Reviewer's report 
 Inhibition of Gastric H,K-ATPase Activity and Gastric Epithelial Cell IL-8 Secretion by the Title: 
Pyrrolizine Derivative ML 3000 
 1  5 November 2003 Version: Date: 
 Shinji Asano Reviewer: 
Reviewer's report: 
General 
In this paper, the authors reported that a novel NSAID, ML 3000, which causes little damage to the 
gastric mucosa, reversibly inhibited H,K-ATPase activity and IL-8 secretion. The finding is interesting 
and the paper is suitable for publication. However, unfortunately, some experimental data are 
premature at present and should be performed more precisely. Some conclusions and discussion 
are not adequately supported by the data. Therefore, I think that this paper is acceptable for publication after major compulsory revisions. I hope that the authors revise the paper adequately, and the paper is finally accepted for publication. 
----- 
Discretionary Revisions (which the author can choose to ignore) 1) Page 6 
According to the text, the preparation (microsomal preparation) contains 95 % latent K-ATPase 
activity. Is it necessary to add ionophores or detergent (or permeabilize the preparation) to measure 
the maximal activity of H,K-ATPase (140 - 170 micromoles/mg/h) ? ------- Minor Compulsory Revisions (such as missing labels on figures, or the wrong use of a term, which 
the author can be trusted to correct) 
1) Check spelling and writing Page 5 last line: ?modification? Page 8, line 18, and Page 12, line 24, ?sub-unit? ? ?subunit? 
Page 12, line 3, a-subunit ? alpha (?a? in Greek letter)-subunit 
2) Missing label on the Y (vertical)-axis of Fig. 1B 
3) Figure legends of Figure 2 
There is no explanation for Figure 2A. The authors should mention how ML3000 was acidified and neutralized before ATPase assay. ---- 
Major Compulsory Revisions (that the author must respond to before a decision on publication can 
be reached) 1) Figure 3A 
The authors reported arachidonic acid dose-dependently inhibited H,K-ATPase activity with an IC50 
value of 30 microM (page 8, line 21). However, in Fig. 3A, there is no point between 10 and 100 microM (of arachidonic acid). The inhibition percentage is 15 % and 95 % for 10 and 100 microM of arachidonic acid. Therefore, the IC50 value of 30 microM is unreliable. The authors should perform 
this experiment using arachidonic acid at a concentration(s) between 10 and 100 microM (such as 
20, 50 microM). 
2) 
Figure 4A 
The authors reported TEDBC inhibited H,K-ATPase activity with an IC50 of 3.3 microM (page 8, last 
line). However, in Fig. 4A, there is no point between 1 and 10 microM (of TEDBC). The authors 
should perform this experiment using TEDBC at a concentration(s) between 1 and 10 microM. 
3) Page 5, 2nd paragraph 
They should add reference(s) indicating that H. pyroli increases the rate and amplitude of IL-8 
secretion both in vitro and in vivo. 
4) Page 5, line 15 
In this paper, there is no data showing that ML 3000 inhibits H. pyroli-stimulated IL-8 secretion. 
5) Page 8, line 2 
The IC50 values for ML 3000 and omeprazole were 16.6 uM and 1 uM, respectively. The former is 16-fold larger than the latter. I do not think that these two drugs display comparable IC50 values. 
6) Page 12 lines 10-12 
As the authors mentioned in the text, inhibitory effect of omeprazole depends on pre-treatment 
conditions; pH, incubation time and intactness of vesicle preparation. Therefore, the authors should 
explain the conditions of omeprazole pre-treatment in figure legend (Fig. 1B) as well as in the 
Method section. 
7) Page 12 line 21 
I cannot understand how the authors explain the difference in the effect of PGE2 on H,K-ATPase 
from the difference of specific activity between the author?s group and Im?s group. 
8) I strongly recommend the authors to mention the in vitro inhibitory effect of reversible inhibitors of 
H,K-ATPase such as SCH 28080 as a control because ML 3000 is a reversible inhibitor for gastric 
H,K-ATPase. 
 Unable to decide on acceptance or rejection until the authors have responded to the What next?: 
major compulsory revisions 
 An article whose findings are important to those with closely related research Level of interest: 
interests 
 Acceptable Quality of written English: 
 No Statistical review: 
Declaration of competing interests: 
'None'
Reviewer's report 
 Inhibition of Gastric H,K-ATPase Activity and Gastric Epithelial Cell IL-8 Secretion by the Title: 
Pyrrolizine Derivative ML 3000 
 1  5 November 2003 Version: Date: 
 Shinji Asano Reviewer: 
Reviewer's report: 
General 
In this paper, the authors reported that a novel NSAID, ML 3000, which causes little damage to the 
gastric mucosa, reversibly inhibited H,K-ATPase activity and IL-8 secretion. The finding is interesting 
and the paper is suitable for publication. However, unfortunately, some experimental data are 
premature at present and should be performed more precisely. Some conclusions and discussion 
are not adequately supported by the data. Therefore, I think that this paper is acceptable for publication after major compulsory revisions. I hope that the authors revise the paper adequately, and the paper is finally accepted for publication. 
----- 
Discretionary Revisions (which the author can choose to ignore) 1) Page 6 
According to the text, the preparation (microsomal preparation) contains 95 % latent K-ATPase 
activity. Is it necessary to add ionophores or detergent (or permeabilize the preparation) to measure 
the maximal activity of H,K-ATPase (140 - 170 micromoles/mg/h) ? ------- Minor Compulsory Revisions (such as missing labels on figures, or the wrong use of a term, which 
the author can be trusted to correct) 
1) Check spelling and writing Page 5 last line: ?modification? Page 8, line 18, and Page 12, line 24, ?sub-unit? ? ?subunit? 
Page 12, line 3, a-subunit ? alpha (?a? in Greek letter)-subunit 
2) Missing label on the Y (vertical)-axis of Fig. 1B 
3) Figure legends of Figure 2 
There is no explanation for Figure 2A. The authors should mention how ML3000 was acidified and neutralized before ATPase assay. ---- 
Major Compulsory Revisions (that the author must respond to before a decision on publication can 
be reached) 1) Figure 3A 
The authors reported arachidonic acid dose-dependently inhibited H,K-ATPase activity with an IC50 
value of 30 microM (page 8, line 21). However, in Fig. 3A, there is no point between 10 and 100 microM (of arachidonic acid). The inhibition percentage is 15 % and 95 % for 10 and 100 microM of arachidonic acid. Therefore, the IC50 value of 30 microM is unreliable. The authors should perform 
this experiment using arachidonic acid at a concentration(s) between 10 and 100 microM (such as 
20, 50 microM). 
2) 
Figure 4A 
The authors reported TEDBC inhibited H,K-ATPase activity with an IC50 of 3.3 microM (page 8, last 
line). However, in Fig. 4A, there is no point between 1 and 10 microM (of TEDBC). The authors 
should perform this experiment using TEDBC at a concentration(s) between 1 and 10 microM. 
3) Page 5, 2nd paragraph 
They should add reference(s) indicating that H. pyroli increases the rate and amplitude of IL-8 
secretion both in vitro and in vivo. 
4) Page 5, line 15 
In this paper, there is no data showing that ML 3000 inhibits H. pyroli-stimulated IL-8 secretion. 
5) Page 8, line 2 
The IC50 values for ML 3000 and omeprazole were 16.6 uM and 1 uM, respectively. The former is 16-fold larger than the latter. I do not think that these two drugs display comparable IC50 values. 
6) Page 12 lines 10-12 
As the authors mentioned in the text, inhibitory effect of omeprazole depends on pre-treatment 
conditions; pH, incubation time and intactness of vesicle preparation. Therefore, the authors should 
explain the conditions of omeprazole pre-treatment in figure legend (Fig. 1B) as well as in the 
Method section. 
7) Page 12 line 21 
I cannot understand how the authors explain the difference in the effect of PGE2 on H,K-ATPase 
from the difference of specific activity between the author?s group and Im?s group. 
8) I strongly recommend the authors to mention the in vitro inhibitory effect of reversible inhibitors of 
H,K-ATPase such as SCH 28080 as a control because ML 3000 is a reversible inhibitor for gastric 
H,K-ATPase. 
 Unable to decide on acceptance or rejection until the authors have responded to the What next?: 
major compulsory revisions 
 An article whose findings are important to those with closely related research Level of interest: 
interests 
 Acceptable Quality of written English: 
 No Statistical review: 
Declaration of competing interests: 
'None'
Please review this paper.
user
Reviewer's report 
 Inhibition of Gastric H,K-ATPase Activity and Gastric Epithelial Cell IL-8 Secretion by the Title: 
Pyrrolizine Derivative ML 3000 
 1  5 November 2003 Version: Date: 
 Shinji Asano Reviewer: 
Reviewer's report: 
General 
In this paper, the authors reported that a novel NSAID, ML 3000, which causes little damage to the 
gastric mucosa, reversibly inhibited H,K-ATPase activity and IL-8 secretion. The finding is interesting 
and the paper is suitable for publication. However, unfortunately, some experimental data are 
premature at present and should be performed more precisely. Some conclusions and discussion 
are not adequately supported by the data. Therefore, I think that this paper is acceptable for publication after major compulsory revisions. I hope that the authors revise the paper adequately, and the paper is finally accepted for publication. 
----- 
Discretionary Revisions (which the author can choose to ignore) 1) Page 6 
According to the text, the preparation (microsomal preparation) contains 95 % latent K-ATPase 
activity. Is it necessary to add ionophores or detergent (or permeabilize the preparation) to measure 
the maximal activity of H,K-ATPase (140 - 170 micromoles/mg/h) ? ------- Minor Compulsory Revisions (such as missing labels on figures, or the wrong use of a term, which 
the author can be trusted to correct) 
1) Check spelling and writing Page 5 last line: ?modification? Page 8, line 18, and Page 12, line 24, ?sub-unit? ? ?subunit? 
Page 12, line 3, a-subunit ? alpha (?a? in Greek letter)-subunit 
2) Missing label on the Y (vertical)-axis of Fig. 1B 
3) Figure legends of Figure 2 
There is no explanation for Figure 2A. The authors should mention how ML3000 was acidified and neutralized before ATPase assay. ---- 
Major Compulsory Revisions (that the author must respond to before a decision on publication can 
be reached) 1) Figure 3A 
The authors reported arachidonic acid dose-dependently inhibited H,K-ATPase activity with an IC50 
value of 30 microM (page 8, line 21). However, in Fig. 3A, there is no point between 10 and 100 microM (of arachidonic acid). The inhibition percentage is 15 % and 95 % for 10 and 100 microM of arachidonic acid. Therefore, the IC50 value of 30 microM is unreliable. The authors should perform 
this experiment using arachidonic acid at a concentration(s) between 10 and 100 microM (such as 
20, 50 microM). 
2) 
Figure 4A 
The authors reported TEDBC inhibited H,K-ATPase activity with an IC50 of 3.3 microM (page 8, last 
line). However, in Fig. 4A, there is no point between 1 and 10 microM (of TEDBC). The authors 
should perform this experiment using TEDBC at a concentration(s) between 1 and 10 microM. 
3) Page 5, 2nd paragraph 
They should add reference(s) indicating that H. pyroli increases the rate and amplitude of IL-8 
secretion both in vitro and in vivo. 
4) Page 5, line 15 
In this paper, there is no data showing that ML 3000 inhibits H. pyroli-stimulated IL-8 secretion. 
5) Page 8, line 2 
The IC50 values for ML 3000 and omeprazole were 16.6 uM and 1 uM, respectively. The former is 16-fold larger than the latter. I do not think that these two drugs display comparable IC50 values. 
6) Page 12 lines 10-12 
As the authors mentioned in the text, inhibitory effect of omeprazole depends on pre-treatment 
conditions; pH, incubation time and intactness of vesicle preparation. Therefore, the authors should 
explain the conditions of omeprazole pre-treatment in figure legend (Fig. 1B) as well as in the 
Method section. 
7) Page 12 line 21 
I cannot understand how the authors explain the difference in the effect of PGE2 on H,K-ATPase 
from the difference of specific activity between the author?s group and Im?s group. 
8) I strongly recommend the authors to mention the in vitro inhibitory effect of reversible inhibitors of 
H,K-ATPase such as SCH 28080 as a control because ML 3000 is a reversible inhibitor for gastric 
H,K-ATPase. 
 Unable to decide on acceptance or rejection until the authors have responded to the What next?: 
major compulsory revisions 
 An article whose findings are important to those with closely related research Level of interest: 
interests 
 Acceptable Quality of written English: 
 No Statistical review: 
Declaration of competing interests: 
'None'
assistant