late. 1960's, polyribonucleotide complexes such as Poly inosinic:polycytidylic acid. (Poly. I:C) were identified as potent antitumor and antiviral agents. [15] whose.
Cytokine
induction
Michael J. Reiter, Mark A. Tomai’ Dept
of Pharmacology,
in mice by the immunomodulator
Traci 3M
L. Testerman,
Pharmaceuticals,
Richard St.
Paul,
led to diminished after the final
production dose. Elevated
of IFN levels
in mice of serum
as meatumor
induced significantly higher amounts of IFN but lower levels of TNF and IL-6 than imiquimod. Imiquimod stimulated significantly higher levels of IFN when compared with 2-amino-5-bromo-6-phenyl-4(3H)-pyrimidinone (ABPP) and similar levels of IFN when compared with tilorone. Neither ABPP nor tilorone induced TNF or IL-6. Finally, imiquimod stimulated TNF, IFN, and IL-6 production in cultures of mouse spleen and bone marrow cells. These studies demonstrate that imiquimod induces not only IFN but other cytokines as well, all of
Key tumor
may contribute 55: 234-240; Words:
interleukin
to its biological 1994. .
interferon
-
activity.J.
cytokine
.
.
anti-
INTRODUCTION Since the early 1960s, interferons (IFN) have been found to exhibit a number of potentially beneficial activities including antiviral and antitumor activities [i-4]. The IFNs can be separated into immune IFN, or IFN-gamma (IFN-’y), and non-immune IFN, or IFN-alpha/beta (IFN-a). IFN-’y is produced by activated T lymphocytes and natural killer cells
cells.
[5, 6] whereas IFN-a is produced mainly by leukocytes, ineluding monocytes/macrophages, dendritic cells, and B cell lines [7-10]. IFN-a is actually a family of related proteins of which at least 15 different subtypes have been identified [ii]. Besides having potent antiviral and antitumor activity, IFNa also enhances MHC class I expression on cells [12], enhances natural killer cell activity [13], and augments macrophage activation [14]. All of these properties may contribute to the overall biological activity. These beneficial immunomodulating properties have resulted in the approval of IFNa for the treatment ofcertain tumors and viral infections [4]. In recent years, a number of synthetic molecules have been shown to induce IFN-a as well as other cytokines. In
234
Journal
of Leukocyte
Biology
Volume
55,
February
and
2-furyi)-2-thiazolyl] formamide-induced chemical carcinogenesis and actually cured mice of the FCB bladder tumor [25]. Using monoclonal antibodies, it was demonstrated that IFN-a was an important, but not sole, mediator of the antitumor activity of imiquimod [24]. The mechanism by which IFN-a and imiquimod inhibit tumor growth is not known; however, both agents seem to inhibit tumor-induced angiogenesis which may partially explain these effects [26]. The present study was undertaken to evaluate imiquimod for its ability to induce IFN-a and other cytokines in mice. Results were compared to other known IFN-a inducers as well as the immunomoduiator bacterial lipopolysaccharide (LPS). We report that imiquimod induces not only IFN-a but also interleukin (IL)-6 and tumor necrosis factor (TNF)a in mice and in cultures of mouse spleen and bone marrow
Leukoc.
antiviral
E. Weeks,
the late 1960’s, polyribonucleotide complexes such as Poly inosinic:polycytidylic acid (Poly I:C) were identified as potent antitumor and antiviral agents [15] whose biological activity has been attributed mainly to the induction of IFNa. In addition, a number of low molecular weight compounds including the fluorenones [16], pyrimidinones [17], and anthraquinones [18] stimulate IFN-a production in a number of animal species. However, none of the above agents is capable of inducing high levels of IFN-a in humans [4]. Therefore, a potent, orally active inducer of IFN-cx in humans may be of clinical value. Recently, a new class of immunomodulating agents, represented by the molecule i-(2-methylpropyl)-iH-imidazo[4,5-C] quinolin-4-amine (R-837, imiquimod) was found to protect guinea pigs from herpes simplex virus (HSV) infection [19-21]. Further studies showed that this agent had no direct antiviral activity, but that efficacy correlated with the induction of IFN-a [21]. Imiquimod was not only effective against primary HSV infection in guinea pigs but was also effective against recurrent HSV infection and a number ofother viral infections including cytomegalovirus infection in guinea pigs [22] and arbovirus infection in mice [23]. In addition to its antiviral activity, imiquimod was found to inhibit tumor growth in a number of murine models including significantly inhibiting the growth of the MC-26 colon carcinoma, RIF-i sarcoma, and Lewis lung carcinoma in mice [24, 25]. Imiqisimod also inhibited N-[4-(5-nitro-
necrosis factor (TNF)-a and interleukin (IL)-6 but not IL-la were found in serum from mice treated with imiquimod. Imiquimod produced significantly higher levels of IFN but lower levels of TNF and IL-6 and IL-la than lipopolysaccharide. Polyinosinic acid:polycytidylic acid
which Biol.
Charles
Minnesota
Abstract: Imiquimod has been identified as a potent antiviral and antitumor agent in animal models. The biological activity associated with imiquimod has been attributed to its induction of interferon (IFN)-a. The present studies evaluated imiquimod administered orally for its ability to stimulate production of IFN and other cytokines in mice. The cytokine profile induced by imiquimod was compared with other known immunomodulators. Imiquimod was found to stimulate increased serum IFN in mice. Daily dosing of imiquimod for five consecutive days sured
L. Miller,
imiquimod
Abbreviations:
HSV,
herpes
polysaccharide;
ABPP, 2-amino-5-bromo-6-phenyl-4(3H)pyrimidinone; simplex virus; IFN, interferon; IL, interleukin; PBMC,
inosinic:polycytidylic Reprint requests: 270-25-06, St. Paul, Received July 22,
1994
peripheral
blood
acid; TNF, tumor Mark A. Tomai, MN 55144. 1993;
accepted
mononuclear
necrosis factor. 3M Pharmaceuticals,
September
27,
LPS,
lipo-
Poly
I:C,
cells;
1993.
3M
Center
MATERIALS
1FN (UImi)
AND METHODS
160C
Mice 140(
Male River
CFW Labs,
mice (22-28g) Wilmington,
were MA.
purchased
from
Charles
120(
loot
Reagents LPS
80
from MO)
Louis,
animals solubilized neal by
Salmonella.
typhimurium
was
solubilized
iv.
Poly I:C in water, cooling to
in
(Sigma Chemical, water before challenging
St.
(Pharmacia LKB, Milwaukee, WI) was heated to 56#{176}C,and then allowed to anroom temperature before challenge of
mice i.p. or iv. The pyrimidinone, 2-amino-5-bromo-6phenyl-4(3H)-pyrimidinone (ABPP), was suspended in saline and the fluorenone tilorone was solubilized in water before challenging mice p.o. Preliminary studies were performed to determine optimal doses of LPS, Poly I:C, ABPP, and tilorone for cytokine induction and these doses were used throughout this study. Imiquimod was suspended in water or saline before
challenging
Cytokine
mice
induction
60 404 20
lx
in mice
Mice were challenged with the above agents and blood was collected from the retrobulbar plexus at various times after stimulation. Blood was pooled from similarly treated animals (3-4 per treatment) and the serum was collected following centrifugation. The serum samples were stored at 70#{176}Cuntil assayed for cytokine activities.
Fig.
2. Induction
administered
spleen
and bone marrow
cultures
Mice were sacrificed, spleens removed from 2-3 mice, and cells isolated by passage through an 80 mesh screen. Single cell suspensions were made by passing cell clumps through a 19 gauge needle. Cells were then washed twice with Hanks’ balanced salt solution (HBSS; Sigma) and finally diluted to 2
x
106
cells/ml
in
RPMI
medium
(Sigma)
fetal calf serum, 2 mM L-glutamine, ethanol, and penicillin/streptomycin plete). Bone marrow cells were isolated mice by forcing HBSS through the
containing
5 x 10 solution
10%
M 2-mercapto(RPMI com-
doses and
the femurs of the bone.
of 2-3 Single
of
a series
by
four
2 h. Serum
Results
are
of
thee
ofimiquimod. times was
Imiquimod
at
3 mg/kg
collected
expressed
2 h after
in U/ml
was
each
time. the
oflFN-a.
final
Results
experiments.
assay
[27]
using
mouse
L929
cephalomyocarditis U of IFN/ml based kocyte IFN standard. was
determined
serum
to
TNF-a from
cell
San
monolayers
Diego, CA. The and Kronenberg
challenged
with
en-
virus. Results are presented as reference on the value obtained for the mouse leuIn some experiments, the type of IFN
using
antiserum
to
IFN-a/13
and
IL-6
as
well
as
anti-
IFN-f3.
of TNF-a,
IL-la,
and IL-la activity were assessed using ELISA Genzyme, Cambridge, MA; results are expressed IL-6 concentrations were assessed using an ELISA Endogen, Boston, MA; again, results are presented
kits in kit in
RESULTS
1000
100
‘a 1.0
2.0
Time 1
Fig.
1. IFN-a
given
orally
8, and Results
presented
3
10,
30,
induction at
1, 3,
=
....
24 h after treatment. represent the mean
as leukocyte
reference IFN
value
100
8.0
Stimulation
(hr)
...
.e-
oral
dose
mg/kg
30
based
100
serum
on
the
(mg/kg)
collected
for IFN experiments.
value
was I, 2, 4,
by bioassay. Results are
obtained
IFN-a in mice. Groups p.o. and then sacrificed
set
of studies
tested
imiquimod
for
its
ability
to in-
of3-4 mice were given imiquiat 1, 2, 4, 8, and 24 h after drug
as
1 h after
treatment,
with
peak
levels
generally
ring at 2 h after treatment. An optimal dosage ranged between 10-100 mg/kg, with higher 250) inducing similar levels (data not shown).
Imiquimod was
first
duce mod
early
24.0
R-837
ofimiquimod. and
Serum was analyzed of one to eleven
U of IFN/ml standard.
After
10
by a single and
4.0
The
challenge. Serum was collected, pooled, and assayed for IFN activity. Results in Figure 1 show that imiquimod stimulated a dose-dependent increase in serum levels of IFN, with doses as low as 3 mg/kg being active. IFN levels were detected as
-
.5
mouse
IFN.
or
IFN was assessed by Lee Biomolecular, method used was described by Brennan
from pg/ml.
10000
separated for
doses
three,
cell suspensions were made as described above and cells were diluted to 2 x 106 cells/mi in RPMI complete. Spleen and bone marrow cells were incubated separately with the various stimuli for 24 h at 37#{176}Cand 5% CO2. Supernatants were then collected, filter sterilized, and stored at - 70#{176}C until they were analyzed for cytokines.
Assessment from center
(U/mi)
10
were
analyzed
two,
4x
of R-837
by multiple
one,
representative
pg/mi. IFN
ofIFN-ct orally
Multiple dose
IFN
Mouse
3x Doses
are
p.o.
2x
for
the
bodies to neutralize in the serum after Several multi-dose
the IFN imiquimod dosing
tion of 3 mg/kg
mice.
Rezier
IFN-a in imiquimod,
et al.
Imiquimod
One, separated
induced
activity, challenge regimens
the
type of IFN found was >97% IFN-a. were tested for induc-
two, three, or four by 2 h, were given
cytokine
occur-
of imiquimod dosages (150Using anti-
production
doses of p.o. and
235
8000
tion
5000 3000
2000
LI ____I
100
30mg/kg
.. (IX)
30mg/kg
(5X)
#{149}i hrLIl2hr4hr
Time
3. Effects
of multiple
Imiquimod
was
tive
Serum
doses.
measured
daily
administered
as
was
Post
doses orally
collected
described.
mg/kg
1, 2, are
and
to evaluate imiquimod
once a day 1, 2, and
IFN-a induc(Fig. 3). Mice
for five consecutive 4 h after the final
days dose.
an increase in TNF. However, the kinetics were different than those observed for IFN-a induction since elevated levels ofTNF were observed as early as 30 mm after administration (1700 pg/ml), peaked at 1 h (2300 pg/mi), and returned to baseline as early as 2-4 h after treatment (Fig. 4B). Imiquimod induced significant levels of IL-6 (Fig. 4C) and the response was similar to that observed for IFN-a in that the peak was detected at 2 h and the levels had returned to baseline by 4 h. No increases in serum IL-ia levels were detected (Fig. 4D). Parenterally administered LPS induced all of the above cytokines, including IL-la. The kinetics for enhancement of IFN (Fig. 4A) and TNF (Fig. 4B) were similar to those obslightly
on once
performed with
stimulated
(hrj
of imiquimod at 30
at
Results
Dose
IFN-a
production.
or daily
4 h after
representative
of
for
5 consecu-
the
final
dose
a
series
of
separate
experiments.
serum multiple found
was collected 2 h after the final dose. Using dosing regimens, enhanced levels of IFN-a at 2 h after the final dose as shown in Figure
IFN
were dosing
Results demonstrated that IFN-a levels in mice dosed daily for five days were dramatically reduced compared with those after a single 30 mg/kg dose. This phenomenon was probably not due to a shift in IFN-a kinetics since levels were reduced at all time points. To evaluate the production of other cytokines induced by imiquimod, mice were dosed with 30 mg/kg of imiquimod orally or the immunomodulator bacterial LPS administered i.p. Mice were then bled at various times and the serum was analyzed for IFN, TNF-a, IL-ia, and IL-6. Again, imiquimod produced an increase in circulating levels ofIFN-a with the 2 h time point being maximal (Fig. 4A). Imiquimod also
2.4000
Fig.
studies daily
received 30 mg/kg p.o. and serum was collected
6000
:
Further after
and three
these were 2A.
(U/mi)
TNF
4000
(pg/mi)
10000-
3500 3000 2500 2000 1500
1000 500
0 Time
e- imiqulmod
After
Stimulation
30mg/kg po
-a
1 Time
(hr) LPS
1 mg/kg
iv
Imiqulmod iL-k.
IL.6
30mg/kg
2 After
Stimuiation
po
4
(hr)
-‘-
LPS
1 mg/kg
iv
(pg/mi)
D
120
100
80
60
40
20
a0 0
1 Time
Imiquimod
Fig. was
4. Cytokine collected
production.
236
induction at
(B)
Journal
2 After
30mg/kg
Stimuiation
4 h after
TNF-a
production.
of Leukocyte
dosing (C)
(hr)
.9.
imiquimod
30mg/kg
po
-I..
4
(hr) LPS
1 mg/kg
iv
-5.
P0
by imiquimod
1, 2, and
2
Time After Stimulation
4
and and IL-6
Biology
LPS.
Imiquimod
(30 mg/kg)
and
for
levels
as described.
analyzed production.
Volume
cytokine (D)
55,
IL-la
February
production.
1994
LPS
(1 mg/kg) Results
were are
administered
representative
to mice ofa
series
p.o. and ofthree
iv.,
respectively.
experiments.
Serum (A)
IFN-a
served for imiquimod; however, LPS induced significantly lower levels of IFN and higher levels of TNF compared to imiquimod. LPS stimulated dramatically higher levels of IL-6 compared to imiquimod. LPS stimulated dramatically higher levels of IL-6 compared to imiquimod, and these levels were sustained for at least 4 h after treatment (Fig. 4C). Levels were greater than 6000 pg/mi; after further analysis, levels were actually found to be approximately 100,000 pg/ml. Unlike imiquimod, LPS induced low but significant serum levels 2 h after treatment
of IL-la (Fig. 4D).
in mice
with
levels
peaking
1FN (U/mi) or TNF (pg/mi)
at
A number of other agents known to induce IFN-a were then compared with imiquimod for cytokine induction. As seen in Figure 5A, when Poly I:C was given i.p., it produced significantly higher levels of serum IFN-a than imiquimod. Levels were elevated as early as 2 h and continued to be elevated at least until 8 h. Tilorone also induced higher levels of IFN-a; however, significant levels were not detected until 4 h postdose and these levels continued to rise until peaking at 24-30 h after administration. Peak levels of IFN-a were somewhat greater than those after imiquimod. ABPP was also capable ofstimulating IFN-a with increased levels being
(R-837J
(pg/mi)
.,,.iFN iL-6
...TNF
(pg/mi)
B
4000#{149}
3500 3000
2500 IFN (U/mi)
2000
12000 1500 10000
-
1000500-
8000
06000
0
0.1
0.05
1.0
e- iL.6
4000
Fig. 6. Cytokine induction Spleen cells (1 x 106/ml)
2000
ofimiquimod. TNF, Time .
imiquimod
..‘-
After
Poiy
Stimulation
Culture
and
IL-6
(A)
by imiquimod were cultured supernatants
Spleen
in cultures for 24 h with
were
IFN
and
TNF
collected
results.
of mouse spleen cells. various concentrations and
(B)
analyzed
Spleen
for
IL-6
IFN-a,
results.
(hr)
i:C
ABPP
..-
Tilorone
TNF (pg/mi) 3000
2500
2000
1500
detected at 2-4 h after drug treatment. Peak levels of serum IFN-a induced by ABPP were lower than those observed for imiquimod. The above agents were then evaluated for induction of TNF (Fig. SB). Imiquimod produced detectable levels of TNF as early as 0.5 h after dosing with peak levels being found usually around 1 h. Poly I:C also stimulated production of TNF; however, the peak levels were detected later at 4 h. Tilorone and ABPP were ineffective at inducing significant serum levels of TNF in mice. Imiquimod was tested for its ability to stimulate cytokine release in vitro in splenic cultures (Fig. 6). In spienic cultures, imiquimod induced a significant increase in TNF, IL-6, and IFN-cr in these cultures; however, a true doseresponse was not seen for IFN-a. Similar induction of TNF, IL-6, and IFN-a was observed in bone marrow cultures (Fig. 7). At the higher concentrations of imiquimod, levels
1000
500
2
Time After 6-
Fig.
0.5
(pg/mi)
fR837
imiquimod
-‘-
Poiy
of IFN-a declined and levels sharp contrast to the linear
4
Stimulation
(hr)
i:C
ABPP
of TNF plateaued, which dose-response of IL-6.
is in
...
5. Comparison of imiquimod with Poly I:C, ABPP, and tilorone for IFN-a and TNF induction. Imiquimod (30 mg/kg p.o.), Poly I:C (100 Lg/mouse i.p.), ABPP (250 mg/kg p.o.) or tilorone (250 mg/kg p.o.) were given to mice. Serum was collected 1, 2, 4, 6, 8, 24, or 30 h after treatment and analyzed for (A) effects on serum IFN-a levels (B) effects on serum TNF-a levels. Results are representative of a series of 3 separate experiments.
DISCUSSION Imiquimod has been shown to be a potent antiviral and antitumor agent in a number of animal models [19-25]. This compound is not directly active against viruses and tumors; rather, its activity is mediated by immune stimulation.
Reiter
et al.
Imiquimod
induced
cytokine
production
237
iFN (U/mi) or TNF (pg/mi)
quimod induces only low levels ofIL-ia that, due to its short half-life, are below detectable limits of the assay. The cytokine pattern induced by imiquimod is somewhat similar to that observed for LPS stimulation of monocyte/ macrophages. Therefore it is reasonable to hypothesize that imiquimod is stimulating cells of the monocyte/macrophage lineage to secrete these cytokines. Indeed, imiquimod has been shown to stimulate cells of the promonocytic cell line THP-i to secrete TNF, IL-i, IL-6, and IL-8 and the mouse macrophage cell line 264.7 to secrete TNF (MA. Tomai, unpublished observations). Thus, one of the cellular
800
RAW
targets for imiquimod is cells from the phage lineage. Despite certain similarities in cytokine some significant differences between LPS(pg/mi)
[R-837) .e.iFN
-TNF
0
0.5
is.
Fig.
7. Cytokine
cells.
Bone
induction
marrow
concentrations
(1
of imiquimod.
lyzed for IFN-a, ( B) Bone marrow periments.
TNF, IL-6
Much
antiviral
of the
iL-6
by imiquimod
cells
x
in cultures
l06/ml)
were
Culture
ofmouse
cultured
supernatants
and IL-6. (A) results. Results
and
1.0
(pg/mi)
[R.837]
for were
Bone marrow are representative
antitumor
marrow
h with
collected
IFN
activity
bone 24
and oftwo
has
various and
ana-
TNF results. separate cx-
been
at-
tributed to the induction ofIFN, specifically IFN-a. Indeed, antibodies to IFN-ct inhibit much of the antitumor activity [24]; however, the induction of IFN-a cannot solely explain the biological activity of imiquimod. This study demonstrates that in a mouse model, imiquimod induces not only IFN-a but also TNF and IL-6. Likewise, in a rat model, imiquimod stimulated production of both IFN-a and TNF, albeit with slightly differentkinetics (data not shown). These results are similar to those observed by Weeks and Gibson in which they observed TNF, IL-6, and IFN-a in cultures of human peripheral blood mononuclear cells (PBMC) that had been treated with imiquimod (C.E. Weeks, S.J. Gibson, submitted). They also observed induction of other cytokines, including IL-l/3 and IL-8, in these cultures. Thus, the induction of IL-i, IL-6, and TNF as well as other cytokines that are known to have antitumor and antiviral effects may contribute The
substantially fact that
to the imiquimod
biological did not
activity induce
of imiquimod. detectable levels
of IL-ia in mice is not totally surprising since this form of IL-I remains largely within the cell [28] or is membrane bound [29]. We have demonstrated high levels of IL-ia inside human PBMC that were stimulated with imiquimod as well as low levels in the culture filtrate (MA. Tomai,unpublished observation). Another explanation may be that imi-
238
Journal
of Leukocyte
Biology
Volume
55,
February
1994
monocyte/macroprofile, there and imiquimod-
are
induced cytokine production. First, imiquimod stimulated significantly higher levels of IFN, both in vivo and in vitro, than did LPS. In fact, the amount of IFN induced by LPS may actually be a combination of IFN-a and IFN-’y since it is known that LPS also induces IFN-’y [30]. Another difference is that LPS produced significantly higher levels of TNF, IL-ia, and IL-6 when compared to imiquimod. Thus, although these agents share some common features for cytokine induction, they are functionally distinct. With regard to Poly I:C, the cytokine profile is, again, distinct from imiquimod. Poly I:C given i.p. stimulated significantly higher levels of IFN-a than either imiquimod or LPS. In addition, Poly I:C induced IFN-a levels which peaked at 6-8 h, while imiquimod levels peaked at 2 h. This difference in kinetics can be explained, in part, by the route of Poly I:C administration. Poly I:C also induced TNF and IL-6 production, but in significantly lower amounts than those found with imiquimod or LPS. When Poly I:C was given i.v. rather than i.p. the kinetics of IFN-a, TNF, and IL-6 induction were quite similar to that of imiquimod. Thus, imiquimod stimulated a distinct pattern of cytokines when compared with Poly I:C. When comparing the cytokine profiles of imiquimod and two other orally active IFN inducers, ABPP and tilorone, an entirely different pattern emerges. ABPP induced only low levels of IFN-a compared to imiquimod. With regard to tilorone, the kinetics of IFNa production were quite different from that induced by imiquimod in that peak levels were seen at least 24 h after treatment. The lag in IFN-a production may indicate an indirect rather than a direct induction of IFN-a by tilorone. Another significant difference between imiquimod and the other orally active IFN-a inducers was that ABPP and tilorone did not induce detectable levels of either IL-6 or TNF. Thus, cytokine production by imiquimod is quite different from that observed with ABPP and tilorone. The fact that spleen cells and bone marrow cells produced significant levels of IFN-a, TNF, and IL-6 in response to imiquimod and LPS indicated that these tissues may contribute to the overall levels ofcytokines seen in the serum. As was seen in vivo, LPS stimulated both spleen and bone marrow cells to produce more IL-6 and TNF and less IFN than did imiquimod. On a per cell basis bone marrow and spleen cell preparations produced similar amounts ofeach cytokine. As observed in Figure 6A, levels of IFN-a in spleen cell preparations increased with increasing concentrations of R-837 up to 0.1 jzg/ml. With higher concentrations, levels actually declined, which was somewhat surprising since this was not observed in bone marrow cultures receiving lower concentrations of drug (data not shown). The possibility exists that at higher concentrations of drug (> 0.5 eg/ml) it is toxic; however, this does not seem to be the case since via-
bilities These levels higher
Studies are in progress It is not too surprising mod Poly
the antitumor effect of interferon in mice. Nature (Lond.) 239, 167-168. 3. Brunda, M.J., Rosenbaum, D., Stern, L. (1984) Inhibition of experimentally induced murine metastases by recombinant a interferon: correlation between the modulatory effect of interferon treatment on natural kilier activity and inhibition of metastases. Int. J. Cancer 34, 421-426. 4. Dianzani, F. (1992) Interferon treatments: How to use an endogenous system as a therapeutic agent. j Interferon Res. Special Issue 12, 109-118.
for drug treated and untreated cultures were similar. results may have implications in vivo in that low blood of R-837 may give optimal levels of IFN-a, whereas blood levels may actually give lower levels of IFN-a.
induced I:C and
32]. The responsiveness Stringfellow
to address that daily
a hyporesponsive LPS also exhibit
this possibility. administration
state this
mechanism by which these is not fully known; [32] and others suggest
of imiqui-
in mice. It is known same phenomenon
that [31,
agents induce hypohowever, studies by that prostaglandins of
5.
IFN-a is produced Because of the
endogenously problems with
by the injectable
active inducer ofendogenous IFN-a may The polynucleotides, represented by the
host. IFN-a,
an
be clinically agent Poly
orally
useful. I:C, are
potent inducers of IFN-a; however, most are inherently toxic. Another problem is that although Poly I:C is very potent in animal species, it is inactive in humans due to degradation by nucleases in the serum. A derivative of Poly I:C, Poly IC:LC, is active in humans; however, it still has the disadvantage of being toxic. The orally active IFN-a inducers, ABPP and tilorone, definitely induce IFN-a in a number of animal species; however, they both have been ineffective at inducing IFN-a in humans. Also, these agents induced only low levels of IFN-a in mice when compared with Poly I:C and imiquimod. In conclusion, although the above agents have some inherently beneficial activities, the clinical use of these compounds has not yet been advocated. Imiquimod has demonstrated potent antiviral and antitumor agent in a number of animal models. This molecule offers the advantages of being a small, chemically-defined, orally active molecule in a number of animal species inciuding mice, rats, guinea pigs and primates. In vitro, imiquimod is also capable of that may contribute to PBM. These activities mod in humans. Most to be effective using healthy results indicate ing in humans.
inducing IFN-a and the antiviral activities provided the impetus recently, imiquimod
at inducing human subjects that imiquimod
IFN-a in phase 1 clinical [33, 34]. Taken together, warrants further clinical
(1979)
9.
Zoon,
K.C.,
Samuel,
G.E.
(1987)
Inter-
Virus-induced
interferon
production
by
human
macro-
Feldman,
M.,
Bocarsly,
P.F.
(1990)
Sequential
enrichment
and
immunocytochemical visualisation of human IFN-a producing cells. j Interferon Res. 10, 435-446. 10. Strander, H., Mogensen, K.E., Cantell, K. (1975) Production of human lymphoblastoid interferon.j Clin. MicrobwL 1, 116-124. 11. Weissman, C., Weber, H. (1986) The interferon genes. Prog. Nucleic Acid Res. Mol. Biol. 33, 251-300. 12. Heron, I., Hokiand, M., Berg, K. (1978) Enhanced expression of
f32-microglobuiin
and
HLA
antigens
on
human
lymphoid
cells by IFN. Proc. Nati. Acad. Sci. USA 75, 6215-6219. 13. Trinchieri, G., Santoli, D. (1978) Antiviral activity induced culturing lymphocytes cells. Enhancement
by
with tumor derived or virus transformed of human natural killer cell activity by
in-
terferon and antagonistic inhibition of susceptibility of target cells to lysis. j Exp. Med. 147, 1314-1333. 14. Grossberg, SE. (1987) Interferons: an overview of their biological and biochemical properties. In Mechanisms ofinterferon Action (L.M. Pfeffer, ed), CRC Press, Boca Raton, Fla. 1-32. 15. Fields, AK., Tytell, A.A., Lampson, G.P., Hilleman, EM. (1967) Inducers of interferon and host resistance II multistranded synthetic polynucleotide inducers. Proc. NatI. Acad. Sci. USA 58, 1004-1010. 16. Mayer, GD., Krueger, R.F. (1970) Tilorone hydrochloride: mode of action. Science 169, 1214-1215. 17.
Nichol,
FR.,
Weed,
S.D.,
Underwood,
G.E.
(1976)
of murine interferon by a substituted pyrimidine. Agents Chemother. 9, 433-439. 18. Stringfeilow, D.A., Weed, S.D., Underwood, G.E.
i9.
trials these test-
Langer,J.A.,
phages.j Immunol. 123, 365-369. 8. Hayes, M.P., Enterline, J.C., Gerrard, T.L., Zoon, K.C. (1991) Regulation of interferon production by human monocytes: requirements for priming for lipopolysaccharide-induced production. j Leukoc. Biol. 50, 176-181.
viral
other cytokines seen in human to test imiquihas been shown
S.,
ferons and their action. Annu. Rev. Biochern. 56, 727-777. 6. Bancroft, G.J., Sheehan, K.C.F., Schreiber, RD., Unanue, ER. (1989) Tumor necrosis factor is involved in the T cellindependent pathway of macrophage activation in scid mice. J. Irnrnunol. 143, 127-130. 7. Roberts, NJ., Douglas, R.G., Simons, R.M., Diamond, ME.
the E series can reverse the hyporesponsiveness. Further studies are being designed to delineate the mechanism of induction of the hyporesponsive state induced by imiquimod. Injectable IFN-a has only recently been used clinically in the treatment of certain viral diseases and cancer [4]. However, problems with the administration of IFN-a remain. Like most cytokines, IFN-a has only a short half-life and acts only over a very short distance and, therefore, must be given in very high quantities to observe clinically beneficial effects. In addition, a number of patients given IFN-a have developed antibodies that may neutralize its activity. Finally, the injection of exogenous IFN-a may bypass the appropriate regulatory elements that would normally be induced when
Pestka,
20.
and
interferon
inducing
properties
of
1,5
Stimulation
Antirnicrob. (1979)
Anti-
diaminoanthra-
quinones. Antirnicrob. Agents Chernother. 15, 111-118. Miller, R.L., Imbertson, L.M., Reiter, MJ., Schwartmiller, D.H., Pecore, SE., Gerster,J.E (1985) Inhibition ofherpes simplex virus infection in a guinea pig model by 5-26308. ASM Interscience Conference on Antimicrob. Agents Chemother. 235. Bernstein, DI., Harrison, CJ. (1989) Effects of the immunomodulating agent R-837 on acute and latent herpes simplex virus type 2 infections. Antirnicrob. Agents Chemoiher 33, 1511-1515.
ACKNOWLEDGMENT We
thank
Ms.
Mary
Nilan
for excellent
secretarial
21.
Harrison, CJ., Jenski, L., Voychehovski, (1988) Modification of immunological disease during topical R-837 treatment tion. Antiviral Res. 10, 209-224.
22.
Chen,
assistance.
M.,
Efficacy
REFERENCES 23.
1. Isaacs, A., Lindenmann, J. (1957) Virus interference. I. The terferon. Proc. R. Soc. B. 147, 258-267. 2. Gresser, I., Maury, C., Brouty-Boye, D. (1972) Mechanism
inof
24.
Griffith,
B.P.,
Lucia,
H.L.,
T., Bernstein, DI. responses and clinical of genital HSV-2 infecHsuing,
GD.
(1988)
5-26308 against guinea pig cytomegalovirus. Antirnicrob. Agents Chemother. 32, 678-683. Kende, M., Lupton, H.W., Canonico, PG. (1988) Treatment of experimental viral infections with immunomodulators. Adv. Biosci. 68, 51-63. Sidky, Y.A., Borden, E.C., Weeks, G.E., Reiter, MJ., Hatcher,
Reiter
et al.
of
Imiquimod
induced
cytokine
production
239
Bryan, G.T. (1992) Inhibition of murine tumor growth by an interferon inducing imidazoquinoline. Cancer Res. 52, 3528-3533. Sidky, Y.A., Bryan, G.T, Weeks, G.E., Hatcher, J.M., Borden, E.G. (1990) Effects of treatment with an oral interferon inducer,
J.E,
25.
imidazoquinolinamine
(R-837),
on
the
growth
of mouse
30. Le, J., Jian-Xin, L., Henriksen-DeStefano, Bacterial lipopolysaccharide-induced j Immunol. 136, 4525-4530. 31. Madonna,
bladder
carcinoma
FCB. j Interferon Res. 10, (Suppl. 1), 5123. 26. Sidky, Y.A., Borden, E.G., Weeks, G.E. (1992) Inhibition of tumorinduced angiogenesis by the interferon inducer imiquimod. Proc. Am. Ass. Cancer Res. 33, 458. 27. Brennan, G.L., Kronenburg, L.H. (1983) Automated bioassay of interferons in microtest plates. Biotechniques 1, 78-82. 28. Dinarello, CA. (1991) Interleukin-1 and interleukin-1 antagonism. Blood 77, 1627-1652. 29. Kurt-Jones, E.A., Beiler, DI., Mizel, SB., Unanue, ER. (1985) Identification ofa membrane-associated interleukin I in macrophages. Proc. NatI. Acad Sci. USA 82, 1204-1208.
240
Journal
of Leukocyte
Biology
Volume
55,
February
G.S.,
associated
with
precursor
poois.
Vogel,
SN.
alterations
j
Immunol.
(1985) in bone
135,
D.,
Vilcek,
interferon--y Early
endotoxin
marrow-derived
J. (1986) production. tolerance
is
macrophage
3763-3771.
32.
Stringfellow, D.A. (1981) Induction lar weight compounds: fluorenone pyrimdinones. Methods Enzymol.
ofinterferon with low molecuesters, ethers (tilorone), and 78, 262-284.
33.
Wick, K.A., Kvam, D.C., Weeks, G.E., Dixon, R.M., Hafstad, K., Witt, P.L. (1991) Oral R-837 induces interferon in healthy volunteers. Proc. Amer. Ass. Cancer Res. 32, 257.
34. Borden, E., Witt, P., Kvam, D., Wick, K., Ritch, P., Anderson, T., Owens, M., Sidky, Y., Grossberg, S. (1991) An effective oral inducer ofinterferons in humans.]. Interferon Res. 11 (suppl. 1), S92.
1994
