FXR agonist in post-liver transplantation patients: a randomized open-labeled study.

Anila K N; Saraswathy S Nair; Dinesh Balakrishnan`; Unnikrishnan G; Binoj S T; Ramachandran Narayana Menon; Christi Titus Varghese; Shweta Mallick; Krishnanunni Nair; Madhu Srinivasan Durairaj; Guhan V; Nafiya Zackariah; Haritha Rajakrishnan; Arun Valsan; Johns Shaji Mathew; Cyriac Abby Philips; Christopher Watson; Sudheer O V; Sudhindran S

doi:10.12688/f1000research.159202.2

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Study Protocol

Revised

FXR agonist in post-liver transplantation patients: a randomized open-labeled study.

[version 2; peer review: 2 approved with reservations]

Anila K N ¹, Saraswathy S Nair¹, Dinesh Balakrishnan`¹, [...] Unnikrishnan G¹, Binoj S T¹, Ramachandran Narayana Menon¹, Christi Titus Varghese¹, Shweta Mallick¹, Krishnanunni Nair¹, Madhu Srinivasan Durairaj¹, Guhan V¹, Nafiya Zackariah¹, Haritha Rajakrishnan¹, Arun Valsan², Johns Shaji Mathew³, Cyriac Abby Philips⁴, Christopher Watson⁵, Sudheer O V¹, Sudhindran S ¹

Anila K N ¹, Saraswathy S Nair¹, [...] Dinesh Balakrishnan`¹, Unnikrishnan G¹, Binoj S T¹, Ramachandran Narayana Menon¹, Christi Titus Varghese¹, Shweta Mallick¹, Krishnanunni Nair¹, Madhu Srinivasan Durairaj¹, Guhan V¹, Nafiya Zackariah¹, Haritha Rajakrishnan¹, Arun Valsan², Johns Shaji Mathew³, Cyriac Abby Philips⁴, Christopher Watson⁵, Sudheer O V¹, Sudhindran S ¹

PUBLISHED 27 Jun 2025

Author details Author details

¹ GI Surgery, Amrita Institute of Medical Sciences & Research Centre, Kochi, Amrita Vishwa Vidyapeetham, Coimbatore, Tamil Nadu, India
² Hepatology, Division of Gastromedicine, Amrita Institute of Medical Sciences & Research Centre, Amrita Vishwa Vidyapeetham, Coimbatore, Tamil Nadu, India
³ Department of Multiorgan transplant and HPB Surgery, Burjeel Hospital, Abu Dhabi, Abu Dhabi, United Arab Emirates
⁴ Department of Clinical and Translational Hepatology, The Liver Institute, Rajagiri Hospital, Aluva, Kerala, India
⁵ Addenbrookes Hospital, Cambridge University Hospitals NHS Foundation Trust, Cambridge, CB2 OQQ, UK

Anila K N
Roles: Data Curation, Formal Analysis, Investigation, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing

Saraswathy S Nair
Roles: Data Curation, Methodology

Dinesh Balakrishnan`
Roles: Conceptualization, Supervision, Writing – Review & Editing

Unnikrishnan G
Roles: Conceptualization, Methodology, Supervision, Writing – Review & Editing

Binoj S T
Roles: Writing – Review & Editing

Ramachandran Narayana Menon
Roles: Conceptualization

Christi Titus Varghese
Roles: Writing – Review & Editing

Shweta Mallick
Roles: Writing – Review & Editing

Krishnanunni Nair
Roles: Resources, Writing – Review & Editing

Madhu Srinivasan Durairaj
Roles: Writing – Review & Editing

Guhan V
Roles: Writing – Review & Editing

Nafiya Zackariah
Roles: Methodology

Haritha Rajakrishnan
Roles: Methodology

Arun Valsan
Roles: Writing – Review & Editing

Johns Shaji Mathew
Roles: Conceptualization, Methodology

Cyriac Abby Philips
Roles: Writing – Review & Editing

Christopher Watson
Roles: Writing – Review & Editing

Sudheer O V
Roles: Writing – Review & Editing

Sudhindran S
Roles: Conceptualization, Funding Acquisition, Methodology, Resources, Supervision, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

Abstract

Liver diseases cause nearly 2 million deaths worldwide each year, with approximately 1 million deaths from cirrhosis complications and another 1 million from viral hepatitis and liver cancer, according to WHO estimates. Liver transplantation (LT) remains the primary curative option, boasting success rates of 85% in the first year and 75% at five years post-transplant. Despite high costs, LT is considered cost-effective, especially for younger patients with active work years remaining. However, post-transplant complications, particularly intrahepatic cholestasis, present notable challenges. This complication arises from factors such as ischemia-reperfusion injury, infections, immunological rejection, and surgical complications, all contributing to impaired bile flow and liver damage. Current medical therapies for post-LT cholestasis are limited, with ursodeoxycholic acid (UDCA) frequently used, despite questionable efficacy. Obeticholic acid (OCA), a potent Farnesoid X Receptor (FXR) agonist approved by the FDA for treating primary biliary cholangitis (PBC), has shown potential benefits in reducing elevated cholestatic liver enzymes. Given its significant effects on liver health, OCA may offer therapeutic value in managing post-transplant cholestasis and improving graft survival.

This randomized controlled trial (RCT) aims to evaluate OCA’s efficacy compared to UDCA in reducing cholestatic injury and enhancing graft function post-LT. The primary outcomes will focus on a 15% reduction in alkaline phosphatase and gamma-glutamyl transferase levels at 3, 6, and 12 months from baseline one month. Secondary outcomes include molecular markers, biliary complications, graft rejection, quality of life, and cost-effectiveness. Results are anticipated to demonstrate that OCA could improve graft survival, reduce complications, and enhance quality of life, potentially setting a new standard in post-LT care if found beneficial.

Keywords

Liver transplantation, cholestasis, ursodeoxycholic acid, obeticholic acid, alkaline phosphatase.

Corresponding authors: Anila K N, Sudhindran S

Competing interests: No competing interests were disclosed.

Grant information: This work has recently received seed grant funding from the institution’s Research fund.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2025 K N A et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: K N A, S Nair S, Balakrishnan` D et al. FXR agonist in post-liver transplantation patients: a randomized open-labeled study. [version 2; peer review: 2 approved with reservations]. F1000Research 2025, 14:9 (https://doi.org/10.12688/f1000research.159202.2) First published: 02 Jan 2025, 14:9 (https://doi.org/10.12688/f1000research.159202.1) Latest published: 27 Jun 2025, 14:9 (https://doi.org/10.12688/f1000research.159202.2)

Revised Amendments from Version 1

This revised version (Version 2) of the article incorporates all the suggestions and recommendations provided by the two reviewers. Based on their valuable feedback, several sections have been revised for clarity, accuracy, and improved scientific rigor. Specific modifications include updates to the methodology for better transparency. We sincerely acknowledge the reviewers’ constructive input, which has significantly enhanced the quality and clarity of the manuscript.

See the authors' detailed response to the review by Gregory Guthrie
See the authors' detailed response to the review by Palak J Trivedi

Introduction

Intrahepatic cholestasis is a major complication following liver transplantation, caused by multiple factors. Ischemia-reperfusion injury during organ retrieval and transplantation leads to cellular damage and postoperative cholestasis.^7,8 Sepsis from infections worsen cholestasis through inflammatory responses.⁹ Essential post-transplant drugs, including immunosuppressants and antibiotics, can interfere with bile formation, causing liver injury.^10,11 Immunological reactions such as T-cell and B-cell-mediated rejection and lymphocytic cholangitis also contribute to cholestasis.¹² Surgical complications like stenosis or obstruction of the biliary and vascular anastomosis can also result in intra- and extrahepatic cholestasis.^13–15

It is usually administered for three months post-transplant at a dose of 10-15 mg/kg thrice daily. In 2016, the FDA approved obeticholic acid (OCA), a strong selective Farnesoid X Receptor (FXR) agonist, to treat primary biliary cholangitis (PBC) in conjunction with UDCA.^16,17 Research has demonstrated that over 12 months, OCA, either in combination with UDCA or on its own, dramatically lowers levels of total bilirubin and alkaline phosphatase.^18,19 In individuals with advanced liver disease, the FDA has cautioned against administering OCA at doses of 10–50 mg.²⁰ Studies comparing OCA’s effects on cholestasis, rejection, regeneration, and cost-effectiveness to the current standard UDCA are being conducted to assess the role of OCA in the postoperative recovery of liver transplant patients.²¹ Our proposed project is a clinical trial between OCA and the current standard of care, UDCA, in managing post-liver transplant cholestasis and improving graft survival.²²

Rationale

Following LT, a major morbidity mitigating long-term liver functions is intrahepatic cholestasis due to a multitude of reasons. The only drug available for the treatment of intrahepatic cholestasis currently is UDCA.²³ FXR is a newly detected receptor gene, playing multiple roles in liver homeostasis, including detoxification of bile acid excess, stimulation of bile salt export from hepatocytes, reduction of inflammation, delaying fibrosis, and perhaps may even assist liver regeneration.^24,25 A promising alternative is obeticholic acid, that has got its approval by the FDA for cholestatic conditions like primary biliary cholangitis (PBC).²⁶ Research shows OCA significantly reduces key liver enzymes like alkaline phosphatase, gamma glutamyl transferase and total bilirubin in cholestatic liver disease, though its effects specifically in LT patients have not been well-studied.^27,28

This study aims to fill an essential gap by evaluating the efficacy of obeticholic acid compared to ursodeoxycholic acid in managing post-liver transplant cholestasis. By conducting specific biochemical tests to assess cholestasis, this research will provide insights into the effectiveness of OCA relative to the current standard, UDCA.²⁹ Additionally, this study will examine the potential effects of OCA on rejection, tissue regeneration, and cost-effectiveness—areas where knowledge is currently limited.^30,31 The ultimate goal of this study is to determine whether OCA can emerge as a new standard of care in post-LT management, offering cost-effective solutions to enhance long-term outcomes for liver transplant patients.

Primary outcomes

We are assessing the effects of obeticholic acid compared to ursodeoxycholic acid in post-liver transplant patients in ameliorating cholestatic injury with primary endpoints, a 15% reduction in alkaline phosphatase and gamma-glutamyl transferase levels at 3, 6, and 12 months from one-month baseline between groups.

Secondary outcomes

• Assessment of biochemical markers (Liver function tests, metabolic profile, CRP, tacrolimus levels).
• Molecular markers (fibroblast growth factor 19 (FGF-19), transforming growth factor β (TGF-β) level, Cytokeratin 18 level, Serum autotaxin level, Bile Salt Export Pump levels (BSEP), Plasma bile acid levels.
• Biopsy-proven acute or chronic rejection.
• Incidence of biliary complications.
• All-cause mortality.
• Any adverse events.
• Death due to acute or chronic rejection.
• Quality of life.

Methods

This RCT is being conducted in the Department of GI Surgery, Amrita Institute of Medical Sciences, Kochi.

Study design and participants

The study is being conducted as an investigator-initiated, parallel-group, open-label randomized controlled trial in patients undergoing live donor liver transplantation since December 2021 in the Department of Gastrointestinal Surgery at a tertiary care teaching hospital.

Eligibility & exclusion criteria

All adult patients undergoing live donor liver transplantation during the study period are being included. Recipients who are less than 18 years of age, those undergoing deceased donor liver transplants, ABO-incompatible transplants, retransplant cases, those not surviving beyond 30 days, and patients lost to follow-up during the study period, are being excluded. We excluded patients who were randomized into the trial but died within the first 30 days. This decision was based on the fact that early mortality in this period is most commonly attributable to sepsis or technical complications. Including such patients would not be appropriate for evaluating interventions aimed at the management of cholestasis, which usually occur 2 to 3 weeks after surgery. Mortality within 30 days, purely secondary to the adverse effect of the drug, with no other technical (vasculo-biliary), septic or immunological cause is extremely unlikely.

Recruitment & randomization

A clinical pharmacist is randomizing study participants admitted to the GI Surgery ICU following liver transplantation. A permuted block sequence of computer-generated random numbers is being used to enroll subjects into either the control arm (C) or the study arm (T)—i.e., to receive either the standard liver protectant ursodeoxycholic acid [URSETOR^® of Torrent Pharma] 10–15 mg/kg TID (control) or obeticholic acid 5 mg OD [OCANASH^® of Macleods Pharmaceutical Limited]. The sequence is being kept in sealed opaque envelopes and is being opened by the pharmacist on the second day following liver transplantation.

Given the difference in tablet size and dosing frequency (three times daily for UDCA vs once daily for OCA), blinding is not being done.

Follow-up visits for physical examinations and safety assessments are being scheduled during the study period at the following intervals: one month, three months, six months, and twelve months post-transplant ( Figure 1). Liver function tests, C-reactive protein (CRP), platelet count, prothrombin time/international normalized ratio (PT/INR), fasting blood sugar (FBS), tacrolimus concentration, and other relevant parameters are being measured at each follow-up visit. Lipid profile—including total cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL), very low-density lipoprotein (VLDL), and triglycerides (TG)—as well as glycosylated hemoglobin (HbA1c) levels, are being assessed at pre-transplant admission, and at three, six, and twelve months post-transplant.

Figure 1. Flow chart showing study methodology.

We selected a 15% reduction in both alkaline phosphatase (ALP) and gamma-glutamyl transferase (GGT) as our primary biochemical endpoint based on evidence from previous trials in cholestatic liver disease. Specifically, the landmark study by Nevens et al., published in The New England Journal of Medicine (2016), evaluated obeticholic acid in patients with primary biliary cholangitis and used a composite endpoint that included a ≥15% reduction in ALP as part of the therapeutic response criteria.

Although this trial was not conducted in post–liver transplant patients, it provided a strong precedent for using ALP-based endpoints to assess treatment response in cholestatic conditions. We extended this rationale by including GGT alongside ALP, as the combination of these two enzymes provides a more comprehensive and sensitive assessment of cholestasis, particularly in the post-transplant setting where bile duct injury and graft function are dynamic.

Given the lack of clinical trials of OCA in the liver transplant population, this combined biochemical endpoint serves as a reasonable and evidence-informed surrogate marker of response in our study.

Secondary endpoint serum markers are being assessed using an enzyme-linked immunosorbent assay (ELISA) [ELK Biotechnology™] at the centralized biochemistry laboratory. Blood samples collected during the one-month and twelfth-month post-transplant follow-up visits are being used for ELISA testing.

In addition, demographics, patient characteristics, and other critical study details (e.g., medication history, medical history, social habits, family history, and use of other medical systems) are being collected through direct patient or bystander interviews, doctor consultation notes, hospital health information systems, and case records. The collected data is being transcribed into a pre-designed proforma (data collection form) by the investigator.

Biopsy and rejection evaluation

A biopsy is being performed when suspected rejection (unexplained rise in transaminases) is observed. The biopsy results are being evaluated based on the Rejection Activity Index score and the Banff criteria. Data on the frequency and duration of the first biopsy-proven acute rejection (BPAR) episode (within six months of transplantation) that requires therapy are being recorded. Any biliary system-related strictures, leaks, or anastomosis issues are being documented. Early allograft dysfunction (EAD) is being assessed using the criteria developed by Olthoff et al.^7,32

Adverse reactions and causality assessment

Adverse events are being captured by direct interview of patients during the routine follow-up visits. We are capturing the pruritus by physical examination, vital signs and 5D pruritus questionnaire and documented systematically using structured Case Report Forms (CRFs) to ensure standardized data collection and analysis. The Naranjo algorithm will be used to establish causality and then classify them as definite, probable, possible, or unlikely.

Quality of life (QOL) measurement

During patient interviews, a validated Chronic Liver Disease Questionnaire (CLDQ) is being administered to assess quality of life (QOL).³³ The copyright license for the English version of the questionnaire is being obtained from the authors. The QOL questionnaire is being completed both pre-transplant and at the one-year post-transplant follow-up. Patients are completing the questionnaire in either Malayalam or English.

The CLDQ includes six domains:

• Abdominal symptoms (AB): Items 1, 5, 17
• Fatigue (FA): Items 2, 4, 8, 11, 13
• Systemic symptoms (SY): Items 3, 6, 21, 23, 27
• Activity (AC): Items 7, 9, 14
• Emotional functions (EM): Items 10, 12, 15, 16, 19, 20, 24, 26
• Worry (WO): Items 18, 22, 25

The CLDQ overall score is the sum of the scores from each domain, ranging from 0 to 203. Lower scores indicate poorer quality of life.

Sample size

The sample size was calculated based on a pilot study comparing the effect of obeticholic acid (OCA) and ursodeoxycholic acid (UDCA) on cholestatic injury in post-liver transplant patients. In the pilot study, the difference in alkaline phosphatase (5.131 ± 39.639 vs 12.496 ± 52.139) was observed between the two groups. With 80% power and 95% confidence, the minimum sample size required was determined to be 108 patients in each group, totaling 216 samples. We will be recruiting around 110 to 120 patients per arm.

Study status

The study is currently in the recruitment phase.

Statistical analysis

• To compare the means of continuous variables between groups (Drug A vs Drug B), an independent sample t-test will be used at each time point.
• To compare the changes in continuous variables from pre- (1 month) to post-transplant (3, 6, and 12 months), a paired t-test will be applied.
• To compare the means of variables from baseline to subsequent time points within each group, repeated measures ANOVA will be used if significant. The Bonferroni multiple comparison test will be applied for pairwise comparisons.

Expected results

We expect a significant reduction in cholestatic liver enzymes following LDLT in the OCA group compared to the UDCA group, with improvements observed from one month to subsequent time points (3, 6, and 12 months). We hypothesize that the potent mechanism of action of OCA will ameliorate cholestatic injury and enhance graft survival. Additionally, we anticipate a reduction in biliary complications, all-cause mortality, and an increase in quality of life for OCA-receiving patients. This may reduce long-term financial expenditure for patients. Given that there are currently no drugs to prevent damage to the transplanted liver, a positive outcome could be a significant advancement for the transplant population. If beneficial, this study could lead to the adoption of OCA as a standard of care following liver transplantation.

Conclusion

Obeticholic acid (OCA) is a farnesoid X receptor (FXR) agonist, with significantly more potency than UDCA in modulating bile acid metabolism. While OCA has shown promise in treating cholestatic liver diseases, its effects post-liver transplantation (LT) remain unclear due to the lack of high-quality trials. Our study aims to evaluate whether OCA’s strong FXR activation can benefit patients with post-LT cholestasis. We hypothesize that OCA will improve graft function, reduce biliary complications, lower associated costs, and enhance overall quality of life and survival for post-LT patients.

Confidentiality

All information about participants enrolled in this clinical trial are treated with the utmost confidentiality to protect their privacy and adhere to ethical standards. Personal identifying information are securely stored and accessible only to authorized personnel involved in the study. Data collected are anonymized and coded to maintain confidentiality during analysis and reporting. Any publication or presentation of results will not include identifiable information unless explicit consent is obtained from participants.

Access to data

Access to data collected during this clinical trial will be managed according to established protocols and regulatory requirements. Authorized personnel, including investigators, statisticians, and regulatory authorities, will have access to raw data for analysis, monitoring, and verification. Data-sharing agreements will be established to govern access by external parties, ensuring compliance with data protection regulations and participant confidentiality. Requests for access to study data from external researchers or institutions will be considered on a case-by-case basis and subject to review by the study sponsor and relevant ethics committees.

Data dissemination

Upon completion of the study, data will be disseminated through peer-reviewed journal publications, conference presentations, and participant communication. Findings will be shared transparently with participants, addressing risks and benefits. Regulatory reporting will ensure compliance with obligations to relevant agencies. Additionally, data-sharing agreements and repositories will facilitate access for other researchers while maintaining participant confidentiality.

Ethical considerations

The study protocol was initially approved by the Institutional Ethics Committee, AIMS (IEC-AIMS-2021-GISURG-262) on 13-10-2021 and later modified for a title change on 15-06-2022 (modified ethics number—IEC-AIMS-2022-GISURG-160). This study involves liver transplant patients aged 18 years or older admitted to our hospital’s gastrointestinal surgery department. Before the study’s initiation, patients gave written informed consent. The consent form was prepared both in English and the local language Malayalam. The study has received ethical clearance from our Institutional Ethics Committee. The committee also approved the informed consent in both languages. This study was registered with the Clinical Trials Registry of India. Our CTRI registration number is CTRI/2021/11/038218 dated on 23/11/2021. The study was conducted according to the principles of the Declaration of Helsinki.

Author contributions

• S. Sudhindran, Unnikrishnan G, Dinesh Balakrishnan, Johns Shaji Mathew, and Shweta Mallick contributed to formulating the concepts.
• Implementation of study including randomization, and data collection: Anila KN, Saraswathy S Nair, Nafiya Zackariah, Haritha Rajakrishnan.
• Drafting manuscript: Anila K N.
• S. Sudhindran, OV Sudheer, Arun Valsan, Binoj ST, Ramachandran Narayana Menon, Krishnanunni Nair, Madhu Srinivasan Durairaj, V Guhan, Christi Titus Varghese contributed to giving valuable inputs and critical revisions.

Data availability statement

No data are associated with this article.

Extended data

Open Science Frame (OSF): FXR agonist in post Liver transplantation patients: A randomized open-labeled study, DOI: 10.17605/OSF.IO/JDXBF³⁴

This project contains the following underlying data:

1. CLD malayalam questionnaire. Pdf
2. CLD QUESTIONNAIRE english.pdf
3. Data collection CRF.pdf
4. Ethics docs.zip
5. Fillable-SPIRIT-Outcomes-2022-Checklist-with-SPIRIT-2013.pdf
6. INFORMED CONSENT DOCUMENT English final.pdf
7. Informed consent Malayalam modified.pdf

Data are available under the terms of the Creative Commons Zero “No Rights Reserved” data waiver (CC0 1.0 Public Domain Dedication).

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Version 2

VERSION 2 PUBLISHED 02 Jan 2025

Author details Author details

Anila K N
Roles: Data Curation, Formal Analysis, Investigation, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing

Saraswathy S Nair
Roles: Data Curation, Methodology

Dinesh Balakrishnan`
Roles: Conceptualization, Supervision, Writing – Review & Editing

Unnikrishnan G
Roles: Conceptualization, Methodology, Supervision, Writing – Review & Editing

Binoj S T
Roles: Writing – Review & Editing

Ramachandran Narayana Menon
Roles: Conceptualization

Christi Titus Varghese
Roles: Writing – Review & Editing

Shweta Mallick
Roles: Writing – Review & Editing

Krishnanunni Nair
Roles: Resources, Writing – Review & Editing

Madhu Srinivasan Durairaj
Roles: Writing – Review & Editing

Guhan V
Roles: Writing – Review & Editing

Nafiya Zackariah
Roles: Methodology

Haritha Rajakrishnan
Roles: Methodology

Arun Valsan
Roles: Writing – Review & Editing

Johns Shaji Mathew
Roles: Conceptualization, Methodology

Cyriac Abby Philips
Roles: Writing – Review & Editing

Christopher Watson
Roles: Writing – Review & Editing

Sudheer O V
Roles: Writing – Review & Editing

Sudhindran S
Roles: Conceptualization, Funding Acquisition, Methodology, Resources, Supervision, Writing – Review & Editing

Competing interests

No competing interests were disclosed.

Grant information

This work has recently received seed grant funding from the institution’s Research fund.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Article Versions (2)

version 2

Revised

Published: 27 Jun 2025, 14:9

https://doi.org/10.12688/f1000research.159202.2

version 1

Published: 02 Jan 2025, 14:9

https://doi.org/10.12688/f1000research.159202.1

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K N A, S Nair S, Balakrishnan` D et al. FXR agonist in post-liver transplantation patients: a randomized open-labeled study. [version 2; peer review: 2 approved with reservations]. F1000Research 2025, 14:9 (https://doi.org/10.12688/f1000research.159202.2)

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Open Peer Review

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PUBLISHED 02 Jan 2025

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Reviewer Report 15 May 2025

Gregory Guthrie, Baylor College of Medicine, Houston, Texas, USA

Approved with Reservations

https://doi.org/10.5256/f1000research.174896.r377804

This manuscript addresses an important clinical question: the optimal management of post-liver transplant (LT) cholestasis, comparing Obeticholic Acid (OCA) with the current standard, Ursodeoxycholic Acid (UDCA). The rationale for exploring FXR agonists like OCA in this setting is sound, given their known roles in bile acid homeostasis, inflammation, and potentially regeneration.
However, for the manuscript to be considered for indexed, several weaknesses need to be addressed, and significant clarifications and additions are required.

Major Weaknesses and Areas Requiring Clarification/Addition:

Study Timeline and Design Clarity (Critical Flaw):
- Contradiction: The "Methods" section states the study "was conducted... from December 2014 to 2021." However, the "Study status" section says, "The study is currently in the recruitment phase." Furthermore, ethics approval is dated 2021/2022, and CTRI registration is from 2021. This is a fundamental contradiction. My understanding is that it is patients that received LT in these years that will be recruited to the study, but the constant change is present and past tense, and the wording makes this unclear. Furthermore, exclusion criteria for patients that died within 30 days would also suggest a difference in timeline, as a patient who died in 2014 after 30 days post LT would certainly not be available to include in a study taking place in 2025. Otherwise, is this exclusion criteria for patients that are started on the study and die within this time frame, in which case this could exclude patients experiencing severe early adverse events or graft failure potentially related to the interventions. A rationale for this exclusion is needed.
Rationale for OCA Dosage and Safety in LT Population:
- FDA Warning Context: The introduction mentions, "In individuals with advanced liver disease, the FDA has cautioned against administering OCA at doses of 10–50 mg." Liver transplant recipients, by definition, have end-stage liver disease pre-transplant and a recovering, vulnerable graft post-transplant. While the study uses 5 mg OD, which is lower, the potential for OCA-related complications (decompensation, worsening LFTs before improvement, severe pruritus) in this specific population needs more thorough discussion.
Primary Endpoint Justification and Baseline:
- 15% Reduction: The primary endpoint is a "15% reduction in alkaline phosphatase and gamma-glutamyl transferase levels." What is the clinical significance of a 15% reduction in these enzymes in post-LT cholestasis? Is this threshold based on previous studies in LT or other cholestatic conditions demonstrating improved long-term outcomes? This needs robust justification.
- One-Month Baseline: The baseline for assessing the primary endpoint is "one-month" post-transplant. Significant cholestasis and LFT fluctuations can occur much earlier post-LT. Why was a 1-month baseline chosen? This might miss early effects or be confounded by stabilization that might occur irrespective of the study drug. A rationale for this time point is crucial.
  - Omission of Bilirubin: Total bilirubin is a key marker of cholestasis severity and liver function. Why is bilirubin not included as a primary or at least a key secondary endpoint for cholestasis assessment?
Sample Size Calculation and Pilot Study Data:
- The pilot study data used for sample size calculation ("difference in alkaline phosphatase (5.131 ± 39.639 vs 12.496 ± 52.139)") shows extremely large standard deviations relative to the means. This suggests massive variability or heterogeneity in the pilot data, or potentially a very small effect size, making the observed difference potentially unreliable for powering a larger trial. A difference of ~7 U/L (assuming these are absolute values) seems very small.
Exclusion Criteria and Analysis Population:
- "patients who discontinued study drugs were excluded." Excluding patients who discontinue the drug from the analysis can introduce significant bias. An intention-to-treat (ITT) analysis, including all randomized patients in the groups to which they were originally assigned, is standard for RCTs and should be the primary analysis. A per-protocol analysis can be secondary. This needs to be specified in the statistical analysis section.
- See earlier comment on exclusion criteria of patients that died <30 days.

Secondary Weaknesses and Areas for Clarification/Addition:

UDCA Dosing:
- - UDCA is dosed at "10-15 mg/kg TID." Typically, UDCA is administered BID in post-transplant settings. Provide a rationale for the TID dosing regimen for UDCA.
Measurement of Specific Secondary Endpoints:
- - "Bile Salt Export Pump levels (BSEP)": How will BSEP levels be measured? Perhaps I am mistaken, but BSEP is a membrane-bound transporter, so will only be measured with biopsies, as in you methods, will only be done in the suspicion of rejection. This is currently too vague.
- "Plasma bile acid levels":
  - Clarification Needed: Will this be total plasma bile acids, or will specific bile acid species be fractionated and quantified (e.g., via LC-MS/MS)? The latter would be much more informative regarding FXR target engagement and changes in bile acid pool composition.
Definition of "Cellular Level" Assessment:
- The Rationale mentions "specific biochemical tests to assess cholestasis at the cellular level."
  - Clarification Needed: Which specific tests (beyond standard LFTs and the listed secondary markers) are being referred to that assess cholestasis at a "cellular level"? This phrasing needs to be more precise or linked directly to specific planned assays (e.g., markers of oxidative stress, apoptosis, or specific transport protein expression if biopsies are analyzed beyond histology).
Introduction and Conclusion Phrasing:
- Introduction: "Although its usefulness is questionable, many practitioners utilize ursodeoxycholic acid (UDCA)..." While there are debates, phrasing it as "questionable" without immediate specific referencing for the LT context might be too strong. Perhaps "its efficacy for preventing all forms of post-LT cholestasis or improving long-term graft survival has limitations" or similar.
- Conclusion: "Obeticholic acid (OCA) is a potent farnesoid X receptor (FXR) agonist, with 100 times the efficacy of the current standard treatment, UDCA..." This is misleading. OCA is ~100 times more potent as an FXR agonist than chenodeoxycholic acid (a primary bile acid), not UDCA. UDCA is not a potent FXR agonist; it has different mechanisms of action (choleretic, cytoprotective, immunomodulatory). This statement needs correction for accuracy.

The study addresses a relevant clinical need. However, the current manuscript contains fundamental inconsistencies and lacks necessary detail and justification in several key areas. Addressing these points comprehensively will be essential for its scientific validity and potential for indexing.

Is the rationale for, and objectives of, the study clearly described?

Yes
Is the study design appropriate for the research question?

Yes
Are sufficient details of the methods provided to allow replication by others?

No
Are the datasets clearly presented in a useable and accessible format?

Partly

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: My research is focused on mechanisms and treatment of cholestatic liver diseases.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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Author Response 11 Sep 2025

Anila K N, GI Surgery, Amrita Institute of Medical Sciences & Research Centre, Kochi, Amrita Vishwa Vidyapeetham, Coimbatore, India

11 Sep 2025

Author Response

Thank you very much for the very pertinent comments.

Major Weaknesses and Areas Requiring Clarification/Addition:
Query Study Timeline and Design Clarity (Critical Flaw):
Contradiction: The "Methods" section states ... Continue reading Thank you very much for the very pertinent comments.

Major Weaknesses and Areas Requiring Clarification/Addition:
Query Study Timeline and Design Clarity (Critical Flaw):
Contradiction: The "Methods" section states the study "was conducted... from
December 2014 to 2021." However, the "Study status" section says, "The study is
currently in the recruitment phase." Furthermore, ethics approval is dated 2021/2022,
and CTRI registration is from 2021. This is a fundamental contradiction. My
understanding is that it is patients that received LT in these years that will be
recruited to the study, but the constant change is present and past tense, and the
wording makes this unclear.

Reply: Thank you for highlighting these points. We apologize for the mistake in the dates.
The Institutional Ethics Committee approval for the study was obtained in August 2021, and the CTRI registration was obtained on 23/11/2021. The study commenced thereafter. The data collection period for the pilot study was mistakenly reported as December 2014 to 2021." and has now been corrected as given below. We have also addressed the inconsistencies in verb tense throughout the manuscript. We appreciate your careful review and constructive feedback.

Correction in the manuscript:

Study design and participants
The study is being conducted as an investigator-initiated, parallel-group, open-label randomized controlled trial in patients undergoing live donor liver transplantation since December 2021 in the Department of Gastrointestinal Surgery at a tertiary care teaching hospital.” under Study design and participants.

Ethical considerations
The study protocol was initially approved by the Institutional Ethics Committee, AIMS (IEC-AIMS-2021-GISURG-262) on 13-10-2021 and later modified for a title change on 15-06-2022 (modified ethics number—IEC-AIMS-2022-GISURG-160). This study involves liver transplant patients aged 18 years or older admitted to our hospital’s gastrointestinal surgery department. Before the study’s initiation, patients gave written informed consent. The consent form was prepared both in English and the local language Malayalam. The study has received ethical clearance from our Institutional Ethics Committee. The committee also approved the informed consent in both languages. This study was registered with the Clinical Trials Registry of India. Our CTRI registration number is CTRI/2021/11/038218 dated on 23/11/2021. The study was conducted according to the principles of the Declaration of Helsinki.

Query: Furthermore, exclusion criteria for patients that died within 30 days would also suggest a difference in timeline, as a patient who died in 2014 after 30 days post LT would certainly not be available to include in a study taking place in 2025. Otherwise, is this exclusion criteria for patients that are started on the study and die within this time frame, in which case this could exclude patients experiencing severe early adverse events or graft failure potentially related to the interventions. A rationale for this exclusion is needed.

Explanation: Thank you once again for pointing out these observations.
The confusion is partly due to the mistake in the date, as the study started only in Dec 21. (which we have corrected as above)
We excluded patients who were randomized into the trial but died within the first 30 days. This decision was based on the fact that early mortality in this period is most commonly attributable to sepsis or technical complications. Including such patients would not be appropriate for evaluating interventions aimed at the management of cholestasis, which usually occur 2 to 3 weeks after surgery. Mortality within 30 days, purely secondary to the adverse effect of the drug, with no other technical ( vasculo-biliary), septic or immunological cause is extremely unlikely. Furthermore, we require at least 4 to 6 weeks of drug therapy for any noticeable effect on cholestasis. Patients dying within 30 days will not get this time period.

We have added this in the manuscript s follows: We excluded patients who were randomized into the trial but died within the first 30 days. This decision was based on the fact that early mortality in this period is most commonly attributable to sepsis or technical complications. Including such patients would not be appropriate for evaluating interventions aimed at the management of cholestasis, which usually occur 2 to 3 weeks after surgery. Mortality within 30 days, purely secondary to the adverse effect of the drug, with no other technical ( vasculo-biliary), septic or immunological cause is extremely unlikely.

Query: Rationale for OCA Dosage and Safety in LT Population:
FDA Warning Context: The introduction mentions, "In individuals with advanced liver
disease, the FDA has cautioned against administering OCA at doses of 10–50 mg."
Liver transplant recipients, by definition, have end-stage liver disease pre-transplant
and a recovering, vulnerable graft post-transplant. While the study uses 5 mg OD,
which is lower, the potential for OCA-related complications (decompensation,
worsening LFTs before improvement, severe pruritus) in this specific population
needs more thorough discussion.

Reply: Thank you very much for your valuable comment.

The FDA Blackbox warning was issued on the higher doses of obeticholic acid (10–50 mg) in individuals with advanced liver disease. As you correctly pointed out, liver transplant recipients represent a unique and vulnerable population with recovering graft function, and careful attention to OCA-related safety concerns is warranted.

In our study, we have used a conservative dose of 5 mg once daily, which is well below the threshold associated with reported complications. Nevertheless, as this is an open-label trial, we are closely monitoring for potential adverse effects, including hepatic decompensation, transient worsening of liver function tests, and pruritus. These safety parameters are being actively assessed throughout the study duration and will be discussed comprehensively in the final analysis and manuscript.

Query: Primary Endpoint Justification and Baseline:
15% Reduction: The primary endpoint is a "15% reduction in alkaline phosphatase
and gamma-glutamyl transferase levels." What is the clinical significance of a 15%
reduction in these enzymes in post-LT cholestasis? Is this threshold based on
previous studies in LT or other cholestatic conditions demonstrating improved long
term outcomes? This needs robust justification.

Reply: We selected a 15% reduction in both alkaline phosphatase (ALP) and gamma-glutamyltransferase (GGT) as our primary biochemical endpoint based on evidence from previous trials in cholestatic liver disease. Specifically, the landmark study by Nevens et al., published in The New England Journal of Medicine (2016), evaluated obeticholic acid in patients with primary biliary cholangitis and used a composite endpoint that included a ≥15% reduction in ALP as part of the therapeutic response criteria.
Although this trial was not conducted in post–liver transplant patients, it provided a strong precedent for using ALP-based endpoints to assess treatment response in cholestatic conditions. We extended this rationale by including GGT alongside ALP, as the combination of these two enzymes provides a more comprehensive and sensitive assessment of cholestasis, particularly in the post-transplant setting where bile duct injury and graft function are dynamic.
Given the lack of clinical trials of OCA in the liver transplant population, this combined biochemical endpoint serves as a reasonable and evidence-informed surrogate marker of response in our study.

Query: One-Month Baseline: The baseline for assessing the primary endpoint is "one
month" post-transplant. Significant cholestasis and LFT fluctuations can occur much
earlier post-LT. Why was a 1-month baseline chosen? This might miss early effects or
be confounded by stabilization that might occur irrespective of the study drug. A
rationale for this time point is crucial.

Reply: We chose to initiate study assessments after one-month post-transplant because a significant proportion of our liver transplant recipients are high MELD patients who typically present with markedly elevated bilirubin and cholestatic enzyme levels at baseline. Additionally, in the immediate post-transplant period, transient elevations in liver function tests are commonly observed due to ischemia–reperfusion injury, surgical factors, sepsis, or early graft rejection.
In live donor liver transplant for high MELD recipients, these fluctuations are often pronounced during the first few weeks following transplantation. Therefore, a 4-week window was provided to allow for stabilization of liver function parameters and resolution of early postoperative complications, ensuring that subsequent measurements more accurately reflect the effect of the study intervention rather than early perioperative variability.

Query: Omission of Bilirubin: Total bilirubin is a key marker of cholestasis severity
and liver function. Why is bilirubin not included as a primary or at least a key
secondary endpoint for cholestasis assessment?
Reply: Total bilirubin has been retained as an important secondary endpoint, given its clinical relevance in assessing hepatocellular function and persistent cholestasis. Indeed, liver function tests including bilirubin levels are listed as the first secondary end point.

Query: Sample Size Calculation and Pilot Study Data:
The pilot study data used for sample size calculation ("difference in alkaline
phosphatase (5.131 ± 39.639 vs 12.496 ± 52.139)") shows extremely large standard
deviations relative to the means. This suggests massive variability or heterogeneity in
the pilot data, or potentially a very small effect size, making the observed difference
potentially unreliable for powering a larger trial. A difference of ~7 U/L (assuming
these are absolute values) seems very small.

Reply: Regarding the sample size calculation, we acknowledge that data specific to the post–liver transplant population receiving obeticholic acid is extremely limited. Apart from our pilot study, we were unable to identify any previously published data in this specific setting to guide formal power calculations. Based on the effect sizes and variability observed in our pilot data, our statistician advised that this sample size would be adequate to provide preliminary evidence and inform the design of a larger, adequately powered trial in the future.

Exclusion Criteria and Analysis Population:
"patients who discontinued study drugs were excluded." Excluding patients who
discontinue the drug from the analysis can introduce significant bias. An intention-to
treat (ITT) analysis, including all randomized patients in the groups to which they
were originally assigned, is standard for RCTs and should be the primary analysis. A
per-protocol analysis can be secondary. This needs to be specified in the statistical
analysis section.
See earlier comment on exclusion criteria of patients that died <30 days.

Reply: Thank you very much for your observation. We are in fact conducting this trial as an intention-to-treat (ITT) analysis approach. Accordingly, all randomized patients—including those who discontinue the study prematurely—will be included in the final analysis. Efforts will be made to document and analyze the reasons for discontinuation, as this information is critical for understanding both the tolerability and the real-world applicability of the intervention.

We have modified the exclusion criteria after removing this sentence as follows:
We excluded patients who were randomized into the trial but died within the first 30 days. This decision was based on the fact that early mortality in this period is most commonly attributable to sepsis or technical complications. Including such patients would not be appropriate for evaluating interventions aimed at the management of cholestasis, which usually occur 2 to 3 weeks after surgery. Mortality within 30 days, purely secondary to the adverse effect of the drug, with no other technical ( vasculo-biliary), septic or immunological cause is extremely unlikely.

Query: Secondary Weaknesses and Areas for Clarification/Addition:
UDCA Dosing:
UDCA is dosed at "10-15 mg/kg TID." Typically, UDCA is administered BID in
post-transplant settings. Provide a rationale for the TID dosing regimen for
UDCA.

Reply: Thank you for your comment regarding dosing. The typical recommended dosage for managing cholestasis—particularly with ursodeoxycholic acid (UDCA)—is 10 to 15 mg/kg/day, usually administered in three divided doses. Based on this standard recommendation, we have selected the dose accordingly in our study to ensure therapeutic efficacy while maintaining safety and consistency with existing clinical guidelines.
Several peer-reviewed studies and reviews that report the use of ursodeoxycholic acid (UDCA) at doses of 10–15 mg/kg/day in post–liver transplant patients for prophylaxis and early graft protection (Ref : Pedersen, Mark R.^*,1; Greenan, Garrett²; Arora, Sumant³; Murali, Arvind R.³; Mayo, Marlyn J.¹. Ursodeoxycholic Acid Decreases Incidence of Primary Biliary Cholangitis and Biliary Complications After Liver Transplantation: A Meta‐Analysis. Liver Transplantation 27(6):p 866-875, June 2021. | DOI: 10.1002/lt.25935). For an adult with normal/average body weight, the doses of UDCA thus may be divided to three times a day.

Query: Measurement of Specific Secondary Endpoints:
"Bile Salt Export Pump levels (BSEP)": How will BSEP levels be measured?
Perhaps I am mistaken, but BSEP is a membrane-bound transporter, so will
only be measured with biopsies, as in you methods, will only be done in the
suspicion of rejection. This is currently too vague.

Reply: Thank you for your thoughtful feedback. You’re absolutely right—BSEP is a membrane‑bound canalicular transporter and, under normal conditions, is not released into the bloodstream in its intact form. In our study, however, we specifically sought to identify and quantify membrane‑derived fragments of BSEP that may be shed into the circulation during post‑transplant hepatocellular injury. To achieve this, we used a commercial sandwich ELISA kit validated for human serum and plasma. It is probable that the usefulness may not be as much as measuring the tissue BSEP levels, which will entail multiple liver biopsies in our patient. Our study results will hopefully give a better clue.

Query: "Plasma bile acid levels":
Clarification Needed: Will this be total plasma bile acids, or will specific bile
acid species be fractionated and quantified (e.g., via LC-MS/MS)? The latter
would be much more informative regarding FXR target engagement and
changes in bile acid pool composition.

Reply: Thank you for raising this important point. In our study, total plasma bile acid levels were quantified using commercially available ELISA kits. While we fully agree that fractionated bile acid profiling using LC-MS/MS would provide deeper insights into FXR target engagement and alterations in bile acid pool composition, this approach was not feasible in our current setting due to budgetary constraints and limited access to mass spectrometry facilities.
Should we secure adequate funding in the future, we certainly plan to incorporate detailed bile acid species quantification via LC-MS/MS to enhance the mechanistic depth of our analysis.

Definition of "Cellular Level" Assessment:
The Rationale mentions "specific biochemical tests to assess cholestasis at the cellular
Clarification Needed: Which specific tests (beyond standard LFTs and the
listed secondary markers) are being referred to that assess cholestasis at a
"cellular level"? This phrasing needs to be more precise or linked directly to
specific planned assays (e.g., markers of oxidative stress, apoptosis, or specific
transport protein expression if biopsies are analyzed beyond histology).

Thank you very much for the comment. We have removed the usage of “cellular” from the context

The text in the manuscript modified as below:
This study aims to fill an essential gap by evaluating the efficacy of obeticholic acid compared to ursodeoxycholic Acid in managing post-liver transplant cholestasis. By conducting specific biochemical tests to assess cholestasis, this research will provide insights into the effectiveness of OCA relative to the current standard, UDCA.

Query: Introduction and Conclusion Phrasing:
Introduction: "Although its usefulness is questionable, many practitioners utilize
ursodeoxycholic acid (UDCA)..." While there are debates, phrasing it as "questionable"
without immediate specific referencing for the LT context might be too strong.
Perhaps "its efficacy for preventing all forms of post-LT cholestasis or improving long
term graft survival has limitations" or similar.

Reply: Thank you very much for the comment. We have changed the sentence according to your suggestions.
While many practitioners utilize UDCA as a hepatoprotective drug, its efficacy for preventing all forms of post liver transplant cholestasis or improving long-term graft server has limitations.

Query: Conclusion: "Obeticholic acid (OCA) is a potent farnesoid X receptor (FXR) agonist,
with 100 times the efficacy of the current standard treatment, UDCA..." This is
misleading. OCA is ~100 times more potent as an FXR agonist than chenodeoxycholic
acid (a primary bile acid), not UDCA. UDCA is not a potent FXR agonist; it has different
mechanisms of action (choleretic, cytoprotective, immunomodulatory). This
statement needs correction for accuracy.

Reply:Thank you very much for the comment. We have changed the sentence according to your suggestions.

Sentence now reads as’
Obeticholic acid (OCA) is a farnesoid X receptor (FXR) agonist, with significantly more potency than UDCA in modulating bile acid metabolism.
Thank you very much for the very pertinent comments.

Major Weaknesses and Areas Requiring Clarification/Addition:
Query Study Timeline and Design Clarity (Critical Flaw):
Contradiction: The "Methods" section states the study "was conducted... from
December 2014 to 2021." However, the "Study status" section says, "The study is
currently in the recruitment phase." Furthermore, ethics approval is dated 2021/2022,
and CTRI registration is from 2021. This is a fundamental contradiction. My
understanding is that it is patients that received LT in these years that will be
recruited to the study, but the constant change is present and past tense, and the
wording makes this unclear.

Reply: Thank you for highlighting these points. We apologize for the mistake in the dates.
The Institutional Ethics Committee approval for the study was obtained in August 2021, and the CTRI registration was obtained on 23/11/2021. The study commenced thereafter. The data collection period for the pilot study was mistakenly reported as December 2014 to 2021." and has now been corrected as given below. We have also addressed the inconsistencies in verb tense throughout the manuscript. We appreciate your careful review and constructive feedback.

Correction in the manuscript:

Study design and participants
The study is being conducted as an investigator-initiated, parallel-group, open-label randomized controlled trial in patients undergoing live donor liver transplantation since December 2021 in the Department of Gastrointestinal Surgery at a tertiary care teaching hospital.” under Study design and participants.

Ethical considerations
The study protocol was initially approved by the Institutional Ethics Committee, AIMS (IEC-AIMS-2021-GISURG-262) on 13-10-2021 and later modified for a title change on 15-06-2022 (modified ethics number—IEC-AIMS-2022-GISURG-160). This study involves liver transplant patients aged 18 years or older admitted to our hospital’s gastrointestinal surgery department. Before the study’s initiation, patients gave written informed consent. The consent form was prepared both in English and the local language Malayalam. The study has received ethical clearance from our Institutional Ethics Committee. The committee also approved the informed consent in both languages. This study was registered with the Clinical Trials Registry of India. Our CTRI registration number is CTRI/2021/11/038218 dated on 23/11/2021. The study was conducted according to the principles of the Declaration of Helsinki.

Query: Furthermore, exclusion criteria for patients that died within 30 days would also suggest a difference in timeline, as a patient who died in 2014 after 30 days post LT would certainly not be available to include in a study taking place in 2025. Otherwise, is this exclusion criteria for patients that are started on the study and die within this time frame, in which case this could exclude patients experiencing severe early adverse events or graft failure potentially related to the interventions. A rationale for this exclusion is needed.

Explanation: Thank you once again for pointing out these observations.
The confusion is partly due to the mistake in the date, as the study started only in Dec 21. (which we have corrected as above)
We excluded patients who were randomized into the trial but died within the first 30 days. This decision was based on the fact that early mortality in this period is most commonly attributable to sepsis or technical complications. Including such patients would not be appropriate for evaluating interventions aimed at the management of cholestasis, which usually occur 2 to 3 weeks after surgery. Mortality within 30 days, purely secondary to the adverse effect of the drug, with no other technical ( vasculo-biliary), septic or immunological cause is extremely unlikely. Furthermore, we require at least 4 to 6 weeks of drug therapy for any noticeable effect on cholestasis. Patients dying within 30 days will not get this time period.

We have added this in the manuscript s follows: We excluded patients who were randomized into the trial but died within the first 30 days. This decision was based on the fact that early mortality in this period is most commonly attributable to sepsis or technical complications. Including such patients would not be appropriate for evaluating interventions aimed at the management of cholestasis, which usually occur 2 to 3 weeks after surgery. Mortality within 30 days, purely secondary to the adverse effect of the drug, with no other technical ( vasculo-biliary), septic or immunological cause is extremely unlikely.

Query: Rationale for OCA Dosage and Safety in LT Population:
FDA Warning Context: The introduction mentions, "In individuals with advanced liver
disease, the FDA has cautioned against administering OCA at doses of 10–50 mg."
Liver transplant recipients, by definition, have end-stage liver disease pre-transplant
and a recovering, vulnerable graft post-transplant. While the study uses 5 mg OD,
which is lower, the potential for OCA-related complications (decompensation,
worsening LFTs before improvement, severe pruritus) in this specific population
needs more thorough discussion.

Reply: Thank you very much for your valuable comment.

The FDA Blackbox warning was issued on the higher doses of obeticholic acid (10–50 mg) in individuals with advanced liver disease. As you correctly pointed out, liver transplant recipients represent a unique and vulnerable population with recovering graft function, and careful attention to OCA-related safety concerns is warranted.

In our study, we have used a conservative dose of 5 mg once daily, which is well below the threshold associated with reported complications. Nevertheless, as this is an open-label trial, we are closely monitoring for potential adverse effects, including hepatic decompensation, transient worsening of liver function tests, and pruritus. These safety parameters are being actively assessed throughout the study duration and will be discussed comprehensively in the final analysis and manuscript.

Query: Primary Endpoint Justification and Baseline:
15% Reduction: The primary endpoint is a "15% reduction in alkaline phosphatase
and gamma-glutamyl transferase levels." What is the clinical significance of a 15%
reduction in these enzymes in post-LT cholestasis? Is this threshold based on
previous studies in LT or other cholestatic conditions demonstrating improved long
term outcomes? This needs robust justification.

Reply: We selected a 15% reduction in both alkaline phosphatase (ALP) and gamma-glutamyltransferase (GGT) as our primary biochemical endpoint based on evidence from previous trials in cholestatic liver disease. Specifically, the landmark study by Nevens et al., published in The New England Journal of Medicine (2016), evaluated obeticholic acid in patients with primary biliary cholangitis and used a composite endpoint that included a ≥15% reduction in ALP as part of the therapeutic response criteria.
Although this trial was not conducted in post–liver transplant patients, it provided a strong precedent for using ALP-based endpoints to assess treatment response in cholestatic conditions. We extended this rationale by including GGT alongside ALP, as the combination of these two enzymes provides a more comprehensive and sensitive assessment of cholestasis, particularly in the post-transplant setting where bile duct injury and graft function are dynamic.
Given the lack of clinical trials of OCA in the liver transplant population, this combined biochemical endpoint serves as a reasonable and evidence-informed surrogate marker of response in our study.

Query: One-Month Baseline: The baseline for assessing the primary endpoint is "one
month" post-transplant. Significant cholestasis and LFT fluctuations can occur much
earlier post-LT. Why was a 1-month baseline chosen? This might miss early effects or
be confounded by stabilization that might occur irrespective of the study drug. A
rationale for this time point is crucial.

Reply: We chose to initiate study assessments after one-month post-transplant because a significant proportion of our liver transplant recipients are high MELD patients who typically present with markedly elevated bilirubin and cholestatic enzyme levels at baseline. Additionally, in the immediate post-transplant period, transient elevations in liver function tests are commonly observed due to ischemia–reperfusion injury, surgical factors, sepsis, or early graft rejection.
In live donor liver transplant for high MELD recipients, these fluctuations are often pronounced during the first few weeks following transplantation. Therefore, a 4-week window was provided to allow for stabilization of liver function parameters and resolution of early postoperative complications, ensuring that subsequent measurements more accurately reflect the effect of the study intervention rather than early perioperative variability.

Query: Omission of Bilirubin: Total bilirubin is a key marker of cholestasis severity
and liver function. Why is bilirubin not included as a primary or at least a key
secondary endpoint for cholestasis assessment?
Reply: Total bilirubin has been retained as an important secondary endpoint, given its clinical relevance in assessing hepatocellular function and persistent cholestasis. Indeed, liver function tests including bilirubin levels are listed as the first secondary end point.

Query: Sample Size Calculation and Pilot Study Data:
The pilot study data used for sample size calculation ("difference in alkaline
phosphatase (5.131 ± 39.639 vs 12.496 ± 52.139)") shows extremely large standard
deviations relative to the means. This suggests massive variability or heterogeneity in
the pilot data, or potentially a very small effect size, making the observed difference
potentially unreliable for powering a larger trial. A difference of ~7 U/L (assuming
these are absolute values) seems very small.

Reply: Regarding the sample size calculation, we acknowledge that data specific to the post–liver transplant population receiving obeticholic acid is extremely limited. Apart from our pilot study, we were unable to identify any previously published data in this specific setting to guide formal power calculations. Based on the effect sizes and variability observed in our pilot data, our statistician advised that this sample size would be adequate to provide preliminary evidence and inform the design of a larger, adequately powered trial in the future.

Exclusion Criteria and Analysis Population:
"patients who discontinued study drugs were excluded." Excluding patients who
discontinue the drug from the analysis can introduce significant bias. An intention-to
treat (ITT) analysis, including all randomized patients in the groups to which they
were originally assigned, is standard for RCTs and should be the primary analysis. A
per-protocol analysis can be secondary. This needs to be specified in the statistical
analysis section.
See earlier comment on exclusion criteria of patients that died <30 days.

Reply: Thank you very much for your observation. We are in fact conducting this trial as an intention-to-treat (ITT) analysis approach. Accordingly, all randomized patients—including those who discontinue the study prematurely—will be included in the final analysis. Efforts will be made to document and analyze the reasons for discontinuation, as this information is critical for understanding both the tolerability and the real-world applicability of the intervention.

We have modified the exclusion criteria after removing this sentence as follows:
We excluded patients who were randomized into the trial but died within the first 30 days. This decision was based on the fact that early mortality in this period is most commonly attributable to sepsis or technical complications. Including such patients would not be appropriate for evaluating interventions aimed at the management of cholestasis, which usually occur 2 to 3 weeks after surgery. Mortality within 30 days, purely secondary to the adverse effect of the drug, with no other technical ( vasculo-biliary), septic or immunological cause is extremely unlikely.

Query: Secondary Weaknesses and Areas for Clarification/Addition:
UDCA Dosing:
UDCA is dosed at "10-15 mg/kg TID." Typically, UDCA is administered BID in
post-transplant settings. Provide a rationale for the TID dosing regimen for
UDCA.

Reply: Thank you for your comment regarding dosing. The typical recommended dosage for managing cholestasis—particularly with ursodeoxycholic acid (UDCA)—is 10 to 15 mg/kg/day, usually administered in three divided doses. Based on this standard recommendation, we have selected the dose accordingly in our study to ensure therapeutic efficacy while maintaining safety and consistency with existing clinical guidelines.
Several peer-reviewed studies and reviews that report the use of ursodeoxycholic acid (UDCA) at doses of 10–15 mg/kg/day in post–liver transplant patients for prophylaxis and early graft protection (Ref : Pedersen, Mark R.^*,1; Greenan, Garrett²; Arora, Sumant³; Murali, Arvind R.³; Mayo, Marlyn J.¹. Ursodeoxycholic Acid Decreases Incidence of Primary Biliary Cholangitis and Biliary Complications After Liver Transplantation: A Meta‐Analysis. Liver Transplantation 27(6):p 866-875, June 2021. | DOI: 10.1002/lt.25935). For an adult with normal/average body weight, the doses of UDCA thus may be divided to three times a day.

Query: Measurement of Specific Secondary Endpoints:
"Bile Salt Export Pump levels (BSEP)": How will BSEP levels be measured?
Perhaps I am mistaken, but BSEP is a membrane-bound transporter, so will
only be measured with biopsies, as in you methods, will only be done in the
suspicion of rejection. This is currently too vague.

Reply: Thank you for your thoughtful feedback. You’re absolutely right—BSEP is a membrane‑bound canalicular transporter and, under normal conditions, is not released into the bloodstream in its intact form. In our study, however, we specifically sought to identify and quantify membrane‑derived fragments of BSEP that may be shed into the circulation during post‑transplant hepatocellular injury. To achieve this, we used a commercial sandwich ELISA kit validated for human serum and plasma. It is probable that the usefulness may not be as much as measuring the tissue BSEP levels, which will entail multiple liver biopsies in our patient. Our study results will hopefully give a better clue.

Query: "Plasma bile acid levels":
Clarification Needed: Will this be total plasma bile acids, or will specific bile
acid species be fractionated and quantified (e.g., via LC-MS/MS)? The latter
would be much more informative regarding FXR target engagement and
changes in bile acid pool composition.

Reply: Thank you for raising this important point. In our study, total plasma bile acid levels were quantified using commercially available ELISA kits. While we fully agree that fractionated bile acid profiling using LC-MS/MS would provide deeper insights into FXR target engagement and alterations in bile acid pool composition, this approach was not feasible in our current setting due to budgetary constraints and limited access to mass spectrometry facilities.
Should we secure adequate funding in the future, we certainly plan to incorporate detailed bile acid species quantification via LC-MS/MS to enhance the mechanistic depth of our analysis.

Definition of "Cellular Level" Assessment:
The Rationale mentions "specific biochemical tests to assess cholestasis at the cellular
Clarification Needed: Which specific tests (beyond standard LFTs and the
listed secondary markers) are being referred to that assess cholestasis at a
"cellular level"? This phrasing needs to be more precise or linked directly to
specific planned assays (e.g., markers of oxidative stress, apoptosis, or specific
transport protein expression if biopsies are analyzed beyond histology).

Thank you very much for the comment. We have removed the usage of “cellular” from the context

The text in the manuscript modified as below:
This study aims to fill an essential gap by evaluating the efficacy of obeticholic acid compared to ursodeoxycholic Acid in managing post-liver transplant cholestasis. By conducting specific biochemical tests to assess cholestasis, this research will provide insights into the effectiveness of OCA relative to the current standard, UDCA.

Query: Introduction and Conclusion Phrasing:
Introduction: "Although its usefulness is questionable, many practitioners utilize
ursodeoxycholic acid (UDCA)..." While there are debates, phrasing it as "questionable"
without immediate specific referencing for the LT context might be too strong.
Perhaps "its efficacy for preventing all forms of post-LT cholestasis or improving long
term graft survival has limitations" or similar.

Reply: Thank you very much for the comment. We have changed the sentence according to your suggestions.
While many practitioners utilize UDCA as a hepatoprotective drug, its efficacy for preventing all forms of post liver transplant cholestasis or improving long-term graft server has limitations.

Query: Conclusion: "Obeticholic acid (OCA) is a potent farnesoid X receptor (FXR) agonist,
with 100 times the efficacy of the current standard treatment, UDCA..." This is
misleading. OCA is ~100 times more potent as an FXR agonist than chenodeoxycholic
acid (a primary bile acid), not UDCA. UDCA is not a potent FXR agonist; it has different
mechanisms of action (choleretic, cytoprotective, immunomodulatory). This
statement needs correction for accuracy.

Reply:Thank you very much for the comment. We have changed the sentence according to your suggestions.

Sentence now reads as’
Obeticholic acid (OCA) is a farnesoid X receptor (FXR) agonist, with significantly more potency than UDCA in modulating bile acid metabolism.
Competing Interests: nil Close
Report a concern
Respond or Comment

COMMENTS ON THIS REPORT

Author Response 11 Sep 2025

Anila K N, GI Surgery, Amrita Institute of Medical Sciences & Research Centre, Kochi, Amrita Vishwa Vidyapeetham, Coimbatore, India

11 Sep 2025

Author Response

Thank you very much for the very pertinent comments.

Major Weaknesses and Areas Requiring Clarification/Addition:
Query Study Timeline and Design Clarity (Critical Flaw):
Contradiction: The "Methods" section states ... Continue reading Thank you very much for the very pertinent comments.

Major Weaknesses and Areas Requiring Clarification/Addition:
Query Study Timeline and Design Clarity (Critical Flaw):
Contradiction: The "Methods" section states the study "was conducted... from
December 2014 to 2021." However, the "Study status" section says, "The study is
currently in the recruitment phase." Furthermore, ethics approval is dated 2021/2022,
and CTRI registration is from 2021. This is a fundamental contradiction. My
understanding is that it is patients that received LT in these years that will be
recruited to the study, but the constant change is present and past tense, and the
wording makes this unclear.

Reply: Thank you for highlighting these points. We apologize for the mistake in the dates.
The Institutional Ethics Committee approval for the study was obtained in August 2021, and the CTRI registration was obtained on 23/11/2021. The study commenced thereafter. The data collection period for the pilot study was mistakenly reported as December 2014 to 2021." and has now been corrected as given below. We have also addressed the inconsistencies in verb tense throughout the manuscript. We appreciate your careful review and constructive feedback.

Correction in the manuscript:

Study design and participants
The study is being conducted as an investigator-initiated, parallel-group, open-label randomized controlled trial in patients undergoing live donor liver transplantation since December 2021 in the Department of Gastrointestinal Surgery at a tertiary care teaching hospital.” under Study design and participants.

Ethical considerations
The study protocol was initially approved by the Institutional Ethics Committee, AIMS (IEC-AIMS-2021-GISURG-262) on 13-10-2021 and later modified for a title change on 15-06-2022 (modified ethics number—IEC-AIMS-2022-GISURG-160). This study involves liver transplant patients aged 18 years or older admitted to our hospital’s gastrointestinal surgery department. Before the study’s initiation, patients gave written informed consent. The consent form was prepared both in English and the local language Malayalam. The study has received ethical clearance from our Institutional Ethics Committee. The committee also approved the informed consent in both languages. This study was registered with the Clinical Trials Registry of India. Our CTRI registration number is CTRI/2021/11/038218 dated on 23/11/2021. The study was conducted according to the principles of the Declaration of Helsinki.

Query: Furthermore, exclusion criteria for patients that died within 30 days would also suggest a difference in timeline, as a patient who died in 2014 after 30 days post LT would certainly not be available to include in a study taking place in 2025. Otherwise, is this exclusion criteria for patients that are started on the study and die within this time frame, in which case this could exclude patients experiencing severe early adverse events or graft failure potentially related to the interventions. A rationale for this exclusion is needed.

Explanation: Thank you once again for pointing out these observations.
The confusion is partly due to the mistake in the date, as the study started only in Dec 21. (which we have corrected as above)
We excluded patients who were randomized into the trial but died within the first 30 days. This decision was based on the fact that early mortality in this period is most commonly attributable to sepsis or technical complications. Including such patients would not be appropriate for evaluating interventions aimed at the management of cholestasis, which usually occur 2 to 3 weeks after surgery. Mortality within 30 days, purely secondary to the adverse effect of the drug, with no other technical ( vasculo-biliary), septic or immunological cause is extremely unlikely. Furthermore, we require at least 4 to 6 weeks of drug therapy for any noticeable effect on cholestasis. Patients dying within 30 days will not get this time period.

We have added this in the manuscript s follows: We excluded patients who were randomized into the trial but died within the first 30 days. This decision was based on the fact that early mortality in this period is most commonly attributable to sepsis or technical complications. Including such patients would not be appropriate for evaluating interventions aimed at the management of cholestasis, which usually occur 2 to 3 weeks after surgery. Mortality within 30 days, purely secondary to the adverse effect of the drug, with no other technical ( vasculo-biliary), septic or immunological cause is extremely unlikely.

Query: Rationale for OCA Dosage and Safety in LT Population:
FDA Warning Context: The introduction mentions, "In individuals with advanced liver
disease, the FDA has cautioned against administering OCA at doses of 10–50 mg."
Liver transplant recipients, by definition, have end-stage liver disease pre-transplant
and a recovering, vulnerable graft post-transplant. While the study uses 5 mg OD,
which is lower, the potential for OCA-related complications (decompensation,
worsening LFTs before improvement, severe pruritus) in this specific population
needs more thorough discussion.

Reply: Thank you very much for your valuable comment.

The FDA Blackbox warning was issued on the higher doses of obeticholic acid (10–50 mg) in individuals with advanced liver disease. As you correctly pointed out, liver transplant recipients represent a unique and vulnerable population with recovering graft function, and careful attention to OCA-related safety concerns is warranted.

In our study, we have used a conservative dose of 5 mg once daily, which is well below the threshold associated with reported complications. Nevertheless, as this is an open-label trial, we are closely monitoring for potential adverse effects, including hepatic decompensation, transient worsening of liver function tests, and pruritus. These safety parameters are being actively assessed throughout the study duration and will be discussed comprehensively in the final analysis and manuscript.

Query: Primary Endpoint Justification and Baseline:
15% Reduction: The primary endpoint is a "15% reduction in alkaline phosphatase
and gamma-glutamyl transferase levels." What is the clinical significance of a 15%
reduction in these enzymes in post-LT cholestasis? Is this threshold based on
previous studies in LT or other cholestatic conditions demonstrating improved long
term outcomes? This needs robust justification.

Reply: We selected a 15% reduction in both alkaline phosphatase (ALP) and gamma-glutamyltransferase (GGT) as our primary biochemical endpoint based on evidence from previous trials in cholestatic liver disease. Specifically, the landmark study by Nevens et al., published in The New England Journal of Medicine (2016), evaluated obeticholic acid in patients with primary biliary cholangitis and used a composite endpoint that included a ≥15% reduction in ALP as part of the therapeutic response criteria.
Although this trial was not conducted in post–liver transplant patients, it provided a strong precedent for using ALP-based endpoints to assess treatment response in cholestatic conditions. We extended this rationale by including GGT alongside ALP, as the combination of these two enzymes provides a more comprehensive and sensitive assessment of cholestasis, particularly in the post-transplant setting where bile duct injury and graft function are dynamic.
Given the lack of clinical trials of OCA in the liver transplant population, this combined biochemical endpoint serves as a reasonable and evidence-informed surrogate marker of response in our study.

Query: One-Month Baseline: The baseline for assessing the primary endpoint is "one
month" post-transplant. Significant cholestasis and LFT fluctuations can occur much
earlier post-LT. Why was a 1-month baseline chosen? This might miss early effects or
be confounded by stabilization that might occur irrespective of the study drug. A
rationale for this time point is crucial.

Reply: We chose to initiate study assessments after one-month post-transplant because a significant proportion of our liver transplant recipients are high MELD patients who typically present with markedly elevated bilirubin and cholestatic enzyme levels at baseline. Additionally, in the immediate post-transplant period, transient elevations in liver function tests are commonly observed due to ischemia–reperfusion injury, surgical factors, sepsis, or early graft rejection.
In live donor liver transplant for high MELD recipients, these fluctuations are often pronounced during the first few weeks following transplantation. Therefore, a 4-week window was provided to allow for stabilization of liver function parameters and resolution of early postoperative complications, ensuring that subsequent measurements more accurately reflect the effect of the study intervention rather than early perioperative variability.

Query: Omission of Bilirubin: Total bilirubin is a key marker of cholestasis severity
and liver function. Why is bilirubin not included as a primary or at least a key
secondary endpoint for cholestasis assessment?
Reply: Total bilirubin has been retained as an important secondary endpoint, given its clinical relevance in assessing hepatocellular function and persistent cholestasis. Indeed, liver function tests including bilirubin levels are listed as the first secondary end point.

Query: Sample Size Calculation and Pilot Study Data:
The pilot study data used for sample size calculation ("difference in alkaline
phosphatase (5.131 ± 39.639 vs 12.496 ± 52.139)") shows extremely large standard
deviations relative to the means. This suggests massive variability or heterogeneity in
the pilot data, or potentially a very small effect size, making the observed difference
potentially unreliable for powering a larger trial. A difference of ~7 U/L (assuming
these are absolute values) seems very small.

Reply: Regarding the sample size calculation, we acknowledge that data specific to the post–liver transplant population receiving obeticholic acid is extremely limited. Apart from our pilot study, we were unable to identify any previously published data in this specific setting to guide formal power calculations. Based on the effect sizes and variability observed in our pilot data, our statistician advised that this sample size would be adequate to provide preliminary evidence and inform the design of a larger, adequately powered trial in the future.

Exclusion Criteria and Analysis Population:
"patients who discontinued study drugs were excluded." Excluding patients who
discontinue the drug from the analysis can introduce significant bias. An intention-to
treat (ITT) analysis, including all randomized patients in the groups to which they
were originally assigned, is standard for RCTs and should be the primary analysis. A
per-protocol analysis can be secondary. This needs to be specified in the statistical
analysis section.
See earlier comment on exclusion criteria of patients that died <30 days.

Reply: Thank you very much for your observation. We are in fact conducting this trial as an intention-to-treat (ITT) analysis approach. Accordingly, all randomized patients—including those who discontinue the study prematurely—will be included in the final analysis. Efforts will be made to document and analyze the reasons for discontinuation, as this information is critical for understanding both the tolerability and the real-world applicability of the intervention.

We have modified the exclusion criteria after removing this sentence as follows:
We excluded patients who were randomized into the trial but died within the first 30 days. This decision was based on the fact that early mortality in this period is most commonly attributable to sepsis or technical complications. Including such patients would not be appropriate for evaluating interventions aimed at the management of cholestasis, which usually occur 2 to 3 weeks after surgery. Mortality within 30 days, purely secondary to the adverse effect of the drug, with no other technical ( vasculo-biliary), septic or immunological cause is extremely unlikely.

Query: Secondary Weaknesses and Areas for Clarification/Addition:
UDCA Dosing:
UDCA is dosed at "10-15 mg/kg TID." Typically, UDCA is administered BID in
post-transplant settings. Provide a rationale for the TID dosing regimen for
UDCA.

Reply: Thank you for your comment regarding dosing. The typical recommended dosage for managing cholestasis—particularly with ursodeoxycholic acid (UDCA)—is 10 to 15 mg/kg/day, usually administered in three divided doses. Based on this standard recommendation, we have selected the dose accordingly in our study to ensure therapeutic efficacy while maintaining safety and consistency with existing clinical guidelines.
Several peer-reviewed studies and reviews that report the use of ursodeoxycholic acid (UDCA) at doses of 10–15 mg/kg/day in post–liver transplant patients for prophylaxis and early graft protection (Ref : Pedersen, Mark R.^*,1; Greenan, Garrett²; Arora, Sumant³; Murali, Arvind R.³; Mayo, Marlyn J.¹. Ursodeoxycholic Acid Decreases Incidence of Primary Biliary Cholangitis and Biliary Complications After Liver Transplantation: A Meta‐Analysis. Liver Transplantation 27(6):p 866-875, June 2021. | DOI: 10.1002/lt.25935). For an adult with normal/average body weight, the doses of UDCA thus may be divided to three times a day.

Query: Measurement of Specific Secondary Endpoints:
"Bile Salt Export Pump levels (BSEP)": How will BSEP levels be measured?
Perhaps I am mistaken, but BSEP is a membrane-bound transporter, so will
only be measured with biopsies, as in you methods, will only be done in the
suspicion of rejection. This is currently too vague.

Reply: Thank you for your thoughtful feedback. You’re absolutely right—BSEP is a membrane‑bound canalicular transporter and, under normal conditions, is not released into the bloodstream in its intact form. In our study, however, we specifically sought to identify and quantify membrane‑derived fragments of BSEP that may be shed into the circulation during post‑transplant hepatocellular injury. To achieve this, we used a commercial sandwich ELISA kit validated for human serum and plasma. It is probable that the usefulness may not be as much as measuring the tissue BSEP levels, which will entail multiple liver biopsies in our patient. Our study results will hopefully give a better clue.

Query: "Plasma bile acid levels":
Clarification Needed: Will this be total plasma bile acids, or will specific bile
acid species be fractionated and quantified (e.g., via LC-MS/MS)? The latter
would be much more informative regarding FXR target engagement and
changes in bile acid pool composition.

Reply: Thank you for raising this important point. In our study, total plasma bile acid levels were quantified using commercially available ELISA kits. While we fully agree that fractionated bile acid profiling using LC-MS/MS would provide deeper insights into FXR target engagement and alterations in bile acid pool composition, this approach was not feasible in our current setting due to budgetary constraints and limited access to mass spectrometry facilities.
Should we secure adequate funding in the future, we certainly plan to incorporate detailed bile acid species quantification via LC-MS/MS to enhance the mechanistic depth of our analysis.

Definition of "Cellular Level" Assessment:
The Rationale mentions "specific biochemical tests to assess cholestasis at the cellular
Clarification Needed: Which specific tests (beyond standard LFTs and the
listed secondary markers) are being referred to that assess cholestasis at a
"cellular level"? This phrasing needs to be more precise or linked directly to
specific planned assays (e.g., markers of oxidative stress, apoptosis, or specific
transport protein expression if biopsies are analyzed beyond histology).

Thank you very much for the comment. We have removed the usage of “cellular” from the context

The text in the manuscript modified as below:
This study aims to fill an essential gap by evaluating the efficacy of obeticholic acid compared to ursodeoxycholic Acid in managing post-liver transplant cholestasis. By conducting specific biochemical tests to assess cholestasis, this research will provide insights into the effectiveness of OCA relative to the current standard, UDCA.

Query: Introduction and Conclusion Phrasing:
Introduction: "Although its usefulness is questionable, many practitioners utilize
ursodeoxycholic acid (UDCA)..." While there are debates, phrasing it as "questionable"
without immediate specific referencing for the LT context might be too strong.
Perhaps "its efficacy for preventing all forms of post-LT cholestasis or improving long
term graft survival has limitations" or similar.

Reply: Thank you very much for the comment. We have changed the sentence according to your suggestions.
While many practitioners utilize UDCA as a hepatoprotective drug, its efficacy for preventing all forms of post liver transplant cholestasis or improving long-term graft server has limitations.

Query: Conclusion: "Obeticholic acid (OCA) is a potent farnesoid X receptor (FXR) agonist,
with 100 times the efficacy of the current standard treatment, UDCA..." This is
misleading. OCA is ~100 times more potent as an FXR agonist than chenodeoxycholic
acid (a primary bile acid), not UDCA. UDCA is not a potent FXR agonist; it has different
mechanisms of action (choleretic, cytoprotective, immunomodulatory). This
statement needs correction for accuracy.

Reply:Thank you very much for the comment. We have changed the sentence according to your suggestions.

Sentence now reads as’
Obeticholic acid (OCA) is a farnesoid X receptor (FXR) agonist, with significantly more potency than UDCA in modulating bile acid metabolism.
Thank you very much for the very pertinent comments.

Major Weaknesses and Areas Requiring Clarification/Addition:
Query Study Timeline and Design Clarity (Critical Flaw):
Contradiction: The "Methods" section states the study "was conducted... from
December 2014 to 2021." However, the "Study status" section says, "The study is
currently in the recruitment phase." Furthermore, ethics approval is dated 2021/2022,
and CTRI registration is from 2021. This is a fundamental contradiction. My
understanding is that it is patients that received LT in these years that will be
recruited to the study, but the constant change is present and past tense, and the
wording makes this unclear.

Reply: Thank you for highlighting these points. We apologize for the mistake in the dates.
The Institutional Ethics Committee approval for the study was obtained in August 2021, and the CTRI registration was obtained on 23/11/2021. The study commenced thereafter. The data collection period for the pilot study was mistakenly reported as December 2014 to 2021." and has now been corrected as given below. We have also addressed the inconsistencies in verb tense throughout the manuscript. We appreciate your careful review and constructive feedback.

Correction in the manuscript:

Study design and participants
The study is being conducted as an investigator-initiated, parallel-group, open-label randomized controlled trial in patients undergoing live donor liver transplantation since December 2021 in the Department of Gastrointestinal Surgery at a tertiary care teaching hospital.” under Study design and participants.

Ethical considerations
The study protocol was initially approved by the Institutional Ethics Committee, AIMS (IEC-AIMS-2021-GISURG-262) on 13-10-2021 and later modified for a title change on 15-06-2022 (modified ethics number—IEC-AIMS-2022-GISURG-160). This study involves liver transplant patients aged 18 years or older admitted to our hospital’s gastrointestinal surgery department. Before the study’s initiation, patients gave written informed consent. The consent form was prepared both in English and the local language Malayalam. The study has received ethical clearance from our Institutional Ethics Committee. The committee also approved the informed consent in both languages. This study was registered with the Clinical Trials Registry of India. Our CTRI registration number is CTRI/2021/11/038218 dated on 23/11/2021. The study was conducted according to the principles of the Declaration of Helsinki.

Query: Furthermore, exclusion criteria for patients that died within 30 days would also suggest a difference in timeline, as a patient who died in 2014 after 30 days post LT would certainly not be available to include in a study taking place in 2025. Otherwise, is this exclusion criteria for patients that are started on the study and die within this time frame, in which case this could exclude patients experiencing severe early adverse events or graft failure potentially related to the interventions. A rationale for this exclusion is needed.

Explanation: Thank you once again for pointing out these observations.
The confusion is partly due to the mistake in the date, as the study started only in Dec 21. (which we have corrected as above)
We excluded patients who were randomized into the trial but died within the first 30 days. This decision was based on the fact that early mortality in this period is most commonly attributable to sepsis or technical complications. Including such patients would not be appropriate for evaluating interventions aimed at the management of cholestasis, which usually occur 2 to 3 weeks after surgery. Mortality within 30 days, purely secondary to the adverse effect of the drug, with no other technical ( vasculo-biliary), septic or immunological cause is extremely unlikely. Furthermore, we require at least 4 to 6 weeks of drug therapy for any noticeable effect on cholestasis. Patients dying within 30 days will not get this time period.

We have added this in the manuscript s follows: We excluded patients who were randomized into the trial but died within the first 30 days. This decision was based on the fact that early mortality in this period is most commonly attributable to sepsis or technical complications. Including such patients would not be appropriate for evaluating interventions aimed at the management of cholestasis, which usually occur 2 to 3 weeks after surgery. Mortality within 30 days, purely secondary to the adverse effect of the drug, with no other technical ( vasculo-biliary), septic or immunological cause is extremely unlikely.

Query: Rationale for OCA Dosage and Safety in LT Population:
FDA Warning Context: The introduction mentions, "In individuals with advanced liver
disease, the FDA has cautioned against administering OCA at doses of 10–50 mg."
Liver transplant recipients, by definition, have end-stage liver disease pre-transplant
and a recovering, vulnerable graft post-transplant. While the study uses 5 mg OD,
which is lower, the potential for OCA-related complications (decompensation,
worsening LFTs before improvement, severe pruritus) in this specific population
needs more thorough discussion.

Reply: Thank you very much for your valuable comment.

The FDA Blackbox warning was issued on the higher doses of obeticholic acid (10–50 mg) in individuals with advanced liver disease. As you correctly pointed out, liver transplant recipients represent a unique and vulnerable population with recovering graft function, and careful attention to OCA-related safety concerns is warranted.

In our study, we have used a conservative dose of 5 mg once daily, which is well below the threshold associated with reported complications. Nevertheless, as this is an open-label trial, we are closely monitoring for potential adverse effects, including hepatic decompensation, transient worsening of liver function tests, and pruritus. These safety parameters are being actively assessed throughout the study duration and will be discussed comprehensively in the final analysis and manuscript.

Query: Primary Endpoint Justification and Baseline:
15% Reduction: The primary endpoint is a "15% reduction in alkaline phosphatase
and gamma-glutamyl transferase levels." What is the clinical significance of a 15%
reduction in these enzymes in post-LT cholestasis? Is this threshold based on
previous studies in LT or other cholestatic conditions demonstrating improved long
term outcomes? This needs robust justification.

Reply: We selected a 15% reduction in both alkaline phosphatase (ALP) and gamma-glutamyltransferase (GGT) as our primary biochemical endpoint based on evidence from previous trials in cholestatic liver disease. Specifically, the landmark study by Nevens et al., published in The New England Journal of Medicine (2016), evaluated obeticholic acid in patients with primary biliary cholangitis and used a composite endpoint that included a ≥15% reduction in ALP as part of the therapeutic response criteria.
Although this trial was not conducted in post–liver transplant patients, it provided a strong precedent for using ALP-based endpoints to assess treatment response in cholestatic conditions. We extended this rationale by including GGT alongside ALP, as the combination of these two enzymes provides a more comprehensive and sensitive assessment of cholestasis, particularly in the post-transplant setting where bile duct injury and graft function are dynamic.
Given the lack of clinical trials of OCA in the liver transplant population, this combined biochemical endpoint serves as a reasonable and evidence-informed surrogate marker of response in our study.

Query: One-Month Baseline: The baseline for assessing the primary endpoint is "one
month" post-transplant. Significant cholestasis and LFT fluctuations can occur much
earlier post-LT. Why was a 1-month baseline chosen? This might miss early effects or
be confounded by stabilization that might occur irrespective of the study drug. A
rationale for this time point is crucial.

Reply: We chose to initiate study assessments after one-month post-transplant because a significant proportion of our liver transplant recipients are high MELD patients who typically present with markedly elevated bilirubin and cholestatic enzyme levels at baseline. Additionally, in the immediate post-transplant period, transient elevations in liver function tests are commonly observed due to ischemia–reperfusion injury, surgical factors, sepsis, or early graft rejection.
In live donor liver transplant for high MELD recipients, these fluctuations are often pronounced during the first few weeks following transplantation. Therefore, a 4-week window was provided to allow for stabilization of liver function parameters and resolution of early postoperative complications, ensuring that subsequent measurements more accurately reflect the effect of the study intervention rather than early perioperative variability.

Query: Omission of Bilirubin: Total bilirubin is a key marker of cholestasis severity
and liver function. Why is bilirubin not included as a primary or at least a key
secondary endpoint for cholestasis assessment?
Reply: Total bilirubin has been retained as an important secondary endpoint, given its clinical relevance in assessing hepatocellular function and persistent cholestasis. Indeed, liver function tests including bilirubin levels are listed as the first secondary end point.

Query: Sample Size Calculation and Pilot Study Data:
The pilot study data used for sample size calculation ("difference in alkaline
phosphatase (5.131 ± 39.639 vs 12.496 ± 52.139)") shows extremely large standard
deviations relative to the means. This suggests massive variability or heterogeneity in
the pilot data, or potentially a very small effect size, making the observed difference
potentially unreliable for powering a larger trial. A difference of ~7 U/L (assuming
these are absolute values) seems very small.

Reply: Regarding the sample size calculation, we acknowledge that data specific to the post–liver transplant population receiving obeticholic acid is extremely limited. Apart from our pilot study, we were unable to identify any previously published data in this specific setting to guide formal power calculations. Based on the effect sizes and variability observed in our pilot data, our statistician advised that this sample size would be adequate to provide preliminary evidence and inform the design of a larger, adequately powered trial in the future.

Exclusion Criteria and Analysis Population:
"patients who discontinued study drugs were excluded." Excluding patients who
discontinue the drug from the analysis can introduce significant bias. An intention-to
treat (ITT) analysis, including all randomized patients in the groups to which they
were originally assigned, is standard for RCTs and should be the primary analysis. A
per-protocol analysis can be secondary. This needs to be specified in the statistical
analysis section.
See earlier comment on exclusion criteria of patients that died <30 days.

Reply: Thank you very much for your observation. We are in fact conducting this trial as an intention-to-treat (ITT) analysis approach. Accordingly, all randomized patients—including those who discontinue the study prematurely—will be included in the final analysis. Efforts will be made to document and analyze the reasons for discontinuation, as this information is critical for understanding both the tolerability and the real-world applicability of the intervention.

We have modified the exclusion criteria after removing this sentence as follows:
We excluded patients who were randomized into the trial but died within the first 30 days. This decision was based on the fact that early mortality in this period is most commonly attributable to sepsis or technical complications. Including such patients would not be appropriate for evaluating interventions aimed at the management of cholestasis, which usually occur 2 to 3 weeks after surgery. Mortality within 30 days, purely secondary to the adverse effect of the drug, with no other technical ( vasculo-biliary), septic or immunological cause is extremely unlikely.

Query: Secondary Weaknesses and Areas for Clarification/Addition:
UDCA Dosing:
UDCA is dosed at "10-15 mg/kg TID." Typically, UDCA is administered BID in
post-transplant settings. Provide a rationale for the TID dosing regimen for
UDCA.

Reply: Thank you for your comment regarding dosing. The typical recommended dosage for managing cholestasis—particularly with ursodeoxycholic acid (UDCA)—is 10 to 15 mg/kg/day, usually administered in three divided doses. Based on this standard recommendation, we have selected the dose accordingly in our study to ensure therapeutic efficacy while maintaining safety and consistency with existing clinical guidelines.
Several peer-reviewed studies and reviews that report the use of ursodeoxycholic acid (UDCA) at doses of 10–15 mg/kg/day in post–liver transplant patients for prophylaxis and early graft protection (Ref : Pedersen, Mark R.^*,1; Greenan, Garrett²; Arora, Sumant³; Murali, Arvind R.³; Mayo, Marlyn J.¹. Ursodeoxycholic Acid Decreases Incidence of Primary Biliary Cholangitis and Biliary Complications After Liver Transplantation: A Meta‐Analysis. Liver Transplantation 27(6):p 866-875, June 2021. | DOI: 10.1002/lt.25935). For an adult with normal/average body weight, the doses of UDCA thus may be divided to three times a day.

Query: Measurement of Specific Secondary Endpoints:
"Bile Salt Export Pump levels (BSEP)": How will BSEP levels be measured?
Perhaps I am mistaken, but BSEP is a membrane-bound transporter, so will
only be measured with biopsies, as in you methods, will only be done in the
suspicion of rejection. This is currently too vague.

Reply: Thank you for your thoughtful feedback. You’re absolutely right—BSEP is a membrane‑bound canalicular transporter and, under normal conditions, is not released into the bloodstream in its intact form. In our study, however, we specifically sought to identify and quantify membrane‑derived fragments of BSEP that may be shed into the circulation during post‑transplant hepatocellular injury. To achieve this, we used a commercial sandwich ELISA kit validated for human serum and plasma. It is probable that the usefulness may not be as much as measuring the tissue BSEP levels, which will entail multiple liver biopsies in our patient. Our study results will hopefully give a better clue.

Query: "Plasma bile acid levels":
Clarification Needed: Will this be total plasma bile acids, or will specific bile
acid species be fractionated and quantified (e.g., via LC-MS/MS)? The latter
would be much more informative regarding FXR target engagement and
changes in bile acid pool composition.

Reply: Thank you for raising this important point. In our study, total plasma bile acid levels were quantified using commercially available ELISA kits. While we fully agree that fractionated bile acid profiling using LC-MS/MS would provide deeper insights into FXR target engagement and alterations in bile acid pool composition, this approach was not feasible in our current setting due to budgetary constraints and limited access to mass spectrometry facilities.
Should we secure adequate funding in the future, we certainly plan to incorporate detailed bile acid species quantification via LC-MS/MS to enhance the mechanistic depth of our analysis.

Definition of "Cellular Level" Assessment:
The Rationale mentions "specific biochemical tests to assess cholestasis at the cellular
Clarification Needed: Which specific tests (beyond standard LFTs and the
listed secondary markers) are being referred to that assess cholestasis at a
"cellular level"? This phrasing needs to be more precise or linked directly to
specific planned assays (e.g., markers of oxidative stress, apoptosis, or specific
transport protein expression if biopsies are analyzed beyond histology).

Thank you very much for the comment. We have removed the usage of “cellular” from the context

The text in the manuscript modified as below:
This study aims to fill an essential gap by evaluating the efficacy of obeticholic acid compared to ursodeoxycholic Acid in managing post-liver transplant cholestasis. By conducting specific biochemical tests to assess cholestasis, this research will provide insights into the effectiveness of OCA relative to the current standard, UDCA.

Query: Introduction and Conclusion Phrasing:
Introduction: "Although its usefulness is questionable, many practitioners utilize
ursodeoxycholic acid (UDCA)..." While there are debates, phrasing it as "questionable"
without immediate specific referencing for the LT context might be too strong.
Perhaps "its efficacy for preventing all forms of post-LT cholestasis or improving long
term graft survival has limitations" or similar.

Reply: Thank you very much for the comment. We have changed the sentence according to your suggestions.
While many practitioners utilize UDCA as a hepatoprotective drug, its efficacy for preventing all forms of post liver transplant cholestasis or improving long-term graft server has limitations.

Query: Conclusion: "Obeticholic acid (OCA) is a potent farnesoid X receptor (FXR) agonist,
with 100 times the efficacy of the current standard treatment, UDCA..." This is
misleading. OCA is ~100 times more potent as an FXR agonist than chenodeoxycholic
acid (a primary bile acid), not UDCA. UDCA is not a potent FXR agonist; it has different
mechanisms of action (choleretic, cytoprotective, immunomodulatory). This
statement needs correction for accuracy.

Reply:Thank you very much for the comment. We have changed the sentence according to your suggestions.

Sentence now reads as’
Obeticholic acid (OCA) is a farnesoid X receptor (FXR) agonist, with significantly more potency than UDCA in modulating bile acid metabolism.
Competing Interests: nil Close
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Reviewer Report 03 Feb 2025

Palak J Trivedi, University of Birmingham, Birmingham, UK

Approved with Reservations

https://doi.org/10.5256/f1000research.174896.r355921

The rationale for the study is reasonable, but depth of secondary endpoints being captured is limited. Additionally, which adverse events will be captured, in what way, and how will they be recorded?

The dose of UDCA is also lower than what would be considered standard practice.

How will pruritus be captured? Highly relevant for OCA-treated patients.

The pivotal phase III trial of obeticholic acid needs to be cited (Nevens et al. NEJM).

As the authors may be aware, the EMA and FDA are in the process of revoking approval for obeticholic acid in Europe and the United States, respectively. Whilst such regulators do not necessarily have jurisdiction in India, how do the authors feel that these decisions will affect access to OCA?

Is the rationale for, and objectives of, the study clearly described?

Yes
Is the study design appropriate for the research question?

Yes
Are sufficient details of the methods provided to allow replication by others?

No
Are the datasets clearly presented in a useable and accessible format?

Partly

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Cholestasis, liver transplantation, clinical trials.

CITE

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Author Response 11 Sep 2025

Anila K N, GI Surgery, Amrita Institute of Medical Sciences & Research Centre, Kochi, Amrita Vishwa Vidyapeetham, Coimbatore, India

11 Sep 2025

Author Response

The rationale for the study is reasonable, but depth of secondary endpoints being captured is limited.

Thank you very much for the comments.

Our secondary endpoints include all ... Continue reading The rationale for the study is reasonable, but depth of secondary endpoints being captured is limited.

Thank you very much for the comments.

Our secondary endpoints include all biochemical parameters (Liver function tests including Gamma Glutamyl transferase), post-transplant molecular markers fibroblast growth factor 19 (FGF-19), transforming growth factor β (TGF-β) level, Cytokeratin 18 level, Serum autotaxin level, Bile Salt Export Pump levels (BSEP), Plasma bile acid levels that assess biliary secretion. We did not include the pharmacodynamics and pharmacokinetics of obeticholic acid as it has already been done. We have also included health related quality of life assessment.
We did not include any exploratory endpoints to formulate due to cost constraints.
Which adverse events will be captured, in what way, and how will they be recorded?
&
How will pruritus be captured? Highly relevant for OCA-treated patients.

Thank You for the insightful query.

Adverse events will be captured by direct interview of patients during the routine follow-up visits. The Naranjo algorithm will be used to establish causality. We will capture the pruritus by physical examination, vital signs and 5D pruritus questionnaire and will be documented systematically using structured Case Report Forms (CRFs) to ensure standardized data collection and analysis. The most common adverse event observed is pruritis.
Pruritus associated with Obeticholic Acid (OCA) is known to be dose-dependent. However, in this study, we are using a lower dose of 5 mg once daily (OD), which is expected to minimize the incidence and severity of pruritus. Nonetheless, any pruritus reported by patients will be systematically assessed and documented in CRFs for consistency in data collection.

The text will now read as:
Adverse events are being captured by direct interview of patients during the routine follow-up visits. We are capturing the pruritus by physical examination, vital signs and 5D pruritus questionnaire and documented systematically using structured Case Report Forms (CRFs) to ensure standardized data collection and analysis. The Naranjo algorithm will be used to establish causality and then classify them as definite, probable, possible, or unlikely.

The dose of UDCA is also lower than what would be considered standard practice?

Thank you for this comment.

In fact, our recommended dose is 10 to 15 milligram per kilogram TID. Unfortunately, at one point the dose was mistakenly written as 10 to 15 mg/kg twice daily which is lower than the recommended dose, but shall be corrected.

The text in the manuscript now reads as:

It is usually administered for three months post-transplant at a dose of 10-15 mg/kg thrice daily. In 2016, the FDA approved obeticholic acid (OCA), a strong selective Farnesoid X Receptor (FXR) agonist, to treat primary biliary cholangitis (PBC) in conjunction with UDCA. ¹⁶^,¹⁷Research has demonstrated that over 12 months, OCA, either in combination with UDCA or on its own, dramatically lowers levels of total bilirubin and alkaline phosphatase. ¹⁸^,¹⁹In individuals with advanced liver disease, the FDA has cautioned against administering OCA at doses of 10–50 mg. ²⁰Studies comparing OCA’s effects on cholestasis, rejection, regeneration, and cost-effectiveness to the current standard UDCA are being conducted to assess the role of OCA in the postoperative recovery of liver transplant patients. ²¹Our proposed project is a clinical trial between OCA and the current standard of care, UDCA, in managing post-liver transplant cholestasis and improving graft survival.

As the authors may be aware, the EMA and FDA are in the process of revoking approval for obeticholic acid in Europe and the United States, respectively. Whilst such regulators do not necessarily have jurisdiction in India, how do the authors feel that these decisions will affect access to OCA?

Thank you for the comment. We are aware of the recent FDA revoke for approval of obeticholic acid for further trials in steatohepatitis. However, we are not aware of any change in FDA approval for primary sclerosing cholangitis or primary biliary scleroses or similar cholestatic liver diseases.

In any case, the caution against its use was for patients with advanced liver disease. In our scenario of post-liver transplant patients, that liver function is essentially normal and the dose that we use is much lower than the toxic doses that has been reported. We are using 5 milligram once a day. We hope that results of our trial may give further insight into the effectiveness and adverse effects of this drug in post-transplant population.
The rationale for the study is reasonable, but depth of secondary endpoints being captured is limited.

Thank you very much for the comments.

Our secondary endpoints include all biochemical parameters (Liver function tests including Gamma Glutamyl transferase), post-transplant molecular markers fibroblast growth factor 19 (FGF-19), transforming growth factor β (TGF-β) level, Cytokeratin 18 level, Serum autotaxin level, Bile Salt Export Pump levels (BSEP), Plasma bile acid levels that assess biliary secretion. We did not include the pharmacodynamics and pharmacokinetics of obeticholic acid as it has already been done. We have also included health related quality of life assessment.
We did not include any exploratory endpoints to formulate due to cost constraints.
Which adverse events will be captured, in what way, and how will they be recorded?
&
How will pruritus be captured? Highly relevant for OCA-treated patients.

Thank You for the insightful query.

Adverse events will be captured by direct interview of patients during the routine follow-up visits. The Naranjo algorithm will be used to establish causality. We will capture the pruritus by physical examination, vital signs and 5D pruritus questionnaire and will be documented systematically using structured Case Report Forms (CRFs) to ensure standardized data collection and analysis. The most common adverse event observed is pruritis.
Pruritus associated with Obeticholic Acid (OCA) is known to be dose-dependent. However, in this study, we are using a lower dose of 5 mg once daily (OD), which is expected to minimize the incidence and severity of pruritus. Nonetheless, any pruritus reported by patients will be systematically assessed and documented in CRFs for consistency in data collection.

The text will now read as:
Adverse events are being captured by direct interview of patients during the routine follow-up visits. We are capturing the pruritus by physical examination, vital signs and 5D pruritus questionnaire and documented systematically using structured Case Report Forms (CRFs) to ensure standardized data collection and analysis. The Naranjo algorithm will be used to establish causality and then classify them as definite, probable, possible, or unlikely.

The dose of UDCA is also lower than what would be considered standard practice?

Thank you for this comment.

In fact, our recommended dose is 10 to 15 milligram per kilogram TID. Unfortunately, at one point the dose was mistakenly written as 10 to 15 mg/kg twice daily which is lower than the recommended dose, but shall be corrected.

The text in the manuscript now reads as:

It is usually administered for three months post-transplant at a dose of 10-15 mg/kg thrice daily. In 2016, the FDA approved obeticholic acid (OCA), a strong selective Farnesoid X Receptor (FXR) agonist, to treat primary biliary cholangitis (PBC) in conjunction with UDCA. ¹⁶^,¹⁷Research has demonstrated that over 12 months, OCA, either in combination with UDCA or on its own, dramatically lowers levels of total bilirubin and alkaline phosphatase. ¹⁸^,¹⁹In individuals with advanced liver disease, the FDA has cautioned against administering OCA at doses of 10–50 mg. ²⁰Studies comparing OCA’s effects on cholestasis, rejection, regeneration, and cost-effectiveness to the current standard UDCA are being conducted to assess the role of OCA in the postoperative recovery of liver transplant patients. ²¹Our proposed project is a clinical trial between OCA and the current standard of care, UDCA, in managing post-liver transplant cholestasis and improving graft survival.

As the authors may be aware, the EMA and FDA are in the process of revoking approval for obeticholic acid in Europe and the United States, respectively. Whilst such regulators do not necessarily have jurisdiction in India, how do the authors feel that these decisions will affect access to OCA?

Thank you for the comment. We are aware of the recent FDA revoke for approval of obeticholic acid for further trials in steatohepatitis. However, we are not aware of any change in FDA approval for primary sclerosing cholangitis or primary biliary scleroses or similar cholestatic liver diseases.

In any case, the caution against its use was for patients with advanced liver disease. In our scenario of post-liver transplant patients, that liver function is essentially normal and the dose that we use is much lower than the toxic doses that has been reported. We are using 5 milligram once a day. We hope that results of our trial may give further insight into the effectiveness and adverse effects of this drug in post-transplant population.
Competing Interests: No competing interests were disclosed. Close
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COMMENTS ON THIS REPORT

Author Response 11 Sep 2025

Anila K N, GI Surgery, Amrita Institute of Medical Sciences & Research Centre, Kochi, Amrita Vishwa Vidyapeetham, Coimbatore, India

11 Sep 2025

Author Response

The rationale for the study is reasonable, but depth of secondary endpoints being captured is limited.

Thank you very much for the comments.

Our secondary endpoints include all ... Continue reading The rationale for the study is reasonable, but depth of secondary endpoints being captured is limited.

Thank you very much for the comments.

Our secondary endpoints include all biochemical parameters (Liver function tests including Gamma Glutamyl transferase), post-transplant molecular markers fibroblast growth factor 19 (FGF-19), transforming growth factor β (TGF-β) level, Cytokeratin 18 level, Serum autotaxin level, Bile Salt Export Pump levels (BSEP), Plasma bile acid levels that assess biliary secretion. We did not include the pharmacodynamics and pharmacokinetics of obeticholic acid as it has already been done. We have also included health related quality of life assessment.
We did not include any exploratory endpoints to formulate due to cost constraints.
Which adverse events will be captured, in what way, and how will they be recorded?
&
How will pruritus be captured? Highly relevant for OCA-treated patients.

Thank You for the insightful query.

Adverse events will be captured by direct interview of patients during the routine follow-up visits. The Naranjo algorithm will be used to establish causality. We will capture the pruritus by physical examination, vital signs and 5D pruritus questionnaire and will be documented systematically using structured Case Report Forms (CRFs) to ensure standardized data collection and analysis. The most common adverse event observed is pruritis.
Pruritus associated with Obeticholic Acid (OCA) is known to be dose-dependent. However, in this study, we are using a lower dose of 5 mg once daily (OD), which is expected to minimize the incidence and severity of pruritus. Nonetheless, any pruritus reported by patients will be systematically assessed and documented in CRFs for consistency in data collection.

The text will now read as:
Adverse events are being captured by direct interview of patients during the routine follow-up visits. We are capturing the pruritus by physical examination, vital signs and 5D pruritus questionnaire and documented systematically using structured Case Report Forms (CRFs) to ensure standardized data collection and analysis. The Naranjo algorithm will be used to establish causality and then classify them as definite, probable, possible, or unlikely.

The dose of UDCA is also lower than what would be considered standard practice?

Thank you for this comment.

In fact, our recommended dose is 10 to 15 milligram per kilogram TID. Unfortunately, at one point the dose was mistakenly written as 10 to 15 mg/kg twice daily which is lower than the recommended dose, but shall be corrected.

The text in the manuscript now reads as:

It is usually administered for three months post-transplant at a dose of 10-15 mg/kg thrice daily. In 2016, the FDA approved obeticholic acid (OCA), a strong selective Farnesoid X Receptor (FXR) agonist, to treat primary biliary cholangitis (PBC) in conjunction with UDCA. ¹⁶^,¹⁷Research has demonstrated that over 12 months, OCA, either in combination with UDCA or on its own, dramatically lowers levels of total bilirubin and alkaline phosphatase. ¹⁸^,¹⁹In individuals with advanced liver disease, the FDA has cautioned against administering OCA at doses of 10–50 mg. ²⁰Studies comparing OCA’s effects on cholestasis, rejection, regeneration, and cost-effectiveness to the current standard UDCA are being conducted to assess the role of OCA in the postoperative recovery of liver transplant patients. ²¹Our proposed project is a clinical trial between OCA and the current standard of care, UDCA, in managing post-liver transplant cholestasis and improving graft survival.

As the authors may be aware, the EMA and FDA are in the process of revoking approval for obeticholic acid in Europe and the United States, respectively. Whilst such regulators do not necessarily have jurisdiction in India, how do the authors feel that these decisions will affect access to OCA?

Thank you for the comment. We are aware of the recent FDA revoke for approval of obeticholic acid for further trials in steatohepatitis. However, we are not aware of any change in FDA approval for primary sclerosing cholangitis or primary biliary scleroses or similar cholestatic liver diseases.

In any case, the caution against its use was for patients with advanced liver disease. In our scenario of post-liver transplant patients, that liver function is essentially normal and the dose that we use is much lower than the toxic doses that has been reported. We are using 5 milligram once a day. We hope that results of our trial may give further insight into the effectiveness and adverse effects of this drug in post-transplant population.
The rationale for the study is reasonable, but depth of secondary endpoints being captured is limited.

Thank you very much for the comments.

Our secondary endpoints include all biochemical parameters (Liver function tests including Gamma Glutamyl transferase), post-transplant molecular markers fibroblast growth factor 19 (FGF-19), transforming growth factor β (TGF-β) level, Cytokeratin 18 level, Serum autotaxin level, Bile Salt Export Pump levels (BSEP), Plasma bile acid levels that assess biliary secretion. We did not include the pharmacodynamics and pharmacokinetics of obeticholic acid as it has already been done. We have also included health related quality of life assessment.
We did not include any exploratory endpoints to formulate due to cost constraints.
Which adverse events will be captured, in what way, and how will they be recorded?
&
How will pruritus be captured? Highly relevant for OCA-treated patients.

Thank You for the insightful query.

Adverse events will be captured by direct interview of patients during the routine follow-up visits. The Naranjo algorithm will be used to establish causality. We will capture the pruritus by physical examination, vital signs and 5D pruritus questionnaire and will be documented systematically using structured Case Report Forms (CRFs) to ensure standardized data collection and analysis. The most common adverse event observed is pruritis.
Pruritus associated with Obeticholic Acid (OCA) is known to be dose-dependent. However, in this study, we are using a lower dose of 5 mg once daily (OD), which is expected to minimize the incidence and severity of pruritus. Nonetheless, any pruritus reported by patients will be systematically assessed and documented in CRFs for consistency in data collection.

The text will now read as:
Adverse events are being captured by direct interview of patients during the routine follow-up visits. We are capturing the pruritus by physical examination, vital signs and 5D pruritus questionnaire and documented systematically using structured Case Report Forms (CRFs) to ensure standardized data collection and analysis. The Naranjo algorithm will be used to establish causality and then classify them as definite, probable, possible, or unlikely.

The dose of UDCA is also lower than what would be considered standard practice?

Thank you for this comment.

In fact, our recommended dose is 10 to 15 milligram per kilogram TID. Unfortunately, at one point the dose was mistakenly written as 10 to 15 mg/kg twice daily which is lower than the recommended dose, but shall be corrected.

The text in the manuscript now reads as:

It is usually administered for three months post-transplant at a dose of 10-15 mg/kg thrice daily. In 2016, the FDA approved obeticholic acid (OCA), a strong selective Farnesoid X Receptor (FXR) agonist, to treat primary biliary cholangitis (PBC) in conjunction with UDCA. ¹⁶^,¹⁷Research has demonstrated that over 12 months, OCA, either in combination with UDCA or on its own, dramatically lowers levels of total bilirubin and alkaline phosphatase. ¹⁸^,¹⁹In individuals with advanced liver disease, the FDA has cautioned against administering OCA at doses of 10–50 mg. ²⁰Studies comparing OCA’s effects on cholestasis, rejection, regeneration, and cost-effectiveness to the current standard UDCA are being conducted to assess the role of OCA in the postoperative recovery of liver transplant patients. ²¹Our proposed project is a clinical trial between OCA and the current standard of care, UDCA, in managing post-liver transplant cholestasis and improving graft survival.

As the authors may be aware, the EMA and FDA are in the process of revoking approval for obeticholic acid in Europe and the United States, respectively. Whilst such regulators do not necessarily have jurisdiction in India, how do the authors feel that these decisions will affect access to OCA?

Thank you for the comment. We are aware of the recent FDA revoke for approval of obeticholic acid for further trials in steatohepatitis. However, we are not aware of any change in FDA approval for primary sclerosing cholangitis or primary biliary scleroses or similar cholestatic liver diseases.

In any case, the caution against its use was for patients with advanced liver disease. In our scenario of post-liver transplant patients, that liver function is essentially normal and the dose that we use is much lower than the toxic doses that has been reported. We are using 5 milligram once a day. We hope that results of our trial may give further insight into the effectiveness and adverse effects of this drug in post-transplant population.
Competing Interests: No competing interests were disclosed. Close
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Palak J Trivedi, University of Birmingham, Birmingham, UK
Gregory Guthrie, Baylor College of Medicine, Houston, USA

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15 May 2025 | for Version 1

Gregory Guthrie, Baylor College of Medicine, Houston, Texas, USA

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Approved With Reservations

Study Timeline and Design Clarity (Critical Flaw):
- Contradiction: The "Methods" section states the study "was conducted... from December 2014 to 2021." However, the "Study status" section says, "The study is currently in the recruitment phase." Furthermore, ethics approval is dated 2021/2022, and CTRI registration is from 2021. This is a fundamental contradiction. My understanding is that it is patients that received LT in these years that will be recruited to the study, but the constant change is present and past tense, and the wording makes this unclear. Furthermore, exclusion criteria for patients that died within 30 days would also suggest a difference in timeline, as a patient who died in 2014 after 30 days post LT would certainly not be available to include in a study taking place in 2025. Otherwise, is this exclusion criteria for patients that are started on the study and die within this time frame, in which case this could exclude patients experiencing severe early adverse events or graft failure potentially related to the interventions. A rationale for this exclusion is needed.
Rationale for OCA Dosage and Safety in LT Population:
- FDA Warning Context: The introduction mentions, "In individuals with advanced liver disease, the FDA has cautioned against administering OCA at doses of 10–50 mg." Liver transplant recipients, by definition, have end-stage liver disease pre-transplant and a recovering, vulnerable graft post-transplant. While the study uses 5 mg OD, which is lower, the potential for OCA-related complications (decompensation, worsening LFTs before improvement, severe pruritus) in this specific population needs more thorough discussion.
Primary Endpoint Justification and Baseline:
- 15% Reduction: The primary endpoint is a "15% reduction in alkaline phosphatase and gamma-glutamyl transferase levels." What is the clinical significance of a 15% reduction in these enzymes in post-LT cholestasis? Is this threshold based on previous studies in LT or other cholestatic conditions demonstrating improved long-term outcomes? This needs robust justification.
- One-Month Baseline: The baseline for assessing the primary endpoint is "one-month" post-transplant. Significant cholestasis and LFT fluctuations can occur much earlier post-LT. Why was a 1-month baseline chosen? This might miss early effects or be confounded by stabilization that might occur irrespective of the study drug. A rationale for this time point is crucial.
  - Omission of Bilirubin: Total bilirubin is a key marker of cholestasis severity and liver function. Why is bilirubin not included as a primary or at least a key secondary endpoint for cholestasis assessment?
Sample Size Calculation and Pilot Study Data:
- The pilot study data used for sample size calculation ("difference in alkaline phosphatase (5.131 ± 39.639 vs 12.496 ± 52.139)") shows extremely large standard deviations relative to the means. This suggests massive variability or heterogeneity in the pilot data, or potentially a very small effect size, making the observed difference potentially unreliable for powering a larger trial. A difference of ~7 U/L (assuming these are absolute values) seems very small.
Exclusion Criteria and Analysis Population:
- "patients who discontinued study drugs were excluded." Excluding patients who discontinue the drug from the analysis can introduce significant bias. An intention-to-treat (ITT) analysis, including all randomized patients in the groups to which they were originally assigned, is standard for RCTs and should be the primary analysis. A per-protocol analysis can be secondary. This needs to be specified in the statistical analysis section.
- See earlier comment on exclusion criteria of patients that died <30 days.

Secondary Weaknesses and Areas for Clarification/Addition:

UDCA Dosing:
- - UDCA is dosed at "10-15 mg/kg TID." Typically, UDCA is administered BID in post-transplant settings. Provide a rationale for the TID dosing regimen for UDCA.
Measurement of Specific Secondary Endpoints:
- - "Bile Salt Export Pump levels (BSEP)": How will BSEP levels be measured? Perhaps I am mistaken, but BSEP is a membrane-bound transporter, so will only be measured with biopsies, as in you methods, will only be done in the suspicion of rejection. This is currently too vague.
- "Plasma bile acid levels":
  - Clarification Needed: Will this be total plasma bile acids, or will specific bile acid species be fractionated and quantified (e.g., via LC-MS/MS)? The latter would be much more informative regarding FXR target engagement and changes in bile acid pool composition.
Definition of "Cellular Level" Assessment:
- The Rationale mentions "specific biochemical tests to assess cholestasis at the cellular level."
  - Clarification Needed: Which specific tests (beyond standard LFTs and the listed secondary markers) are being referred to that assess cholestasis at a "cellular level"? This phrasing needs to be more precise or linked directly to specific planned assays (e.g., markers of oxidative stress, apoptosis, or specific transport protein expression if biopsies are analyzed beyond histology).
Introduction and Conclusion Phrasing:
- Introduction: "Although its usefulness is questionable, many practitioners utilize ursodeoxycholic acid (UDCA)..." While there are debates, phrasing it as "questionable" without immediate specific referencing for the LT context might be too strong. Perhaps "its efficacy for preventing all forms of post-LT cholestasis or improving long-term graft survival has limitations" or similar.
- Conclusion: "Obeticholic acid (OCA) is a potent farnesoid X receptor (FXR) agonist, with 100 times the efficacy of the current standard treatment, UDCA..." This is misleading. OCA is ~100 times more potent as an FXR agonist than chenodeoxycholic acid (a primary bile acid), not UDCA. UDCA is not a potent FXR agonist; it has different mechanisms of action (choleretic, cytoprotective, immunomodulatory). This statement needs correction for accuracy.

Is the rationale for, and objectives of, the study clearly described?

Yes
Is the study design appropriate for the research question?

Yes
Are sufficient details of the methods provided to allow replication by others?

No
Are the datasets clearly presented in a useable and accessible format?

Partly

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

My research is focused on mechanisms and treatment of cholestatic liver diseases.

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Responses (1)

Author Response

11 Sep 2025

Anila K N, GI Surgery, Amrita Institute of Medical Sciences & Research Centre, Kochi, Amrita Vishwa Vidyapeetham, Coimbatore, India

Thank you very much for the very pertinent comments.

Major Weaknesses and Areas Requiring Clarification/Addition:
Query Study Timeline and Design Clarity (Critical Flaw):
Contradiction: The "Methods" section states the study "was conducted... from
December 2014 to 2021." However, the "Study status" section says, "The study is
currently in the recruitment phase." Furthermore, ethics approval is dated 2021/2022,
and CTRI registration is from 2021. This is a fundamental contradiction. My
understanding is that it is patients that received LT in these years that will be
recruited to the study, but the constant change is present and past tense, and the
wording makes this unclear.

Reply: Thank you for highlighting these points. We apologize for the mistake in the dates.
The Institutional Ethics Committee approval for the study was obtained in August 2021, and the CTRI registration was obtained on 23/11/2021. The study commenced thereafter. The data collection period for the pilot study was mistakenly reported as December 2014 to 2021." and has now been corrected as given below. We have also addressed the inconsistencies in verb tense throughout the manuscript. We appreciate your careful review and constructive feedback.

Correction in the manuscript:

Study design and participants
The study is being conducted as an investigator-initiated, parallel-group, open-label randomized controlled trial in patients undergoing live donor liver transplantation since December 2021 in the Department of Gastrointestinal Surgery at a tertiary care teaching hospital.” under Study design and participants.

Ethical considerations
The study protocol was initially approved by the Institutional Ethics Committee, AIMS (IEC-AIMS-2021-GISURG-262) on 13-10-2021 and later modified for a title change on 15-06-2022 (modified ethics number—IEC-AIMS-2022-GISURG-160). This study involves liver transplant patients aged 18 years or older admitted to our hospital’s gastrointestinal surgery department. Before the study’s initiation, patients gave written informed consent. The consent form was prepared both in English and the local language Malayalam. The study has received ethical clearance from our Institutional Ethics Committee. The committee also approved the informed consent in both languages. This study was registered with the Clinical Trials Registry of India. Our CTRI registration number is CTRI/2021/11/038218 dated on 23/11/2021. The study was conducted according to the principles of the Declaration of Helsinki.

Query: Furthermore, exclusion criteria for patients that died within 30 days would also suggest a difference in timeline, as a patient who died in 2014 after 30 days post LT would certainly not be available to include in a study taking place in 2025. Otherwise, is this exclusion criteria for patients that are started on the study and die within this time frame, in which case this could exclude patients experiencing severe early adverse events or graft failure potentially related to the interventions. A rationale for this exclusion is needed.

Explanation: Thank you once again for pointing out these observations.
The confusion is partly due to the mistake in the date, as the study started only in Dec 21. (which we have corrected as above)
We excluded patients who were randomized into the trial but died within the first 30 days. This decision was based on the fact that early mortality in this period is most commonly attributable to sepsis or technical complications. Including such patients would not be appropriate for evaluating interventions aimed at the management of cholestasis, which usually occur 2 to 3 weeks after surgery. Mortality within 30 days, purely secondary to the adverse effect of the drug, with no other technical ( vasculo-biliary), septic or immunological cause is extremely unlikely. Furthermore, we require at least 4 to 6 weeks of drug therapy for any noticeable effect on cholestasis. Patients dying within 30 days will not get this time period.

We have added this in the manuscript s follows: We excluded patients who were randomized into the trial but died within the first 30 days. This decision was based on the fact that early mortality in this period is most commonly attributable to sepsis or technical complications. Including such patients would not be appropriate for evaluating interventions aimed at the management of cholestasis, which usually occur 2 to 3 weeks after surgery. Mortality within 30 days, purely secondary to the adverse effect of the drug, with no other technical ( vasculo-biliary), septic or immunological cause is extremely unlikely.

Query: Rationale for OCA Dosage and Safety in LT Population:
FDA Warning Context: The introduction mentions, "In individuals with advanced liver
disease, the FDA has cautioned against administering OCA at doses of 10–50 mg."
Liver transplant recipients, by definition, have end-stage liver disease pre-transplant
and a recovering, vulnerable graft post-transplant. While the study uses 5 mg OD,
which is lower, the potential for OCA-related complications (decompensation,
worsening LFTs before improvement, severe pruritus) in this specific population
needs more thorough discussion.

Reply: Thank you very much for your valuable comment.

The FDA Blackbox warning was issued on the higher doses of obeticholic acid (10–50 mg) in individuals with advanced liver disease. As you correctly pointed out, liver transplant recipients represent a unique and vulnerable population with recovering graft function, and careful attention to OCA-related safety concerns is warranted.

In our study, we have used a conservative dose of 5 mg once daily, which is well below the threshold associated with reported complications. Nevertheless, as this is an open-label trial, we are closely monitoring for potential adverse effects, including hepatic decompensation, transient worsening of liver function tests, and pruritus. These safety parameters are being actively assessed throughout the study duration and will be discussed comprehensively in the final analysis and manuscript.

Query: Primary Endpoint Justification and Baseline:
15% Reduction: The primary endpoint is a "15% reduction in alkaline phosphatase
and gamma-glutamyl transferase levels." What is the clinical significance of a 15%
reduction in these enzymes in post-LT cholestasis? Is this threshold based on
previous studies in LT or other cholestatic conditions demonstrating improved long
term outcomes? This needs robust justification.

Reply: We selected a 15% reduction in both alkaline phosphatase (ALP) and gamma-glutamyltransferase (GGT) as our primary biochemical endpoint based on evidence from previous trials in cholestatic liver disease. Specifically, the landmark study by Nevens et al., published in The New England Journal of Medicine (2016), evaluated obeticholic acid in patients with primary biliary cholangitis and used a composite endpoint that included a ≥15% reduction in ALP as part of the therapeutic response criteria.
Although this trial was not conducted in post–liver transplant patients, it provided a strong precedent for using ALP-based endpoints to assess treatment response in cholestatic conditions. We extended this rationale by including GGT alongside ALP, as the combination of these two enzymes provides a more comprehensive and sensitive assessment of cholestasis, particularly in the post-transplant setting where bile duct injury and graft function are dynamic.
Given the lack of clinical trials of OCA in the liver transplant population, this combined biochemical endpoint serves as a reasonable and evidence-informed surrogate marker of response in our study.

Query: One-Month Baseline: The baseline for assessing the primary endpoint is "one
month" post-transplant. Significant cholestasis and LFT fluctuations can occur much
earlier post-LT. Why was a 1-month baseline chosen? This might miss early effects or
be confounded by stabilization that might occur irrespective of the study drug. A
rationale for this time point is crucial.

Reply: We chose to initiate study assessments after one-month post-transplant because a significant proportion of our liver transplant recipients are high MELD patients who typically present with markedly elevated bilirubin and cholestatic enzyme levels at baseline. Additionally, in the immediate post-transplant period, transient elevations in liver function tests are commonly observed due to ischemia–reperfusion injury, surgical factors, sepsis, or early graft rejection.
In live donor liver transplant for high MELD recipients, these fluctuations are often pronounced during the first few weeks following transplantation. Therefore, a 4-week window was provided to allow for stabilization of liver function parameters and resolution of early postoperative complications, ensuring that subsequent measurements more accurately reflect the effect of the study intervention rather than early perioperative variability.

Query: Omission of Bilirubin: Total bilirubin is a key marker of cholestasis severity
and liver function. Why is bilirubin not included as a primary or at least a key
secondary endpoint for cholestasis assessment?
Reply: Total bilirubin has been retained as an important secondary endpoint, given its clinical relevance in assessing hepatocellular function and persistent cholestasis. Indeed, liver function tests including bilirubin levels are listed as the first secondary end point.

Query: Sample Size Calculation and Pilot Study Data:
The pilot study data used for sample size calculation ("difference in alkaline
phosphatase (5.131 ± 39.639 vs 12.496 ± 52.139)") shows extremely large standard
deviations relative to the means. This suggests massive variability or heterogeneity in
the pilot data, or potentially a very small effect size, making the observed difference
potentially unreliable for powering a larger trial. A difference of ~7 U/L (assuming
these are absolute values) seems very small.

Reply: Regarding the sample size calculation, we acknowledge that data specific to the post–liver transplant population receiving obeticholic acid is extremely limited. Apart from our pilot study, we were unable to identify any previously published data in this specific setting to guide formal power calculations. Based on the effect sizes and variability observed in our pilot data, our statistician advised that this sample size would be adequate to provide preliminary evidence and inform the design of a larger, adequately powered trial in the future.

Exclusion Criteria and Analysis Population:
"patients who discontinued study drugs were excluded." Excluding patients who
discontinue the drug from the analysis can introduce significant bias. An intention-to
treat (ITT) analysis, including all randomized patients in the groups to which they
were originally assigned, is standard for RCTs and should be the primary analysis. A
per-protocol analysis can be secondary. This needs to be specified in the statistical
analysis section.
See earlier comment on exclusion criteria of patients that died <30 days.

Reply: Thank you very much for your observation. We are in fact conducting this trial as an intention-to-treat (ITT) analysis approach. Accordingly, all randomized patients—including those who discontinue the study prematurely—will be included in the final analysis. Efforts will be made to document and analyze the reasons for discontinuation, as this information is critical for understanding both the tolerability and the real-world applicability of the intervention.

We have modified the exclusion criteria after removing this sentence as follows:
We excluded patients who were randomized into the trial but died within the first 30 days. This decision was based on the fact that early mortality in this period is most commonly attributable to sepsis or technical complications. Including such patients would not be appropriate for evaluating interventions aimed at the management of cholestasis, which usually occur 2 to 3 weeks after surgery. Mortality within 30 days, purely secondary to the adverse effect of the drug, with no other technical ( vasculo-biliary), septic or immunological cause is extremely unlikely.

Query: Secondary Weaknesses and Areas for Clarification/Addition:
UDCA Dosing:
UDCA is dosed at "10-15 mg/kg TID." Typically, UDCA is administered BID in
post-transplant settings. Provide a rationale for the TID dosing regimen for
UDCA.

Reply: Thank you for your comment regarding dosing. The typical recommended dosage for managing cholestasis—particularly with ursodeoxycholic acid (UDCA)—is 10 to 15 mg/kg/day, usually administered in three divided doses. Based on this standard recommendation, we have selected the dose accordingly in our study to ensure therapeutic efficacy while maintaining safety and consistency with existing clinical guidelines.
Several peer-reviewed studies and reviews that report the use of ursodeoxycholic acid (UDCA) at doses of 10–15 mg/kg/day in post–liver transplant patients for prophylaxis and early graft protection (Ref : Pedersen, Mark R.^*,1; Greenan, Garrett²; Arora, Sumant³; Murali, Arvind R.³; Mayo, Marlyn J.¹. Ursodeoxycholic Acid Decreases Incidence of Primary Biliary Cholangitis and Biliary Complications After Liver Transplantation: A Meta‐Analysis. Liver Transplantation 27(6):p 866-875, June 2021. | DOI: 10.1002/lt.25935). For an adult with normal/average body weight, the doses of UDCA thus may be divided to three times a day.

Query: Measurement of Specific Secondary Endpoints:
"Bile Salt Export Pump levels (BSEP)": How will BSEP levels be measured?
Perhaps I am mistaken, but BSEP is a membrane-bound transporter, so will
only be measured with biopsies, as in you methods, will only be done in the
suspicion of rejection. This is currently too vague.

Reply: Thank you for your thoughtful feedback. You’re absolutely right—BSEP is a membrane‑bound canalicular transporter and, under normal conditions, is not released into the bloodstream in its intact form. In our study, however, we specifically sought to identify and quantify membrane‑derived fragments of BSEP that may be shed into the circulation during post‑transplant hepatocellular injury. To achieve this, we used a commercial sandwich ELISA kit validated for human serum and plasma. It is probable that the usefulness may not be as much as measuring the tissue BSEP levels, which will entail multiple liver biopsies in our patient. Our study results will hopefully give a better clue.

Query: "Plasma bile acid levels":
Clarification Needed: Will this be total plasma bile acids, or will specific bile
acid species be fractionated and quantified (e.g., via LC-MS/MS)? The latter
would be much more informative regarding FXR target engagement and
changes in bile acid pool composition.

Reply: Thank you for raising this important point. In our study, total plasma bile acid levels were quantified using commercially available ELISA kits. While we fully agree that fractionated bile acid profiling using LC-MS/MS would provide deeper insights into FXR target engagement and alterations in bile acid pool composition, this approach was not feasible in our current setting due to budgetary constraints and limited access to mass spectrometry facilities.
Should we secure adequate funding in the future, we certainly plan to incorporate detailed bile acid species quantification via LC-MS/MS to enhance the mechanistic depth of our analysis.

Definition of "Cellular Level" Assessment:
The Rationale mentions "specific biochemical tests to assess cholestasis at the cellular
Clarification Needed: Which specific tests (beyond standard LFTs and the
listed secondary markers) are being referred to that assess cholestasis at a
"cellular level"? This phrasing needs to be more precise or linked directly to
specific planned assays (e.g., markers of oxidative stress, apoptosis, or specific
transport protein expression if biopsies are analyzed beyond histology).

Thank you very much for the comment. We have removed the usage of “cellular” from the context

The text in the manuscript modified as below:
This study aims to fill an essential gap by evaluating the efficacy of obeticholic acid compared to ursodeoxycholic Acid in managing post-liver transplant cholestasis. By conducting specific biochemical tests to assess cholestasis, this research will provide insights into the effectiveness of OCA relative to the current standard, UDCA.

Query: Introduction and Conclusion Phrasing:
Introduction: "Although its usefulness is questionable, many practitioners utilize
ursodeoxycholic acid (UDCA)..." While there are debates, phrasing it as "questionable"
without immediate specific referencing for the LT context might be too strong.
Perhaps "its efficacy for preventing all forms of post-LT cholestasis or improving long
term graft survival has limitations" or similar.

Reply: Thank you very much for the comment. We have changed the sentence according to your suggestions.
While many practitioners utilize UDCA as a hepatoprotective drug, its efficacy for preventing all forms of post liver transplant cholestasis or improving long-term graft server has limitations.

Query: Conclusion: "Obeticholic acid (OCA) is a potent farnesoid X receptor (FXR) agonist,
with 100 times the efficacy of the current standard treatment, UDCA..." This is
misleading. OCA is ~100 times more potent as an FXR agonist than chenodeoxycholic
acid (a primary bile acid), not UDCA. UDCA is not a potent FXR agonist; it has different
mechanisms of action (choleretic, cytoprotective, immunomodulatory). This
statement needs correction for accuracy.

Reply:Thank you very much for the comment. We have changed the sentence according to your suggestions.

Sentence now reads as’
Obeticholic acid (OCA) is a farnesoid X receptor (FXR) agonist, with significantly more potency than UDCA in modulating bile acid metabolism.

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Competing Interests

nil

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Reviewer Report

22 Views

03 Feb 2025 | for Version 1

Palak J Trivedi, University of Birmingham, Birmingham, UK

22 Views Cite this report Responses(1)

Approved With Reservations

Is the rationale for, and objectives of, the study clearly described?

Yes
Is the study design appropriate for the research question?

Yes
Are sufficient details of the methods provided to allow replication by others?

No
Are the datasets clearly presented in a useable and accessible format?

Partly

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Cholestasis, liver transplantation, clinical trials.

Respond to this report

Responses (1)

Author Response

11 Sep 2025

Anila K N, GI Surgery, Amrita Institute of Medical Sciences & Research Centre, Kochi, Amrita Vishwa Vidyapeetham, Coimbatore, India

The rationale for the study is reasonable, but depth of secondary endpoints being captured is limited.

Thank you very much for the comments.

Our secondary endpoints include all biochemical parameters (Liver function tests including Gamma Glutamyl transferase), post-transplant molecular markers fibroblast growth factor 19 (FGF-19), transforming growth factor β (TGF-β) level, Cytokeratin 18 level, Serum autotaxin level, Bile Salt Export Pump levels (BSEP), Plasma bile acid levels that assess biliary secretion. We did not include the pharmacodynamics and pharmacokinetics of obeticholic acid as it has already been done. We have also included health related quality of life assessment.
We did not include any exploratory endpoints to formulate due to cost constraints.
Which adverse events will be captured, in what way, and how will they be recorded?
&
How will pruritus be captured? Highly relevant for OCA-treated patients.

Thank You for the insightful query.

Adverse events will be captured by direct interview of patients during the routine follow-up visits. The Naranjo algorithm will be used to establish causality. We will capture the pruritus by physical examination, vital signs and 5D pruritus questionnaire and will be documented systematically using structured Case Report Forms (CRFs) to ensure standardized data collection and analysis. The most common adverse event observed is pruritis.
Pruritus associated with Obeticholic Acid (OCA) is known to be dose-dependent. However, in this study, we are using a lower dose of 5 mg once daily (OD), which is expected to minimize the incidence and severity of pruritus. Nonetheless, any pruritus reported by patients will be systematically assessed and documented in CRFs for consistency in data collection.

The text will now read as:
Adverse events are being captured by direct interview of patients during the routine follow-up visits. We are capturing the pruritus by physical examination, vital signs and 5D pruritus questionnaire and documented systematically using structured Case Report Forms (CRFs) to ensure standardized data collection and analysis. The Naranjo algorithm will be used to establish causality and then classify them as definite, probable, possible, or unlikely.

The dose of UDCA is also lower than what would be considered standard practice?

Thank you for this comment.

In fact, our recommended dose is 10 to 15 milligram per kilogram TID. Unfortunately, at one point the dose was mistakenly written as 10 to 15 mg/kg twice daily which is lower than the recommended dose, but shall be corrected.

The text in the manuscript now reads as:

It is usually administered for three months post-transplant at a dose of 10-15 mg/kg thrice daily. In 2016, the FDA approved obeticholic acid (OCA), a strong selective Farnesoid X Receptor (FXR) agonist, to treat primary biliary cholangitis (PBC) in conjunction with UDCA. ¹⁶^,¹⁷Research has demonstrated that over 12 months, OCA, either in combination with UDCA or on its own, dramatically lowers levels of total bilirubin and alkaline phosphatase. ¹⁸^,¹⁹In individuals with advanced liver disease, the FDA has cautioned against administering OCA at doses of 10–50 mg. ²⁰Studies comparing OCA’s effects on cholestasis, rejection, regeneration, and cost-effectiveness to the current standard UDCA are being conducted to assess the role of OCA in the postoperative recovery of liver transplant patients. ²¹Our proposed project is a clinical trial between OCA and the current standard of care, UDCA, in managing post-liver transplant cholestasis and improving graft survival.

As the authors may be aware, the EMA and FDA are in the process of revoking approval for obeticholic acid in Europe and the United States, respectively. Whilst such regulators do not necessarily have jurisdiction in India, how do the authors feel that these decisions will affect access to OCA?

Thank you for the comment. We are aware of the recent FDA revoke for approval of obeticholic acid for further trials in steatohepatitis. However, we are not aware of any change in FDA approval for primary sclerosing cholangitis or primary biliary scleroses or similar cholestatic liver diseases.

In any case, the caution against its use was for patients with advanced liver disease. In our scenario of post-liver transplant patients, that liver function is essentially normal and the dose that we use is much lower than the toxic doses that has been reported. We are using 5 milligram once a day. We hope that results of our trial may give further insight into the effectiveness and adverse effects of this drug in post-transplant population.

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Competing Interests

No competing interests were disclosed.

Alongside their report, reviewers assign a status to the article:

Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions

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FXR agonist in post-liver transplantation patients: a randomized open-labeled study.

Abstract

Abstract

Keywords

Revised Amendments from Version 1

Introduction

Rationale

Primary outcomes

Secondary outcomes

Methods

Study design and participants

Eligibility & exclusion criteria

Recruitment & randomization

Figure 1. Flow chart showing study methodology.

Biopsy and rejection evaluation

Adverse reactions and causality assessment

Quality of life (QOL) measurement

Sample size

Study status

Statistical analysis

Expected results

Conclusion

Confidentiality

Access to data

Data dissemination

Ethical considerations

Author contributions

Data availability statement

Extended data

References

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