Abstract
Linomide (quinoline-3-carboxamide) is a synthetic immunomodulator that increases the natural killer cell activity. We previously demonstrated that linomide effectively inhibited the clinical and histopathologic signs of acute and chronic relapsing experimental autoimmune encephalomyelitis. We report a double-blind, placebo-controlled study to evaluate tolerability and to obtain preliminary indications of the clinical efficacy of linomide on secondary progressive MS. Thirty patients suffering from clinically definite and laboratory-supported secondary progressive MS, with an expanded disability status scale (EDSS) of 3.0 to 7.0, were included in this study. Patients were treated daily with linomide (2.5 mg) or placebo orally and were followed up for side effects and changes in their neurologic status; monthly MRI scans were taken throughout the treatment period. Twenty-four patients completed at least 6 months of treatment. Mild to moderate side effects, including muscle pains, arthralgia, and edema, were present in 11 of the 15 patients receiving placebo and in 13 of the 15 patients treated with linomide. At 24 weeks, the mean shift in EDSS was +0.272 +/- 0.156 in the placebo group versus -0.166 +/- 0.167 in the linomide group (p = 0.0451). The percentage of patients with evidence of ``activity'' on their MRI (new, enlarging, or new gadolinium diethylenetriaminepentaacetic acid [Gd-DTPA]-enhancing lesions) throughout the treatment period was 75% in the placebo group and 33% in the linomide group (p = 0.0205). The mean total number of new Gd-DTPA-enhancing lesions per MRI scan for the same period was 0.42 +/- 0.143 in the placebo group and 0.19 +/- 0.114 in the linomide group (p = 0.0387). In this study, linomide proved to be safe and well tolerated in patients with secondary progressive MS. In addition, our results indicate that linomide tends to inhibit the progression of the disease, especially preventing the appearance of new active lesions in the MRI scans. Based on these results, two multicenter phase III trials are currently under way in the United States and in Europe and Australia.
NEUROLOGY 1996;47: 341-346
MS is predominantly a disease that occurs in young adults; it is characterized by multifocal inflammatory and demyelinating lesions of the white matter of the CNS, leading to chronic disability, mainly due to pyramidal, cerebellar, and optic tract involvement. [1] It is manifested clinically by recurrent attacks or by a chronic progressive course of neurologic dysfunction. The pathogenetic mechanisms of MS remain largely obscure. Several immunologic studies have provided evidence for a possible autoimmune pathogenesis. [1,2] Common therapeutic approaches, therefore, involve the use of immunosuppressive modalities such as azathioprine, cyclophosphamide, and cyclosporine. [3,4]
Several investigators have demonstrated immunoregulatory abnormalities in patients with MS, including defective suppressor cell activity and interferon-alpha (IFN-alpha) production, reduced natural killer (NK) cell activity, and increased IFN-gamma and transforming growth factor-alpha production. [5-8] Because of these observations, the design of new treatments for the disease has been focused on immunomodulatory agents such as IFN-beta, high-dose immunoglobulins, Copolymer 1 (Copaxone), and oral tolerization techniques. [4,9-12]
Linomide is a novel immunomodulator that increases the NK cell activity and induces consistent and extensive activation of several other lymphocytic subpopulations in experimental animals and humans. [13-16] The reported enhancement of NK cell activity was not associated with increased IFN production. [14,15] Linomide exclusively stimulated lytically inactive bone marrow NK progenitors, but did not affect mature NK cells in the spleen. [14] In a model of ocular B16F10 melanoma, linomide drastically inhibited metastatic disease. [17,18] The in vitro proliferative response of lymphocytes to T-cell mitogens was enhanced by linomide, with a concomitant increase in interleukin-2 production. [19]
Despite its immunostimulatory effects, linomide profoundly inhibited both spontaneously developing and experimentally induced autoimmune diseases in several animal models. It reduced the autoantibody production and ameliorated the clinical manifestations of systemic lupus erythematosus-like disease in MRL-lpr and New Zealand black and white mice, of insulin-dependent diabetes mellitus in NOD mice, and of experimental autoimmune myasthenia gravis in albino rabbits and Lewis rats. [20-24] The drug also exhibited anti-inflammatory properties in coxsackievirus B3-induced myocarditis in BALB/c mice. [25]
In the animal models of MS--experimental autoimmune encephalomyelitis (EAE) and chronic relapsing EAE--we previously showed that linomide effectively prevented and reversed paralytic signs and inhibited further relapses, even when given at advanced stages of the disease. [26,27] Moreover, it totally prevented the development of CNS inflammatory and demyelinating histopathologic lesions of EAE, without inducing generalized immunosuppressive effects.
In this double-blind clinical pilot trial, we tested the safety and efficacy of linomide in patients with secondary progressive MS. As suggested by many investigators, even a small number of patients (20 to 40) and a relatively short period of follow-up (6 months) may be sufficient to grossly evaluate the efficacy of an experimental drug in the disease, when serial brain MRI tests are performed. [28] Detection of MRI activity (especially the appearance of new gadolinium diethylenetriaminepentaacetic acid [Gd-DTPA]-enhancing lesions) shows a good correlation with changes in functional disability status [29] and is an objective means for evaluating MS activity. [29,30]
Methods.
Patients and treatment protocol.
Thirty patients (16 men and 14 women) with clinically definite and laboratory-supported MS (according to the criteria of Poser et al. [31]) of the secondary progressive type (progressive clinical deterioration for at least 2 years following an initial relapsing-remitting course) were included in this study. Patients were 25 to 55 years old, with evidence of deterioration of at least one degree according to the expanded disability status scale (EDSS) [32] in the 2 years prior to the study, and at least three lesions on the screening MRI scan or at least one Gd-DTPA-enhancing lesion. The patients had not been treated with immunosuppressive treatments for 3 months, or with corticosteroids for 1 month prior to inclusion. The final protocol of the study was approved by the Helsinki Ethics Committee of Hadassah University Hospital and by the Israel Ministry of Health. All patients signed an informed consent form and were assigned a randomized number. Treatment was initiated with linomide or placebo, 2.5 mg/day PO, for 6 months. Sealed bottles containing the similarly shaped and colored pills of linomide and placebo were provided by Pharmacia. Each patient received a numbered bottle corresponding to a computerized randomization list. Treatment was continued for 6 to 12 months, unless adverse reactions appeared. When side effects of grade 2 or 3 (according to the World Health Organization [WHO]) were seen, the dosage was reduced to 3 times per week or to once per week, respectively. Screening tests, including lumbar puncture (LP) and evaluation of the initial reaction to the treatment, were carried out during a 5-day hospital period. Once a week thereafter, the patients were examined for side effects. Blood tests (WBC and complete biochemistry) were performed weekly during the first month and on a monthly basis for the remainder of the study period. A complete neurologic examination, including EDSS/functional systems (FS), was performed at baseline and once every 3 months by two experienced neurologists. A second LP was done after 6 months of treatment. Brain MRI scans were performed every month.
The primary objective of the study was to evaluate the safety of linomide, given daily for 6 months, in patients with secondary progressive MS. The secondary aim was to evaluate its efficacy in preventing MRI ``activity'' (especially the appearance of new Gd-DTPA-enhancing lesions on serial MRI scans) and in inhibiting the clinical deterioration of MS, as evidenced by changes in the EDSS/FS scores in follow-up.
In case of a relapse or clinical deterioration of more than 1 degree on the EDSS scale, which, according to the opinion of the treating physician, required treatment with corticosteroids, the patient was withdrawn from the study. A patient who underwent treatment for at least 4 months without missing more than one MRI scan was considered eligible for full evaluation. Six patients were withdrawn from the study: three in the linomide group (two because of a relapse during the first 20 days of treatment and one because of the appearance of edema in the legs) and three in the placebo group (two because of severe neurologic deterioration requiring treatment with corticosteroids and one because of the occurrence of a subdural hematoma after falling). One patient from the placebo group was not included in the per protocol evaluation of the EDSS score owing to discontinuation of treatment on week 22 because of epileptic seizures (no EDSS data on week 24), but this patient was included in the MRI evaluation.
When all 24 patients had completed 6 months of treatment, all data from the case report files were entered into a computer database and ``locked.'' Only then were the code numbers bared. The demographic data revealed an equal distribution of clinical variables (duration, severity of disease) between the two groups Table 1. There was a difference in the mean baseline number of Gd-DTPA-enhancing lesions (linomide group: 1.54 +/- 0.82 per patient; placebo group: 0.50 +/- 0.36 per patient) (see Table 1).
Table 1. Demography
MRI protocol.
MRI was performed in a 2.0-tesla unit (Elscint, Haifa, Israel). Sagittal T1-weighted images were used to plan the axial T2-weighted and postcontrast T1-weighted images. Axial images were obtained parallel to the inferior surfaces of the genu and splenium of the corpus callosum. Slice thickness was 5 mm, with a 2-mm gap. The axial T2-weighted images were obtained with a relaxation time (TR) of 3,000 msec and echo times (TEs) of 20 msec and 80 msec. Axial T1-weighted images were obtained with a TR/TE of 450/10 msec. Gd-DTPA (Magnevist, Schering, Germany), at a dosage of 0.15 mmol/kg, was used as contrast material. An MRI test was performed 1 to 15 days before initiation of treatment (baseline) and once a month thereafter. Positioning of the patient was easily reproducible, and identical sections were obtained for each patient and at every time point. Analysis of the MRI scans was performed by two experienced neuroradiologists in a blinded manner.
To evaluate the effect of treatment on the MRI activity, we focused on the Gd-DTPA-enhancing lesions and on the ``active'' lesions, as described by Miller et al. [28] According to these investigators' indications, the clinical disease activity is best correlated with new Gd-DTPA-enhancing lesions, enlarging lesions, and newly appearing nonenhancing lesions.
Statistics.
The proportion of patients developing active MRI lesions in the two groups was compared using the chi-square test. The mean number of active lesions per patient was analyzed using the Wilcoxon rank-sum test. The changes in the EDSS score were also compared using the Wilcoxon rank-sum test. All tests were one-sided, testing whether linomide was superior to placebo in halting the progression of the disease (inhibiting the appearance of active lesions on the MRI scan and decreasing the EDSS score) based on the ``per protocol'' analysis.
Results.
Side effects.
Adverse events were reported in 11 of 15 patients in the placebo group (28 total events) and in 13 of 15 patients in the linomide group (44 total events). There were no major side effects (WHO grade 3 or higher) in the linomide-treated patients. The most frequently observed side effects were arthralgia/myalgia (in four of the linomide-treated patients vs. two from the placebo group), peripheral or facial edema (in three of the linomide-treated patients), transient diarrhea (in three of the linomide-treated patients), and a mild (less than threefold) and temporary (<14 days) increase in liver enzyme levels (in two of the linomide-treated patients). Three serious adverse events were reported in the placebo group: One patient had severe urinary tract infection that required hospitalization; one patient developed a bilateral subdural hematoma after a head trauma; and one patient stopped treatment on week 22 because of epileptic seizures. A complete list of all the side effects reported in this study has been submitted to the National Auxiliary Publication Service (NAPS) and is available to any reader. We must mention that in the two other MS trials with linomide (in Sweden and the United States), two incidents of aseptic pericarditis have been observed in the linomide group (Anders Linde, personal communication). In various clinical trials including patients with acute myeloid leukemia (AML) and in healthy volunteers, 13 cases of pericarditis have been reported (possibly or probably related to linomide) among a total of 754 patients participating in these trials. In almost all of these cases, the symptoms resolved after treatment with nonsteroidal anti-inflammatory drugs and corticosteroids.
A dose reduction following adverse events was similar (approximate 10%) in both groups, as shown by the mean dosage per patient, which was 2.29 +/- 0.14 mg/day for the placebo group and 2.23 +/- 1.08 mg/day for the linomide group; this indicates an equal distribution of intolerable adverse events in the two groups.
Effect of linomide on the clinical course of MS.
During the 24-week period, six patients from the placebo group (eight patients, including those who were withdrawn and treated with steroids on week 16) showed clinical deterioration (increased EDSS score), as compared with only three patients in the linomide group. Five patients showed clinical improvement in the linomide group (a decrease in EDSS scale), as compared with two patients in the placebo group. A detailed table of the EDSS changes in each patient has been submitted to the NAPS and is available to any reader. The mean change in the EDSS score (Delta EDSS = EDSS on week 24 minus baseline EDSS) was +0.272 +/- 0.156 in the placebo group (deterioration) and -0.166 +/- 0.167 in the linomide group (improvement) (p = 0.0451 by the Wilcoxon rank-sum test).
Effect of linomide treatment on MRI activity.
According to the baseline MRI (before the start of treatment), 16% of the patients in the placebo group and 33% of the patients in the linomide group had Gd-DTPA-enhancing lesions. These percentages were reversed in the course of the 6-month follow-up. During the entire 4- to 24-week treatment period, 75% of the placebo-treated patients displayed ``activity'' on the MRI scan (new Gd-DTPA-enhancing lesions plus enlarging lesions plus newly appearing nonenhancing lesions), as compared with 33% of the linomide-treated patients (p = 0.0205) Figure 1A. The number of MRIs showing ``active'' lesions (according to the above definition) was 24 in the placebo group (mean 2.00 +/- 0.50 per patient) and 11 in the linomide group (mean 0.92 +/- 0.49 per patient) (p = 0.0382) Figure 1B. The mean total number of new Gd-DTPA-enhancing lesions per MRI scan for the same period was 0.42 +/- 0.143 in the placebo group and 0.19 +/- 0.114 in the linomide group (p = 0.0387) Figure 1C.
Figure 1. Effect of linomide treatment on MRI activity. (A) Percenlage of patients with MRI activity (new Gd-DTPA-enhancing lesions plus enlarging lesions plus newly appearing lesions) in all six MRI tests (throughout the entire treatment period, from week 4 to week 24). (B) Mean number of MRI scans per patient that showed evidence of activity (at least one active lesion, as defined in the Methods section). (C) Mean number of new Gd-DTPA-enhancing lesions per patient per scan.
Since the appearance of new enhancing lesions on the MRI scan may be the best indicator of clinical relapses and because clinical deterioration usually follows the appearance of a new lesion, [29] we demonstrated the MRI scans containing new Gd-DTPA-enhancing lesions as black spots Figure 2 in order to visualize disease activity over time. Twenty-four MRI scans with new enhancing lesions were encountered in the placebo group, as compared with nine in the linomide group (p = 0.0258 by the chi-square test).
Figure 2. MRI scans with new Gd-DTPA-enhancing lesions in each patient over the 24 weeks of observation. Every MRI scan on which at least one newly appearing Gd-DTPA-enhancing lesion was found is represented in the graph by a dark spot.
Laboratory results.
Analysis of several immunologic functions was performed at four time points. Preliminary analysis of these data revealed some interesting changes, indicating that linomide acts as an immunomodulator rather than an immunosuppressor. The peripheral WBC counts were significantly increased in the linomide group, gradually rising from a baseline value of 7.78 +/- 0.27 times 103/mm3 to a peak value of 11.9 +/- 0.79 times 103/mm3 at week 16 (p < 0.0002). In addition, fluorescence-activated cell sorter analysis of the lymphocyte surface molecule expression revealed a significant increase in CD45-Ra+ lymphocytes (in G1 gating for lymphocytes). These data will be presented in detail in a separate report. The mean blood levels of linomide in the treated group were 1.185 +/- 0.142 mmol/liter at week 4, 1.108 +/- 0.115 mmol/liter at week 12, and 0.844 +/- 0.139 mmol/liter at week 24.
Discussion.
Several investigators have shown that MRI follow-up in MS may detect disease activity much more often than the clinical relapse rate. [28,30] This allows us to test the efficacy of an experimental drug in a small group of patients (20 to 40) during a short period of observation by performing monthly MRI scans (preferably with injection of Gd-DTPA). [28-30] The suggested design, by Nauta et al., [28] is a double-blind, parallel group study with a baseline correction scan, as was performed in our trial.
We have shown here that orally administered linomide was well tolerated, giving rise to only mild to moderate side effects. This treatment seems to inhibit MS activity, as indicated by the reduced number of newly appearing Gd-DTPA-enhancing lesions in the linomide group and by the differences between the placebo and linomide groups observed in the functional disability status scale.
Linomide does not induce generalized immunosuppression, and it seems to enhance immunoregulatory cells (i.e., NK cells). Preliminary immunologic analysis revealed that linomide increased the number of leukocytes and specifically upregulated the proportion of CD45-Ra+ cells, without affecting the CD45-Ro+ lymphocytes. The CD45-Ra+ cells were previously shown to function as inducers of suppressor lymphocytes, [33] and their proportion was reduced in progressive MS, [34] correlating reciprocally with disease activity and with the appearance of active lesions on MRI. [35]
In previous studies, we have shown that linomide was one of the most potent agents for the regulation of several experimental autoimmune diseases. In vitro, linomide was found to interfere with antigen presentation, probably by strengthening NK, cytotoxic, and suppressor cells, which downregulate the functional capability of monocytes to present autoantigens to lymphocytes (Lehmann et al., submitted for publication).
In conclusion, despite the small size of this first pilot study, the results of the monthly MRI evaluations suggest the efficacy of linomide in halting the progression of MS of the secondary progressive type. These positive results agree with our previous experimental data and provide a basis for further investigation of this medication in MS and in other autoimmune diseases. The current trend for treating MS, and probably other autoimmune diseases as well, is likely to involve immunomodulatory rather than immunosuppressive agents. To this direction, linomide may play an important role as an alternative nontoxic therapeutic approach for MS. Two multicenter, phase III trials are currently under way in the United States and in Europe and Australia.
- Copyright 1996 by Advanstar Communications Inc.
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