Single-Culture Hepatocyte Biliary Excretion Assay
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Solution Overview
Problem
Current methods for assessing biliary excretion of chemical entities are inefficient, requiring multiple cultures of primary hepatocytes and relying on indirect and inaccurate assays, which are costly and prone to variability.
Innovation Solution
A single-culture in vitro method that involves forming bile canaliculi in hepatocyte cultures, allowing direct measurement of chemical entity uptake and excretion by disrupting canaliculi without lysing cells, using techniques like calcium-free media or latrunculin A, and employing LC-MS/MS for detection, enabling accurate and reproducible biliary clearance assessment.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If a two-culture assay format is used to measure biliary excretion, then biliary accumulation can be determined, but the number of primary hepatocytes required increases and cost increases
Solution Approach 1:
The patent combines the measurement of both biliary accumulation and cytoplasmic accumulation into a single culture system. By using a single culture of primary hepatocytes and sequentially measuring total accumulation followed by cytoplasmic accumulation, the method eliminates the need for parallel cultures while maintaining measurement capability. This merging of functions directly reduces the quantity of primary hepatocytes required from two cultures to one culture.
2Measurement precision
If a two-culture assay format is used to measure biliary excretion, then biliary accumulation can be determined, but assay variability increases
Solution Approach 1:
The patent combines the measurement of both biliary accumulation and cytoplasmic accumulation into a single culture system. By using a single culture of primary hepatocytes and sequentially measuring total accumulation followed by cytoplasmic accumulation, the method eliminates the need for parallel cultures while maintaining measurement capability. This merging of functions directly reduces the quantity of primary hepatocytes required from two cultures to one culture.
Solution Approach 2:
The patent employs a sequential measurement approach where the first measurement of total accumulation informs the second measurement of cytoplasmic accumulation. By using the results from the first measurement to guide the second measurement in the same culture, the method creates a feedback loop that reduces variability compared to independent parallel measurements. The subtraction of cytoplasmic accumulation from total accumulation provides a more reliable biliary accumulation value.
3Quantity of substance
If indirect marker compound assays are used to assess biliary excretion, then a single culture can be used, but measurement accuracy decreases
Solution Approach 1:
The patent extracts the direct measurement capability from indirect marker compound assays. Instead of relying on marker compounds to indirectly indicate biliary excretion, the method directly measures the accumulation of the test chemical entity itself in both total and cytoplasmic compartments. This extraction of direct measurement capability eliminates the accuracy limitations of indirect methods while maintaining the efficiency of using a single culture.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This method significantly reduces the need for primary hepatocytes, enhances accuracy, and provides direct measurements of biliary accumulation and cytoplasmic accumulation, improving the characterization of biliary excretion with lower variability compared to traditional assays.
Implementation Method 1
disrupting the at least one bile canaliculus without lysing the hepatocytes... by using techniques like calcium-free media
Implementation Method 2
disrupting the at least one bile canaliculus without lysing the hepatocytes... using techniques like... latrunculin A
Implementation Method 3
employing LC-MS/MS for detection, enabling accurate and reproducible biliary clearance assessment
Data Source
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AI summary
An in vitro methods of characterizing biliary excretion of a chemical entity using a single hepatocyte culture. Comprising providing cell culture comprising hepatocytes forming at least one bile canaliculus; contacting the cell culture with a first chemical entity for a time sufficient to allow uptake of the chemical entity by hepatocytes in the culture; disrupting the at least one bile canaliculus without lysing the hepatocytes and detecting the amount (if any) of the first chemical entity and/or a metabolite thereof released by the at least one bile canaliculus; and lysing the hepatocytes and detecting the amount of the first chemical entity and/or a metabolite thereof released by the hepatocytes.