The rate of oxidation of dimethylarsinous acid to dimethylarsinic acid is pH dependent: Implications for the analysis and toxicology of arsenic metabolites in urine

Article abstract of DOI:10.1007/s10120-006-0371-x

A ¹H NMR spectroscopic study has shown the rate oxidation of dimethylarsinous acid to dimethylarsinic acid in buffered aqueous solutions to depend upon pH. Dimethylarsinous acid has been reported to be a highly toxic arsenical metabolite and component of the urine of persons exposed to inorganic arsenic, particularly through drinking water. As the pH of human urine can range from 4.5 to 8, the pH dependence of the oxidation rate of dimethylarsinous acid to dimethylarsinic acid has profound implications for the detection and analysis of these compounds in urine samples, and for the relevance of the experimental toxicology of dimethylarsinous acid.

Full text of DOI:10.1007/s10120-006-0371-x

Gastric Cancer (2006) 9: 185–191
DOI 10.1007/s10120-006-0371-x
” 2006 by
International and
Japanese Gastric
Cancer Associations
Original article
A phase II study of combined chemotherapy with methotrexate,
5-fluorouracil, and low-dose cisplatin (MFP) for histologically
diffuse-type advanced and recurrent gastric cancer (KDOG9501)
Norisuke Nakayama, Wasaburo Koizumi, Satoshi Tanabe, Tohru Sasaki, and Katsunori Saigenji
Kitasato Digestive Disease and Oncology Group (KDOG) / Department of Gastroenterology, Kitasato University School of Medicine,
1-15-1 Kitasato, Sagamihara, Kanagawa 228-8555, Japan
Abstract
ations of tumor cell metabolism. Sequential treatment
with methotrexate and 5-fluorouracil, a regimen based
on the principle of biochemical modulation, was pro-
posed by Bertino et al. [1] in 1977 for the management
of colorectal cancer. This regimen was introduced to
Japan in 1980 for the treatment of gastric cancer [2] and
has been widely used and confirmed to be beneficial.
The combination of cisplatin and 5-fluorouracil is, simi-
larly, based on the concept of biochemical modulation.
Cisplatin not only acts as an effector, but also augments
the antitumor activity of 5-fluorouracil [3,4].
Several clinical trials have evaluated the response of
advanced gastric cancer to sequential methotrexate and
5-fluorouracil. Dickinson et al. [5] reported a response
rate of 22% (5/23) and a median survival of 11 months.
Phase II studies done by Blijham et al. [6] reported
a response rate of 18% (4/22) and a median survival
of 4 months. A previous Japanese phase II study
of sequential methotrexate/5-fluorouracil therapy
against advanced gastric cancer demonstrated that low-
and intermediate-dose methotrexate/5-fluorouracil
regimens achieved response rates of 23% (13 partial
responses [PRs]/56 patients) and 41% (15PRs/37 pa-
tients), respectively [7]. Sasaki et al. [8] reported that an
intermediate-dose methotrexate/5-fluorouracil regimen
achieved a response rate of 17.9% (10PRs/56 patients),
and Konishi et al. [2] reported a response rate of 23.7%
(9/38). These findings suggest that sequential metho-
trexate/5-fluorouracil therapy may be effective in ad-
vanced gastric cancer.
Background. Histologically diffuse-type gastric cancer is well
known to have a poor prognosis and is often complicated with
abdominal and pleural effusions. We evaluated the efficacy of
a low dose of cisplatin combined with methotrexate and 5-
fluorouracil (MFP therapy) in diffuse-type advanced gastric
cancer.
Methods. The study group comprised 47 patients. Patients
with pleural effusion or ascites were actively enrolled. Meth-
otrexate (30mg/m2 per day) plus 5-fluorouracil (600mg/m2 per
day) was administered on days 1 and 8. Cisplatin (6mg/m2 per
day) was given daily for 14 days after which there was 14 day’s
rest. Calcium leucovorin (15mg) was administered after
methotrexate.
Results. The overall response rate was 38.3%. Ascites disap-
peared or decreased in 15 of 23 patients (disappeared in 9).
Pleural effusion disappeared or decreased in 6 of 8 patients
(disappeared in 4). Of the 21 patients with poor oral intake
at study entry, 15 (71.4%) were able to orally ingest food.
Hematologic toxicity of grade 3 or higher comprised leuko-
penia in 10 patients (21.3%) and neutropenia in 9 (19.1%).
Nonhematologic toxicity included anorexia and nausea/vom-
iting, in 1 patient each (2.1%). The median survival time was
211 days.
Conclusion. MFP therapy is useful for the management of
diffuse-type inoperable and recurrent gastric cancer, even in
patients with conditions such as pleural effusion, ascites, or
lymphangitis carcinomatosa who have a poor prognosis or
cannot eat solid food.
Key words Histologically diffuse-type gastric cancer · Combi-
nation chemotherapy
Combination chemotherapy with cisplatin and 5-
fluorouracil has also been evaluated in patients with
advanced gastric cancer. A phase II trial done by
Lacave et al. [9] reported a response rate of 41% (22/
53), with a median survival of 10.6 months. A phase
III study comparing 5-fluorouracil plus cisplatin (FP)
with 5-fluorouracil alone and a combination of 5-
fluorouracil, doxorubicin, and mitomycin C, done by
Kim et al. [10], obtained a response rate of 51% for 5-
Introduction
Biochemical modulation refers to the enhancement of
antitumor activity by drug-induced biochemical alter-
Offprint requests to: N. Nakayama
Received: August 5, 2005 / Accepted: February 21, 2006

186
N. Nakayama et al.: MTX, 5-FU, and CDDP for gastric cancer
fluorouracil plus cisplatin, with a median survival of 8
months. A phase II study done by Rouger et al. [11]
reported a response rate of 43% (36/83) and a median
survival of 9 months. Ohtsu et al. [12] performed a
phase II study and reported a response rate of 43% (17/
40) and a median survival of 7 months.
Treatment methods
An intravenous injection of methotrexate (30mg/m2)
was followed 3h later by an intravenous infusion of
5-fluorouracil (600mg/m2), given over the course of
15min. Twenty-four h after the treatment with metho-
trexate, calcium leucovorin (15mg) was administered,
at 6-h intervals, for a total of six times. To prevent renal
dysfunction due to methotrexate, patients received
an intravenous infusion of 7% sodium bicarbonate
(120ml), given at 6-h intervals, starting 3h before the
injection of methotrexate. Immediately after the treat-
ment with methotrexate, acetazolamide (250mg) was
given by intravenous injection, to alkalinize the urine.
This treatment was given on days 1 and 8. Cisplatin
(6mg/m2 per day, 1-h infusion) was administered daily
on days 1 to 14. No chemotherapy was given on days 15
to 28. This cycle was repeated.
Histologically diffuse-type gastric cancer is well
known to have a poor prognosis and is often compli-
cated with abdominal and pleural effusions. Few studies
support the usefulness of chemotherapy for the treat-
ment of diffuse-type gastric cancer. But Konishi et al.
[2] reported that histologically diffuse-type gastric
cancer showed a better response than the intestinal
type when treated with sequential methotrexate and
5-fluorouracil. Moreover, in-vitro experiments have
demonstrated that diffuse-type gastric cancer is sensi-
tive to cisplatin, suggesting antitumor activity in this
type [13]. These findings provide evidence that sequen-
tial methotrexate and 5-fluorouracil combined with
cisplatin may be effective clinically against histologi-
cally diffuse-type gastric cancer. We assessed the re-
sponse of inoperable and recurrent histologically
diffuse-type gastric cancer to sequential methotrexate
and 5-fluorouracil given concurrently with cisplatin
(MFP), serving as an effector. The primary endpoint
planned for this study was the tumor response rate.
Secondary endpoints were overall survival, response
rate in patients with asctes and pleural effusion, and
response rate in patients with poor oral intake.
Evaluation of response
The tumor response was evaluated based on changes
in the size of measurable lesions and assessment of
evaluable lesions. Measurable lesions and assessment of
evaluable lesions were evaluated according to the Japa-
nese Criteria for Evaluating the Efficacy of Chemo-
therapy and Radiation Therapy in the Treatment of
Gastric Cancer [14].
A complete response (CR) was defined as the disap-
pearance of all evidence of cancer for at least 4 weeks,
and a PR was defined as less than complete, but more
than 50% reduction of tumor volume for at least 4
weeks, without any evidence of new lesions or progres-
sion. No change (NC) was defined as less than a 50%
reduction or less than a 25% increase without any new
lesions. Progressive disease (PD) was defined as a more
than 25% increase in a solitary lesion or the appearance
of new lesions. Tumor measurements were performed
every 4 weeks, using CT scans, plain chest X-ray films,
upper gastrointestinal endoscopy, and ultrasonography.
Primary lesions were classified into the following three
categories based on X-ray and endoscopic findings:
measurable, not measurable but evaluable, and diffuse
infiltration. Ascites and pleural effusion were evaluated
by CT, as disappeared (no evidence of ascites or effu-
sion for 4 consecutive weeks), decreased (a decrease for
4 consecutive weeks), or no response. The responses
of effusions and lymphangitis were not counted in the
overall response. Adverse events were evaluated ac-
cording to World health Organization (WHO) criteria
[15].
Patients and methods
Patient eligibility
The study group comprised patients with inoperable or
recurrent gastric cancer treated at our hospital between
1995 and 2001. Eligibility criteria were as follows: (1) all
patients had a histopathological diagnosis of diffuse-
type adenocarcinoma, as confirmed by endoscopic bi-
opsy; (2) unresectable or recurrent disease; (3) no other
cancers were identified; (4) the patient was not more
than 85 years of age at entry; (5) performance status
was 0 to 2 according to the World Health Organization
(WHO) criteria; (6) adequate organ function; (7) there
was no serious concurrent disease; and (8) informed
consent was obtained from all patients prior to entry.
Patients with pleural effusion or ascites (including mas-
sive ascites) and lymphangitis carcinomatosa were also
actively enrolled. Malignant ascites and pleural effusion
were confirmed by computed tomography (CT) and
cytology. Effusions were not drained prior to or during
the course of the study.
Statistical considerations
The required sample size for this study was calculated
based on a target response rate of 40% and a minimum

N. Nakayama et al.: MTX, 5-FU, and CDDP for gastric cancer
187
Table 1. Patient characteristics (n = 47)
response rate of 20%, with an α error of 0.1 and a β
error of 0.1. But, during the study, because the response
rate was high, we enlarged the sample size. Survival was
defined as the interval from the day of starting the MFP
therapy to death. Survival was estimated according to
the Kaplan-Meier method [16].
n
Percentage
Age (years)
Median (range)
62 (28–84)
Sex
Male
Female
29
18
61.7
38.3
Performance status
0
10
13
24
21.3
27.7
51.0
Results
1
2
Patient characteristics
Histology
Diffuse
Intestinal
47
0
100
0
Forty-seven patients (29 men and 18 women) were
enrolled in this study. Histologically, all tumors were
diffuse type. Eight patients had received prior chemo-
therapy (5-fluorouracil alone, n = 1; 5-fluorouracil
and cisplatin, n = 2; cisplatin, mitomycin C, and
doxifluridine, n = 1; 5-fluorouracil, mitomycin C, and
cisplatin, n = 1; capecitabine, n = 1; tegafur-uracil [UFT],
n = 1; and UFT and cisplatin, n = 1). Median age was 62
years (range, 28–84 years). Performance status ranged
from 0 to 2 (0, n = 10; 1, n = 13; 2, n = 24). Twenty-one
patients could not orally ingest food. Ascites was
present in 23 patients (48.9%) and pleural infusion in 8
(17.0%). Lymphangitis carcinomatosa was present in 2
patients (4.3%). The demographic characteristics of all
patients are shown in Table 1.
Prior chemotherapy
None
5-FU
39
1
82.9
2.1
4.3
2.1
2.1
2.1
2.1
2.1
FP (5-FU, CDDP)
CDDP, MMC, 5′DFUR
FMP (5-FU, MMC, CDDP)
Capecitabine
UFT
2
1
1
1
1
UFT + CDDP
Surgical resection
Yes
1
4
8.5
91.5
No
43
Oral intake
Good
26
21
55.3
44.7
Poor
Metastasis
Liver
Lymph nodes
Ovary
Lung
Ascites
Pleural effusion
Lymphangitis carcinomatosa
No. of treatment cycles
Median (range)
9
39
4
19.1
82.9
8.5
Response to chemotherapy and survival
2
4.3
23
8
48.9
17.0
4.3
In total, 141 administrations of MFP therapy were per-
formed in 47 patients. The median number of adminis-
trations was 3 (range, 1–7). Among the 47 patients with
evaluable lesions, the overall response rate was 38.3%
(18/47; 95% confidence interval [CI], 24.4%–52.2%).
Ascites disappeared or decreased in 15 of 23 patients
(65.2%; 95% CI, 45.8–84.7%) including complete
disappearance in 9. Pleural effusion disappeared or de-
creased in 6 of 8 patients (75.0%) including complete
disappearance in 4 (Table 2). Oral ingestion of food
became possible in 15 of the 21 patients (71.4%) who
were previously unable to orally ingest food. The sur-
vival curve is shown in Fig. 1. Median survival time was
211 days (95% CI, 145–277 days) and the 1- and 2-year
survival rates were 28.9 and 4.4%, respectively. The
overall response rate and median survival time in the
23 patients with ascites were 43.5% (10/23; 95% CI,
23.2%–63.7%) and 211 days (95% CI, 114–308 days),
respectively.
2
3 (1–7)
Performance status according to the grading system of the World
Health Organization
leukopenia in 10 patients (21.3%) and neutropenia in
9 (19.1%). Nonhematologic toxicity of grade 3 or higher
was shown for anorexia and nausea/vomiting in 1 pa-
tient each (2.1%). Hematologic toxicity was controlled
through the use of granulocyte colony–stimulating
factor (G-CSF), and no major problems occurred.
There were no treatment-related deaths.
Discussion
Methotrexate is a folic acid antagonist that blocks
dihydrofolate reductase (DHFR), an enzyme cat-
alyzing the conversion of dihydrofolate (FH2) to
tetrahydrofolate (FH4) [17]. Nucleic acid synthesis is,
consequently, blocked. However, monotherapy with
methotrexate is of limited effectiveness for gastric can-
Adverse events
The major treatment-induced hematologic and non-
hematologic adverse events are shown in Table 3.
Hematologic toxicity of grade 3 or higher comprised

188
N. Nakayama et al.: MTX, 5-FU, and CDDP for gastric cancer
Table 2. Responses
CR
PR
NC
PD
Response rate
Overall
Primary
Liver
Lymph nodes
Lung
Ovary
0
0
1
3
0
0
18
17
4
14
1
2
20
26
2
16
0
2
9
4
2
6
1
0
38.3% (18/47)
36.2% (17/47)
55.6% (5/9)
43.6% (17/39)
50.0% (1/2)
50.0% (2/4)
Disappeared
Decreased
No response
Ascites
Pleural effusion
Lymphangitis
9
4
6
2
1
8
2
1
65.2% (15/23)
75.0% (6/8)
50.0% (1/2)
CR, complete response; PR, partial response; NC, no change; PD, progressive disease
Table 3. Toxicity
WHO grade
1
2
3
4
3 or 4
Leukopenia
Neutropenia
Anemia
Thrombocytopenia
AST
ALT
4
2
5
1
2
2
2
1
8
4
3
3
1
1
10
9
7
6
3
3
21.3%
19.1%
3
9
1
1
Alkaline phosphatase
Creatinine
1
Nausea/Vomiting
Anorexia
2
2
2
1
1
2.1%
2.1%
Diarrhea
Stomatitis
Alopecia
Peripheral neuropathy
WHO, World Health Organization; AST, aspartate aminotransferase;
ALT, alanine aminotransferase
Fig. 1. Overall cumulative survival rate after treatment. The
median survival time was 211 days (95% confidence interval
[CI]; 145–277 days)
cer [18]. The mechanism of sequential methotrexate
and 5-fluorouracil, a regimen based on the principle of
biochemical modulation, has been described by Bertino
et al. [1] and Fernandes and Bertino [19]. Pretreat-
ment with the folic acid antagonist methotrexate in-
creases the production of dihydrofolate polyglutamate,
which strongly binds to 5-fluoro-2′-deoxyuridine
5′-monophosphate (5-FdUMP) and thymidylate
synthetase (TS), forming a terminal complex that
inactivates TS, thereby attenuating DNA synthesis.
Cadman et al. [20,21] have reported that methotrexate
triggers the blockade of purine synthesis, resulting in
the intracellular accumulation of phosphoribosyl pyro-
phosphate (PRPP). Because PRPP serves as a substrate
for the conversion of 5-fluorouracil into 5-fluorouridine-
monophosphate (5-FUMP), a reaction catalyzed by
orotate phosphoribosyl transferase (OPRT), metho-
trexate pretreatment promotes the conversion of
FUMP to 5-fluorouridine 5′-triphosphate (FUTP).
Large amounts of 5-FUTP are then incorporated into
RNA, enhancing antitumor activity.
Sequential methotrexate and 5-fluorouracil has been
shown to be effective against histologically diffuse-type
gastric cancer [2,22,23]. TS activity is high in diffuse-
type gastric cancer and low in the highly differentiated
type, whereas thymidine kinase (TK) activity is low in
diffuse-type gastric cancer and high in the highly differ-
entiated type [23]. Studies examining the relation be-
tween TS expression and the response to leucovorin
plus 5-fluorouracil have reported that high expression is
associated with a low response rate [24]. DNA synthesis

N. Nakayama et al.: MTX, 5-FU, and CDDP for gastric cancer
189
is suppressed in cells with high TS activity and is dis-
turbed in cells with low TS activity [25]. Available evi-
dence thus suggests that sequential methotrexate and
5-fluorouracil acts by functionally impairing RNA, and
is thereby effective against diffuse cancer.
ferentiated carcinoma. Konishi et al. [2] reported that
histologically diffuse-type gastric cancer showed a bet-
ter response than the intestinal type to sequential meth-
otrexate/5-fluorouracil treatment. They reported that
this sequential therapy was effective even in patients
with ascites, with a response rate in patients with ascites
of 57% (8/14) [35].
We concomitantly administered a low dose of
cisplatin with sequential methotrexate and 5-
fluorouracil. Cisplatin inhibits DNA synthesis by bind-
ing, in a bridge-like fashion, to bases between the double
strands of DNA, killing tumor cells. The response rate of
gastric cancer to cisplatin monotherapy ranges from
18% to 33% [26–30]. In-vitro experimental studies have
shown that diffuse-type cancer is sensitive to cisplatin,
suggesting antitumor effectiveness in this type [13].
Clinically, Lacave et al. [31] reported that remissions
achieved by cisplatin monotherapy in patients with gas-
tric cancer were obtained not only in the intestinal type
but also in the diffuse type. Hainsworth et al. [32] recom-
mended cisplatin-based combination chemotherapy for
the treatment of poorly differentiated carcinoma and
adenocarcinoma of an unknown primary site. The re-
sults of these studies suggest that cisplatin is effective for
diffuse-type gastric cancer. However, large doses of
cisplatin can cause severe toxicity, including gastrointes-
tinal symptoms and renal dysfunction. Moreover, renal
toxicity requires the administration of large volumes of
infusion solutions. Drewinko et al. [33] showed that the
exposure of lymphoma cells to 50µg/ml of cisplatin for
1h or to 5µg/ml of cisplatin for 8h had similar cytocidal
activity in vitro. Belliveau et al. [34] reported that con-
tinuous infusion of cisplatin for 5 days had a 40 times
lower maximal plasma concentration (Cmax) and an
approximately two times greater area under the plasma-
concentration-time curve than did an equivalent dose
given as a single 30-min infusion. These reports suggest
that the administration of cisplatin in small divided
doses reduces the risk of toxicity such as renal dysfunc-
tion, by lowering the peak plasma concentration of
cisplatin, without compromising antitumor effective-
ness. In Japan, the dose of cisplatin used in monotherapy
for gastric cancer is 70–90mg/m2 per month. One-day
administration of the same dosage requires a large
amount of infusion to prevent cisplatin-induced renal
injury. This is, however, inappropriate for patients who
have peritoneal dissemination and retention of ascites,
because such patients may have renal impairment or
poor performance status. We therefore fixed the dose of
cisplatin at 6mg/m2 per day for 14 days (84mg/m2 per
month). Because intermediate-dose methotrexate has
greater toxicity than low-dose methotrexate, and be-
cause MFP is a triple chemotherapy, we adopted low-
dose methotrexate and cisplatin, because we wanted to
reduce the risk of toxicity.
We therefore combined low-dose cisplatin with se-
quential methotrexate and 5-fluorouracil in patients
with histologically diffuse-type gastric cancer, which
is often associated with ascites and pleural effusion,
conditions in which large infusion volumes are
contraindicated.
Our overall response rate with MFP was 38.3% (18/
47). The overall response rate in the 23 patients with
inoperable or recurrent gastric cancer who had ascites
was 43.5% (10/23), with 15 patients showing a distinct
decrease in ascitic fluid (including 9 with complete dis-
appearance of ascites). Yamao et al. [36] reported that
sequential methotrexate and 5-fluorouracil distinctly
decreased ascites in 13 of 37 patients (35.1%, including
3 with complete disappearance of ascites) who had in-
operable or recurrent gastric cancer with malignant
ascites. Tahara et al. [37] demonstrated a clear-
cut decrease in ascites in 13 of 26 patients with gastric
cancer (50.0%; 5 with complete disappearance of
ascites) who received sequential methotrexate and 5-
fluorouracil. Pleural effusion distinctly decreased in 6 of
8 patients (75.0%), including 4 with complete resolu-
tion. These results indicate that MFP is effective in pa-
tients who have ascites and pleural effusion associated
with advanced gastric cancer.
Konishi et al. [38] gave sequential methotrexate and
5-fluorouracil to patients who had gastric cancer with
ascites; they reported that the serum methotrexate con-
centration was maintained at higher levels in the pres-
ence of ascites than in the absence of ascites. The
concentration of methotrexate in ascitic fluid was higher
than that in serum from 6 to 72h after treatment. Simi-
lar methotrexate levels are expected in patients given
MFP. High penetration of methotrexate to ascitic fluid
results in delayed excretion, but is considered respon-
sible for the decreases in ascites and pleural effusion,
with the attendant improvement in symptoms. We
therefore recommend MFP for patients with ascites or
pleural effusion.
Of our 21 patients who were unable to eat solid food
at study entry, 15 (71.4%) were able to orally ingest
food after MFP. This regimen can thus be used in
patients who cannot orally ingest food or anticancer
agents, and our results suggest that the activities of daily
living are improved substantially. To reduce the risk of
toxicity, the patients’ urine was alkalinized by treatment
with sodium bicarbonate or acetazolamide to promote
methotrexate excretion. On the days that patients
Peritoneal dissemination is more frequent in patients
with undifferentiated carcinoma than in those with dif-

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with ascites was also 211 days (95% CI, 114–308 days).
This result was compatible with the results in studies by
Yamao et al. [36] and Tahara et al. [37], who reported
the median survival times of patients with ascites as 155
days and 244 days after MF tharapy, respectively.
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