Reviewer’s report
Title: 
Overexpression of P-glycoprotein, MRP2, and CYP3A4 impairs intestinal
absorption of octreotide in rats with portal hypertension
Version: 
0 
Date: 
07 Apr 2019
Reviewer's report:
Sun et. al. performed an extensive investigation on intestinal absorption
mechanisms of octreotide in healthy and cirrhotic rats. The research is well
performed and experiments appropriately chosen. However, there are some
points, which need to be addressed by the authors:
MAJOR points:
1) The background is poorly written and contains inaccurately descriptions.
1a) Please refer to current PH treatment guidelines/standards, variceal bleeding
prophylaxis and the role of OCT for treatment of acute variceal bleeding.
1b) "Accordingly, our previous study showed that hepatic first-pass effects are
diminished during PH due to increased collateral circulation (unpublished
observation)." Citing an unpublished observation in the background part of a
manuscript is unusual, especially since you state in the discussion "A previous
study showed that the establishment of a portosystemic shunt decreased hepatic
first-pass effects under PH conditions [1]." Hence I suggest to either show your
own data or include reference [1] in the background as well.
1c) "P-gp and MRP2 largely control drug uptake, distribution and excretion."
Since these molecules are efflux pumps, they cannot 'control' drug uptake or
distribution.
1d) "OCT can regulate blood circulation and vascular resistance in the liver,
thereby reducing PH." - Please specify that OCT mainly regulates 'splanchnic'
blood flow. Moreover it does not change intrahepatic resistance!
[Gastroenterology. 1981 Mar;80(3):518-25.]
1e) "Due to its polypeptidic structure, OCT
exhibits low systemic bioavailability upon ingestion" This statement is in
contrast with reference [11].
2) The authors include the ARRIVE guidelines in the supplements but do not
fully adhere to them:
2a) The authors do not provide a primary study aim and a sample-size
calculation is lacking.
2b) Please specify how ether anaesthesia was performed/dosed/controlled.
2c) Please specify how post-surgical gentamicin was dosed (drug concentration?,
adapted for bodyweight?).
2d) Please provide details of materials and consumables used (e.g. "INDICATE
CONTRAST MEDIUM USED HERE")
3) DSA analysis is performed by catheterizing the hepatic artery and imaging
porto-systemic collaterals? Please explain how this is possible. Moreover this
analysis and Figure 1 does not add valuable information for this manuscript. I
suggest to perform microbead injection to assess and quanitfy shunting [Am J
Physiol. 1981 May;240(5):G371-5. & Digest Dis Sci (1991) 36: 209] or exclude
this part from the manuscript.
4) Please explain why you switch between different P-gp inhibitors (verapamil,
rhodamine) in your experiments and do not adhere to one throughout your
investigations.
5) Authors showed that OCT did not inhibit CYP3A4, however it has been
shown that OCT inhibits MRP2 (J Pharmacol Exp Ther. 2015 Nov;355(2):145-
51.). This has not been discussed.
6) While rat experiments were carried out in the jejunum, cellular experiments
used Caco-2 cells which derive from colon - a very different part of the intestine.
This has not been discussed.
7) "Thus, inhibition of P-gp, MRP2, and CYP3A4 may be a plausible strategy to
effectively decrease PH via oral administration of OCT." - Translational research
is of great importance, however this statement is a huge exaggeration, because it
(1) ignores the importance of these transporters for hepatic/renal excretion of
OCT and (2) neglects that most cirrhotic patients take mutiple drugs where
inhibting such central enzymes might cause drug-drug interactions and
complicate appropriate drug-therapy titration.
MINOR comments:
1) I may suggest to change the wording of the control group from "intact" to
"healthy".
2) Please show the full Western Blots as supplements.
3) I suggest to move Table 1 to the supplements.
4) It might help the readers to include a Figure 1 graphically explaining all
performed experiments/methos with respective groups.
5) "in the absence of severe liver dysfunction in our BDL model." - How was
liver dysfunction assessed?
Are the methods appropriate and well described?
If not, please specify what is required in your comments to the authors.
Yes
Does the work include the necessary controls?
If not, please specify which controls are required in your comments to the
authors.
Yes
Are the conclusions drawn adequately supported by the data shown?
If not, please explain in your comments to the authors.
Yes
Are you able to assess any statistics in the manuscript or would you
recommend an additional statistical review?
If an additional statistical review is recommended, please specify what aspects
require further assessment in your comments to the editors.
I am able to assess the statistics
Quality of written English
Please indicate the quality of language in the manuscript:
Needs some language corrections before being published
Declaration of competing interests
Please complete a declaration of competing interests, considering the following
questions:
1
. 
Have you in the past five years received reimbursements, fees, funding, or
salary from an organisation that may in any way gain or lose financially
from the publication of this manuscript, either now or in the future?
2
. 
Do you hold any stocks or shares in an organisation that may in any way
gain or lose financially from the publication of this manuscript, either now
or in the future?
3
. 
Do you hold or are you currently applying for any patents relating to the
content of the manuscript?
4
. 
Have you received reimbursements, fees, funding, or salary from an
organization that holds or has applied for patents relating to the content of
the manuscript?
5
. 
Do you have any other financial competing interests?
6
. 
Do you have any non-financial competing interests in relation to this
paper?
If you can answer no to all of the above, write 'I declare that I have no competing
interests' below. If your reply is yes to any, please give details below.
I declare that I have no competing interests
I agree to the open peer review policy of the journal. I understand that my name
will be included on my report to the authors and, if the manuscript is accepted
for publication, my named report including any attachments I upload will be
posted on the website along with the authors' responses. I agree for my report to
be made available under an Open Access Creative Commons CC-BY license
(
http://creativecommons.org/licenses/by/4.0/
). I understand that any comments
which I do not wish to be included in my named report can be included as
confidential comments to the editors, which will not be published.
I agree to the open peer review policy of the journal
Reviewer’s report
Title: 
Overexpression of P-glycoprotein, MRP2, and CYP3A4 impairs intestinal
absorption of octreotide in rats with portal hypertension
Version: 
0 
Date: 
07 Apr 2019
Reviewer's report:
Sun et. al. performed an extensive investigation on intestinal absorption
mechanisms of octreotide in healthy and cirrhotic rats. The research is well
performed and experiments appropriately chosen. However, there are some
points, which need to be addressed by the authors:
MAJOR points:
1) The background is poorly written and contains inaccurately descriptions.
1a) Please refer to current PH treatment guidelines/standards, variceal bleeding
prophylaxis and the role of OCT for treatment of acute variceal bleeding.
1b) "Accordingly, our previous study showed that hepatic first-pass effects are
diminished during PH due to increased collateral circulation (unpublished
observation)." Citing an unpublished observation in the background part of a
manuscript is unusual, especially since you state in the discussion "A previous
study showed that the establishment of a portosystemic shunt decreased hepatic
first-pass effects under PH conditions [1]." Hence I suggest to either show your
own data or include reference [1] in the background as well.
1c) "P-gp and MRP2 largely control drug uptake, distribution and excretion."
Since these molecules are efflux pumps, they cannot 'control' drug uptake or
distribution.
1d) "OCT can regulate blood circulation and vascular resistance in the liver,
thereby reducing PH." - Please specify that OCT mainly regulates 'splanchnic'
blood flow. Moreover it does not change intrahepatic resistance!
[Gastroenterology. 1981 Mar;80(3):518-25.]
1e) "Due to its polypeptidic structure, OCT
exhibits low systemic bioavailability upon ingestion" This statement is in
contrast with reference [11].
2) The authors include the ARRIVE guidelines in the supplements but do not
fully adhere to them:
2a) The authors do not provide a primary study aim and a sample-size
calculation is lacking.
2b) Please specify how ether anaesthesia was performed/dosed/controlled.
2c) Please specify how post-surgical gentamicin was dosed (drug concentration?,
adapted for bodyweight?).
2d) Please provide details of materials and consumables used (e.g. "INDICATE
CONTRAST MEDIUM USED HERE")
3) DSA analysis is performed by catheterizing the hepatic artery and imaging
porto-systemic collaterals? Please explain how this is possible. Moreover this
analysis and Figure 1 does not add valuable information for this manuscript. I
suggest to perform microbead injection to assess and quanitfy shunting [Am J
Physiol. 1981 May;240(5):G371-5. & Digest Dis Sci (1991) 36: 209] or exclude
this part from the manuscript.
4) Please explain why you switch between different P-gp inhibitors (verapamil,
rhodamine) in your experiments and do not adhere to one throughout your
investigations.
5) Authors showed that OCT did not inhibit CYP3A4, however it has been
shown that OCT inhibits MRP2 (J Pharmacol Exp Ther. 2015 Nov;355(2):145-
51.). This has not been discussed.
6) While rat experiments were carried out in the jejunum, cellular experiments
used Caco-2 cells which derive from colon - a very different part of the intestine.
This has not been discussed.
7) "Thus, inhibition of P-gp, MRP2, and CYP3A4 may be a plausible strategy to
effectively decrease PH via oral administration of OCT." - Translational research
is of great importance, however this statement is a huge exaggeration, because it
(1) ignores the importance of these transporters for hepatic/renal excretion of
OCT and (2) neglects that most cirrhotic patients take mutiple drugs where
inhibting such central enzymes might cause drug-drug interactions and
complicate appropriate drug-therapy titration.
MINOR comments:
1) I may suggest to change the wording of the control group from "intact" to
"healthy".
2) Please show the full Western Blots as supplements.
3) I suggest to move Table 1 to the supplements.
4) It might help the readers to include a Figure 1 graphically explaining all
performed experiments/methos with respective groups.
5) "in the absence of severe liver dysfunction in our BDL model." - How was
liver dysfunction assessed?
Are the methods appropriate and well described?
If not, please specify what is required in your comments to the authors.
Yes
Does the work include the necessary controls?
If not, please specify which controls are required in your comments to the
authors.
Yes
Are the conclusions drawn adequately supported by the data shown?
If not, please explain in your comments to the authors.
Yes
Are you able to assess any statistics in the manuscript or would you
recommend an additional statistical review?
If an additional statistical review is recommended, please specify what aspects
require further assessment in your comments to the editors.
I am able to assess the statistics
Quality of written English
Please indicate the quality of language in the manuscript:
Needs some language corrections before being published
Declaration of competing interests
Please complete a declaration of competing interests, considering the following
questions:
1
. 
Have you in the past five years received reimbursements, fees, funding, or
salary from an organisation that may in any way gain or lose financially
from the publication of this manuscript, either now or in the future?
2
. 
Do you hold any stocks or shares in an organisation that may in any way
gain or lose financially from the publication of this manuscript, either now
or in the future?
3
. 
Do you hold or are you currently applying for any patents relating to the
content of the manuscript?
4
. 
Have you received reimbursements, fees, funding, or salary from an
organization that holds or has applied for patents relating to the content of
the manuscript?
5
. 
Do you have any other financial competing interests?
6
. 
Do you have any non-financial competing interests in relation to this
paper?
If you can answer no to all of the above, write 'I declare that I have no competing
interests' below. If your reply is yes to any, please give details below.
I declare that I have no competing interests
I agree to the open peer review policy of the journal. I understand that my name
will be included on my report to the authors and, if the manuscript is accepted
for publication, my named report including any attachments I upload will be
posted on the website along with the authors' responses. I agree for my report to
be made available under an Open Access Creative Commons CC-BY license
(
http://creativecommons.org/licenses/by/4.0/
). I understand that any comments
which I do not wish to be included in my named report can be included as
confidential comments to the editors, which will not be published.
I agree to the open peer review policy of the journal
Please review this paper.
user
Reviewer’s report
Title: 
Overexpression of P-glycoprotein, MRP2, and CYP3A4 impairs intestinal
absorption of octreotide in rats with portal hypertension
Version: 
0 
Date: 
07 Apr 2019
Reviewer's report:
Sun et. al. performed an extensive investigation on intestinal absorption
mechanisms of octreotide in healthy and cirrhotic rats. The research is well
performed and experiments appropriately chosen. However, there are some
points, which need to be addressed by the authors:
MAJOR points:
1) The background is poorly written and contains inaccurately descriptions.
1a) Please refer to current PH treatment guidelines/standards, variceal bleeding
prophylaxis and the role of OCT for treatment of acute variceal bleeding.
1b) "Accordingly, our previous study showed that hepatic first-pass effects are
diminished during PH due to increased collateral circulation (unpublished
observation)." Citing an unpublished observation in the background part of a
manuscript is unusual, especially since you state in the discussion "A previous
study showed that the establishment of a portosystemic shunt decreased hepatic
first-pass effects under PH conditions [1]." Hence I suggest to either show your
own data or include reference [1] in the background as well.
1c) "P-gp and MRP2 largely control drug uptake, distribution and excretion."
Since these molecules are efflux pumps, they cannot 'control' drug uptake or
distribution.
1d) "OCT can regulate blood circulation and vascular resistance in the liver,
thereby reducing PH." - Please specify that OCT mainly regulates 'splanchnic'
blood flow. Moreover it does not change intrahepatic resistance!
[Gastroenterology. 1981 Mar;80(3):518-25.]
1e) "Due to its polypeptidic structure, OCT
exhibits low systemic bioavailability upon ingestion" This statement is in
contrast with reference [11].
2) The authors include the ARRIVE guidelines in the supplements but do not
fully adhere to them:
2a) The authors do not provide a primary study aim and a sample-size
calculation is lacking.
2b) Please specify how ether anaesthesia was performed/dosed/controlled.
2c) Please specify how post-surgical gentamicin was dosed (drug concentration?,
adapted for bodyweight?).
2d) Please provide details of materials and consumables used (e.g. "INDICATE
CONTRAST MEDIUM USED HERE")
3) DSA analysis is performed by catheterizing the hepatic artery and imaging
porto-systemic collaterals? Please explain how this is possible. Moreover this
analysis and Figure 1 does not add valuable information for this manuscript. I
suggest to perform microbead injection to assess and quanitfy shunting [Am J
Physiol. 1981 May;240(5):G371-5. & Digest Dis Sci (1991) 36: 209] or exclude
this part from the manuscript.
4) Please explain why you switch between different P-gp inhibitors (verapamil,
rhodamine) in your experiments and do not adhere to one throughout your
investigations.
5) Authors showed that OCT did not inhibit CYP3A4, however it has been
shown that OCT inhibits MRP2 (J Pharmacol Exp Ther. 2015 Nov;355(2):145-
51.). This has not been discussed.
6) While rat experiments were carried out in the jejunum, cellular experiments
used Caco-2 cells which derive from colon - a very different part of the intestine.
This has not been discussed.
7) "Thus, inhibition of P-gp, MRP2, and CYP3A4 may be a plausible strategy to
effectively decrease PH via oral administration of OCT." - Translational research
is of great importance, however this statement is a huge exaggeration, because it
(1) ignores the importance of these transporters for hepatic/renal excretion of
OCT and (2) neglects that most cirrhotic patients take mutiple drugs where
inhibting such central enzymes might cause drug-drug interactions and
complicate appropriate drug-therapy titration.
MINOR comments:
1) I may suggest to change the wording of the control group from "intact" to
"healthy".
2) Please show the full Western Blots as supplements.
3) I suggest to move Table 1 to the supplements.
4) It might help the readers to include a Figure 1 graphically explaining all
performed experiments/methos with respective groups.
5) "in the absence of severe liver dysfunction in our BDL model." - How was
liver dysfunction assessed?
Are the methods appropriate and well described?
If not, please specify what is required in your comments to the authors.
Yes
Does the work include the necessary controls?
If not, please specify which controls are required in your comments to the
authors.
Yes
Are the conclusions drawn adequately supported by the data shown?
If not, please explain in your comments to the authors.
Yes
Are you able to assess any statistics in the manuscript or would you
recommend an additional statistical review?
If an additional statistical review is recommended, please specify what aspects
require further assessment in your comments to the editors.
I am able to assess the statistics
Quality of written English
Please indicate the quality of language in the manuscript:
Needs some language corrections before being published
Declaration of competing interests
Please complete a declaration of competing interests, considering the following
questions:
1
. 
Have you in the past five years received reimbursements, fees, funding, or
salary from an organisation that may in any way gain or lose financially
from the publication of this manuscript, either now or in the future?
2
. 
Do you hold any stocks or shares in an organisation that may in any way
gain or lose financially from the publication of this manuscript, either now
or in the future?
3
. 
Do you hold or are you currently applying for any patents relating to the
content of the manuscript?
4
. 
Have you received reimbursements, fees, funding, or salary from an
organization that holds or has applied for patents relating to the content of
the manuscript?
5
. 
Do you have any other financial competing interests?
6
. 
Do you have any non-financial competing interests in relation to this
paper?
If you can answer no to all of the above, write 'I declare that I have no competing
interests' below. If your reply is yes to any, please give details below.
I declare that I have no competing interests
I agree to the open peer review policy of the journal. I understand that my name
will be included on my report to the authors and, if the manuscript is accepted
for publication, my named report including any attachments I upload will be
posted on the website along with the authors' responses. I agree for my report to
be made available under an Open Access Creative Commons CC-BY license
(
http://creativecommons.org/licenses/by/4.0/
). I understand that any comments
which I do not wish to be included in my named report can be included as
confidential comments to the editors, which will not be published.
I agree to the open peer review policy of the journal
assistant