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  20 November 2003 Version: Date: 
 Ian Beales Reviewer: 
Reviewer's report: 
General 
The authors have studied a novel anti-inflammatory compound (ML 3000) and demonstrated that the 
lack of ulcerogenic activity might be due to the compound’s acid-inhibitory or cytokine suppressive 
actions. 
The paper is interesting and the studies are generally appropriately performed and reported. The 
data provide an interesting insight into how this new compound may act and deserve wide dissemination as that is likely to stimulate further research. The findings are likely to be of wide interest to many interested in NSAIDs and gastric pharmacology and pathophysiology. The paper is 
generally well written, although there are several errors that require correcting. The authors’ line of 
argument about the importance of studying IL-8 secretion in relation to NSAID gastropathy is somewhat hard to follow. Several sections of the Methods could be improved, as the methodology would be difficult to 
replicate from the information given. 
Discretionary Revisions (which the author can choose to ignore) 
1. Add the species used to the title 
2. Remove the repeated use of “identical reaction conditions” from the figure legends. Surely by inference a repeated or duplicate experiment always uses identical conditions? 
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. Use consistent terminology throughout: gastric parietal cells OR gastric glands (see Abstract). 
2. Explain in 1st paragraph of Background why inhibiting LT synthesis would be expected to minimize mucosal injury; otherwise the logic is difficult to follow. 3. Background paragraph 2, line 9 “to rats indicate ML 3000 undergoes…..(something seems 
missing from that sentence) 
4. Background para 4, line 11, esomeprazole is incorrectly spelt. 5. Background para 5 is difficult to follow. It is accepted that H. pylori pathogenesis involves IL-8 (and this should be referenced) but the authors should provide more information as to why IL-8 
secretion is important in NSAID pathogenesis, to support their line of experimentation. 
6. Please provide information on yield and purity of parietal cell preparation. 7. Within statistical analysis, please provide number of experiments and duplicates per experiment. 
8. Both a results sub-heading and figure 8 legend quote “Helicobacter pylori-induced IL-8 secretion.” 
As far as I can see, no H. pylori were used anywhere in the study. 
9. Please provide reference for the statement that “AGS cells do not express HK-ATPase.” I believe 
the authors have shown this previously. 10. Amend the statement at the end of Discussion, 1st paragraph. The data presented, especially 
the effects on IL-1b-stimulated IL-8 secretion do not really support an effect via 5-lipooxygenase. 
The authors report this themselves later in the Discussion. 
11. Reference 24 does not support the statement made about omeprazole and gastric cancers. The 
study referred to showed omeprazole increased gastric inflammation and achlorhydria and induced a 
hyperproliferative mucosa but that study was too short to actually demonstrate cancer development. 
The relevant sentence should be altered to illustrate this or an alternative reference cited. 12. The less prolonged acid-inhibition of ML 3000 may not be sufficient to prevent NSAID-ulcers in 
humans. H2 receptor antagonists are less effective prophylaxis than PPIs. The authors could 
acknowledge this uncertainty. 13. Last sentence of Discussion. Why is TNF-a stimulated IL-8 secretion of such interest in this model, surely the mechanism is the same as IL-1b? 
14. What measure of variance is used for the error bars in the figures? 
15. Clarify legends to Figure 2. It is ambigious as to when the dilution step occurred, before or after the addition to microsomes? 16. Does figure 4A shows typical experimental data from one of exactly 3 or at least 3 experiments? 
17. The figure 8D legend refers to ATPase activities being measured: this figure concerns IL-8. 
Surely there is an error here? 
Major Compulsory Revisions (that the author must respond to before a decision on publication can 
be reached) 
1. The Methods section should be revised. The sources of the chemicals and their in vitro 
manipulation described in more detail. ML 3000 is a novel compound, yet the manuscript does not 
quote the source. Equally the provider of the other enzyme inhibitors and eicosanoids should be available. As the authors allude to, many of these compounds are difficult to use in vitro and required special methods to solubilise them. What methods were used and what controls were 
applied for the effects of the solvents? 
2. The methodology for the AGS cell experiment is confusing. One sentence suggests 18 hours 
incubation was used, the next sentence suggests 24 hours and the results section suggests 6 hours. 
This needs to be clarified. The Methods establish the experiments in 6 well culture plates with > 3 
x105 cells, yet the results report data with less cells and I assume less volume of culture medium. I 
cannot reconcile these different results and a uniform methodology must be described throughout. 3. The section on acidification of omeprazole in the discussion section is irrelevant to the current 
study and can be omitted. However the essential nugget of information describing pre-acidification of 
omeprazole before the HK-ATPase studies should be within the Methods and not the discussion. 
Similarly the Methods do not make it clear whether the ML 3000 was pre-acidified or the whole assay was acidified (Fig 2B), I assume the former but this methodology should be made explicit, to allow replication. 
 Accept after minor compulsory 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  20 November 2003 Version: Date: 
 Ian Beales Reviewer: 
Reviewer's report: 
General 
The authors have studied a novel anti-inflammatory compound (ML 3000) and demonstrated that the 
lack of ulcerogenic activity might be due to the compound’s acid-inhibitory or cytokine suppressive 
actions. 
The paper is interesting and the studies are generally appropriately performed and reported. The 
data provide an interesting insight into how this new compound may act and deserve wide dissemination as that is likely to stimulate further research. The findings are likely to be of wide interest to many interested in NSAIDs and gastric pharmacology and pathophysiology. The paper is 
generally well written, although there are several errors that require correcting. The authors’ line of 
argument about the importance of studying IL-8 secretion in relation to NSAID gastropathy is somewhat hard to follow. Several sections of the Methods could be improved, as the methodology would be difficult to 
replicate from the information given. 
Discretionary Revisions (which the author can choose to ignore) 
1. Add the species used to the title 
2. Remove the repeated use of “identical reaction conditions” from the figure legends. Surely by inference a repeated or duplicate experiment always uses identical conditions? 
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. Use consistent terminology throughout: gastric parietal cells OR gastric glands (see Abstract). 
2. Explain in 1st paragraph of Background why inhibiting LT synthesis would be expected to minimize mucosal injury; otherwise the logic is difficult to follow. 3. Background paragraph 2, line 9 “to rats indicate ML 3000 undergoes…..(something seems 
missing from that sentence) 
4. Background para 4, line 11, esomeprazole is incorrectly spelt. 5. Background para 5 is difficult to follow. It is accepted that H. pylori pathogenesis involves IL-8 (and this should be referenced) but the authors should provide more information as to why IL-8 
secretion is important in NSAID pathogenesis, to support their line of experimentation. 
6. Please provide information on yield and purity of parietal cell preparation. 7. Within statistical analysis, please provide number of experiments and duplicates per experiment. 
8. Both a results sub-heading and figure 8 legend quote “Helicobacter pylori-induced IL-8 secretion.” 
As far as I can see, no H. pylori were used anywhere in the study. 
9. Please provide reference for the statement that “AGS cells do not express HK-ATPase.” I believe 
the authors have shown this previously. 10. Amend the statement at the end of Discussion, 1st paragraph. The data presented, especially 
the effects on IL-1b-stimulated IL-8 secretion do not really support an effect via 5-lipooxygenase. 
The authors report this themselves later in the Discussion. 
11. Reference 24 does not support the statement made about omeprazole and gastric cancers. The 
study referred to showed omeprazole increased gastric inflammation and achlorhydria and induced a 
hyperproliferative mucosa but that study was too short to actually demonstrate cancer development. 
The relevant sentence should be altered to illustrate this or an alternative reference cited. 12. The less prolonged acid-inhibition of ML 3000 may not be sufficient to prevent NSAID-ulcers in 
humans. H2 receptor antagonists are less effective prophylaxis than PPIs. The authors could 
acknowledge this uncertainty. 13. Last sentence of Discussion. Why is TNF-a stimulated IL-8 secretion of such interest in this model, surely the mechanism is the same as IL-1b? 
14. What measure of variance is used for the error bars in the figures? 
15. Clarify legends to Figure 2. It is ambigious as to when the dilution step occurred, before or after the addition to microsomes? 16. Does figure 4A shows typical experimental data from one of exactly 3 or at least 3 experiments? 
17. The figure 8D legend refers to ATPase activities being measured: this figure concerns IL-8. 
Surely there is an error here? 
Major Compulsory Revisions (that the author must respond to before a decision on publication can 
be reached) 
1. The Methods section should be revised. The sources of the chemicals and their in vitro 
manipulation described in more detail. ML 3000 is a novel compound, yet the manuscript does not 
quote the source. Equally the provider of the other enzyme inhibitors and eicosanoids should be available. As the authors allude to, many of these compounds are difficult to use in vitro and required special methods to solubilise them. What methods were used and what controls were 
applied for the effects of the solvents? 
2. The methodology for the AGS cell experiment is confusing. One sentence suggests 18 hours 
incubation was used, the next sentence suggests 24 hours and the results section suggests 6 hours. 
This needs to be clarified. The Methods establish the experiments in 6 well culture plates with > 3 
x105 cells, yet the results report data with less cells and I assume less volume of culture medium. I 
cannot reconcile these different results and a uniform methodology must be described throughout. 3. The section on acidification of omeprazole in the discussion section is irrelevant to the current 
study and can be omitted. However the essential nugget of information describing pre-acidification of 
omeprazole before the HK-ATPase studies should be within the Methods and not the discussion. 
Similarly the Methods do not make it clear whether the ML 3000 was pre-acidified or the whole assay was acidified (Fig 2B), I assume the former but this methodology should be made explicit, to allow replication. 
 Accept after minor compulsory 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  20 November 2003 Version: Date: 
 Ian Beales Reviewer: 
Reviewer's report: 
General 
The authors have studied a novel anti-inflammatory compound (ML 3000) and demonstrated that the 
lack of ulcerogenic activity might be due to the compound’s acid-inhibitory or cytokine suppressive 
actions. 
The paper is interesting and the studies are generally appropriately performed and reported. The 
data provide an interesting insight into how this new compound may act and deserve wide dissemination as that is likely to stimulate further research. The findings are likely to be of wide interest to many interested in NSAIDs and gastric pharmacology and pathophysiology. The paper is 
generally well written, although there are several errors that require correcting. The authors’ line of 
argument about the importance of studying IL-8 secretion in relation to NSAID gastropathy is somewhat hard to follow. Several sections of the Methods could be improved, as the methodology would be difficult to 
replicate from the information given. 
Discretionary Revisions (which the author can choose to ignore) 
1. Add the species used to the title 
2. Remove the repeated use of “identical reaction conditions” from the figure legends. Surely by inference a repeated or duplicate experiment always uses identical conditions? 
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. Use consistent terminology throughout: gastric parietal cells OR gastric glands (see Abstract). 
2. Explain in 1st paragraph of Background why inhibiting LT synthesis would be expected to minimize mucosal injury; otherwise the logic is difficult to follow. 3. Background paragraph 2, line 9 “to rats indicate ML 3000 undergoes…..(something seems 
missing from that sentence) 
4. Background para 4, line 11, esomeprazole is incorrectly spelt. 5. Background para 5 is difficult to follow. It is accepted that H. pylori pathogenesis involves IL-8 (and this should be referenced) but the authors should provide more information as to why IL-8 
secretion is important in NSAID pathogenesis, to support their line of experimentation. 
6. Please provide information on yield and purity of parietal cell preparation. 7. Within statistical analysis, please provide number of experiments and duplicates per experiment. 
8. Both a results sub-heading and figure 8 legend quote “Helicobacter pylori-induced IL-8 secretion.” 
As far as I can see, no H. pylori were used anywhere in the study. 
9. Please provide reference for the statement that “AGS cells do not express HK-ATPase.” I believe 
the authors have shown this previously. 10. Amend the statement at the end of Discussion, 1st paragraph. The data presented, especially 
the effects on IL-1b-stimulated IL-8 secretion do not really support an effect via 5-lipooxygenase. 
The authors report this themselves later in the Discussion. 
11. Reference 24 does not support the statement made about omeprazole and gastric cancers. The 
study referred to showed omeprazole increased gastric inflammation and achlorhydria and induced a 
hyperproliferative mucosa but that study was too short to actually demonstrate cancer development. 
The relevant sentence should be altered to illustrate this or an alternative reference cited. 12. The less prolonged acid-inhibition of ML 3000 may not be sufficient to prevent NSAID-ulcers in 
humans. H2 receptor antagonists are less effective prophylaxis than PPIs. The authors could 
acknowledge this uncertainty. 13. Last sentence of Discussion. Why is TNF-a stimulated IL-8 secretion of such interest in this model, surely the mechanism is the same as IL-1b? 
14. What measure of variance is used for the error bars in the figures? 
15. Clarify legends to Figure 2. It is ambigious as to when the dilution step occurred, before or after the addition to microsomes? 16. Does figure 4A shows typical experimental data from one of exactly 3 or at least 3 experiments? 
17. The figure 8D legend refers to ATPase activities being measured: this figure concerns IL-8. 
Surely there is an error here? 
Major Compulsory Revisions (that the author must respond to before a decision on publication can 
be reached) 
1. The Methods section should be revised. The sources of the chemicals and their in vitro 
manipulation described in more detail. ML 3000 is a novel compound, yet the manuscript does not 
quote the source. Equally the provider of the other enzyme inhibitors and eicosanoids should be available. As the authors allude to, many of these compounds are difficult to use in vitro and required special methods to solubilise them. What methods were used and what controls were 
applied for the effects of the solvents? 
2. The methodology for the AGS cell experiment is confusing. One sentence suggests 18 hours 
incubation was used, the next sentence suggests 24 hours and the results section suggests 6 hours. 
This needs to be clarified. The Methods establish the experiments in 6 well culture plates with > 3 
x105 cells, yet the results report data with less cells and I assume less volume of culture medium. I 
cannot reconcile these different results and a uniform methodology must be described throughout. 3. The section on acidification of omeprazole in the discussion section is irrelevant to the current 
study and can be omitted. However the essential nugget of information describing pre-acidification of 
omeprazole before the HK-ATPase studies should be within the Methods and not the discussion. 
Similarly the Methods do not make it clear whether the ML 3000 was pre-acidified or the whole assay was acidified (Fig 2B), I assume the former but this methodology should be made explicit, to allow replication. 
 Accept after minor compulsory 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