Glatiramer acetate (Copaxone) treatment
`in relapsing–remitting MS
`Quantitative MR assessment
`
`Y. Ge, MD; R.I. Grossman, MD; J.K. Udupa, PhD; J. Fulton, MD; C.S. Constantinescu, MD, PhD;
`F. Gonzales–Scarano, MD; J.S. Babb, PhD; L.J. Mannon, RT; D.L. Kolson, MD, PhD; and J.A. Cohen, MD
`
`Article abstract—Objective: To evaluate the efficacy of glatiramer acetate (GA, Copaxone; Teva Pharmaceutical Indus-
`tries, Ltd., Petah Tiqva, Israel) by MRI-based measures in patients with relapsing–remitting (RR) MS. Methods: Twenty-
`seven patients with clinically definite RR-MS were treated with either 20 mg of GA by daily subcutaneous self-injection
`(n 5 14) or placebo (n 5 13) for approximately 24 months. Axial dual-echo fast-spin-echo T2-weighted images and
`T1-weighted images before and after gadolinium (Gd) were acquired at 1.5 tesla and transferred into an image processing
`computer system. The main outcome measures were the number of Gd-enhanced T1 and T2 lesions and their volume as
`well as brain parenchyma volume. Results: The values of age, disease duration, Expanded Disability Status Scale (EDSS)
`score, the number of T1- and T2-weighted lesions, and their volume were similar between GA- and placebo-receiving
`groups at the entry of this study. There was a decrease in the number of T1-enhanced lesions (p 5 0.03) and a significant
`percent annual decrease of their volume in GA recipients compared with those of placebo recipients. There were no
`significant differences between changes in the two groups in the number of T2 lesions and their volume. The loss of brain
`tissue was significantly smaller in the GA group compared with that of the placebo group. Conclusions: These results show
`that GA treatment may decrease both lesion inflammation and the rate of brain atrophy in RR-MS. Key words:
`Glatiramer acetate—Relapsing-remitting MS—Expanded Disability Status Scale score—MR—Volume measurement.
`NEUROLOGY 2000;54:813–817
`
`The traditional outcome measures in therapeutic
`tasks in MS are most often relapse frequency or pro-
`gression in disability, as measured by standard dis-
`ability indexes such as Expanded Disability Status
`Scale (EDSS) score.1 Although these clinical markers
`still are emphasized as the primary outcome mea-
`sures of definitive therapeutic or Phase III clinical
`trials,2,3 neuroimaging techniques such as MRI may
`offer a more direct measure of pathologic changes
`within the CNS, and MRI is being used increasingly
`as an outcome criterion in many new treatments for
`MS in recent years.4-6 Because MRI provides a more
`direct measure of the extent of pathologic changes,
`the potential advantages include the quantitative
`nature of the data, ability to detect subclinical activ-
`ity, and sensitivity to the long-term subclinical accu-
`mulation of disease in the brain.3 MRI criteria for
`MS treatment trials have been presented in the neu-
`rologic literatures in recent years7 and typically are
`based on routine conventional or fast-spin-echo pro-
`ton density (PD)- and T2-weighted as well as en-
`hanced T1-weighted images. Recent advances in
`computer-assisted brain lesion load quantification8-10
`may help in accurately assessing the total volume of
`
`signal abnormalities on either long or short repeti-
`tion time (TR) images. More recently, studies of mea-
`surements of cerebellum,11 spinal cord,12 and cerebral
`atrophy13 have shown a good correlation between the
`degree of atrophy and clinical disability. Their roles
`in monitoring in therapeutic trials, however, remain
`unknown.
`Glatiramer acetate (GA, Copaxone; Teva Pharma-
`ceutical Industries, Ltd., Petah Tiqva, Israel), previ-
`ously called copolymer 1,14 is the most recently
`approved drug in the United States for the treat-
`ment of MS.15 Published results from a multicenter,
`double-masked, placebo-controlled trial showed its
`efficacy against relapsing-remitting (RR) MS in sig-
`nificantly decreasing the frequency of relapses by an
`average of 30%.16,17 The work presented here reports
`the results of MRI studies performed on a cohort of
`patients at one site in the multicenter Phase III trial.
`We have quantitated both the changes in the volume of
`brain lesions and the absolute number of lesions over
`time based on gadolinium (Gd)-enhanced T1- and T2-
`weighted images. We have also quantitated the whole
`brain parenchyma volume to analyze the effect of treat-
`ment on the development of brain atrophy.
`
`From the Departments of Radiology (Drs. Ge, Grossman, Udupa, and Fulton, and L.J. Mannon) and Neurology (Drs. Constantinescu, Gonzales–Scarano, and
`Kolson), Hospital of the University of Pennsylvania, Philadelphia, PA; the Mellen Center for MS Treatment and Research (Dr. Cohen), Cleveland Clinic
`Foundation, Cleveland, OH; and the Department of Biostatistics (Dr. Babb), Fox Chase Cancer Center, Philadelphia, PA.
`Supported in part by grants R01 NS 29029 and NS 37172 from the National Institutes of Health.
`Received April 1, 1999. Accepted in final form October 15, 1999.
`Address correspondence and reprint requests to Dr. Robert I. Grossman, Department of Radiology, Hospital of the University of Pennsylvania, Founders,
`3400 Spruce Street, Philadelphia, PA 19104-4283; e-mail: grossman@oasis.rad.upenn.edu
`
`Copyright © 2000 by the American Academy of Neurology 813
`
` EXHIBIT NO. 1025 Page 1
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`
`

`
`Materials and methods. Patients. The study design,
`patient enrollment criteria, baseline clinical and demo-
`graphic data, and overall results of the multicenter Phase
`III trial were published previously.16,17 The cohort enrolled
`at the University of Pennsylvania included 23 women and
`4 men, ages 25 to 45. All had clinically definite RR-MS
`with disease duration of 1 to 17 years. Baseline EDSS
`ranged from 1.0 to 5.0. Fourteen subjects were randomized
`to receive active drug (GA, 20 mg subcutaneously daily),
`and 13 received placebo. Four placebo patients did not
`complete the entire 2-year studies but instead were on
`study for between 0.54 and 1.39 years. The reasons the
`patients cited for dropping out were unrelated to
`treatment-induced adverse effects and included movement
`to another state (1), excessive attention (2), and continued
`clinical signs (1). Study medication was supplied by Teva
`Pharmaceutical Industries, Ltd (Petah Tiqva, Israel) under
`a manufacturing protocol approved by the Institutional
`Review Board of the University of Pennsylvania, and
`informed consent was obtained from all patients before
`enrollment.
`MR imaging and analysis. MR studies were performed
`at the scheduled study accompanied by masked clinical
`evaluation. All brain MRI scans during this trial period
`were performed at 1.5 T (General Electric, Milwaukee, WI)
`using a quadrature head coil. Three-millimeter contiguous,
`interleaved axial dual-echo fast-spin-echo (PD and T2) im-
`ages were collected from all patients according to the fol-
`lowing protocol: TR 5 2,500 msec; echo time (TE) 5 18/90
`msec; echo train length 5 8; 192 3 256 matrix; number of
`excitations 5 1; field of view 5 22 cm2. The T1-weighted
`spin-echo (TR 5 600 msec/TE 5 27 msec) images were
`acquired before and after injection of Gd-DTPA at 0.1
`mmol/kg with similar field of view and slice thickness to
`the dual-echo images.
`All patient studies were transferred electronically to
`our medical image processing laboratory, where the total
`number and volume of T2 lesions and Gd-enhancing T1
`(Gd-T1) lesions were computed. The associated image pro-
`cessing was performed using an internal version of the
`3DVIEWNIX software system18 on a Sun Sparc 20 (Sun
`Microsystems, Mountain View, CA) workstation. The algo-
`rithms are based on a theory of object definition in images
`that has been well described previously with very low in-
`trareader and inter-reader variability.19,20 Various aspects
`of the theory as well as the algorithms were described in
`detail elsewhere.21 The brain parenchyma volume was also
`calculated using 3DVIEWNIX. The process begins with
`excluding the extracranial contents based on segmentation
`of fuzzy connected three-dimensional objects (gray matter,
`white matter, and CSF) to get the intracranial contents
`including brain parenchyma and CSF.19,20 An angle image
`of CSF is then produced from segmented T2- and PD-
`weighted data sets.20 In brief, this technique creates a
`voxel-by-voxel image using the following formula: Iangle 5
`tan-1 (I T2/I PD), where Iangle, I T2, and I PD are the intensi-
`ties of the corresponding voxels from the angle and from
`the T2- and PD-weighted images. The resulting angle im-
`age has relatively homogeneous CSF intensity values,
`which can be easily thresholded to produce a CSF-only
`image and volume. The total brain parenchyma image and
`volume are obtained by subtracting CSF image and vol-
`ume from the image of the intracranial contents. This
`814 NEUROLOGY 54 February (2 of 2) 2000
`
`method has been routinely used in our department for
`brain segmentation and has shown .99% reproducibility.
`The interoperator and intraoperator coefficient of variation
`for total T2 lesion volume in this system has been found to
`be #0.9%.19,20 The 95% CI of the false-negative volume
`fraction (i.e., the volume of missed lesions as a fraction of
`the total volume of true lesions) has been 0% to 22.8%. For
`enhanced lesion assessment,9 the system has no interop-
`erator and intraoperator variations, with only 1 missed
`enhancing lesion of 38 true lesions.
`Statistical analysis. The Mann–Whitney–Wilcoxon
`test was used to compare the two treatment groups with
`respect to the change per year in each of the outcome
`variables. The change in each outcome variable was com-
`puted as the last observation minus the baseline response
`so that negative values correspond to a decline in response
`over the course of the study. For outcome variables that
`were strictly positive at study onset, the yearly change in
`response is reported as a percentage of the baseline mea-
`surement. A Bonferroni correction for multiple hypothesis
`tests was used to ensure an overall type-I error rate no
`greater than 5% when comparing the two treatment
`groups. Specifically, with respect to each outcome variable,
`the treatment groups were declared significantly different
`at the 5% level only if the probability value for the relevant
`statistical test was ,0.05/6 5 0.0083.
`
`Results. All 27 patients were observed for a period of at
`least 6 months with a median time on study equal to 1.99
`(range, 1.54 to 2.17) and 2.03 (range, 0.54 to 2.64) years in
`the GA and placebo groups, respectively. The demograph-
`ics, clinical characteristics, and MRI measurements of the
`two treatment groups at the entry of this study are sum-
`marized in table 1. To determine the impact of group dif-
`ferences in these factors on our results, we repeated our
`primary group comparisons with respect to the outcome
`variables after including all baseline variables as covari-
`ates. The impact on our results was minimal; therefore, only
`the results of the adjusted analyses are presented here.
`The annual changes in EDSS, brain parenchyma vol-
`ume, as well as the T1 and T2 lesion number and volume
`are summarized in table 2. Clinical outcomes for our pa-
`tient cohort differed from those of the study cohort as a
`whole in showing no significant reduction in relapse rate
`or change in EDSS around 2 years. There were 24 relapses
`in both the 14 GA-treated patients and the 13 placebo-
`treated patients. The mean percentage change of EDSS
`was 5.9% in the GA group and -1.3% in the placebo group
`( p 5 0.08). There was a significant difference between GA-
`and placebo-treated groups with respect to the annual
`change in both brain parenchyma volume and Gd-T1 vol-
`ume. Specifically, the placebo-treated patients exhibited a
`nearly threefold greater annual decline in brain volume
`than GA-treated patients. The Gd-T1 enhanced lesion
`number showed a decrease trend ( p 5 0.03) in the GA
`group compared with that of the placebo group after using
`Bonferroni correction for multiple hypothesis tests. No
`other difference between the two treatments was signifi-
`cant at an overall significance level of 5%.
`
`Discussion. GA is a mixture of synthetic random
`polypeptides made from alanine, glutamic acid, ly-
`sine, and tyrosine15 that has shown a clear benefit in
`experimental allergic encephalomyelitis (EAE) and
`
` EXHIBIT NO. 1025 Page 2
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`

`
`Table 1 Baseline characteristics of GA- and placebo-treated patients with MS
`
`Mean
`
`Median
`
`SD
`
`Characteristic
`
`Age, y
`Disease duration, y
`EDSS
`Gd-T1 number
`Gd-T1 volume (mm3)
`T2 number
`T2 volume (mm3)
`
`Placebo
`
`34.4
`4.2
`2.4
`1.5
`93.8
`28.7
`8654.1
`
`GA
`
`37.1
`5.2
`3.0
`3.1
`215.0
`21.3
`6521.2
`
`Placebo
`
`33.8
`4.0
`2.5
`1.0
`42.0
`17.0
`6438.1
`
`GA
`
`36.4
`5.0
`3.0
`1.0
`74.0
`22.0
`3610.0
`
`Placebo
`
`6.3
`3.0
`0.8
`1.7
`177.4
`12.8
`6279.0
`
`GA
`
`6.1
`3.7
`1.0
`5.0
`391.0
`22.1
`8678.0
`
`p Value*
`
`0.28
`0.22
`0.13
`0.25
`0.31
`0.30
`0.48
`
`* p Value is the significance level obtained from a Mann–Whitney test of the difference between the GA and placebo treatment groups.
`
`GA 5 glatiramer acetate; EDSS 5 Expanded Disability Status Scale; Gd-T1 5 gadolinium-enchancing lesion.
`
`MS.22,23 Clinical and laboratory-based studies sug-
`gest direct interference by GA with antigen (myelin
`basic protein) presentation by antigen-presenting
`cells to effector T lymphocytes and ultimately at
`least partial blocking of the putative demyelinating
`cascade.24-26 In the aforementioned multicenter GA
`trial,17 the therapeutic activity of GA showed a re-
`duction of the mean relapse rate (.2 years) and a
`slower decline of EDSS in GA patients compared
`with placebo patients, although we failed to see a
`similar significant clinical benefit in our small co-
`hort. However, the MRI analysis was not reported in
`the previous large multicenter GA trial. A recent
`MRI study27 examined the effect of GA on MRI
`changes in 10 patients and showed the frequency of
`new Gd-enhancing lesions decreased during the
`treatment period compared with that of the pretreat-
`ment period. The study assessed lesion area instead
`of lesion volume and did not have a control group.
`In our study, the MRI-based measurements showed
`that treatment with GA has a favorable impact on
`enhancing T1 lesions and whole brain parenchyma
`atrophy. The different change of T1-enhancing lesion
`volume (283.5 mm3 versus 147.5 mm3, mean) and
`
`brain volume (20.6% versus 21.8%, mean) was signif-
`icantly different between GA-treated and placebo-
`treated patients with RR-MS. This MRI-based
`systematic study for evaluating the efficacy of a ran-
`domized, placebo-controlled GA trial in RR-MS objec-
`tively used a highly reliable brain and lesion volume
`quantitation technique that may provide insight into
`the events involved in lesion development and brain
`parenchyma loss in MS.
`In MS, the lesions seen on MRI principally reflect
`a histopathologic spectrum including the presence
`edema, demyelination, remyelination, and inflamma-
`tion. The two most commonly used MRI measures of
`MS lesions are the new lesions in Gd-enhanced T1-
`weighted images and the amount of abnormal brain
`tissue as seen in PD- and T2-weighted images. One
`of the early abnormalities believed to occur during
`the formation of the MS plaques is the breakdown of
`the blood–brain barrier, which can be detected by
`Gd-enhanced MR images.28-31 It is believed that the
`systematic use of repeated Gd-enhanced scans can
`reveal a very high degree of activity in which there is
`a good correlation with clinical activity compared
`with the PD- and T2-weighted scans.32,33 Recent data
`
`Table 2 Change per year from baseline to endpoint
`
`Mean
`
`Median
`
`SD
`
`Measurements
`
`Placebo
`
`EDSS (%)
`Brain volume (%)
`Gd-T1 number
`Gd-T1 volume
`T2 number (%)
`T2 volume (%)
`
`21.3
`21.8
`0.5
`147.5
`18.7
`16.8
`
`GA
`
`5.9
`20.6
`21.2
`283.5
`18.3
`53.0
`
`Placebo
`
`0
`21.6
`0.5
`50.6
`14.0
`8.9
`
`GA
`
`8.6
`20.5
`20.2
`29.5
`0.6
`19.8
`
`Placebo
`
`11.3
`1.8
`1.0
`226.1
`25.8
`26.7
`
`GA
`
`20.6
`0.9
`2.7
`185.3
`43.2
`76.3
`
`p Value*
`
`0.08
`0.0078
`0.03
`0.003
`0.34
`0.51
`
`* p Value is the significance level obtained from a Mann–Whitney test of the difference between the GA and placebo treatment groups.
`
`EDSS 5 Expanded Disability Status Scale; GA 5 glatiramer acetate; Gd-T1 5 gadolinium-enhancing lesion.
`
`February (2 of 2) 2000 NEUROLOGY 54 815
`
` EXHIBIT NO. 1025 Page 3
`
` AMNEAL
`
`

`
`have also suggested that the volume of tissue rather
`than the number of lesions might be a better crite-
`rion for evaluating the disease activity in MS.34 In
`our study, there was a significant difference of an-
`nual change for Gd-T1 enhanced lesion volume be-
`tween GA and placebo group, both when considering
`using Mann–Whitney–Wilcoxon test and using Bon-
`ferroni correction for multiple hypothesis tests. Our
`results confirmed the effect of GA on Gd-enhanced
`lesions observed by another group,27 although the
`yearly change of the number of enhancing lesion did
`not reach significant difference between GA- and
`placebo-treated groups after using correction of mul-
`tiple hypothesis tests. The effect of GA on Gd-T1
`lesion volume may
`reflect
`short-term anti-
`inflammatory effects in improving the integrity of
`the blood– brain barrier in patients with RR-MS,
`however, whereas measures of brain parenchyma
`volume likely reflect largely irreversible tissue loss.
`Changes in T2 lesion load have been used fre-
`quently to monitor clinical trials as the long-term
`progression measure in patients and likely reflect
`both reversible and irreversible pathologic change.
`Unlike enhancement, T2-weighted abnormalities
`may persist indefinitely and likely represent perma-
`nent focal myelin loss, gliosis, and some neuronal
`cell loss, as well as some reversible edema and in-
`flammation.35 However, T2 lesion number and T2
`lesion volume may not reflect global parenchyma
`loss and global treatment effect. In our study, the
`number of T2 lesions and their volume between two
`treatment groups did not reach a significant differ-
`ence, whereas the measures of whole brain paren-
`chyma atrophy did. This may reflect the small
`sample size of our study, as well as the partially
`reversible nature of some T2 lesions. The mecha-
`nisms of the therapeutic benefits of GA in T1-
`enhanced and T2 lesion in patients with RR-MS are
`still unknown but may be similar to its effect of
`suppressing EAE in the acute phase.36
`Brain atrophy has long been found in patients
`with MS.37,38 Because of the multifocal nature of MS
`lesions dispersed throughout the entire brain, the
`relation between MR lesion load and clinical disabil-
`ity was shown to be variable and complex. Recently,
`quantitative brain parenchyma measurements have
`shown good correlation with disability measures.13
`We used a highly reliable brain segmentation tech-
`nique to follow the treatment effects of GA and pla-
`cebo. Preservation of brain parenchyma by a specific
`therapeutic regimen represents an important mea-
`sure of treatment efficacy. We have shown a differ-
`ent percent change of brain volume (brain atrophy)
`in two treatment groups. Our data provide evidence
`that GA may slow progressive brain atrophy (i.e., cell
`loss) in RR-MS ( p 5 0.0078), even in these two small
`sample groups. Such precise, quantitative measures
`of brain volume may offer a more sensitive measure
`of drug efficacy in MS than currently used MR as-
`sessment measures.
`816 NEUROLOGY 54 February (2 of 2) 2000
`
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`sis. AJNR 1998;19:1251–1265.
`36. Bornstein MB, Miller A, Slagle S, et al. A pilot trial of copoly-
`mer 1 in exacerbation-remitting multiple sclerosis. N Engl
`J Med 1987;317:408–414.
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`Edinburgh 1916;50:517–740.
`38. Zimmerman HM, Netsky MG. The pathology of multiple scle-
`rosis. Res Publ Assoc Nerv Ment Dis 1950;28:271–312.
`
`Neuro Images
`
`Figure. (A) Punctate calcifications in brain CT scan (arrows). (B) Vertical section of heart with large MAC cavity between left atrium (a)
`and left ventricle (V). (C) H-E stain of mitral annulus wall embedded with dark spicules of calcium (thick arrow) and paler, amorphous
`substance (thin arrow). Original magnification 3100 before 96% reduction. (D) Embolic calcific material in subarachnoid artery, original
`magnification 3160 before 96% reduction, Luxol Fast Blue stain.
`
`Mitral annulus calcareous brain emboli
`Maryam Mohammadkhani, MD, Pamela Schaefer, MD,
`Walter Koroshetz, MD, and E. Tessa Hedley-Whyte, MD,
`Boston, MA
`This 86-year-old woman came to the emergency room
`with 2 days of visual flashing lights and “floaters.” Within
`hours, she developed myocardial infarction, right hemipare-
`sis, back pain, and coma and died. Radiographs showed ex-
`tensive mitral annular calcification (MAC) and multiple
`punctate calcifications in the brain. Autopsy showed erosion
`of a massive MAC with extrusion into the left atrium. Mate-
`rial identical to the MAC content was found in vessel lumens
`of all organs sampled except the lungs. Calcareous matter
`occluded multiple subarachnoid and brain parenchymal ves-
`sels.
`Calcific embolization is regarded as a rare complication of
`this relatively common cardiac condition. Calcareous sys-
`
`temic embolization from MAC has been well documented
`pathologically but not radiologically. Previously, a small, cal-
`cific density on a brain CT scan was suggested to represent
`calcific embolus from the mitral valve, but without pathologic
`documentation.1 Our case provides pathologic confirmation
`that the punctate calcifications in the brain images corre-
`spond to the fatal shower of calcific emboli. The exact inci-
`dence of calcific emboli from MAC is unknown. A suggested
`frequency of 11.3% indicates under-recognition and under-
`reporting of this complication.2 This case illustrates that rec-
`ognition of embolic calcifications on brain imaging enables
`antemortem diagnosis of MAC rupture.
`
`1. Katsamakis G, Lukovits TG, Gorelick PB. Calcific cerebral embolism in
`systemic calciphylaxis. Neurology 1998;51:295–297.
`2. Lin C, Schwartz IS, Chapman I. Calcification of the mitral annulus
`fibrosus with systemic embolization: a clinicopathologic study of 16
`cases. Arch Pathol Lab Med 1987;111:411–414.
`
`February (2 of 2) 2000 NEUROLOGY 54 817
`
` EXHIBIT NO. 1025 Page 5
`
` AMNEAL