ARTICLE
Auteur(s) : Tsuyoshi MITSUISHIa, Kazumi
IIDAb, Seiji KAWANAa
a Department of Dermatology, Nippon Medical
School, 1‐1‐5, Sendagi, Bunkyo‐ku, Tokyo, 113‐8603, Japan.
b Research Institute of Vaccine Therapy for Tumors
and Infectious Diseases, Nippon Medical School, 1‐1‐5, Sendagi,
Bunkyo‐ku,Tokyo, 113‐8603, Japan.
Reprints: T. Mitsuishi, Fax: (+81)‐3‐3823‐6731 E‐mail:
tmitsunms.ac.jp
Article accepted on 16\06\03
Cimetidine is a histamine receptor antagonist commonly used to
treat gastrointestinal reflux and gastric ulcers in adults and
children [1‐3]. Recent investigations have recognized the
immunological effects of high or low doses of cimetidine against
multiple or recalcitrant warts [4‐7]. Those reports have suggested
that cimetidine, through various immunomodulating mechanisms, can
lead to the remission of viral warts [4‐7]. In contrast, one study
reported that a low dose of cimetidine does not have a significant
effect in the treatment of multiple warts [8]. The mechanism of the
effect of cimetidine on warts is still unclarified at the cytokine
level. In general, liquid nitrogen (LN2) cryotherapy is usually
used for the treatment of multiple warts. According to Ahmed et
al., of 86 patients, only 24 (27.9%) responded to
cryotherapy and there was no correlation between the type of human
papillomavirus (HPV) in the warts and the response to cryotherapy
[9]. Cell mediated immunity is important for controlling HPV
infection and HPV associated tumors in experimental models [10]. T
helper (Th) 1 cells are effective in the host defense against
viral infections and tumors [11]. In general, cimetidine promotes
cellular immunity by activating natural killer (NK) cells,
eliciting an increase in IL‐2 production [11, 12]. In
addition, cimetidine promotes the expression and subsequent release
of IL‐12 from monocytes or macrophages in vivo [13] and
may enhance Th cell and NK cell activity, and the production of
IL‐2 and interferon‐(IFN)‐γ [11, 12]. IFN‐γ is produced not
only by NK cells but also by Th 1 cells [11]. While
IL‐18 stimulates IFN‐γ production by Th cells and by NK cells,
there is limited information on the contribution of this to
immunity in patients with various infectious diseases [14]. In this
study, we examined whether oral cimetidine therapy is effective in
treating various types of multiple warts in two treatment groups
(low dose and high dose) within 4 months (maximum). We
analyzed the quantity and change in local expression of mRNAs for
IL‐2, IL‐18 and IFN‐γ before and during treatment of viral
warts using real time PCR.
Materials and methods
Patients and doses of oral cimetidine
Fifty‐five non‐immunosuppressed patients, 6 to
77 years of age (mean age; 27.6 years), were enrolled in
an open‐label study. All patients with multiple viral warts (at
least 5 to more than 50) were treated with oral cimetidine
alone. Some patients had been previously treated with other
methods, i.e., LN2 cryotherapy and desiccation. In this study,
55 patients with verruca vulgaris (22 cases), genital
warts (10 cases), verruca vulgaris\verruca plantaris
(9 cases), verruca plantaris (8 cases) and verruca planae
(6 cases) were selected and received 20 mg\kg
(group A) or 30 to 40 mg\kg (group B) cimetidine for
10 days to 16 weeks, as shown in Table
I. As an average dose of oral cimetidine, group A received
15.3 mg\day of the drug, while group B received
33.2 mg\day of the drug. No other therapies were applied
during the study. Patients received the maximum daily dose up to
3 months based on the Food and Drug Administration‐‐approved
level of 2400 mg\day. We then compared the effects of
cimetidine therapy between group A and group B.Table
I. Clinical characteristics of patients with cimetidine
therapy of group A and group B
|
Groupe A ( 20 mg\kg) ‡ |
Groupe B (30 mg\kg to 40
mg\kg) ‡‡ |
| No. patients: n ∓ 26 |
CR: n ∓ 7 (26.9%) |
PR: n ∓ 4 (15.3%) |
NR: n ∓ 15 (57.7%) |
No. patients:
n ∓ 29 |
CR: n ∓ 12 (41.3%) |
PR: n ∓ 9 (31.0%) |
NR: n ∓ 8 (27.5%) |
| Age (years) |
|
|
|
|
|
|
|
|
| Mean |
30 |
|
|
|
25.4 |
|
|
|
| Range |
12‐77 |
|
|
|
6‐71 |
|
|
|
| Type of viral warts |
|
|
|
|
|
|
|
|
| Verruca Vulgaris |
10 |
4 (40%) |
2 (20%) |
4 (40%) |
12 |
7 (58.3%) |
2 (17%) |
3 (25%) |
| Genital Warts |
5 |
0 (0%) |
0 (0%) |
5 (100%) |
5 |
0 (0%) |
1 (20%) |
4 (80%) |
| Verruca Vulgaris\Verruca Plantaris |
5 |
1 (20%) |
1 (20%) |
3 (60%) |
4 |
1 (25%) |
3 (75%) |
0 (0%) |
| Verruca Plantaris |
4 |
2 (50%) |
0 (0%) |
2 (50%) |
4 |
2 (50%) |
2 (50%) |
0 (0%) |
| Verruca Planae |
2 |
0 (0%) |
1 (50%) |
1 (50%) |
4 |
2 (50%) |
1 (25%) |
1 (25%) |
CR: complete response: 100% reduction in the size and number of
lesions
PR: partial response: more than 50% reduction in the size and\or
number of lesions
NR: no response: less than 50% reduction in the size and\or number
of lesions
‡: medium cimetidine dose: 15.3 mg\kg
‡‡: medium cimetidine dose: 33.2 mg\kg
.
Cytokine assays
Ten patients had two skin punch biopsies (using a 5 mm
disposable punch) taken before and during the treatment. Biopsies
were repeated when the warts were reduced in number and\or
4 months later. The biopsy specimens were immediately stored
at ‐‐80°C. Messenger RNA was extracted from those specimens as
detailed below and levels of local IL‐2, IL‐18, and IFN‐γ mRNAs
were measured using real time PCR.
Briefly, total RNA was extracted from 5 mg tissue by the acid
guanidinium thiocyanate‐phenol‐chloroform extraction (AGPC) method
using Isogen (WAKO Junyaku, Osaka, Japan) and DNase I (Takara,
Shiga, Japan), and was collected by precipitation in ethanol. To
prepare standard RNA, each PCR product was cloned into the
pBluescript vector (Stratagene, CA, USA) and was linearised to
prevent any activity at the T3 promoter site. Standard RNA was
synthesized using T7 RNA polymerase and was purified by Isogen and
DNase I treatment. The PCR reaction mixture was prepared using a
TaqMan Universal reagent kit (PE Applied Biosystems, CA, USA)
according to the manufacturer‘s instructions [15]. The reaction
mixture (50 µL) was prepared containing 1x Universal PCR
buffer, 0.25 µM forward primer and backward primer and
0.1 µM probe. A real time PCR system (ABI PRISM
7700 Sequence Detection System: PE Applied Biosystems)
provides essential information used to quantify the initial target
copy number, because it can draw an amplification curve. Using 5‘
nuclease activity, a specific fluorescent signal is generated and
is measured at every cycle during a run. The primer set to amplify
IL‐2 mRNA was designed according to GenBank J00264 accession
using primers for exon 1‐2: 5‘‐AAA GAA AAC ACA GCT ACA AC‐3‘ and
for exon 2‐3: 5‘‐TGA AG TGT TTC AGT TCT GTG GCC‐3‘. The probe (exon
2): 5‘‐AGA ATC CCA AAC TCA CCA GGA TGC TCA CAT T‐3‘ was designed to
target an internal region between these primers. The primer set to
amplify IL‐18 mRNA was designed according to GenBank E17138,
using primers for exon 2: 5‘‐CCT GGA ATC AGA TTA CTT TGG C‐3‘ and
for exon 3: 5‘‐ACA GCC ATA CCT CTA GGC TGG‐3‘ and using a probe for
exon 2: 5‘‐GAA TGA CCA AGT TCT CTT CAT TGA CCA AGG‐3‘. The primer
set to amplify IFN‐γ mRNA was designed according to GenBank
AF375790, using primers for exon 3: 5‘‐CGA GAT GAC TTC GAA AAG CTG
ACT‐3‘ and for exon 4: 5‘‐TCC TTT TTC GCT TCC CTG TTT TA‐3‘ and
using a probe for exon 4: 5‘‐CAA GTG ATG GCT GAA CTG TCG CCA GC‐3‘.
The primer set for amplification of glyceraldehyde‐3‐phosphate
dehydrogenase (G3PDH) mRNA, as an internal control, was designed
according to GenBank NM008084, using primers for exon 3: 5‘‐TCA ACG
ACC CCT TCA TTG ACC TCA‐3‘ and for exon 5: 5‘‐GTG AAG ACA CCA GTA
GAC TCC AC‐3‘. The probe of G3PDH‐P was designed for exon 4: 5‘‐ACG
GCA CAG TCA AGG CCG AGA ATG GG‐3‘. Each PCR reaction was performed
for 50 cycles (95°C for 30 sec, 60°C for 40 sec,
72°C for 30 sec) using this real‐time PCR system. The probe
was labeled with fluorescent dyes at both ends of the probe:
fluorescein as a reporter dye and rhodamine as a quencher dye.
After annealing the probe onto the internal locus of the amplicon,
the probe was cleaved with the 5‘ exonuclease activity of
thermostable DNA polymerase.
After cleavage of the probe, the reporter dye emission no longer
transferred efficiently to the quencher dye, resulting in an
increase in the reporter dye fluorescent emission spectra. The
fluorogenic samples were excited with a laser (488 nm), and
were recorded using a charge‐coupled device camera during the PCR
amplification by an ABI PRISM 7700 Sequence Detection
System.
Statistical methods
Proportions were compared with the Chi‐square test or with
Fisher‘s exact test using a statistical software package (Stat View
5.0 for Windows, SAS Institute Inc., Cary, NC, USA). P values
were considered to be statistically significant at
P < 0.05.
Results
Clinical effects of oral cimetidine to treat multiple
warts
Fifty‐five non‐suppressed patients with multiple warts,
26 in group A (low dose) and 29 in group B (high dose),
were evaluated in this study (Table I).
Clinical aspects of both groups, and a comparison of patients
showing complete (CR) or partial (PR) responses, are also
summarized in Table I. Seven CR and
4 PR (more than 50% reduction in the size and\or number of
lesions) were obtained in group A, while in group B, 12 CR and
9 PR were observed. Overall, 4 patients (2 with
verruca vulgaris, 1 with verruca planae, and 1 with
verruca plantaris) showed dramatic rapid improvement within
2 weeks.
Genital warts had a relatively low response (10%: 1\10) compared
with other types of warts (68.9%: 31\45). In group B,
5 patients reported side effects, nausea, abdominal pain,
dizziness or elevation of transaminase, however, these side effects
were all reversible. While no side effects were noted in group
A.
Quantification of IL‐2, IL‐18 and IFN‐γ mRNA
expression
Real time PCR was used to quantify mRNA levels of IL‐2,
IL‐18 and IFN‐γ before and during treatment in 10 cases.
The amounts of mRNA for IL‐2 and IFN‐γ were significantly
higher (p < 0.05) in tissues during treatment than
before treatment (Fig.
1a,b). In some cases before cimetidine treatment,
IL‐2 and\or IFN‐γ had been already released in the lesional
skin. In contrast, the production of IL‐18 mRNA was
significantly lower (p < 0.05) during treatment than
before treatment (Fig.
1c). One case (a flat wart: no response) was treated with
oral cimetidine (30.7 mg\kg\day) for 4 months and showed
no remarkable changes in mRNA levels of IL‐2, IL‐18, or IFN‐γ in
the tissues before and during treatment, indicating that the levels
of cytokines change in parallel with the clinical course.
.
Discussion
Surgical, chemical, immunological, and\or pharmacological
therapies are usually used to treat multiple or recalcitrant warts.
Among those, one type of immunological therapy, oral cimetidine,
has been reported in recent investigations [4‐7]. In fact,
aggressive surgical therapy or LN2 cryotherapy is painful and
stressful and is sometimes not effective for treating warts [9]. In
addition, such therapy occasionally leads to the recurrence of
warts. We examined whether a low dose of cimetidine therapy is
effective in treating various types of multiple warts compared with
a high dose of cimetidine therapy within 4 months (maximum),
and we analyzed the local mRNA expression of cytokine mRNAs before
and during treatment. Of 55 patients, 34.5% showed complete
resolution, 23.6% showed partial resolution and 41.8% had no
response. The present study showed a statistically significant
improvement between low and high doses of cimetidine
(P ∓ 0.031), and complete resolution did not depend on
the number of warts. According to Gooptu C et al., of
47 patients who received oral cimetidine (30 to
40 mg\kg) for 3 months, 87% of children and 68% of adults
improved with that drug [6]. Orlow S et al. reported that of
32 children with multiple warts who received oral cimetidine
(25 to 40 mg\kg), 26 (81%) improved with the treatment
within 2 months [4]. Moreover, Glass and Solomon examined
18 adult patients with recalcitrant warts; 16 of those
patients (84%) had dramatic clinical improvement or complete
resolution of their warts after 3 months of cimetidine therapy
[5].
Cimetidine promotes the release of IL‐12 mRNA expression from
monocytes or from macrophages in vivo [13]and also activates
some types of cells, especially Th1 cells [12], which release
IL‐2 and IFN‐γ. Cimetidine also stimulates NK cells, resulting
in increased levels of IFN‐γ. Th1 cells are effective in host
defenses against viral infections and tumors [11]. In addition
IFN‐γ may immunomodulate keratinocytes with HPV. In this study,
high levels of IL‐2 mRNA were detected in warts during treatment
using real time PCR (Fig. 1a). This evidence
supports the hypothesis that cimetidine activates Th1 cells and
that those cells then release IL‐2, and also IFN‐γ. However, in
some cases before cimetidine treatment, IL‐2 and\or IFN‐γ had
been already released in the lesional skin. This result suggests
that Th1 cells already existed in the viral wart tissues and
delayed the keratinocytes with HPV in order to maintain
homeostasis.Our data revealed that the expression of IL‐18
decreases in parallel with reductions in the sizes of viral warts,
indicating that keratinocytes infected with HPV express IL‐18.
There are now more than 90 different known types of HPV. HPV
associated with skin lesions are classified as cutaneous HPV, which
are different from the genital HPV associated with gynecological
lesions. The difference between cutaneous types and genital types
of HPV is that the cutaneous types do not have the E5 open reading
frame (except for a few cutaneous types) compared with the genital
types [16]. One study examined low dose therapy of oral cimetidine
for treating children with verruca plantaris and analyzed the HPV
genotypes. However, there were no differences in cimetidine effect
between any cutaneous HPV genotypes [8]. In our study, the fact
that about 41.8% of the viral warts treated with cimetidine were
resistant indicates that high viral loads were present in those
lesions and\or that they were highly keratinized, acantholytic
lesions. Based on the different responses between groups A and B,
the dose of cimetidine therapy is important in order to reduce the
size of warts. Additionally, our data show that genital warts
(condyloma acuminatum) were resistant to cimetidine therapy,
suggesting that the E5 gene in genital types may play an important
role in protecting against decreasing viral load.
In summary, our study demonstrates that oral cimetidine therapy of
viral warts activates Th1 cells and that IL‐2\IFN‐γ released by
those Th1 cells immunomodulates keratinocytes infected with HPV
in vivo. In contrast, cimetidine does not enhance
IL‐18 expression during treatment of viral warts, suggesting
that keratinocytes infected with HPV release IL‐18. The successful
use of high doses of cimetidine in treating multiple or
recalcitrant viral warts may encourage the use of this therapy to
treat other HPV‐‐related lesions, especially those high risk HPV
which contain the E5 gene, and the examination of the efficacy of
this treatment.
Acknowledgements. We thank N Kumi for her excellent technical
assistant. This study was supported in part by the Ministry of
Education, Science, Sports and Culture, Japan (Grant‐in‐Aid for
Encouragement of Young Scientists 12770465).
References
1 . P Bardhan KD. Six years of continuous cimetidine
treatment in peptic ulcer disease: efficacy and safety. Anglo‐Irish
Cimetidine Long‐Term Study Group. Aliment Pharmacol Ther
1988; 2: 395‐405.
2 . Rhodes LE, Tingle MD, Park BK et al. Cimetidine
improves the therapeutic\toxic ratio of dapsone in patients on
chronic dapsone therapy. Br J Dermatol 1995; 132:
257‐62.
3 . Raju GS, Bardhan KD, Royston C, Beresford J. The
management of refractory gastric ulcer using H2‐receptor
antagonists. Aliment Pharmacol Ther 1996; 10: 387‐96.
4 . Orlow SJ, Paller A. Cimetidine therapy for multiple
warts in children. J Am Acad Dermatol 1993; 28: 794‐6.
5 . Glass AT, Solomon BA. Cimetidine therapy for
recalcitrant warts in adults. Arch Dermatol 1996; 132:
680‐2.
6 . Gooptu C, Higgins CR, James MP. Treatment of viral
warts with cimetidine: an open‐label study. Clinical
Dermatol 2000
; 25: 183‐5.
7 . Parsad D, Pandhi R, Juneja A, Negi KS. Cimetidine and
levamisole versus cimetidine alone for recalcitrant warts in
children. Pediatric Dermatol 2001; 18: 349‐52.
8 . Lee SH, Rose B, Thompson CH, Cossart Y. Plantar warts
of defined aetiology in adults and unresponsiveness to low dose
cimetidine. Australas J Dermatol 2000; 42: 220‐1.
9 . Ahmed IA, Bell GL, Berth‐Jones J, Sterling JC. Common
warts: no correlation human papillomavirus type with duration of
wart or response to cryotherapy. Br J Dermatol 2001; 145
(Suppl.59): 52.
10 . Wu TC. Immunology of human papilloma virus in relation
to cancer. Curr Opin Immunol 1994; 6: 746‐54.
11 . EI‐Sherif AM, Seth R, Tighe PJ et al.
Quantitative analysis of IL‐10 and IFN‐γ mRNA levels in normal
cervix and human papillomavirus type 16 associated cervical
precancer. J Pathol 2001; 195: 179‐85.
12 . Mosmann TR, Sad S. The expanding universe of T‐cell
subsets: Th1, Th2 and more. Immunol Today 1996; 17:
138‐46.
13 . Ishikura H, Fukui H, Takeyama N, Tanaka T. Cimetidine
activates interleukin‐12, which enhances cellular immunity.
Blood 1999; 93: 1782‐3.
14 . Vankayalapati R, Benjamin W, Weis SE, Samten B, Girard
WM, Barnes PF. Production of interleukin‐18 in human
tuberculosis. J Infect Dis 2000; 182: 234‐9.
15 . Hisatomi H, Nagao K, Wakita K, Kohno N. ARHI\NOEY2
inactivation may be important in breast tumor pathogenesis.
Oncology 2001; 183: 130‐3.
16 . Matsukura T, Sugase M: Relationships between
80 human papillomavirus genotypes and different grades of
cervical intraepithelial neoplasia: Association and causality.
Virology 2001; 283: 139‐47.
|
Printable version
Version PDF
