Mechanism of Action and Clinical Effects of Beta-Interferon in MS (Venice Workshop 1999)

Proceedings of the MS ForumModern Management Workshop

Venice, March 1999

Mechanisms ofAction and

Clinical Effects ofBeta Interferon inMultiple Sclerosis

Chairmen: Kees Lucas; Barry Arnason

The MS Forum is supported by an unrestricted educational grant fromSchering AG, Berlin, Germany.

Produced by PPS Europe, Worthing UK, a division of PAREXEL MMS Europe Ltd, under an educational grantfrom Schering AG, Berlin, Germany.

The opinions expressed in this educational programme are not necessarily those of the producers of the publication or Schering AG.

Although great care is taken to ensure accuracy, those involved in producing the publication cannot be liable for any errors or inaccuracies. All dosages referred to should be checked against the relevant data sheets

for the products.

1999 PPS Europe. All rights reserved, including that of translation into other languages. No part of this book maybe reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying,

recording, or any information storage and retrieval systems, without permission of the copyright holder.

The MS Forum is supported by an unrestricted educational grant fromBerlex Laboratories, Richmond, California, USA.

Produced by PPS Europe, Worthing UK, a division of PAREXEL MMS Europe Ltd, under an educational grantfrom Berlex Laboratories, Richmond, California, USA.

The opinions expressed in this educational programme are not necessarily those of the producers of the publication or Berlex Laboratories.


for the products.



The MS Forum is supported by an unrestricted educational grant fromBerlex Canada Inc., Lachine, Quebec, Canada.

Produced by PPS Europe, Worthing UK, a division of PAREXEL MMS Europe Ltd, under an educational grantfrom Berlex Canada Inc., Lachine, Quebec, Canada.

The opinions expressed in this educational programme are not necessarily those of the producers of the publication or Berlex Canada Inc.


for the products.



Proceedings of the MS ForumModern Management Workshop

Venice, March 1999

Mechanisms ofAction and

Clinical Effects ofBeta Interferon inMultiple Sclerosis

Chairmen: Kees Lucas Barry ArnasonLeiden, Chicago, USAThe Netherlands

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Workshop ParticipantsProfessor Barry Arnason (Chairman) Chicago, USA

Professor Kees Lucas (Chairman) Leiden, The Netherlands

Dr David Bates Newcastle-upon-Tyne, UK

Professor Michel Clanet Toulouse, France

Dr Frank Dahlke Berlin, Germany

Professor George Ebers Oxford, UK

Professor Cesare Fieschi Rome, Italy

Dr Joseph Frank Bethesda, USA

Dr Sten Fredrikson Stockholm, Sweden

Professor Reinhard Hohlfeld Munich, Germany

Professor Ioannis Milonas Thessaloniki, Greece

Professor Mathias Mller Vienna, Austria

Dr Lex Nagelkerken Leiden, The Netherlands

Professor Chris Polman Amsterdam, The Netherlands

Dr Richard Ransohoff Cleveland, USA

Dr Anthony Reder Chicago, USA

Professor Nancy Ruddle New Haven, USA

Dr Takahiko Saida Kyoto, Japan

THE MS FORUMThe MS Forum is an initiative funded by Berlex Laboratorieswhich has been established to improve the awareness andunderstanding of multiple sclerosis on an international basis.Founded in 1993, the MS Forum draws its direction from anExecutive Committee of internationally renowned opinionleaders. It is committed to encouraging debate and exchange ofknowledge in all aspects of patient care, from which will emergeclear and practical guidelines for the care of people withmultiple sclerosis.



















THE MS FORUMThe MS Forum is an initiative funded by Schering AG which hasbeen established to improve the awareness and understandingof multiple sclerosis on an international basis. Founded in 1993,the MS Forum draws its direction from an Executive Committeeof internationally renowned opinion leaders. It is committed toencouraging debate and exchange of knowledge in all aspects ofpatient care, from which will emerge clear and practicalguidelines for the care of people with multiple sclerosis.

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THE MS FORUMThe MS Forum is an initiative funded by Berlex Canada Incwhich has been established to improve the awareness andunderstanding of multiple sclerosis on an international basis.Founded in 1993, the MS Forum draws its direction from anExecutive Committee of internationally renowned opinionleaders. It is committed to encouraging debate and exchange ofknowledge in all aspects of patient care, from which will emergeclear and practical guidelines for the care of people withmultiple sclerosis.

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ContentsPage

Workshop Participants ii

Introduction iv

Chapter One

The Clinical Evidence for Beta Interferon in 1Multiple Sclerosis

Chapter Two

Implications of Beta Interferon Clinical Trial Results 5

Chapter Three

Multiple Sclerosis Immunopathology and 8Immunomodulation

Chapter Four

Biological Markers of Beta Interferon Activity 12

Chapter Five

Kinetics of Beta Interferon Treatment 16

Chapter Six

Clinical Significance of Magnetic Resonance Imaging 19Findings with Beta Interferon

Chapter Seven

Beta Interferon-mediated Intracellular Signalling 23

Chapter Eight

Effect of Beta Interferon on Immune Regulation 27

Chapter Nine

Effect of Beta Interferon on Mediators of Inflammation 31

Chapter Ten

Effect of Beta Interferon on Immune Activation 34

Chapter Eleven

Effect of Beta Interferon on the Natural History of 37Multiple Sclerosis

Concluding Remarks 41

i v

IntroductionToday, the clinical course of both relapsing/remitting and secondary progressive forms of multiple sclerosis (MS) canfavourably be altered using beta interferon. This is the result of almost 20 years of clinical development, beginningwith a study by Jacobs et al that was published in 1981.1 This report provided the first indication that natural humanfibroblast (beta) interferon, administered intrathecally, was capable of reducing relapse rates in people withrelapsing/remitting MS.

Since this initial report, numerous further clinical studies, including several large phase III clinical trials, haveconfirmed the benefit of beta interferon in two clinical forms of MS. However, there is one important question thathas yet to be answered. How does beta interferon mediate its effects both on relapse rate and on diseaseprogression in MS?

The original rationale for exploring the effects of the interferons in MS was based on the premise that MS was avirally mediated disease. Viraemic episodes frequently presage a clinical MS relapse, and it was hoped thatadministering the bodys natural antiviral agents would reduce the impact of the viral episode and thereby influencedisease activity. However, this view turned out to be simplistic. A trial of gamma interferon, another innate antiviralagent, dramatically worsened MS, suggesting that this agent has a role in the pathological processes underlying MS.2

Perhaps one of the greater challenges facing researchers trying to explain the mechanism of action of beta interferonin MS is the complex pathological process that causes the disease. It is clear that MS has an autoimmune basis, but itis less clear how these autoimmune reactions originate, nor exactly how the immune system causes the damage thatresults in the disease. There are many possible mechanisms that could be involved, and therefore many possible theoretical points for therapeutic intervention.

Similarly, researchers are showing that beta interferon has many diverse effects on the immune system. Some ofthese may inhibit the pathological processes underlying MS, whereas others may do the opposite. The challenge is toidentify the points of interaction between MS pathology and beta interferon activity, thereby revealing themechanism by which beta interferon exerts its beneficial effects.

The following chapters examine the available evidence for clues that may reveal the most important of theseinteractions. Under consideration are the outcomes of the major clinical trials, the MRI findings in these and otherstudies, and investigations of the biological effects of the drug. Also examined are the underlying biology of betainterferon, and its activity in relation to other inflammatory and anti-inflammatory immune mediators.Understanding these many aspects of the biology of beta interferon, and the implications of the findings, may offerthe opportunity to enhance beneficial effects, minimise potentially deleterious ones, and, overall, improve thetreatments currently available for people with MS.

References1. Jacobs L, OMalley J, Freeman A, Ekes R. Intrathecal interferon reduces exacerbations of multiple sclerosis. Science 1981; 214: 10261028.

2. Panitch HS, Hirsch AL, Haley AS, Johnson KP. Exacerbations of multiple sclerosis in patients treated with gamma interferon. Lancet 1987; i:893895.

O N E

C H A P T E R O N E

The Clinical Evidence for Beta Interferonin Multiple Sclerosis

Beta interferon was the first agent proven to favourably alter the course of multiple sclerosis (MS). Several largeclinical trials, and many smaller trials, have demonstrated this effect using both clinical and magnetic resonanceimaging (MRI) parameters. Before considering the mechanisms underlying the observed clinical benefit, it is useful toreview recent trial outcomes to assess the magnitude of this effect in different types of MS.

Efficacy of Beta Interferon in Relapsing/Remitting MSTo date, three preparations of beta interferon have been studied in large Phase III clinical trials, and these are nowavailable for use in the clinics of many countries to treat people with relapsing/remitting MS. Numerous smaller trialshave also been conducted using these three agents.

Table 1 outlines the designs of three clinical trials of beta interferon.15 Each was unique in design, treatmentprotocol, enrolled patient population and clinical outcomes. This prevents direct comparison of the trials.Nevertheless, a review of the clinical trial outcomes may provide useful insights into the mechanism of action.

Relapse-related outcomes of the three clinical trials are outlined in table 2. These outcomes demonstrate the clinicaleffect of beta interferon on disease activity, and on related clinically important consequences. The implications ofthese findings will be discussed in chapter two.

IFN MS Study Group13 MSCRG4 PRISMS5Duration 2 years plus 1-year extensiona Up to 2 years 2 years

Preparation Interferon beta-1b Interferon beta-1a Interferon beta-1a

Treatment protocol 8 MIU versus 1.6 MIU 6 MIU versus placebo 12 MIU versus 6 MIUversus placebo versus placebo

Frequency and route Every other day, sc Once a week, im Three times a week, scof administration

EDSS range 05.5 1.03.5 05.0

Primary outcome measures Relapse rate Time to onset of Relapse rateProportion of patients relapse-free confirmed progression

Secondary outcome measures Time to first relapse Relapse rate Time to first relapseRelapse duration and severity Time to first relapse Proportion of patients Hospitalisations relapse-freeChange in EDSS Relapse severityProportion of patients with Steroid useconfirmed progression Hospitalisations

Ambulation indexArm function testsChange in EDSS

MRI outcome measures Disease activity, Disease activity, Disease activity,disease burden disease burden disease burden

a rolling recruitment enabled 5-year data to be obtained in some patients

Table 1: Design of three clinical trials of beta interferon in relapsing/remitting MS

Interferon beta-1a4

Outcome Placebo Interferon beta-1a 6 MIU P value

Annual relapse rate 0.82 0.67 (18%) 0.04

Median time to first relapse (days) 253 331 (+31%) ns

Proportion of patients relapse-freea (%) 26 38 nr

ns not significant; nr not reported; a subset of patients on study for 104 weeks

Table 2: Relapse-related outcomes in relapsing/remitting MS trials (continued overleaf)

Disability-related outcomes were assessed in all of the studies. In each study, progression was defined as an increaseof 1 EDSS point, confirmed after 3 or 6 months. Other disability-related outcomes were also assessed. These findingsare outlined in table 3; again, the implications of these findings are discussed in chapter two.

MRI outcomes were particularly important in all the trials. These are outlined in chapter six, together with theimplications of those findings for the mechanism of action of beta interferon.

T W O

Interferon beta-1a5

Placebo Interferon beta-1a

6 MIU 12 MIU

Relapses per patient (mean)c 2.56 1.82 (27%) 1.73 (33%)

Efficacy of Beta Interferon in Secondary Progressive MSOnly one trial of beta interferon in people with secondary progressive MS has been published.6 Further trials areunderway or are awaiting publication in this patient population. Unlike all but one trial in relapsing/remitting MS,the primary outcome measure was disability-related. Table 4 outlines the design of the trial, and disability-relatedoutcomes are presented in table 5.

Additional points of interest include:

no effect of baseline EDSS score on the proportion of patients with confirmed progression no effect of relapses prior to, or during, the study on the primary endpoint. However, relapses during the study

slightly increased the rate of progression in all patients.

Relapse-related outcomes are described in table 6 overleaf. Due consideration is given to the behaviour ofsecondary progressive MS some patients do not experience overt clinical relapses, and in those that do, relapserate typically is much less than in relapsing/remitting MS.

MRI outcome measures are discussed in chapter six. The outcomes of studies using the newer MR techniques,including magnetisation transfer imaging and atrophy measurements, have not yet been published.

A second trial of beta interferon in secondary progressive MS has recently been completed. Initial reports suggestthat interferon beta-1a at doses of 6 MIU and 12 MIU three times a week failed to show a significant treatmenteffect on the primary outcome measure of time to onset of confirmed progression.7 On secondary outcomemeasures related to relapses, the high dose showed significant treatment effects. MRI disease activity was alsoreduced, and a doseresponse effect was seen. However, the full implications of these findings must awaitpublication of the study results.

T H R E E

Duration 3 years, interim analysis after all subjects had completed 24 months treatment

Treatment protocol 8 MIU interferon beta-1b versus placeboFrequency and route of administration Every other day, sc

EDSS range 3.06.5Primary outcome measures Time to confirmed 1 pointa progression of EDSSSecondary clinical outcome measures Time taken to become wheelchair-bound

Annual relapse rateTertiary outcome measures Proportion of patients with confirmed progression

EDSS at endpointTime to first relapseProportion of patients with moderate or severe relapses

MRI outcome measures Disease activityDisease burdenOther MR techniques

a 0.5 point between EDSS 6.07.0

Table 4: Design of the clinical trial of interferon beta-1b in secondary progressive MS

Outcome Placebo Interferon beta-1b 8 MIU P value

Time to confirmed progression (40th percentile) Delayed up to 12 months

SummaryThree preparations of beta interferon have proven to be effective in relapsing/remitting MS. The benefits arereasonably consistent, with disease activity being reduced by 1834% depending on the trial and dosing arm. Atsimilar weekly doses, comparable clinical benefit was observed. Treatment benefit is also apparent on relapse-related outcomes including hospitalisations and steroid use. All the preparations reduced the number of people withprogression of disability, significantly so in two trials.

The effectiveness of beta interferon in secondary progressive MS has the support of a single published trial, althoughthese results are convincing. Both the time to confirmed progression and the time taken to become wheelchair-bound were significantly delayed after only 1 year of treatment. Both completed trials confirm that the effects ofbeta interferon on disease activity in secondary progressive MS reflect the observations in relapsing/remitting disease.

Beta interferon is, therefore, an effective treatment for people with relapsing/remitting MS, and there is convincingdata from one published study that interferon beta-1b also offers clinically meaningful benefit in people withsecondary progressive MS.

References1. The IFN Multiple Sclerosis Study Group. Interferon beta-1b is effective in relapsing-remitting multiple sclerosis. I. Clinical results of a

multicenter, randomized, double-blind, placebo-controlled trial. Neurology 1993; 43: 655661.

2. Paty DW, Li DK, University of British Columbia MS/MRI Study Group and the IFN Multiple Sclerosis Study Group. Interferon beta-1b iseffective in relapsing-remitting multiple sclerosis. II. MRI analysis results of a multicenter, randomized, double-blind, placebo-controlled trial.Neurology 1993; 43: 662667.

3. The IFN Multiple Sclerosis Study Group and The University of British Columbia MS/MRI Analysis Group. Interferon beta-1b in the treatmentof multiple sclerosis: Final outcome of the randomized controlled trial. Neurology 1995; 45: 12771285.

4. Jacobs LD, Cookfair DL, Rudick RA, et al. Intramuscular interferon beta-1a for disease progression in relapsing multiple sclerosis. Ann Neurol1996; 39: 285294.

5. PRISMS Study Group. Randomised double-blind placebo-controlled study of interferon beta-1a in relapsing/remitting multiple sclerosis. Lancet1998; 352: 14981504.

6. European Study Group on interferon beta-1b in secondary progressive MS. Placebo-controlled multicentre randomised trial of interferon beta-1b in treatment of secondary progressive multiple sclerosis. Lancet 1998; 352: 14911497.

7. Paty DW, the SPECTRIMS Study Group. Secondary progressive efficacy clinical trial of recombinant interferon beta-1a in MS (Abstract).Presented at the 9th ENS meeting, Milan, Italy, 1999.

F O U R

Outcome Placebo Interferon beta-1b 8 MIU P value

Annual relapse rate

overall 0.64 0.44 (31.3%) 0.0002

with prior relapsesab 0.77 0.56 (27.2%)

without prior relapsesab 0.33 0.19 (42.4%)

Median time to first relapse (days) 403 644 0.003

Annual rate of moderate and severe relapsesb 0.5 0.33 (34%) 0.001

Proportion of patients with moderate or 53.1 43.6 0.0083severe relapses (%)

Proportion of patients receiving steroid therapy (%) 67.9 53.6

F I V E

C H A P T E R T W O

Implications of Beta Interferon ClinicalTrial Results

The results of the clinical trials in relapsing/remitting and secondary progressive multiple sclerosis (MS) clearlydemonstrate that beta interferon is beneficial for people with these forms of the disease. Careful consideration of thetrial results, however, reveals features that may have important implications for the mechanism of action of betainterferon in MS. This chapter will consider a number of the features and will speculate on their influence on ourunderstanding of the mechanism of action of beta interferon.

Implications from Relapsing/Remitting MS TrialsThe predominant features of relapsing/remitting MS are the random, sometimes frequent, bouts of inflammation,demyelination and other pathological processes that result in clinically apparent signs. Magnetic resonance imaging(MRI) also shows foci of inflammation in the brain that appear and resolve with a frequency greater than that ofclinically apparent signs. Changes in disability are less pronounced, but accumulation of MRI-determined burden ofdisease is a relentless process. Thus, there are several, apparently disparate, aspects to the clinical picture ofrelapsing/remitting MS.

The main trials of beta interferon in this form of MS15 are reasonably consistent in that they show:

approximately one third reduction in relapse rate at higher doses minor doseresponse effects on most outcome measures (figure 1) rapid onset of effect within 1 year for relapse rate and within a few weeks for MRI disease activity disproportionately large effects on inflammation as measured by MRI disease activity slowing in the accumulation of MRI burden of disease a tendency to reduce the number of patients with observed progression of disability.

The doseresponse effect is observedclinically (chapter one), and can also beseen when the activity of beta interferonis assessed using biological responsemarkers (chapter four). Increasing thedose brings greater benefits in those trialswith two dose arms, but it is also clearthat there may be a plateau effect atapproximately 3035% reduction inrelapse rate. In the PRISMS study, thedoubling of the dose did not achieve aproportionate reduction in the relapserate. In fact, increasing the dose tends toincrease the incidence of side-effects,hinting that greater doses will adverselyaffect tolerability with little clinical gain.

The observations that severe relapseswere less frequent, and that the clinicaleffect on severe relapses was greater than on relapses overall in the interferon beta-1b trial, suggest that one effect ofbeta interferon is to reduce the aggressiveness of ongoing inflammatory events. This is in addition to reducing thenumber of such events.

None of the trial reports indicate whether beta interferon reduces the duration of relapses. This outcome is difficultto measure, but it has implications for the mechanism of action of beta interferon. If relapse duration is unchanged,it would suggest that once the inflammatory event has started, beta interferon does not enhance the downregulatory

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mechanisms involved in resolving the lesion. If relapses are shortened by treatment, this would suggest thatresolution of lesions is accelerated, and that beta interferon acts on both of these aspects.

An important observation from trials involving high doses of beta interferon is that the clinical benefit begins within12 months of starting treatment indeed, an indication of the clinical benefit of beta interferon in the interferonbeta-1b trial was apparent after 2 months. Time to maximum clinical effect may be a further doseresponse effect,implying some cumulative effect to treatment. However, the marked reduction in relapse rate after 1 year with 1.6MIU interferon beta-1b every other day, in contrast with the lesser effect of 6 MIU interferon beta-1a once a week,may suggest an alternative explanation that beta interferon levels need to be maintained above baselinethroughout the week to offer a treatment effect at these relatively low doses. Also of note is that, in the interferonbeta-1b trial, the magnitude of the reduction in relapse rate was sustained for up to 5 years, suggesting a long-termbenefit. What remains to be answered is whether clinical benefit persists after treatment has stopped, and if so, forhow long?

In all of the large trials, MRI parameters were measured, and the results largely supported the clinical findings.25,6

Essentially, both disease activity and the accumulation of disease burden were reduced by appropriately dosed betainterferon. These findings are discussed in greater detail in chapter six.

Each of the beta interferon trials showed that fewer treated patients accumulated permanent neurological deficit.Even today, the findings, especially at the lower end of the EDSS, remain controversial given the relative insensitivityof the assessment scales and clinical uncertainty of minor changes in disability. Nevertheless, since all the trials hintat such a benefit, and given that relapse rate and disability progression are weakly correlated, the suggestion is thatbeta interferon may act directly on certain aspects of the pathology underlying disease progression, and can slowprogression of MS in the early stages of the disease. In more advanced, secondary progressive MS, the trial ofinterferon beta-1b provided robust evidence to show a slowing down in the progression of disability.7

Implications from the Secondary Progressive MS TrialTo date, only one trial report of beta interferon in secondary progressive MS has been published.7 The findings haveimportant implications for the underlying pathology of secondary progressive MS, the mechanism of action of betainterferon, and for the treatment of all people with this type of MS. A second trial has been completed, and thepreliminary findings presented,8 but an assessment of the implications of these findings for the mechanism of actionof MS must await publication of the trial report. The results of this study will not be discussed in this chapter.

The most important clinical finding of the interferon beta-1b trial7 was a marked delay in the time it took to developconfirmed disability progression. This supports the suggestion from relapsing/remitting studies that beta interferoncan beneficially act on the pathology that underlies this aspect of the disease. From the mechanism of actionperspective, the most important finding was that this beneficial effect was observed regardless of the occurrence ofrelapses during, or in the 2 years prior to, the study. This suggests that the pathology underlying disability is distinctfrom the pathology that causes relapses, although the slightly more rapid progression of patients treated or not

Observation Implications

Doseresponse effect High, tolerable doses are probably essential to obtain maximum benefit Plateau effect Beta interferon does not address all relapse-related pathology Reduction in relapse severity Beta interferon may downgrade the severity of inflammation in active lesions No reported reduction in relapse duration Beta interferon may not accelerate the resolution of ongoing

inflammatory lesions

Rapid onset of activity Beta interferon acts directly on an early step in pathology Sustained clinical effect MS pathology remains sensitive to sustained beta interferon treatment Poor correlation between relapses Suggestion of disparate but linked pathology underlying each clinical aspect

and progression of disability

Table 7: Beta interferon mechanism of action: implications from the relapsing/remitting MS studies

S E V E N

with relapses does indicate some overlap. Thus the mechanism of action of beta interferon is likely to be sufficientlydiverse to target both pathologies.

Other clinical outcomes include the clinical effect of beta interferon on relapse rate (which was consistent with thatobserved in relapsing/remitting MS an approximate 30% reduction in relapses), a dramatic reduction in MRIdisease activity, and a reduction in MRI disease burden that contrasted with an annual increase in the placebogroup.

SummaryOutcomes of large clinical trials of beta interferon all point to the same fact: this agent is effective inrelapsing/remitting MS and one agent is proven to be effective in secondary progressive MS. They also suggest that thepathology of MS is complex, and that beta interferon has positive effects on various components of this diversepathology, either by targeting one common aspect, or by targeting several diverse features. The mechanism of actionis also likely to be consistent between the two stages of the disease and not influenced by pre-existing pathology.These, and other interpretations of the trial outcomes, may prove to be useful in elucidating the most importanteffects of this agent in MS.

References1. The IFN Multiple Sclerosis Study Group. Interferon beta-1b is effective in relapsing-remitting multiple sclerosis. I. Clinical results of a

multicenter, randomized, double-blind, placebo-controlled trial. Neurology 1993; 43: 655661.

2. The IFN Multiple Sclerosis Study Group and The University of British Columbia MS/MRI Analysis Group. Interferon beta-1b in the treatmentof multiple sclerosis: Final outcome of the randomized controlled trial. Neurology 1995; 45: 12771285.



5. Freedman MS, for the OWIMS Study Group. Dose-dependent clinical and magnetic resonance imaging efficacy of interferon beta-1a (Rebif) inmultiple sclerosis. Ann Neurol 1998; 44: 992. Abstract 9.

6. Paty DW, Li DK, University of British Columbia MS/MRI Study Group and the IFN Multiple Sclerosis Study Group. Interferon beta-1b iseffective in relapsing-remitting multiple sclerosis. II. MRI analysis results of a multicenter, randomized, double-blind, placebo-controlled trial.Neurology 1993; 43: 662667.

7. European Study Group on interferon beta-1b in secondary progressive MS. Placebo-controlled multicentre randomised trial of interferon beta-1b in treatment of secondary progressive multiple sclerosis. Lancet 1998; 352: 14911497.

8. Paty DW, the SPECTRIMS Study Group. Secondary progressive efficacy clinical trial of recombinant interferon beta-1a in MS (Abstract).Presented at the 9th ENS meeting, Milan, Italy, 1999.

Further ReadingNew treatments for multiple sclerosis a review of clinical trials. Proceedings of the MS Forum Symposium, Istanbul, Turkey, 1997. Worthing: PPS Europe, 1998.

Observation Implications

Marked effect on disability progression Confirmation that beta interferon can slow the progression of the underlying pathology Effect on relapses consistent Consistent relapse-related pathology between the disease types

with relapsing/remitting MS trials

Benefit on progression Disparate but overlapping pathologies responsible for relapses and progression ofindependent of relapses disability

Table 8: Beta interferon mechanism of action: implications from the secondary progressive MS trial

E I G H T

C H A P T E R T H R E E

Multiple Sclerosis Immunopathology andImmunomodulation

Although the factors that induce multiple sclerosis (MS) or trigger relapses remain obscure, the pathological processesinvolved in causing demyelination are generally well understood. What are less well understood are the mechanismsthat translate demyelination into sustained neurological deficit. Research continues to clarify the fine details of theseprocesses, and also tries to identify potential avenues by which these pathological processes could be modulated tobring about clinical benefit. This chapter will summarise our current understanding both of the immunopathology ofMS and of the potential therapeutic approaches.

The Inflammatory Basis of MSIt is now generally agreed that the initial step in an MS episode is the activation of T cells specific for myelinantigens. Many hypotheses have been put forward to explain this activation of autoreactive T cells, includingbystander activation, superantigen stimulation and cross-reaction based on molecular mimicry, but as yet none canbe considered to be definitive. Nevertheless, activated autoreactive T cells can be found within the circulation(figure 2),1 where they may, in certain individuals, subsequently generate an autoimmune response.

Activated T cells all share the abilityto cross the bloodbrain barrier. This is normal behaviour, and does not usually incur anyconsequences. However, autoreactivecells that subsequently encountertheir target antigen in the appropriatecontext within the central nervoussystem (CNS) will respond byproducing cytokines and chemo-attractants that will stimulate aninflammatory response. Clonalexpansion of the autoreactive T cells,and the migration of accessory cellsinto the inflamed region, enhance theinflammatory process and lead totissue injury.

These inflammatory episodes are self-limiting. The activation of pro-inflammatory cells also results instimulation of immunomodulatorypathways, including the production ofinhibitory cytokines, which suppress the activity of autoreactive T cells, macrophages and other accessory cells. Thisresults in a resolution of the inflammation within several days to a few weeks.

The Pathological Consequences of InflammationThe active inflammatory lesion can have several pathological consequences. The first, and probably the one withminimal long-term consequences, is direct conduction block of nerve signals by pro-inflammatory cytokines (figure 3).2 This can rapidly induce neurological dysfunction. However, the increasing concentrations ofimmunomodulatory cytokines, and the consequent reduction of inflammatory mediators towards the end of theinflammatory episode, relieve the conduction block and restore function to otherwise undamaged axons.

Systemiccirculation

Bloodbrainbarrier

Central nervoussystem

Recruitment of cells Myelin injury

Conduction block

MicrogliaAstrocyte

Activation ofendothelium

Antigen-presenting cellAutoreactiveT cell

T cell

Activation bysuperantigens, molecular

mimicry, or unknownmechanisms

Loss of peripheraltolerance

Activatedautoreactive T cell

Chemokines

Cytokines

Figure 2: Pathogenesis of MS. Reproduced with permission from Rudick et al. New Engl J Med 1997.1

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The second and most widely recognisedconsequence of inflammation isdemyelination (figure 4).3 Loss of theinsulating myelin sheath decreases theefficiency of action potential conduction.This leads to reduced conduction velocityand, in severe cases, conduction block.4

This also has less obvious but equallyimportant implications for the health ofthe axon itself. Loss of myelin destabilisesthe molecular structure of the axonalcytoskeleton, possibly predisposing thenerve cell to further injury.5

Axonal recovery following demyelinationis a two-stage process. The first stage isaxonal adaptation, which takes placeover several days and involvesredistribution of sodium channels, aprocess that may be under the control ofastrocytes. Within a week, the sustained

but slow transmission of action potentials begins across the demyelinated segment, restoring some neurologicalfunction. However, allied to this is electrical instability. Ectopic impulses may be generated, and these can travel ineither direction along the naked axon. Such ectopicimpulses may lead to tingling or pain sensations. Thesecond stage of recovery is remyelination, mediated bysurviving oligodendrocytes or maturing oligodendrocyteprecursors within the brain parenchyma. Remyelinatedaxons typically show shortened internodes and thin myelin.This process re-establishes stable conduction of actionpotentials at speeds approaching normal, leading, over aperiod of some weeks, to partial or complete restoration ofneurological function.

The loss and subsequent restoration of myelin and neural functionprovides a good model for the relapsing/remitting nature of early MS.However, it is not a useful explanation of the progressive disease. Thefinal direct pathological consequence axonal transection (figure 5) mayprovide a good explanation of the progressive worsening of MS.

In a recent study, active and chronic MS lesions were examined forpathological axonal changes using immunohistochemistry.6 The firstimportant observation was extensive pathological changes to the axonalneurofilament in MS lesions. In acutely active lesions, these injured axonswere identified throughout the inflamed area, whereas in chronic lesionsthey tended to be located at the margins, where inflammation wasongoing. The second observation, confirmed by three-dimensionalconfocal microscopy, was the presence of terminal ovoids markingtransected axons. These ovoids were located in areas of activedemyelination. Table 9 shows the number of transected axons in differenttypes of lesion. These findings demonstrate that axonal transection is anongoing process even at the earliest stage of MS. Axonal loss may be amajor contributing factor to permanent deficit and cerebral atrophy,which are both features of progressive MS.3

NORMAL CONDUCTIONBLOCK

RECOVERY

Figure 3: Conduction block by inflammatorycytokines

NORMAL DEMYELINATION ADAPTATION REMYELINATION

Figure 4: Demyelination, axonal recovery andremyelination

Tissue sample Lesions Transected axons/mm3analysed (mean SE)

Active lesions 5 11,236 2275

Chronic active lesions 13

edge 3138 688

core 875 246

Non-lesion white matter 11 17 2.8

Control white matter 5 0.7 0.7

Table 9: Distribution and extent of axonal transection in MS lesions

NORMAL DEMYELINATION and TRANSECTION NEURONAL DEATH

Figure 5: Demyelination and axonal transection

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Potential Targets for ImmunomodulationThere are many potential targets for immunomodulatory activity in the immunopathological process underlying MS.These include:

activation of pro-inflammatory autoreactive T cells reactivation of autoreactive T cells by antigen-presenting cells (APCs) within the CNS production of pro-inflammatory cytokines within the CNS conduction blockade by pro-inflammatory cytokines activation of bloodbrain barrier endothelium and bloodbrain barrier transit by activated autoreactive T cells recruitment of accessory cells myelin injury axonal damage, transection and loss.

In addition to these processes, the innate immunomodulatory mechanisms that self-limit the inflammatory responsemay be enhanced or supplemented.

Central to any immune response is the interaction between the T cell and the APC. This is a complex event thatincludes antigen recognition, accessory molecule signalling, adhesion molecule interaction, and cytokine productionand reception (figure 6).7 Any of these may impact on the outcome of the event and drive both the APC and the T cell along a variety of outcome pathways that will influence the character of the ensuing immune response. Betainterferon may sufficiently influence the expression of any of these molecules to modify the outcome of thisinteraction. For example, it may bias the commitment of nave T cells towards the Th1 or Th2 phenotypes (chaptereight), or it may lead to the presentation of autoantigen in a non-inflammatory context.

A major feature of the inflammatoryprocess is the recruitment ofaccessory cells into the lesion;without this influx, disease is verylimited. Studies using gadolinium-enhanced magnetic resonanceimaging (which reveals loss ofbloodbrain barrier integrity andinflammation) show that betainterferon treatment almost immedi-ately halts the development of newlesions (chapter six).8 This anti-inflammatory activity is confirmed bya decline in leucocyte count withinthe cerebrospinal fluid of treatedpatients.9 How this occurs is not fullyunderstood, but it may involveredirection of leucocyte trafficking,which would imply an influence ofbeta interferon on specific adhesionmolecules including the integrins.

In one study, levels of soluble VCAM-1 (a membrane ligand for VLA-4 that can be shed into the circulation,probably from endothelial cells) were rapidly increased in people starting beta interferon treatment.10 VLA-4 isbelieved to be important in the mechanism by which inflammatory cells migrate into active lesions. Declining VLA-4levels, and increasing soluble VCAM-1 levels, suggest a reduced potential for T cells to localise within inflammatorylesions.11 In a second study, the ability of leucocytes to cross the bloodbrain barrier was reduced by beta interferontreatment. This correlated with a reduction in the expression of matrix metalloproteinase-9 and the ability of thisenzyme to degrade fibronectin.12

CD40

CD4

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TCR

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LFA-3

CD2

I-CAM-1I-CAM-2

VCAM

VLA-4

LFA-1CD40-L

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CD28CTLA-4

Cytokines

Cytokines

Cytokinereceptors

Cytokinereceptors

ANTIGEN-PRESENTING CELL

T CELL

Integrin Ig Super-family member

Costimulation T cell/MHCcomplex

Adhesion

Figure 6: The T cell:APC interaction

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E L E V E N

Thus, beta interferon may have many potential immunomodulatory effects in MS, and several of those protectingbloodbrain barrier integrity, altering cellular trafficking and reducing the capacity of cells to cross the bloodbrainbarrier have been demonstrated. Many more are under investigation.

SummaryThe inflammatory mechanism that induces demyelination may be responsible for more of the pathologicalconsequences of MS than previously thought. Three possible outcomes can be envisaged conduction block,demyelination and recovery, or axonal transection and there is good evidence that axonal transection occurs earlyin the disease. Thus, MS may now be considered a neurodegenerative disease, different from others in that theearly, normally clinically silent, progression is illuminated by clinically apparent signs of ongoing inflammation.

Beta interferon has many effects on the ongoing inflammatory process. Perhaps the most important are protectingbloodbrain barrier integrity, altering inflammatory cell homing to the CNS, and reducing the ability of inflammatorycells to enter the CNS. Early treatment with beta interferon, with the aim of reducing the early accumulation ofpermanent neurological damage, and searching for ways to potentiate the protective effects of beta interferon, mayoffer even more effective ways to reduce the effects of this disease.

References1. Rudick RA, Cohen JA, Weinstock-Guttman B, et al. Management of multiple sclerosis. New Engl J Med 1997; 337: 16041611.

2. Bornstein MB, Crain SM. Functional studies of cultured human brain tissues as related to demyelinative disorders. Science 1965; 148:12421244.

3. Trapp BD, Ransohoff RM, Fisher E, Rudick RA. Neurodegeneration in multiple sclerosis: Relationship to neurological disability. Neuroscientist1999; 5: 4857.

4. Waxman SG. Pathophysiology of demyelinated and remyelinated axons. In: Cook SD (ed). Handbook of multiple sclerosis. 2nd Edn. NewYork: Marcel Dekker, 1996, 257294.

5. Kirkpatrick LL, Brady ST. Modulation of the axonal microtubule cytoskeleton by myelinating Schwann cells. J Neurosci 1994; 14: 74407450.

6. Trapp BD, Peterson J, Ransohoff RM, et al. Axonal transection in the lesions of multiple sclerosis. New Engl J Med 1998; 338: 278285.

7. Hohlfeld R. Biotechnological agents for the immunotherapy of multiple sclerosis: Principles, problems and perspectives. Brain 1997; 120:865916.

8. Calabresi PA, Stone LA, Bash CN, et al. Interferon beta results in immediate reduction of contrast-enhanced MRI lesions in multiple sclerosispatients followed by weekly MRI. Neurology 1997; 48: 14461448.

9. Rudick R, Cookfair D, Simonian N, et al. Cerebrospinal fluid abnormalities in a phase III trial of Avonex (IFN beta-1a) for relapsing multiplesclerosis. J Neuroimmunol 1999; 93: 814.

10. Calabresi PA, Tranquill LR, Dambrosia JM, et al. Increases in soluble VCAM-1 correlate with a decrease in MRI lesions in multiple sclerosistreated with interferon beta-1b. Ann Neurol 1997; 41: 669674.

11. Calabresi PA, Pelfrey CM, Tranquill LR, et al. VLA-4 expression on peripheral blood lymphocytes is downregulated after treatment of multiplesclerosis with interferon beta. Neurology 1997; 49: 11111116.

12. Stve O, Dooley NP, Uhm JH, et al. Interferon beta-1b decreases the migration of T lymphocytes in vitro: Effects on matrix metalloproteinase-9.Ann Neurol 1996; 40: 853863.

Biological Markers of Beta InterferonActivity

Beta interferon reduces multiple sclerosis (MS) disease activity following subcutaneous or intramuscularadministration. One of the challenges of identifying the mechanism of action is that of relating the clinical benefit inthe central nervous system to the peripheral administration. Even immediately following administration, it is difficultto detect beta interferon in the circulation with sufficient reliability. Thus, traditional assessments of the kinetics oftreatment have not been forthcoming.

An alternative approach to assessing the kinetics of beta interferon is to study its effects on biological responsemarkers (BRMs), levels of which respond to beta interferon administration. This chapter will review beta interferonBRMs and discuss the implications for treatment and the mechanism of action of beta interferon.

What are Biological Response Markers?Beta interferon has a widerange of effects on the body.One of these is to prepare boththe immune system and normalbody tissue to combat viralinfection (table 10). As part ofthis process, the expression of arange of cell-surface moleculesand soluble mediators, andproduction of metabolites, isaltered. Expression of some ofthese substances is highlysensitive to the presence of betainterferon, and their expressionmay increase or declinedramatically. Among thosemolecules with a short half-life,rapid expression and high peaklevels are those that areconsidered to be the bestmarkers.

A number of BRMs have beenused in studies of betainterferon (table 11). Because ofthe diverse influence of beta interferon on the immune system, and the uncertainty surrounding the precisemechanism of action of this agent in MS, the BRMs cannot be considered surrogate markers of efficacy in thisdisease. Nevertheless, they can be useful in providing insight into the many characteristics of this agent.

C H A P T E R F O U R

T W E L V E

Process Effect

ActivationMajor histocompatibility complex (MHC) class I expressionMHC class II expression B7.1 expression on B cells B7.2 expression on monocytes Suppressor cell function a

RecruitmentCirculating lymphocyte numbers Lymphocyte entry to lymph nodes

ExpansionInterleukin (IL)-2 production by Th1 cells bIL-2 receptor expression bT cell proliferation

TraffickingLymphocyte exit from lymph nodes Endothelial cell adhesionmolecule expression bGamma interferon production

Process Effect

Tissue damageLymphotoxin production by Th1 cells Tumour necrosis factor alpha production by macrophages bMacrophage cytolytic activity bRelease of mitogen and oxygen intermediates by macrophages bCD14 expression on macrophages b

RecoveryIL-10 production by macrophages bTransforming growth factor beta-1 productionby peripheral blood mononuclear cells bProstaglandin E2 release from macrophages b

OtherAntibody synthesis Cytotoxic T cell function Cytotoxic natural killer cell function Cytostasis

Table 10: The myriad effects of beta interferon

Biological response marker Source and function

Cellular 2',5'-oligoadenylate synthase Interferon-induced enzyme that degrades viral RNA

Serum neopterin A metabolite produced by interferon-activated macrophages

Serum 2-microglobulin A component of MHC class I antigens, upregulated by interferonsIL-10 An immunomodulatory cytokine

Serum human MxA protein An inhibitor of viral replication

Serum soluble IL-2 receptor Soluble antagonist of the pro-inflammatory IL-2

IL-12 receptor Cellular response to IL-12

Table 11: Some beta interferon biological response markers

a restored to normal in people with MS; b in vitro; increased; decreased; variable

T H I R T E E N

Insights from Biological Response Marker StudiesBRM studies have demonstrated a dose-response to beta interferon. In one study of asingle administration of interferon beta-1b orinterferon beta-1a at a range of doses androutes of administration, the response of threeBRMs appeared to be related to dose.1 Datafor human MxA are given in table 12. Thisstudy also showed that there was no apparentdifference in the biological activity of the twobeta interferons at the approved dose androute of administration.

Another aspect relating to dose is the durationof biological response to beta interferon. In astudy comparing the biological response tointerferon beta-1a and interferon beta-1busing approved doses and regimens, it wasdemonstrated that once-a-week dosing wasinadequate to maintain most BRMs abovebaseline throughout the week, whereasfrequent dosing sustained the BRM response(figure 7).2

The influence of the route of administration onthe biological activity of beta interferon has been extensively debated. Several studies have reported contradictoryfindings (table 13).1,35 Nevertheless, the available evidence favours the view that subcutaneous and intramuscularroutes of administration are equivalent in terms of their influence on beta interferon biological activity.

Treatment MIU Human MxA

Cmaxc AUCc(ng/ml SD) (ng.k/ml SD)

Interferon beta-1a, im 1 54.9 32.1 3942 28563 86.6 13.8a 6232 2406a

6b 107.0 17.5 9246 29939 106.3 26.5 8947 2652

12 159.6 11.5 14507 3614

Interferon beta-1a, sc 1 49.2 27.7 2715 21543 96.6 20.7 8971 36566 102.8 17.6 9925 37289 130.0 11.3 12316 3154

12 134.8 10.1 12775 2679

Interferon beta-1b, sc 2 81.1 17.3 5177 27974 85.0 34.7 6046 32328b 111.8 13.5 9424 396212 137.1 37.1 12157 393416 126.5 12.4 11423 1500

a n = 4 (otherwise, n = 5); b clinically utilised dose and route of administration; AUC area under the curve; c relative to baseline

Table 12: Doseresponse of the biological response marker, human MxA, to beta interferon

5A

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##

#

#

#

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*#

*#

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*#*# *#

*#

*#

*#

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*#

*#

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*# #

Interferon beta-1bInterferon beta-1a

Drug administration

*P

F O U R T E E N

The Biological Response to TreatmentIn addition to the specific BRMs used to assess the kinetics of beta interferon treatment, there are many otherbiological effects seen as a direct consequence of beta interferon treatment. Among these are the clinical outcomes changes in relapse rate and MRI activity in particular but also effects on cytokines, adhesion molecules and otherparameters of the immune system, as well as side-effects, injection-site reactions and neutralising antibody.

In people receiving long-term beta interferon treatment, there is evidence to support an influence on gammainterferon and gamma interferon-secreting cells; an increase in interleukin (IL)-10 production, with a possiblecorrelation with clinical response; a reduction in tumour necrosis factor alpha production; and other changes. Theseall point towards a shifting of the immune response towards the Th2 phenotype (chapter eight), although it is not yetclear whether the change in IL-10 production is a feature of T cells or monocytes.

Several groups have examined the change in IL-10 production in people treated with beta interferon. Kinetic studiesafter the first and fifth injection of interferon beta-1a induced significant elevations of serum IL-10 within 48 hours.6

In the same study, levels of IL-10 in the cerebrospinal fluid were increased in people with a favourable clinicaloutcome at 2 years, compared with those that continued to show progression of disease. Also reported in this studywas an immediate increase in the production of IL-4 but a slightly reduced gamma interferon production byperipheral blood mononuclear cells. The IL-10 observations are important since this cytokine is key in promotingand mediating Th2 responses, and levels appear to be correlated with clinical outcome in MS.

Others have reported different findings. In one series, IL-10 production in people starting treatment increasedmarkedly, but returned towards baseline within a few months.7 There were differences between individuals in termsof the IL-10 response, and some evidence to suggest an association between high IL-10 production and improvedclinical outcome. Overall, there is evidence that MS disease activity is related to elevated IL-12 and reduced IL-10levels, and that beta interferon acts by normalising levels of both cytokines and shifting the immune system towardsthe Th2 phenotype (chapter eight). However, there are many outstanding questions to be resolved.

Evidence for a shift towards the Th2 phenotype comes from reports of increased production of autoantibodies. Thisis a well-recognised consequence of alpha interferon treatment, and some studies suggest that this is also true forbeta interferon.8 Autoantibodies also tend to be more frequent in autoimmune diseases regardless of treatment. Itremains to be determined whether development of these autoantibodies is a common event and if they will lead toclinically manifest disease in some treated individuals. Isolated case reports suggest autoantibody-mediatedalterations to liver and thyroid function.

Neutralising antibodies to beta interferon are a recognised phenomenon in people receiving this treatment.Depending on the assay and the threshold criteria used to assess neutralising antibody titres, between 10% and 30%of people will, at some time, develop neutralising antibody. However, it is not yet clear whether different preparationsare more or less antigenic, nor is the influence of altering route or frequency of administration understood.

These antibodies may have in vivo consequences. In a study of people receiving interferon beta-1a once-weekly bysubcutaneous injection, the presence of neutralising antibody was correlated with a reduced response of neopterin

Study Findings

Strzebecher et al1 Doseresponse effect; interferon beta-1a 6 MIU im, interferon beta-1a 6 MIU sc and interferon beta-1b 8 MIU sc were equivalent

Salmon et al3 Extent and duration of the biological response of 2,5-oligoadenylate synthase to interferon beta-1a 6 MIU sc or im was independent of route of administration

Alam et al4 Interferon beta-1a 6 MIU, formulated for and administered im, gave a significantly greater biological response than interferon beta-1a 6 MIU, formulated for and administered sc

Munafo et al5 Interferon beta-1a 6 MIU formulated for sc and administered sc and im was equivalent to interferon beta-1a 6 MIU that had been formulated for and administered im

Table 13: Biological response marker study findings

F I F T E E N

and 2-microglobulin. There was also a trend towards reduced treatment benefit on MRI outcomes.9 In the NorthAmerican trial of interferon beta-1b in relapsing/remitting MS, presence of neutralising antibody suggested reducedclinical efficacy in cross-sectional analyses for relapse reduction and MRI activity.10 However, when neutralisingantibody titres were assessed longitudinally, many patients reverted to an antibody-negative status. Longitudinalanalysis found only an indication of attenuated treatment effect on relapse rate. No significant correlation betweenantibody status and progression or MRI outcomes at the 8 MIU dose were found.11 It is interesting to note that manyindividuals who develop antibody revert to, and remain, neutralising antibody-negative. In one study that followedindividuals treated with interferon beta-1b for over 8 years, neutralising antibody disappeared in the majority ofpatients.12

SummaryBRMs might be a useful tool to monitor the kinetics of beta interferon treatment. They support the view thatintramuscular and subcutaneous routes of administration are equivalent, and have been used to demonstrate adoseresponse effect and a sustained effect only with frequent dosing. The biological response to treatment alsosuggests that IL-10 levels may be increased at least over the short term and that this may bias the immune systemtowards a Th2 phenotype. Evidence of increasing autoantibody levels supports this view. Neutralising antibody isalso a biological reaction that may, over the short term, influence clinical efficacy of beta interferon, but long-termfollow-up suggests that the majority of treated individuals revert to antibody-negative status.

References1. Strzebecher S, Maibauer R, Heuner A, et al. Pharmacodynamic comparison of single doses of interferon beta-1a and interferon beta-1b in

healthy volunteers. J Interferon Cytokine Res 1999; 19: in press.

2. Williams GJ, Witt PL. Comparative study of the pharmacodynamic and pharmacologic effects of Betaseron and AVONEX. J InterferonCytokine Res 1998; 18: 967975.

3. Salmon P, Le Cotonnec J-Y, Galazka A, et al. Pharmacokinetics and pharmacodynamics of recombinant human interferon-beta in healthy malevolunteers. J Interferon Cytokine Res 1996; 16: 759764.

4. Alam J, Goelz S, Rioux P, et al. Comparative pharmacokinetics and pharmacodynamics of two recombinant human interferon beta-1a productsadministered intramuscularly in healthy male and female volunteers. Pharmaceutical Res 1997; 14: 546549.

5. Munafo A, Lugan-Trinchard I, Nguyen TXQ, Buraglio M. Comparative pharmacokinetics and pharmacodynamics of recombinant humaninterferon beta-1a after intramuscular and subcutaneous administration. Eur J Neurol 1998; 5: 187193.

6. Rudick RA, Ransohoff RM, Lee J-C, et al. In vivo effects of interferon beta-1a on immunosuppressive cytokines in multiple sclerosis. Neurology1998; 50: 12941300.

7. Rep MHG, Schrijver HM, van Lopik T, et al. Interferon-beta treatment enhances CD95 and IL-10 expression but reduces interferon-gammaproducing T cells in MS patients. J Neuroimmunol 1999; 96: 92100.

8. Durelli L, Ferrero B, Oggero A, et al. Autoimmune events during interferon beta-1b treatment for multiple sclerosis. J Neurol Sci 1999; 162:7483.

9. Rudick RA, Simonian NA, Alam JA, et al. Incidence and significance of neutralising antibodies to interferon beta-1a in multiple sclerosis.Neurology 1998; 50: 12661272.

10. The IFN MS Study Group, the University of British Columbia MS/MRI Analysis Group. Neutralising antibodies during treatment of multiplesclerosis with interferon beta-1b: Experience during the first three years. Neurology 1996; 47: 889894.

11. Petkau J, White R. Neutralising antibodies and the efficacy of interferon beta-1b in relapsing-remitting multiple sclerosis (Abstract). Mult Scler1997; 3: 402.

12. Rice GP, Pazner B, Oger J, et al. The evolution of neutralising antibodies in multiple sclerosis patients treated with interferon beta-1b.Neurology 1999; 52: 12771279.

S I X T E E N

C H A P T E R F I V E

Kinetics of Beta Interferon TreatmentBeta interferon has many effects within the body. As an immune modulator, it is capable of inducing the expression ofproteins in both cells of the immune system and normal tissue cells as part of the overall programme to controlinfection. Of the many events that are influenced by beta interferon either directly, or indirectly as a consequence ofthe activity of induced proteins only a small proportion are likely to be responsible for the clinical benefit obtained bybeta interferon treatment in multiple sclerosis (MS). This chapter will examine key features of the response to betainterferon and highlight the clues that could tell us more about the mechanism of action of this agent.

Clinical Observations with Beta Interferon TreatmentIn almost all trials of beta interferon, the observed clinical effect has been measurable within weeks of commencingtreatment. However, it is not immediate on all outcome measures. Reductions in relapse rate tend to becomeapparent from the second month of treatment, reaching a sustained maximum treatment effect from the thirdmonth. This suggests that the clinically beneficial effect is not immediate either because a period of sustainedtreatment is required to reduce the onset of relapses, or because relapses initiated before treatment was startedcontinue to cause clinical disease.

This is in contrast with the observed effects on gadolinium-enhanced magnetic resonance imaging (MRI) scans,which reveal a rapid decline in the total number of active, enhancing lesions. MRI findings generally reveal amaximal effect within 2 months of starting treatment and may be much faster.1

Flu-like SymptomsAnother clear consequence of beta interferon treatment is the occurrence of flu-like symptoms. These symptomsoccur following administration of alpha, beta or gamma interferon. Severity appears dose-dependent and inverselyrelated to body size. Flu-like symptoms also show kinetics:

in the short term, with symptoms appearing within 46 hours and fading after a further 48 hours in the longer term, where the incidence of flu-like symptoms declines from over 56% of patients experiencing

these symptoms to approximately 10% of patients by 3 months of treatment, although they may recur at anytime.

Beta interferon

A

B

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E

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+

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H

I

J

K

L

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+

+

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Positive effect on disease

Negative effect on disease

A...R Consequences*

Time (hours) Time (weeks)

*such as elevated cytokine production, reduced cell activity, etc.

How does beta interferon exert its clinical effect?

The complexity of the bodys response to beta interferon enables the expression of many proteins that maythemselves have either a direct or an indirect impact on the disease. By carefully studying the cascade of eventsfollowing beta interferon administration, some clues may be found that tell us which of these effects are duedirectly to beta interferon, or are due to one of its induced products.

The short-term kinetics of flu-like symptoms related to beta interferon are similar to those of alpha and gammainterferon. They suggest a direct, or almost direct, stimulation by beta interferon of endogenous pyrogens such asinterleukin (IL)-1, IL-6 and tumour necrosis factor alpha (TNF). It is interesting to note that beta interferon caninduce gamma interferon and that this can induce IL-6 and TNF. It is also of note that neopterin, a useful biologicalresponse marker, responds far more sensitively to gamma interferon than to beta interferon, and that levels of HLA-DR an antigen of the major histocompatibility complex are elevated on circulating monocytes 12 weeksafter starting treatment. This is another gamma interferon-mediated response.

In the longer term, the decline in beta interferon-related side-effects reflects changes in the levels of gammainterferon-secreting cells in the circulation. Immediately following initiation of beta interferon treatment, levels ofcirculating gamma interferon-secreting cells increase in approximately 60% of patients.2 Longer-term follow-upshows that the number of these circulating cells had returned to normal within 3 months. The longer-term profile offlu-like reactions to alpha interferon reflects that of beta interferon; however, in patients receiving gamma interferonthere is no decline in the frequency of symptoms over time.

Injection-site ReactionsInjection-site reactions are also typical in people injecting beta interferon via the subcutaneous route. Again, theyhave kinetics that can provide clues to their aetiology. Onset of these reactions occurs 1224 hours after injection,peaks within 12 weeks and they resolve after 34 weeks. The erythema is largely due to vasodilatation, and may bea consequence of a depot effect of administered beta interferon.

Histology of these lesions suggests that cellular infiltration begins within 18 hours of beta interferon administration.Recruited cells include CD8+ T cells, activated macrophages and a limited number of natural killer cells. In terms ofthe inducing agent, vasodilatation occurs in response to nitric oxide, which is itself produced in response to eithergamma interferon, TNF or lipopolysaccharide. CD8+ cells are preferentially activated by alpha and beta interferon,and macrophages mature and express HLA-DR in response to gamma interferon.

Implications for the Mechanism of ActionBeta interferon administration has many effects. Some are mediated directly, such as the preferential recruitment ofCD8+ T cells and expression of 2-microglobulin, and some are mediated indirectly. Many of the clinicalconsequences of beta interferon administration, such as flu-like symptoms or injection-site reactions, and molecularchanges such as neopterin production, macrophage maturation, HLA-DR expression, and pyrogen production, maybe due to the increased expression of gamma interferon, which itself is directly enhanced by beta interferon.Perhaps part of the mechanism of action of beta interferon in MS consists of a normalisation of gamma interferonresponses an effect that becomes fully established after approximately 3 months.

The effects of beta interferon on CD8+ T cells could have far-reaching consequences. Proliferation of CD8+ T cells,in part, reflects the antiviral activities of a subset of these cells in a response that supplements the innate antiviralproperties of beta interferon. However, CD8+ CD28 T cells are regulatory in nature under the appropriatecircumstances, they can produce immunosuppressive molecules such as transforming growth factor beta and therecruitment of these cells may also enhance immune suppression.

In animal models, CD8+ T cells are implicated in the prevention of relapses in experimental allergicencephalomyelitis, but not in recovery from a relapse; CD8 knockout mice typically exhibit relapsing and remittingdisease, whereas normal mice generally experience monophasic disease.3 In people with MS, CD8+ T cell functionappears to be disturbed, and this may reflect one aspect of the immune dysfunction responsible for MS.4 In vitro andclinical evidence suggests that beta interferon normalises CD8+ T cell function, and this may enhance protectionagainst onset of new relapses.

S E V E N T E E N

SummaryThe biological response to beta interferon is diverse and can be revealed not only by changes in molecular biologicalresponse markers, but also by clinical effects including MRI outcomes and side-effects of treatment. These all suggesta cascade of events following beta interferon administration, several of which may contribute to the clinical benefitof the agent. Among these events are some that are likely to be mediated by gamma interferon, including some ofthe less desirable outcomes, and some directly mediated by beta interferon, such as restoration of poor suppressor T cell function. These events provide solid evidence for the activity of beta interferon, and suggest important routesby which beta interferon may exert clinical benefit.

References1. Calabresi PA, Stone LA, Bash CN, et al. Interferon beta results in immediate reduction of contrast-enhanced MRI lesions in multiple sclerosis

patients followed by weekly MRI. Neurology 1997; 48: 14461448.

2. Dayal AS, Jensen MA, Lledo A, Arnason BGW. Interferon-gamma-secreting cells in multiple sclerosis patients treated with interferon beta-1b.Neurology 1995; 45: 21732177.

3. Arnason BGW, Dayal A, Qu Z-X, et al. Mechanisms of action of beta interferon in multiple sclerosis. Semin Immunopathol 1996; 18: 125148.

4. Antel JP, Arnason BGW, Medof ME. Suppressor cell function in multiple sclerosis: Correlation with clinical disease activity. Ann Neurol 1979;5: 338342.

E I G H T E E N

Beta interferon

Induce CD8+ T cells

Induce MHC class I

Induce IL-10, IL-4

Induce gamma interferon

Downregulate adhesionmolecules

Normalise CD8+ suppressor activity

Increase viral surveillance

Divert immune responsestowards a Th2 phenotype

Antagonise gamma interferon

Induce neopterin

Induce pyrogens and fever

Reduce inflammatory cellhoming to the CNS

Normalise gamma interferonproduction

+

+

+

+

+

?

?

+ Positive effect on disease Negative effect on disease ? Uncertain effect on disease

How beta interferon may exert its clinical effect possible effects

The consequences and kinetics of events following beta interferon administration suggest several that maycontribute to the clinical benefit of the agent.

N I N E T E E N

C H A P T E R S I X

Clinical Significance of MagneticResonance Imaging Findings with

Beta InterferonMagnetic resonance imaging (MRI) is an immensely valuable technique that can objectively assess changes in thecentral nervous system (CNS). In multiple sclerosis (MS), it is a powerful tool that allows quantification of diseaseactivity, disease burden and pathological and biochemical changes as the disease progresses. It is of greatestimportance as a tool for use in clinical trials as an efficacy outcome measure, but it may also provide useful insightsinto the mechanism of action of beta interferon in MS.

MRI in the Beta Interferon TrialsIn the clinical trials of the three beta interferon preparations, MRI proved to be very informative. All the trialsassessed both changes in T2-weighted burden of disease and disease activity according to the number of new, activeor enlarging lesions. These two techniques assess different aspects of MS pathology, much as EDSS and relapse rateassess different clinical aspects. Each can provide information relating to the mechanism of action of beta interferon.Several trials also used gadolinium (Gd)-enhancement as a measure of disease activity. This technique very likelyenables bloodbrain barrier disruption to be visualised.

Despite the variety of protocols used, the MRI findings are reasonably consistent between the various trials (tables14ad).16 MRI-determined disease activity is suppressed by 6090%. This depends on a number of factors,including the dose used and the assessment technique, and contrasts with the modest 3035% reduction in clinicalrelapse rate. Also notable is the rapid onset of effect, which typically reaches maximum within 3 months. Theseobservations suggest that the pathological processes visualised by MRI and those responsible for clinical relapsesoverlap, but that they are not identical. This may simply be due to the inability of MRI to elucidate all relapse-associated pathology. However, it does indicate that different aspects of the mechanism of action of beta interferonare being observed using clinical and MRI assessments.

Measure Placebo Interferon beta-1a 6 MIU

n Mean SD n Mean SD P value

Gd-enhancing lesion number

baseline 132 2.32 0.37 141 3.17 0.62 ns

at 1 year 123 1.59 0.31 134 1.04 0.28 0.02

at 2 years 82 1.65 0.48 83 0.80 0.22 0.05

Gd-enhancing lesion volume (mm3)

baseline 132 219.0 36.2 140 255.0 45.1 ns

at 1 year 123 96.5 21.2 134 70.0 24.9 0.02

at 2 years 82 122.4 48.5 82 74.1 38.3 0.03

n Median (range) n Median (range)

T2 lesion volume (mm3)

baseline 12,075 (nr) 9238 (nr) 0.01

change at 1 year 113 455 120 152 ns

(765019,035) (10,45517,655)

baseline 13,620 (nr) 10,210 (nr) ns

change at 2 years 80 1410 78 628 ns

(988530,645) (563030,430)

ns not significant; nr not reported

Table 14a: MRI outcomes of the beta interferon trials interferon beta-1a3,4

MRI burden of disease continues to increase in untreated individuals at approximately 5% per year. This is indicativeof the accumulation of residual pathology. However, there was a very poor correlation between this accumulationand the increase in clinically apparent neurological impairment again indicating a disparity between clinical andMRI parameters. This was further emphasised by the almost complete suppression of MRI disease burdenaccumulation in the interferon beta-1b trial over 5 years, and the failure to demonstrate convincingly a slowing ofdisease progression in this trial. Thus, until the pathological consequences of MRI disease burden are more clearlyunderstood, the implications of the treatment effect of beta interferon on this parameter are unclear.

Although most of the trialsincluded people withr e l a p s i n g / r e m i t t i n gdisease, the one publishedtrial in people withsecondary progressive MSreported similar MRIfindings. Disease activityover the first 6 months ofthe study, and betweenmonths 18 and 24, was

substantially reduced, and progression of disease burden was comparable with that in the relapsing/remitting studiesdespite a substantially greater baseline disease burden.

Overall, there are a number of implications that can be drawn from these findings:

newly active MRI lesions are a consistent feature of untreated people, reinforcing the view that MS is an ongoing,rather than an episodic, inflammatory disease

beta interferon has a marked treatment effect on disease activity in people receiving no treatment, a steady accumulation of disease burden of 510% per year is observed, reflecting

gross damage within the brain. This finding appears to be true in both mild and more severe disease cases beta interferon can slow, halt or slightly reverse accumulation of disease burden, and this can be sustained over

several years. However, the bulk of existing burden remains, suggesting that much of the observed diseaseburden is permanent or escapes the effect of beta interferon.

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Measure Placebo Interferon beta-1a

6 MIU 12 MIU P Value

T2 lesion burden (% median change over 2 years) 22 1.2 3.8

Further MRI Studies with Beta InterferonOne of the challenges of large clinical trials is that the MRI outcome measures are normally assessed in a cross-sectional fashion. Longitudinal assessment of MRI parameters can provide detailed insight into the behaviour of MSover time. As part of a natural history study, one group with MS was monitored from early in the disease everymonth using several MRI techniques.7,8 These studies show that the number of Gd-enhanced lesions correlating withbloodbrain barrier disruption can vary quite dramatically from month to month within individuals. There is a goodcorrelation between bursts of MRI activity and clinically apparent relapses. However, each individual had acharacteristic level of disease activity when averaged over several months. By contrast, the level of disease activitybetween individuals could vary considerably. Similarly, total T2-weighted disease burden was shown to vary frommonth-to-month, but overall, it increased with time. Both the intra- and inter-individual variations observed in theseserial studies may explain why the correlations between disease burden and EDSS scores in cross-sectional studiestend to be weak.

The treatment effect of beta interferon on individuals assessed in this way is marked. In the group of 14 individualsstudied by Stone et al, mean total contrast-enhancing lesion number in the 7 months prior to starting interferon beta-1b was 3.46 0.83, and during the 6 months following treatment was 0.48 0.16, representing an 86%reduction in disease activity.8 In longer-term follow-up, suppression of disease activity remains substantial, but thereis some variability after 23 years (figure 8).9 Similarly, T2 lesion load shows initial reductions that are followed byincreased variability and a trend towards increase by 3 years. This variability may be due to a number of factors.

Part of the variability in these observationsderives from individual responses totreatment. Some individuals respondimmediately and completely to interferonbeta-1b, while others continue to develop areduced number of new enhancing lesions.Over time, some individuals fail ontreatment, and they present with increasingnumbers of enhancing lesions that canapproach baseline levels. In most cases, thisis largely correlated with the appearance ofneutralising antibodies. However, thepresence of neutralising antibodies does notnecessarily imply a return of MRI lesions some individuals with particularly highneutralising antibody titres continue to showan almost complete absence of enhancinglesions. In addition to treatment failure, asmall proportion of individuals simply fail torespond to beta interferon treatment with areduction in MRI disease activity.

One interesting observation from serial studies is that cessation of beta interferon treatment in individuals respondingto treatment does not necessarily lead to an immediate rebound in disease activity or continued accumulation ofdisease burden. This may have implications both for the clinical management of people on treatment and for themechanism of action of beta interferon.

The advent of newer MRI techniques is also adding to the information on the dynamics of disease activity inindividuals. Magnetisation transfer ratio imaging provides a good marker of permanent damage resulting frominflammatory lesions, while atrophy measurement is providing some evidence that beta interferon can slow thedecline in brain volume.

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NIH baseline vs treatment interferon beta-1b trial (32 RRMS Pts)10

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SummaryMRI provides an objective tool to assess both disease activity and accumulation of disease burden in people withMS. It is apparent that MS is an ongoing, active disease, even in the early stages. Lesions are characterised by a lossof bloodbrain barrier integrity and subsequent inflammation. There is also a notable heterogeneity in MRI activitywithin the population. Treatment with beta interferon can almost completely suppress the appearance of newinflammatory lesions within a matter of a few weeks, and there is some evidence to suggest that the benefit oftreatment can be sustained for some weeks after treatment itself has ceased. However, some individuals fail ontreatment, and this may be due to the presence of neutralising antibody. Newer MRI techniques will provide greaterpathological specificity, which, it is hoped, will be able to shed more light on the mechanism of action of betainterferon.

References1. Paty DW, Li DKB, the University of British Columbia MS/MRI Analysis Group. Interferon beta-1b is effective in relapsing-remitting multiple

sclerosis. II. MRI analysis results of a multicenter, randomized, double-blind, placebo-controlled trial. Neurology 1993; 43: 662667.

2. The IFN MS Study Group, the University of British Columbia MS/MRI Analysis Group. Interferon beta-1b in the treatment of multiplesclerosis: Final outcome of the randomized controlled trial. Neurology 1995; 45: 12771285.


4. Simon JH, Jacobs LD, Campion M, et al. Magnetic resonance studies of intramuscular interferon beta-1a for relapsing multiple sclerosis. AnnNeurol 1998; 43: 7987.


6. European Study Group on Interferon beta-1b in secondary progressive MS. Placebo-controlled multicentre randomised trial of interferon beta-1b in treatment of secondary progressive multiple sclerosis. Lancet 1998; 352: 14911497.

7. Harris JO, Frank JA, Patronas N, et al. Serial gadolinium-enhanced magnetic resonance imaging scans in patients with early, relapsing-remittingmultiple sclerosis: Implications for clinical trials and natural history. Ann Neurol 1991; 29: 548555.

8. Stone LA, Frank JA, Albert PS, et al. The effect of interferon beta on bloodbrain barrier disruptions demonstrated by contrast-enhancedmagnetic resonance imaging in relapsing/remitting multiple sclerosis. Ann Neurol 1995; 37: 611619.

9. Frank JA, McFarland HF. Personal communication.

Further ReadingImaging in Multiple Sclerosis. Proceedings of the MS Forum Modern Management Workshop, 1997, Aylesbury, UK. Worthing: PPS Europe, 1997.

New Treatments for Multiple Sclerosis A Review of Clinical Trials. Proceedings of the MS Forum Symposium, 1997, Istanbul, Turkey. Worthing:PPS Europe, 1998.

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C H A P T E R S E V E N

Beta Interferon-mediatedIntracellular Signalling

At the most fundamental level, the mechanism of action of beta interferon will depend on the intracellular signallingpathway that translates the presence of the molecule at the cell surface into a pattern of gene expression within thecell nucleus. The specific response of the beta interferon receptor, overlap between intracellular signalling pathwaysof beta interferon and other mediators, and the resultant pattern of gene expression, will all contribute to ourunderstanding of the biological activity of this agent. This chapter will consider all these aspects of intracellularsignalling in response to beta interferon, and suggest possible mechanisms of action.

Intracellular Signalling The Model PathwayOver recent years, a prototype model has beendeveloped to describe the signalling pathway thatconveys a chemical signal the presence of acytokine, a hormone or other signalling molecule or a receptor-mediated intercellular signal at thesurface of the cell into an altered pattern of geneexpression within the cell nucleus. The followingevents form the basis of this signalling pathway(figure 9):

binding of a chemical messenger or receptorligand to a specific receptor at the cell surface,resulting in a signal

transduction of the signal across the cellmembrane, either directly or via an accessorymolecule

phosphorylation of a specific intracellularprotein by the receptor or accessory molecule

transport of the phosphorylated intracellularsignalling protein, or transduction of the signalvia additional molecules, to the nucleus

interaction between the phosphorylatedsignalling protein and transcription controlelements, leading to modified gene expression.

In reality, there are many different pathways by which a signal can be transduced to the cell nucleus, and many ofthese pathways overlap and interact. This allows for great flexibility in the cellular response to different stimuli, butgreat complexity when attempting to understand the specific responses to a single stimulus.

Beta Interferon Intracellular SignallingOnly in the last few years has the signalling pathway used by the interferons been identified, and work continues toaddress many outstanding questions. Cloning of the human genes for the type I (alpha and beta) and type II (gamma)interferon receptors enabled researchers to show that none of the receptor chains contained intrinsic tyrosine kinaseactivity within their intracellular domains an essential function of signal transducers at the cell surface. This impliesthat, in order to activate an intracellular signalling pathway, an accessory molecule with this activity must beassociated with these receptor chains.1

The Janus family of tyrosine kinases (JAK) was first discovered using genetic screening techniques that identifiedgenes with tyrosine kinase catalytic domains.2 Further analysis revealed a family of four mammalian JAK kinases withcommon features (see box: The JAK family of tyrosine kinases). It was found that members of the JAKs bound

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Mechanism of Action and Clinical Effects of Beta-Interferon in MS (Venice Workshop 1999)

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