Immunomodulatory action of levamisole - II. Enhancement of concanavalin A response by levamisole is associated with an oxidation degradation product of levamisole formed during lymphocyte culture


Hanson, K.A.; Heidrick, M.L.

International Journal of Immunopharmacology 13(6): 669-676

1991


Previously we determined that levamisole (LMS), when stored for a period of time, breaks down to three degradation products at neutral and alkaline pH. At low concentrations (10(-6) M), Product 1 inhibits the lymphocyte response to concanavalin A (Con A). Product 2 enhances the response and Product 3 has no effect. At higher concentrations (10(-5) M) all three products inhibit the response. To determine if these products are formed in culture media under culture conditions (e.g. in RPMI-1640 bicarbonate buffered medium, 37 degrees C, pH 7.0-7.5, during a 72 h culture period), we added freshly prepared LMS solutions to culture media with and without lymphocytes present and maintained the pH at 7.0, 7.25 or 7.5 by varying the amount of CO2 present. Periodically over a 72 h period, aliquots of the media were removed and analyzed for the presence of LMS and the three degradation products. Within 4 h, two of the degradation product began to form in culture media with or without lymphocytes present. Product No. 1, 3-(2-mercaptoethyl)-5-phenylimidazolidine-2-one or dl-2-oxy-3-(2-mercaptoethyl)-5-phenylimidazolidine (OMPI), which inhibits the lymphocyte response to concanavalin A (Con A) at concentrations above 0.4 micrograms/ml, was formed at pH 7.0, 7.25 and 7.5, but the compound did not reach inhibitory concentrations in the lymphocyte cultures during the 72 h culture period. Product No. 2, 6-phenyl-2,3-dihydroimidazo (2,1-b) thiazole, which enhances the Con A response between concentrations of 0.5 and 10 micrograms/ml, was detected at concentrations between 2.5 and 3.5 micrograms/ml at pH 7.25 and 7.5. Product 2 was not detected in cultures at pH 7.0 and subsequently when we cultured lymphocytes with freshly prepared LMS and maintained the pH at 7.0, no significant enhancement of the Con A response was observed. Product No. 3, bis[3-(2-oxo-5-phenylimidazolidin-1-y1) ethyl] disulfide was not detected in the culture media during the 72 h period. A solution of LMS which had been stored for 2 weeks in RPMI-1640 medium at 4°C (in which Product 2 was detected) significantly enhanced the Con A response of cells cultured at pH 7.0, while a similar LMS solution which had been stored for 2 weeks at a higher temperature, 37°C (which contained both Products 1 and 2), inhibited the response. The results indicate that the enhancement of the Con A response by LMS is primarily due to an oxidation product of LMS which is formed in small, but stimulatory amounts during the 72 h culture period and suggest that the varied results reported with LMS in cell culture (enhancement, no effect or inhibition) may be due to the formation of these stimulatory and inhibitory products, the relative concentration of which may vary depending upon the culture conditions used and/or the method of preparing and/or storing LMS solutions.

Int.
J.
Immunopharmac.,
Vol.
13,
No.
6,
pp.
669
676,
1991.
Printed
in
Great
Britain.
0192
0561/91
$3.00
+
.00
Pergamon
Press
plc.
International
Society
for
Immunopharmacology.
IMMUNOMODULATORY
ACTION
OF
LEVAMISOLE
-
II.
ENHANCEMENT
OF
CONCANAVALIN
A
RESPONSE
BY
LEVAMISOLE
IS
ASSOCIATED
WITH
AN
OXIDATION
DEGRADATION
PRODUCT
OF
LEVAMISOLE
FORMED
DURING
LYMPHOCYTE
CULTURE
KIMBERLY
A.
HANSON*
1
and
MARGARET
L.
HEIDRICK**
*Department
of
Biochemistry,
University
of
Nebraska
Medical
Center,
Omaha,
NE
68198-4525,
U.S.A.
(Received
15
June
1990
and
in
final
form
30
January
1991)
Abstract
Previously
we
determined
that
levamisole
(LMS),
when
stored
for
a
period
of
time,
breaks
down
to
three
degradation
products
at
neutral
and
alkaline
pH.
At
low
concentrations
(10'
M),
Product
1
inhibits
the
lymphocyte
response
to
concanavalin
A
(Con
A),
Product
2
enhances
the
response
and
Product
3
has
no
effect.
At
higher
concentrations
(10
-5
M)
all
three
products
inhibit
the
response.
To
determine
if
these
products
are
formed
in
culture
media
under
culture
conditions
(e.g.
in
RPMI-1640
bicarbonate
buffered
medium,
37°C,
pH
7.0
7.5,
during
a
72
h
culture
period),
we
added
freshly
prepared
LMS
solutions
to
culture
media
with
and
without
lymphocytes
present
and
maintained
the
pH
at
7.0,
7.25
or
7.5
by
varying
the
amount
of
CO,
present.
Periodically
over
a
72
h
period,
aliquots
of
the
media
were
removed
and
analyzed
for
the
presence
of
LMS
and
the
three
degradation
products.
Within
4
h,
two
of
the
degradation
product
began
to
form
in
culture
media
with
or
without
lymphocytes
present.
Product
No.
1,
3-(2-mercaptoethyl)-5-
phenylimidazolidine-2-one
or
d/-2-oxy-3-(2-mercaptoethyl)-5-phenylimidazolidine
(OMPI),
which
inhibits
the
lymphocyte
response
to
concanavalin
A
(Con
A)
at
concentrations
above
0.4
vg/ml,
was
formed
at
pH
7.0,
7.25
and
7.5,
but
the
compound
did
not
reach
inhibitory
concentrations
in
the
lymphocyte
cultures
during
the
72
h
culture
period.
Product
No.
2,
6-phenyl-2,3-dihydroimidazo
(2,1-b)
thiazole,
which
enhances
the
Con
A
response
between
concentrations
of
0.5
and
10
µg/ml,
was
detected
at
concentrations
between
2.5
and
3.5
µg/ml
at
pH
7.25
and
7.5.
Product
2
was
not
detected
in
cultures
at
pH
7.0
and
subsequently
when
we
cultured
lymphocytes
with
freshly
prepared
LMS
and
maintained
the
pH
at
7.0,
no
significant
enhancement
of
the
Con
A
response
was
observed.
Product
No.
3,
bis[3-(2-oxo-5-phenylimidazolidin-
1
-y1)
ethyl]
disulfide
was
not
detected
in
the
culture
media
during
the
72
h
period.
A
solution
of
LMS
which
had
been
stored
for
2
weeks
in
RPMI-1640
medium
at
4°C
(in
which
Product
2
was
detected)
significantly
enhanced
the
Con
A
response
of
cells
cultured
at
pH
7.0,
while
a
similar
LMS
solution
which
had
been
stored
for
2
weeks
at
a
higher
temperature,
37°C
(which
contained
both
Products
1
and
2),
inhibited
the
response.
The
results
indicate
that
the
enhancement
of
the
Con
A
response
by
LMS
is
primarily
due
to
an
oxidation
product
of
LMS
which
is
formed
in
small,
but
stimulatory
amounts
during
the
72
h
culture
period
and
suggest
that
the
varied
results
reported
with
LMS
in
cell
culture
(enhancement,
no
effect
or
inhibition)
may
be
due
to
the
formation
of
these
stimulatory
and
inhibitory
products,
the
relative
concentration
of
which
may
vary
depending
upon
the
culture
conditions
used
and/or
the
method
of
preparing
and/or
storing
LMS
solutions.
Levamisole
(LMS),
originally
developed
as
an
antihelminthic
drug,
was
first
described
as
an
immunomodulator
by
Renoux
&
Renoux
(1971).
Since
that
time,
numerous
studies
with
LMS
as
an
immunotherapeutic
drug
have
been
undertaken
(for
review
see
Symoens
&
Rosenthal,
1977;
Amery
&
Gough,
1981)
and
currently,
LMS
is
being
used
in
humans
as
an
immunotherapeutic
agent
in
cancer,
rheumatoid
arthritis
and
viral
infections
(Horsley
-
Petersen,
Bentsen,
Engstrom
-Laurent,
Junker,
Halberg
&
Lorenzen,
1988;
Veys,
Mielants
&
Verbruggen,
1987;
Klefstrom,
Holsti,
Grohn,
tPresent
address:
Department
of
Anesthesia,
Stanford
University
School
of
Medicine,
Stanford,
CA
94305,
U.S.A.
*Author
to
whom
correspondence
should
be
addressed.
669
670
K.
A.
HANSON
and
M.
L.
HEIDRICK
Heinonen
&
Holsti,
1985;
Balaram,
Padmanabhan
&
Vasudevan,
1988;
Hoofnagle,
1987;
Steele
&
Charlton,
1987;
Laurie,
Moertel,
Fleming,
Wieand,
Leigh,
Rubin,
McCormack,
Gerstner,
Krook,
Malliard,
Twito,
Morton,
Tschetter
&
Barlow,
1989;
Moertel,
Fleming,
MacDonald,
Haller,
Laurie,
Goodman,
Ungerleider,
Emerson,
Tormey,
Glick,
Veeder
&
Mailliard,
1990).
LMS
appears
to
act
primarily
on
macrophages
and
T
-lymphocytes
influencing
cell
-mediated
immunity
but
the
exact
mechanism
of
action
remains
undefined.
In
our
laboratory,
we
observed
that
solutions
of
LMS
stored
4°C
consistently
elevated
the
lymphocyte
proliferative
response
to
Con
A
to
a
greater
extent
than
freshly
prepared
LMS
solutions.
In
the
previous
paper
(Hanson,
Nagel
&
Heidrick,
1991),
we
reported
that
LMS
was
found
to
decompose
during
storage
in
aqueous
solutions
at
neutral
and
alkaline
pH
to
form
three
products.
The
three
products
were
purified,
identified
and'
their
effects
on
lymphocyte
response
to
Con
A
were
found
to
be
both
stimulatory
and
inhibitory.
In
this
study
we
determined
the
amount
of
the
three
products
formed
from
LMS
during
cell
culture
conditions
(i.e.
LMS
dissolved
in
RPMI-1640
media
with
fetal
calf
serum
and
maintained
at
37°C
for
a
72
h
period
in
a
CO2
humidified
incubator,
both
with
and
without
lymphocytes
present).
The
amount
of
CO,
present
was
varied
to
produce
pH
measurements
of
7.0,
7.25
or
7.5
in
the
culture
media.
EXPERIMENTAL
PROCEDURES
Quantitative
determination
of
LMS
degradation
products
The
three
LMS
degradation
products
were
purified
from
LMS
solutions
by
high
pressure
liquid
chromatography
and
their
structures
determined
by
infrared
spectrophotometry,
proton
nuclear
magnetic
resonance
spectrometry,
carbon
nuclear
magnetic
resonance
and
homo-
and
heteronuclear
two
dimensional
nuclear
magnetic
resonance
(see
companion
paper
Hanson
et
al.,
1991).
Standard
curves
for
the
products
were
prepared
by
injecting
0.1
10
pg
of
the
purified
products
onto
an
analytical
high
pressure
liquid
chromatography
column.
For
all
three
products,
there
was
a
linear
relationship
between
the
pg
added
to
the
high
pressure
liquid
chromatography
column
and
the
area
of
the
peak.
The
amount
of
the
three
degradation
products
present
in
stored
solutions
of
LMS
and
culture
media
was
then
determined
by
comparing
the
peaks
from
the
unknown
solutions
with
the
standard
curves.
Study
of
LMS
stability
during
lymphocyte
culture
Lymphocytes
were
prepared
as
previously
described
(Hanson
et
al.,
1991)
and
after
appropriate
dilution
in
RPMI-1640
with
bicarbonate
buffer,
500
µl
aliquots
containing
2.5
x
10
5
cells
were
transferred
to
the
wells
of
tissue
culture
24
-well
plates,
containing
60
I.41
of
medium.
Four
hundred
microliters
of
a
freshly
prepared
LMS
solution
were
then
added
to
appropriate
culture
wells
[final
concentration
of
LMS
was
100
pig/nil
(4.89
x
10'
M)1.
After
1
h
incubation,
40
ill
of
heat
-inactivated
fetal
calf
serum
was
added
to
each
culture
(total
volume
of
culture
was
1
ml).
Similar
wells
were
prepared
without
lymphocytes.
The
cultures
were
maintained
at
37°C
in
three
separate
humidified
gas-paks
(BBL
Microbiology
Sys.,
Cockeysville,
MD,
U.S.A.)
gassed
with
the
appropriate
CO,
air
mixture
to
produce
a
final
pH
measurement
of
either
7.0,
7.25
or
7.5.
At
times
0,
4,
12,
24,
48
and
72
h,
the
contents
of
three
wells
were
removed
and
analyzed
individually
for
the
presence
of
the
three
LMS
degradation
products.
Media
from
wells
containing
lymphocytes
were
centrifuged
at
200
g
for
15
min
to
remove
the
cells.
The
supernatants
were
extracted
twice
with
5
ml
of
chloroform,
the
chloroform
extracts
combined
and
vacuum
evaporated
to
dryness.
The
residues
were
resuspended
in
1
ml
of
methanol
water
ammonium
hydroxide
(60
+
39.9
+
0.1)
for
high
pressure
liquid
chromatography
analysis.
The
high
pressure
liquid
chromatography
method
of
analysis
was
the
same
as
previously
described
(Hanson
et
al.,
1991)
except
a
100
ill
(10
p
g
LMS)
aliquot
of
each
solution
was
injected
onto
the
high
pressure
liquid
chromatography
analytical
column.
The
concentration
of
the
products
formed
was
determined
by
utilizing
standard
curves
prepared
with
known
amounts
of
the
purified
products.
Study
of
LMS
stability
when
stored
in
tissue
culture
media
with
bicarbonate
buffer
To
determine
the
stability
of
LMS
stored
in
tissue
culture
media,
LMS
was
dissolved
in
RPMI-1640
medium
with
bicarbonate
buffer,
at
a
concentration
of
2.4
mg/ml.
The
solutions
were
stored
under
sterile
conditions
at
either
4
or
37°C
for
2
weeks,
during
which
time
the
pH
of
the
solutions
increased
from
7.0
to
8.0
8.5
due
to
the
bicarbonate
buffer.
The
medium
was
extracted
twice
with
5
ml
of
chloroform,
the
chloroform
extracts
were
combined
Immunomodulatory
Action
of
Levamisole—II
and
vacuum
rotoevaporated
to
dryness.
The
residues
were
resuspended
in
5
ml
of
methanol
water
ammonium
hydroxide
(60.0
+
39.9
+
0.1)
for
high
pressure
liquid
chromatography
analysis.
Twenty-
one
microliter
aliquots
were
injected
onto
the
high
pressure
liquid
chromatography
analytical
column
and
the
concentrations
of
the
degradation
products
present
calculated
using
the
standard
curves
prepared
with
known
amounts
of
the
purified
products.
Lymphocyte
proliferation
assay
Spleen
cells
were
prepared
from
BC3F,
mice
and
cultured
with
Con
A
as
previously
described
(Hanson
et
al.,
1991).
The
culture
plates
were
incubated
at
37°C
in
a
humidified
CO,
incubator
with
the
CO,
flow
adjusted
to
maintain
the
pH
of
the
culture
media
at
7.0.
LMS
(freshly
prepared
in
RPMI-1640
medium
or
after
storage
in
RPMI-1640
medium
for
2
weeks
at
4
or
37°C)
was
added
at
a
final
well
concentration
of
100
1.4g/ml.
Forty-eight
hours
after
adding
Con
A,
the
cultures
were
pulsed
with
[
3
H]-thymidine,
harvested
and
counted
as
previously
described
(Hanson
et
al.,
1991).
Statistical
analysis
of
data
Data
were
analyzed
by
Student's
t
-test
with
a
P<0.05
chosen
as
the
criterion
for
significance.
RESULTS
Standard
curves
for
purified
LMS
degradation
products
Various
concentrations
of
purified
preparations
of
Products
1,
2
and
3
were
analyzed
by
analytical
high
pressure
liquid
chromatography
and
standard
curves
prepared
by
plotting
the
peak
area
vs
1.4g
of
compound
injected.
For
all
three
products
there
was
a
linear
correlation
between
the
peak
area
and
the
amount
of
product
injected
over
the
range
of
1
10
pg
(data
not
shown).
These
standard
curves
were
then
used
to
determine
the
amount
of
the
degradation
products
present
in
unknown
samples
of
culture
media
and
stored
solutions
of
LMS.
Determination
of
LMS
degradation
products
formed
during
72
h
culture
period
with
and
without
lymphocytes
Since
we
previously
determined
that
decomposition
of
LMS
occurred
under
conditions
in
which
cell
culture
is
normally
done
(37°C,
pH
7.0
7.5)
it
is
possible
that
the
enhancement
.5
.4
.3
.2
.1
U
tz
A
WITHOUT
CELLS
pH
7.0
pH
7.25
O
671
11•01,C1.1
3
511
B
WITH
CELLS
pH
7.25
..
.................
.....
....
pH
T.
5
pH
7.0
12
24
36
TIME
HOURS
48
60
72
Fig.
1.
Concentrations
of
Product
I
formed
from
LMS
during
lymphocyte
culture
at
37°C
over
72
h.
The
stability
of
LMS
was
determined
in
the
absence
(A)
and
presence
(B)
of
lymphocytes.
The
cultures
were
maintained
at
either
pH
7.0,
7.25
or
7.5
as
indicated.
The
original
LMS
concentration
was
100
µg/ml.
Values
are
the
mean
of
three
wells
±
S.D.
6
A
WITHOUT
CELLS
atys
5
PH
7.5
4
3
/25
2
1
I
I
pH
7.0
0
I
+
5
B
WITH
CELLS
pH
7.5
a
4
4
...
I:
-
-.a.
......
a..
...............
......
pH
7.25
1
il
pH
7.0
0
1
0
12
24
36
48
60
72
TIME
HOURS
Fig.
2.
Concentrations
of
Product
2
formed
from
LMS
during
lymphocyte
culture
at
37°C
over
72
h.
The
stability
of
LMS
was
determined
in
the
absence
(A)
and
presence
(B)
of
lymphocytes.
The
cultures
were
maintained
at
either
pH
7.0,
7.25
or
7.5
as
indicated.
The
original
LMS
concentration
was
100
µg/ml.
Values
are
the
mean
of
three
wells
±
S.D.
672
K.
A.
HANSON
and
M.
L.
HEIDRICK
Table
1.
Concentrations
of
Products
I,
2
and
3
formed
from
LMS
stored
in
RPMI-1640
with
bicarbonate
buffer
for
two
weeks
at
37
and
4°C*
Product
concentration
1.1g/m1
4°C
37°C
Product
1
0.0
13.3
±
1.4t
Product
2
7.5
±
0.1
15.0
±
0.1
Product
3
0.0 0.0
*LMS
concentration,
2.4
mg/ml.
tValues
are
the
mean
of
three
aliquots
analyzed
separate-
ly
±
S.D.
observed
with
freshly
prepared
solutions
of
LMS
is
due
to
the
formation
of
Product
2
in
the
culture
medium.
Therefore,
we
investigated
the
stability
of
LMS
under
actual
cell
culture
conditions.
Freshly
prepared
LMS
was
added
to
culture
media
and
incubated
for
various
time
periods
up
to
72
h
with
and
without
lymphocytes
present.
Periodically,
media
from
three
wells
was
removed
and
analyzed
individually
for
the
presence
of
the
LMS
degradation
products,
The
results
showed
that
within
4
h,
two
of
the
degradation
products
(Products
1
and
2)
were
detectable
in
the
media.
The
amount
of
Product
1
measured
in
the
media
is
shown
in
Fig.
1.
In
the
culture
media
without
cells
present
(Fig.
1A)
the
formation
of
Product
1
was
pH
dependent
with
greater
degradation
occurring
as
the
pH
increased.
The
pH
effect
was
not
as
apparent
in
the
cultures
with
cells
present.
The
concentration
of
Product
1
did
not
reach
inhibitory
concentrations
(0.4
µg/m1)
in
the
cultures
with
the
cells
present
at
any
time
during
the
72
h
period
and
a
decline
in
the
amount
of
the
compound
present
was
observed
after
12
24
h.
In
cultures
maintained
at
pH
7.5
without
cells,
the
concentration
of
Product
1
reached
0.54
1.4g/ml.
The
formation
of
Product
2
in
the
culture
media
with
and
without
cells
present
is
shown
in
Fig.
2.
Product
2
was
detectable
within
4
h
at
pH
7.25
and
7.5,
but
was
never
detected
at
pH
7.0
throughout
the
72
h
period.
In
the
presence
or
absence
of
lymphocytes,
the
concentration
of
Product
2
formed
during
the
first
48
h
was
in
the
range
of
2
4µg/ml,
well
within
the
stimulatory
range
of
0.5
10
Kg/ml.
In
the
absence
of
lymphocytes,
the
concentration
of
Product
2
began
to
decrease
after
48
h.
In
the
presence
of
lymphocytes
(Fig.
2B),
the
concentration
of
Product
2
remained
fairly
constant
for
the
72
h
culture
period.
In
the
presence
or
absence
of
lymphocytes,
the
formation
of
Product
2
from
LMS
was
dependent
on
pH,
with
greater
degradation
occurring
at
pH
7.5
than
7.25.
Analysis
of
LMS
solutions
stored
in
RPMI-1640
at
4
or
37°C
for
2
weeks
Our
original
observation
was
that
LMS
solutions
in
RPMI-1640
stored
at
4°C
were
more
stimulatory
than
were
freshly
prepared
solutions
of
LMS.
To
determine
if
this
observation
could
be
attributed
to
formation
of
stimulatory
concentrations
of
Product
2,
we
stored
solutions
of
LMS
in
RPMI-1640
at
4
and
also
at
37°C
and
after
2
weeks
analyzed
aliquots
on
the
high
pressure
liquid
chromatography
for
the
presence
of
the
degradation
products.
The
results
(Table
1)
show
that
at
4°C,
Product
2
but not
Product
1
is
formed.
The
concentration
of
Product
2
detected
(7.5
µg/ml)
when
diluted
(0.01
ml
to
0.240
ml)
would
be
0.31
µg/ml,
which
is
in
the
stimulatory
range
based
on
our
assessment
of
the
stimulatory
activities
of
this
compound
(Hanson
et
al.,
1991).
Analysis
of
the
solution
stored
at
37°C
showed
that
both
Product
1
and
Product
2
were
formed
at
the
higher
temperature
and
were
present
at
concentrations
of
13.3
and
15.0
µg/ml,
respectively.
When
diluted
in
culture,
the
concentrations
would
be
0.554
µg/ml
for
Product
1
(inhibitory)
and
0.625
µg/ml
for
Product
2
(stimulatory).
Comparison
of
freshly
prepared
and
stored
solutions
of
LMS
on
lymphocytes
cultures
at
pH
7.0
The
observation
that
the
stimulatory
degradation
product,
Product
2,
was
not
formed
during
culture
at
pH
7.0,
allowed
us
to
address
the
question
of
whether
the
stimulation
observed
with
freshly
prepared
LMS
was
entirely
due
to
formation
of
Product
2
or
if
undegraded
LMS
has
enhanced
activity.
Four
separate
experiments
were
set
up
with
lymphocytes
cultured
at
pH
7.0.
To
these
cultures
were
added
(1)
freshly
prepared
LMS,
from
which
no
Product
2
would
be
formed
and
the
amount
of
Product
1
formed
would
be
neither
stimulatory
nor
inhibitory,
so
any
stimulation
observed
should
be
due
to
undegraded
LMS;
(2)
LMS
stored
at
4°C,
which
when
diluted
contained
0.31
µg/ml
of
Product
2,
but
no
Product
1
and
should
be
stimulatory;
or
(3)
LMS
stored
at
37°C,
which
when
diluted
contained
0.554
pig/m1
of
Product
1
(inhibitory)
and
0.625
pig/
ml
of
Product
2
(stimulatory).
The
results
(Table
2)
showed
that
slight
(but
not
significant)
enhancement
was
observed
with
freshly
prepared
LMS
in
three
of
the
four
experiments,
significant
stimulation
was
observed
in
all
four
experiments
with
LMS
stored
at
4°C
and
inhibition
was
observed
with
LMS
stored
at
37°C.
Immunomodulatory
Action
of
Levamisole-II
Table
2.
Effect
of
LMS
stored
at
various
temperatures
on
lymphocyte
proliferative
response
673
Additions
to
culture
Counts/min/culture
(stimulation
index)
1
Experiment
2
3
4
None
1358
1192
945
1532
LMS
fresh
3181*
2672*
3465* 3254*
LMS
4°C
4040*
3168*
3116*
3647*
LMS
37°C
5629**
4166*I
4643**
4806**
Con
A
189,016
113,552
157,797
148,856
Con
A
+
LMS
fresh
191,136
(1.0)*
115,892
(1.0)
140,751
(0.9)
154,754
(1.0)
Con
A
+
LMS
4°C
203,575
(1.1)
141,677'
(1.2)
172,147'
(1.1)
170,872
°
(1.1)
Con
A
+
LMS
37°C
146,458"
(0.7)
101,095"
(0.8)
127,136"
(0.8)
106,440"
(0.7)
*Significantly
higher
than
none
(P<0.001).
*Significantly
higher
than
none,
LMS
fresh
and
LMS
4°C
(P<0.05).
*Stimulation
index
=
[(Con
A
+
LMS)
-
LMS
alone]
/(Con
A
-
none).
'Significantly
higher
than
Con
A
(P<0.05).
"Significantly
lower
than
Con
A
(P<0.05).
Table
3.
Effect
of
various
combinations
of
Product
1
and
Product
2
on
lymphocyte
proliferation
response
to
Con
A
Product
1
concentration
(Ng/ml)
0.0
0.5
1.0
2.0
3.0
4.0
0.0
1.00*
1.11
1
1.13
5
*
1.19ft 1.25ft
1.29ft
0.1
1.01
1.05
(+.01)
°
1.06
(+.01)
1.04k
(+.06)
I.
(+
.04)
1.21**
(+.06)
0.2
0.97
0.98
(-
.06)
1.02
(+.03)
1.23
1
(+.15)
1.16
1-
(+.05)
1.18**
(+.05)
0.4
0.87**
0.88
(
-
.11)
0.88
(-.12)
0.90
(-.13)
0.82
(-
.24)
1.01
(
-
.07)
0.6
0.83**
0.79tt
(-
.18)
0.84
1-4
(-.14)
0.85tt
(-
.16)
0.88
(-
.16)
1.00
(
-
.06)
0.8
0.81ft
0.78**
(
-
.18)
0.84ft
(-
.13)
0.74ft
(-
.26)
0.87ft
(-.16)
0.94
(-.11)
*Results
are
expressed
as
stimulation
index:
Stimulation
index
=
(Con
A
+
Products)
-
Products
alone
(Con
A-
none)
The
values
are
the
average
stimulation
indexes
of
two
separate
experiments
(triplicate
wells).
Average
counts/min/culture
of
Con
A
stimulated
cultures
for
Exp.
No.
1
were
143,655
±
9928
and
for
Exp.
No.
2,
155,379
±
3621.
*Significantly
different
from
Con
A
stimulated
culture
in
Exp.
No.
1
(P<0.05).
*Significantly
different
from
Con
A
stimulated
culture
in
Exp.
No.
2
(P<0.05).
'Difference
between
SI
if
additive
(SI
of
Product
1
+
SI
of
Product
2/2)
and
actual
SI
(SI
of
Product
1
+
Product
2).
Product
2
concentration
(pig/m1)
Effect
of
various
combinations
of
Product
1
and
Product
2
on
lymphocyte
proliferative
response
to
Con
A
To
further
investigate
why
the
LMS
solution
stored
at
37°C
was
markedly
inhibitory
although
stimulatory
concentrations
of
Product
2
were
present,
we
investigated
the
effect
of
various
combinations
of
purified
Product
I
and
Product
2
on
the
proliferation
assay.
The
results
(Table
3)
indicate
that
concentrations
of
Product
1
greater
than
0.4
pig/m1
abolished
the
stimulatory
effect
of
Product
2.
674
K.
A.
HANSON
and
M.
L.
HEIDRICK
DISCUSSION
In
the
previous
paper
(Hanson
et
al.,
1991)
we
found
that
LMS
non
-enzymatically
decomposed
when
stored
under
mild
conditions
to
form
three
products.
The
three
products
were
purified,
their
structures
determined
and
their
individual
effect
on
Con
A
stimulated
lymphocyte
proliferation
was
assessed.
Products
1,
2
and
3
were
identified
as
3-(2-mercaptoethyl)-5-phenylimidazolidine-2-
one,
6-phenyl-2,3-dihydroimidazo
(2,1-b)
thiazole
and
bis
[3-(2-oxo-5-phenylimidazolidin-1
-y1)
ethyl]
disulfide,
respectively.
Product
2
enhanced
the
lymphocyte
proliferative
responses
to
Con
A
at
concentrations
between
0.5
and
10.0
µg/ml,
but
inhibited
the
responses
at
concentrations
greater
than
10.0.
Products
1
and
3
did
not
enhance
the
response
to
Con
A
at
any
of
the
concentrations
tested,
but
inhibited
the
responses
at
concentrations
>0.4
and
10.0
pg/ml,
respectively.
In
this
study
we
found
that
LMS
decomposes
under
actual
cell
culture
conditions
in
a
very
short
period
of
time
to
form
two
of
the
three
products,
Product
1
and
Product
2,
and
that
the
amount
of
each
product
formed
is
influenced
by
slight
changes
in
pH.
The
two
products
were
found
in
the
culture
media
regardless
of
whether
or
not
lymphocytes
were
present,
but
the
concentration
of
both
products
was
relatively
less
in
the
cultures
with
lymphocytes.
This
suggests
that
the
lymphocytes
are
not
responsible
for
the
degradation,
but
that
the
cells
may
bind
or
further
metabolize
the
breakdown
products,
leading
to
a
lower
concentration
of
the
products
in
the
medium.
Ogawa,
Nakayama
&
Tsubura
(1983)
reported
that
nil
LMS
binds
to
lymphocytes,
granulocytes
and
thymocytes
and
that
the
binding
is
saturatable
and
temperature
dependent
suggesting
a
receptor
or
binding
carrier
protein
for
LMS
is
present.
Their
use
of
a
general
label
LMS]
does
not
rule
out
the
possibility
that
the
labeled
LMS
may
have
first
been
converted
to
Product
1
or
Product
2
before
binding
to
the
cells.
The
studies
of
Hadden,
Coffey,
Hadden,
Lopez-Corrales
&
Sunshine
(1975);
Sunshine,
Lopez-Corrales,
Hadden,
Coffey,
Wanebo,
Hadden
&
Rojas
(1977)
and
Anderson,
Glover,
Koornhof
&
Rabson
(1976)
showing
that
LMS
increases
cyclic-GMP
and
decreases
cyclic
-
AMP
also
suggests
that
LMS
or
its
degradation
products
or
metabolites
act
by
binding
to
the
cell
membrane.
The
formation
of
both
stimulatory
and
inhibitory
products
and
the
influence
of
pH
and
temperature
upon
their
relative
concentrations
explains
why
the
stored
solutions
of
LMS
produce
both
stimulation
and
inhibition
of
the
Con
A
response.
The
greater
enhancement
observed
with
solutions
of
LMS
stored
in
RPMI-1640
for
2
weeks
at
4°C,
can
be
attributed
to
formation
of
Product
2,
which
reaches
stimulatory
concentrations.
The
formation
of
Product
2
would
be
favored
by
the
alkaline
pH
which
develops
with
time
due
to
the
bicarbonate
buffer
in
the
medium.
The
formation
of
Product
1
(inhibitory)
did
not
occur
at
this
temperature.
In
the
solutions
of
LMS
stored
at
the
higher
temperature,
37°C,
the
formation
of
both
products
was
greatly
accelerated.
The
inhibitory
effect
of
this
solution
can
be
accounted
for
by
the
high
concentration
of
Product
1
and
the
fact
that
after
Product
1
reaches
the
concentration
of
0.4
pg/ml
it
appears
to
negate
the
stimulatory
effect
of
Product
2.
How
this
occurs
is
not
clear
but
could
involve
competition
for
a
receptor.
We
and
others
(Woods,
Siegel
&
Chirigos,
1974;
Hiestand
&
Strasser,
1985)
have
found
LMS
to
have
mitogenic
activity
by
itself
on
murine
lymphocytes.
In
these
studies,
purified
Product
1
consistently
showed
mitogenic
activity
on
non
-Con
A
-stimulated
lymphocytes
at
concentrations
of
0.4
µg/ml
and
above,
even
though
the
compound
inhibited
the
Con
A
response
at
these
concentrations.
Product
3
was
also
mitogenic
by
itself
(Hanson
et
al.,
1991)
but
the
formation
of
this
product
was
not
observed
under
actual
cell
culture
conditions.
Thus
the
mitogenic
activity
associated
with
LMS
is
probably
at
least
partially
due
to
formation
of
Product
1.
The
concept
of
LMS
immunopotentiation
and
immunoinhibition
being
due
to
the
action(s)
of
a
degradation
product(s)
or
metabolite(s)
is
also
supported
by
in
vivo
data.
In
vivo,
LMS
is
extensively
metabolized
with
a
half-life
of
1
4
h,
while
LMS
metabolites
have
a
much
longer
half-life.
Graziani
&
De
Martin
(1977)
studied
the
metabolism
of
LMS
in
the
rat
and
found
four
initial
reactions.
The
most
significant
quantitative
reaction
was
formation
of
an
intermediate
produced
by
the
oxidative
introduction
of
a
new
double
bond
into
the
imidazoline
ring
(i.e.
Product
2),
while
the
formation
of
OMPI
(Product
1)
was
the
least
important
pathway
from
a
quantitative
standpoint.
Renoux,
Kassel,
Renoux,
Fiore,
Guillamin
&
Palat
(1977)
reported
that
serum
taken
from
mice
or
rabbits,
24
h
after
injection
with
LMS,
contained
no
LMS,
but
did
contain
a
dialyzable,
heat
resistant
factor
which
enhanced
the
response
to
SRBC
of
untreated
recipient
mice.
Their
studies
suggest
the
immunomodulating
effects
may
be
due
to
the
longer
lived
metabolites
rather
than
to
the
parent
drug.
Immunomodulatory
Action
of
Levamisole—II
675
Our
overall
conclusion
from
these
studies
is
that
the
Con
A
-enhancing
activity
attributed
to
LMS
may
be
due
to
a
metabolite
or
degradation
product
of
LMS,
Product
2,
and
that
the
amount
of
enhancement
observed
may
be
influenced
by
the
formation
of
an
antagonistic
metabolite
or
degradation
product,
Product
1.
The
continued
use
of
LMS
for
treatment
of
cancer
and
rheumatoid
arthritis
justify
further
investigation
of
the
actions
of
these
metabolites.
Acknowledgements
We
thank
Linda
Hendricks
for
her
technical
assistance
and
Dr
Sidney
Mirish
for
use
of
his
high
pressure
liquid
chromatography
system.
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