Multiple Sclerosis

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Safety and immunogenicity of a new formulation of interferon -1a (Rebif® New Formulation) in a
Phase IIIb study in patients with relapsing multiple sclerosis: 96-week results
G Giovannoni, O Barbarash, F Casset-Semanaz, J King, L Metz, G Pardo, J Simsarian, PS Sørensen, B Stubinski and on
behalf of the Rebif® New Formulation Study Group
Mult Scler 2009 15: 219 originally published online 28 August 2008
DOI: 10.1177/1352458508097299
The online version of this article can be found at:
http://msj.sagepub.com/content/15/2/219

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RESEARCH PAPER Multiple Sclerosis 2009; 15: 219–228

Safety and immunogenicity of a new formulation of

interferon β-1a (Rebif® New Formulation) in a Phase IIIb
study in patients with relapsing multiple sclerosis:
96-week results
G Giovannoni1, O Barbarash2, F Casset-Semanaz3, J King4, L Metz5, G Pardo6, J Simsarian7,
PS Sørensen8 and B Stubinski3; on behalf of the Rebif® New Formulation Study Group

Background A new formulation of subcutaneous (s.c.) interferon-β-1a has been developed (Rebif®
New Formulation, RNF), produced without fetal bovine serum and without human serum albumin as
an excipient, with the aim of improving injection tolerability, and reducing immunogenicity.
Objectives This article reports 96-week analyses of a Phase IIIb, open-label study of the safety and
immunogenicity of RNF compared with historical (EVIDENCE study) and recent (REGARD study)
data on the original formulation.
Methods Patients with relapsing multiple sclerosis (McDonald criteria) and an Expanded Disability
Status Scale score < 6.0 received RNF, 44 μg s.c. three times weekly.
Results The proportion of neutralizing antibody-positive (NAb+) patients (serum NAb status ≥20
neutralizing units/mL) at week 96 (last observation carried forward; primary endpoint) was 17.4%
(exact 95% confidence interval [CI]: 13.0–22.5), compared with 21.4% (95% CI: 17.2–26.2) in the
EVIDENCE study, and 27.3% (95% CI: 22.8–32.1) in the REGARD study. The proportion of patients
NAb+ at any time during the 96 weeks was 18.9% (95% CI: 14.4–24.2), compared with 27.1% (95%
CI: 22.4–32.2) and 33.7% (95% CI: 28.9–38.7), respectively. Most pre-specified categories of
adverse events were reported by patients in the RNF study at a similar or lower proportion than in
the EVIDENCE and REGARD studies. Injection-site reactions were experienced by fewer patients
than in the EVIDENCE and REGARD studies.
Conclusions RNF has improved overall immunogenicity and safety profiles compared with the origi-
nal formulation. Multiple Sclerosis 2009; 15: 219–228. http://msj.sagepub.com

Key words: immunogenicity; injection-site reactions; interferon-β-1a; multiple sclerosis; Rebif® New
Formulation; safety

Introduction with interferon (IFN)-β in patients with multiple sclerosis

(MS) can be associated with the develop-ment of
As has been observed with other therapeutically neutralizing antibodies (NAbs) [1–4]. Although the full
administered recombinant proteins, treatment clinical impact of NAbs is still to

1Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, London, UK
2State Educational Institute of Higher Professional Education, Kemerovo State Medical Academy, Kemerovo, Russia
3
Merck Serono International S.A.*, Geneva, Switzerland
4Department of Neurology, Royal Melbourne Hospital, Melbourne, Australia
5Department of Clinical Neurosciences, University of Calgary, Calgary, Alberta, Canada
6MS Center of Oklahoma, Mercy NeuroScience Institute, Oklahoma City, Oklahoma, USA
7
Neurology Center of Fairfax, Hamaker Ct Ste 400, Fairfax, Virginia, USA
8Department of Neurology, Danish MS Research Center, Copenhagen University Hospital, Rigshospitalet,
Copenhagen, Denmark
*An affiliate of Merck KGaA, Darmstadt, Germany
Correspondence to: Gavin Giovannoni, Institute of Cell and Molecular Science, Barts and the London School of
Medicine and Dentistry, London EC1A 7BE, UK. Email:
g.giovannoni@qmul.ac.uk Received 5 June 2008; accepted 6 August 2008

© SAGE Publications 2009 10.1177/1352458508097299

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220 G Giovannoni et al.
be determined, the presence of persistently high titers of
NAbs to IFN-β reduces the therapeutic effect of treatment
[5–7]. The immunogenicity of thera-peutic proteins may
also be influenced by a variety of other factors, including
formulation [8,9].
Therapeutic benefit can also be compromised by lack of
patient adherence to therapy. Injection-site reactions are
among the most frequently reported local-tissue adverse
events (AEs) associated with subcutaneous (s.c.)
administration of IFN-β in MS [10,11].
A new formulation of s.c. IFN-β-1a (Rebif®) has been
developed (Rebif® New Formulation, RNF) that is
produced without fetal bovine serum (FBS) and without
human serum albumin (HSA) as an excipient, with the aim
of improving injection tol-erability and reducing
immunogenicity. The safety and immunogenicity of RNF
have been evaluated in a 96-week, single-arm, Phase IIIb,
open-label study (the RNF study). Results of planned 24-
and 48-week interim analyses have been reported
previously [12]. This article reports the final results of
analyses conducted on 96-week data. To evaluate the
safety and immunogenicity of RNF, data were compared
descriptively with historical and recent IFN-β-1a data
reported in the EVidence of Interferon Dose– response:
European North American Comparative Efficacy
(EVIDENCE) study and the REbif® versus Glatiramer
Acetate in Relapsing MS Disease (REGARD) study [13–
16]. The EVIDENCE study was used for comparison
because it was the most recent, large, completed clinical
study of the previ-ous s.c. IFN-β-1a HSA-containing
formulation when the RNF study was designed. It also
used procedures for reporting and recording AEs that were
similar to the RNF study, and, furthermore, was the basis
of the sample size estimations for the RNF study. The
REGARD study is now the most recent, large clinical
study of s.c. IFN-β-1a, and was used for comparison
because it was conducted in parallel with the RNF study,
had the same duration, used identical meth-ods, and,
importantly, had the same laboratory to test for binding
antibodies (BAbs) and NAbs. This is important because
the development of NAbs and NAb titers has been shown
to vary according to the method used to analyze them.
Methods
Study design
The RNF study was carried out from January 2005 to June
2007. The design of this study has been described in detail
previously [12]. In brief, this was a 96-week, single-arm,
Phase IIIb, open-label study (protocol 25632; US National
Institutes of
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Health identifier: NCT00110396; European Clinical Trials
Database number: 2004-003799-13) con-ducted at 47
centers in 12 countries to evaluate the safety and
immunogenicity profiles of RNF, 44 μg s.c. three times
weekly (t.i.w.), in patients with relapsing forms of MS.
The primary objective of the study was to com-pare the
immunogenicity of RNF with historical data for the
previous formulation of IFN-β-1a, 44 μg s.c. t.i.w.
Secondary objectives included examining the time course
of NAb development in patients with relapsing forms of
MS newly exposed to RNF, determining the overall safety
profile of RNF com-pared with the previous Rebif®
formulation, describing changes in pharmacodynamic
markers, and assessing patients’ clinical status (Expanded
Disability Status Scale [EDSS] score, relapses) throughout
the study.
Patients and interventions
Eligible patients were aged 18–60, IFN-β-naive, with an
EDSS score <6.0, and a diagnosis of relapsing MS
according to McDonald criteria [17,18]. No other approved
disease-modifying drug for MS or any cytokine or anti-
cytokine therapy could be used within the 3 months before
the study, and no immunomodulatory or
immunosuppressive ther-apy could be used within the 12
months before the study. Further exclusion criteria have
been described previously [19].
Patients self-injected RNF, 44 μg s.c. t.i.w., after a
standard dose-titration period of 4 weeks (in accor-dance
with the labeling for Rebif®).
Assessments
Blood samples were collected at baseline and at weeks 12,
24, 36, 48, 60, 72, and 96 for analysis of BAbs and NAbs
(using an enzyme-linked immuno-sorbant assay and
cytopathic-effect assay, respec-tively). Details of these
methods have been described previously [19]. AEs were
recorded up to the last assessment. Laboratory assessments
were conducted at baseline, study day 1, and weeks 4, 12,
24, 48, 72, and 96.
The safety population was defined as all patients who
received at least one dose of trial medication. The
intention-to-treat (ITT) population was defined as all
patients who received at least one dose of trial medication
and had at least one post-baseline NAb assessment.
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 Safety and immunogenicity of Rebif® New Formulation
Statistical methods
The primary endpoint of the study was the propor-tion of
patients who were NAb-positive (NAb+) at week 96, last
observation carried forward (LOCF).
Secondary immunogenicity endpoints included the
proportion of patients who were NAb+ at any post-baseline
visit up to week 96 (‘anytime’ positive). Safety was also a
secondary endpoint, evaluated by monitoring AEs, serious
AEs (SAEs), and laboratory parameters. Pre-specified
categories of AEs that were identified as being commonly
or occasionally associ-ated with IFN-β-1a administration
were summarized and analyzed by combining Medical
Dictionary for Regulatory Activities-preferred terms and
have been described previously [12]. Another secondary
end-point was the percent change in pharmacodynamic
markers (neopterin and β-2-microglobulin) over time up to
week 96.
Secondary efficacy endpoints included the pro-portion
of patients free from relapse, relapse rates, and mean
change from baseline in EDSS score. Effi-cacy was
assessed as an exploratory analysis by recording the
number of relapses and EDSS score. Relapses before the
study were assessed retrospec-tively on an MS relapse
history report. Relapses dur-ing the study were collected
on an MS relapse report. Within patient differences in the
number of relapses between time periods were calculated
to compare the proportions of patients who had fewer,
equal, or more relapses during the study period compared
with 2 years before study entry. The sign test was used to
test the hypothesis that patients had an equal number of
relapses in both periods.
Immunogenicity and safety results were com-pared
with data from patients who received the previous
formulation of IFN-β-1a, 44 μg s.c. t.i.w., in the
EVIDENCE and REGARD studies [14,15]. Comparisons
with historical data were descriptive, based on point
estimators and corresponding 95% exact confidence
intervals (CI), with no formal hypothesis testing
performed. The 96-week data from the EVIDENCE study
are based on patients who received IFN-β-1a treatment up
to 96 weeks and extrapolation of data for the remaining
patients. Post-hoc sensitivity analysis on the pri-mary
endpoint was performed using an alternative imputation
method to LOCF to assess the impact of using LOCF
imputation on the primary endpoint. To conduct the
alternative imputation, transition probabilities for NAb
status at each time point were calculated using the
available immunogenicity data in each study. Simulations
were conducted (1000 times each) applying these
transition proba-bilities. From these simulations, the
proportion of NAb+ patients at week 96 was estimated.
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221
In addition, a post-hoc meta-analysis approach was
used to obtain pooled estimates of the propor-tion of NAb+
patients at week-96 LOCF and the proportion of anytime
NAb+ patients (and corre-sponding 95% CI) for IFN-β-1a
treatment in the EVIDENCE and REGARD studies. Fixed-
effects mod-els with the proportions as the response
variable were applied.
Comparator studies
The EVIDENCE and REGARD studies were both pro-
spective, randomized, and assessor-blinded compar-ative
trials in patients with relapsing–remitting MS (RRMS): the
EVIDENCE study compared s.c. IFN-β-1a, 44 μg t.i.w.,
with intramuscular IFN-β-1a, 30 μg once weekly, over 48
weeks, and the REGARD study compared s.c. IFN-β-1a,
44 μg t.i.w., with s.c. glatiramer acetate, 20 mg once daily,
over 96 weeks.
Results
Patients
All 260 patients enrolled were included in the safety
population and 259 patients were included in the ITT
population (one patient did not have a post-baseline NAb
assessment). Of the 260 patients who enrolled in the study,
207 (79.6%) completed the study on treatment and 224
(86.2%) completed the study regardless of treatment
completion (Figure 1).
A total of 31 patients (11.9%) withdrew from treatment
because of AEs. The remaining withdra-wals were due to
protocol violation (two patients, 0.8%), lack of efficacy
(two patients, 0.8%), being lost to follow-up (one patient,
0.4%), pregnancy (three patients, 1.2%), or other reasons
(14 patients, 5.4%). Patient demographics and baseline
disease characteristics were largely similar between the
studies (Table 1). The median duration of MS was slightly
longer in the RNF study (5.5 years) com-pared with the
EVIDENCE (4.0 years) and REGARD (3.7 years) studies.
Both the RNF and REGARD studies used the
McDonald criteria for diagnosis of relapsing MS at
inclusion, whereas the Poser criteria were used in the
EVIDENCE study. The RNF study had broader inclusion
criteria (relapsing forms of MS) than the REGARD and
EVIDENCE studies (RRMS). Therefore, six patients with
secondary progressive MS with superimposed relapses and
one patient with pro-gressive relapsing MS were also
included in the RNF study.
The median (range) time on treatment was 95.7
(3.6–97.3) weeks. Patients received a median (range)
Multiple Sclerosis 2009; 15: 219–228
222 G Giovannoni et al.

patients in the REGARD study. At week 96, using LOCF

for missing data imputation, the proportion of patients who
were NAb+ was 17.4% (45/259; exact 95% CI: 13.0–22.5;
primary endpoint) com-pared with 21.4% (72/336; 95%
CI: 17.2–26.2) in the EVIDENCE study and 27.3%
(102/374; 95% CI: 22.8–32.1) in the REGARD study
(Figure 2).
A pooled estimate of the proportion of NAb+ patients at
week-96 LOCF in the EVIDENCE and REGARD studies
combined was 24.3% (95% CI: 21.1–27.4) (Figure 2).

An a posteriori sensitivity analysis was performed to

Figure 1 Patient disposition. One patient did not have a
post-baseline neutralizing antibody assessment and so was
not included in the intention-to-treat (ITT) population. aIn-
cluded: refusal to participate, withdrawal of consent,
patient’s decision. bIncluded: administration of plasmophor-
esis, travel, initiation of treatment with mitoxantrone,
patient’s decision, pregnancy, planned participation in
another study.
of 285 (11–291) injections of study medication. The
median (range) total cumulative dose was 12,196.8
(172.0–12,531.0) μg.
Immunogenicity
By week 96, 28.6% (74/259) of patients developed BAbs
compared with 36.9% (124/336) of patients in the
EVIDENCE study and 37.7% (141/374) of
assess the impact of using LOCF imputation on the
primary endpoint given that in the RNF and EVIDENCE
studies the proportion of patients who were missing a
week-96 assessment differed (13.5% vs 42.3%,
respectively; the proportion of patients who did not have a
week-96 assessment in the REGARD study was 18.2%).
The higher proportion of patients in the EVIDENCE study
without a week-96 assessment is due to the original trial
design being for 48 weeks. At the end of the comparative
phase of the study (maximum 64 weeks of treat-ment),
patients could enter an open-label extension phase with
IFN-β-1a, 44 μg s.c. t.i.w. The a posteriori sensitivity
analysis used an imputation method for missing data that
was different from LOCF, but sup-ported the findings from
the LOCF method: the proportion of NAb+ patients at
week 96 was 18.9% in the RNF study compared with
23.9% in the EVI-DENCE study and 28.3% in the
REGARD study.
The proportion of patients who were anytime NAb+
during the 96 weeks was 18.9% (49/259; 95% CI: 14.4–
24.2), compared with 27.1% (91/336; 95% CI: 22.4–32.2)
of patients in the EVI-DENCE study and 33.7% (126/374;
95% CI: 28.9–38.7) of patients in the REGARD study. A
pooled estimate of the proportion of anytime NAb+
patients in the EVIDENCE and REGARD studies
combined was 30.4% (95% CI: 27.0–33.7).

Table 1 Baseline patient demographics and disease characteristics in the Rebif ® New Formulation (RNF), EVIDENCE and REGARD
studies (intention-to-treat populations)

RNF (n = 260) EVIDENCE (n = 339) REGARD (n = 386)

Median age, years (range) 34.0 (18–58) 39.0 (18–55) 36.0 (17–60)
Women, n (%) 186 (71.5) 254 (74.9) 267 (69.2)
Race, n (%)
White 253 (97.3) 313 (92.3) 360 (93.3)
Black 3 (1.2) 13 (3.8) 16 (4.1)
Other 4 (1.5) 13 (3.8) 10 (2.6)
Median duration of MS, years (range) 5.5 (0.2–33.2) 4.0 (0.4–37.5) 3.7 (0.2–41.5)
Median number of relapses within 1 year 1 (0–4) 2 (0–5) 1 (0–5)
before study (range)
Median EDSS score at baseline (range) 2.0 (0–5.5) 2.0 (0–5.5) 2.0 (0–5.5)

EDSS, Expanded Disability Status Scale.

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 Safety and immunogenicity of Rebif® New Formulation 223

Figure 2 Proportion of neutralizing antibody-positive (NAb+) patients (titer >20 neutralizing units/mL; exact 95% confi-dence
interval) at week-96 last observation carried forward in the REGARD, EVIDENCE, and Rebif ® New Formulation (RNF)
studies.

The majority of patients in the LOCF analysis Safety

experienced NAbs at more than one visit; during 96 weeks,
8.5% (4/47; 95% CI: 2.4–20.4) of patients who were
anytime NAb+ up to week 72 in the RNF study
seroreverted compared with 22.7% (20/88; 95% CI: 14.5–
32.9) in the EVIDENCE study and 19.8% (24/121; 95%
CI: 13.1–28.1) in the REGARD study. The proportion of
patients with NAb titers ≥1000 neutralizing units (NU)/mL
at week 96 was 8.1% (95% CI: 5.1–12.1) in the RNF
study, 9.8% (95% CI: 6.9–13.5) in the EVIDENCE study,
and 13.1% (95% CI: 9.9–17.0) in the REGARD study. The
proportion of NAb+ patients with NAb titers <200 NU/mL
at week 96 was 28.9% (95% CI:
16.4–44.3) in the RNF study, 27.8% (95% CI:
17.9–39.6) in the EVIDENCE study, and 22.5%
(95% CI: 14.9–31.9) in the REGARD study.
A total of 226/260 patients (86.9%) reported at least one of
the pre-specified AE categories of interest (Table 2). The
proportion of patients experiencing injection-site reactions
was approximately threefold lower than in the EVIDENCE
study and also lower than in the REGARD study. The
incidence of flu-like symptoms was higher in the RNF
study. There was considerable heterogeneity in the
proportions of patients who reported flu-like symptoms by
coun-try. Compared with the EVIDENCE and REGARD
studies, a similar or lower proportion of patients in the
RNF study reported hepatic disorders, depres-sion and
suicidal ideation, skin rashes, hypersensi-tivity reactions or
thyroid disorders. Cytopenia was reported in a similar
proportion of patients to the

Table 2 Pre-specified groups of adverse events (AEs) at week 96 in the Rebif® New Formulation (RNF), EVIDENCE, and REGARD
studies

Incidence, n (%) of patients

RNF (n = 260) EVIDENCE (n = 339) REGARD (n = 381)
AE group of interest 226 (86.9) 323 (95.3) 264 (69.3)
Injection-site reactionsa 80 (30.8) 291 (85.8) 157 (41.2)
Flu-like symptomsb 186 (71.5) 166 (49.0) 137 (36.0)
Hepatic disorders 37 (14.2) 63 (18.6) 38 (10.0)
Cytopenia 35 (13.5) 44 (13.0) 29 (7.6)
Depression and suicidal ideation 17 (6.5) 77 (22.7) 36 (9.4)
Skin rashes 16 (6.2) 56 (16.5) 28 (7.3)
Hypersensitivity reactions 15 (5.8) 19 (5.6) 18 (4.7)
Thyroid disorders 11 (4.2) 25 (7.4) 11 (2.9)
aInjection-site reactions comprised injection-site desquamation, erythema, hemorrhage, induration, inflammation, irritation, necrosis,
edema, pain, pruritus, rash, reaction, and swelling.
bFlu-like symptoms included all reported events of ‘influenza-like illness’ as well as at least two pre-specified preferred terms repre-
senting typical flu-like symptoms (e.g., headache, chills, myalgia, arthralgia, body temperature increase, pyrexia, fatigue, and
malaise) that occurred within a 48-h interval.

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224 G Giovannoni et al.

Table 3 Most frequent treatment-emergent adverse events (AEs; preferred term, occurring in >10% of patients in total in the Rebif ®
New Formulation [RNF] study) at week 96 in the RNF study (by severity) and incidence of these AEs in the EVIDENCE and
REGARD studies

Incidence, n (%) of patients

RNF study (n = 260) EVIDENCE study REGARD study
(n = 339) (n = 381)

Milda Moderatea Severea Totalb Totalb Totalb

Any AE 225 (86.5) 158 (60.8) 37 (14.2) 247 (95.0) 338 (99.7) 346 (90.8)
Influenza-like illness 129 (49.6) 64 (24.6) 7 (2.7) 176 (67.7) 153 (45.1) 119 (31.2)
Headache 74 (28.5) 33 (12.7) 6 (2.3) 98 (37.7) 141 (41.6) 74 (19.4)
Injection-site erythema 60 (23.1) 3 (1.2) 0 (0.0) 63 (24.2) 158 (46.6) 121 (31.8)
a
A patient may report the same AE under more than one category of severity.
b
The total refers to the proportion of patients with AEs regardless of severity.

EVIDENCE study, but a higher proportion of patients than
in the REGARD study.
A total of 247/260 patients (95.0%) reported at least
one treatment-emergent AE (TEAE) (Table 3). The most
frequent TEAEs (by preferred term, reported by >10% of
patients) were influenza-like illness, headache, and
injection-site erythema. The majority of influenza-like
illness was mild (49.6%) and transient (median duration of
24 weeks using a worst-case approach to estimation of
duration). At the week-96 visit, 8.5% of patients were
experienc-ing flu-like symptoms. The incidence of
injection-site erythema at week 96 was 24.2% and in most
patients (23.1%) was of mild severity. In compari-son, the
incidence of injection-site erythema was 46.6% in the
EVIDENCE study and 31.8% in the REGARD study. The
majority of patients experienc-ing AEs reported events of
mild or moderate severity (Table 3). A total of 15 patients
(5.8%) reported 20 SAEs. Only one preferred term was
reported as serious in more than one patient (depression:
three events in three patients). In comparison, the
proportion of patients reporting SAEs in the EVI-DENCE
and REGARD studies was 8.6% and 7.6%, respectively. A
total of 31 patients (11.9%) experi-enced AEs that led to
permanent discontinuation of treatment, compared with
5.6% in the EVI-DENCE studya and 8.1% in the
REGARD study. No deaths were reported during the
study.
There were no unexpected findings on laboratory
assessments of liver enzymes or hematology. The majority
of cytopenias and elevations in liver enzymes that were
recorded were of low toxicity grade (based on the common
terminology criteria for AEs). In terms of liver enzymes,
grade 3 toxicities were recorded for serum glutamic
oxaloacetic transaminase (aspartate aminotransferase)
(seven patients, 2.7%) and for serum glutamic pyruvic
trans-
a
The low rate in the EVIDENCE study could have been biased by
those patients who did not continue from the comparative phase
into the extension phase.
aminase (alanine aminotransferase) (11 patients, 4.2%).
One grade 4 toxicity was recorded for each of these
variables (one patient, 0.4%, for each; the same patient). In
terms of hematology variables, grade 3 toxicities were
recorded for hemoglobin (two patients, 0.8%), white-cell
count (three patients, 1.2%), lymphocyte count (two
patients, 0.8%), and neutrophil count (22 patients, 8.5%).
Grade 4 toxic-ity was reported for neutrophil count only
(six patients, 2.3%).
Neopterin and β-2-microglobulin levels over 96 weeks
indicated a sustained pharmacodynamic response. Median
neopterin levels were 6.6 nmol/L at baseline and 10.1
nmol/L at week 96. Median β-2-microglobulin levels were
1.5 mg/L at baseline and 1.8 mg/L at week 96.
Efficacy
Relapses remained well controlled over the 96 weeks of
the study. The proportion of patients who were relapse free
at week 96 was 53.3% (95% CI: 47.0–59.5). The mean
number of relapses per patient (annualized) was lower
during the 96 weeks on study compared with during the 2
years before study entry (knowing that this comparison is
indirect as the information on relapses before informed
consent was collected retrospectively at baseline; Figure
3). Median EDSS score at week 96 (2.0; range: 0.0–6.5)
was unchanged from baseline (2.0; range: 0.0–5.5);
median change was 0.0 (range: −2.5 to 4.5).
Discussion
This 96-week, Phase IIIb, open-label, single-arm,
multicenter study evaluated the safety and immu-
nogenicity of RNF in patients with relapsing forms of MS.
On the basis of the proportion of patients developing NAbs
at week 96, results indicate that the overall immunogenic
profile of RNF is improved

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 Safety and immunogenicity of Rebif® New Formulation
Figure 3 Mean number of relapses per patient per year
before and during the Rebif® New Formulation study. aData
collected retrospectively.
compared with that of the previous formulation of IFN-β-
1a s.c. t.i.w., based on data from the EVI-DENCE and
REGARD studies. In the RNF study, 17.4% of patients
were NAb+ at week-96 LOCF (primary endpoint),
compared with 21.4% in the EVIDENCE study and 27.3%
in the REGARD study. Sensitivity analyses to address
missing values showed a similar trend in the data.
The proportion of patients who were anytime NAb+
also showed a trend towards being lower in the RNF study
than in the EVIDENCE and REGARD studies. The
proportion of patients with persistent NAbs (NAb+ at week
72 and week 96, no LOCF) was 17.0%.
High NAb titers impact the clinical efficacy of IFN-β
therapy [6,7]. The proportion of patients with high NAb
titers (>200 NU/mL) was similar to that in the EVIDENCE
study, but lower than that observed in the REGARD study.
Patients with high NAb titers are also less likely to
serorevert over the longer term [20,21]. The proportion of
patients with NAb titers ≥1000 NU/mL was lower in the
RNF study than in both of the historical studies. These
results support the data from interim analyses [12].
The results from this study and the trends observed
versus the comparator studies suggest that RNF has
reduced immunogenicity compared with the previous IFN-
β-1a s.c. t.i.w. formulation. This may be a consequence of
the removal of
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225
serum-derived components (HSA and FBS) from the
preparation. However, other factors can also influence the
immunogenicity of biopharmaceuti-cals, including protein
modifications, impurities or contaminants, patient
characteristics, and route of administration [8,9]. Some of
these factors (other than patient characteristics and route of
administra-tion) have also been refined in the production
process, and therefore the improvement in immu-
nogenicity cannot be attributed to one factor.
Development of NAbs may block the binding of IFN-β
to its receptor [3,22], thus preventing its bio-logical effects
and potentially reducing treatment efficacy [23,24].
Results from the EVIDENCE study, however, did not
show an effect of NAbs on relapse outcomes over 12
months [13]. Although IFN-β pre-parations may differ in
their ability to induce NAbs [25,26], and although it has
previously been sug-gested in Europe that patients with
persistently high NAb titers should be offered an
alternative ther-apy [5], it is recommended that treatment
decisions should still be made primarily on clinical
grounds and not on NAb status alone. No new safety con-
cerns were identified during the study, based on
comparison with previous trials using the HSA-containing
formulation of IFN-β-1a. Results showed notable
improvements to particular aspects of the safety and
tolerability profile typical of injectable s.c. IFN-β-1a. In
particular, patients were nearly three times less likely to
experience injection-site reactions compared with those in
the EVIDENCE study; injection-site reactions also
occurred in a lower proportion of patients than in the
REGARD study. As injection-site reactions are among the
most frequently reported local-tissue AEs associated with
s.c. administration of IFN-β [10,11], this improvement is
noteworthy and may result in improved patient satisfaction
with, and adherence to, treatment. Tolerability and side-
effects of MS medications are important factors in
treatment com-pliance and adherence [27,28]. There was
also a notable decrease in the proportion of patients report-
ing injection-site reactions in the REGARD study
compared with the EVIDENCE study, as well as between
the RNF study and both comparator studies. This is likely
to be due to a number of improvements in administration
and formulation over the years, such as the introduction of
a thinner gauge (29G/ 5-bevel) needle, an auto-injector
device, and the development of RNF. Patients have
reported less pain with the thinner needle (compared with
the previous 27G/3-bevel needle) [29], but a comparison of
RNF with the previous formulation, which used the same
29G/5-bevel needle design for all groups, found that RNF
was still associated with reduced application-site erythema
and improvements in local tolerability [30]. Thus, the
reduction in injection-site reactions in the RNF study is not
due
Multiple Sclerosis 2009; 15: 219–228
226 G Giovannoni et al.
simply to improvements in needle design. In the RNF
study, an auto-injector device was available as an optional
aid at all study sites. A study investi-gating the local
tolerability of IFN-β-1a s.c. t.i.w., however, showed that
injection-site reactions were experienced by 85.4% of
patients with manual injec-tions compared with 78.7% of
patients using the auto-injector [31]. Thus, the reduction in
injection-site reactions in the RNF study could only partly
be explained by the use of the auto-injector. Injection-site
reactions in the RNF study did still occur in more than
10% of patients. Nevertheless, the observed improvement
is favorable in terms of the benefit-to-risk profile of s.c.
IFN-β-1a therapy.
Depression and suicidal ideation were experi-enced by
fewer patients than in either comparator study.
Although the proportion of patients experienc-ing
cytopenia as an AE was higher compared with the
REGARD study, it was similar to that in the EVIDENCE
study. Furthermore, there were no unexpected findings on
laboratory assessments of hematology. The majority of
cytopenias that were recorded were of a low toxicity grade.
Flu-like symptoms were mostly mild in severity and
transient, occurring in 71.5% of patients. A similar
incidence was also seen with other HSA-free preparations
of IFN-β-1a (Avonex®, 88%) [32]. Flu-like symptoms can
be managed prophylactically or pro re nata with
concomitant medications (e.g., paracetamol and ibuprofen)
[33]. Indeed, the RNF study protocol stated that, at the
discretion of the physician, paracetamol or non-steroidal
anti-inflammatory drugs (NSAIDs) may be administered
prophylactically to ameliorate constitutional symp-toms
(e.g., fever, myalgia, and flu-like symptoms) as required
throughout the study. Only 38% of patients received
prophylactic anilides/NSAIDS on study day 1 before
initiation of IFN therapy. Increasing the proportion of
patients who receive such prophylactic treatment may
reduce the inci-dence of flu-like symptoms. There was
considerable heterogeneity by country in both the
proportion of patients who reported flu-like symptoms and
in the use of prophylactic anilides/NSAIDS at study entry.
The remaining pre-specified AE groups of interest
occurred in a similar proportion of patients in the RNF
study compared with the EVIDENCE and REGARD
studies. Overall, the favorable safety pro-file of RNF
observed at week 48 was maintained.
Although the study was not designed to assess efficacy,
RNF showed an apparent efficacy at week 96 in line with
what was expected based on results from other studies.
Limitations of the RNF study have also been described
previously [12]. The main limitation is that there was no
placebo control group. A regres-sion to the mean effect for
relapse outcomes should
Multiple Sclerosis 2009; 15: 219–228
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also be considered. The study was also open-label.
Comparing results between studies should always be done
with some degree of caution, but the stud-ies chosen for
comparison used the same treatment dose with a similar
population and similar schedule of assessments. The
objective nature of the primary endpoint (laboratory data,
all performed by the same laboratory) also increases the
validity of the comparisons.
On the basis of comparisons with historical and recent
data, the results of this 96-week study indi-cate that RNF
may offer the treatment benefits of high-dose, high-
frequency IFN-β-1a coupled with improvements in both
the overall safety and immu-nogenicity profiles compared
with the previous formulation.
Acknowledgments
This study was sponsored by Merck Serono Interna-tional
S.A., Geneva, Switzerland (an affiliate of Merck KGaA,
Darmstadt, Germany). The authors thank the many patients
and site personnel and the RNF clinical trial team at Merck
Serono Interna-tional S.A., which included the following
indivi-duals: E. Abgrall, S. Brunati, P. Cornelisse, C.
Filmer, S. Gerin, B. Granados, D. Issard, M. Lopez-
Bresnahan, P. Perrone, and J. Weiner. The authors also
thank Matthew Evans, PhD (supported by Merck Serono
International S.A., Geneva, Switzer-land), for assistance
with the preparation of the manuscript.
References
1. Durelli, L, Ricci, A. Anti-interferon antibodies in multiple
sclerosis. Molecular basis and their impact on clinical efficacy.
Front Biosci 2004; 9: 2192–2204.
2. Ross, C, Clemmesen, KM, Svenson, M, et al. Immunoge-nicity
of interferon-beta in multiple sclerosis patients: influence of
preparation, dosage, dose frequency, and route of administration.
Danish Multiple Sclerosis Study Group. Ann Neurol 2000; 48:
706–712.
3. Goodin, DS, Frohman, EM, Hurwitz, B, et al. Neutralizing
antibodies to interferon beta: assessment of their clinical and
radiographic impact: an evidence report. Report of the
Therapeutics and Technology Assessment Subcom-mittee of the
American Academy of Neurology. Neurology 2007; 68: 977–
984.
4. Sorensen, PS, Ross, C, Clemmesen, KM, et al. Clinical
importance of neutralising antibodies against interferon beta in
patients with relapsing-remitting multiple sclero-sis. Lancet
2003; 362: 1184–1191.
5. Sorensen, PS, Deisenhammer, F, Duda, P, et al. Guide-lines on
use of anti-IFN-beta antibody measurements in multiple
sclerosis: report of an EFNS Task Force on IFN-beta antibodies
in multiple sclerosis. Eur J Neurol 2005; 12: 817–827.
6. Perini, P, Calabrese, M, Biasi, G, Gallo, P. The clinical impact of
interferon beta antibodies in relapsing-remitting MS. J Neurol
2004; 251: 305–309.
http://msj.sagepub.com
 Safety and immunogenicity of Rebif® New Formulation
7. Petkau, AJ, White, RA, Ebers, GC, et al. Longitudinal analyses
of the effects of neutralizing antibodies on inter-feron beta-1b in
relapsing-remitting multiple sclerosis.
Mult Scler 2004; 10: 126–138.
8. Schellekens, H. The immunogenicity of biopharmaceuti-cals.
Neurology 2003; 61: S11–S12.
9. Schellekens, H, Casadevall, N. Immunogenicity of recombinant
human proteins: causes and consequences.
J Neurol 2004; 251(Suppl. 2): II4–II9.
10. Gottberg, K, Gardulf, A, Fredrikson, S. Interferon-beta treatment
for patients with multiple sclerosis: the patients’ perceptions of
the side-effects. Mult Scler 2000; 6: 349–354.
11. Walther, EU, Hohlfeld, R. Multiple sclerosis: side effects of
interferon beta therapy and their management. Neuro-logy 1999;
53: 1622–1627.
12. Giovannoni, G, Barbarash, O, Casset-Semanaz, F, et al.
Immunogenicity and tolerability of an investigational
formulation of interferon-beta1a: 24- and 48-week interim
analyses of a 2-year, single-arm, historically con-trolled, phase
IIIb study in adults with multiple sclerosis.
Clin Ther 2007; 29: 1128–1145.
13. Panitch, H, Goodin, D, Francis, G, et al. Benefits of high-dose,
high-frequency interferon beta-1a in relapsing-remitting multiple
sclerosis are sustained to 16 months: final comparative results of
the EVIDENCE trial. J Neurol Sci 2005; 239: 67–74.
14. Panitch, H, Goodin, DS, Francis, G, et al. Randomized,
comparative study of interferon beta-1a treatment regi-mens in
MS: the EVIDENCE trial. Neurology 2002; 59: 1496–1506.
15. Mikol, DD, Barkhof, F, Chang, P, et al. Comparison of
subcutaneous interferon beta-1a with glatiramer acetate in
patients with relapsing multiple sclerosis (the REbif vs
Glatiramer Acetate in Relapsing MS Disease [REGARD] study):
a multicentre, randomised, parallel, open-label trial. Lancet
Neurol 2008; in press.
16. Mikol, D, Barkhof, F, Chang, P, et al. REGARD Study Group.
The REGARD trial: a randomised assessor-blinded trial
comparing interferon beta-1a and glatira-mer acetate in
relapsing-remitting multiple sclerosis. Mult Scler 2007;
13(Suppl. 2): 119.
17. Kurtzke, JF. Rating neurologic impairment in multiple sclerosis:
an expanded disability status scale (EDSS).
Neurology 1983; 33: 1444–1452.
18. McDonald, WI, Compston, A, Edan, G, et al. Recom-mended
diagnostic criteria for multiple sclerosis: guide-lines from the
International Panel on the diagnosis of multiple sclerosis. Ann
Neurol 2001; 50: 121–127.
19. Francis, GS, Rice, GP, Alsop, JC. Interferon beta-1a in MS:
results following development of neutralizing antibodies in
PRISMS. Neurology 2005; 65: 48–55.
20. Gneiss, C, Reindl, M, Lutterotti, A, et al. Interferon-beta: the
neutralizing antibody (NAb) titre predicts reversion to NAb
negativity. Mult Scler 2004; 10: 507–510.
21. Bellomi, F, Scagnolari, C, Tomassini, V, et al. Fate of neu-
tralizing and binding antibodies to IFN beta in MS patients
treated with IFN beta for 6 years. J Neurol Sci 2003; 215: 3–8.
22. Sorensen, PS, Koch-Henriksen, N, Bendtzen, K. Are ex vivo
neutralising antibodies against IFN-beta always det-rimental to
therapeutic efficacy in multiple sclerosis. Mult Scler 2007; 13:
616–621.
23. Malucchi, S, Sala, A, Gilli, F, et al. Neutralizing antibodies
reduce the efficacy of beta IFN during treatment of mul-tiple
sclerosis. Neurology 2004; 62: 2031–2037.
24. Hartung, HP, Polman, C, Bertolotto, A, et al. Neutralising
antibodies to interferon beta in multiple sclerosis: expert panel
report. J Neurol 2007; 254: 827–837.
http://msj.sagepub.com
Downloaded from msj.sagepub.com at PACE UNIV LIBRARY on December 1, 2013
227
25. Files, JG, Hargrove, D, Delute, L, Cantillon, M. Measured
neutralizing titers of IFN-beta neutralizing antibodies (NAbs) can
depend on the preparations of IFN-beta used in the assay. J
Interferon Cytokine Res 2007; 27: 637–642.
26. Sominanda, A, Rot, U, Suoniemi, M, Deisenhammer, F, Hillert,
J, Fogdell-Hahn, A. Interferon beta preparations for the treatment
of multiple sclerosis patients differ in neutralizing antibody
seroprevalence and immunogenic-ity. Mult Scler 2007; 13: 208–
214.
27. Mohr, DC, Goodkin, DE, Likosky, W, et al. Therapeutic
expectations of patients with multiple sclerosis upon ini-tiating
interferon beta-1b: relationship to adherence to treatment. Mult
Scler 1996; 2: 222–226.
28. Mohr, DC, Likosky, W, Boudewyn, AC, et al. Side effect profile
and adherence to in the treatment of multiple sclerosis with
interferon beta-1a. Mult Scler 1998; 4: 487–489.
29. Jaber, A, Bozzato, GB, Vedrine, L, et al. A novel needle for
subcutaneous injection of interferon β-1a: Effect on pain in
volunteers and satisfaction in patients with multiple sclerosis.
BMC Neurol 2008; in press.
30. Brearley, C, Jaber, A, Bertolino, M, Priestley, A, Seiberling, M.
Assessment of the safety, tolerability, and PK/PD properties of
two new formulations of subcutane-ously administered IFN-
beta1a: a double-blind, placebo-controlled comparison with the
currently available for-mulation. Int J Clin Pharmacol Ther
2007; 45: 307–318.
31. Mikol, D, Lopez-Bresnahan, M, Taraskiewicz, S, Chang, P,
Rangnow, J. A randomized, multicentre, open-label, parallel-
group trial of the tolerability of interferon beta-1a (Rebif)
administered by autoinjection or manual injec-tion in relapsing-
remitting multiple sclerosis. Mult Scler
2005; 11: 585–591.
32. Phillips, JT, Rice, G, Frohman, E, et al. A multicenter, open-
label, phase II study of the immunogenicity and safety of a new
prefilled syringe (liquid) formulation of Avonex in patients with
multiple sclerosis. Clin Ther 2004; 26: 511–521.
33. Reess, J, Haas, J, Gabriel, K, Fuhlrott, A, Fiola, M. Both
paracetamol and ibuprofen are equally effective in man-aging
flu-like symptoms in relapsing-remitting multiple sclerosis
patients during interferon beta-1a (AVONEX) therapy. Mult
Scler 2002; 8: 15–18.
Appendix
The RNF study group comprises the following indi-
viduals: Argentina: A. Carra, Hospital Británico de Buenos
Aires, Ciudad de Buenos Aires; E. Cristiano, Hospital
Italiano de Buenos Aires, Ciudad de Buenos Aires; G.
Luetic, Instituto de Neurociencias de Rosario, Rosario,
Santa Fe; A. Rodriguez Alfici, ‘Insti-tuto Medico
Rodriguez Alfici’, Godoy Cruz, Men-doza; S. Vetere,
Hospital Interzonal de Agudos ‘Gral. San Martín’, La Plata,
Buenos Aires; Australia: J. King, Royal Melbourne
Hospital, Melbourne; J. Pollard, Royal Prince Alfred
Hospital, Sydney; K. Boundy, The Queen Elizabeth
Hospital, Adelaide; L. Sedal, St Vincent’s Hospital,
Melbourne; H. Butzkueven, The Box Hill Hospital
Multiple Sclerosis Centre, Mel-bourne; R. Macdonnell,
Austin Hospital, Austin Health, Melbourne; Canada: L.M.
Metz, University of Calgary, Calgary, Alberta; R.K.
Zabad, University of Calgary, Calgary, Alberta; Denmark:
P. Soelberg
Multiple Sclerosis 2009; 15: 219–228
228 G Giovannoni et al.
Sørensen, Copenhagen University Hospital, Rigshos-
pitalet, Copenhagen; Ireland: O. Hardiman, Beau-mont
Hospital, Dublin; N. Tubridy, St Vincent’s Private
Hospital, Dublin; Israel: D. Karussis, Haddas-sah
University Hospital, Jerusalem; A. Achiron, MS Centre,
Sheba MC, Tel-Hashomer, Tel-Hashomer; Lithuania: A.
Vaitkus, Hospital of Kaunas University of Medicine,
Kaunas; R. Kizlaitiene, Vilnius Univer-sity Hospital,
Santariskiu Clinics, Vilnius; Russia: O. Barbarash, State
Educational Institution of Higher Professional Education
‘Kemerovo State Medical Academy’, Kemerovo; O.
Lesnyak, State Institution of Public Health ‘Sverdlovsk
Regional Clinical Hospital #1’, Ekaterinburg; L.
Zaslavsky, Leningrad Regional Clinical Hospital, St
Petersburg; I. Poveren-nova, Samara State Medical
University, Samara; D. Khasanova, State Educational
Institution of Higher Professional Education ‘Kazan State
Medical University of Ministry of Health of Russian
Federa-tion’, Kazan; P. Sidorov, State Educational
Institution of Higher Professional Education ‘North State
Medical University’, Arkhangelsk; V. Alifirova, Siber-ian
State Medical University, Tomsk; M. Sherman, Municipal
Institution of Public Health ‘Kirov Clin-ical Hospital #1’,
Kirov; V. Balyazin, State Educa-tional Institution of
Higher Professional Education ‘Rostov State Medical
University’, Rostov-on-Don; M. Lutsky, Regional Clinical
State Institution of
Multiple Sclerosis 2009; 15: 219–228
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Public Health ‘Voronezh Regional Clinical Hospi-tal #1’,
Voronezh; Spain: B. Casanova, Consulta Externa de
Neurología, Hospital La Fe, Valencia;
A. Rodríguez-Antigüedad, Hospital de Basurto, Bil-bao; J.
García-Merino, Hospital Puerta de Hierro, Madrid; R.
Arroyo, Hospital Clinico San Carlos, Madrid; Sweden: T.
Olsson, Karolinska University Hospital, Stockholm; UK:
D. Barnes, St George’s Hospital, London; G. Giovannoni,
Institute of Cell and Molecular Science, Barts and the
London
School of Medicine and Dentistry, London;
C. Young, Walton Centre, Liverpool; J. O’Riordan,
Ninewells Hospital, Dundee; O. Malik, Charing Cross
Hospital, London; USA: R. Armstrong, Ashe-ville
Neurology Specialists, PA, Asheville, NC; C. Sheppard,
Oak Clinic for Multiple Sclerosis, Uniontown, OH; J.
Simsarian, Neurology Center of Fairfax, Fairfax, VA; B.
Thrower, Shepherd Cen-ter, Inc., Atlanta, GA; S. Wray,
Baptist West Hospi-tal, Knoxville, TN; J. Gross,
Associated Neurologists
of Southern Connecticut, P.C., Fairfield, CT; D. Wynn,
Consultants in Neurology, Northbrook, IL; T. Miller,
Advanced Neurology, Ft. Collins, CO; G. Pardo, MS
Center of Oklahoma, Mercy NeuroScience Institute,
Oklahoma City, OK; G. Garmany, Alpine Clinical
Research Center, Boulder, CO.
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