Intestine related DMPK analysis using RepliGut® Planar Models

Verify your Caco-2 results using multiple donors and regions for reducing late-stage failures

Importance of intestinal contribution to DMPK

In vitro modeling of intestinal absorption plays a pivotal role in advancing preclinical drug development by bridging the gap between laboratory research and clinical application. Predictions of bioavailability derived from cell culture models serve to refine drug design, optimize formulations, and flag potential safety issues. The RepliGut^® Planar model is the first intestinal epithelial model that accurately reflects human intestinal contribution to metabolism and transport in a format similar to Caco-2 cells, enabling scientists to further close the gap between in vitro and in vivo bioavailability prediction and reduce late-stage clinical failures.

BCS permeability classification in accordance with the ICH M9 guidance criteria

RepliGut® Planar- Jejunum distinguishes between high and low BCS classification drugs. Correlation between RepliGut® Planar-Jejunum Papp and human absorption. Dotted lines indicate threshold between high and low permeability drugs

RepliGut® Planar- Jejunum distinguishes between high and low BCS classification drugs. Fourteen (14) compounds were tested in accordance with ICH M9 guidance criteria for Papp and compared with known human fraction absorbed (Fa).

High Permeability

Propranolol
Antipyrene
Metoprolol
Desipramine
Theophylline
Carbamazepine
Ketoprofen

Low Permeability

Terbutaline
Atenolol
Enalapril Maleate
Lisinopril
Nadolol
Ranitidine
Furosemide

Correlation of Papp measurements to Caco-2

Comparison of passive permeability between RepliGut® Planar-Jejunum and Caco-2 cells. The apparent permeability of 14 drugs showed a strong correlation, with an R² value of 0.83.

How RepliGut® Planar compares

Ensuring accuracy in intestinal absorption modeling necessitates the presence and functionality of pertinent influx and efflux transporters along with metabolic enzymes. While Caco-2 cells have conventionally served as the benchmark cell culture model for in vitro absorption investigations, their fidelity to native human intestinal tissues is compromised by unregulated proliferation, physiologically inaccurate differentiation processes, and altered expression of drug-related transporters and enzymes, thereby impeding their reliability in mimicking in vivo drug absorption and metabolism.

Feature	Caco-2 Cells	RepliGut® Planar	In Vivo
Human origin
Polarized epithelial monolayer
Multiple cell lineages
Regional specificity maintained
Interindividual variability
Mucus excretion
Phase I and II metabolism
96-well format

Unlike Caco-2 cells, RepliGut^®models are comprised of multiple cell lineages found in the in vivo gut, which better reflect in vivo DMPK processes. The cell populations in RepliGut^® models are confirmed to be ALP⁺, MUC2⁺ and CHGA⁺ positive and form a polarized monolayer with intact tight junctions.¹ Furthermore, RepliGut® Jejunum models express more physiologically relevant levels of several genes encoding phase I and phase II metabolic enzymes reducing the chance of overpredicting bioavailability compared to Caco-2 cells

RepliGut^® Models enable you to assess region-specific DMPK endpoints, from more than one donor, for a more thorough vetting of drug candidate bioavailability.

Capabilities Summary

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Apparent permeability (P_app) and efflux ratios

Human jejunum epithelial stem cells are cultured and differentiated on semi-permeable membrane inserts in a 96-well plate to facilitate access to both the apical and basal sides of the cell monolayer. Test compounds are introduced into either compartment to assess their transport in both directions. Accurate mass spectrometry analysis of compound concentrations in each compartment allows for the calculation of permeability (P_app) and efflux ratios. Controls such as atenolol and propranolol, representing low and high permeability respectively, can be included. Additionally, transporter inhibitors can be applied to assess compound interactions with specific transporters. These assays are adaptable to specific research needs.

Typical study design
Cell Culture Timeline	10-11 days
Number of Replicates	Typically 3 wells per treatment
Incubation Time	Up to 120 min
Incubation Buffer	HBSS + 10mM HEPES + 10mM Glucose, pH 7.4
Barrier Integrity Assessment	Automated TEER using EVOM™ Auto (WPI) measured before and after compound incubation
Control Drugs	Atenolol and propranolol
Analysis Method	Accurate mass spectrometry measurement using LC/MS
Data Readout	Papp (apparent permeability coefficient), Efflux ratio, TEER

RepliGut® Planar- Jejunum accurately models passive and active drug transport. (A) Passive and (B) active transport drugs were applied to either the apical or basal compartment and drug concentration was measured in the receiver compartment at 2 hours post exposure. Digoxin and E3S transport were conducted in the presence and absence of P-gp inhibitor, tariquidar, and BCRP inhibitor, Ko143, respectively. Efflux ratios (green) were calculated as the ratio of B→A (pink) to A→B (blue).

Permeability markers tested in RepliGut® Planar-Jejunum

Absorption Group	Drug	RepliGut^® A→B Papp (10^-6 cm/s)	Human Fraction Absorbed (F_a)
High absorption (F_a ≥ 85%)	Antipyrine	14.7 ±2.3	97%
	Metoprolol	10.5 ±5.3	95%
	Propranolol	22.1 ±1.0	90%
	Carbamazepine	12.8 ±3.2	85%
Moderate absorption (F_a = 50 – 84%)	Ranitidine	0.98 ±0.2	50%
Moderate absorption (F_a = 50 – 84%)	Atenolol	0.97 ±0.9	50%
Low absorption (F_a < 50%)	Nadolol	0.47 ±0.29	34%
Low absorption (F_a < 50%)	Lisinopril	0.17 ±0.14	25%

Absorption Group	Drug	Transporter	Efflux Ratio
Efflux Substrates	Antipyrine	P-gp	12.5 ±5.8
Efflux Substrates	Estrone-3-sulphate	BCRP	30.7 ±14.4

Intestinal contribution to metabolic stability

Drug metabolism and transporter gene expression analysis

Transcriptomic analysis of RepliGut® Planar Systems can provide insights into gut-specific metabolic activity and potential for drug-drug interactions in the GI tract. We offer QuantiGene™ Plex Gene Expression Assays or TaqMan probe RT-PCR to provide targeted gene expression analysis to focus on the impact to your genes of interest. A reference RNAseq data set is available to confirm expression of your gene of interest in RepliGut® Planar- Jejunum.

Accurate mass spectrometry method development with quantitative or qualitative bioanalysis

Altis has partnered with BioAgilytix Labs to offer method development and bioanalysis using state of the art LC/MS instrumentation including a high-throughput SCIEX Echo® MS+ coupled to a SCIEX ZenoTOF 7600.

Comparisons between intestinal regions and donors

We have isolated epithelial stem cells from multiple intestinal regions obtained from multiple donors, allowing researchers to study colon or small intestine-specific metabolism and transport in demographically diverse backgrounds (e.g. age, sex, blood type). Our cryopreserved biobank enables the ability to work with the same donor as your project progresses or have a variety of donors to evaluate donor differences.

Companion barrier integrity monitoring

Accurate measurement of paracellular transport requires a consistently tight and intact cell culture barrier throughout the entire experiment. RepliGut® Planar barrier integrity is monitored in real time using Transepithelial Electrical Resistance (TEER). TEER is measured using the high-throughput and automated EVOM™ Auto (WPI) before and after compound incubation to ensure barrier integrity is not compromised.

References:

1. Pike CM, Zwarycz B, McQueen BE, et al. Characterization and optimization of variability in a human colonic epithelium culture model. ALTEX. Published online April 18, 2024. doi:10.14573/altex.2309221

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Intestine related DMPK analysis using RepliGut® Planar Models

Importance of intestinal contribution to DMPK

BCS permeability classification in accordance with the ICH M9 guidance criteria

Correlation of Papp measurements to Caco-2

How RepliGut® Planar compares

RepliGut^® Models enable you to assess region-specific DMPK endpoints, from more than one donor, for a more thorough vetting of drug candidate bioavailability.

Capabilities Summary

Apparent permeability (P_app) and efflux ratios

Permeability markers tested in RepliGut® Planar-Jejunum

Intestinal contribution to metabolic stability

Drug metabolism and transporter gene expression analysis

Accurate mass spectrometry method development with quantitative or qualitative bioanalysis

Comparisons between intestinal regions and donors

Companion barrier integrity monitoring

References:

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Intestine related DMPK analysis using RepliGut® Planar Models

Importance of intestinal contribution to DMPK

BCS permeability classification in accordance with the ICH M9 guidance criteria

Correlation of Papp measurements to Caco-2

How RepliGut® Planar compares

RepliGut® Models enable you to assess region-specific DMPK endpoints, from more than one donor, for a more thorough vetting of drug candidate bioavailability.

Capabilities Summary

Apparent permeability (Papp) and efflux ratios

Permeability markers tested in RepliGut® Planar-Jejunum

References:

Ready to Go With Your Gut?

Get in touch with us

Have a question?

4.9 out of 5 stars from 47 reviews

Quick Links

Important Links

Let’s Connect!

RepliGut^® Models enable you to assess region-specific DMPK endpoints, from more than one donor, for a more thorough vetting of drug candidate bioavailability.

Apparent permeability (P_app) and efflux ratios