Stuart D. Cook

A Placebo-Controlled Trial of Oral Cladribine for Relapsing Multiple Sclerosis

BACKGROUND Cladribine provides immunomodulation through selective targeting of lymphocyte subtypes. We report the results of a 96-week phase 3 trial of a short-course oral tablet therapy in patients with relapsing-remitting multiple sclerosis. METHODS We randomly assigned 1326 patients in an approximate 1:1:1 ratio to receive one of two cumulative doses of cladribine tablets (either 3.5 mg or 5.25 mg per kilogram of body weight) or matching placebo, given in two or four short courses for the first 48 weeks, then in two short courses starting at week 48 and week 52 (for a total of 8 to 20 days per year). The primary end point was the rate of relapse at 96 weeks. RESULTS Among patients who received cladribine tablets (either 3.5 mg or 5.25 mg per kilogram), there was a significantly lower annualized rate of relapse than in the placebo group (0.14 and 0.15, respectively, vs. 0.33; P<0.001 for both comparisons), a higher relapse-free rate (79.7% and 78.9%, respectively, vs. 60.9%; P<0.001 for both comparisons), a lower risk of 3-month sustained progression of disability (hazard ratio for the 3.5-mg group, 0.67; 95% confidence interval [CI], 0.48 to 0.93; P=0.02; and hazard ratio for the 5.25-mg group, 0.69; 95% CI, 0.49 to 0.96; P=0.03), and significant reductions in the brain lesion count on magnetic resonance imaging (MRI) (P<0.001 for all comparisons). Adverse events that were more frequent in the cladribine groups included lymphocytopenia (21.6% in the 3.5-mg group and 31.5% in the 5.25-mg group, vs. 1.8%) and herpes zoster (8 patients and 12 patients, respectively, vs. no patients). CONCLUSIONS Treatment with cladribine tablets significantly reduced relapse rates, the risk of disability progression, and MRI measures of disease activity at 96 weeks. The benefits need to be weighed against the risks. (ClinicalTrials.gov number, NCT00213135.)

250 μg or 500 μg interferon beta-1b versus 20 mg glatiramer acetate in relapsing-remitting multiple sclerosis: a prospective, randomised, multicentre study

BACKGROUND The aim of the Betaferon Efficacy Yielding Outcomes of a New Dose (BEYOND) trial was to compare the efficacy, safety, and tolerability of 250 microg or 500 microg interferon beta-1b with glatiramer acetate for treating relapsing-remitting multiple sclerosis. METHODS Between November, 2003, and June, 2005, 2447 patients with relapsing-remitting multiple sclerosis were screened and 2244 patients were enrolled in this prospective, multicentre, randomised trial. Patients were randomly assigned 2:2:1 by block randomisation with regional stratification to receive one of two doses of interferon beta-1b (250 microg or 500 microg) subcutaneously every other day or 20 mg glatiramer acetate subcutaneously every day. The primary outcome was relapse risk, defined as new or recurrent neurological symptoms separated by at least 30 days from the preceding event and that lasted at least 24 h. Secondary outcomes were progression on the expanded disability status scale (EDSS) and change in T1-hypointense lesion volume. Clinical outcomes were assessed quarterly for 2.0-3.5 years; MRI was done at screening and annually thereafter. Analysis was by per protocol. This study is registered, number NCT00099502. FINDINGS We found no differences in relapse risk, EDSS progression, T1-hypointense lesion volume, or normalised brain volume among treatment groups. Flu-like symptoms were more common in patients treated with interferon beta-1b (p<0.0001), whereas injection-site reactions were more common in patients treated with glatiramer acetate (p=0.0005). Patient attrition rates were 17% (153 of 888) on 250 microg interferon beta-1b, 26% (227 of 887) on 500 microg interferon beta-1b, and 21% (93 of 445) for glatiramer acetate. INTERPRETATION 500 microg interferon beta-1b was not more effective than the standard 250 microg dose, and both doses had similar clinical effects to glatiramer acetate. Although interferon beta-1b and glatiramer acetate had different adverse event profiles, the overall tolerability to both drugs was similar. FUNDING Bayer HealthCare Pharmaceuticals.

Genome-wide meta-analysis identifies novel multiple sclerosis susceptibility loci

To perform a 1‐stage meta‐analysis of genome‐wide association studies (GWAS) of multiple sclerosis (MS) susceptibility and to explore functional consequences of new susceptibility loci.

Serologic evidence of previous Campylobacter jejuni infection in patients with the Guillain-Barré syndrome

The Guillain-Barre syndrome, sometimes called acute inflammatory polyneuropathy, is an inflammatory demyelinating disease of peripheral nerves characterized by various degrees of weakness, sensory abnormalities, and autonomic dysfunction [1, 2]. Since the marked decline in poliomyelitis, the Guillain-Barre syndrome has become the most common cause of acute neuromuscular paralysis in adults and children in the United States and has an annual incidence of 1.7 per 100 000 people [3, 4]. Epidemiologic studies in all parts of the world have confirmed the association between the Guillain-Barre syndrome and previous acute infection, especially of the respiratory or gastrointestinal tracts [4-8]. Most report that between 50% and 75% of patients have an infectious illness 1 to 3 weeks before onset of neurologic symptoms; previous diarrheal illness occurs in 10% to 30% of patients [4-6]. Bacteria of the genus Campylobacter are important human pathogens and are common causes of gastrointestinal illness in both developed and developing countries [9]. Extraintestinal complications of Campylobacter jejuni infection such as reactive arthritis, pancreatitis, and carditis are well described [10], and in the last decade, clinical and epidemiologic evidence has suggested that infection with C. jejuni may be a precipitating factor for development of the Guillain-Barre syndrome. In small serologic studies of patients with the Guillain-Barre syndrome, as many as one third to one half of patients had increased levels of C. jejuni antibodies at the time of onset of neurologic symptoms [11-13]. Routine stool cultures of eight patients with the Guillain-Barre syndrome in Japan yielded C. jejuni in seven (88%) patients [14]. In an important U.S. investigation of 106 patients with the Guillain-Barre syndrome, C. jejuni was isolated from 4 (44%) of 9 patients who had antecedent clear-cut diarrheal illness; however, cultures had not been obtained from 97 other patients [15]. To assess the extent to which the Guillain-Barre syndrome is associated with recent C. jejuni infection, we did serologic screening with defined assays to determine the frequency of C. jejuni antibodies in a large group of patients with the syndrome and in two control groups. Methods Study Population Case Patients Case patients included 126 persons with the Guillain-Barre syndrome admitted to the University of Maryland Medical Center, the University of Medicine and Dentistry of New Jersey, or to one of several hospitals in St. Louis between 1983 and 1990. A consecutive sample was used. All patients met the National Institute of Neurologic and Communicative Disorders and Stroke [16] criteria for diagnosis of the Guillain-Barre syndrome; none had any exclusion criteria. Most of the patients had demyelinating polyneuropathy, although some had axonal damage. All had a monophasic illness that progressed during 1 to 4 weeks, plateaued, and almost all patients recovered to some extent; one patient died. Blood samples were collected within 3 weeks after the onset of neurologic symptoms from 118 of the patients; 8 patients had blood samples collected from 24 days to more than 3 months after onset of neurologic symptoms (median, 10 weeks). Because antibody response to C. jejuni infection is usually transient [17], specimens from these eight patients are not included in the analysis. Histories of symptoms of gastrointestinal or respiratory infections before onset of the Guillain-Barre syndrome were not available. Controls Two groups of controls were studied. One control group, a convenience sample, consisted of 56 patients with neurologic diseases other than the Guillain-Barre syndrome who were admitted to the neurology services at the University of Maryland, the University of Medicine and Dentistry of New Jersey, or one of the St. Louis hospitals. Diagnoses of these patients included optic neuritis, multiple sclerosis, amyotrophic lateral sclerosis, polyneuropathy associated with IgM paraproteinemia, chronic inflammatory demyelinating polyneuropathy, Charcot-Marie-Tooth disease, unexplained peripheral neuropathy, ischemic neuropathy, neuropathy associated with diabetes, and drug-induced neuropathy. The second control group, a voluntary sample, included 18 healthy employees of the neurology departments of the three universities and 29 healthy family members of case patients and of disease controls. Medical histories of healthy controls were not obtained. Sera and Data Collection Sera from case patients and controls was stored frozen at the collection sites until the serologic assays were done. Data collected included disease severity of case patients, sex, age, and date of sera collection. Information about race was available only from the patients at the University of Medicine and Dentistry of New Jersey. Serologic Methods Presence of serum IgA, IgG, and IgM antibodies specific for C. jejuni was determined by enzyme-linked immunosorbent assay (ELISA) as previously described [17]. Antigens from three C. jejuni strains of Penner (O-) types 1, 2, and 3, which are commonly isolated from humans, were prepared as described by McCoy and colleagues [18]; they contained a mixture of acid-extracted proteins common to C. jejuni strains [17, 19, 20]. The serum dilutions used were as follows: 1:50 for IgA, 1:200 for IgG, and 1:400 for IgM. The class-specific second antibody used (and the use dilution) was peroxidase-conjugated goat anti-human IgA (1:2000), IgG (1:2500), or IgM (1:1000) (Tago Inc; Burlingame, California). The plates were read at 414 nm on a Dynatech ELISA reader (Alexandria, Virginia), and results were expressed as an optical density value for each well. All sera had been coded and were assayed blindly three times on different days in duplicate wells for each run. Calculation of Optical Density Ratios The assays were standardized using sera from a patient with the Guillain-Barre syndrome and culture-proven C. jejuni infection (positive control) and sera from 38 healthy children in New York who had had no recent history of diarrheal illness (negative controls). Optical density values were determined serially for plates containing these sera, and the mean optical density of the positive control was plotted versus the mean optical density + 1 SD of the negative controls. For each immunoglobulin class, a regression line was calculated and then used each day the assay was done (using the same positive control serum) to generate a threshold value for determination of the optical density ratio. (Regression analysis for IgA assay, r = 0.973, P < 0.001; regression analysis for the IgG assay, r = 0.998, P < 0.001; regression analysis for the IgM assay, r = 0.981, P < 0.001.) To control for day-to-day assay variation, the optical density ratio was defined as the ratio of the optical density of each unknown serum sample to the observed threshold on that day. A similar method has been used to standardize Helicobacter ELISA determination [21]. Case Definitions and Statistical Methods A positive serologic response was defined as an optical density ratio greater than or equal to a specified threshold in one or more immunoglobulin classes. Differences in the proportion of positive responses among case patients and controls were ascertained using an optical density ratio 1, 2, or 3 as the threshold. Statistical analyses were done in a univariate manner using the Mantel Haenszel Chi-squared analysis or the Fisher exact test when indicated and by the calculation of odds ratios. The sensitivity and specificity of the assays were determined as follows. Optical density ratios were determined as described above for 17 patients in the convalescent phase of culture-proven C. jejuni infection. These were U.S. soldiers who had become infected while on military exercise in Thailand in 1988; convalescent sera were obtained 4 weeks after onset of diarrhea. The comparison group comprised 19 healthy adults who were employees of or visitors to the Infectious Diseases Division of Vanderbilt University and included 20 healthy children in Nashville who had no recent history of diarrheal illness. Results Patients Demographic characteristics of the 118 case patients and 103 controls are shown in (Table 1). Age was known for 112 (95%) case patients and 93 (90%) controls; gender was known for 116 (98%) case patients and for 99 (96%) controls. Information about the month blood was drawn was available for 116 (98%) case patients and 80 (78%) controls. The groups were similar except that controls were somewhat younger (median age, 33 years compared with 41 years for case patients) and were more likely to have had their blood drawn during the summer months. Race of patients and controls (from the University of Medicine and Dentistry of New Jersey only) also was similar in the two groups. In both groups there were more male patients. Table 1. Demographic Characteristics of 118 Patients with the Guillain-Barre Syndrome and of 103 Controls, Including 56 Controls with Disease and 47 Healthy Controls Serologic Assays in Persons with Campylobacter jejuni Infection and in Controls Among 17 persons with culture-proven C. jejuni infections, in the IgA assay, 15 (88%) had an optical density ratio 1. Increasing the threshold for defining a positive serologic response to an optical density ratio 2 and 3 decreased the sensitivity of the test (Table 2). The specificity of the test was evaluated by the serologic responses in the control group. Defining a positive serologic response as an optical density ratio 1, the IgA assay was 85% specific (specificity determined by subtracting the percentage of controls exceeding threshold from 100%) (Table 2). Increasing the threshold to an optical density ratio 2 or 3 improved the specificity of the test. The sensitivity and specificity of the IgG and IgM assays are also shown in Table 2. As expected, raising the stringency by increasing the optical density ratios or numbers of positive classes

Efficacy of treatment of MS with IFNβ-1b or glatiramer acetate by monthly brain MRI in the BECOME study

Background: There are no published MRI studies comparing interferon beta 1b (IFNβ-1b) and glatiramer acetate (GA) for treatment of relapsing multiple sclerosis (MS). Objective: To compare the efficacy of IFNβ-1b and GA for suppression of MS disease activity as evidenced on frequent brain MRI. Methods: A total of 75 patients with relapsing-remitting MS or clinically isolated syndromes were randomized to standard doses of IFNβ-1b or GA and followed by monthly brain MRI for up to 2 years with a protocol optimized to detect enhancement. The primary outcome was the number of combined active lesions (CAL) per patient per scan during the first year, which included all enhancing lesions and nonenhancing new T2/fluid-attenuated inversion recovery (FLAIR) lesions. Secondary outcomes were the number of new lesions and clinical exacerbations over 2 years. Results: Baseline characteristics were similar between the groups. The primary outcome showed similar median (75th percentile) CAL per patient per scan for months 1–12, 0.63 (2.76) for IFNβ-1b, and 0.58 (2.45) for GA (p = 0.58). There were no differences in new lesion or clinical relapses for 2 years. Only 4.4% of CAL on monthly MRI scans were nonenhancing new T2/FLAIR lesions. Conclusion: Patients with relapsing multiple sclerosis randomized to interferon beta 1b or glatiramer acetate showed similar MRI and clinical activity.

Antibodies to acidic glycolipids in Guillain-Barré syndrome and chronic inflammatory demyelinating polyneuropathy

Using an enzyme-linked immunosorbent assay and a thin-layer chromatography-immunostaining procedure, we detected serum antibodies against acidic glycolipids in 36 of 53 patients with Guillain-Barré syndrome (GBS) and 8 of 16 patients with chronic inflammatory demyelinating polyneuropathy (CIDP). Although we also found anti-acidic glycolipid antibodies in 4 of 13 patients with other neurological diseases; 2 of 10 patients with multiple sclerosis; 8 of 33 patients with inflammatory, infectious, allergic or autoimmune disorders and 3 of 32 healthy subjects, the levels of antibodies in these controls were much lower than in GBS patients. There were several patterns of reactivity of GBS sera including antibodies to LM1 and HexLM1, GM1 or GD1b or both, various other gangliosides, sulfated glycolipids, and as yet unidentified glycolipids. Sera from 30% of GBS patients had antibodies against two or more antigenically distinct acidic glycolipid antigens. Levels of anti-acidic glycolipid antibodies correlated with clinical symptoms in 9 of 11 GBS patients. While the increased incidence of antibodies to acidic glycolipids in patients with GBS (P less than 0.001) and CIDP (P less than 0.025) compared to controls could be an epiphenomenon, anti-acidic glycolipid antibodies may play a role in nerve injury in some GBS and CIDP patients.

Cell death and birth in multiple sclerosis brain

The hallmark of the brain pathology in multiple sclerosis is the white matter plaque, characterized by myelin destruction and oligodendrocyte loss. To examine the role that cell death plays in the development of MS lesions, we used the in situ TUNEL technique, a method that sensitively detects DNA fragmentation associated with death at the single cell level. We found that patchy areas within acute MS lesions have massive numbers of inflammatory and glial cells undergoing cell death. The punched out areas of some long-standing chronic lesions also had labeled glial cells showing that the attack was not a single event. Immunocytochemical identification of the dying cells with glial specific marker co-labeling showed that 14-40% were the myelin-sustaining oligodendroglial cell. Confocal microscopic evaluation of fluorescein-labeled TUNEL positive cells revealed nuclei with morphologic characteristics of apoptosis, and electrophoresed MS brain DNA produced a ladder characteristic of apoptotic DNA cleavage confirming that substantial numbers of labeled cells, but not necessarily all, were dying by apoptotic mechanisms rather than cell necrosis. Companion studies using a marker for cell proliferation on MS lesions revealed that unexpectedly large populations of perivascular inflammatory cells and parenchymal glial cells had entered the cell proliferation cycle. These findings establish that two opposing glial cell responses - relentless cell death and coincident brisk cellular proliferation - are important features of MS pathology. In the end, however, glial cell loss prevails, and we suspect apoptosis may be the critical death mechanism responsible for the depletion of myelin observed in this condition.

Sustained disease-activity-free status in patients with relapsing-remitting multiple sclerosis treated with cladribine tablets in the CLARITY study: a post-hoc and subgroup analysis

BACKGROUND On the basis of various clinical and MRI measurements, the phase 3 Cladribine Tablets Treating Multiple Sclerosis Orally (CLARITY) study in patients with relapsing-remitting multiple sclerosis (RRMS) showed that short-course oral treatment with cladribine at cumulative doses of 3·5 and 5·25 mg/kg over 96 weeks was more effective than placebo. Achieving sustained freedom from disease activity is becoming a viable treatment goal in RRMS; we therefore aimed to assess the effects of cladribine on this composite outcome measure by doing a post-hoc analysis of data from the CLARITY study. METHODS Freedom from disease activity is composed of three components that are commonly used individually as endpoints in clinical trials; it is defined as the patient having no relapse, no 3-month sustained change in expanded disability status scale (EDSS) score, and no new MRI lesions (no T1 gadolinium-enhancing or active T2 lesions) over a specified period. We assessed the effect of two doses of cladribine tablets versus placebo on the proportion of patients who were free from disease activity based on the individual components, all pair-wise combinations, and the composite of the three components (freedom from disease activity). Freedom from disease activity was analysed at 24, 48, and 96 weeks, and in subgroups of patients stratified according to baseline demographic and disease characteristics (age, disease duration, previous treatment with disease-modifying therapy, T1 gadolinium-enhancing lesion number, T2 lesion volume, EDSS score, number of previous relapses, and highly active disease). FINDINGS Of the 1326 patients randomly assigned to treatment in the CLARITY study, 1192 were assessable for freedom from disease activity at 96 weeks. Over 24 weeks, 266 (67%) of 395 patients in the cladribine 3·5 mg/kg group and 283 (70%) of 406 in the cladribine 5·25 mg/kg group were free from disease activity, versus 145 (39%) of 373 in the placebo group (odds ratio [OR] 3·31, 95% CI 2·46-4·46 for the 3·5 mg/kg group; and 3·68, 2·73-4·97 for the 5·25 mg/kg group; both p<0·0001). Over 48 weeks, 208 (54%) of 384 patients in the cladribine 3·5 mg/kg group and 222 (56%) of 396 patients in the cladribine 5·25 mg/kg group were free from disease activity, versus 86 (24%) of 360 patients in the placebo group (OR 3·80, 2·77-5·22 for the 3·5 mg/kg group; 4·13, 3·02-5·66 for the 5·25 mg/kg group; both p<0·0001). Over 96 weeks, 178 (44%) of 402 patients in the cladribine 3·5 mg/kg group and 189 (46%) of 411 patients in the cladribine 5·25 mg/kg group were free from disease activity, versus 60 (16%) of 379 patients in the placebo group (OR 4·28, 3·05-6·02 for the 3·5 mg/kg group; 4·62, 3·29-6·48 for the 5·25 mg/kg group; both p<0·0001). The effects of cladribine tablets on freedom from disease activity were significant across all patient subgroups. INTERPRETATION Treatment with cladribine tablets significantly increased the proportion of patients with sustained freedom from disease activity over 96 weeks compared with placebo. Sustained freedom from disease activity could become an important measure of therapeutic response in RRMS. FUNDING Merck Serono SA-Geneva, Switzerland; an affiliate of Merck, Darmstadt, Germany.

Multiple sclerosis and viruses: An overview

The evidence for a viral etiology of MS has been reviewed. The strongest evidence favoring a virus is based primarily on epidemiologic considerations. Less convincing evidence comes from pathologic studies, serology, lymphocyte reactivity to viral antigens, and reports of identification of virus in MS tissues. Animal models of viral demyelination exist, which may provide insight into possible etiologic agents and pathogenetic mechanisms. Considering all the data, it is clear that no agent can be convincingly linked to MS at the present time. If a single virus causes the majority of cases of MS, then a morbilliform virus—canine distemper—is a leading contender, because this agent is consistent with the epidemiologic and serologic findings and is highly neurovirulent for animals ranging from mice to primates. Since no virus fulfills the usual criteria for linking an infectious agent to a disease, other possibilities must be considered. If MS is caused by a single virus, it may be a common virus not presently considered as being associated with MS, or an agent as yet unidentified. It is also conceivable that multiple agents, acting alone or in concert, initiate the MS process, perhaps through a common immune-mediated pathway. In this regard, another human demyelinating disease—the Guillain-Barré syndrome—which may in some instances become a chronic remitting and relapsing disorder, is thought to be initiated by multiple infectious agents but to have an immunologic pathogenesis.

Beneficial effect of erythropoietin on experimental allergic encephalomyelitis

We have known for a long time that erythropoietin signaling plays a key role in bone marrow erythrocyte proliferation. However, recent studies have indicated that erythropoietin also may have protective effects on the nervous system. This unexpected role remains incompletely characterized. To investigate the potential neuroprotective role of erythropoietin in the central nervous system, we assessed its effects on a well‐characterized autoimmune demyelinating model of multiple sclerosis–myelin oligodendrocyte glycoprotein‐induced experimental autoimmune encephalomyelitis (EAE) in the mouse. We found that erythropoietin administered intravenously for 14 days after the onset of symptoms reduced both disease severity and duration of maximum impairment at dose levels as low as 50U/kg (p < 0.001). We assessed the neuropathology of diseased spinal cords and found that erythropoietin‐treated EAE animals had reduced axonal damage, inflammatory cell infiltration and demyelination, and diminished blood–brain barrier leakage when compared with saline‐treated EAE controls. Moreover, the pronounced upregulation of spinal cord major histocompatibility complex (MHC) class II expression found in saline‐treated EAE was significantly reduced in erythropoietin‐treated animals, a finding we replicated in vitro, using microglial cultures. The notion that short‐term erythropoietin therapy might be of clinical benefit in human autoimmune demyelinating diseases needs investigation. Ann Neurol 2004; 56: 767‒777