Development of interferon-β as a therapy for multiple sclerosis

Drug Development Strategy

10.1517/14728214.9.2.363 © 2004 Ashley Publications Ltd ISSN 1472-8214 363

Ashley Publicationswww.ashley-pub.com

1. Introduction

2. Preclinical development

3. Clinical development

4. Side effects of interferon-β

therapy

5. Expert opinion

Central & Peripheral Nervous Systems

Development of interferon-β as a therapy for multiple sclerosisClyde MarkowitzUniversity of Pennsylvania, Department of Neurology, 3400 Spruce Street, 3W Gates Building,

Philadelphia, PA 19104, USA

The introduction of interferon (IFN) therapy represents a milestone in multi-ple sclerosis (MS) treatment. This class of drugs is part of an evolving area ofMS therapy that utilises immunomodulating agents to alter the immuneprocesses thought to be integral to the development of MS. IFN-β has beendeveloped in three different formulations that have proven benefit in reduc-ing exacerbations in relapsing-remitting MS. Two of the formulations consistof the naturally-occurring amino acid sequence of IFN-β and are referred toas IFN-β1a. One is delivered intramuscularly (IM IFN-β1a), and the second formof IFN-β1a is delivered subcutaneously (SC IFN-β1a). The third formulation ofIFN-β, known as IFN-β1b, consists of a modified amino acid sequence contain-ing a cysteine to serine mutation at amino acid 17 and a deletion of theamino terminal methionine. This review describes the evolution of IFNs astherapeutics for MS.

Keywords: disease-modifying therapy, drug development, interferon-β, multiple sclerosis, neutralising antibodies

Expert Opin. Emerg. Drugs (2004) 9(2):363-374

1. Introduction

The development of interferon (IFN) therapy for multiple sclerosis (MS) representsone of the first examples of the emergence of biologicals as therapeutic agents. Theuse of IFN stems from insight gained into the disease over the past 40 years, sincethe recognition of the essential role that the immune system plays in the aetiology ofMS. Early treatments used prior to 1950 were ineffective. The first treatment forMS used in the modern medical era was corticotrophin and cortisone, whichreduced the time to remission in patients suffering acute exacerbations [1,2]. Thedevelopment of the synthetic steroid methylprednisolone shifted the emphasis inMS therapy to this relatively new class of drug [3,4]. The effectiveness of these agentswas instrumental in verifying a role for the immune system in MS and in the recog-nition that inflammatory or immune processes were involved in MS.

The rationale for the initial testing of IFNs as therapeutic agents for MS was theview that MS may be caused by a latent viral infection in patients with subclinicaldeficiencies in immune function or who had been exposed during a critical period inlife. The concept that a viral infection may play a role in the aetiology of MS has beenexamined at several times during the history of the study of this disease [5]. Viral agentshave been suspected for several reasons, including epidemiological data and evidencefrom animal models of MS. However, a viral cause for MS has never been established.

In terms of epidemiology, relatively high rates of MS in some regions have beeninterpreted to indicate an infectious agent. For example, the high rate of MS follow-ing the presence of British soldiers on the Faroe Islands during World War II hasbeen examined periodically and suggests that an infectious agent was introducedthat resulted in periodic episodes of MS [6]. Other epidemiological evidence, such asregional differences in MS prevalence by latitude, twin studies, and potential

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Development of interferon-β as a therapy for multiple sclerosis

364 Expert Opin. Emerg. Drugs (2004) 9(2)

association of exacerbations with acute infections, suggestsmultifactorial influences on the risk of developing MS,including the potential role of an infectious agent in asusceptible population [7].

Evidence from animal models also suggests a possible rolefor an infectious agent. The most widely accepted murinemodel of MS is experimental allergic encephalomyelitis(EAE). EAE is characterised by a chronic demyelinationincited by inoculation with CNS homogenate, myelin pro-teins or encephalitogenic proteins, along with an adjuvantinto genetically susceptible mice. A number of viral infectionscan also induce inflammation of the CNS and demyelinationin mice, including Theiler’s mouse encephalomyelitis virus,Semliki Forest virus and mouse hepatitis virus [8].

The discovery of IFN as a ‘viral interfering agent’, pro-duced by infected cells following the introduction of foreignnucleic acids, by Isaacs and Lindenmann in 1957, opened thepossibility of a therapeutic use for such an agent in MS [9,10].It is now known that the IFNs are a family of proteins withantiviral properties. IFNs are divided into Type I and II,which are structurally unrelated and act through separate cellsurface receptors [11]. Type I IFNs include IFN-α, IFN-β,IFN-ω and IFN-τ. The genes for Type I IFNs are clustered onthe short arm of chromosome 9, and there are 21 IFN-αgenes and 1 IFN-β gene in humans. Initially known as fibro-blast IFN, IFN-β is the predominant IFN produced by non-haematopoietic cells. IFN-α, formerly known as leukocyteIFN, and IFN-ω are produced by haematopoietic cells. IFN-γis the only member of the Type II family and is produced bycells of the immune system [11].

The synthesis of IFNs is activated by an increase in IFNgene transcription following the introduction of double-stranded RNA. The secreted IFNs bind to specific cell surfacereceptors. Type I and Type II IFNs have distinct receptors.Both receptor types, the Type I or α/β-receptor and the TypeII or γ-receptor, are heterodimers, consisting of two trans-membrane proteins that signal primarily through the Januskinase signal transducer and activator of transcriptionsystem [12]. IFNs induce the synthesis of > 300 proteins, andthe exact pattern of response depends upon the cell type andthe type of IFN. One of the major actions of IFNs is to sup-press protein synthesis through the action of a double-stranded RNA-dependent protein kinase (PKR). PKR synthe-sis is induced by IFN, and subsequently phosphorylates thetranslation initiation factor eIF-2 to inhibit translation, andIκB to activate NF-κB [12].

In the immune system, IFN-β has been shown to reducemajor histocompatibility complex II expression on mono-cytes, inhibit proliferation of T cells, reduce the expression ofinterleukin-12 (IL-12) and enhance the production of IL-10[13]. In addition, IFN-β reduces the transcription of severalmembers of the matrix metalloprotease (MMP) family of pro-teases [14]. The MMPs are important in lymphocyte migrationacross the blood–brain barrier, and reduced levels of theseimportant extracellular proteases has been proposed as a

potential mechanism underlying the beneficial effects ofIFN-β in MS [15].

2. Preclinical development

Prior to the availability of recombinant IFN-β, investigatorsutilised IFNs purified from mammalian cells treated withagents that simulate viral infection. These forms of IFN willbe referred to as natural IFN (-α, -β or -γ) for the purposes ofthis discussion. Three different formulations of recombinantIFN-β have been developed. The first form to be producedhas been modified by deletion of the amino terminal methio-nine and at amino acid 17 (cysteine to serine). It was pro-duced in bacteria and thus is not glycosylated. Two otherforms of IFN-β containing the naturally-occurring aminoacid sequence were produced in Chinese hamster ovary cells.These forms are glycosylated by virtue of their synthesis inmammalian cells. One formulation has been delivered byintramuscular injection (IM IFN-β1a) and the second formu-lation has been delivered by subcutaneous injection (SCIFN-β1a). There is some evidence that glycosylation decreasesaggregate formation and may serve to decrease theimmunogenicity of the protein [14].

The biological activity of recombinant IFN was first exam-ined using IFN-β1b, which was the first recombinant formproduced. The availability of biomarkers for IFN-β activityprovided quantifiable end points for pharmacodynamic stud-ies. For example, serum levels of neopterin (6-D-erythro-1,2,3-dihydroxypropylpterin) produced during activation ofcell-mediated immunity are used as a measurement of theeffect of IFN-β. Two other biomarkers used to assess IFNactivity are β2-microglobulin and 2′,5′-oligoadenylate syn-thetase activity. Dose-response studies indicated that doses inthe range of 1 – 5 million international units (MIU) providedmaximal biological response in terms of neopterin,β2-microglobulin, and 2′,5′-oligoadenylate synthetaseactivity [15,16].

Phase III trials for SC IFN-β1a and IM IFN-β1a benefitedfrom strong preclinical studies providing pharmacokineticsdata and dosing levels. Preclinical studies to determine the bestroute of delivery for these forms of IFN-β were carried out, andinitial studies indicated that intramuscular delivery producedthe best biological response as judged by neopterin and β2-microglobulin measurements in response to a single injectionof IFN-β [17]. Other studies found no advantage to intramus-cular delivery, and concluded that intramuscular and subcuta-neous delivery were equivalent in terms of bioavailability [18].

The dose of IM IFN-β1a used in the pivotal Phase III trialwas determined from a pilot study designed to identify thehighest dose that could be used in a double-blind study usingparacetmol (acetaminophen) for side effects [19]. The once-weekly dosing schedule was based on observations that a sin-gle dose of IM IFN-β1a produced a significant elevation ofβ2-microglobulin that was sustained over 6 days. The nextlower dose of IM IFN-β1a produced an elevation in

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Expert Opin. Emerg. Drugs (2004) 9(2) 365

β2-microglobulin that declined after 48 h; the next higherproduced no significantly greater response [19].

In a Phase I study, the clinical tolerance and biologicalproperties of recombinant SC IFN-β1a were compared withnatural IFN-β in healthy volunteers. The production of neop-terin and β2-microglobulin were used as markers of IFN activ-ity [20]. The biomarkers were measured up to 5 days followingadministration of a single injection of SC IFN-β1a. Bothneopterin and β2-microglobulin levels were significantlyincreased by natural IFN-β and SC IFN-β1a for up to 72 h.Neopterin levels were not significantly increased beyond 48 h;the elevated levels of β2-microglobulin were maintained to96 h, but not beyond. These results indicate that the effects ofSC IFN-β are stable for 72 – 96 h.

A randomised, double-blind, pilot study using one of therecombinant forms of IFN, SC IFN-β1b, enrolled 30 patientsto test safety and obtain information on dosing and efficacy.

SC IFN-β1b was delivered at four different doses, three timesweekly. At 6 months, the data showed a dose-related effect,with no relapses in the high-dose group (90 MIU). Althoughside effects of injection-site erythema, fever and fatigue weresevere enough to preclude continued use of the highest dose,the lower doses (4.5, 22.5 and 45 MIU) were welltolerated [21]. The MIU designation at the time of this studydiffers from the current MIU designation for IFN-β1b, andthe 45 MIU dose is equivalent to the 8 MIU dose used in thePhase III trial of IFN-β1b.

3. Clinical development

As summarised in the following sections and Table 1, earlyclinical studies using IFN-α, IFN-γ and IFN-β were carriedout to determine their potential as therapeutic agents in MS,initially based on their antiviral effects and influence on the

Table 1. Clinical trials of IFN in MS.

Study IFNAdministration route

Treatment duration

No. of patients

Clinical type Design Results

Knobler et al., 1984 [22] nIFN-α i.m. 6 months 24 15 RR,9 RP

DB, PC crossover

Reduced attacks in RR group No effect on RP

Camenga et al., 1986 [23] rIFN-α2 s.c. 12 months 98 72 RR,26 RP

DB, PC No difference between IFN and placebo

AUSTIMS research group, 1989 [24]

nIFN-α s.c. 3 years 182* 166 RR, 16 CP

DB, PC No effect on disability, relapses, VER or CSF

Panitch et al., 1987 [26] rIFN-γ i.v. 1 month 18 RR Single-blind

Attacks in 7 patients during treatment

Ververken et al., 1979 [27] nIFN-β i.m. 2 weeks 3 CP Open No effect

Huber et al., 1988 [28] nIFN-β i.v. 6 months 9 RR Open No effect

Baumhefner et al., 1987 [29]

nIFN-β i.v. 3 months 6 CP Open Improvement in 4 patients

Jacobs et al., 1981, 1982, 1985 [30-32]

nIFN-β i.t. 6 months 20 8 RR, 4 CP, 8 SR

Open Reduced attacks inRR group

Jacobs et al., 1986 [33] nIFN-β i.t. 6 months 69 RR DB, PC Reduced attacks

Johnson et al., 1990 [21] rIFN-β s.c. 6 months 30 RR DB, PC Dose-related reduced attacks

IFNB MS study group, 1993 [34]

rIFN-β s.c. 2 years 372 RR DB, PC Fewer exacerbationsImprovement on MRI

Jacobs et al., 1996 [36] rIFN-β i.m. 2 years 301 RR DB, PC Delayed progressionFewer exacerbations Improvement on MRI

PRISMS study group, 1998 [38]

rIFN-β s.c. 2 years 560 RR DB, PC Delayed progression Fewer exacerbationsImprovement on MRI

*Includes 61 patients who received transfer factor.AUSTIMS: Australian trial of transfer factor and interferon as treatment for MS; CP: Chronic progressive; CSF: Cerebrospinal fluid; DB: Double-blind; IFN: Interferon; i.m.: Intramuscular; i.t.: Intrathecal; i.v.: Intravenous; MRI: magnetic resonance imaging; MS: multiple sclerosis; nIFN-α: Natural IFN-α; nIFN-β: Natural IFN-β; PC: Placebo-controlled; PRISMS: prevention of relapses and disability by Interferon β1a subcutaneously in multiple sclerosis; rIFN-α: Recombinant IFN-α; rIFN-β: Recombinant IFN-β; rIFN-γ: Recombinant IFN-γ; RP: Relapsing progressive; RR: Relapsing-remitting; s.c.: Subcutaneous; SR: Stable with residua; VER: Visual-evoked responses.



immune system. Natural IFN-α was tested in several studiesinvolving patients with MS. The initial study using IFN-αenrolled 24 patients with relapsing-remitting MS (RRMS) orprogressive MS. The study was a double-blind, placebo-con-trolled crossover design, and IFN-α was delivered by intra-muscular injection [22]. The end points were exacerbation rateand disability (on the Expanded Disability Status Scale[EDSS]) over a 6-month period. Although the reduction inrelapse rates did not achieve statistical significance comparedwith placebo, the results prompted larger controlled trials ofIFN-α. Camenga and colleagues [23] treated 98 patients withrecombinant IFN-α or placebo for up to 52 weeks, followedby a 3-month observation period. A total of 98 patients wereenrolled in this double-blind, placebo-controlled study. Notherapeutic benefit of IFN-α treatment was observed. A largerstudy was carried out involving 121 patients by the AUS-TIMS (Australian Trial of Transfer Factor and Interferon asTreatment for MS) group. Patients were treated with naturalIFN-α or placebo for up to 3 years. IFN was delivered subcu-taneously in this double-blind, placebo-controlled study. Theprimary outcome was progression of disability. No differencein progression of disability, as measured by the Kurtzke Disa-bility Severity Scale, was found and IFN-α was poorly toler-ated [24]. A double-blind, randomised trial of IFN-α involving20 patients with RRMS was carried out over a 6-monthperiod. Patients received 9 × 106 IU delivered via intramuscu-lar injection every other day. There was a significant decreasein the exacerbation rate (0.17 versus 1.00 for placebo,p < 0.03), and 10 of 12 patients receiving IFN-α were exacer-bation free during the treatment period compared with 2 of 8in the placebo group. Side effects of IFN-α treatment weresignificant. Of the 12 patients in the IFN-α group, 8 experi-enced fever, 3 experienced hair loss and a ‘flu-like syndrome’(fever, headache, arthalgias and myalgias) occurred in severalpatients. The most severe side effect was chronic fatigue,which affected 50% of IFN-α-treated patients and was severeenough to affect employment in two cases [25]. On the basis ofthese studies, IFN-α has not been enthusiastically pursued asa treatment option for MS.

Clinical studies using IFN-γ in MS were initiated becauseof the antiviral properties that IFN-γ shares with other IFNs.However, investigators noted that IFN-γ could also induceclass II histocompatibility antigens on several cell types andactivate macrophages, properties that could potentially inducean autoimmune response and contribute to MS. A baseline-controlled trial using IFN-γ enrolled 18 patients with RRMS.In this single-blind study, IFN-γ was delivered twice-weeklyintravenously over a period of 1 month. The primary out-come was exacerbation rate. During this study, seven patientsreceiving IFN-γ experienced exacerbations. Thus, patientstreated with IFN-γ experienced a significantly higher rate ofexacerbations compared with the pre-study exacerbation rate[26]. In addition, increases were observed in activated mono-cytes and lymphocyte proliferation during IFN-γ treatment.These results suggested a role for IFN-γ in naturally-occurring

exacerbations of MS and ended attempts to use IFN-γ as atherapeutic agent for MS. The lack of efficacy of IFN-γ treat-ment was surprising given the fact that ventricular administra-tion of IFN-γ suppressed EAE in Lewis rats and anti-IFN-γantibodies exacerbated EAE in C57BL6J mice. However, theeffects of IFN-γ in humans may be due, at least in part, to thefact that IFN-γ is a pro-inflammatory cytokine. Increased lev-els of IFN-γ would thus be expected to lead to further activa-tion of immune responses, which may be detrimental inpatients with MS.

In the first investigation of IFN-β for the treatment of MS,natural IFN-β was administered intramuscularly on alternatedays for 2 weeks to three patients with chronic progressiveMS. During the 9- to 18-month follow-up period, no toxic-ity was observed. However, clinical benefit also was not seen[27]. In a second study, natural IFN-β was delivered intrave-nously twice-weekly for 4 weeks and then twice-monthly for5 months. A total of nine patients with RRMS were enrolled,and the primary outcome measurement was exacerbationrate. The treatments were well tolerated; however, no changein exacerbation rate was observed during a follow-up of> 1 year [28].

Subsequently, a beneficial effect of intravenous naturalIFN-β was reported in a small open-label study involving sixpatients with chronic progressive MS. Natural IFN-β(3 MIU) was administered once-weekly for 12 weeks intra-venously. The patients also received physical therapy 5 daysa week. Improvements in coded neurological examinationswere seen in four of the six patients. However, no effect onMRI lesions seen on magnetic resonance imaging (MRI) wasfound [29].

Intrathecal delivery of natural IFN-β was employed in sev-eral studies based on the rationale that IFNs do not readilycross the blood–brain barrier, and thus, intrathecal deliverymay improve efficacy. Jacobs and co-workers [30-32] were ableto utilise natural IFN-β that was being produced at theRoswell Park Cancer Institute for intrathecal delivery to can-cer patients. In this open-label trial, 20 patients with MSreceived IFN-β via lumbar punctures twice-weekly for thefirst 4 weeks, and once-weekly for the next 5 months. Theinclusion criteria included patients with both RRMS andchronic progressive MS. The primary outcome was exacerba-tion rate. Over an average follow-up of 1.9 years, patientswith RRMS who received IFN-β showed a significant reduc-tion in exacerbation rate (p < 0.01 versus controls). From 0.6to 6 exacerbations were predicted based on previous diseaseseverity. However, in eight of these patients no exacerbationsoccurred. The exacerbation rate continued to decline over amean follow-up of 4.4 years (1.8/year pre-study – 0.16/yearat 4.4 years). Headaches were the most frequent side effectand were experienced by all patients on at least one occasion.Low-grade fever also occurred in six patients.

A randomised, double-blind, placebo-controlled study wassubsequently carried out using natural IFN-β delivered byintrathecal infusion. A total of 69 patients with RRMS were

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enrolled. IFN-β was administered weekly for the first 4 weeksand then monthly for the next 5 months. The primary outcomemeasurement was time to progression (≥1 point on the EDSSscale). A significant delay in the time to sustained progressionwas observed in patients receiving IFN-β. In addition, a signifi-cant reduction in exacerbation rate was seen among recipients ofIFN-β compared with the placebo group (p < 0.05). Over2 years of follow-up, the exacerbation rate in the IFN-β groupdeclined from 1.79 to 0.76/year; the exacerbation rate in theplacebo group declined from 1.98 to 1.48/year [33].

The ability to produce large quantities of recombinantIFN-β1b facilitated the development of this formulation, lead-ing rapidly to Phase III trials following the successful Phase IIstudies. The impetus for the rapid development of IFN-β1b

was in part driven by the positive results that had beenobtained using the natural IFN-β by intrathecal delivery. APhase III study using IFN-β1b was initiated in 1988 at11 centres in the US and Canada [34]. The study included372 patients with RRMS who had EDSS scores of 0 – 5.5,and ≥ 2 exacerbations in the previous 2 years. Patients wererandomised to receive IFN-β1b at 1.6 MIU, 8 MIU or pla-cebo. IFN-β1b or placebo was administered subcutaneouslyevery other day. The primary efficacy end points were theannual exacerbation rate and proportion of exacerbation-freepatients. Serial MRI was performed yearly on all patients; asubset underwent MRI scan every 6 weeks. At 2 years, annualexacerbation rates were significantly lower in both IFN-β1b

groups compared with placebo: 1.27 for placebo versus 1.17for the 1.6 MIU group and 0.84 for the 8 MIU group (p< 0.05). Similarly, the percentage of patients who were exacer-bation-free was significantly greater in patients treated withIFN: 36% in the high-dose group, 21% in the low-dose groupand 16% in the placebo group (8 MIU p = 0.007 versus pla-cebo, 1.6 MIU p = 0.076 versus placebo). The exacerbationseverity was also reduced in the high-dose group, and thesepatients had a greater lag time to first exacerbation. The timeto first exacerbation was 147 days in the placebo group versus199 and 264 in the 8 MIU and the 1.6 MIU group, respec-tively. A significant decrease (0.1%) in total MRI lesion areawas seen in the IFN-β1b 8 MIU group after 2 years, whereasthe placebo group showed a 20% increase in lesion area (p <0.001) [34,35]. The 8 MIU group showed an 80% reduction innew, recurrent or enlarging T2 lesions compared with placebo(p = 0.0082) [35]. Both IFN-β1b groups showed significantreductions in annual accumulation of lesion activity duringthe extension of the Phase III trial (p = 0.001 versus placebo).

The use of MRI as a biomarker in MS was a critical ele-ment in the development of IFN-β1b. Measurement of brainlesions provides a measure of acute disease activity that is notpossible to detect by clinical evaluation. Thus, the MRI meas-urements provided a quantitative measure of drug efficacy andhad the potential to detect subclinical disease activity duringthe pivotal clinical trials of IFN-β. Although clinical efficacyremained the primary outcome for drug approval, a reliablesecond measurement of disease activity improved confidence

in study results. It was the combination of clinical efficacy andMRI data that led to the approval of IFN-β1b by the Food andDrug Administration (FDA) in 1993. The drug was grantedorphan drug status due to the relatively small number ofpatients with MS in the US. Orphan drug status is providedby the FDA based on the Orphan Drug Act passed in 1983.Under this law, ‘orphans’ are defined as drugs targeting rarediseases. These drugs may offer small profits to the manufac-turer, but large benefits to people with the rare diseases. Tofoster the development of these drugs, firms are allowed totake tax deductions for a portion of the cost of their clinicalstudies and given exclusive marketing rights for 7 years. Thedevelopment of IFN-β for MS is one example of the positiveimpact that this legislation has had on patient populationssuffering from rare disorders.

A randomised, double-blind, placebo-controlled study wasinitiated in 1990 at four US centres to determine whetherintramuscular IFN-β1a could slow the progressive course ofRRMS [36]. A total of 301 patients were randomised to receiveeither placebo or IM IFN-β1a. Patients were eligible for partic-ipation in the Phase III trial provided they had a baselineEDSS score of 1.0 – 3.5 and had experienced ≥ 2 exacerba-tions in the previous 3 years. The primary efficacy outcome,time to onset of sustained worsening in disability, was definedas deterioration from baseline by ≥ 1.0 point on the EDSSsustained for ≥ 6 months. Secondary outcomes includedrelapse rate, number and volume of gadolinium-enhanced(Gd+) lesions and number and volume of T2 lesions. Patientsin the IFN-β1a group had a significant reduction in time tosustained progression of disability compared with patientsgiven placebo. At 104 weeks, the proportion of patients withdisease progression was 34.9% in the placebo group and21.9% in the IM IFN-β1a group (p = 0.02). The annual exac-erbation rate was 0.82 for the placebo group and 0.67 for theIM IFN-β1a group (p < 0.05) [36]. In terms of MRI measure-ments, the number of Gd+ lesions was significantly reducedin the active treatment group (p = 0.05) [36]. Treatment withIM IFN-β1a produced a significant reduction in T2 lesion vol-ume after 1 year (p = 0.02); however, this difference was notsignificant at 2 years.

The FDA approved the IM IFN-β1a formulation for use intreating RRMS in 1996. The approval was granted despite theorphan drug status that had been granted to IFN-β1b. Thereason for breaking of the orphan drug status for IFN-β1b wasthe improved safety profile of IM IFN-β1a. Specifically, thereduction in injection-site reactions was cited as the primaryadvantage of IM IFN-β1a over SC IFN-β1b [37].

A Phase III study of the third recombinant form of IFN,SC IFN-β1a, involving 560 patients with RRMS was initiatedin 1994 at 22 centres across 9 countries [38]. The EDSS scoresof the participants were in the range of 0 – 5.0. Patientsreceived either placebo or one of two doses of SC IFN-β1a, 22or 44 µg. SC IFN-β1a or placebo was administered three timesweekly. Patients were followed for 2 years in the initial study.The primary efficacy measure was number of relapses over the



course of the study. Secondary outcome measures includedproportion of relapse-free patients, progression to disabilityand disease activity on MRI scan. Relapses were significantlyreduced in both active treatment groups compared with pla-cebo. The percentage of reduction versus placebo was 27% forthe 22 µg dose and 33% for the 44 µg dose (p < 0.05). Thepercentage of patients who were relapse-free over both yearswas increased dose-dependently, 22% for placebo versus 37%for the 22 µg dose and 45% for the 44 µg dose. Time to pro-gression of disability was also significantly increased in thehigh-dose SC IFN-β1a group from 11.9 months in the pla-cebo group to 18.5 months in the 22 µg group and 21.3 inthe 44 µg group (p < 0.05 versus placebo for both doses). Theburden of disease, as measured by T2 lesions on MRI scan,increased by 10.9% in the placebo group over the course ofthe study. Patients receiving both doses of SC IFN-β1a showeda significant reduction in T2 lesions on MRI scan (1.2% and3.8% for the 22 µg and 44 µg doses, respectively, bothp < 0.0001 versus placebo) [38,39]. Both SC IFN-β1a doses alsoproduced a significant reduction in the number of T2 activelesions, 67% reduction for the 22 µg dose and 78% reductionfor the 44 µg dose (p < 0.0001 versus placebo), with a doseeffect in favour of the 44 µg dose (p = 0.0003).

Subcutaneous IFN-β1a provided improvement in relapserates and MRI measures of disease activity. However, theorphan drug status of IFN-β1b and IM IFN-β1a had to be chal-lenged to market SC IFN-β1a in the US. This required demon-stration of greater safety (as in the case of IM IFN-β1a) orgreater efficacy in head-to-head comparisons. A head-to-headcomparison of SC IFN-β1a and IM IFN-β1a in 677 patientswas carried out over a 6-month period. This study was specifi-cally designed to challenge the orphan drug status of IMIFN-β1a. At 24 weeks, 74% of patients receiving SC IFN-β1a

remained exacerbation free, whereas 63.3% of patients receiv-ing IM IFN-β1a were exacerbation free (p < 0.001) and therewere fewer active lesions on MRI scans (1.2 versus 0.8) for IMIFN-β1a versus SC IFN-β1a. At 48 weeks, 62% of patients

receiving SC IFN-β1a remained exacerbation free, whereas52% of patients receiving IM IFN-β1a were exacerbation free(p = 0.006) and there were fewer T2 lesions on MRI scans (1.4to 0.9) for IM IFN-β1a versus SC IFN-β1a [58].

The reduced exacerbation rate provided a basis for thebreaking of the orphan drug status for IM IFN-β1a [40]. Thedifference in the per cent of exacerbation-free patients wasreduced in follow-up studies at 12 and 16 months. Theapproval of SC IFN-β1a in the US in March of 2003 providedthree forms of IFN-β for clinical use in the US. A summary ofthe physical measures of disease activity from the pivotal PhaseIII trials for the three forms of IFN-β is presented in Table 2.

The progressive nature of MS suggests that initiation of ther-apy early in the disease will maximise benefits to patients. Thisidea is also supported by the modest response to IFN-β therapyin secondary progressive MS (SPMS). SC IFN-β1b has beenexamined in a study of 718 patients with SPMS and EDSSscores of 3.0 – 6.5. Patients were randomised to either placeboor 8 MIU IFN-β1b every other day. The primary outcomemeasurement was time to confirmed EDSS progression of≥ 1.0 point. In this study, a significant delay in EDSS progres-sion was reported. At study end point, 56% of patients in theplacebo group had experienced confirmed progressioncompared with 47% of patients who received SC IFN-β1b [41].

The same formulation of IFN-β1b was used in a double-blind trial of 939 patients with SPMS at North American cen-tres. Patients were randomised to placebo or to IFN-β1b eitherat a dose of 8 MIU or a dose of IFN-β1b adjusted for bodyweight and size. Each dose of IFN-β1b was administered everyother day. The primary end point of the study was time to con-firmed progression of disability defined as a > 1 point increasein EDSS score sustained for > 6 months. The study wasstopped due to a lack of efficacy in the primary end point [42].

A study to evaluate the efficacy of two doses of SC IFN-β1a,22 and 44 µg, three times weekly, in SPMS, enrolled618 patients. Time to sustained progression, as measured byEDSS, was used as the primary outcome measure. No

Table 2. Summary of results on physical measures of disease activity from pivotal Phase III clinical trials.

IFN Dosage Relapse rate Reduction in relapses (%)*

Relapse-free patients (%)*

Reduction in disease progression (%)*†Placebo IFN-β

SC IFN-β1b [34] 8 MIU (250 µg) every other day

1.27 0.84 31 31 29

IM IFN-β1a [36] 30 µg 1 × week 0.82 (all patients)0.90 (patients treated for 2 years)

0.67 (all patients)0.61 (patients treated for 2 years)

18 (all patients)32 (patients treated for 2 years)

38 37

SC IFN-β1a [38] 22 µg 3 × week 1.28 0.91 29 27 23

44 µg 3 × week - 0.865 32 32 31*Total over 2 years.†A worsening of ≥ 1 point on the Expanded Disability Status Scale sustained for 6 months (IFN-β1a-Avonex trial) or 3 months (IFN-β1b-Betaseron and IFN-β1a-Rebif trials).IM: Intramuscular; IFN: Interferon; MIU: Million international units; SC: Subcutaneous; wk: Week.

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difference in disability progression using the standard disabil-ity measure, EDSS, was found; however, an effect on therelapse rate was seen. The annual relapse rate was 0.71 in theplacebo group, 0.50 in the 22 µg group and 0.50 in the 44 µggroup (p < 0.001) [43].

A recent study of the efficacy of low-dose IFN-β1a in SPMSenrolled 371 patients with EDSS scores of < 7.0 (mean 4.8).Patients were randomised to placebo or either SC IFN-β1a

22 or 44 µg three times a week. The primary outcome meas-urement was time to progression on the EDSS, as defined byan increase from baseline of ≥ 1 (≥ 0.5 if the baseline EDSSscore was ≥ 5.5). No significant effect of low-dose IFN-β1a

was observed on time to progression. At study end, 44% ofpatients in the placebo group experienced disease progression,and 43% in the IFN-β1a group experienced disease progres-sion [44]. Similarly, the relapse rate was not significantlyaffected by IFN-β1a treatment (0.27 versus 0.25, placeboversus IFN-β1a, respectively).

A two-arm trial was carried out at 42 centres (31 in the US,4 in Canada and 7 in Europe) involving 436 patients withSPMS. Patients were randomised to IM IFN-β1a (60 µgweekly) or placebo. The primary outcome measure waschange on the Multiple Sclerosis Functional Composite(MSFC) score from baseline to 24 months. Secondary out-comes included disease progression as measured by EDSSscores and relapse rates. The disease progressed in both groupsover the course of the study; however, the IFN-β1a IM groupshowed a 40% reduction in change from baseline in MSFCscore (-0.495 versus -0.32, placebo versus IM IFN-β1a, respec-tively, p = 0.033). No significant difference was seen in thechange from baseline in EDSS score; however, a 33% reduc-tion was observed in the relapse rate (0.3 versus 0.2, placeboversus IM IFN-β1a, respectively, p = 0.008) [45].

The variable results of IFN-β therapy in patients withSPMS are in contrast to the more robust response seen inpatients with RRMS and suggest that this type of therapy maybe more effective when initiated early in the disease.

The efficacy of IFN-β therapy in primary progressive MS(PPMS) has also been examined but the studies done so farhave been limited by small patient numbers. A randomisedtrial of 73 patients compared patients receiving 8 MIU SCIFN-β1b with placebo. The primary outcome measurementwas time to sustained progression (defined as an increase inEDSS score of 1 in patients with an EDSS score of ≥ 5, or 0.5in patients with an EDSS score of 5 – 5.5). Additional out-comes included MSFC scores. There was no significant dif-ference in the primary outcome (time to sustainedprogression), but there was a significant improvement inMSFC scores compared with placebo [46]. A negative resultfor IFN-β therapy was also noted in a trial involving50 patients treated with either 30 or 60 µg IM IFN-β1a com-pared with placebo. The primary end point in this study wasalso time to sustained progression, and secondary outcomemeasures included lesions on MRI scans and nine-hole pegtests. There was no significant effect on the primary end

point although some improvement in the lesion load on MRIscan was observed [47].

The impact of early IFN therapy on disease progressionhas been evaluated in controlled trials. A 3-year, randomised,double-blind study enrolled 383 patients with a first demy-elinating event. The primary outcome measure was time toclinically definite MS. A diagnosis of MS required the occur-rence of a new neurological event lasting > 48 h. Patientswho received treatment with IM IFN-β1a showed a signifi-cantly lower cumulative probability of developing clinicallydefinite MS (p = 0.002 versus placebo) [48]. At 18 months,patients who received IM IFN-β1a at a first event also showeda significant reduction in volume of brain lesions, fewer newor enlarging lesions and fewer Gd+ lesions (all p < 0.001versus placebo).

Another study of SC IFN-β1a was performed using a2-year, randomised, double-blind design. This study enrolled375 patients presenting with a first episode of neurologicaldysfunction, and 307 patients were randomised. Patientsreceived IFN-β1a at 22 µg once-weekly. The primary endpoint was the conversion to clinically definite MS, as definedby the occurrence of a second exacerbation, with new neuro-logical symptoms lasting ≥ 24 h. In this study, treatment withSC IFN-β1a significantly decreased the probability of conver-sion to clinically definite MS, and provided benefits, as seenon MRI (p < 0.05 versus placebo) [49].

In aggregate, the available evidence suggests that earlyintervention with IFN therapy is beneficial. First, IFN ther-apy provides a clear benefit to patients with RRMS. Second, amodest effect of IFN therapy was seen on later stages of thedisease. Third, studies in patients with the initial presentingsymptoms of MS indicate that the appearance of clinicallydefinite disease is significantly delayed by IFN therapy.Finally, one of the most consistent effects of IFN therapy is toreduce the number of new and active lesions, as assessed byMRI. These results, when taken together, provide a rationalefor early intervention in patients with MS.

3.1 Utility of magnetic resonance imaging as a surrogate marker of disease progression in multiple sclerosisA critical element in the clinical development of IFN-β wasthe use of MRI for imaging of the CNS. Advancements inMRI techniques during the 1980s and 90s allowed direct vis-ualisation of MS disease activity in the brain. Lesions withcontrast enhancement on T1-weighted MRI after Gd admin-istration indicate breakdown of the blood–brain barrier andare thought to represent active disease [50,51]. On MRI, newGd+ lesions may have predictive value regarding the short-term course of MS [52]. Longitudinal studies have suggested acorrelation between lesion burden, that is, the number oflesions on MRI, and disease progression. A study of18 patients who were followed for 1 year found a 0.46 corre-lation between EDSS score and cumulative number of lesions(p = 0.002) and a slightly stronger correlation between the



ambulatory index (AI) and cumulative number of lesions(0.056, p < 0.001) [53].

A study of 60 patients with SPMS who were followed for9 months found that patients with fewer new Gd+ lesionswere more likely to be relapse free. The correlation betweenthe number of new Gd+ lesions and relapses during the9-month study was 0.62 (p ≤ 0.02) [54]. Areas of high signalon T2-weighted MRI, the T2 lesion load, are used as a meas-ure of disease activity and are often referred to as the ‘burdenof disease’ [55]. Other MRI lesions, known as T1-hypointenselesions or ‘black holes’, reflect axonal loss, gliosis, loss of intra-cellular matrix and demyelination, indicative of moredestructive focal CNS damage [55-57].

The availability of reliable measurements of disease onMRI facilitated the development of IFN-β in MS for severalreasons. First, investigators were able to use MRI measure-ments in early studies to assess the acute impact of treatment.Second, MRI measurements provide valuable confirmation ofclinical measurements. Improvements both in clinical meas-ures and on MRI allow independent confirmation of clinicalbenefit. Thus, MRI outcomes have been included assecondary measures in the Phase III trials of IFN-β.

4. Side effects of interferon-β therapy

Side effects that were reported during the Phase III trial of IFN-β1b included fever, chills, myalgia, sweating and malaise in addi-tion to injection-site reactions (Table 3). Mild neutropoenia,anaemia and thrombocytopoenia were also reported, but werenot statistically significant. Of patients who received IFN-β1b

1.6 MIU, 63% reported injection-site inflammation, while69% of patients who received IFN-β1b 8 MIU reported thiscompared with 6% of patients given placebo.

Adverse events that occurred during the pivotal Phase IIItrial of IM IFN-β1a were flu-like symptoms (61 versus 40% inplacebo, p < 0.01), muscle aches (34 versus 15% in placebo),fever (23 versus 13% in placebo) and chills (21 versus 7% inplacebo) (Table 3). Anaemia occurred in 3% of IM IFN-β1a

recipients and 1% of placebo recipients, but was not severeenough to require discontinuation of the study drug or bloodtransfusion. No significant differences were seen in the

incidence of depression, menstrual disorders or injection-sitereactions. No reports of leukopoenia, thrombocytopoenia orelevated liver enzyme levels occurred in patients treated withIM IFN-β1a [36], although it should be noted that later studiesdid observe mild increases in liver enzyme levels in 18% ofpatients, and three patients discontinued therapy due toelevations in liver enzyme levels [58].

Adverse events reported in first 3 months of the Phase IIItrial of SC IFN-β1a are presented in Table 3. Injection-sitereactions occurred significantly more often (p ≤ 0.05) inpatients given SC IFN-β1a (61 – 62%) compared with thosegiven placebo (22%). Adverse events that occurred signifi-cantly more often (p ≤ 0.05) in the high-dose group comparedwith the placebo group included lymphopoenia, increasedalanine aminotransferase (ALT) levels, leukopoenia and granu-locytopoenia. No significant differences were seen among thethree treatment groups in the incidence of depression [38].

Development of neutralising antibodies (NAbs) has beendocumented for all current formulations of IFN. The inci-dence of NAbs has been reported in the Phase III trials to bein the range 38 – 47% with IFN-β1b, 12 – 24% with SCIFN-β1a, and 5 – 22% with IM IFN-β1a [34,36,38]. Compari-sons across studies are difficult due to differences in method-ology and patient populations, among other variables. Therehave been several attempts to provide direct comparisonsbetween different IFN-β formulations. In a direct comparisonwith a relatively large patient population (n = 677), NAbsdeveloped in 25% of patients receiving SC IFN-β1a whereas2% of patients receiving IM IFN-β1a developed NAbs [58].This difference in the incidence of NAbs seems to be due todifferences in drug formulation rather than route of deliverybecause patients receiving IM delivery of the SC IFN-β1a for-mulation developed NAbs at the same rate as patients whoreceived SC IFN-β1a via the normal route of delivery [59]. Sim-ilarly, altering the route of delivery may reduce, but not elimi-nate, the incidence of NAbs in patients receivingIFN-β1b [60,61].

The presence of NAbs seems to impact biological activityand clinical efficacy of IFN-β, although an impact on clinicalparameters has not been observed in every study. In the com-parator study examining SC IFN-β1a and IM IFN-β1a

Table 3. Adverse events reported with different formulations of IFN-β therapy.

IFN Injection-site inflammation

Flu-like symptoms

Myalgia Fever Chills Mild neutro-poenia

Lympho-poenia

Leuko-poenia

Granulocyto-poenia

↑ ALT

IFN-β1b X X X X X

IM IFN-β1a X X X X

SC IFN-β1a X X X X X X X

Adverse events listed are those reported to occur at significantly greater frequency in IFN-β-treated patients, p < 0.05 compared with placebo.ALT: Alanine aminotransferase; IM: Intramuscular; MIU: Million international units; SC: Subcutaneous.Data represent the incidence of adverse events during a 2-year treatment period with an 83% completion rate [34].IM IFN-β1a data represent the incidence of adverse events over a 2-year treatment period [36].

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formulations, the presence of NAbs did not affect relapse ratesover the 48-week course of the study [58]. The effect of NAbson efficacy was also examined at the conclusion of the PhaseIII trial of SC IFN-β1a in a 2-year extension period [61]. Theplacebo group from the original trial was randomised to oneof the two doses of SC IFN-β1a (22 or 44 µg three times/week) and comprised a crossover group (n = 172). Patientswho had originally received SC IFN-β1a (n = 167) continuedwith treatment. Patients exhibiting antibody titres > 20 neu-tralising units were considered positive. The relapse rates weresignificantly higher among patients who were antibody posi-tive (0.81 versus 0.5, p = 0.002). A more recent longitudinalanalysis of data from the pivotal Phase III trial of IFN-β1b

indicates that during periods when NAb titres are high, therelapse rate increases. Patients were tested for NAbs on a quar-terly basis over a 60-month period. When NAb-positivepatients enter periods of reduced NAb titres, relapse rates arealso reduced [62]. A similar increase in relapse rates duringNAb-positive periods was reported in a Scandinavian cohort[63]. More recent studies to examine the biological impact ofNAbs on IFN-β responses have employed sensitive measuressuch as the expression of the IFN acute response gene myxovi-rus resistance protein A (MxA). The expression of MxA issharply increased in response to IFN-β injection in healthysubjects. This response is abrogated in patients who are per-sistently NAb positive [64]. Similarly, the suppression of MMPin response to IFN-β therapy is reduced in patients who areNAb positive [65].

The persistence of NAbs has been examined in several stud-ies. During the 2-year extension period of the SC IFN-β1b

Phase III trial, most patients who developed antibodies toIFN-β1b were positive at the next test. An analysis of the totaltime that NAb-positive patients were positive for NAbs indi-cated that these patients produce significant titres of NAbs75 – 80% of the time on treatment [62]. Differing results werereported by Rice et al [66] who followed a small group ofpatients involved in the pivotal trial for IFN-β1b. A total of59 patients, of whom 24 were positive for NAbs, were testedfor NAb status over an average of 8 years. At this time a signif-icant number of NAb-positive patients (17 of 24) hadconverted to a NAb-negative state.

An examination of NAbs in 541 patients treated with SCIFN-β1a (n = 265), IM IFN-β1a (n = 82), and IFN-β1b

(n = 194) every 12 months for 60 months following initiationof therapy indicated that most NAb-positive patients developNAbs to IFN-β1b during the first 6 – 18 months of therapywhereas the majority of patients NAb positive for IFN-β1

developed antibodies during the first 12 – 18 months of ther-apy [63]. In addition, most patients who developed NAbs didso during the first 12 months of therapy. Few patients (5% ofthe total population), converted from NAb negative to NAbpositive after the initial 12 months of therapy. The relative percent of NAb-positive patients who converted to a NAb-nega-tive state varied between treatments. Patients receivingIFN-β1b who became NAb positive were more likely to revert

to a NAb-negative state than were patients receiving IFN-β1a

who became NAb positive.

5. Expert opinion

The development of IFN-β as a therapeutic agent for MSunderscores both the enormous progress made in understand-ing complex interactions between the immune system and theCNS and the enormous amount of work that will be requiredto achieve the goal of a curative therapy for MS. Most clinicaltrials are of relatively short duration compared with the pro-gression of MS, which occurs over decades. Thus, the resultsof these trials are, by necessity, subject to extrapolation, andlonger follow-up is required for patients who are currently onIFN therapy to assess the impact of IFN.

Although IFN-β therapy can delay disease progression andreduce relapse rates, the mechanism of action has not beencompletely elucidated. Several molecular responses to IFN-βhave been described, such as changes in cytokine production,decreased expression in adhesion molecules, suppression ofMMPs and inhibition in cell migration. However, it is notclear how these changes relate to the clinical effects of IFN-β.Furthermore, a need still exists for additional markers for dis-ease activity to monitor the effectiveness of therapy prior toirreparable tissue destruction signified by disease relapses andfurther neurological deficiencies.

An area that requires further attention is the issue of MS inpaediatric populations. There is a growing awareness that MSis not strictly an adult disease. Paediatric cases of MS represent2 – 5% of the total MS cases and these patients seem torespond to therapy in a manner similar to adult patients, butfurther study is required [67]. It is clear that MS is notexclusively an adult disease.

Over the last 10 years it has become clear that early therapyis important to the long-term management of patients withMS, because current therapies do not reverse damage, but serveto limit exacerbations. Initial studies examining the impact ofintervention following the first clinically evident demyelinatingevent suggest that a significant delay in the appearance of clini-cally definite MS can be achieved [48,49]. Thus, early interven-tion with appropriate therapy may be critical to limitneurological deterioration and delay disease progression.

It will also be critical to establish the impact of NAbs and tocreate formulations that will decrease the incidence of NAbsfurther. It is possible that the use of immune agents, whichlimit immune response, may decrease the incidence of NAbsand thus may be useful as adjunct therapy to IFN-β. For exam-ple, administration of corticosteroids on a monthly basis hasbeen reported to reduce the incidence of NAbs to IFN-β1b [68].Current assays for NAbs are not standardised, nor is it clear if athreshold level of NAbs exists. Thus, low levels of NAbs maynot be clinically relevant; however, no good data address thisissue. In addition, the existence of patients who exhibit a reduc-tion in NAb titre suggests that in some patients continued ther-apy will be beneficial even in the presence of NAbs. These



patients cannot be identified with our present level of under-standing. In summary, the data are clear that NAbs to IFN-βdevelop during long-term therapy with IFN-β and that theseNAbs impact clinical efficacy. However, the issues of the titre ofNAbs that signifies clinical significance and the persistence ofNAbs have not been adequately studied to date.

The issue of the impact of NAbs on efficacy must be con-sidered in the context of the clinical course of the disease. Lossof efficacy may not be distinguishable on MRI scans until ayear or more has passed, and a decrease in efficacy may not benoted in clinical outcomes for a greater time (≥3 years). Thus,the short-term studies that have examined the clinical impactof NAbs are unlikely to provide an accurate assessment oftheir significance. Although the weight of the evidence indi-cates that NAbs do significantly impact clinical efficacy, thereis still a need for more information from long-term studies.However, the current working hypothesis should be thatNAb-positive patients are likely to obtain less benefit fromIFN therapy than NAb-negative patients.

New applications of MRI technology will provide oppor-tunities for more accurate analysis of disease activity. Theuse of volumetric magnetisation transfer imaging can pro-vide an assessment of structural brain damage that may berelated to cognitive decline [69]. A second recent application

of MRI technology, magnetisation transfer ratio histogramanalysis, can provide a quantitative analysis of tissue damagethat is not detected by T2-weighted MRI [70]. Calculation ofthe brain parenchymal fraction using computer-aided analy-sis has been employed to monitor disease activity [71,72].Additional advances in MRI technology, such as diffusiontensor imaging, which allows observation of molecular dif-fusion in tissues, perfusion imaging and spectroscopy, willalso improve the ability to monitor disease activity. As MRItechnologies continue to advance, a more comprehensiveanalysis of neurological degeneration will be available toguide treatment.

In summary, the impact of IFN-β on MS cannot be under-stated, yet it is clear that this therapy represents only the firststep in the evolution of disease-modifying therapies for MS.The impact of IFN-β on disease progression is measurable,but modest. It appears that IFN-β treats the inflammatoryaspect of the disease, but does not address damage to brain tis-sue. Future therapies are likely to involve combination ther-apy to address both of these aspects of MS. In this regard,multiple compounds are in development that have muchgreater target specificity than IFN-β. The advantages of thistarget specificity will lie in the reduction of side effects andpotentially greater efficacy than existing therapy.

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AffiliationClyde MarkowitzUniversity of Pennsylvania, Department of Neurology,3400 Spruce Street,3W Gates Building 5,Philadelphia, PA 19104, USATel: +1 215 662 3381; Fax: +1 215 349 5579;E-mail: [email protected]

Development of interferon-β as a therapy for multiple sclerosis

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