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Double-blind placebo controlled crossover evaluation of levamisole in rheumatoid arthritis.

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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 alter antibody responses to tetanus and typhoid antigens,
lymphocyte blastogenesis to phytohemagglutinins, and
the number of null cells in peripheral blood. Agranulocytosis 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 preferential 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 Rheumatology, 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 Professor 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 Veteran Avenue, Los Angeles, California 90024.
Submitted for publication June 28, 1979; accepted in revised
form October 22, 1979.
Arthritis and Rheumatism, Vol. 23, No. 2 (February 1980)
whose rheumatoid arthritis has been refractory to conventional 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 correlation 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. Crossover 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), immunosuppressive agents (7 patients), or corticosteroids ( 5 patients).
The 5 patients on corticosteroids had been on a stable or tapering daily dosage of 10 mg or less of prednisone or its equivalent 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 immunosuppressive 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.
EVALUATION OF LEVAMISOLE IN RA
173
Table 1. Patient characteristics at baseline (median and range)
Characteristic
No. of patients
Age, years
Sex M:F
Weight, kg
Disease duration, years
No. tender or swollen joints
Pain score (0-100%)
Morning stiffness, minutes
Grip strength
Ring size
50-foot walking time
Westergren ESR
Latex fixation titer’
Levamisole group
10
50.5 (38-65)
1:9
61 (44.5-109)
13.5 (2-23)
19.5 (10-29)
54 (30-90)
165 (30-720)
90 (57-140)
8.9 (5.5-9.5)
11.5 (9-22.7)
74.5 (30-105)
4.5 -+ 0.9
Placebo group
10
55.0 (37-68)
0 10
57 (45-69.5)
10.0 (1-29)
20.0 (12-28)
57 (20-99)
135 (0-240)
88.5 (49-176)
8.0 (6.25-1 1.25)
11.1 (7.4-23.5)
41.0 (14-85)
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 underwent 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 timetable 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, histoplasmin, 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% dinitrochlorobenzene (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 irritation 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). Lymphocytes lacking the markers of B or T lymphocytes were considered “non-B, non-T lymphocytes” or “null cells.” Lymphocyte 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-
MILLER ET AL
174
Table 2. Parallel comparison: efficacy of levamisole versus placeb-linical
variables weeks 0 to 16 (median and range)
Placebo'
(n = 10)
Variable?
Morning stiffness, minutes
Grip strength, mm Hg
Joint count11
Pain scale (0-100%)
Ring size??
50-foot walking time (secs)
ESR, mm/hr
Week 0
130
(0-240)
88.5
(47- 176)
20.0
(12-30)
57
(20-99)
8.0
(6.25-1 1.25)
11.1
(7.4-23.5)
41.0
(14-85)
4.3 f 0.9
Levamisole*
(n= 10)
Week 16
Week 0
63
(0-480)§
92
(62-169)#
19.5
(10-26)§
45
(30-86)§
7.25
(5.25-1 1.75)s
9.5
(5.6-20.2)$$
45.0
(7-80)§
4.5 f 0.9s
165
(3LL720)
89
(57- 140)
19.5
( 1&29)
54
(3&90)
8.85
(5.5-13.0)
11.5
(9-22.7)
74.5
(3& 105)
5.1 k 1.1
Week 16
22.5
~330)n
109
(75-150)n
16.0
(5-26)''
19
(0-7211
7.65
(4
13.o)n
10.3
(7.7- 18.3)~
45.0
(9-77)n
3.4 6.8.;
P value
(A levamisole
compared
A placebo)+
= 0.05
= 0.05
NS
< 0.025
NS
NS
< 0.01
*
< 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.
Latex§§
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
these cases number of patients analyzed = 9.
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
1: 10,240 = 9; 1 :20,480 = 10.
+
tween observations at baseline and at the end of the initial 16week 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 medication 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 o n 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 016 improvement for both these variables was significantly greater in the levamisole group.
EVALUATION OF LEVAMISOLE IN RA
175
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)
Variable
Week 16
(end of levamisole)
Week 32
(end of placebo)
22.5
(0-210)
109.5
(75- 150)
16
(5-26)
19.0
(0-72)
7.65
(4-13)
10.3
(7.7-1 8.3)
45
(9-77)
3.40 & 2.64
75
(5-720)
107
(56-1 16)
18
(11-26)
25
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
*P
=
P*
NS
< 0.025
NS (< 0.1 > 0.05)
< 0.01
(4-85)
7.5
(3.75-12.5)
11.5
(6.7-22.0)
62
( 18-80)
5.5 & 1.05
NS
< 0.05
< 0.05
< 0.01
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
Morning stiffness, minutes
Grip strength, mm Hg
Joint count
Pain scale, 0-100%*
Ring size
50-foot walking time
ESR, mm/hr
Latext
Pre-levamisole
108
(30-720)
91
(57- 169)
19
(10-29)
50
(30-90)
8.25
(5.25-13.0)
10.5
(5.6-22.7)
52.5
(7-105)
4.52 f 0.77
16 weeks levamisole
22.0
(0-720)
111
(75-183)
15.5
(5-26)
26.5
(0-72)
7.25
(4-13.0)
10.4
(5.05-18.3)
39.5
(4-89)
3.05 f.0.55
~
* 100% = worst possible pain.
t For conversion see Materials and Methods. Mean and standard error.
P
<0.05
<0.005
t0.025
<0.005
~0.005
~0.025
~0.005
<0.005
MILLER ET AL
176
P1 acebo
100
100
40
1500
Levami sol e
P1 acebo
J
CH50
!T
1
-f
IqG
1400
1300
1200
-=
1100
140 J
0
mained unchanged. No patient achieved a full remission. 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. Injection 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. Furthermore, 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
0.001). 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-
32
16
1s
0
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 unusually high.
EVALUATION OF LEVAMISOLE IN RA
177
Table 5. Effect of levamisole on lymphocyte subpopulations
Lymphocyte su bpopulation
Total lymphocyte count'
B lymphocyte*
T lymphocyte*
Non-B, non-T lyrnphocytet
Percent
100
11 + 2
66 f 3
22
Pre-levamisole
After 16 weeks of levamisole
Absolute
concentration,
per mm3
Absolute
concentration,
per mm3
1973 f 206
145 f 21
I139 f 160
692 f 56
Percent
100
lo+ I
67 f 3
20
1830 f 246
167 & 29
1192 f 180
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. Titers peaked at 2 to 4 weeks then began to decline. Following 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 o u t h e s 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. Anorexia, 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 hospitalization 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
MILLER ET AL
178
Levami s o l e
CRQSSOMR
+
TETANUS
1024
51 2
256
OH
I
-
128,
32 -
64
I
..
' 0 NORMAL RESPONSE
160 1
#
2
0
I
4
160'
80-
1
I
8
12
I
16
17
I
I
I
18
19
20
\
I
I
24
TYPHOID H
/*\
#'
1
22
*,
\
TL
0
2
4
8
12
16
17
18
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.
EVALUATION OF LEVAMISOLE IN RA
Table 6. Adverse reactions during double-blind study of 20 patients
Adverse effect
Placebo
Levamisole
Both*
Agranulocytosis
Skin rash
Stomatitis
Depression
Insomnia
Chills
Gastrointestinaldisturbance
0
3*
0
8
5
It
0
2
0
5
2
0
2
1
3
5§
2
9
4
2
5
* 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 participate 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. Objectively, the number of tender and swollen joints increased. This subjective and objective deterioration occurred within 4-8 weeks after the dose was reduced.
179
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 Schuermans (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 retained 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. Antiimmunoglobulin (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 involved in this previously undescribed booster phenome-
180
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, differentiate 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 contributes 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 nonrheumatic 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. Agranulocytosis 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
MILLER ET AL
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 usefulness 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 experience 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 recommended 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-
EVALUATION OF LEVAMISOLE IN RA
sistance, Marilyn Rogers for assistance with statistical analysis
and Janssen R & D Inc. for kindly supplying levamisole and
placebo.
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