More MS news articles for May 2002

Glatiramer acetate

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Neurologia 2002 May;17(5):244-58
Comi G, Moiola L.
Head of the Departments of Neurology and Neurophysiology. University Vita-Salute San Raffaele. Milan. Italy.

ABSTRACT

Glatiramer acetate (GA) is a mixture of synthetic polypeptides composed of four aminoacids. GA is very effective in suppression of experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS). Various mechanisms of action of GA have been proposed, but the most important is probably the induction of antigen-specific suppressor T cells. Class one clinical trials have demonstrated that GA reduces the relapse rate and the accumulation of disability in relapsing-remitting (RR) MS. The positive effects on disease activity and disease progression are explained by the reduction of the number and volume of the active lesions as showed by Magnetic resonance imaging (MRI) studies. Moreover new MRI techniques suggest that GA may also have some neuroprotective effects. The drug is usually well tolerated with modest side effects. In vitro and in vivo animal studies have shown that GA is devoid of teratogenic or mutagenic effects. GA is a good alternative to interferon beta for treatment of RR-MS.

BACKGROUND

Glatiramer acetate (GA), previously known as copolymer-1 (trade name Copaxone® ), is not a new drug, even though it was first licensed in the United states and in a few other countries only in 1997; its history is very interesting and reveals the slowness with which a drug may pass from the investigative phase to clinical availability. GA is the acetate salt of a mixture of synthetic polypeptides with average molecular weights ranging from 4,700 to 13,000 Da. The polypeptides consist of random sequences of four naturally occurring aminoacids, L-alanine, L-glutamic acid, L-lysine and L-tyrosine in a defined molar ratio of 4.2:1.4:3.4:1.0, respectively [1,2] (table 1). In the 1960s Drs. Arnon, Sela, Teitelbaum and their colleagues at the Weizmann Institute of Science in Israel were involved in studies on the immunological properties of a series of polymers and copolymers developed to resemble myelin basic protein (MBP). They were interested in evaluating whether these polypeptides alone or in combination with various lipids could simulate the ability of MBP and of regions and fragments of the MBP molecule to induce experimental allergic encephalomyelitis (EAE), the best animal model of multiple sclerosis (MS) [3-6]. None of the series was capable of inducing EAE, but several of the synthetic copolymers showed marked efficacy in suppressing EAE in guinea-pigs and the most active was GA [1,3]. Subsequent studies demonstrated the effectiveness of GA in preventing or decreasing the severity of EAE not only in rodents but also in several other species [6] (table 2). Abramsky et al [7] first treated a group of severe relapsing-remitting (RR) MS patients with intramuscular GA, 2-3 mg every 2-3 days for 3 weeks, then weekly for 2-5 months. No conclusion could be drawn regarding drug efficacy but there were no significant undesirable side reactions. In the 1980s three clinical trials were performed: a preliminar trial in MS patients with RR or secondary progressive (SP) courses and two pilot trials, one in RR and the other in SP patients [8-11]. These studies showed some evidence of the efficacy of GA, particularly in RR patients, and a good safety profile. However, the results of these studies must be interpreted with some caution because before 1991 the production of the drug was not standardized [2,12] ; different batches of the drug had variable suppressive effects on EAE which could also imply variable effects in MS patients. In 1991 a phase III multicenter trial was started in the USA, supported by TEVA Pharmaceuticals Limited, Petah Tiqva, Israel, the Federal Food and Drug Administration (FDA) Orphan Drug Program and the National Multiple Sclerosis Society. In this double-blind placebo-controlled study a highly standardized preparation of GA was utilized. This study demonstrated that GA significantly reduces the relapse rate (by 29%) without significant side-effects [13]. In 1996 GA (Copaxone, Teva Pharmaceuticals) was approved by the US FDA as a treatment for ambulatory patients with active RR MS and in the last years it has been approved in many other countries.

MECHANISMS OF ACTION

Glatiramer acetate (GA) and interferon (IFN)-b are both used for the immunommodulatory treatment of multiple sclerosis, but they act in different ways. IFN-b exerts its multiple immunomodulatory effects in an antigen-nonspecific way, in contrast GA is believed to selectively down-modulate the immune response to myelin autoantigens [14-15]. GA has been shown to exert a marked suppressive and protective effect on EAE induced in various animal species including primates [1,3,4,16]. GA ameliorated chronic relapsing EAE [17] as well as EAE induced by various encephalitogens, e.g., myelin basic protein (MBP) [3,4] , and myelin oligodendrocyte glycoprotein (MOG) peptide [18] , proteolipid protein (PLP) peptides [19]. Due to the random sequence and size of the peptides that comprise GA, it is likely to have pleiotropic effects on the immune system in MS. The site of action of GA is at the trimolecular complex consisting of major histocompatibility complex (MHC) molecules, processed antigen, and the T cell receptor (TCR). Neuhaus and colleagues, in a recent review [20] , after an exhaustive analysis of the major studies on EAE and MS, identified four major effects of GA on trimolecular complex:

1. Direct competition between GA peptides and central nervous system (CNS) myelin antigens for MHC binding

Results in vitro suggest that GA binds directly to MHC class II antigens displayed on intact or fixed antigen-presenting cells (APC) and that this binding is of high avidity being demonstrated for all common MS-associated DR haplotypes ("promiscuous binding") [21]. GA does not require antigen-processing for its binding to class II antigens and subsequent presentation to T-cells [22]. In vitro experiments have demonstrated that GA competitively inhibits the binding of MBP [22-25] , PLP [19] and MOG [18] and displaces those antigens when they are already bound to MHC [22,26]. The cross-reactivity of GA and MBP is also displayed by the observations that GA reacts with monoclonal antibodies raised against MBP and viceversa [27] and that T-cell lines and clones generated to either glatiramer or MBP where frequently inhibited when cross-stimulated by either antigen [24-25]. The binding of GA to MHC on APCs inhibits the activation of T cell lines and clones reactive with the antigens in question [25-26]. This mechanism is unlikely to play a role in vivo because after s.c. administration GA is quickly degraded to free amino acids and small oligopeptides [28]. Therefore it is unlikely that significant amount of GA or GA small peptides can reach the CNS where they could compete with the relevant auto-antigens for MHC-binding.

2. Competition of GA/MHC with MBP/MHC for binding to the antigen-specific surface receptor of MBP-specific T cells ("TCR antagonism")

The experimental evidence supporting this effect is controversial. GA was reported to act as an antagonist of the antigenic peptide MBP 82-100 but not MBP 1-11 and proteolipid protein (PLP) [29]. A recently published report demonstrated that inhibition of MBP (87-99)-induced TCR downmodulation was comparable for GA and influenza hemagglutinin peptide HA (306-318), indicating that most if not all the inhibitory effect of GA was primarily due to blocking of peptide binding rather than partial agonist or antagonist activity on the TCR [30]. If this mechanism occurs in vitro, it is unlikely to be relevant in vivo because GA is unlikely to reach sites where it could compete with MBP.

3. Anergy induction of MBP-specific T cells by GA acting as an altered peptide ligand (APL) relative to MBP

A peptide that is able to engage a specific TCR may be able to inhibit its subsequent activation by a stimulatory peptide [31]. Gran and colleagues in a recent paper [30] demonstrated that when MBP specific T cell clones were stimulated with different concentrations of GA, their subsequent response to the nominal antigen MBP was turned off, i.e., the cells had been anergized by GA, although not to the same extent as the native peptide. This is consistent with previous observations showing that APLs based on MBP (83-89) were able to induce anergy but less strongly than native peptide [32]. This suggests that a specific TCR engagement by GA does occur and that functional anergy may contribute to the inhibitory effect of GA on myelin antigen specific T cells. This mechanism can occur in the periphery at the injections sites or in their draining lymph nodes where the MBP specific cells might be confronted with GA. The mechanism of anergy can explain why the frequency of GA-specific T cell lines appears to decline in MS patients during GA treatment [33]. The used regimen of daily subcutaneous injection may favor the induction of anergy rather than a full immunization that require longer intervals between doses.

4. GA treatment induces a Th1-to-Th2 shift in GA-reactive T cells in vivo

T helper cells can be divided into several types based on their characteristic cytokine secretion patterns and effector functions [34-37]. In organ-specific autoimmune diseases like MS and its animal model EAE, CD4 [+] T cells of Th1 type play a central role in the destructive immunopathology. When stimulated by their antigen these cells produce proinflammatory cytokines such as interleukin-2 (IL-2), IL-12, interferon gamma (IFN-a ), and tumor necrosis factor alpha (TNF-a ). To regulate this response, a target organ antigen-specific subset of T cells of the CD4 [+] Th2 type must be induced or expanded. When stimulated by their antigen the Th2 cells produce downregulatory cytokines such as IL-4, IL-5, IL-6, IL-10, IL-13 and transforming growth factor beta (TGF-b ). Local cytokine production varies significantly during disease and changes in sets of cytokines are associated with acute response or recovery/chronic phases of the disease [38,39]. In this regard, the Th1/Th2 balance plays a key role in MS/EAE regulation. The induction of regulatory/suppressive-glatiramer acetate specific Th2 cells has been identified in both animals and humans [40-42]. It has been demonstrated that the unresponsiveness to EAE induced by GA is regulated by T suppressor (Ts) cells, because it can be adoptively transferred to normal recipients and abrogated by pretreatment with cyclophosphamide, an immunological manipulation recognized to selectively deplete Ts cells [16]. GA specific Ts cells lines and Ts hybridomas were established from spleens of mice that had rendered unresponsive to EAE by GA and these T cells or their supernatants inhibited in vitro the response of an encephalitogenic line to MBP when cocultered [43]. Furthermore they prevented the development of EAE induced by whole mouse spinal cord homogenate (MSCH) in vivo, indicating that they are regulatory suppressor cells confined to Th2 pathway [40]. The GA induced T cells had never been exposed to the autoantigen MBP, nevertheless, they responded to MBP by secretion of Th2 immmunosuppressive cytokines [40]. This cross reactivity of GA with the natural autoantigen MBP was previously demonstrated [23,27] and it has been shown to be essential for GA suppressive activity in vivo [44]. Indeed, as mentioned above, the GA/MBP specific lines protected mice against the development of EAE induced by MSCH [40]. However, MSHC contains in addition to MBP other encephalitogenic antigens implicated in disease induction [45-47]. These findings suggested that GA specific cells can suppress the encephalitogenic processes induced by other antigens and not only MBP. The phenomenon of T cells specific to one antigen which suppress the immunological response induced by another antigen, has been previously described and termed bystander suppression [48-49]. In the case of EAE and MS, bystander suppression must be due to the propinquity of the antigens within the myelin sheat. Bystander suppression is especially important in the treatment of both MS and EAE because of the antigen/epitope spreading which has been demonstrated for these diseases [50]. Further support for the proposed protective role of GA reactive regulatory cells comes from the recent demonstration that GA-specific Th2 cells are present in CNS of GA treated mice [51] by isolation of GA specific Th2 cells from the brain of treated mice and by following the appearance in the CNS of adoptively transferred labelled GA-specific cells. A shift from Th1-b iased cytokine profile toward a Th2-biased profile has been observed also in human subjects. GA treatment was shown to increase serum IL-10 levels and TGF-b and IL-4 mRNA in peripheral blood lymphocytes (PBL), whereas it suppressed TNF-a mRNA [42]. Using intracellular double-immunofluorescence flow cytometry it has been shown that long-term GA reactive T-cells lines (TCL) from untreated MS patients and healthy controls predominantly produce IFN-a and are to be classified as Th1 cells, whereas GA-reactive TCL from GA treated MS patients predominantly produce IL-4, acting like Th2 cells [52]. Recent observations on short term [33,53] and on long term GA reactive [54] are consistent with these findings. A recent study conducted by Farina and colleagues [55] demonstrated, by using an automated, computer-assisted enzyme-linked immunoadsorbent spot assay (Elispot) for detecting GA-induced IFN-a and IL-4-producing cells, that patients treated with GA compared with healthy controls and untreated MS patients showed a) a significant reduction of GA-induced T cells proliferation, b) a positive IL-4 Elispot response mediated predominantly by CD4 cells after stimulation with a wide range of GA concentrations and c) an elevated IFN-a response partially mediated by CD8 cells after stimulation with high GA concentrations. All three effects were GA specific as they were not observed with control antigens; they were consistent over time and allowed correct identification of GA treated and untreated patients. The authors proposed these criteria to monitor the immunological response to GA [55].

In summary it appears that, following injection, GA is rapidly bound to class II molecules present on the APC that are found in subcutaneous tissues. These cells, either locally or following circulation to draining lymph nodes or spleen, present unprocessed GA to CD4 T cells. GA induces a population of Th2 cells which cross-react with MBP and other myelin antigens released during CNS inflammation/demyelina-tion [2,24,56]. GA treatment induces an increasing GA-specific Th2 polarized T-cell repertoire that recognizes antigen in a more degenerate fashion [33]. GA reactive Th2 cells are able to cross the blood brain barrier because they are activated by daily immunization [57]. Inside the CNS, in the presence of MBP and other products of myelin turnover presented by local APC [58] , these cross-reactive regulatory Th2 cells limit immune-mediated damage through local downregulation of the immune response by producing anti-inflammatory cytokines such as IL4, IL-6, IL-10 and even neurotro-phic factors [59-61] and thereby suppressing autoaggressive, pathogenic Th1 cells via a bystander mechanism [40,41]. This scenario is proposed in the figure 1 [20].

Fig. 1. Schematic view of the putative mechanism of action of glatiramer acetate (GA). In the periphery, outside the CNS, GA initially stimulates a population of Th1-like cells. During treatment, the properties of the GA-stimulated T cells change, and they become more Th2-like (dotted arrow). The activated GA-specific T cells enter the CNS, where they encounter CNS antigens like MBP bound to MHC class II and presented on the surface of microglia cells. The GA-reactive T cells are stimulated to secrete downmodulatory cytokines like IL-4, which exert a bystander suppressive effect on other T cells. TCR=T-cell receptor; MHC=major histocompatibility complex; Ag=antigen.Reproduced from Neuhaus (Neurology 2001;56:702-8).

PHARMACOKINETICS

There are few studies in vivo on the pharmacological profile of GA. The pharmacokinetic profile of GA was evaluated in mice, rats and monkeys using a radiolabel ling technique because no reliable methods exist for measuring unmodified GA in biological fluid [28]. The results of these studies must be cautiously interpreted because of the possibility of de-iodination and incorporation of amino acids from GA in other peptides. The drug, after subcutaneous injection, is quickly absorbed with only about 10% remaining at the injection site after 1 hour. The plasma concentration reaches a peak after 1-2 hours in rats and after 2-4 hours in monkeys. GA undergoes rapid degradation to amino acids and shorter peptides. The curves of plasma radioactivity are similar after oral administration and after intramuscular and subcutaneous injections. Long-term administration in rats did not affect the pharmacokinetic parameters of GA. The distribution of iodinated material showed the highest level in stomach and thyroid and the lowest in the brain, probably because the penetration through the blood-brain barrier is impeded by the high polarity and the hydrophilic nature of GA. Urinary excretion is the major elimination pathway for the radioactive labels, and faeces contained only trace amounts.

CLINICAL STUDIES

Preliminary trials

In the late 1970s and early 1980s four early exploratory open studies were performed in order to obtain indications on safety and dosing of GA [7-9,62]. In total, 41 patients with RR or SP MS were involved in these studies; injected doses of GA, treatment schedule and treatment duration were quite variable from study to study. The maximum dose utilized was 20 mg/day; this dose was well tolerated and the safety profile of GA was benign in all these studies.

Phase II pilot trial in relapsing-remitting multiple sclerosis

The evidence of some potential therapeutic benefit encouraged Bornstein and colleagues [10] to conduct a double-blind, placebo-controlled, pilot trial. Fifty patients with RR MS entered the study and self-injected subcutaneously either 20 mg of GA dissolved in 1 ml of saline or just saline, daily, for 2 years. Seven patients did not complete the 2 years trial, three in the active arm and four in the placebo arm; two of these patients, both in the placebo arm, were excluded from the analysis owing to unusable data. There were 62 relapses in the placebo group (average 2.7) and 16 among patients in the GA group (average 0.6). In addition, the proportion of relapse-free patients was twice as great in the GA group as in the placebo group. Five patients receiving active drug and 11 patients receiving placebo had a confirmed progression of disability, defined as an increase of one or more units on the Disability Status Scale [63] , this difference being statistically significant (p = 0,005). The effects on relapse rate and on disability were more pronounced in patients who had the least disability at entry (an interesting finding which was reproduced also in the subsequent GA- trials). This study also reported the occurrence of a post-injection systemic reaction in two patients, the relevant adverse effect of GA treatment which will be discussed later on.

Phase II pilot study in secondary progressive multiple sclerosis

A subsequent double-blind, randomized study in patients with chronic progressive MS was conducted at Albert Einstein College of Medicine and Baylor College of Medicine in the mid-1980s [11]. One hundred six patients with a chronic progressive course for at least 18 months, no more than two exacerbations in the previous 2 years and entry disability between 2 and 6.5 on Expanded Disability Status Scale (EDSS), were randomized to receive subcutaneously injections of GA (15 mg twice a day) or placebo. Interestingly no distinction was made between primary progressive (PP) and SP courses or between progression due to incomplete recovery from a relapse and progression unrelated to relapses. According to the more recent classification of MS courses [64] , patients recruited in this study belonged to all four main types of courses. Eighty-six patients completed the study. For purposes of statistical analysis, the 20 withdrawals were counted as follows: 17 (eight on GA and nine on placebo) who did not meet progression criteria were censored at the time of withdrawal and three patients (two on GA and one on placebo) were counted as confirmed progression at the time of withdrawal. There were 23 who had confirmed progression of whom nine (17.6%) had received GA and 14 (25.5%) had received placebo. The differences between the overall survival curves were not significant. The progression rate at 12 and 24 months was non-significantly higher for the placebo group (p = 0.09) with 2 year probabilities of progressing of 20.4% for GA and 29.5% for placebo. Subgroup analysis by center showed that for the Einstein Center, at the time point of 24 months, there was a 21.4% chance of progression with GA and 38.5% chance of progressing with placebo (p = 0.04). For the Baylor Center the probability of progressing was 19% in both treatment arms. The authors had no explanations for the intertrial or the intercenter differences in placebo response. A possible interpretation of these findings is that more PP patients with a typical slower progression than SP were included at the Baylor Center than at the Einstein Center. In conclusion there was no evidence of efficacy of GA in patients with progressive MS, although, as in the study of RR MS, a favorable trend was more marked in those whose disability was less at the time of beginning treatment.

Phase III definitive trial in relapsing-remitting multiple sclerosis

On the basis of the encouraging results of the previous studies a definitive phase III trial was designed to assess more comprehensively the efficacy of GA in patients with RR MS. Considerable efforts were made to standardize the preparation of the drug to eliminate the great variability in biological activity between vials, observed in the old preparations. The trial was conducted at 11 US medical centers from 1991 to 1994 and the results were published in 1995 [13]. A total of 251 RR MS patients 18-45 years of age, with at least two relapses in the 2 years before the trial, with mild to moderate disability (EDSS < 5.0) were randomized to receive either GA (Copaxone, TEVA), 20 mg subcutaneously daily, or placebo for 24 months. The primary end-point of the study was relapse rate, relapses being confirmed by objective changes on neurological examination. Clinical evaluations were performed every 3 months. MRI was performed in only a small cohort of patients at a single center [65]. The patients' baseline characteristics were well matched in the two arms except for EDSS, which was slightly higher in the GA group. During the course of the trial 36 patients, 19 (15.2%) from the GA and 17 (13.5%) from the placebo group, withdrew for various reasons. There was no difference between the groups in the time of withdrawal. The mean annualized relapse rates were 0.59 for the GA group and 0.84 for the placebo group, a 29% reduction which was statistically significant (p = 0.007). Additional supporting relapse data are shown in table 3. The proportion of relapse-free patients (33.6% on GA and 27.0% on Placebo group) and the median time to the first relapse from baseline (287 days on GA vs. 198 on Placebo) were both greater in the GA group, although the differences were not statistically significant. Patients in both groups with higher EDSS scores at entry had a higher relapse rate, while the largest reduction in relapse rate between groups occurred in patients with a baseline EDSS lower: 33% reduction in patients with EDSS 0-2 versus 22% in patients with an EDSS score at entry > 2.0. The effect of treatment was constant throughout the study duration (fig. 2). The proportion of progression-free patients was similar in the two arms: 78.4% in the GA group and 75.4% in the placebo group. However, more GA-treated patients improved by at least one EDSS unit and more placebo-treated worsened by at least one EDSS unit by the trial's original planned end (p = 0.037; fig. 3). The mean change of EDSS score was +0.21 in the placebo group and ­0.05 in the GA group, this difference being statistically significant (p = 0.023) but of dubious clinical significance. In summary, secondary outcome measures showed constant trends favoring GA over placebo, but the differences were usually marginal [12].

Fig. 2.Number of relapses over time per semester in the double-blind, placebo-controlled, phase III trial on the effects of GA in RR-MS.

Fig. 3. Effects of glatiramer acetate treatment on neurologic disability in the phase III trial (p = 0.037). Effects of glatiramer acetate treatment on neurologic disability in the phase III trial (p = 0.037). Patients were considered improved or worsened if the EDSS score decresased or increased respectively of >= 1 point. (from Johnson et al Neurology, 1995).

MRI studies were carried out in 27 patients, 14 receiving GA and 13 receiving placebo. Patients underwent MRI evaluations at months 0, 1, 3, 6, 12, 18, 20, 22 and 24. There was a trend toward benefit with GA in terms of reduced number of gadolinium-enhancing lesions starting at 12 months of treatment. However, because of the small number of evaluated patients and the high variability both within and between groups, no conclusions can be drawn from MRI data [65].

Extension of the phase III double-blind placebo controlled trial in relapsing-remitting multiple sclerosis

The double-blind, placebo-controlled phase III trial was extended in a double-blind fashion up to 35 months to 203 patients in order to obtain more safety and efficacy data [66]. Ninety-seven out of the 99 completers in the GA arm and 97 out of the 104 completers in the placebo arm completed the extension study. A significantly beneficial effect was observed on the relapse rate with a reduction of 32% in GA treated group compared to placebo group (p = 0,002). The percentage reduction of relapse rate was largest in patients with a baseline EDSS of 0-2 (36%). The proportion of patients classified as improved, not changed or worsened by one or more EDSS steps between baseline and last measurement, showed a statistically significant treatment effect favoring GA (p = 0,024). The favorable safety profile was also maintained during the extension [12,66].

Extension in an open label fashion of the phase III double-blind placebo controlled study in relapsing-remitting multiple sclerosis: clinical benefits after six years of observation

To obtain long-term safety, tolerability and efficacy data of GA, patients randomized to the pivotal double-blind, placebo-controlled trial where offered the opportunity to participate in an organized open-label extension, in which patients who had received GA from onset were designated group A, whereas patients who had received placebo from onset were switched to GA treatment and were designated group B. The patients' characteristics at entry into the open-label phase were well matched between group A and B with exception of the mean number of relapses during the 2 years before entry. Obviously, the mean number of relapses was greater (p = 0.022) for patient in placebo group. The modalities of drug administration were the same as in the double-blind phase: 20 mg/day of GA was administered s.c. by self injection. The patients were evaluated at 6 month intervals and during suspected relapses. The observation period lasted approximately 6 years, including the double-blind phase of up to 35 months and the open-label phase of over 36 months. The results of this study were focused especially on group A, i.e. patients who had received the drug since randomization. The mean number or relapses for group A during the entire study period was 2.23 with a mean annual relapse rate of 0,42. This result represents a 72% reduction in the annual relapse rate for patients treated wi th GA compared with the documented experience [13] before entering the trial (p = 0.0001). Furthermore 25,7% of patient of group A remained relapse free. The relapse rate per year of group A has continued to drop and for sixth year was 0,23, that it means that risk of relapse become less than one over 4 years (table 4). The majority of patients in group A experienced no confirmed progression of the disease. Based on a definition of worsening > 1 EDSS step sustained for over 90 days, 40.6% of patients of group A worsened during the 6 year period of observation. When the EDSS scores at the end of the 6 years were compared with the EDSS at their randomization, 50.5% of patients of group A were neurologically unchanged (EDSS change of + 0.,5 step) and 18,8% had improved (EDSS score decrease by one ore more steps). In fact, the risk of worsening by > 1.5 EDSS steps becomes less likely the longer the patient remains on therapy. While there can be no long-term comparison with a placebo group now that therapies of documented value are available, the results of this study were compared with published data on similar groups. The experience of group A was better than similar group treated with IFN-b for 5 years [67] but with the difference that IFN-b trial was placebo-controlled with patients and examiners blinded to their study medication. Another available comparison is with the natural history cohort studied by Weinshenker et al [68]. The natural history data would have predicted that 77% patients would have worsened of at least 1 or more EDSS steps, while in the GA study only 30.6% showed unsustained progression and 40.6% experienced progression by > 1 EDSS step sustained for over 90 days, but the later definition of progression was not used in the studies of natural history where the definition was less precise. Results of the open label study must be interpretated with great caution because of the absence of a real control group; however they confirm the excellent safety profile of GA [69].

New recent clinical studies

A multicenter, double-blind, placebo-controlled trial of GA in primary progressive MS was initiated in march 1999 in North America, France and United Kingdom. Nine hundred patients with progressive disease in the absence of relapses, EDSS entry of 3.0-6.5 have been randomized to receive 20 mg of GA s.c. or placebo. The primary endpoint of this study is to determine whether GA slows the confirmed progression of disease.

Recent studies have suggested that feeding glatiramer orally to rats before the active induction of EAE protects animals from disease [70] and that in phase I trial in patients with RR-MS GA administered orally was safe and well tolerated [71]. A multicenter, double-blind, placebo-controlled trial phase III trial testing the effects of two doses of oral GA (5 and 50 mg) in 1650 RR-MS patients has been terminated. Results will be available in early 2002.

MAGNETIC RESONANCE IMAGING STUDIES

The effects of the interferons on the pathological process of the disease are strongly supported by MRI data, but information on the effect of GA on MRI changes was limited until few years ago [65]. A preliminary open-label cross-over trial in ten RR-MS patients suggested that GA decreases the frequency of new gadolinium (Gd)-enhanced lesions found on monthly MRI [72] ; therefore in last years many studies have been conducted to better define MRI changes during GA treatment.

Phase III trial in relapsing-remitting multiple sclerosis patient: effects on MRI parameters

A large European/Canadian multicenter, double blind, randomized, placebo controlled study was conducted to determine the effect, onset and durability of any effect of GA on MRI-monitored disease activity in patients with relapsing-remitting MS. Two hundred thirty-nine eligible patients with relapsing remitting MS were randomized to receive either 20 mg GA (n = 119), or placebo (n = 120) by daily subcutaneous injection. Eligibility required one or more relapses in the two years prior to entry and at least one Gd-enhanced lesion on a screening MRI scan. The study consisted of two preplanned phases: a randomized, double blind, placebo controlled phase during which all patients studied underwent monthly MRI scans and rigorous clinical assessments over nine months, followed by a nine month, open-label phase during which all patients were treated with GA and followed at three month intervals. The primary outcome measure was the total number of Gd-enhanced lesions on T1-weighted images. Secondary outcome measures included the proportion of patients with Gd-enhanced lesions, the number of new Gd-enhanced lesions and change in their volume; the number of new lesions detected on T2-weighted images and change in their volume, and the change in volume of hypointense lesions seen on unenhanced T1-weighted images. Clinical measures of disease activity were also evaluated. The results from the 9-month, double blind phase of the study have been analyzed and recently published [73]. The active treatment and placebo groups were comparable at entry for all demographic, clinical and MRI variables. Treatment with GA showed a significant reduction in the total number of Gd-enhanced lesions on T1-weighted images compared to placebo (p = 0.003) with a reduction of 29% (fig. 4). Consistent differences favoring treatment with GA were seen for almost all secondary end-points examined: the number of new Gd-enhanced lesions with a reduction of 33% (p < 0.003), the monthly change in the volume of Gd-enhanced lesions (p = 0.01; fig. 5), and the change in the volume (p = 0.006) and the number of new lesions seen on T2-weighted images (p < 0.003; fig. 6). The relapse rate was also significantly reduced by 33% for GA treated patients (p = 0.012). All effects increased over time.

Fig. 4. The mean number of enhanced lesions identified on all post-gadolinium enhanced T1-weighted images over the nine months of the controlled phase of the study is displayed. The data are shown using the last available observation carried forward (LOCF) when MRI data were missing for any specific time interval for a given patient. They are also shown using all available data (As Is) without an adjustment for missing values.

Fig. 5. The median cumulative change in enhanced lesion volume from randomization is displayed in L. Statistically significant differences first emerged after six months of continued therapy from randomization.

Fig. 6. The cumulative mean number of new lesions observed on the T2-weighted images at each month on study is displayed. Statistically significant differences first emerged after five months of continued therapy from randomization.

The time of the appearance of both the clinical and MRI-monitored therapeutic effects in this trial are consistent with the apparent principal mechanism of action of GA, i.e. the induction of a population of GA-specific suppressor Th2 cells which secrete anti-inflammatory cytokines and cross-react with myelin-basic protein [33,40,52]. A previous longitudinal study performed in ten RR-MS patients treated with GA demonstrated a reduction in proinflammatory cytokines and an increase of anti-inflammatory cytokines [42]. Also in this trial anti-inflammatory cytokine levels peaked during the first six months of treatment and then gradually decreased, while proinflammatory cytokine levels continued to decrease. The time dependent effect of treatment with GA seen in the present trial is consistent with some delay in the induction and expansion of an appropriate protective GA-specific regulatory T cell population. It is also of interest that the magnitude of the effect on the inflammatory aspect of the disease as monitored by Gd-enhancement on MRI is modest, possibly consistent with a major site of action of these cells within the CNS that limits the extent of tissue damage. The latter is reflected by the parallel effect on new lesions seen on T2-weighted images and on the accumulated disease burden. This trial has shown that the magnitude of the effects of GA on clinical and MRI measures of disease activity is comparable to that of the various formulations of interferon-b [74-76] with the difference that, at most clinically effective doses, IFN-b therapy is associated with a substantial reduction of Gd-enhancement that is rapid in onset, being established within weeks of initiation of therapy [77]. The uncertain relationship between frequency of enhancement and accumulation of irreversible disability [78] does not allow to define whether the ability of the drug to suppress inflammation is one of the fundamental mechanisms through GA has shown to reduce the accumulation of disability in patients with RRMS [13,66,69]. This study also confirms the excellent safety and tolerability profile of GA.

Preliminar data of the second phase of this trial have been presented at the 17 [th] World Congress of Neurology (London, June 2001) [79]. The total mean number of enhancing lesions in the patients switched from placebo to GA dropped from 12.6 in the double-blind phase to 5.9 (­53%; p < 0.0001) in the open-label phase, while the effect in those patients receiving GA from the start was sustained and showed a reduction from 7.7 to 6.2 (­20%; n.s.). Similarly, the mean volume of enhancing lesions fell from 1.8 to 0.8 ml (­56%; p < 0.0001) in the patients originally randomized to placebo, and from 1.1 to 0.8 ml (­27%; n.s.) in those in the continuous active treatment group. At the final evaluation, the median percent increase in T2-lesion load from baseline was 17.4% in the patients initially randomized to placebo and 13% in those treated with GA from the outset (p = 0.018). Annualized relapse rates, which had shown a decrease in favor of GA in the placebo-controlled phase, dropped further during the open-label phase: from 0.73 to 0.33 (p = 0.0001) in the patients originally assigned to placebo and from 0.52 to 0.26 (p = 0.003) in those receiving GA from the start. The open-label extension of the double-blind European Canadian GA study showed that the positive effect of GA on MRI disease activity and burden is persistent also after a longer period of time.

OTHER MRI STUDIES

The assessment of brain volume change with serial MRI provides an objective measure of irreversible damage due to MS. Rovaris and colleagues, in a recent paper [80] , investigated if GA has a beneficial effect on the development of brain atrophy in the two groups of RR-MS patients of the European/Canadian multicenter, double blind, randomized, placebo controlled study on MRI-monitored disease activity. The reduction in brain volume in the first phase of the study was 0.8% and 0.7% in GA-treated and in placebo patients, respectively. In the second phase brain volume continued to decrease, however, by only 0.4% for patients always on GA and 0.6% for those originally on placebo. Although these differences between the treatment groups were not statistically significant, a possible late trend for treatment with GA to retard the loss of brain volume is suggested.

New lesions are usually hypointense in T1 weighted images when they appear; then about 2/3 become isointense and 1/3 remain hypointense (permanent black holes) [81]. The evolution to permanent black holes indicates a failure of reparative mechanisms. Among the patients in the European/Canadian trial, 1722 new lesions were identified. Fewer (about 30% less) new lesions appeared in GA-treated than in placebo patients (p = 0.05), while enhancement duration was similar in both groups. The percentage of new lesions which disappeared on subsequent T2-weighted scans, was slightly, albeit not significantly, higher in GA-treated (4.1%) than in placebo (2.7%) patients. The frequency of lesions re-enhancing on subsequent scans was lower in GA- (5.0%) than in placebo-treated (8.5%) patients (p = 0.002). Most importantly, the percentage of lesions which evolved into permanent black holes was lower by almost 50% in GA-treated compared to placebo patients, being 8 months after their appearance 15.6% vs. 31.4%, respectively (p = 0.002) [82]. These results suggest t he ability of GA to enhance the recovery mechanisms in MS lesions, an interpretation supported by some recent in vivo studies [83].

SAFETY

Toxicological studies, conducted in many animal species (rats, mice, dogs, rabbits), indicated that the drug is safe at the current dosage regimen (20 mg/day), with an adequate safety margin. Reproduction studies performed in rats and rabbits (Teva Pharmaceutical Industries Ltd, internal reports) showed no effects on fetal loss or fetal abnormalities or toxic effects during the post-natal development. In vitro and in vivo studies demonstrated that GA is devoid of any mutagenic or carcinogenic potential. Furthermore studies in vivo have been conducted to verify the potential effects of GA on the cardiovascular system and it was found that, only after e.v. injection of high dose of GA, there can be a hypotensive effect while there is no such effect if GA is administered s.c. and at dosage of 20 mg. Of some interest is the observation that rat peripheral mast cells exposed to GA release histamine; it is probable that the local reaction, often seen at the injection site of treated patients, is caused by the induction of histamine release from dermal mast cells (Teva Pharmaceutical Industries Ltd, internal reports).

The safety data have been obtained from the observation of more than 3500 MS patients treated with GA in controlled and uncontrolled studies (cumulative data from Teva Pharmaceutical Industries Ltd, internal reports) at the dosage of 20 mg s.c. Overall withdrawal from GA therapy due to clinical adverse experiences occurred in 8.4% of the patients. The most frequent reasons for treatment withdrawal were dyspnea and vasodilatation, each of which occurred in about 2% of the patients. No patients were withdrawn because of laboratory abnormalities. The most commonly observed adverse experience associated with the use of GA is local reactions at the injection site which are detailed in table 5. Some of the most common local reactions are erythema (41.6%), pain (37%), inflammation (24.5%), pruritus (19.5%) and swelling (12.8%). Generally these local reactions decline over time and there is no skin necrosis. It has recently been described a localized lipoatrophy after prolonged treatment with GA [84,85]. Other adverse events reported from more than 5% of patients are: vasodilatation (14%), dyspnea (10%), pain (10%), cephalea (10%), asthenia (10%), urinary infections (8%), unsteadiness (8%), chest pain (8%), paresthesia (7%), rash (7%), depression (6%), nausea (5%), anxiety (5%), rachialgia (5%), fever (5%), allergic reactions (1%). The most remarkable adverse event that can affect patients treated with GA is a systemic post-injection reaction that occurs in about 10% of the patients. This reaction consists of a variable combination of flushing, chest tightness, palpitations, dyspnea, tachycardia and anxiety. This reaction is sporadic and unpredictable: it occurs within seconds or few minutes after the injection, lasts between 30 seconds-30 minutes and resolves spontaneously without sequelae, but in some cases it can last for more than 1 hour. Most patients who had this reaction reported only one episode, but a few patients experienced more episodes [86]. They tend to occur more frequently during the first months of treatment. The cause of the systemic reaction is unknown. Routine electrocardiographic monitoring did not reveal any cardiac anormalities [13]. The observed symptomatology suggests a dysfunction of the autonomic nervous system. Because the systemic reaction is self-limited and without relevant consequences there is no reason to stop treatment. Patients should be informed that this reaction can occur, in order to reduce the emotional impact; moreover, the re-injection, after such a reaction, should preferably be done under medical observation to reassure the patient. Anaphylaxis can be associated with the administration of any foreign peptide. Two out of 3,736 patients treated in open label trials experienced non fatal anaphylactoid reactions (cumulative data from Teva Pharmaceutical Industries Ltd, internal reports). Controlled studies demonstrated that GA treatment does not provoke hematological abnormalities, elevation of hepatic enzymes, flu-like syndrome, depression and abnormalities on blood pressure [13]. There is also no evidence of relevant drug interactions. A total of 38 women receiving GA in clinical trials became pregnant. Twenty-one pregnancies were terminated prematurely. Five pregnancies culminated in birth of which four were healthy babies. One baby had a cleft lip; however he was, during pregnancy, exposed also to carbamazepine (TEVA Pharmaceutical Industries, Internal Reports). No information was available on the outcome of the additional 12 pregnancies. In conclusion, the adverse events observed during GA treatment are few and mild and none of the deaths occurred during controlled or opened studies have been related to GA treatment.

ANTI-GLATIRAMER ACETATE ANTIBODIES

Experimental studies in rat and monkey showed, by radioimmunoassay, the presence of anti-GA antibodies in most of the animals. These antibodies did not reduce the effect of GA in mice with EAE or the proliferation of a T-cell line specific to GA [87]. All the patients receiving GA in the double-blind placebo-controlled trial in RR MS developed GA-reactive antibodies [66,87]. Maximal levels were attained after an average treatment duration of 3-4 months; thereafter antibodies declined, but remained positive. No such antibodies were detected in placebo patients. The presence of anti-GA antibodies does not influence the clinical efficacy of the drug. These antibodies belong to the immunoglobulin G class and do not exhibit neutralizing activity in several in vitro and in vivo systems.

Evidence of sustained efficacy of GA for more than 6 years in an open-label study [69] support the conclusion that neutralizing antibodies do not impair the clinical effectiveness of GA over time.

In a recent study conducted by Brenner and colleagues [88] high antibody titers were demonstrated in relapse-free patients and therefore no correlation was found between changes in EDSS and the existence or level of these antibodies. No correlation was found between anti-GA antibodies and the appearance of local or systemic adverse events. No cross reactivity was found between these antibodies and MBP and these later findings are similar to those previously described in animal studies [23,27]. The GA-antibodies were also isotyped and IgG1 levels were 2-3 folds higher than those of IgG2: this preferential production of IgG1 may indicate that Th2 responses are involved in mediating the clinical effect of GA, as explained in me-chanisms of action of GA.

POST-MARKETING EXPERIENCE

Copaxone has been authorized, as a treatment for ambulatory patients with RR MS, in many countries: at the end of 1996 in Israel and USA and Byelorussia, in 1997 in Argentina, Russia, Slovenia, Canada e Hungaria, in 1998 in Romania, Brazil, Ukraine, in 1999 in Australia, Switzerland, in 2000 in United Kingdom and in 2001 in most of the European countries.

At the end of 2001 about 40,000 MS patients are treated with GA worldwide. New adverse events have not been reported. A few other patients had non fatal anaphylactoid reactions. There have been other 195 reports of pregnancies. One hundred and seventeen pregnancies were lost to follow-up, 24 pregnancies resulted in abortions (3 elective abortions and 21 miscarriages for unknown reasons), 47 resulted in healthy infants (2 of which were premature), 4 babies had congenital abnormalities, one pregnancy resulted in a stillbirth and there was one case of twins with anencephaly requiring abortion (cumulative data from Teva Pharmaceutical Industries Ltd, internal reports).

THE PLACE OF GLATIRAMER ACETATE IN MULTIPLE SCLEROSIS THERAPY AND FUTURE DEVELOPMENTS

In conclusion the extensive experience collected until now (about 40,000 patients) clearly demonstrates that GA is a specific treatment for MS patients having favorable effects on clinical parameters (reduction of relapses and slowing disability) and on MRI parameters (reduction of new lesions, prevention of re-enhancement of old lesions and potentiation of reparative mechanisms). The full effect of the drug requires 4-6 months and do not decline over time. This drug is safe and usually well tolerated because side-effects are minimal. The very good safety profile is supported by in vitro and in vivo animal studies which showed that the drug has no teratogenic or mutagenic effects. However the available data on births from women exposed to the drug are still insufficient to confirm the absence of teratogenic or mutagenic effects. Another interesting aspect emerging from the studies performed is that GA seems to be more effective in the early phases of MS.

At the present the drugs approved for clinical use in multiple sclerosis are four: the glatiramer acetate (Copaxone), IFN-b 1b (Betaseron, Schering- Berlex), IFN-b 1a (Avonex, Biogen), IFN-b 1a (Serobif, Serono). Direct comparison among these four drugs is not possible because there are many differences (methodological, dose, route of injection, patient selection) between the clinical trials which explored and demonstrated the efficacy of the drugs. However, the effects on relapse rate and MRI parameters were quite similar in all the trials, suggesting that there are not crucial differences between the drugs.

These drugs can improve the disease course and the scientific community of neurologists agrees on the opinion that these treatments should be started at the beginning of the disease when the accumulated clinical disability is minimal or absent.

The neurologist has to face with at least three questions:

1. Which treatment first?

2. How to evaluate if a patient is responsive to one treatment and when to shift from one type of treatment to another one?

3. Will a patient benefit from the combination of GA with one of the three IFN-B or from the combination of GA or IFNb with an immunosuppressive drug?

No answer is available for these questions at present. There have been some attempts to define better what constitutes a responder and non-responder [64] , but the definition in the single patient is almost impossible. Although the precise mechanisms of action of GA and IFN-b are not completely understood, there are converging indications that they work through different immunological pathways. As a consequence, if a patient does not respond to interferon beta it is possible that he responds to GA and vice versa. GA treatment is suggested, because of the absence of hematological abnormalities and of interaction with other drugs, for patients who present association with other diseases, as hepatic or thyroid dysfunctions. Comparing GA with IFN-b, the advantage of GA treatment is the absence of important side effects like the flu-like syndrome but the disadvantage is the daily administration. Limited in vitro data [89] suggest that the combination of an interferon beta and glatiramer may have additive effects. However data in vivo are not concordant: Miziachi-Koll [90] demonstrated a synergistic effects of the two drugs in hyperacute EAE, while Brod [91] demonstrated that combinations of the two drugs at doses where either one is individually effective in blocking the induction of EAE, are ineffective when combined. To what extent these laboratory observations have relevance to humans is uncertain; trials in patients have been started to test first if the combination is safe before starting larger studies to determine if the combination is additive or synergistic in benefits. In conclusion we would emphasize that none of these drugs is able to stop disease activity completely and that the effects on disability progression appear at best to be modest. Even though they represent substantial progress in treatment of multiple sclerosis, a definitive cure is lacking and it might be possible only when the etiology of this disease will be clarified.

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