comparison of two dosing frequencies of subcutaneous interferon beta-1a in patients with a first clinical demyelinating event suggestive of multiple sclerosis (REFLEX)

Comparison of two dosing frequencies of subcutaneous interferon beta-1a in patients with a first clinical demyelinating event suggestive of multiple sclerosis (REFLEX): a phase 3 randomised controlled trial The Lancet Neurology - Volume 11, Issue 1 (January 2012)  -

Articles
Comparison of two dosing frequencies of subcutaneous interferon beta-1a in patients with a first clinical demyelinating event suggestive of multiple sclerosis (REFLEX): a phase 3 randomised controlled trial


Giancarlo Comi, Prof, MD^a,* 
Nicola De Stefano, Prof, MD^b 
Mark S Freedman, Prof, MD^c 
Frederik Barkhof, Prof, MD^d 
Chris H Polman, Prof, MD^e 
Bernard MJ Uitdehaag, Prof, MD^e 
Florence Casset-Semanaz, MSc^f 
Brian Hennessy, MSc^f 
Margaretha Stam Moraga, MSc^f 
Sanda Rocak, PhD^f 
Bettina Stubinski, MD^f 
Ludwig Kappos, Prof, MD^g 

---

^a  Department of Neurology, Ospedale San Raffaele, Milan, Italy 
^b  Department of Neurological and Behavioural Sciences, University of Siena, Siena, Italy 
^c  Multiple Sclerosis Research Unit, Ottawa Hospital-General Campus, Ottawa, ON, Canada 
^d  Diagnostic Radiology and Image Analysis Centre, VU Medical Center, Amsterdam, Netherlands 
^e  Department of Neurology, VU Medical Center, Amsterdam, Netherlands 
^f  Merck Serono SA, Geneva, Switzerland 
^g  Neurology and Department of Biomedicine, University Hospital Basel, Basel, Switzerland

---

* Correspondence to: Prof Giancarlo Comi, Department of Neurology, Ospedale San Raffaele, Via Olgettina, 60, 20132 Milan, Italy

---

E-mail address:  g.comi@hsr.it

PII S1474-4422(11)70262-9

---

Summary

 Background

In patients presenting with a first clinical demyelinating event that is suggestive of multiple sclerosis (MS), treatment with interferon beta can delay the occurrence of further attacks and the onset of MS. We investigated the effects of two dosing frequencies of subcutaneous interferon beta-1a in patients with a first clinical demyelinating event.

Methods

We undertook a multicentre phase 3 study (REbif FLEXible dosing in early MS [REFLEX]) that included patients (aged 18–50 years) with a single clinical event suggestive of MS, and at least two clinically silent T2 lesions on brain MRI. Participants were randomly assigned in a 1:1:1 ratio by use of a centralised interactive voice response system to receive the serum-free formulation of subcutaneous interferon beta-1a 44 μg three times a week or once a week (plus placebo twice a week for masking), or placebo three times a week for up to 24 months. Patients and physicians were masked to group allocation. The primary endpoint was time to a diagnosis of MS as defined by the 2005 McDonald criteria and the main secondary endpoint was time to clinically definite MS (CDMS) as defined by the Poser criteria. Analysis was by intention to treat. The study is registered with ClinicalTrials.gov, numberNCT00404352.

Findings

517 patients were randomly assigned (171 to subcutaneous interferon beta-1a three times a week, 175 to subcutaneous interferon beta-1a once a week, and 171 to placebo) and 515 were treated. The 2-year cumulative probability of McDonald MS was significantly lower in patients treated with subcutaneous interferon beta-1a (three times a week 62·5%, p<0 0="" 2-year="" 20="" 21="" 75="" a="" adverse="" beta-1a.="" beta-1a="" both="" cdms="" ci="" conversion="" dosing="" established="" events="" for="" hazard="" hr="" interferon="" lower="" of="" once="" p="" placebo="" profile="" rates="" ratio="" regimens="" subcutaneous="" than="" the="" three="" times="" to="" versus="" week="" were="" within="">Interpretation

Both regimens of subcutaneous interferon beta-1a delayed clinical relapses and subclinical disease activity. The potential differences between the regimens warrant longer-term study.

Funding

Merck Serono SA, Geneva, Switzerland.

---

Introduction

The first clinical manifestation of multiple sclerosis (MS) is often a single neurological event consistent with one or more white matter lesions in the CNS, such as optic neuritis.^[1] This event is usually followed by the recurrent attacks that characterise relapsing MS. ^[2] , [3] The recurrence of attacks consistent with CNS demyelination has traditionally been used to make a definitive diagnosis of MS (clinically definite MS [CDMS]). ^[4] , [5] The criteria introduced by Poser and colleagues^[5] in 1983 required two clinical attacks for a diagnosis of CDMS, although clinical, paraclinical, and laboratory evidence could be taken into account for a diagnosis of probable MS. More recently, advances in MRI techniques have led to the development of the McDonald criteria ^{[6] , [7] , [8]} for an earlier diagnosis of MS (McDonald MS) if the first clinical event is accompanied by specific subclinical MRI features. For a diagnosis of McDonald MS, the MRI evidence of disease must show that CNS lesions are disseminated in time and space. These criteria, first introduced in 2001,^[6] were revised in 2005^[7] and 2010^[8] to improve their sensitivity for early MS diagnosis without compromising specificity.

Many patients are now treated as early as possible after a first clinical event, without the need to wait for a diagnosis of definite MS.^[9] In several studies, treatment with interferon beta or glatiramer acetate at the time of the first clinical event delayed the occurrence of further attacks and the onset of MS. ^{[10] , [11] , [12] , [13] , [14]} Furthermore, early treatment with interferon beta can also affect long-term disease progression. ^{[11] , [15]} In the Early Treatment of MS (ETOMS) study,^[16] subcutaneous interferon beta-1a at 22 μg once a week was effective in delaying MS. However, until now the effect of subcutaneous interferon beta-1a at 44 μg three times a week—the licensed dosing regimen for MS—has not been assessed in people with a clinically isolated event. In our 2-year, double-blind phase 3 study (REbif FLEXible dosing in early MS [REFLEX]), we compared the effects of two dosing frequencies of a serum-free formulation of subcutaneous interferon beta-1a 44 μg with placebo on time to development of MS, as defined in the 2005 version of the McDonald criteria.^[7]

Methods

Patients

Patients from 80 centres in 28 countries were recruited into the phase 3 REFLEX study. They were eligible for inclusion if they were aged 18–50 years and had the following: an expanded disability status scale (EDSS)^[17] score of 0–5·0; a single event suggestive of MS within 60 days before study entry; and at least two clinically silent lesions of 3 mm or more on T2-weighted brain MRI scan, at least one of which was ovoid, periventricular, or infratentorial. Exclusion criteria included diagnosis of MS as per the 2005 McDonald criteria and any other disease that could better account for the signs and symptoms. Previous use of any other immunomodulatory or immunosuppressive therapy was prohibited. Women of childbearing age were required to use contraception.

The study was undertaken in compliance with the Declaration of Helsinki and standards of Good Clinical Practiceaccording to the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. Before initiation of the trial at each centre, the relevant institutional review board or independent ethics committee reviewed and approved the trial protocol, patient information leaflet, informed consent forms, and investigator brochure. All patients provided written informed consent at the screening visit.

Randomisation and masking

The study centre dialled a centralised interactive voice response system (provided by S-Clinica, Brussels, Belgium) to randomly assign patients in a 1:1:1 ratio to the serum-free formulation of subcutaneous interferon beta-1a 44 μg three times a week (the dosing regimen generally recommended for established relapsing MS), the same formulation and dose of interferon beta-1a once a week, or placebo. Patients assigned to the once a week regimen were given two additional subcutaneous injections of placebo per week for masking purposes, and patients assigned to placebo received three subcutaneous injections per week. Placebo was supplied as a transparent sterile solution for injection in prefilled syringes matching the interferon beta-1a prefilled syringes, each containing 0·5 mL. The double-blind period was defined as the time between randomisation and CDMS (defined as either a second event suggestive of MS or an increase of at least 1·5 points in the EDSS^[17] score that was sustained for at least 3 months), until study withdrawal, or 24 months, whichever occurred first.

Dynamic treatment allocation, by use of a minimisation algorithm with an element of chance, was applied to minimise imbalance between the treatment groups. Randomisation was stratified according to baseline factors: age (<30 i="" nbsp="" style="margin: 0px; padding: 0px;" years="">vs

 ≥30 years), steroid use for first event (yes vs no), classification of first event (monofocal vs multifocal), and at least one gadolinium-enhancing lesion (yes vs no).

A treatment kit number, corresponding to the randomisation group, was allocated centrally to each patient for use only by that individual. A two-physician (treating and assessing) model was used to assist with study masking. The treating physician was responsible for supervision of study drug administration and for recording adverse events and safety assessments. The assessing physician was not involved in the care of study participants and was exclusively responsible for all neurological assessments, beginning with the prestudy assessment. Injection sites were covered before a patient saw the assessing physician to maintain masking.

In the active treatment groups, patients received 20% of the full dose for 2 weeks, 50% for a further 2 weeks, and the full dose for the remainder of the study. On conversion to CDMS, patients were switched to open-label subcutaneous interferon beta-1a at 44 μg three times a week until the end of the 24 months, with titration to this dose for all patients to maintain masking to the original randomised treatment group. After conversion to CDMS, patients underwent MRI scans every 6 months.

Procedures

Patients' demographics, and medical history were obtained at the screening visit. Their visits were scheduled every 3 months from baseline to month 24 to gather EDSS, MRI, and other efficacy data, and to report safety. An additional safety visit was made at month 1 for all patients.

Patients were strongly recommended to take ibuprofen (400 mg) or paracetamol (1000 mg) prophylactically with each injection during the first 12 weeks of treatment. Short courses of corticosteroid treatment for MS relapses were permitted according to the trial protocol at the discretion of the treating physician. Any on-study MRI scan had to be done before administration of steroids or at least 7 days after the last steroid dose.

All centres were required to follow an MRI operations protocol to ensure standardised scanning, image acquisition, and data transfer. Each patient was scanned by the same machine throughout the trial whenever possible. When a machine had to be changed, every effort was made to keep the parameters consistent. The protocol specified a preference for 1·5 T scanners. T1-weighted scans without and with administration of a single dose of contrast medium (gadolinium-diethylene triamine pentaacetic acid, 0·2 mL/kg) and T2-weighted double-echo scans were done. Scans were obtained in the axial plane parallel to the line joining the genu and splenium of the corpus callosum and consisted of 46 contiguous images with a slice thickness of 3 mm.

Blood samples for the assessment of binding antibodies and neutralising antibodies to interferon beta were obtained from all patients on study day 1 and then every 6 months until month 24 or study termination. Samples were tested first for binding activity, and those that were positive were tested for neutralising activity using the cytopathic effect assay.^[18]

On completion of the trial, all patients were offered the opportunity to participate in a double-blind extension trial for another 3 years. Patients who had not converted to CDMS at 24 months and decided not to participate in the extension trial were offered an optional additional 1-year open-label treatment with subcutaneous interferon beta-1a at 44 μg three times a week. Patients who declined both these options had a 4-week safety follow-up.

An independent adjudication committee (which remained masked throughout the study and consisted of neurologists and a neuroradiologist with expertise in MS) was responsible for assessing eligibility in all patients, confirming or reclassifying each patient's first event as monofocal or multifocal, reviewing all relapses that contributed to conversion to CDMS, and thus confirming whether the diagnostic criteria for McDonald MS and CDMS were met. The reporting and assessment of relapses were continuous and independent of scheduled visits. A relapse was defined as a new or worsening neurological symptom, in the absence of fever, lasting for at least 24 h, accompanied by an objective change in the relevant (ie, symptomatic) functional system, and preceded by 30 days or more of clinical stability or improvement. MRI scans were assessed centrally at the VU Medical Center, Amsterdam, The Netherlands.

To meet the McDonald criteria for diagnosis of MS, patients had to have evidence of spatial and temporal dissemination of MRI lesions or a second clinical attack. For patients without a second attack, MRI follow-up scans were assessed and lesions were classified qualitatively as persisting, new, or enlarging and the location recorded as infratentorial, juxtacortical, periventricular, or deep white matter. Dissemination in space on MRI was defined as three of the following: at least one gadolinium-enhancing lesion or at least nine T2 hyperintense lesions; at least one infratentorial lesion; at least one juxtacortical lesion; or at least three periventricular lesions. Alternatively, dissemination in space could be defined as at least two MRI lesions consistent with MS plus positive CSF. Dissemination in time was defined as a new gadolinium-enhancing lesion more than 3 months after onset of the first clinical demyelinating event (at a site different from the initial event) or a new T2 lesion at any time compared with a scan at least 30 days after the onset of the initial clinical event.

The safety and tolerability of subcutaneous interferon beta-1a were assessed by documentation of adverse events, laboratory tests, vital signs, electrocardiograms, physical examination, and neutralising antibodies to interferon beta. The following were not judged to be adverse events in the safety analysis: pregnancies, MS symptoms, relapses, and exacerbations or worsening MS, unless the event was judged to be related to the study drug. Adverse events were coded with the Medical Dictionary for Regulatory Activities (MedDRA) and analysed according to the preferred terms. The preferred terms pertaining to prespecified adverse events that were known to be related to interferon beta—flu-like syndrome (influenza-like illness), cytopenia, hepatic disorders, thyroid disorders, hypersensitivity reactions, skin rashes, depression and suicidal ideation, and injection-site reactions—were grouped and analysed together.

Statistical analysis

The primary endpoint was time to conversion to McDonald MS as defined according to the 2005 version of the criteria.^[7] The main secondary endpoint was time to conversion to CDMS and the main MRI secondary endpoint was the mean number of combined unique active lesions per patient per scan. A combined unique active lesion was defined as a new or persisting gadolinium-enhancing lesion on T1 MRI or a new or enlarging lesion on T2 MRI that was non-gadolinium-enhancing on T1 MRI. To control for type 1 error, a hierarchical hypothesis testing strategy was applied, based on the significance of the relevant confirmatory test in the following order: subcutaneous interferon beta-1a 44 μg three times a week versus placebo for time to McDonald MS, time to CDMS, and mean number of combined unique active lesions; then subcutaneous interferon beta-1a 44 μg once a week versus placebo for the same endpoints in the same order. The six confirmatory tests were two-sided and done at the 0·05 significance level in the prespecified sequence; confirmatory testing continued if the null hypothesis in the sequence was rejected. The order of the hierarchical testing was such that the regimen of subcutaneous interferon beta-1a currently recommended in patients with relapsing MS (44 μg three times a week) was tested against all or as many of the primary and secondary endpoints as possible before the once a week regimen. The predefined comparisons between the interferon beta-1a three times a week and once a week dosing regimens were not part of the hypothesis testing and type 1 error control, and were thus exploratory.

The primary analysis was a pair-wise comparison of the treatment groups undertaken for the primary and main secondary endpoints by use of a two-sided stratified log-rank test at the 0·05 significance level. The probability of patients remaining event-free over time (from randomisation) in each of the three treatment groups was shown in the form of survival curves estimated by use of the non-parametric Kaplan-Meier method for the primary and main secondary endpoints. The treatment effect was also assessed by use of hazard ratios with the Cox's proportional hazards model, adjusted by the randomisation stratification factors. For the main MRI endpoint, treatment groups were compared pair-wise at the 0·05 significance level by use of a non-parametric ANOVA on ranks model including treatment and randomisation stratification factors as covariates. The estimates of rate ratios for the main MRI endpoint were calculated by use of a negative binomial model with treatment and randomisation stratification factors as covariates and log number of scans as an offset variable.

150 patients per group were estimated to be needed to achieve 165 events (diagnosis of McDonald MS) for the comparison of subcutaneous interferon beta-1a three times a week versus placebo, and 238 events in total (96 in the placebo group, 73 in the once a week group, and 69 in the three times a week group) to give 90% power for the primary comparison with a two-sided 0·05 ɑ error for detection of a hazard ratio of 0·6 in the primary efficacy endpoint, assuming a 10% dropout rate. This hazard ratio corresponds to an expected proportion of 15% of patients free from McDonald MS at 24 months in the placebo group and 32% in the subcutaneous interferon beta-1a three times a week group, based on a previous study of subcutaneous interferon beta-1b in a similar population.^[19]Assuming a withdrawal rate of 10% over 24 months, the sample size was increased to 160 patients per group.

The intention-to-treat population consisted of all patients who were randomly assigned to treatment (treated or not), analysed according to their original allocation, and was used for the efficacy analyses. The double-blind safety population included all patients who were given at least one dose of treatment, analysed according to the treatment they had received during the double-blind period (up to month 24, study withdrawal, or CDMS, whichever occurred first). As patients were switched to open-label treatment with 44 μg three times a week when they were diagnosed as having converted to CDMS, the open-label safety population consisted of all patients from the double-blind safety population who were given at least one open-label study treatment injection after conversion to CDMS. This analysis also corrected for the variation in the duration of treatment between patients. We noted that no bias had been introduced by this feature, so the data are not reported. The primary endpoint was also analysed in subpopulations defined according to the randomisation stratification factors and predefined subgroups.

The study is registered with ClinicalTrials.gov, number NCT00404352.

Role of the funding source

The study was designed by members of the steering committee (LK, MSF, GC, NDS) and the study sponsor. The study sponsor collected the data, did the analysis, and was involved in the interpretation of the data. The data were available to all authors, and they contributed to the analysis and interpretation of the data. The steering committee was responsible for the final decision to submit this report for publication.

Results

The first patient's first visit was on Nov 16, 2006; the last patient's last visit was on Aug 13, 2010. 701 patients were screened and of these 517 were randomly assigned and included in the intention-to-treat population (subcutaneous interferon beta-1a three times a week, n=171; subcutaneous interferon beta-1a once a week, n=175; and placebo, n=171; figure 1). One patient in each group withdrew consent before treatment, resulting in a total of 514 patients from the intention-to-treat population who were allocated to the study drug or placebo (subcutaneous interferon beta-1a three times a week, n=170; subcutaneous interferon beta-1a once a week, n=174; and placebo, n=170). The groups demographic, clinical, and MRI characteristics at baseline are shown in table 1. One patient allocated to interferon beta-1a once a week was first given a three times a week treatment kit; this patient was included in the once a week group in the intention-to-treat population and the three times a week group for the safety population. Another patient was not randomly assigned because no treatment kit was available within 60 days of screening. Treatment was allocated in a double-blinded manner once a kit was available. To ensure the patient received consistent treatment after this initial kit was received, further treatment allocation was by use of the interactive voice response system so that double blinding was maintained. This patient was included in the safety population but not in the intention-to-treat population. After unblinding, it was revealed that this patient had been given placebo, resulting in a total of 515 patients who were given the study drug or placebo.

Table 1   -- Baseline demographic and disease characteristics of intention-to-treat population

		Interferon beta-1a three times a week (n=171)	Interferon beta-1a once a week (n=175)	Placebo (n=171)	Overall (n=517)
Age (years)		30·6 (8·5)	30·7 (8·1)	30·9 (7·9)	30·7 (8·2)
Women		114 (67%)	106 (61%)	112 (65%)	332 (64%)
EDSS score		1·50 (0–4·0)	1·50 (0–3·5)	1·50 (0–3·5)	1·50 (0–4·0)
Time since first demyelinating event (days)		57·6 (3·7)	57·7 (3·4)	57·6 (4·2)	57·6 (3·8)
Classification of first clinical demyelinating event as monofocal (according to the investigator)		99 (58%)	104 (59%)	97 (57%)	300 (58%)
Classification of first clinical demyelinating event as monofocal (according to the adjudication committee)		96 (56%)	90 (51%)	91 (53%)	277 (54%)
Steroid use at first clinical demyelinating event		121 (71%)	123 (70%)	121 (71%)	365 (71%)
Gadolinium-enhancing lesions
	Patients with at least one T1 gadolinium-enhancing lesion	68 (40%)	72 (41%)	73 (43%)	213 (41%)
	Number of gadolinium-enhancing lesions	1·3 (2·5)	1·5 (3·5)	1·2 (2·7)	1·3 (2·9)
	Gadolinium-enhancing lesion volume (mm3)	156·54 (427·33)	194·15 (593·66)	193·68 (588·50)	181·56 (541·68)
T2 hyperintense lesions
	Number of T2 lesions	22·0 (18·8)	23·6 (21·0)	21·3 (20·2)	22·3 (20·0)
	Patients with at least nine T2 lesions	129 (75%)	126 (72%)	122 (71%)	377 (73%)
	T2 lesion volume (mm3)	3110·53 (3410·74)	3853·12 (4716·71)	3334·92 (3990·41)	3436·11 (4083·90)

Data are number (%), mean (SD), or median (range). Interferon beta-1a was administered subcutaneously. EDSS=expanded disability status scale.

The safety population consisted of 515 patients who were given at least one masked injection, 514 from the intention-to-treat population plus the patient who was not randomly assigned but given placebo (subcutaneous interferon beta-1a three times a week, n=171; subcutaneous interferon beta-1a once a week, n=173; and placebo, n=171). Of these patients, 56 (11%) withdrew from the study treatment during the double-blind period (subcutaneous interferon beta-1a three times a week, 21 [12%]; subcutaneous interferon beta-1a once a week, 15 [9%]; and placebo, 20 [12%]). Reasons for withdrawal were adverse events (15 [27%]), disease progression (five [9%]), loss to follow-up (four [7%]), pregnancy (two [4%]), death (one [2%]), and other reasons (29 [52%]). Of the patients in the double-blind safety population who withdrew from treatment, five (24%) of 21 in the interferon beta-1a three times a week group, four (27%) of 15 in the interferon beta-1a once a week group, and six (30%) of 20 in the placebo group did so because of adverse events. Two deaths occurred, both in the placebo group. 402 (78%) of 517 randomly assigned patients entered the extension trial, 20 (4%) entered the 1-year open-label follow-up, and 26 (5%) did not choose either of these options and completed the 4-week safety follow-up. 130 patients were judged to have converted to CDMS (129 because of a second event that was suggestive of MS, 1 because of an increase in EDSS score of ≥1·5 for ≥3 months), of whom 120 switched to open-label treatment for the rest of the study; 12 of these patients subsequently withdrew from treatment. The mean scheduled dose administered in the double-blind period was 98·67% (SD 3·43) in the subcutaneous interferon beta-1a three times a week group, 98·66% (3·82) in the subcutaneous interferon beta-1a once a week group, and 98·78% (3·25) in the placebo group.

379 patients converted to McDonald MS over 2 years (subcutaneous interferon beta-1a three times a week, n=106; subcutaneous interferon beta-1a once a week, n=129; and placebo, n=144). The 2-year cumulative probability of McDonald MS was significantly lower in patients treated with subcutaneous interferon beta-1a versus that in those treated with placebo (figure 2; table 2). The risk of McDonald MS over 24 months was lower with subcutaneous interferon beta-1a three times a week versus subcutaneous interferon beta-1a once a week (table 2).

F 

Table 2   -- Effect of treatment on conversion to McDonald multiple sclerosis

	Interferon beta-1a three times a week versus placebo	Interferon beta-1a once a week versus placebo	Interferon beta-1a three times a week versus interferon beta-1a once a week
Hazard ratio (95% CI)[*]	0·49 (0·38–0·64)	0·69 (0·54–0·87)	0·71 (0·54–0·91)
Relative risk reduction	51%	31%	29%
Absolute risk reduction (95% CI)	23·3% (12·6–34·0)	10·3% (0·2–20·4)	13·0% (2·0–24·0)
Log-rank p-value[‡]	<0 td="">	0·0080	0·0087

Interferon beta-1a was administered subcutaneously.

*	Adjusted Cox's proportional hazards model with treatment group and randomisation stratification factors as covariates.
‡	Two-sided log-rank test, stratified by randomisation stratification factors.

. 

Table 3   -- Effect of treatment on conversion to clinically definite multiple sclerosis

	Interferon beta-1a three times a week versus placebo	Interferon beta-1a once a week versus placebo	Interferon beta-1a three times a week versus interferon beta-1a once a week
Hazard ratio (95% CI)[*]	0·48 (0·31 to 0·73)	0·53 (0·35 to 0·79)	0·90 (0·56 to 1·43)
Relative risk reduction	52%	47%	10%
Absolute risk reduction (95% CI)	16·9% (5·8 to 28·0)	15·9% (4·7 to 27·1)	1·0% (-8·8 to 10·8)
Log-rank p value[‡]	0·0004	0·0023	0·7737

Interferon beta-1a was administered subcutaneously.

*	Adjusted Cox's proportional hazards model with treatment group and randomisation stratification factors as covariates.
‡	Two-sided log-rank test, stratified by the randomisation stratification factors

The rate ratios for mean number of combined unique active lesions per patient per scan were 0·19 (95% CI 0·14–0·26) for subcutaneous interferon beta-1a three times a week group versus placebo group, 0·37 (0·27–0·50) for subcutaneous interferon beta-1a once a week group versus placebo group, and 0·52 (0·38–0·71) for subcutaneous interferon beta-1a three times a week group versus subcutaneous interferon beta-1a once a week. The mean number of combined unique active lesions per patient per scan was significantly lower with both interferon beta-1a dosing regimens (three times a week 0·60 [SD 1·15], p<0 1="" a="" active="" combined="" lesions="" lower="" mean="" number="" of="" once="" p="0·0015).</p" patient="" per="" placebo="" regimen="" scan="" than="" the="" three="" times="" unique="" was="" week="" with="">

The significance of the effect of interferon beta-1a three times a week regimen was maintained when time to McDonald MS was analysed by selected randomisation stratification factors and subgroups (table 4). This was also true for the once a week regimen, with the exceptions of the subgroups with monofocal presentation, and without gadolinium-enhancing lesions at screening. The treatment effect with the three times a week regimen was greater than that with the once a week regimen in the subgroups of patients with multifocal presentation, no gadolinium-enhancing lesions at screening, and at least nine T2 lesions at screening (table 4).

Table 4   -- 2-year cumulative probability of a diagnosis of McDonald multiple sclerosis by selected randomisation stratification factors and subgroups

	Interferon beta-1a three times a week (n=171)	Interferon beta-1a once a week (n=175)	Placebo (n=171)	Interferon beta-1a three times a week versus placebo		Interferon beta-1a once a week versus placebo		Interferon beta-1a three times a week versus interferon beta-1a once a week
				Hazard ratio (95% CI)	p value	Hazard ratio (95% CI)	p value	Hazard ratio (95% CI)	p value
Monofocal presentation	54%	61%	75%	0·58 (0·40–0·84)	0·0035	0·72 (0·50–1·03)	0·0735	0·83 (0·56–1·21)	0·3280
Multifocal presentation	73%	90%	97%	0·45 (0·31–0·64)	<0 td="">	0·64 (0·47–0·88)	0·0065	0·66 (0·46–0·93)	0·0187
No gadolinium-enhancing lesions at screening	54%	69%	79%	0·49 (0·35–0·70)	0·0001	0·74 (0·53–1·02)	0·0688	0·67 (0·47–0·96)	0·0272
At least one gadolinium-enhancing lesion at screening	75%	85%	94%	0·54 (0·38–0·79)	0·0012	0·66 (0·46–0·93)	0·0185	0·81 (0·56–1·18)	0·2708
Fewer than nine T2 lesions at screening	37%	40%	62%	0·42 (0·22–0·80)	0·0077	0·51 (0·29–0·91)	0·0222	0·84 (0·42–1·68)	0·6239
At least nine T2 lesions at screening	70%	89%	96%	0·46 (0·35–0·62)	<0 td="">	0·71 (0·55–0·93)	0·0119	0·63 (0·47–0·83)	0·0011

Interferon beta-1a was administered subcutaneously. Data are percentages, unless otherwise indicated. Hazard ratios and p values (two-sided Wald test) were obtained from Kaplan-Meier cumulative incidence analyses at 2 years and were calculated by use of a Cox's proportional regression model with treatment as a covariate.

No new or unexpected adverse drug reactions were encountered in either of the active treatment groups during this study. The most frequent treatment-emergent adverse events, grouped according to the MedDRA preferred terms and reported in more than 10% of patients in any treatment group, are shown in the webappendix. Table 5 shows the prespecified adverse events that occurred during the double-blind period. As expected, injection-site reactions, hepatic disorders (increases in liver enzyme levels), hypersensitivity reactions, and cytopenia were more frequent with subcutaneous interferon beta-1a three times a week than with subcutaneous interferon beta-1a once a week or with placebo. Influenza-like illness was less frequent in the interferon beta-1a three times a week group than in the once a week group, but more frequent in both active treatment groups than in the placebo group. Depression and suicidal ideation was reported at a similar incidence in all three groups; no MedDRA-preferred terms relating to suicide were reported for any patient. In all other categories of prespecified adverse events, there was no pattern to indicate an increased frequency in the active treatment groups compared with the placebo group.

Table 5   -- Incidence of prespecified treatment-emergent adverse events (preferred terms) during the double-blind period (safety population, n=515)

	Interferon beta-1a three times a week (n=171)	Interferon beta-1a once a week (n=173)	Placebo (n=171)
Patients with prespecified events	127 (74%)	137 (79%)	69 (40%)
Cytopenia	19 (11%)	9 (5%)	4 (2%)
Depression and suicidal ideation	14 (8%)	11 (6%)	14 (8%)
Influenza-like illness	93 (54%)	122 (71%)	34 (20%)
Hepatic disorders[*]	19 (11%)	16 (9%)	8 (5%)
Hypersensitivity reactions	16 (9%)	10 (6%)	11 (6%)
Injection-site reactions	61 (36%)	42 (24%)	12 (7%)
Skin rashes	16 (9%)	8 (5%)	9 (5%)
Thyroid disorders	11 (6%)	5 (3%)	2 (1%)

Interferon beta-1a was administered subcutaneously.

*

All except four of the preferred terms in this category (hepatic pain [n=1] and increased blood bilirubin [n=1] in the interferon beta-1a once a week group, and hyperbilirubinaemia [n=1] and liver disorder [n=1] in the interferon beta-1a three times a week group) referred to shifts in liver enzyme levels.

Serious adverse events were reported in 26 patients during the double-blind period (interferon beta-1a three times a week, n=6; interferon beta-1a once a week, n=8; placebo, n=12). Serious adverse events that were judged to be possibly related to the study drug were varicella (interferon beta-1a once a week, n=1) and spontaneous abortion (placebo, n=1; interferon beta-1a once a week, n=1). All other serious adverse events were judged to be either unrelated or unlikely to be related to the study drug.

The number of patients who tested positive for neutralising antibodies (titre ≥20 neutralising units/mL) at month 24 or at their last test was 25 of 169 (14·8%, 95% CI 9·8–21·1) in the subcutaneous interferon beta-1a three times a week group and 28 of 168 (16·7%, 11·4–23·2) in the subcutaneous interferon beta-1a once a week group.

Discussion

The serum-free formulation of subcutaneous interferon beta-1a significantly delayed time to McDonald MS and CDMS compared with placebo in patients with a first demyelinating event that was suggestive of MS, thereby further supporting the early treatment model of MS. The effect was consistent across subgroups defined according to baseline characteristics. In this subgroup analysis, the risk reductions in the interferon beta-1a group were around 50%, which can be regarded as clinically relevant.

The present study is, to our knowledge, the first comparison of the efficacy of two dosing frequencies of subcutaneous interferon beta-1a. The three times a week and once a week regimens similarly delayed the occurrence of a clinical relapse, but the three times a week regimen had a more pronounced effect on McDonald MS than did the once a week regimen. This difference might be partly attributable to the inclusion of subclinical disease measures in the McDonald criteria, resulting in an increased power to detect an existing difference through a higher frequency of diagnoses. However, this result might also indicate a more pronounced effect of more frequent dosing on MRI outcomes that is not directly related to functional deficits. This explanation is supported by the difference between the treated groups in the number of combined unique active lesions per patient per scan.

Our study had limitations. How the short-term effects of early treatment translate to later effects on disease progression has not been established.^[20] This limitation also arises in other studies of disease-modifying drugs in similar patient populations (panel; table 6). ^{[11] , [12] , [14]} Longer follow-up might be also needed to detect any differences in effects on clinical relapses between the three times and once a week regimens.

Panel

Interpretation The design of the present study and the patient population included are broadly similar to those of the previous studies of disease-modifying drugs in patients with a first demyelinating event. REbif FLEXible dosing in early multiple sclerosis (REFLEX) is the first study in which the effect of two different dose frequencies of active treatment was assessed. Differences between studies were noted in the proportions of patients with monofocal presentation or steroid use for the first clinical demyelinating event, suggestive of minor differences in the populations of patients. All previous studies included clinically definite multiple sclerosis (CDMS) as the primary endpoint, but the definition of CDMS differed between studies. By contrast, the REFLEX study included CDMS as a secondary endpoint, with McDonald MS as the primary endpoint, consistent with increasing use of the McDonald criteria in clinical practice. Only one study[10] (that of subcutaneous interferon beta-1b) included McDonald MS as a secondary endpoint, but this was the 2001 iteration of the guidelines rather than the 2005 version used here. Despite this difference, the probability of McDonald MS in placebo-treated patients was similar in the two studies and a similar effect of interferon beta treatment (risk reduction) was noted (subcutaneous interferon beta-1a three times a week vs placebo, 51%; subcutaneous interferon beta-1b vs placebo, 46%).[10]The effect of active treatment versus placebo on CDMS in the previous studies was also similar to that in REFLEX. Due to differences in MRI procedures and reporting in the different trials, direct comparisons are not possible, but interferon beta and glatiramer acetate seem to improve MRI outcomes. Taken together, these results confirm that treatment with subcutaneous interferon beta-1a significantly delays the onset of MS as defined according to either McDonald criteria or CDMS, similar to results reported for other interferon beta preparations or glatiramer acetate, and support treatment initiation at the time of the first clinical demyelinating event.

Table 6   -- Comparison of the REFLEX study and other large trials of disease-modifying drugs in patients with a first clinical demyelinating event

	Active treatment	Study design	Conversion to CDMS versus placebo	Notes
Comi et al (REFLEX)	Subcutaneous interferon beta-1a (44 μg three times a week or once a week)	Primary endpoint, McDonald MS (2005 criteria); secondary endpoint, CDMS	Three times a week, adjusted HR 0·48 (95% CI 0·31–0·73; p<0 0="" a="" adjusted="" ci="" hr="" once="" p="0·0023)</td" week="">	Monofocal presentation, 54%; steroid treatment, 71%
Kappos et al (BENEFIT)[10]	Subcutaneous interferon beta-1b (250 μg every other day)	Primary endpoint, CDMS; secondary endpoint, McDonald MS (2001 criteria)	Adjusted HR 0·50 (95% CI 0·36–0·70; p<0 td="">	Similar conversion to McDonald MS in placebo group; similar risk reduction for McDonald MS and CDMS; monofocal presentation, 53%; steroid treatment, 71%
Jacobs et al[12] and O'Connor et al[22](CHAMPS)	Intramuscular interferon beta-1a (30 μg once a week)	Primary endpoint, CDMS	Rate ratio 0·56 (95% CI 0·38–0·81; p=0·002); adjusted rate ratio 0·49 (95% CI 0·33–0·73; p<0 td="">	Monofocal presentation, 70%; steroid treatment, 100%
Comi et al (PRECISE)[14]	Subcutaneous glatiramer acetate (20 mg per day)	Primary endpoint, CDMS	HR 0·55 (95% CI 0·40–0·77; p=0·0005)	Monofocal presentation, 100%; steroid treatment, 61%
Comi et al (ETOMS)[16]	Subcutaneous interferon beta-1a (22 μg once a week)	Primary endpoint, CDMS	Odds ratio 0·61 (95% CI 0·37–0·99; p=0·045)	Monofocal presentation, 61%; steroid treatment, 64%

CDMS=clinically definite multiple sclerosis. REFLEX=REbif FLEXible dosing in early MS. HR=hazard ratio. MS=multiple sclerosis.

The safety data reported here are in accordance with the well established safety profile of subcutaneous interferon beta-1a. However, although the frequencies of most prespecified treatment-emergent adverse events (four are presented)—injection-site reactions, hepatic disorders, hypersensitivity reactions, and cytopenia—were greater with three times a week dosing than with once a week dosing, the proportions of patients who had the most common prespecified treatment-emergent adverse event—influenza-like illness—was lower with three times a week dosing than with once a week dosing. This difference could be due to tachyphylaxis, wherein tolerance of the drug with respect to this adverse event often increases with greater cumulative exposure, as has been noted previously.^[23]For example, in the EVIDENCE (Evidence of Interferon Dose–response: European–North American Comparative Efficacy) study,^[24] influenza-like illness was more common, severe, and persistent in patients treated with intramuscular interferon beta-1a at 30 μg once a week than in patients given subcutaneous interferon beta-1a at 44 μg three times a week.^[24]

The long-term differences between interferon beta-1a three times a week and interferon beta-1a once a week, in terms of the effect on MRI-related disease activity and adverse-event profile, warrants further study and is the focus of the ongoing preplanned, dose-frequency-blinded, 3-year extension of this study.