosing once every2 weeks.

In the tested dose range, Locteron was well tolerated. At an80-�g dose, the pharmacodynamic effects of Locteron weresimilar to those of PEG-Intron (Fig. 6). However, at this dose,influenza-like illness developed in all volunteers treated withPEG-Intron but none of those receiving Locteron.

The nadir in circulating neutrophils occurred later (14 days)in the 320-�g Locteron group, possibly in reflection of thelonger duration of action of this agent compared with PEG-In-tron. Neutrophil levels were less strongly depressed among 320-�g Locteron recipients, none of whom experienced a fall inneutrophils below 1000/�L, as did 2 of 5 PEG-Intron-treatedsubjects with available data. PEG-IFN has been associated witha higher risk of neutropenia compared with standard IFN ther-apy (Kowdley 2005).

The IFN-�2b component of Locteron (BLX-883) is producedin a Lemna aquatic plant expression system. In this report arepresented the first in vivo data directly comparing BLX-883with Intron A, an IFN-�2b preparation synthesized in E. coli.In healthy human subjects, temporal changes in serum IFN-�2band neopterin after 3 � 106 IU BLX-883 were comparable tothose following the same dose of Intron A (Figs. 1 and 2). Fur-thermore, a dose-response relationship was apparent betweenadministered BLX-883 and attained levels of IFN-�2b andneopterin (Figs. 1 and 2). At the same doses (3 � 106 IU), theadverse event profiles of BLX-883 and Intron A were compa-rable (Table 2).

In the present phase I study of healthy human subjects, Loc-teron administration caused prolonged elevations of serum IFN-�2b and biomarkers, suggesting suitability for less frequent dos-ing than that of PEG-Intron. The tolerability profile of Locteronwas favorable. On the basis of these observations, further clin-ical trials of Locteron for treatment of hepatitis C infection arewarranted.

ACKNOWLEDGMENTS

Portions of the research described in this report were carriedout with the collaboration of Xendo Drug Development, Gronin-gen, the Netherlands; Inveresk Research International Ltd, Tra-nent, Scotland, U.K.; and Afforce Healthcare B.V., the Hague,the Netherlands. The guidance of the Biolex Development Ad-visory Board is gratefully acknowledged. We thank Jeroen Beze-mer, Ph.D., for his assistance in the development of Locteron.Mahlon M. Wilkes, Ph.D., contributed to the statistical analysisof the study data and preparation of the manuscript.

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Address reprint requests or correspondence to:Dr. Leo G.J. de Leede

Director Preclinical R&DOctoPlus N.V.

Zernikedreef 122333 CL

LeidenThe Netherlands

Tel: 31 71 5244044Fax: 31 71 5244043

E-mail: deleede@octoplus.nl

Received 27 June 2007/Accepted 13 September 2007

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