Four year double-blind controlled study of levamisole in multiple
sclerosis
Massaro A.R.,* Cioffi R.P.,* Laudisio A.,* Schiavino D.**, Mariani M.***
* Istituto di Neurologia, Università del Sacro Cuore, Roma
**Istituto di Clinica Medica, Università del Sacro Cuore, Roma
* * * CNTS Croce Rossa Italiana, Roma
Summary 41 patients with definite multiple sclerosis (MS) in the stationary phase entered this four year double-blind levamisole-placebo controlled study. 22 patients were treated with levamisole, 150 or 200 mg once a week for a 4 year period, and other 19 with placebo with the same schedule. Patients were put in one of the two groups at random. The treatment was then stopped for those patients who presented a clear exacerbation before the end of the 4 year trial period, and these cases have been considered as negative. Of the group treated with levamisole 8 patients presented an exacerbation during the observation period, and 14 did not. The group treated with placebo presented 14 subjects who had exacerbations and 5 patients who did not. The difference between the two groups was statistically signifìcant. This study demonstrates that levamisole significantly reduced the number of MS patients with acute relapse during the 4 year period of treatment. Nevertheless, not all patients were free from relapse: that could probably suggest that different immunopathological backgrounds may underlie what we usually call MS. It. J. Neurol. Sci. 11: 595-599, 1990
Introduction
Levamisole, the levoisomer of tetramisole, was first marketed as an antihelmintic in 1968. The first evidence of its immunotropic properties was obtained in 1971 [16], and this observation marked the start of modern immunopharmacology[1]. Levamisole induces increased cyclic guanosine monophosphate levels in the immunocytes, and possesses antioxidant properties which may be linked to its immunomodulating functions. Further, levamisole presents indirect thymomimetic activity which is mediated by a serum factor that does not depend on the presence of a functioning thymus. These functions are probably related to multiple effects of the molecule and its metabolite(s) [1].
The immunological abnormalities of multiple sclerosis (MS)[15], as well as the encouraging results of a pilot study previously conducted [11] prompted us to investigate further the effect of the immunomodulator drug levamisole in preventing new attacks and normalizing some immunological parameters in MS patients with typical relapsing-remitting course. The features of the drug [18] and the pathological and immunological aspects of acute MS discouraged us from using levamisole for the treatment of exacerbations. We decided to undertake a long term double-blind placebo controlled study of levamisole in order to find out whether this drug would avoid recurrences of new acute attacks. We did not expect any definite improvement of the patient’s clinical state, but only a prolonged arrest of disease progression.
Patients and method
52 patients with a definite diagnosis of MS, based on widely accepted clinical and laboratory criteria [14, 17, 2], agreed to enter the study after being informed of its experimental nature. They were selected according to the following criteria: they must not be in the acute phase of the disease; they must show a relapsing-remitting clinical course, with at least two relapses in the previous four years; they must not have a high degree of disability; they must not have undergone therapy influencing the immune system within the last two months; they must undertake to comply with the therapy: compliance was evaluated periodically through interviews with the patients and their relatives.
11 patients left the study. 2 of these had to abandon the trial within the fìrst four weeks because of a high fever (39-40°C), which appeared every time shortly after the drug was taken and lasted approximately two days. 2 other patients abandoned the study within one year because of a persistent acnelike skin eruption. The last 4 patients were under therapy with levamisole. No other patient of the study showed any of these side effects. Another 7 patients decided to abandon the study for personal reasons unrealed to the effects of the therapy; 3 of them were treated with levamisole, the remaining 4 with placebo. None of these 11 patients showed any worsening of their status while they were on the therapy.
Table I. Patients groups. Neurological functions (NF) and disability status (DS) mean scores are indicated
| Groups | No. of patients | Males | Females | Age range | mean | median |
| Levamisole | 22 (NF=5.5; DS=3.7) | 8 | 14 | 21-43 years | 32 | 31 |
| Placebo | 19 (NF=5.15; DS=3.9) | 8 | 11 | 18-52 years | 35 | 32 |
| Total | 41 | 16 | 25 | 18-52 years | 33 | 32 |
Of the 41 patients that completed the study, 16 were males and 25 females, aged 18 to 52 years. The onset of the disease varied from 2 to 16 years before the entry in the trial. These patients were randomized into two groups. The first consisted of 22 patients treated with levamisole, 150-200 mg once a week for 4 years. The second consisted of 19 patients who were given placebo pills of identical aspect with the same schedule (Table I). The pill boxes were given by us according to the usual double blind procedures. The treatment was then stopped in those patients who presented a clear exacerbation before the end of the 4 year trial period; such patients were considered as negative results. Other symptomatic side therapies with no known effects on the immune system were continued as necessary. At the beginning of the study, and afterwards every six months, the following parameters were tested: a) neurological examination, including the scores for the neurological functions (NF) and the disability status (DS) scales of Kurtzke [9]; b) phytohemagglutinin (PHA) "in vitro" lymphocytes proliferation test [8] using PHA Blasto-kit (Istituto Sieroterapico Milanese, Italy): c) delayed immunity skin tests with PHA (PHA, Difco), Candida (Candida albicans, Lofarma); Mix Candida Estracts (Dermatophitin, Hallister), Mix Tricophyton (Dermatophitin "O", Hallister), Tubercolin (Protein Purified Derivated, Berna) [7], and active sensitization with Dinitrochlorobenzene (DNCB) [12]; d) cerebrospinal fluid (CSF) IgG and myelin basic protein (MBP). IgG has been tested using LC-partigen immunodiffusion plates (Behring, Italy), and MBP by Myelin B.P.-Ter kit for radioimmunoassay (Biodata, Italy) [10].
Tissue typing of the A, B, C, DR, and DQ antigens was performed using the NIH lymphocytotoxicity test with antibodies produced by the CNTS of the Italian Red Cross, and by Interlabo-Fresenius (GFR): HLA data were statistically analysed using the Fisher test.
We performed the following tests periodically: complete blood count, urine analysis, complete routine blood tests.
Results
Of the group treated with levamisole, 7 patients (32%) presented an exacerbation during the trial period, while 15 (68%) did not. Of these 15, only one patient (case 13) had a higher NF score at the end of the study than at the beginning, while DS remained unchanged and no increase of MBP was detected at the CSF examination we performed every six months. In the placebo group 14 patients (74%) experienced an exacerbation, while 5 (26%) did not. These 5 patients had the same DS and NF scores at the beginning and at the end of the trial period. MBP was used as additional but not main proof of exacerbation. The number of patients who underwent an exacerbation in the study period (including patient 13 who presented only an increase at the final DS score) was significantly lower in the levamisole group compared to placebo (X2 5.899, with p <0.02). These results are shown in Fig. 1 and 2. PHA lymphocytes proliferation test was below normal levels (<30%) in 15 out of 22 patients in the group treated with levamisole, and in 12 out of 19 in the placebo group.
Fig.1. Follow-up of levamisole treated MS patient group. The solid square (v) indicates the time of the exacerbation. The initial neurological functions (NF) and disability status (DS) scores are indicated in the left column, while the final scores are indicated in the right column. The asterisk indicates the cases who had no exacerbation during the entire trial period. Numbers near the solid squares indicate CSF levels of MBP (ng/ml) of the patients who had exacerbations.
Fig. 2. Follow-up of the placebo-group patients. Legend is similar to that of figure 1.
At the end of the study period only 6 out of the 22 patients of the levamisole group had a lower than normal lymphocyte proliferation test, and 13 out of 19 in the placebo group. However, as oscillations in the results of the test were frequent during the follow-up in both groups, this type of laboratory monitoring probably may not follow faithfully the clinical evolution of the disease. Skin tests were below normal in 16 out 22 of the levamisole group, and in 12 out of 19 of the placebo group; at the end of the trial they were still below normal in 13 out of 22 patients of the first group and in 14 out of 19 of the second. The DNCB skin test was below normal in 13 patients of the levamisole group and in 9 of the placebo group; at the end of the trial they were below normal in 10 of the first group and in 10 patients of the second. Also for the skin tests we encountered such variations during the follow-up that we decided to consider these tests only as orientative, not strictly related to the clinical state of the patient. Evidently, the response to the tests must depend upon multifactorial causes, which in turn do not always parallel the clinical evolution of MS. The CSF IgG monitoring did not show significant variations. At the beginning of the study period the mean CSF level of IgG was 5.2 mg/dl in the levamisole group, and 4.9 mg/dl in the placebo group. At the end of the trial they were 4.8 mg/dl in the former and 5.7 mg/dl in the other. Intermediate evaluations of IgG during the follow-up course never showed a lasting normalization of this parameter in those patients who presented such alteration. Levamisole therefore does not seem to influence the local synthesis of IgG within the CNS. HLA data analysis did not show any significant difference between the "placebo" and "levamisole" groups. Furthermore, within the group of patients treated with levamisole there was not any significant difference between the patients who had an exacerbation and those who remained stable. As for MBP, the data are shown in Fig. 1 and 2. This test confirmed the clinical exacerbations in all but two cases, while it was never abnormal in patients who did not show a clinical worsening. The following side-effects (listed in order of frequency) were noticed in the levamisole group: nausea, insomnia, dizziness, agitation, constipation and diarrhea. At least one of these side effects was present in 67% of the patients but rarely was more than one of them present at the same time. In any case they were never such as to induce us to withdraw a patient from the study, and rarely ever lasted more than 24 hours after ingestion of the drug. The side effects that caused the suspension of therapy were listed above. Patients never presented any alteration of the routine analysis we performed periodically: in particular, neither agranulocytosis nor lowering of the platelet count was ever noted.
Discussion and conclusions
This study showed that treatment with levamisole reduces the number of patients with MS who suffer an exacerbation in a 4 year period. Similarly, none of the patients who were free from exacerbations presented a worsening of the neurological scores we monitored during the trial (except patient 13). These results parallel quite closely what we have shown in our one-year pilot study [11] and what Gonsette et al. [6] demonstrated in their three-year levamisole-placebo study. These researchers, using slightly different evaluation criteria, found that "long-term administration of levamisole has a beneficial effect on the annual relapse rate". Unfortunately, a direct comparison between the study by Gonsette et al. and ours is not possible, for we did not continue the treatment in patients who underwent an exacerbation. An additional point of concordance between the study of Gonsette et al. and ours is that neither described any serious side effects. At variance were the inclusion criteria: we selected only patients with a typical relapsing-remitting course, while Gonsette et al. included patients with different patterns of evolution. These different criteria lead also to a different age range between the levamisole treated patients of the two studies: 21-43 years (mean 32) in our study, 29-62 (mean 48) in the other. We think that MS may change immunopathological features as the years pass, and that the response to immunomodulation may differ depending on the clinical state on which it acts. Furthermore, since our study did not include patients with a progressive course, we cannot say whether this drug acts effectively in stopping the evolution of this course of the disease.
In our opinion, a comparison with previous studies of no more than one year duration is not feasible, for the inclusion criteria and the drug dosage are too different [5, 3, 13, 4].
The fact that 7 patients of the levamisole group developed an exacerbation raises the problem of the reasons for such different responses. At this time we are not able to give an answer, essentially because we do not know what the mechanism of action of levamisole in MS could be; we are inclined however to think that what we categorize as "multiple sclerosis" from the clinical and nosological point of view is probably related to different immunopathological backgrounds. It follows that a single form of therapy may not be effective in "all" patients with MS. Unfortunately, HLA analysis did not show any biological factor predictive of therapeutical success.
We agree with Gonsette et al. that "this easily administered and unharmful immunomodulator agent could be a new (we say: reliable) therapeutic approach in patients with a lower disability score and a shorter evolution of the disease".
With regard to the use of CSF MBP for monitoring the evolution of the disease, we think it is an important means of confirming clinical exacerbations, specially in those cases in which doubt may subsist.
Acknowledgements: This study received financial support in part from the Italian M.P.I. (Studio policentrico sulla sclerosi a placche in Italia), and in part from A.I.S.M. (Italian Multiple Sclerosis Society). Prof. Giorgio Macchi’s encouragement, advice and helpful criticism are gratefully acknowledged. ELPIS’ technical support is also akcnowledged. Drs. C. Bisdomini, L. Mottola, and R. Rosati gave us a valuable help in testing HLA patterns.
Sommario
Quarantuno pazienti con sclerosi multipla (SM) certa sono stati introdotti in una sperimentazione in doppio cieco levamisolo/placebo mentre erano nella fase stazionaria della malattia. Ventidue di essi hanno ricevuto levamisolo, 150 o 200 mg una volta a settimana per quattro anni, ed altri 19 hanno ricevuto placebo con lo stesso schema posologico. L’assegnazione ad uno dei due gruppi è stata effettuata a caso. I pazienti che hanno presentato una chiara esacerbazione prima dello scadere dei quattro anni non hanno continuato l’assunzione e sono stati considerati come risultati negativi. Nel gruppo trattato con levamisolo 8 pazienti hanno presentato una esacerbazione e 14 no. Nel gruppo che ha ricevuto placebo 14 hanno presentato una riaccensione e 5 no. Questa differenza è risultata essere statisticamente significativa. In questo studio si dimostra che il levamisolo riduce significativamente il numero dei pazienti che presentano una esacerbazione in un periodo di 4 anni. Tuttavia il fatto che non tutti i pazienti hanno ricevuto un tale beneficio potrebbe suggerire che differenti quadri immunopatologici possono essere alla base di ciò che noi usualmente chiamiamo SM.
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