Reviewer's report 
 Inhibition of Gastric H,K-ATPase Activity and Gastric Epithelial Cell IL-8 Secretion by the Title: 
Pyrrolizine Derivative ML 3000 
 2  9 December 2003 Version: Date: 
 edd rabon Reviewer: 
Reviewer's report: 
General: 
This manuscript presents the pharmacological response of the gastric H,K-ATPase to several classes of compounds involved in gastric acid secretion and inflammation. The manuscript provides a measure of the specificity (or lack of) of the pharmacology of the 
H,K-ATPase to several classes of reagents including NSAIDs, lipoxygenase inhibitors, inflammatory 
cytokines and leukotrienes. This data is not mechanistic in scope and is limited to a catalogue of the 
pharmacological response of the H,K-ATPase. 
The central observation is that the pyrrolizine derivative, ML 3000, exhibits multiple actions including 
that of a reversible inhibitor of the gastric H,K-ATPase and is an inhibitor of gastric gland acid 
secretion. It also functions as an inhibitor of IL-8 secretion in gastric AGS cells. The authors conclude that these actions are consistent with the gastric sparing properties of the drug which have 
been previously noted. 
Discretionary Revisions: 
In figures 1 and 2 ATPase values on the Y axis are represented as a % of baseline activity, but in the 
remainder of the figures are represented as a % of control. Why not be consistent throughout since 
the measurements are presumably all compared to the same SCH28080 sensitive component of the H,K-ATPase ? 
In the 1st paragraph of the Discussion it is unclear what is meant by the statement that ML 3000 
inhibitory activity is independent of pH. Weren’t all measures of activity recorded at the same pH ? 
In general the Discussion could be more focused on the central points that the compound ML 3000 
has multiple effects on the gastric secretory response including its inhibition of the H,K-ATPase and acid secretion in gastric glands as well as IL-8 secretion in AGS cells. The discussion of the mode of action of omeprazole seems unnecessary since this investigation concerns the pharmacology of ML 
3000. 
Minor Compulsory Revisions: 
In Paragraph 1 of the Results section, the authors state that omeprazole and ML3000 have 
comparable efficacy for the H,K-ATPase with IC50s of 16.6 and 1 µM, respectively. To many readers 
these values represent a difference of greater than 1 order of magnitude and would not be 
considered comparable. Perhaps the authors should state the range within the 95% confidence 
interval of the data set to establish that these values actually intersect within that limit. If they do not, 
the text should be revised wherever the identity was claimed. 
There are 2 panels in figure 2 which report experiments testing the effects of acidification of ML3000 
on H,K-ATPase activity. Only figure 2B is described in the legend. If the experiments of these panels 
are designed differently it would be helpful if the legend provided essential detail describing each 
experiment. 
The ATPase inhibition curves presented in figure 3 for arachidonic acid and PGE2 do not appear to 
be calculated from a model. The text describing the inhibition by these inhibitors is very brief and 
would benefit from more information about the experimental design. It would also be informative if 
the method used to calculate the IC50s in this data set was stated. While the standard deviation of 
the IC50 should be stated, the efficacy of these compounds might best be represented by the range of IC50s within the 95% confidence interval of the best fit to the data. 
The presentation of the results concerning the lipooxygenase inhibitors, TEDBC and ZD-2138 is 
awkward because the relationship between lipoxygenase activity and the H,K-ATPase is not clearly defined. Without clarification it seems to me that even if the data set shown in figure 4 does show selective inhibition for the lipooxygenase inhibitors it still does not address their potential role in 
H,K-ATPase activation. Because of the obscure relationship between the pharmacology of the 
lipooxygenase inhibitors and the role of lipooxygenase in the activation of the H,K-ATPase it would be prudent to minimize the speculative prose in this section. 
In panel C of figure 8 it appears that the inhibition of IL-8 secretion by ML 3000 does not conform to 
a well behaved dose response. Is it possible that neither the baseline or IL-1ß stimulated IL-8 response are sensitive to ZD-2136 ? There also appears to be a small component of a ZD-2136 sensitive response in panel D of the same figure. If the data are really different it should be stated 
more carefully in order to clarify the difference in the modeled components. 
Discretionary Revisions: 
In figures 1 and 2 ATPase values on the Y axis are represented as a % of baseline activity, but in the 
remainder of the figures are represented as a % of control. Why not be consistent throughout since 
the measurements are presumably all compared to the same SCH28080 sensitive component of the 
H,K-ATPase ? 
In the 1st paragraph of the Discussion it is unclear what is meant by the statement that ML 3000 
inhibitory activity is independent of pH. Weren’t all measures of activity recorded at the same pH ? 
In general the Discussion could be more focused on the central points that the compound ML 3000 
has multiple effects on the gastric secretory response including its inhibition of the H,K-ATPase and acid secretion in gastric glands as well as IL-8 secretion in AGS cells. The discussion of the mode of 
action of omeprazole seems unnecessary since this investigation concerns the pharmacology of ML 
3000. 
 Accept after minor essential revisions What next?: 
 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 
 2  9 December 2003 Version: Date: 
 edd rabon Reviewer: 
Reviewer's report: 
General: 
This manuscript presents the pharmacological response of the gastric H,K-ATPase to several classes of compounds involved in gastric acid secretion and inflammation. The manuscript provides a measure of the specificity (or lack of) of the pharmacology of the 
H,K-ATPase to several classes of reagents including NSAIDs, lipoxygenase inhibitors, inflammatory 
cytokines and leukotrienes. This data is not mechanistic in scope and is limited to a catalogue of the 
pharmacological response of the H,K-ATPase. 
The central observation is that the pyrrolizine derivative, ML 3000, exhibits multiple actions including 
that of a reversible inhibitor of the gastric H,K-ATPase and is an inhibitor of gastric gland acid 
secretion. It also functions as an inhibitor of IL-8 secretion in gastric AGS cells. The authors conclude that these actions are consistent with the gastric sparing properties of the drug which have 
been previously noted. 
Discretionary Revisions: 
In figures 1 and 2 ATPase values on the Y axis are represented as a % of baseline activity, but in the 
remainder of the figures are represented as a % of control. Why not be consistent throughout since 
the measurements are presumably all compared to the same SCH28080 sensitive component of the H,K-ATPase ? 
In the 1st paragraph of the Discussion it is unclear what is meant by the statement that ML 3000 
inhibitory activity is independent of pH. Weren’t all measures of activity recorded at the same pH ? 
In general the Discussion could be more focused on the central points that the compound ML 3000 
has multiple effects on the gastric secretory response including its inhibition of the H,K-ATPase and acid secretion in gastric glands as well as IL-8 secretion in AGS cells. The discussion of the mode of action of omeprazole seems unnecessary since this investigation concerns the pharmacology of ML 
3000. 
Minor Compulsory Revisions: 
In Paragraph 1 of the Results section, the authors state that omeprazole and ML3000 have 
comparable efficacy for the H,K-ATPase with IC50s of 16.6 and 1 µM, respectively. To many readers 
these values represent a difference of greater than 1 order of magnitude and would not be 
considered comparable. Perhaps the authors should state the range within the 95% confidence 
interval of the data set to establish that these values actually intersect within that limit. If they do not, 
the text should be revised wherever the identity was claimed. 
There are 2 panels in figure 2 which report experiments testing the effects of acidification of ML3000 
on H,K-ATPase activity. Only figure 2B is described in the legend. If the experiments of these panels 
are designed differently it would be helpful if the legend provided essential detail describing each 
experiment. 
The ATPase inhibition curves presented in figure 3 for arachidonic acid and PGE2 do not appear to 
be calculated from a model. The text describing the inhibition by these inhibitors is very brief and 
would benefit from more information about the experimental design. It would also be informative if 
the method used to calculate the IC50s in this data set was stated. While the standard deviation of 
the IC50 should be stated, the efficacy of these compounds might best be represented by the range of IC50s within the 95% confidence interval of the best fit to the data. 
The presentation of the results concerning the lipooxygenase inhibitors, TEDBC and ZD-2138 is 
awkward because the relationship between lipoxygenase activity and the H,K-ATPase is not clearly defined. Without clarification it seems to me that even if the data set shown in figure 4 does show selective inhibition for the lipooxygenase inhibitors it still does not address their potential role in 
H,K-ATPase activation. Because of the obscure relationship between the pharmacology of the 
lipooxygenase inhibitors and the role of lipooxygenase in the activation of the H,K-ATPase it would be prudent to minimize the speculative prose in this section. 
In panel C of figure 8 it appears that the inhibition of IL-8 secretion by ML 3000 does not conform to 
a well behaved dose response. Is it possible that neither the baseline or IL-1ß stimulated IL-8 response are sensitive to ZD-2136 ? There also appears to be a small component of a ZD-2136 sensitive response in panel D of the same figure. If the data are really different it should be stated 
more carefully in order to clarify the difference in the modeled components. 
Discretionary Revisions: 
In figures 1 and 2 ATPase values on the Y axis are represented as a % of baseline activity, but in the 
remainder of the figures are represented as a % of control. Why not be consistent throughout since 
the measurements are presumably all compared to the same SCH28080 sensitive component of the 
H,K-ATPase ? 
In the 1st paragraph of the Discussion it is unclear what is meant by the statement that ML 3000 
inhibitory activity is independent of pH. Weren’t all measures of activity recorded at the same pH ? 
In general the Discussion could be more focused on the central points that the compound ML 3000 
has multiple effects on the gastric secretory response including its inhibition of the H,K-ATPase and acid secretion in gastric glands as well as IL-8 secretion in AGS cells. The discussion of the mode of 
action of omeprazole seems unnecessary since this investigation concerns the pharmacology of ML 
3000. 
 Accept after minor essential revisions What next?: 
 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 
 2  9 December 2003 Version: Date: 
 edd rabon Reviewer: 
Reviewer's report: 
General: 
This manuscript presents the pharmacological response of the gastric H,K-ATPase to several classes of compounds involved in gastric acid secretion and inflammation. The manuscript provides a measure of the specificity (or lack of) of the pharmacology of the 
H,K-ATPase to several classes of reagents including NSAIDs, lipoxygenase inhibitors, inflammatory 
cytokines and leukotrienes. This data is not mechanistic in scope and is limited to a catalogue of the 
pharmacological response of the H,K-ATPase. 
The central observation is that the pyrrolizine derivative, ML 3000, exhibits multiple actions including 
that of a reversible inhibitor of the gastric H,K-ATPase and is an inhibitor of gastric gland acid 
secretion. It also functions as an inhibitor of IL-8 secretion in gastric AGS cells. The authors conclude that these actions are consistent with the gastric sparing properties of the drug which have 
been previously noted. 
Discretionary Revisions: 
In figures 1 and 2 ATPase values on the Y axis are represented as a % of baseline activity, but in the 
remainder of the figures are represented as a % of control. Why not be consistent throughout since 
the measurements are presumably all compared to the same SCH28080 sensitive component of the H,K-ATPase ? 
In the 1st paragraph of the Discussion it is unclear what is meant by the statement that ML 3000 
inhibitory activity is independent of pH. Weren’t all measures of activity recorded at the same pH ? 
In general the Discussion could be more focused on the central points that the compound ML 3000 
has multiple effects on the gastric secretory response including its inhibition of the H,K-ATPase and acid secretion in gastric glands as well as IL-8 secretion in AGS cells. The discussion of the mode of action of omeprazole seems unnecessary since this investigation concerns the pharmacology of ML 
3000. 
Minor Compulsory Revisions: 
In Paragraph 1 of the Results section, the authors state that omeprazole and ML3000 have 
comparable efficacy for the H,K-ATPase with IC50s of 16.6 and 1 µM, respectively. To many readers 
these values represent a difference of greater than 1 order of magnitude and would not be 
considered comparable. Perhaps the authors should state the range within the 95% confidence 
interval of the data set to establish that these values actually intersect within that limit. If they do not, 
the text should be revised wherever the identity was claimed. 
There are 2 panels in figure 2 which report experiments testing the effects of acidification of ML3000 
on H,K-ATPase activity. Only figure 2B is described in the legend. If the experiments of these panels 
are designed differently it would be helpful if the legend provided essential detail describing each 
experiment. 
The ATPase inhibition curves presented in figure 3 for arachidonic acid and PGE2 do not appear to 
be calculated from a model. The text describing the inhibition by these inhibitors is very brief and 
would benefit from more information about the experimental design. It would also be informative if 
the method used to calculate the IC50s in this data set was stated. While the standard deviation of 
the IC50 should be stated, the efficacy of these compounds might best be represented by the range of IC50s within the 95% confidence interval of the best fit to the data. 
The presentation of the results concerning the lipooxygenase inhibitors, TEDBC and ZD-2138 is 
awkward because the relationship between lipoxygenase activity and the H,K-ATPase is not clearly defined. Without clarification it seems to me that even if the data set shown in figure 4 does show selective inhibition for the lipooxygenase inhibitors it still does not address their potential role in 
H,K-ATPase activation. Because of the obscure relationship between the pharmacology of the 
lipooxygenase inhibitors and the role of lipooxygenase in the activation of the H,K-ATPase it would be prudent to minimize the speculative prose in this section. 
In panel C of figure 8 it appears that the inhibition of IL-8 secretion by ML 3000 does not conform to 
a well behaved dose response. Is it possible that neither the baseline or IL-1ß stimulated IL-8 response are sensitive to ZD-2136 ? There also appears to be a small component of a ZD-2136 sensitive response in panel D of the same figure. If the data are really different it should be stated 
more carefully in order to clarify the difference in the modeled components. 
Discretionary Revisions: 
In figures 1 and 2 ATPase values on the Y axis are represented as a % of baseline activity, but in the 
remainder of the figures are represented as a % of control. Why not be consistent throughout since 
the measurements are presumably all compared to the same SCH28080 sensitive component of the 
H,K-ATPase ? 
In the 1st paragraph of the Discussion it is unclear what is meant by the statement that ML 3000 
inhibitory activity is independent of pH. Weren’t all measures of activity recorded at the same pH ? 
In general the Discussion could be more focused on the central points that the compound ML 3000 
has multiple effects on the gastric secretory response including its inhibition of the H,K-ATPase and acid secretion in gastric glands as well as IL-8 secretion in AGS cells. The discussion of the mode of 
action of omeprazole seems unnecessary since this investigation concerns the pharmacology of ML 
3000. 
 Accept after minor essential revisions What next?: 
 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