A step-by-step guide for conducting safe and successful bile duct cannulated

A step-by-step guide for conducting safe and successful bile duct cannulated PK studies

Introduction

In recent years, the biopharmaceutical industry has increasingly focused on reducing animal testing when conducting safety and efficacy studies for new drugs. Ethical considerations, regulatory guidelines, and advancements in alternative testing methods like artificial intelligence have driven this shift. However, certain studies, such as bile duct cannulated (BDC) studies on rats remain unavoidable as they are crucial for understanding the pharmacokinetic profile of drugs, particularly their absorption, distribution, metabolism, and excretion (ADME).

BDC studies are essential for evaluating biliary clearance as they play a significant role in drug excretion.
This is especially true for compounds undergoing extensive hepatic metabolism. By conducting these studies appropriately, biopharma companies can minimize the number of animals used in conformance with regulatory guidelines that emphasize animal welfare and safety. Precisely designed and executed BDC studies not only provide valuable insights into drug behavior but also align with the industry’s commitment to ethical research practices.

In this playbook, we provide a step-by-step account of how to conduct bile duct cannulated studies in
rats, safely and successfully.

Important considerations for designing PK studies

Timing and dosing

Conduct studies at different stages of drug development, including preclinical and clinical phases.
Use appropriate dosing regimens to mimic therapeutic use.

Regulatory guidelines

Follow guidelines from regulatory bodies like the FDA and EMA. For example, the FDA provides detailed recommendations for human radio labeled mass balance studies.
The ICH M3(R2) guideline outlines the requirements for non-clinical safety studies, including pharmacokinetic studies, to support the conduct of human clinical trials and marketing authorization for pharmaceuticals.

Data analysis

Use advanced analytical techniques to quantify drug and metabolite levels.
Apply pharma cokinetic modeling to interpret the data and predict drug behavior in humans.

Reporting

Ensure comprehensive documentation of study design, methods, results, and interpretations.
Submit findings to regulatory authorities as part of the drug approval process.

Excretion in PK studies

Excretion is the process of removing a drug and its metabolites from the body, primarily through urine and feces. The kidneys and liver play significant roles in this process.

Urine excretion:

The kidneys lter drugs and their metabolites from the blood, which are then excreted in the urine. This process is influenced” by factors such as urine pH, renal blood ow, and the drug’s proper ties.

Fecal excretion:

Drugs and metabolites that are secreted into the bile are excreted into the intestine and eliminated through feces. This route is particularly important for drugs that undergo significant hepatic metabolism.

Well-structured BDC studies offer significant insights into drug dynamics while upholding ethical research standards for animal welfare.

Bile is produced by the liver, which helps in the digestion of fat and the elimination of drugs and metabolites. Hepatic metabolism is an essential determinant of drug clearance, bio availability, and excretion. Hepatic drug disposition depends upon organ perfusion rate, protein binding, the activity of drug-metabolizing enzymes, and excretion processes. Rats are the first choice of pre clinical species used to characterize drug clearance, bioavailability, and excretion profile of new chemical entities/drugs. Rats are an ideal model for examining biliary drug disposition as they lack gallbladders, and the bile duct can be cannulated for bile collection.

Biliary clearance

Biliary clearance is an important aspect of drug excretion, particularly for compounds that undergo significant hepatic metabolism. Bile clearance involves the secretion of drugs and their metabolites into the bile, which is then excreted into the intestine and eliminated from the body.
Factors Influencing bile clearance

Drug properties: Lipophilicity, molecular size, and the presence of specific functional groups.
Transporter proteins: Involvement of transporters like P-glycoprotein and bile salt export pump (BSEP).

Understanding bile clearance helps in predicting the overall elimination of the drug and potential drug-drug interactions.

Use advanced analytical techniques to quantify drug and metabolite levels, providing precise insights into drug behavior.

Considerations prior to starting biliary excretion studies

Before beginning a bile duct cannulation (BDC) study, it is essential to consider whether this type of study is the best way to answer scientific questions on drug movement. If yes, one needs to think about how to design the study, including which sex and strain of animals to use and what doses to test. Given below is a decision tree process to help decide whether a BDC study is required or not:

Challenges and Solutions

Selecting between oral BDC study or IV administration study

The recovery in feces following IV administration provides an indication of biliary elimination. This is necessary if quantication of intestinal secretion is required. If not, oral BDC study is sucient.

Inclusion of one or both sexes in BDC study

Inclusion of one sex is justifed where no genuine sex difference can be detected. In such a case, preference should be for male animals as they tend to be larger allowing for more background data to be collected as compared to females.

Selection of doses and number of dose groups

Testing for both high and low dose is justifed only if a notable difference has been identifed between excretion patterns of low and high dose groups for intact animals

Ensuring high success rates once BDC studies are triggered

Once a BDC study is deemed scientifically necessary and the appropriate sex and dose have been selected, measures should then be taken to ensure the welfare of the study animals and to achieve the highest success rates possible. Success rates should routinely exceed 80%. If this level of success is not achieved or if there are fluctuations in the degree of success, troubleshooting should be undertaken to address any underlying problems.

Impact of additional cannulations

Higher success rates can be achieved when the ‘dual cannulations’ method is employed. For this method, both the bile duct and gastrointestinal (GI) tract (duodenum) are cannulated, to allow for the simultaneous collection of bile and reinfusion of bile salt.

Weight of animals selected for the study

Animals should be at least 200-250g (preferably 225-275g) at the time of surgery. This would ensure that the bile ducts are of sucient size for cannulations.

External infiuences

Due care should be taken to ensure the animals are fit following the surgery

Surgical facility and personnel

Surgical facility and personnel

Surgery is to be performed in an isolated area suitable for aseptic procedures.
Personnel should be trained and experienced in the appropriate techniques.
A high standard of asepsis must be maintained throughout the procedure to ensure surgical success. Consistent surgical procedures must be followed to ensure reproducible outcomes and reduced complications.

Surgical techniques

Techniques vary in ensuring how bile is returned to the intestine.
Single cannulations without bile reinfusion are not recommended due to bile depletion.
Dual cannulations are recommended when the bile needs to return to the GI tract rather than the distal bile duct.

Cannula positioning

Suboptimal positioning can lead to leakage, dislodgment, and other issues.
Positioning should be checked if success rates are low.
Post-surgery animal movement can cause twisting or bending of the bile catheter.

Cannula and catheter considerations

Catheters should be of appropriate size for bile duct cannulation.
Silicone or polyurethane catheters can be used, depending on facility and surgeon preference.
Catheter material may a ect success rates.

Anesthesia and analgesia

Examples include Isoflurane in oxygen, carprofen, and buprenorphine.
Use should align with veterinarian recommendations and facility practices.

Use advanced analytical techniques to quantify drug and metabolite levels, providing precise insights into drug behavior.

Syngene's solutions/services

Our BDC studies involve collecting bile from animals at predetermined time points during or after substance administration. These studies are technically complex and require skilled surgeons.

Our scientists can assess the pharmacokinetic profile (ADME) in rats by administering substances either orally or intravenously (IV). If more than 20% of the substance is found in feces after oral dosing, biliary excretion is measured to determine absorption. This is particularly useful for substances that cannot be given intravenously due to formulation issues like low solubility.

Preparation and techniques

Preparation involves invasive surgical techniques.
Dosing and bile collection are intricate, requiring the single housing of animals in metabolism cages.
Cannulas are at risk of twisting or blocking.

Cannula patency

Failure of cannula patency can result in insufficient data, potentially necessitating study repetition.

Study assessment

Assess in advance whether data gathered from the experiment is necessary to answer scientific questions.
Consider alternative methods for obtaining the required information.
Use minimum number of animals to generate usable data.

Study recommendations

Support sta involved in BDC studies should try and maximize generation of useful data.
Ensure the highest possible standards of animal safety.

Ensure cannula positioning is optimal to prevent complications.
Use dual cannulation techniques to avoid bile depletion.
Follow regulatory guidelines for animal welfare and safety.
Employ skilled personnel for surgical procedures.
Use advanced analytical methods for precise data collection.

Perform single cannulations without bile reinfusion.
Neglect the importance of maintaining asepsis during surgery.
Use inappropriate catheter sizes or materials.

Ensure cannula positioning is optimal to prevent complications.
Use dual cannulation techniques to avoid bile depletion.
Follow regulatory guidelines for animal welfare and safety.
Employ skilled personnel for surgical procedures.
Use advanced analytical methods for precise data collection.

Perform single cannulations without bile reinfusion.
Neglect the importance of maintaining asepsis during surgery.
Use inappropriate catheter sizes or materials.

CASE STUDY

QUANTIFICATION OF UNCHANGED PARENT MOLECULES IN BILE, URINE, FECES AND PLASMA

Objective

To quantify the unchanged parent molecules eliminated in bile, urine, and feces after IV and Oral administration in rats and to monitor the putative metabolites in these excretions to understand the metabolite fate of the molecule.

The challenge

Development and availability of precise surgical techniques for bile duct cannulation.
Meticulous collection of samples.
Implementation of advanced analytical methods to ensure the reliability and accuracy of the data obtained.

Solution

Our team conducted a BDC study in rats, which involved:

Performing dual bile duct cannulation to enable the simultaneous collection of bile samples and the infusion of arti cial bile salts.
Administering the drug intravenously/orally to the dual BDC cannulated rats.
Collecting plasma, urine, and feces samples along with bile.
Utilization of advanced analytical techniques to quantify the parent drug and its metabolites in all collected samples.

The Outcome

The BDC study successfully quanti ed the unchanged parent molecules in plasma, bile, urine, and feces. The tested compound showed low biliary and renal excretion, with the percentage of dose excreted in bile and urine being less than 2% after IV and oral administration. Calculated renal clearance and bile clearance after IV administration of the tested compound were 0.12 ml/min/kg and 0.21 mL/min/kg, respectively. The tested compound showed higher recoveries in feces, with 10.3% of the dose unchanged after IV administration and 73% of the dose unchanged after oral administration.

This provided a comprehensive understanding of the drug’s excretion pro le using different metrics. By conducting the study with precision, we ensured high-quality data while adhering to regulatory guidelines for animal safety and welfare. Further, this approach minimized the number of animals used, aligning with the industry’s commitment to ethical research practices.

Syngene advantage in BDC studies

Extensive experience

We have 10+ years of experience in conducting dual BDC studies, with approximately 20 studies (100 surgeries) completed in a year.

High success rates

Our meticulous approach has resulted in reproducible data from over 80% of surgically prepared animals.

Skilled personnel

We have highly skilled and experienced surgeons proficient in complex surgical techniques.
Our expertise ensures consistent and reproducible outcomes, reducing complications and improving study reliability.

Advanced techniques

We employ advanced surgical techniques and innovative solutions to address challenges such as cannula patency and positioning.
Our approach minimizes the risk of cannula twisting or blocking, ensuring continuous bile flow and accurate data collection.

Customized solutions

We provide tailor-made solutions to meet the specific needs of each study, ensuring alignment with project objectives and scientific questions.
We offer flexibility in catheter materials (silicone or polyurethane) and diameters to suit different animal sizes and bile duct conditions.

Comprehensive support

End-to-end support for BDC studies from animal preparation to data analysis.
Highest standards of animal safety ensured while maximizing the output.

Innovative approaches

We ensure continuous innovation to improve surgical techniques and study outcomes.
We ensure incorporating of feedback and iterative improvements to refine methods and enhance success rate

Conclusion

BDC studies remain a critical component of understanding the elimination profiles of new drugs. These studies provide invaluable insights into drug metabolism and excretion, particularly for compounds undergoing significant hepatic metabolism. By conducting BDC studies with precision and care, biopharma companies can ensure they gather essential data while minimizing the use of animals.
Syngene is committed to conducting BDC studies safely and effectively to ensure high success rates while adhering to the highest standards of animal welfare and safety. Our approach includes utilizing skilled surgeons and advanced surgical techniques to perform bile duct cannulation, implementing meticulous sample collection and analytical methods to ensure reliable data, and following regulatory guidelines. By partnering with us, biopharma companies can obtain critical PK data for drug development without compromising on animal safety and welfare.

Know more about our in vivo pharmacokinetic services.

To learn more, contact our experts.

About the authors

Vishwottam Kandikere, Ph.D.

Assistant Vice President, DMPK, Syngene International

Vishwottam has around 25 years of hands-on experience in discovering 40+ drug targets across various therapeutic modalities encompassing small molecules, PROTACs, ADCs, and Peptides. He has assisted in lead discovery and optimization of 13+ NCEs from bench to bedside and is recognized as a key contributor in 13 US patents. He has published more than 90 original research and reviews in journals and presented more than 100 scientific posters at 50+ global scientific conferences.

Amol Raje, Ph.D.

Associate Director, DMPK, Syngene International

Amol has around 24years of experience in providing DMPK support to research programs for more than 30 therapeutic targets. These are across various therapeutic areas and drug modalities like small molecules, large molecules, PROTACs, ADCs, molecular glues, and peptides. As a key contributor, he has successfully led the discovery and optimization of 2 NCEs from bench to Phase-3 clinical trials, besides being an active member of a team that nominated ve clinical candidates for early clinical trials. Amol is interested in translational research, including herb-drug/drug interactions and human PK and dose projections.

Pankaj Paliwal, Ph.D.

Senior Research Investigator, In-Vivo PK, Syngene International

Pankaj leads a team focused on pharmacokinetic studies, contributing to the development of new drugs by designing and performing bioavailability and toxicokinetic studies in various preclinical species. His role involves significant expertise in surgical techniques and the use of advanced software tools for data analysis.

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