NeuroRx威: The Journal of the American Society for Experimental NeuroTherapeutics
`
`Imaging of Multiple Sclerosis: Role in Neurotherapeutics
`
`Rohit Bakshi,* Alireza Minagar,† Zeenat Jaisani,* and Jerry S. Wolinsky‡
`
`*Departments of Neurology and Radiology, Partners MS Center, Center for Neurological Imaging, Brigham and Women’s
`Hospital, Harvard Medical School, Boston, Massachusetts 02115; †Department of Neurology, Louisiana State University Health
`Sciences Center, Shreveport, Louisiana 71130; and ‡Department of Neurology, University of Texas Health Science Center at
`Houston, Houston, Texas 77030
`
`Summary: Magnetic resonance imaging (MRI) plays an ever-
`expanding role in the evaluation of multiple sclerosis (MS).
`This includes its sensitivity for the diagnosis of the disease and
`its role in identifying patients at high risk for conversion to MS
`after a first presentation with selected clinically isolated syn-
`dromes. In addition, MRI is a key tool in providing primary
`therapeutic outcome measures for phase I/II trials and second-
`ary outcome measures in phase III trials. The utility of MRI
`stems from its sensitivity to longitudinal changes including
`those in overt lesions and, with advanced MRI techniques, in
`areas affected by diffuse occult disease (the so-called normal-
`appearing brain tissue). However, all current MRI methodology
`suffers from limited specificity for the underlying histopathol-
`ogy. Conventional MRI techniques, including lesion detection
`and measurement of atrophy from T1- or T2-weighted images,
`
`have been the mainstay for monitoring disease activity in clin-
`ical trials, in which the use of gadolinium with T1-weighted
`images adds additional sensitivity and specificity for areas of
`acute inflammation. Advanced imaging methods including
`magnetization transfer, fluid attenuated inversion recovery, dif-
`fusion, magnetic resonance spectroscopy, functional MRI, and
`nuclear imaging techniques have added to our understanding of
`the pathogenesis of MS and may provide methods to monitor
`therapies more sensitively in the future. However, these ad-
`vanced methods are limited by their cost, availability, complex-
`ity, and lack of validation. In this article, we review the role of
`conventional and advanced imaging techniques with an empha-
`sis on neurotherapeutics. Key Words: Multiple sclerosis, mag-
`netic resonance imaging, brain atrophy, diffusion imaging,
`magnetization transfer, spectroscopy, functional imaging.
`
`INTRODUCTION
`
`Since introduced into clinical medicine, magnetic res-
`onance imaging (MRI) has played expanding roles in the
`evaluation of multiple sclerosis (MS). These include its
`essential place in the initial evaluation of patients sus-
`pected of having the disease to secure and sometimes
`reject the diagnosis of MS,1,2 as a prognostic tool at first
`presentation of symptoms highly suspicious of acute in-
`flammatory CNS demyelination,3 in providing primary
`outcome measures in phase I/II trials, and as a source of
`critical supportive outcome measures in phase III trials
`of MS therapeutics. The utility of MRI in MS in large
`measure stems from its extreme sensitivity to changes in
`regional proton relaxation times that occur with pro-
`cesses that alter tissue water content and constraints on
`hydrogen molecule motion, particularly those associated
`
`Address correspondence and reprint requests to Rohit Bakshi, M.D.,
`FAAN Brigham & Women’s Hospital Harvard Medical School, 77
`Avenue Louis Pasteur, HIM 730, Boston, MA 02115. E-mail:
`rbakshi@bwh.harvard.edu.
`
`with tissue bound and free water molecules. However, all
`current MRI methodology remains insensitive to the un-
`derlying disease processes that give rise to these alter-
`ations. Consequently, the specificity of altered MRI sig-
`nals is limited, and overinterpretation of MRI to imply
`specific histopathologic tissue alterations abounds. Most
`patterns and distributions of lesions found on conven-
`tional and even advanced MRI are neither disease-spe-
`cific nor reflect a specific histopathology. As a result, a
`broad differential diagnosis usually remains when MRI
`is viewed in isolation from the clinical history, physical
`and neurologic findings and laboratory investigations.
`Nevertheless, understanding the sequence of events that
`correlate with conventional MRI-visible lesion forma-
`tion, and the most characteristic topography of lesions in
`the cerebrum, brainstem, and spinal cord help to deter-
`mine the likelihood that a patient has MS and provide
`reasonable markers by which to infer therapeutic effects
`on the evolving underlying disease process.
`Current conventional MRI consists of several series of
`image acquisitions based on generally available pulse
`sequences developed to provide optimal tissue contrast
`
`Vol. 2, 277–303, April 2005 © The American Society for Experimental NeuroTherapeutics, Inc.
`
`277
`
`Mylan Pharms. Inc. Exhibit 1040 Page 1
`
`

`
`278
`
`BAKSHI ET AL.
`
`for routine clinical diagnostic work. In general, these have
`also been the mainstay for addressing disease activity in
`clinical trials. A standardized approach to imaging MS pa-
`tients developed by the Consortium of MS Centers (http://
`www.mscare.org/pdf/MRIProtocol2003.pdf) includes sag-
`ittal fluid attenuated inversion recovery (FLAIR), axial dual
`echo proton density and T2 weighted (TE1 usually ⬍30 ms
`and TE2 ⬎ 80 ms), axial FLAIR and an axial gadolinium
`chelate (Gd) enhanced T1-weighted image series. The post
`Gd T1 series is especially important in suspected MS if
`suspicious lesions are seen on T2-weighted or FLAIR
`images. Advanced MRI including quantification of magne-
`tization transfer ratio (MTR), dual inversion recovery im-
`aging, diffusion tensor imaging, single voxel, two-dimen-
`sional (2D) and three-dimensional (3D) chemical shift
`imaging, and unlocalized spectroscopy signal from whole
`brain magnetic resonance spectroscopy (MRS), among
`other methods have enriched our understanding of conven-
`tional MRI and have added insight into our understanding
`of the pathogenesis of MS.4 However, these more advanced
`methods are not generally available, nor necessary for di-
`agnosis and follow-up evaluations, although they may pro-
`vide additional outcomes through which to determine drug
`efficacy.
`In this article, we will review individual MRI-defined
`changes detected by a broad variety of conventional and
`advanced imaging approaches. We will emphasize their
`specific utility in understanding the pathogenesis of the
`disease and serving as biomarkers for evaluating treat-
`ment effects, including therapeutic effects on tissue pres-
`ervation or repair within the otherwise relatively inac-
`cessible CNS. To lay the groundwork for this review, it
`is important to first understand individual lesion devel-
`opment and maturation as currently viewed using con-
`ventional and advanced imaging.
`
`LESION EVOLUTION
`
`On conventional MRI, new lesions arising in previ-
`ously normal appearing white matter (NAWM) are
`nearly always announced by a nodular area of Gd-en-
`hancement on T1-weighted images (FIG. 1)5 This is
`nearly invariably associated with a hyperintense lesion in
`the same location on T2-weighted images (FIG. 1).6
`Nearly 65% of the larger enhancements correspond to
`hypointense lesions visualized on noncontrast T1-
`weighted images7 (FIG. 2). Most enhancements fade and
`disappear over 4-6 weeks, and 50% of the hypointensi-
`ties spontaneously resolve within 4 weeks. A similar
`proportion of those found at 1 month disappear over the
`next 4 –5 months8 Return to the T1-isointense state or
`mild T1 hypointensity may indicate extensive or partial
`remyelination.9 The extent of the new T2-hyperintense
`lesion usually contracts and its intensity is reduced as
`edema resolves and some tissue repair occurs. However,
`
`NeuroRx威, Vol. 2, No. 2, 2005
`
`most lesions, once evident on T2-weighted images rarely
`disappear unless they are located in the brainstem or
`spinal cord. Potentially more aggressive lesions show
`ring-like propagation of the enhancement over a few
`weeks or longer before the enhancement begins to fade,
`are associated with more complex appearances on T2-
`weighted images, a central spherical hypointensity on
`T1-weighted images, and persistence over time (FIG. 3).
`An incomplete ring of enhancement (“open ring sign”),
`open where the lesion abuts gray matter, is characteristic
`of MS (FIG. 2).10 A complete ring may also be seen,
`particularly when the lesions are confined to the white
`matter (FIG. 3). Careful inspection of the areas surround-
`ing some of the larger T1-hypointense lesion that con-
`tract over time shows this apparent repair to be at the
`expense of surrounding tissue loss. As the center of such
`lesions likely undergoes gliosis and contraction there is
`regional ventricular enlargement and cortical volume
`loss directed toward the lesion. Although the evolution of
`T1-hypointense lesions is intimately associated with en-
`hancements, the relationship must be more complex. En-
`hancement frequency is age dependent, being less fre-
`quent among older (rather than younger) MS patients of
`all disease subtypes.11,12 Yet, hypointense lesions are
`more common with longer disease duration and among
`the progressive disease subtypes. The divergent behavior
`of these seemingly inter-related MRI metrics might sug-
`gest that whereas some hypointense lesions result di-
`rectly from new inflammatory events that are readily
`monitored by enhancements on MRI, other hypointense
`lesions may evolve differently.
`Lesion evolution is more complex when monitored
`with advanced imaging. Newly enhanced lesions that
`form within previously conventional MRI-defined
`NAWM provide informative regions for retrospective
`scrutiny for change that antedates lesion evolution on
`conventional MRI. Retrospective analyses suggest that
`regional abnormalities in MTR develop in NAWM
`months before the enhancement is seen by conventional
`MRI.13,14 Unfortunately, these changes have not been
`robust enough to use prospectively. Focal increases in
`choline and the appearance of signals on MR spectro-
`scopic imaging (MRSI) consistent with alterations in
`lipids or other myelin associated macromolecules also
`precede lesion formation by several months,15,16 to sug-
`gest that focal disruptions of tissue integrity anticipate
`enhancements. It remains unclear whether these changes
`reflect some primary intrinsic tissue process that eventu-
`ally signals a secondary influx of inflammatory cells, or
`whether they reflect microscopic inflammatory change
`beyond the resolution of conventional imaging that must
`first build before a cascading inflammatory response is
`evident. In either case, with enhancement there is a dra-
`matic fall in regional MTR, drop in N-acetylaspartate
`(NAA), increase in choline, the appearance of signals
`
`Mylan Pharms. Inc. Exhibit 1040 Page 2
`
`

`
`IMAGING OF MS
`
`279
`
`FIG. 1. Montage of five patients showing typical MRI features of MS. A: Post-contrast (left) and CSE T2-weighted (right) images are
`shown of a 51-year-old woman with RR MS. Note several enhancing foci
`in the periventricular region bilaterally. Lesions have a
`homogeneous appearance and show corresponding hyperintensity on the T2-weighted image. B: Baseline (left) and 5-year follow-up
`(right) CSE T2-weighted images of a 46-year-old woman with RR MS. EDSS score increased from 2.0 to 3.5 during this time. Note
`progressive number and total volume of T2-hyperintense lesions. C: FLAIR (left) and FSE T2-weighted (right) images of a 41-year-old
`woman with RR MS and EDSS score of 3 illustrates the superiority of FLAIR for the detection of periventricular lesions. Note the
`characteristic appearance of the lesions including an oval/ovoid morphology, size 5 mm or greater in diameter, and tendency to directly
`abut the ventricular margin. D: FLAIR (left) and FSE T2-weighted (right) images of a 51-year-old woman with RR MS and EDSS score
`of 4 shows the superiority of FLAIR for the detection of cortical/juxta-cortical lesions. Note the lesion in the left temporal lobe (arrow)
`seen by FLAIR but not on the T2-weighted image. E: Sagittal FLAIR of a 27-year-old woman with RR MS shows typical perivenular
`orientation of lesions. Note the lesions are perpendicular to the long axis of the lateral ventricles giving an appearance known as
`“Dawson’s fingers.”
`
`from myelin breakdown products, and increases of myo-
`inositol, glutamate ⫹ glutamine (Glx) and lactate.17 The
`biochemical changes are highly dynamic and the concen-
`trations of various metabolites and MTR tend to recover
`toward their normal values with time. Some of the ob-
`served acute changes are in part explained by dilutional
`effects of acute vasogenic edema.18 MTR values do not
`fully normalize, but a return toward normal is accompa-
`nied by partial or complete resolution of associated T1-
`hypointense lesions. Mildly T1-hypointense lesions that
`
`remain with intermediate MTR values correlate with his-
`topathologic evidence of at least partial remyelination.9
`Persistent T1-hypointense lesions show diminished NAA
`indicative of irreversible axonal loss19; they may also
`show increased myo-inositol, possibly indicative of gli-
`osis. Enhanced lesions generally have increased diffu-
`sion, decreased FA, and altered diffusion tensor values,
`and these alterations persist to a variable extent in those
`lesions that have the most severely altered tissue ma-
`trix.20 The specialized anatomy of the brain results in
`
`NeuroRx威, Vol. 2, No. 2, 2005
`
`Mylan Pharms. Inc. Exhibit 1040 Page 3
`
`

`
`280
`
`BAKSHI ET AL.
`
`FIG. 2. Evolution of T1 hypointensities (“black holes”). A 48-year-old woman with RR MS received serial MRI during the pretreatment
`screening period of a clinical trial. Noncontrast (upper row) and post-contrast (lower row) images are shown. Scans were obtained at
`baseline, 1 month, and 2 months later. Note the ring-enhancing lesion appearing at baseline that has corresponding T1 hypointensity
`(solid arrow). The ring enhancement has an incomplete or open ring that is typical of MS.10 The T1 hypointensity resolves 2 months later.
`A second T1 hypointensity develops over 2 months (broken arrow).
`
`alterations at a distance related to disruption along con-
`nected pathways that traverse focal lesions, and Walle-
`rian degeneration along highly organized pathways may
`be reflected in altered diffusion tensor eigen values.21
`These distributed effects may help to explain some of the
`quantitative change that is rather consistently found in
`conventional MRI-defined NAWM.
`
`FIG. 3. T1-weighted post contrast (left) and CSE T2-weighted
`(right) images of a 48-year-old woman with RR MS show a ring
`enhancing lesion and corresponding complex appearance on
`the T2 image.
`
`NeuroRx威, Vol. 2, No. 2, 2005
`
`MRSI defines dynamic metabolite changes compatible
`with alterations in mobile lipids in cortical gray matter.22
`These observations are consistent with the known occur-
`rence of cortical plaques.23 However, intracortical and
`subpial lesions have yet to be adequately resolved by
`conventional MRI at up to 3 Tesla, or with other avail-
`able advanced methods. Recently, postmortem MRI at 8
`Tesla has identified some purely intracortical lesions (K.
`Rammohan, personal communication).
`With this overview of lesion development and evolu-
`tion as defined by MRI, we can turn to a consideration of
`the various conventional and advanced imaging ap-
`proaches applied to MS, with particular attention to their
`current use or potential use in the assessment of MS
`disease modifying therapeutics.
`
`Hyperintense lesions on T2-weighted images
`Technical aspects. The intensity of tissue signals is
`influenced by proton density and the rate at which nu-
`clear MR signals decay in the static magnetic field of the
`scanner following application of a radio-frequency exci-
`tation pulse as characterized by T1 (longitudinal relax-
`ation time) and T2 (transverse relaxation time). These
`three parameters (proton density, T1, and T2 relaxation
`
`Mylan Pharms. Inc. Exhibit 1040 Page 4
`
`

`
`IMAGING OF MS
`
`281
`
`times) determine the MRI appearance; their relative in-
`fluences are affected by scanning parameter changes.
`Proton density and T2-weighted images are generated
`with long repetition time (TR). At relatively short echo
`time (TE), the image appearance is mainly determined by
`proton density, whereas at relatively long TE, the T2
`effect is increased. FLAIR uses an inversion pulse fol-
`lowed by a variable signal recovery time to maximize the
`contrast between tissues with different T1 values. Most
`clinically used FLAIR inversion pulses are designed to
`null the signal from CSF at a long TE to provide a high
`degree of T2 weighting to increase lesion conspicuity,
`particularly for those lesions that abut CSF pathways.
`Neuropathology. The lesions of MS show consider-
`able histopathologic heterogeneity, in part related to evo-
`lution of individual lesions over time and (possibly) to
`fundamental differences in patient-specific pathogenesis
`of lesions.24 However, with the possible exception of
`large ring-enhanced lesions, lesion appearance and pat-
`terns seen by conventional MRI have thus far failed to
`consistently distinguish among these histopathologic
`subtypes. The extent of MRI-defined T2 hyperintense
`lesions seen on postmortem imaging clearly can approx-
`imate the extent of lesions found on direct histopatho-
`logic tissue examination.25,26 Postmortem MRI allows
`for the detection of regions of microscopic inflammation
`or active demyelination otherwise missed by visual in-
`spection of the tissue.27,28 Moreover, there are consider-
`able MRI-defined lesion abnormalities that exceed the
`sensitivity of histopathologically defined plaque bur-
`den.29 These discrepancies are readily explained by the
`basis of the signal generated by T2-weighted image as
`described above, which is highly sensitive to the tissue
`mobility of protons, but influenced to a lesser extent by
`the underlying processes that give rise to altered tissue
`water content and proton mobility. Thus, hyperintensities
`on T2-weighted images in patients with MS are nonspe-
`cific for the relative degree of underlying inflammation,
`edema, demyelination, axonal damage, Wallerian degen-
`eration, and gliosis.
`Clinical correlation. Both cross-sectional and short-
`term longitudinal correlations between T2-hyperintense
`disease burden (T2 BOD) and clinical impairment are
`generally poor.30 –32 The possible reasons for this are
`numerous and readily attributed to the lack of pathologic
`specificity for the extent of tissue destruction and the
`well known limitations of existent clinical rating sys-
`tems, including the benchmark Expanded Disability Sta-
`tus Scale (EDSS).32 One of the main reasons for the lack
`of correlation is that MRI often shows hemispheric in-
`volvement in areas that are clinically silent. However,
`the biologic burden of the disease is probably reflected
`better by the MRI findings. Despite these limitations,
`more focused attention may provide some hope that bet-
`ter correlations may be possible if restricted over appro-
`
`priate phases of the disease. T2 BOD is quite variable
`among patients when evaluated at the earliest clinically
`recognized stage of the disease, with a clinically isolated
`demyelinating syndrome (CIS) highly suggestive of the
`type seen in relapsing-remitting (RR) MS patients. Even
`so, it is at this stage that differences in T2 BOD have
`strong predictive value for distinguishing the subsequent
`short-term clinical course. The best available data sug-
`gest that about a third of patients presenting with CIS
`will have negative cerebral MRI and about 40% will
`have fewer than two lesions. In the Early Treatment of
`Multiple Sclerosis (ETOMS) study of CIS,33 indepen-
`dent of treatment, conversion to clinical definite MS
`(CDMS) occurred in 41% of patients with at least one
`Gd-enhancing lesion or 9 T2-hyperintense lesions, ver-
`sus 11% of those without either. The best data on fol-
`low-up at 5 or more years after presentation come from
`subjects gathered nearly two decades ago.34 A number of
`reports have concentrated on similar patients followed
`for shorter intervals. CIS patients with normal cerebral
`MRI at presentation have only a 5% risk of another
`clinical attack (progressing to CDMS) in the next 1-5
`years; those with cerebral lesions have a considerably
`higher risk. The risk remains below 50% until the cere-
`bral T2 BOD exceeds 1.2 ml; corresponding to about six
`lesions each of about 5 mm in diameter at 5-mm slice
`thickness.35 The risk of progression to CDMS within 10
`years with a negative MRI at presentation remained low
`at 11%, but 2 or more lesions conferred nearly a 90% risk
`of conversion.36 Of those with an abnormal MRI scan,
`31% developed disability equivalent to an EDSS score of
`at least 6.0 within 14 years, and the EDSS score at 14
`years correlated moderately with the increase in lesion
`volume in the initial first 5 years (r ⫽ 0.61).
`International panel MRI criteria for dissemination in
`time require one or more new Gd-enhanced lesions at
`least 3 months after the initial clinical event or a new
`T2-hyperintense lesion identified at least 3 months after
`a baseline set of images obtained after the presenting
`clinical event had stabilized or resolved.1 Admittedly,
`these time intervals are somewhat arbitrary. They were
`originally developed because most new Gd-enhanced le-
`sions will no longer enhance after 6 – 8 weeks, and that
`new lesions continue to appear for some days to weeks in
`association with a single clinical attack.37 Subsequent
`data have generally supported the utility of follow-up
`MRI at 3 months to refine the predictive value of early
`MRI for conversion to CDMS.38,39
`Two clinical trials of different preparations of inter-
`feron ␤ (IFNB)-1a indirectly address the sensitivity of
`the international panel MRI criteria for dissemination in
`space in the early diagnosis of MS. The Controlled High
`Risk Avonex Multiple Sclerosis trial studied patients
`with monosymptomatic CIS.40 Entry criteria were re-
`stricted to subjects with at least two clinically silent
`
`NeuroRx威, Vol. 2, No. 2, 2005
`
`Mylan Pharms. Inc. Exhibit 1040 Page 5
`
`

`
`282
`
`BAKSHI ET AL.
`
`cerebral lesions of ⱖ3 mm diameter on a screening MRI
`scan, one of which needed to be periventricular or ovoid.
`Only 1–3% of all subjects met the minimum number of
`T2-hyperintense lesions, and 70% of all patients had ⱖ9
`lesions at baseline.41 It is not known what proportion of
`these subjects would have met the international panel
`MRI criteria for dissemination in space, but this likely
`exceeded 70% of the entire cohort. Within 6 months of
`follow up, about half of those patients without a second
`clinical attack met international panel MRI criteria for
`dissemination in time for MS, and two-thirds of those
`without an attack by 18 months fulfilled the panel criteria
`for MS, regardless of treatment assignment. The ETOMS
`study enrolled patients with either monosymptomatic or
`polysymptomatic CIS.33 They required patients to have
`at least four cerebral lesions at entry (three sufficed if at
`least one was infratentorial or Gd-enhanced). Only 11%
`of these subjects were free of MRI activity on biannual
`scans over 2 years, regardless of treatment assignment.
`Thus, regardless of whether treatment is initiated at clin-
`ical presentation in subjects with CIS who have at least
`two to three cerebral lesions, imaging to define dissem-
`ination in time by international panel guidelines will
`more rapidly establish a firm diagnosis of MS than will
`clinical criteria. Whether some MRI patterns in isolation
`are adequate for a diagnosis of MS at or before first
`clinical presentation remains unclear. Similarly, the op-
`timal timing and cost effective number of serial MRI
`scans needed to define dissemination in time for patients
`with CIS are not established.
`Role in therapeutic monitoring. Historically, T2
`BOD was first used as potential supportive outcome in
`major clinical trials of cyclosporine versus azathioprine
`in Europe, and versus placebo in North America.42– 45
`Both studies failed to show clinical benefit, and both
`documented progression of MRI-monitored pathology
`that was unaffected by treatment. Several years later, the
`first study used attenuated change in T2 BOD on active
`treatment with IFNB-1b compared with placebo to sup-
`plement the clinical endpoint and support drug approval
`by the FDA.46 In that study, subjective but blinded as-
`sessment of MRI-defined disease showed that the num-
`ber of scans deemed active by the appearance of new
`T2-hyperintense lesions was significantly reduced by ac-
`tive therapy. Moreover, a quantitative measure of the
`area of T2-hyperintense lesions was also reduced 23% by
`treatment compared with placebo.
`The T2-based measures that have evolved to monitor
`treatment are both qualitative and quantitative. An active
`scan designates that one or more new T2-hyperintense
`lesions have appeared as compared with the prior scan,
`and/or previously noted lesions have enlarged (i.e., en-
`larged by ⬃20% in lesional area in a single slice). The
`number of new and/or enlarging T2-hyperintense lesions
`can also be enumerated. These measures are based on
`
`NeuroRx威, Vol. 2, No. 2, 2005
`
`FIG. 4. A quantitative computer-assisted semiautomated meth-
`od170 of determining total brain T2 hyperintense lesion load il-
`lustrated in a patient with MS. Upper panel: raw FLAIR images;
`middle panel: after masking and nulling of skull, other extracra-
`nial tissue, and CSF flow artifacts; lower panel: after threshold-
`ing, the images are segmented into lesion versus nonlesion tis-
`sue; the area and volume of total brain lesions is determined
`based on the number of voxels retained.
`
`subjective assessment of serially obtained images and
`generally require that the images are obtained and dis-
`played in standardized manner. Each image analysis cen-
`ter usually develops its own set of standards on how to
`interpret the suitability of the images for analysis, and for
`defining a new or enlarging lesion. Estimating the T2
`BOD has been performed by manual tracing of lesion
`boundaries, semiautomated thresholding techniques like
`seed growing and contouring (FIG. 4), and more fully
`automated image analysis based on a number of ap-
`proaches. The constraints imposed by the techniques on
`image acquisition and variations in reproducibility vary
`
`Mylan Pharms. Inc. Exhibit 1040 Page 6
`
`

`
`IMAGING OF MS
`
`283
`
`TABLE 1. T2 Activity in Placebo Groups from Selected Controlled Trials
`
`Study
`
`Course
`
`IFNB-1a
`IFNB-1b
`IFNB-1a
`IFNB-1b
`GA
`IFNB-1a
`
`RR
`RR
`SP
`SP
`RR
`CIS
`
`Median Percent Change T2 BOD from
`Baseline by Interval in Months
`
`6
`
`4
`
`1*
`
`18
`
`10.8
`
`3.5*
`
`9
`
`12
`
`6.4
`10.9
`1.8*
`1.6
`
`26
`
`36
`
`15
`10
`11
`
`24
`
`10.9
`16.5
`6.2*
`2.4
`
`8.8
`
`New T2-
`Hyperintense
`Lesions
`
`No T2
`Activity
`
`T2
`Active
`
`Reference
`
`2.25
`
`4.67*
`5.0
`8.0
`6.0*
`
`75%
`
`50%
`84%
`
`8%
`
`24%
`16%
`
`16%
`
`48
`46
`49
`50
`51
`33
`
`*Estimated from data provided in original article; exact metric not in article. Trial duration is indicated by shading of the cells for each trial.
`
`with the methods and among imaging analysis centers.
`Nonetheless, these estimates show sensitivity to change
`over time and for detecting treatment effects.47–51
`As seen in Table 1, the longitudinal changes on vari-
`ous measures of T2-hyperintense lesions in placebo-as-
`signed subjects have varied among studies. Some of
`these differences can be attributed to patient character-
`istics with activity levels over a given time interval
`seemingly higher in the earlier stages of the disease than
`in cohorts selected for secondary progressive (SP) dis-
`ease at entry, by enrichment for subjects with active MRI
`scans at entry into short-term trials, and by improve-
`ments in imaging protocols over time. In all instances
`listed in Table 1, positive effects of treatment on clinical
`endpoints were accompanied by statistically significant
`effects on T2-hyperintense lesions. Most of these studies
`chose sample sizes not based on estimates of the MRI
`effect size, but rather on anticipated effects of the drug
`on the selected clinical outcome variable.
`Molyneux and colleagues52 used data aggregated on
`128 subjects with RR or SP MS from natural history data
`or placebo arms of several studies. The database only
`provided actual observations for 12 months of follow-up,
`but sample size estimates were constructed for studies of
`up to 3 years’ duration on the assumption that increase in
`T2 BOD would remain linear over time. As illustrated in
`Table 1, this assumption does not hold for actual trial
`cohorts. Nevertheless, they projected that if a drug were
`completely effective in containing further increase in T2
`BOD from trial entry, 60 RR MS subjects per arm would
`be needed for a 1-year study and only 12 RRMS subjects
`per arm would be needed for 3 years to have 80% power
`for a treatment effect. Numbers of subjects needed to
`show a 50% reduction in T2 BOD progression were
`estimated at 232 for a 1-year trial and 38 for a 3-year
`trial. If the study were of SP MS, the numbers of subjects
`increased by two- to threefold, reflecting the slower ac-
`cumulation of T2 BOD in more progressive subjects.52
`More precise estimates might be derived from larger inte-
`grated data sets, such those being acquired by the Sylvia
`Lawry Center for MS Research, an international collabora-
`
`tive database containing natural history data from numerous
`clinical studies (http://www.slcmsr.com/).
`
`Gadolinium-enhanced lesions
`Technical aspects. Paramagnetic contrast agents
`markedly shorten the T1 of neighboring water protons.
`As a result, after intravenous bolus administration, they
`locally increase the signal from brain tissue where there
`is normally no blood brain barrier (e.g., the circumven-
`tricular organs, meninges, and choroid plexus), or where
`the barrier is significantly compromised allowing Gd to
`enter the brain abnormally, e.g., in active MS lesions
`(FIGS. 1–3). This effect
`is best monitored on T1-
`weighted images and the conspicuity of enhancement
`can be improved by inclusion of an off resonance mag-
`netization transfer (MT) pulse.53 Protons from water
`molecules that are tightly bound to tissue contribute little
`signal to images acquired with conventional MRI. When
`a narrow bandwidth radio-frequency pulse with a 1–2
`KHz frequency offset from the excitation pulse is used to
`saturate protons associated with the bound water pool,
`magnetization is transferred from the bound to free water
`pool that contributes the most signal to conventional
`MRI. The result is attenuation of the signal proportional
`to the concentration of the bound water molecules, pro-
`viding an estimate of tissue integrity. The MT pulse does
`not directly affect the free water signal. In the case where
`the pulse is applied to post Gd-T1 imaging, the signal
`from brain tissue is reduced and the Gd effect is aug-
`mented by comparison.53 The extent and number of en-
`hancements found may be increased by the dose of con-
`trast selected or by an optimal delay between contrast
`administration and the time to image acquisition.53,54
`Neuropathology. The pathologic correlate of en-
`hancement in MS is altered blood brain barrier perme-
`ability in the setting of acute perivascular inflamma-
`tion.55–57 Transfer of Gd into the CNS is likely complex,
`reflecting components of transendothelial transfer by in-
`creased micropinocytotic activity of activated but struc-
`turally intact endothelium and passive entry through
`structurally damaged endothelial barriers.
`
`NeuroRx威, Vol. 2, No. 2, 2005
`
`Mylan Pharms. Inc. Exhibit 1040 Page 7
`
`

`
`284
`
`BAKSHI ET AL.
`
`TABLE 2. Enhancement Activity and Clinical Effects from Selected Controlled Trials
`
`Course
`
`Effect on Enhancement
`
`Clinical Effect
`
`Study
`
`IFNB-1a
`
`GA
`
`Natalizumab
`
`IFNB-1b
`
`RR
`
`RR
`
`RR
`
`SP
`
`Cladribine
`
`SP & PP
`
`Median new and recurrent en-
`hancements over first 9 months
`on therapy reduced from 8.0 to
`1.4 (22 ␮g) and 1.3 (44 ␮g)
`Median new enhancements over 9
`months on therapy reduced
`from 13.5 to 9.0
`Median new enhancements over 6
`months on therapy reduced
`from 2.0 to 0 (3 mg/kg) and 0
`(6 mg/kg)
`Median new enhancements over
`first 6 months on therapy re-
`duced from 5.0 to 0
`Proportions with enhanced scans
`at 6 and 12 months reduced
`from 33 and 32%, to 12 and
`2% (0.7 mg/kg), and 2 to 6%
`(2.1 mg/kg), respectively
`
`Relapse risk reduction 27% (22
`␮g) and 33% (44 ␮g) at 2
`years
`
`Annualized relapse rate reduced
`from 1.21 to 0.81 over 9
`months
`Proportion with relapses over 6
`months on therapy reduced
`from 38% to 19% (3 mg/kg)
`and 19% (6 mg/kg)
`Odds ratio for confirmed progres-
`sion over 2–3 years was 0.65
`
`No change in time to progression
`by EDSS
`
`Reference
`
`48, 62
`
`51
`
`63
`
`50, 64
`
`65
`
`Clinical correlation. In clinically eloquent regions,
`new clinical symptoms are highly correlated with the
`appearance and resolution of enhancement in that path-
`way. In one study, 94% of 107 patients with acute optic
`neuritis showed enhancement of
`th