double-blind placebo controlled crossover evaluation of levamisole in rheumatoid arthritis

172

DOUBLE-BLIND PLACEBO CONTROLLED CROSSOVER EVALUATION OF LEVAMISOLE IN

RHEUMATOID ARTHRITIS

BRUCE MILLER, PAULA DE MERIEUX, RAMACHANDRAN SRINIVASAN, PHILIP CLEMENTS, PENG FAN, JOSHUA LEVY, and HAROLD E. PAULUS

During levamisole therapy, 14 of 20 patients with previously unresponsive rheumatoid arthritis had significant improvement (P < 0.05) in clinical measures of disease activity, erythrocyte sedimentation rate, and rheumatoid factor titer in a 32-week double-blind placebo controlled crossover trial. Levamisole was shown to al- ter antibody responses to tetanus and typhoid antigens, lymphocyte blastogenesis to phytohemagglutinins, and the number of null cells in peripheral blood. Agranulocy- tosis and rash resulted in discontinuation of the drug in one patient in each group. Though clearly effective, routine use of levamisole as a disease suppressant in rheumatoid arthritis must await more complete clarification of its association with agranulocytosis.

Recent reports suggest that dysfunction of the cellular limb of the immune system may be of significance in the pathogenesis of rheumatoid arthritis (1-13).

Levamisole is an agent capable of potentiating the activity of a suppressed immune system with prefer- ential effects on the cellular limb (14-19). It has been reported to be of benefit in the treatment of patients

From the Department of Medicine, Division of Rheumatol- ogy, University of California, Los Angeles.

Supported in part by USPHS Grant GM 15759, Southern California Chapter of Arthritis Foundation, Kroc Foundation, and Janssen Laboratories.

Bruce Miller, MD: Assistant Clinical Professor of Medicine; Paula de Merieux, MD, FRCP(C): Fellow of the Canadian Arthritis and Rheumatism Society; Ramachandran Srinivasan, MD: Assistant Clinical Professor of Medicine; Philip Clements, MD: Assistant Pro- fessor of Medicine; Peng Fan, MD: Assistant Professor of Medicine; Joshua Levy, MD: Associate Professor of Medicine; Harold E. Paulus, MD: Professor of Medicine.

Address reprint requests to Harold E. Paulus, MD, 1000 Vet- eran Avenue, Los Angeles, California 90024.

Submitted for publication June 28, 1979; accepted in revised form October 22, 1979.

whose rheumatoid arthritis has been refractory to con- ventional modes of therapy. Twenty patients with rheumatoid arthritis were studied to evaluate the effects of levamisole on clinical and laboratory parameters of efficacy and on immunologic variables and their correla- tion with clinical effect. The frequency and types of adverse reactions were also evaluated.

The study was designed not only to assess the overall efficacy of levamisole, but also to evaluate the utility of the crossover design. The spectrum of effects of levamisole versus placebo was assessed in a parallel fashion during the initial 16 weeks of the study. Cross- over from levamisole to placebo permitted careful evaluation under controlled double-blind conditions of the duration of effects of levamisole after its discontinuation.

MATERIALS AND METHODS Twenty patients with classic or definite rheumatoid

arthritis (RA) by American Rheumatism Association (ARA) criteria were studied. All patients participated voluntarily and gave informed consent. Patients had had disease for 1 to 29 years and had continued to have active disease in spite of treatment with one or more of the following: nonsteroidal antiinflammatory agents (20 patients), gold (17 patients), penicillamine (1 patient), antimalarials (4 patients), immuno- suppressive agents (7 patients), or corticosteroids ( 5 patients). The 5 patients on corticosteroids had been on a stable or ta- pering daily dosage of 10 mg or less of prednisone or its equiv- alent for at least 4 months prior to initiation of the study and did not increase their dose during the study. No patients had received gold, penicillamine, antimalarial, or immuno- suppressive agents for at least 3 months prior to starting the study. There were no restrictions on the use of aspirin or any other nonsteroidal antiinflammatory agent, but the dosages of these medications were kept constant throughout the entire study period.

Arthritis and Rheumatism, Vol. 23, No. 2 (February 1980)

EVALUATION OF LEVAMISOLE IN RA 173

Table 1. Patient characteristics at baseline (median and range)

Characteristic Levamisole group Placebo group

No. of patients 10 10 Age, years 50.5 (38-65) 55.0 (37-68) Sex M:F 1:9 0 10 Weight, kg 61 (44.5-109) 57 (45-69.5) Disease duration, years 13.5 (2-23) 10.0 (1-29) No. tender or swollen joints 19.5 (10-29) 20.0 (12-28) Pain score (0-100%) 54 (30-90) 57 (20-99) Morning stiffness, minutes 165 (30-720) 135 (0-240) Grip strength 90 (57-140) 88.5 (49-176) Ring size 8.9 (5.5-9.5) 8.0 (6.25-1 1.25) 50-foot walking time 11.5 (9-22.7) 11.1 (7.4-23.5) Westergren ESR 74.5 (30-105) 41.0 (14-85) Latex fixation titer’ 4.5 -+ 0.9 5.1 f 1.1

* Mean and standard error of latex titer. Latex titers converted as follows: 1:20 = 0; 1:40 = 1; 1:80 = 2; 1:160 = 3; up to 1:10,240 = 9; 1:20,480 = 10.

The study was designed as a double-blind comparison of levamisole versus placebo with crossover comparison after 16 weeks. Ten patients were randomly selected to receive levamisole for the initial 16 weeks and placebo for the second 16 weeks, while the other 10 patients received therapy on the reverse schedule. During the levamisole phase all 20 patients initially received 150 mg levamisole daily. Fourteen patients completed the full 16-week period at this dose. One patient discontinued levamisole temporarily for 2 weeks during weeks 3 and 4 because of rash but then resumed the drug at 150 mg/ day. Two patients discontinued levamisole temporarily for 10 and 11 days because of rash, then the drug was gradually reintroduced at increasing doses to a maximum of 150 mg daily and 100 mg daily, respectively. One patient had levamisole withheld for 1 month while she was hospitalized with meningoencephalitis. Two patients discontinued levamisole permanently, one because of agranulocytosis which developed after 3 weeks and the other because of a severe rash developing after 14 weeks.

Prior to the initiation of therapy, each patient under- went a complete history and physical examination which included documentation of duration of morning stiffness, number of swollen or tender joints (joint count), grip strength, ring size, 50-foot walking time, visual pain scale (20), and global evaluations by both the patient and the physician. These variables were documented at weekly intervals for the first 4 weeks and then every fourth week until the end of the initial 16-week period. After crossover an identical assessment time- table was followed.

Routine laboratory assessments included Westergren sedimentation rate, latex fixation test for rheumatoid factor, antinuclear antibody, anti-DNA, beta 1 -C component of complement, total hemolytic complement, and quantitation of IgG, IgM, and IgA; these were performed at baseline and weeks 0, 8, 16, 24, and 32. Secondary delayed hypersensitivity was assessed by the intradermal injection of coccidiodin, his- toplasmin, trichophyton, Cundidu, and intermediate strength PPD at baseline and at weeks 4, 12, 17, 20, and 32. Primary delayed hypersensitivity was assessed by the intradermal injection of keyhole limpet hemocyanin (KLH) (0.1 mg and 0.01 mg) during the first and third weeks of the initial study period. These times correspond to baseline and 2 weeks after 5 mg of KLH was given intramuscularly. Only those patients

with a negative response to the first intradermal injection (5 10 mm in duration) were considered to be valid candidates for evaluation of a primary immune response and thus received the second injection. In addition, 0.1 cc of 10% dinitrochloro- benzene (DNCB) in acetone was applied to the skin over an area 20 mm in diameter during the first week of drug administration. Minor symptomatic reactions due to chemical irrita- tion usually cleared within 1 week. Three weeks later 0.1 cc applications of 1%, 0.5%, and 0.1% DNCB were placed in the same manner. Reactions to these solutions were recorded throughout the study; itching, erythema, or vesicle formation at the site of application were considered evidence of delayed hypersensitivity.

To assess the function of the humoral immune system typhoid vaccine (USP 0.5 ml), tetanus toxoid (0.5 ml alum precipitated), and KLH (5 mg) were injected intramuscularly 1 week after initiation of the study. Serum antibody titers to these antigens were measured weekly for 6 weeks thereafter. The proportions and absolute concentrations of B lymphocytes (lymphocytes with true membrane immunoglobulin as detected by immunofluorescent technique) and T lymphocytes (lymphocytes which form spontaneous rosettes with sheep erythrocytes) were determined at baseline and at weeks 1, 16, and 32 by methods previously published (21). Lympho- cytes lacking the markers of B or T lymphocytes were considered “non-B, non-T lymphocytes” or “null cells.” Lympho- cyte responses to the mitogen phytohemagglutinin (PHA) were assessed before and after the in vitro addition of levamisole at baseline and at weeks 2, 4, 16, 18, 24, and 32. The methods used in performing these studies have been detailed elsewhere (22).

X-rays of hands and feet were taken at baseline and at weeks 16 and 32 for assessment of erosions.

Results were statistically assessed using the Wilcoxon Matched Pairs Signed Rank test for intragroup changes from weeks 0 to 16 and 16 to 32, while the Mann Whitney U-test was used to assess the significance of the differences in changes between groups. A P value of < 0.05 was considered significant. For the purpose of analysis, latex fixation titers were converted to log values. Thus 1:20 = 0 1:40 = 1; 130 = 2; 1:160 = 3 up to 1:10,240 = 9 and 1:20,480 = 10. The paired data 1-test was used to assess latex titers.

The data were analyzed as follows: 1) differences be-

174 MILLER ET AL

Table 2. Parallel comparison: efficacy of levamisole versus placeb-linical variables weeks 0 to 16 (median and range)

Placebo' Levamisole* P value (n = 10) (n= 10) (A levamisole

Variable? Week 0 Week 16 Week 0 Week 16 A placebo)+ compared

Morning stiffness, minutes 130 63 165 22.5 = 0.05

= 0.05 Grip strength, mm Hg 88.5 92 89 109

Joint count11 20.0 19.5 19.5 16.0 NS

Pain scale (0-100%) 57 45 54 19

NS Ring size?? 8.0 7.25 8.85 7.65

50-foot walking time (secs) 11.1 9.5 11.5 10.3 NS

(0-240) (0-480)§ (3LL720) ~ 3 3 0 ) n

(47- 176) (62-169)# (57- 140) (75-150)n

(20-99) (30-86)§ (3&90) (0-7211

(6.25-1 1.25) (5.25-1 1.75)s (5.5-13.0) (4 13.o)n

(7.4-23.5) (5.6-20.2)$$ (9-22.7) (7.7- 18.3)~

(14-85) (7-80)§ (3& 105) (9-77)n

(12-30) (10-26)§ ( 1&29) (5-26)'' < 0.025

ESR, mm/hr 41.0 45.0 74.5 45.0 < 0.01

Latex§§ 4.3 f 0.9 4.5 f 0.9s 5.1 k 1.1 3.4 * 6.8.; < 0.025

* Statistical significance within each group week 0 compared to week 16.8 = NS; # = P < 0.05; ** = P < 0.025 ++ = P < 0.02; 1 = < 0.005. t Number of patients = 10 for all variables except morning stiffness in placebo group and 50-foot walking time in the levamisole group. In both

+ Comparison of the differences from week 0 to 16 between placebo and levamisole treated groups by Mann-Whitney U-test. 11 Joint swelling or tenderness. tt Ring size for each individual is the mean of all proximal interphalangeal joints. $5 Mean and standard error of latex titer paired data t test. Latex titers converted as follows: 1 : 20 = 0; 1 :40 = I ; 1 : 80 = 2; I : 160 = 3; up to

these cases number of patients analyzed = 9.

1 : 10,240 = 9; 1 :20,480 = 10.

tween observations at baseline and at the end of the initial 16- week period in the levamisole treated group were compared with similar changes in the placebo treated group (16-week parallel study). 2) In the group that initially received levamisole then crossed over to placebo, the status at the end of 16 weeks of levamisole was compared with the status at the end of 16 weeks of placebo. The object of this comparison was to assess the duration of carry over effect of levamisole into the placebo period. 3) Overall efficacy of levamisole was evaluated by comparing pre-levamisole and post-levamisole status in all patients who completed levamisole therapy.

At the end of the 32-week study period, II patients elected to continue with the double-blind trial on the medica- tion they had received during the second 16-week period for an additional 16 weeks. Six patients had been taking placebo and 5 levamisole when they elected to continue. Following completion of this extension of the double-blind study, patients were offered entry to a nonblinded open-ended evaluation of levamisole, and 8 patients accepted. All 8 patients initially received 150 mg on 4 days a week. Because of gastrointestinal intolerance, dosages were lowered in 4 patients. After increasing reports of agranulocytosis associated with levamisole therapy, dosages were later reduced to 150 mg 1 day per week.

RESULTS General characteristics of patient population at

baseline. These are listed in Table 1. Comparison of baseline variables showed no significant differences between the two groups except for a higher mean erythro-

cyte sedimentation rate for the group started on levamisole (70.8 mm/hr) than for the placebo group (46 mm/ hr).

Clinical efficacy of levamisole: parallel study comparing levamisole versus placebo. At 8 weeks there were no significant differences between the two groups in clinical or laboratory variables. Table 2 outlines the comparison of clinical and laboratory data at 16 weeks. There were no statistically significant changes from baseline in duration of morning stiffness, joint count, pain score, or ring size by 16 weeks in the 10 placebo treated patients. In contrast, there was statistically significant improvement in all of these variables in the 10 levamisole treated patients. For each variable except joint count, ring size, and %-foot walking time, the degree of improvement from week 0 to 16 was significantly greater in the levamisole group than in the placebo group. Fifty-foot walking time decreased significantly in both groups.

Clinical laboratory variables showed the same trend (Table 2). No significant changes in the median erythrocyte sedimentation rate occurred in the placebo group. Mean latex titer was also unchanged at week 16. In the levamisole treated group, statistically significant reduction occurred in both these variables. The week 0- 16 improvement for both these variables was significantly greater in the levamisole group.


Table 3. Carry over of levamisole effect: comparison of clinical and laboratory variables during administration of placebo and following 16 weeks of levamisole (median and range)

Week 16 Week 32 Variable (end of levamisole) (end of placebo) P*

Morning stiffness, minutes n = 8

Grip strength, mm Hg n = 9

Joint count n = 7

Pain scale, 0-100% n = 9

Ring size n = 9

50-foot walking time, seconds n = 9

ESR, mm/hr n = 10

Latext n = 10

22.5

109.5

16

19.0

7.65

10.3 (7.7-1 8.3) 45 (9-77) 3.40 & 2.64

(0-210)

(75- 150)

(5-26)

(0-72)

(4-13)

75 (5-720) 107 (56-1 16) 18 (11-26) 25

7.5 (3.75-12.5) 11.5 (6.7-22.0) 62 ( 18-80) 5.5 & 1.05

(4-85)

NS

< 0.025

NS (< 0.1 > 0.05)

< 0.01

NS

< 0.05

< 0.05

< 0.01

* P = Significance of the change from week 16 to week 32. t Mean and standard error

Carryover of levamisole effect. The arthritis of the 10 patients who started with levamisole became somewhat worse after blinded crossover to placebo. Nonetheless, at week 32 the measured parameters continued to be better than at week 0. The results of analysis of carryover effect are given in Table 3. Both grip strength ( P < 0.025) and pain score (P < 0.01) showed significant deterioration after crossover from levamisole to placebo. The ESR and latex fixation titers had increased significantly ( P < 0.05) at 16 weeks of placebo. Joint count and duration of morning stiffness both showed trends toward deterioration. but in neither case

was the degree of deterioration statistically significant. There was not significant increase in ring size or %foot walking time after crossover from levamisole to placebo.

Overall clinical efficacy of levamisole. When the changes from the beginning to the end of levamisole treatment were evaluated for all 18 patients completing levamisole therapy, highly statistically significant improvement occurred in duration of morning stiffness, grip strength, joint count, pain scale, ring size, mean ESR, and latex fixation titers (Table 4). Overall, 14 patients improved (70%), 3 deteriorated (15%)) and l re-

Table 4. Overall efficacy of levamisole: comparison of pre-levamisole values to values after 16 weeks levamisole for all 18 patients (median and range)

Variable Pre-levamisole 16 weeks levamisole P

Morning stiffness, minutes 108 22.0 <0.05

Grip strength, mm Hg 91 111 <0.005

Joint count 19 15.5 t0.025

Pain scale, 0-100%* 50 26.5 <0.005

~ 0 . 0 0 5

50-foot walking time 10.5 10.4 ~ 0 . 0 2 5

ESR, mm/hr 52.5 39.5 ~0.005

<0.005 Latext 4.52 f 0.77 3.05 f. 0.55

(30-720) (0-720)

(57- 169) (75-183)

(10-29) (5-26)

(30-90) (0-72) Ring size 8.25 7.25

(5.25-13.0) (4-13.0)

(5.6-22.7) (5.05-18.3)

(7-105) (4-89)

~

* 100% = worst possible pain. t For conversion see Materials and Methods. Mean and standard error.

176 MILLER ET AL

P1 acebo Levami sol e P1 acebo

100 J CH50

100 ! T

40 1

1500 -f IqG

1400

1300

1200

1100

140 J 0 1 s 32

0 1 6 32

Weeks

Figure 1. Complement and immunoglobulin levels during levamisole and placebo therapy. There were no significant changes in C3, total hemolytic complement, or immunoglobulin levels during levamisole or placebo therapy when compared to baseline, except for a fall in IgG levels during the placebo period in the group started on placebo and a fall in IgM levels during levamisole therapy in the group starting on levamisole (P < 0.05). In both cases baseline values were un- usually high.

mained unchanged. No patient achieved a full remis- sion. Two patients did not complete the study because of adverse effects.

immunologic variables: immunoglobulins and complement. There were no significant differences between the levamisole and placebo treated groups at 16 weeks, or between weeks 0, 16, and 32 in either the levamisole to placebo or placebo to levamisole treatment groups with regard to the third component of complement, total hemolytic complement, or serum IgA levels (Figure 1). When compared to baseline, serum IgG showed a significant reduction at week 16 of placebo in the group treated initially with placebo, and serum IgM showed a significant reduction at week 16 of levamisole in the group treated initially with levamisole (Figure 1). Though statistically significant, these changes are considered to merely reflect the abnormally high initialvalues.

Cell mediated immune responses. Primary delayed hypersensitivity to KLH was assessed at week 3 in the 16 patients with a negative skin test at week 1. Injec- tion of both 0.1 mg and 0.0 1 mg KLH yielded no significant differences in mean induration between the group on levamisole versus the group on placebo P = 0.1 and P -= 0.2 respectively. Similarly, neither the number of responders nor the total amount of induration differed significantly between the levamisole and placebo groups for any of the three concentrations of DNCB used to test for primary sensitization. Analysis of secondary delayed hypersensitivity responses to 5 antigens yielded no significant differences between the two groups or within either group at weeks 0, 16, and 32. Thus, by using total combined induration in response to the 5 antigens pooled, there was no significant difference between levamisole and placebo groups at week 16. Again, there was no significant difference from baseline in total induration in response to these 5 antigens in all 18 patients after 16 weeks of treatment with levamisole. Fur- thermore, in looking at skin test conversion, there was a total of 6 conversions from negative to positive (10 mm induration). Three patients were receiving placebo at the time of conversion and 3 were receiving levamisole. Only one patient converted from negative to positive while on levamisole, then reverted to negative when crossed over to placebo.

In vitro lymphocyte response to suboptimal amounts of PHA was found to be significantly lower at baseline in lymphocytes from these RA patients than from normal controls (1,280 versus 2,600 cpm P c 0.00 1). This decreased responsiveness was found to be enhanced both by the in vitro addition of levamisole at baseline before levamisole therapy and without in vitro addition of drug following 16 weeks of in vivo levami-


Table 5. Effect of levamisole on lymphocyte subpopulations

Pre-levamisole After 16 weeks of levamisole

Absolute Absolute concentration, concentration,

Lymphocyte su bpopulation Percent per mm3 Percent per mm3

Total lymphocyte count' 100 1973 f 206 100 1830 f 246 B lymphocyte* 11 + 2 145 f 21 l o + I 167 & 29 T lymphocyte* 66 f 3 I139 f 160 67 f 3 1192 f 180 Non-B, non-T lyrnphocytet 22 692 f 56 20 460 f 63§

Number of patients = 16. t Number of patients = 9. -$ All values are given as mean f standard error. P < 0.05, 16 weeks of levamisole versus baseline

sole therapy. Moreover, this enhancement was in both instances significantly greater (P < 0.05) in patients who had a moderate to marked clinical improvement on levamisole as opposed to those with minimal or no clinical response. These data have been separately reported (22). In summary, after 16 weeks of levamisole therapy there was 34.5 & 5.5% enhancement over baseline in patients with good clinical response versus 23.2 f 2.8% enhancement for those with poor or no clinical response. Similarly there was a 44.8 & 4.8% enhancement in vitro at baseline for the former group compared with 32.7 & 2.9% for those not responding clinically to levamisole.

Enumeration studies showed no significant changes in absolute number or in percentages of T cells or B cells at any time in either group. However, the number of non-T and non-B (null) cells had decreased significantly at weeks 8 and 16 (P -= 0.05) (Table 5).

Humoral immune responses. Following injection of KLH, tetanus, and typhoid H and 0 antigens, serum antibody titers to at least 3 of these antigens increased in all 20 patients. The magnitude of the increase was similar in both levamisole and placebo treated patients. Ti- ters peaked at 2 to 4 weeks then began to decline. Fol- lowing crossover to the alternate drug, titers continued to decline in all 10 patients crossed from levamisole to placebo; however in 5 of 9 patients crossed from placebo to levamisole, antibody titers to the recall antigens typhoid 0 and H and tetanus began to increase again without further antigen injection (Figure 2). This booster phenomenon was not seen for the primary antigen KLH.

X-ray changes. Joint erosions were difficult to assess serially because of variations in radiographic technique and because of the advanced degree of changes present in many patients at baseline. However, there were no obvious changes discerned in erosions at either week 16 or week 32.

Adverse effects. Table 6 outhes the adverse reactions developing during the study period. Depression, insomnia, and gastrointestinal disturbances were reported with equal frequency during both levamisole and placebo therapy. In several cases patients had the same complaint while taking both placebo and levamisole.

Skin rash prompted discontinuation of levamisole in 4 patients. In 3 cases the drug could be reintroduced either gradually or at the original dosage schedule without recurrence of the rash. In 1 patient, however, the drug could not be administered again. Stomatitis occurred in 2 patients, both on levamisole.

Agranulocytosis developed in 1 patient 3% weeks after initiation of levamisole and while on a daily levamisole schedule. Six days prior to documentation of agranulocytosis and 1 day after a documented normal white blood cell count, the patient developed fever, mild chills, headache, sore mouth, and a sore throat. Ano- rexia, nausea, and a nonproductive cough occurred just prior to admission, and the patient also noted areas of infected skin over her hands and forearms. On admission the patient appeared acutely iU; her temperature was 40°C. WBC was 850/mm3 with 99% lymphocytes, 1% monocytes, 0% neutrophils. Blood and urine cultures grew no organisms. Cultures of infected skin yielded Staphylococcus aureus. The bone marrow showed marked myeloid hypoplasia with only rare myelocytes seen. Colony forming cells were reduced. Upon hospi- talization antibiotics were started and levamisole was discontinued. Her fever responded to antibiotics. Ten days later neutrophils reappeared in the peripheral blood and neutrophil count was normal 3 weeks after discontinuation of levamisole. Following crossover to levamisole, sequential weekly absolute neutrophil counts per cubic mm were: 9204, 5220, 4720,2720, 0, 0, 944, 3530. The patient was found to be HLA-B27 positive. Although no infecting pathogenic organism was

178 MILLER ET AL

Levami s o l e

CRQSSOMR + TETANUS

1024

OH 51 2 I

256 - 128,

64 - 32 - 16 -

I . . '0 NORMAL RESPONSE

0 1 I # I 1 I I I I 1 I

0 2 4 8 1 2 16 1 7 18 1 9 20 22 24

TYPHOID H /*\ #' *,

160-

' \ 80-

I \

TL

0 2 4 8 1 2 1 6 1 7 1 8 19 20 22 24

MEEKS

Figure 2. Booster effect of levamisole on antibody response to recall antigens. Patients were given antigen injections at week 0. The normal response is an initial rise in antibody titer followed by a decline, as shown by the dashed lines (mean values of patients who started with levamisole and then crossed over to placebo). However, patients OH, TR, SP, RW, and MW who were initially treated with placebo and then crossed to levamisole had increases in titers (booster effect) following cross over, without having had another injection of antigen.


Table 6. Adverse reactions during double-blind study of 20 patients

Adverse effect Placebo Levamisole Both*

0 2

Agranulocytosis 0 I t 0

5§ Skin rash 3* Stomatitis 0 Depression 8 9 5 Insomnia 5 4 2 Chills 1 2 0 Gastrointestinal disturbance 3 5 2

2

* Number of patients who experienced the same adverse reaction both on levamisole and on placebo.

t Drug permanently discontinued. + Drug permanently discontinued in 1 patient. 8 Drug discontinued temporarily in 3 patients, permanently in 1,

and rash developed just before crossover to placebo in one patient.

actually identified in blood or urine during the patient’s illness, it was believed that infection associated with the agranulocytosis accounted for her marked chical toxicity.

One patient developed a sterile meningoencephalitis of unknown etiology after 4 weeks of levamisole therapy. The drug was discontinued for one month while the patient was hospitalized. Levamisole was resumed with no adverse reaction and continued for 8 weeks to the end of the trial. This patient continued on levamisole for 16 more weeks during the extended double-blind study and also elected to take part in the open trial.

Open trial. Of the 8 patients electing to partici- pate in the open ended trial, 7 patients developed 10 adverse reactions. Three developed leukopenia with absolute neutrophil counts of 1,944, l,OOO, and 1,025/mm3 for which the drug was discontinued and not reintroduced. None of these patients had developed leukocyte toxicity during levamisole therapy in the double-blind trials. HLA-B27 was sought in 2 of these 3 patients but was absent. In addition, 1 patient had levamisole discontinued because of high fevers and light-headedness that occurred for 2 days after levamisole ingestion on 3 successive weeks. One patient stopped levamisole because of rash. Two patients had nausea and abdominal cramps that lessened with a lower dosage of levamisole, and 1 of the patients with neutropenia developed concomitant blisters on her lip.

During the unblinded segment of the trial, levamisole was reduced from 150 mg on 4 days per week to 150 mg 1 day per week. All 4 patients continuing on the reduced dose noted reduced efficacy of the drug with increased morning stiffness and fatigue. Objec- tively, the number of tender and swollen joints increased. This subjective and objective deterioration occurred within 4-8 weeks after the dose was reduced.

DISCUSSION

The results of this double-blind crossover study clearly demonstrate that levamisole was of benefit in active rheumatoid arthritis in patients who had not responded to other antirheumatic medications. The overall improvement rate of 70% compares favorably to other reports in which 6041% of patients treated for more than 3 months improved (23-32). The onset of action of levamisole was slow. No significant changes were discernable after 8 weeks of therapy, but at 16 weeks significant improvement was present in clinical variables, ESR, and latex fixation titer. This delay in onset of action contrasts with that reported by Schuer- mans (23) who noted striking objective and subjective benefit in an uncontrolled trial within the first month of treatment in 6 patients, but such a delay conforms to the findings of other investigators. Following crossover to placebo, relapse was apparent by 16 weeks. Veys and Mielants (28) noted that 2 of their patients treated with levamisole in an open study for 24 and 6 months re- tained improvement for more than 1 year after discontinuation of the drug. The reason for this longer duration of beneficial effect following discontinuation is unclear but may be related to length of exposure to the drug before its discontinuation, as suggested by Basch et a1 (32). It should be noted, however, that though the deterioration in grip strength and pain score were significant when compared to week 16 of levamisole, these variables still remained improved when compared to baseline values, suggesting some continued benefit of the drug 16 weeks after its discontinuation.

Measures of humoral immunity were variably affected by levamisole. Total immunoglobulin levels have been found by various investigators to increase, decrease, or remain unchanged. These varying findings may be related to differing baseline immunoglobulin levels. In our patients the only significant changes in immunoglobulins were declines in these patients who started at baseline with very high values. Anti- immunoglobulin (rheumatoid factor) levels, however, seem to fall consistently during levamisole therapy. It is not known whether this is a primary effect or a secondary phenomenon reflecting changes in the disease activity. Antibody titers to recall antigens (given at week 1) as measured by tetanus, typhoid 0, and typhoid H antibody titers increased after crossover from placebo to levamisole in spite of initial equal antibody responses to these antigens and progressively decreasing titers at time of crossover in both groups. The mechanisms in- volved in this previously undescribed booster phenome-

180 MILLER ET AL

non are unknown. Possible mechanisms responsible for this phenomenon include direct action on B cells or on immunoglobulin secreting plasma cells, and an indirect effect by enhancing T helper cells or by suppressing the effect of T suppressor cells.

The mechanisms of action of levamisole in rheumatoid arthritis remain unknown. It has been suggested that the drug may correct the hypofunctioning of a subpopulation of regulator T cells postulated to exist in rheumatoid arthritis. In our patients, as in other series, no changes in absolute numbers or percentages of T cells were found. However, null (non-T, non-B) cell numbers decreased significantly as found by Rosenthal et a1 (33). Null cells have been postulated to represent a precursor cell of T and/or B lymphocytes or subpopulations of these lymphocytes. It is possible that these cells may, under the influence of levamisole, dif- ferentiate into a previously deficient subpopulation of regulating lymphocytes with resultant restoration of normal immunoregulation. However, there is presently not enough evidence to support or refute this hypothesis. Our findings that the subnormal blastogenic response of RA lymphocytes to PHA was enhanced by levamisole and that the degree of enhancement correlated with clinical response to the drug may suggest a primary influence of levamisole on the cellular limb of the immune system with secondary effects on humoral immunity. This evidence gives some support to the hypothesis that a primarily cell mediated immune defect contrib- utes to the pathogenesis of RA.

Nonhematologic adverse effects have been reported to occur more frequently during the treatment of the rheumatic disease with levamisole than when non- rheumatic disorders are so treated. We found that frequently these adverse reactions were found in patients on both levamisole and placebo. It is possible that some of the many adverse reactions reported with levamisole therapy are attributable to other medications that these patients take or to the disease itself. However, stomatitis, and in some patients, rash and gastrointestinal disturbance seemed related to levamisole use.

Clearly the most important and serious adverse effect of levamisole is leukocyte toxicity. Agranulocy- tosis or neutropenia occurred at some time in 4 of our 20 patients. Agranulocytosis developed in 1 patient during the double-blind study and was associated with quite severe illness. During the open extension, 3 patients became neutropenic; the dosage of levamisole at the time of neutropenia was 150 mg 4 days per week. It has been suggested that elimination of consecutive day

therapy with white blood cell monitoring several hours after levamisole is taken may reduce the incidence of neutropenia (34). However, this has not yet been clearly established and cases of agranulocytosis during once weekly therapy have been noted. One patient developed agranulocytosis on 50 mg of levamisole weekly (35). Two of 60 patients on once weekly levamisole developed agranulocytosis in one study, and in another open trial, 2 patients developed agranulocytosis while on twice weekly levamisole (36). Thus reduced frequency of administration may not decrease the frequency of this severe adverse effect.

In this study a double-blind crossover placebo controlled design was used. There is some disagreement among investigators and statisticians about the useful- ness of this type of crossover study design. Data from the placebo groups cannot be pooled for analysis because of the interfering carryover effect of active drug in those patients crossing over from drug to placebo. There are, however, some compelling reasons to use this design and these were illustrated in this study. Since no patient is treated with placebo only, the clinician is better able to study patients with severe disease for whom such therapy is indicated. Clinicians are reluctant to commit severely affected patients to the prolonged trials needed to evaluate slowly acting antirheumatic drugs knowing that the patient may only receive a placebo in a non-crossover placebo controlled trial. Moreover, the benefits of parallel study design are not lost because the initial period can be analyzed as a parallel trial. It is well known that patients started on a new drug experi- ence a significant placebo effect. The contribution of this effect can be eliminated by parallel comparison with the placebo group. Finally, the duration of effect of the drug after its discontinuation can be determined under blind controlled conditions in the group that crosses over from test drug to placebo. In other types of drug trials, this information is unavailable.

Levamisole is effective in the treatment of rheumatoid arthritis and may be an alternative to gold, penicillamine, and antimalarials in this disease. However, the problem of leukocyte toxicity associated with its use requires further clarification before it can be recom- mended for noninvestigational use in rheumatoid arthritis (37).

ACKNOWLEDGMENTS We wish to thank Ruth Bangert and Anne Bomberg

for nursing assistance, Edmund Sarkissian for technical assistance, Meredith Zehm and Evelyn Tackles for secretarial as-


sistance, Marilyn Rogers for assistance with statistical analysis and Janssen R & D Inc. for kindly supplying levamisole and placebo.

REFERENCES 1. Yu DTY, Peter JB: Cellular immunological aspects of

rheumatoid arthritis. Semin Arthritis Rheum 4:25-52, 1974

2. Waxman J, Lockshin MD, Schnapp JJ, Doneson IN: Cel- lular immunity in rheumatic diseases. Arthritis Rheum

3. Lloyd TM, Panush RS: Cell mediated immunity in rheumatoid arthritis. J Rheumatol4:23 1-244, 1977

4. Andrianakos AA, Sharp JT, Person DA, Lidsky MD, Duffy J: Cell-mediated immunity in rheumatoid arthritis. Ann Rheum Dis 36: 13-20, 1977

5. Paulus HE, Machleder HI, Levine S, Yu DTY, MacDon- ald NS: Lymphocyte involvement in rheumatoid arthritis. Arthritis Rheum 2 0 1249-1262, 1977

6. Person DA, Sharp JT, Lidsky MD: The cytotoxicity of leukocytes and lymphocytes from patients with rheumatoid arthritis for synovial cells. J Clin Invest 58:69&698, 1976

7. Stastny P, Rosenthal M, Andreis M, ZiKM: Lymphokines in the rheumatoid joint. Arthritis Rheum 18:237-243, 1975

8. Keystone EC, Gladman DD, Urowitz MB, Clarke DA, Falk JA, Osoba D, Gordon DA: Mixed leukocyte reaction in rheumatoid arthritis. Arthritis Rheum 19532-538, 1976

9. Wisloff F, Froland S, Natvig SB: Deficient lymphoid cell mediated PHA-induced cytotoxicity in rheumatoid arthritis patients. Scand J Immunol 4:303-307, 1975

10. Tannenbaum H, Schur PH: The role of lymphocytes in rheumatic diseases. J Rheumatol 1:392412, 1974

11. Lance EM, Knight SC: Immunologic reactivity in rheumatoid arthritis: response to mitogens. Arthritis Rheum 17513-520, 1974

12. Lockshin MD, Eisenhauer AC, Kohn R, Weksler M, Block S, Mushlin SB: Cell mediated immunity in rheumatic diseases. Arthritis Rheum 18:245-250, 1975

13. Silverman HA, Johnson JJ, Vaughan JV, McGlamory JC: Altered lymphocyte reactivity in rheumatoid arthritis. Ar- thritis Rheum 19509-516, 1976

14. Tripodi D, Parks LC, Brugmans J: Drug induced restoration of cutaneous delayed hypersensitivity in anergic patients with cancer. N Engl J Med 289:354357, 1973

15. Symoens J, Rosenthal M: Levamisole in the modulation of the immune response. J Reticuloendothel SOC 21:175- 221, 1977

16. Brugmans J, Schuermans U, DeCock W, Thienpont D, Janssen P, Verhaegen H, Van Nimmen L, Louwagie AC, Stevens E: Restoration of host defense mechanisms in man by levamisole. Life Sci 13:1499-1504, 1973

16~499-506, 1973

17. Golding H, Golding B, Jacobson R, Lomnitzer R, Koorn- hof HJ, Rabson AR: In vitro reversal of cellular unre- sponsiveness induced by levamisole. Clin Exp Immunol 26:295-301, 1976

18. Verhaegen H, De Cree S, DeCock W, Verbruggen F: Res- toration by levamisole of low E-rosette forming cells in patients suffering from various diseases. Clin Exp Immu- no1 27:313-318, 1977

19. Scheinberg MA, Santos L, Mendes NF, Musatti C: De- creased lymphocyte response to PHA, Con-A, and cal- cium inophore in patients with RA and SLE, and reversal with levamisole in rheumatoid arthritis. Arthritis Rheum

20. Huskisson EC: Measurement of pain. Lancet II:1127- 1131, 1974

21. Clements PJ, Levy J: Receptors for IgG (Fc receptors) on human lymphocytes: re-evaluation by multiple tech- niques. Clin Exp Immunol 34:281-287, 1978

22. Levy J: Levamisole and cellular immunity in rheumatoid arthritis--clinical and laboratory correlates. J Rheumatol

23. Schuermans YS: Levamisole in rheumatoid arthritis. Lan- cet I: 11 1, 1975

24. Huskisson EC, Dieppe PA, Scott J, Trapnell J, Balme HW, Willoughby DA: Immunostimulant therapy with levamisole for rheumatoid arthritis. Lancet 1:393-395, 1976

25. Veys EM, Mielants H, de Bussere A, Decrans L, Gabriel P: Levamisole in rheumatoid arthritis. Lancet I:808-809, 1976

26. Rosenthal M, Trabert U, Muller W: Immunotherapy with levamisole in rheumatic diseases. Scand J Rheumatol 5:216-220, 1976

27. Basch CM, Spitler LG, Engleman EG, Engleman EP: Cellular immune reactivity in patients with rheumatoid arthritis and effects of levamisole. J Rheumatol4377-388, 1977

28. Veys EM, Mielants H: Longterm evaluation of inter- mittent levamisole treatment in rheumatoid arthritis. J Rheumatol 4:27-33, 1977

29. Runge LH, Pinals RS, Lourie SH, Tomar RH: Treatment of rheumatoid arthritis with levamisole: a controlled trial. Arthritis Rheum 201445-1448, 1977

30. Multicentre study group: Levamisole in rheumatoid arthritis: a randomized double blind study comparing two dosage regimens of levamisole with placebo. Lancet

3 1. Scott S, Dieppe PH, Huskisson EC: Continuous and inter- mittent levamisole, a controlled trial. Ann Rheum Dis 37:259-261, 1978

32. Basch CM, Spitler LE, Engleman EP: International symposium on levamisole in rheumatoid arthritis: a review of short and long term effects on articular manifestations. J Rheumatol 5(4):11-16, 1978

33. Rosenthal M, Trabert U, Minler W: The effect of levamisole on peripheral blood lymphocytes in patients with

21~326-329, 1978

4(s~ppl):63-68, 1978

II:1007-1012, 1978

182 MILLER ET AL

rheumatoid arthritis. Clin Exp Immunol25:493-496, 1976 34. Mielants H, Veys EM: A study of the hematologic side ef-

fects of levamisole in rheumatoid arthritis with recom- mendations. J Rheumatol 5 (suppl 4):77-83, 1978

35. Felix-Davies DO: International symposium on levamisole

in rheumatoid arthritis. J Rheumatol S(supp1 4):93-95, 1978

36. Janssen R and D Inc: Personal communications 37. Editorial: Levamisole-a cautionary note. Lancet 2:291-

292, 1979

double-blind placebo controlled crossover evaluation of levamisole in rheumatoid arthritis

Documents