Current and Developing In vitro and Ex vivo models for assessing medication permeability into the gut produce a Systemic effect


Zainab Fadhel Alsafar1*, Mohammed Sabar Al-Lami1,2

1College of Pharmacy, University of Basrah, Basrah, Iraq.

2College of Pharmacy, National University of Sciences and Technology, Thi Qar, Iraq.

*Corresponding Author E-mail:



The field of drug permeation assessment concerning the discovery and development of orally administered medications has generated a lot of attention. Inappropriate properties of some drugs such as poor water solubility, limited stability at various pH, being a substrate to efflux transporter and degradation by intestinal enzyme, resulting in inefficient oral administration. In research on improving oral absorption of drugs, the measurement of drug penetration across the intestinal membrane iscritical because it will determine oral absorption. The main question is: what is the best model for studying medication permeation and absorption? This review article answers this question by explaining many methodologies that used to evaluate oral drug permeability/absorption in drug discovery. We address the most common and unique in-vitro and ex vivo models needed to assess drug permeation, the benefits and drawbacks of each model, and the mechanisms of drug absorption that each model may analyze. Moreover, to clarify the improvement of the non-everted rat gut ex vivo technique that is acting as a promising approach in drug permeation orally.


KEYWORDS: Permeation, Intestinal barrier, In-vitro model, Absorption mechanism, Non-everted rat gut sac model.




The oral dosage intake is the most appropriate drug administration regarding patient compliance; convenient, non-invasive, less expensive and can provide prolonged drug release1,2. But, To be absorbed successfully, the medicine has to be proposed in a form capable to cross the gastric intestinal tract (GIT)and passing through many barriers, like pH, degradative enzymes, and membrane permeation, which operate as the principal biological barriers3,4,5. The intestinal mucosal layer connects an outer ward and the human internal environment6. It consists of the enterocyte, goblet cell and Paneth cell.


The intestinal mucosa has a defense function resulting from the mucosal layer, the tight junction between epithelial cells and immune cells present in lamina propria; this function is crucial for the prevention of microorganism and any other foreign substances from entering systemic circulation7,8. One of these substances is API in Biopharmaceutical Classification System (BCS) classes III and IV showed poor intestine penetration, resulting in low oral bioavailability, so that, it is essential to assess drug permeation through the intestinal membrane in the development of a pharmaceutical formulation9,10.


The concentration gradient is the driving force of the diffusion of solute molecules through intestinal membranes5. The diffusion depends on molecular physicochemical properties, including molecular weight, aqueous solubility, lipophilicity, and hydrogen bonding 11,12. Successful simple diffusion necessitates molecular hydrophilicity to give high solubility in intestinal luminal fluid and interact with the membrane's hydrophilic group and hydrophobicity to a specific limit to interact with the membranes phospholipid group13,14.


The transport of substances from the intestinal lumen to the bloodstream occurs by different mechanisms, which include Passive diffusion, which can take place throughout epithelial cells (transcellular) or between epithelial cells (paracellular)4, endocytosis3,  carrier-mediated influx or efflux transport15 as disclosed in figure 1. The permeability assay analyses the flow of drug molecules from the donor site to the acceptor site, which provides an assessment for successful oral drug delivery. The techniques available to study intestinal absorption are continually growing and categorized as cell-free, cell-based, and tissue-based designs, depending on the separating barriers between phases16, 17.


Therefore, selecting the most suitable technique for intestinal permeation studies is difficult. This article discusses the current method used to evaluate drug permeation and development in a non- everted ex vivo rat gut sac permeation model.


Figure 1: Mechanism of absorption drug from small intestine: A-Trans cellar absorption, B-Para cellar, C- carrier-mediated absorption, D-efflux pump, E: endocytosis .adapted from18


The in vitro and ex vivo models for evaluating intestinal drug permeation:

During the period of drug discovery and development of pharmaceutical preparations, numerous models are employed to measure drug absorption, as shown below:


Cell-free models:

Parallel artificial membrane permeability assay (PAMPA):

This technique uses are 96 plates, each of which has filter support and a membrane soaked in phospholipid solvated in an organic solvent, with egg lecithin representing the phospholipid employed in this procedure to approximate the conditions seen in mammalian membranes18,19. In the last 20 years, many types of PAMPA have been applied to develop this technology, with differences in lipid content, filter nature, PH of receptor/donor phase, and sink condition of the receptor phase10. The PAMPA was initially utilized to evaluate the drug permeability through the intestinal epithelium. This model is used to assess absorption of eprosartan mesylate21 and permeation of supersaturated solution of lipophilic drugs22. It has been modified for assessing the permeability through the skin and blood-brain barrier (BBB) because of the versatile composition of the filter membrane. In addition, it can measure drug permeability at different pH gradients to simulate the pH in the GIT. PAMP may also be used as an in vitro method to assess the effect of excipients on drug permeability through biological membranes23. Compared to the caco2 technique, PAMPA has a low cost and is easier to use. Furthermore, the result obtained by measuring permeability using a caco2 technique, which is identical to that obtained by PAMPA24,25.


Phospholipid based vesicle permeation assay (PVPA):

In this technique, each plate composed of liposomes on filter support resembles the phospholipid in the intestinal epithelium. The researchers utilized solvent evaporation and freeze–drying to enhance liposome union, resulting in a tight barrier made up of liposomes that resembled a cell layer or tissue, so the organic solvent was removed in this method. Also, this vesicle was stable at pH range 2-8; this property is essential to simulate the pH range of GIT. A new type of PVPA has been sophisticated, one containing liposomes with a negative charge21 and another named mucus-PVPA, which contains mucus on the upper part of PVPA barrier18. Because the apparent permeability measured by PVPA corresponded well with literature data on human absorption in vivo, it was suggested as a promising method for swiftly screening the passive transport of medicines; the original PVPA might be used as a broad model to approximate a variety of biological absorption barriers, the permeability data acquired from the PVPA model was found to be comparable to the Caco-2 and PAMP-double sink models 14.this model used for assessment permeation of fenofibrate formulated as SNEDDS26,27.



Permeapad has 96 well plates with a barrier separates the upper and lower compartments. Instead of filter support, two cellulose membranes are sandwiched together by a layer of dry phospholipid. It exhibited good storage stability after preparation and can be kept in a dry, light- protected location for one year[18]. Soy bean phosphatidylcholine is used in the Permeapad® model. Furthermore, they become in contact to each other after hydration, so the vesicles remain close and simulate tissue morphology 16. Permeability data derived with Permeapad®was found comparable to the Caco-2 cell assay and PAMPA 15.


The above three cell-free models can assess drug transcellular passive diffusion and not permeability via active carrier and paracellular pathway. It is more costly and time- consuming. Therefore, they use in predicting nanoparticle transport is limited 20.



Figure 2: Represent the plate of cell-free models, which include 1- PAPA, 2:-PVPA, and 3-permeated, adapted from 20.

Cell-based model: Caco2 cell model:

The gold standard for assessing intestinal absorption in vitro is this model. It is made from a monolayer obtained from human colonic cancer. The caco2 cell model requires precise culture environments for the cells to differentiate and grow like a human gut enterocyte. Caco-2 cell monolayers are often cultivated on semi-permeable barrier on plastic support that can be inserted into multi-well culture plates' wells. The drug's permeability is afterward tested by adding it to any of the apical or basolateral sides of the monolayer29. While cells of this model are originated from the e the colonic part of human GIT, this model can be used to assess the drug permeation from the small intestinal lumen to the systemic circulation. Because it has the same brush border, tight junction, uptake, and efflux transporter features of small intestine30. The disadvantage of the caco-2 monoculture is that the top mucosal layer, which covers the intestinal membrane, is missing in this model; we can't use it to analyze the permeation of drugs whose absorption is influenced by passing through the mucosal layer. This limitation was overcome by developing models mimicking in vivo human conditions, such as the HT-29 co-culture (which included mucus-producing cells) and the triple culture (M-cell, goblet, and caco-2 cells) were approved for assessing nanoparticle system permeation. [12]. The limitation of this culture model is that it takes 21 days to discriminate and needs to be maintained every 2–3 days31. It's a complicated and expensive technique [16]. Also, the intra- and inter-laboratory heterogeneity in expression levels; will create variability in drug assessment permeability32.



Figure 3:   Illustrated caco-2 cell permeation model adapted       from 33




This novel system comprises a porous and flexible membrane that connects several chambers and microchannels. The channels, chambers, and other embedded features such as sensors, electrodes, or valves are housed in the chip's body. The membrane is surrounded by two channels, one representing the gut lumen and the other blood vessels as shown in figure 4, resulting in an identical to human situation. The worker uses cell culture (including caco-2 and human intestinal cells) and microfluidic technology in the gut on-chip model. Also, a 3D model with a collagen scaffold on the membrane was to measure para cellar and carrier-mediated molecular transport34. Transepithelial electrical resistance (TEER) has been applied to evaluate the integrity of membrane and barrier function. This model was more sophisticated and included technological hurdles such as developing more biocompatible materials, optimizing microfluidic designs, and finding ways to increase uniformity35.


Figure 4: Illustrated gut-on-chip model adapted from34


Tissue techniques:

Tissue approaches include the permeability chamber model, the rat gut sac model, and the in-situ rat intestinal perfusion technique.


A.   Using chamber model:

The using chamber model is increasingly used in studies of biological and nanoparticulate systems for permeability analysis35,36. This procedure involved filling a chamber with appropriate biological buffers and isolating a sheet or ring of intestinal mucosa from the intestine of a rat or human by cutting the intestine into strips, which were then installed in the chamber for diffusion testing. This method requires that these sheets be supplied with 95 percent O2 and 5% CO2 at 37°C. The integrity of these tissues has to be tested by measuring transepithelial electrical resistance (TEER). The sample should be loaded to the donor chamber at the apical side of the tissue, and the permeability can be measured in samples that drawn from the acceptor chamber at the basolateral at specific time intervals. This model is proper in studying of drug permeation at various regions of the small intestine as well as for controlled-release tablets. The main draw backs is the absent of nerve and blood supply as the segment is isolated from the small intestine, and so may lose the quality of permeation during the experiment37, this technique is used for assessment permeation of microemulsifying drug delivery system38 and labraso39.


Figure 5: illustrated diffusion chamber adapted from40


B.    In situ rat intestinal perfusion technique:

This model can examine drug permeation from all segments of the small intestine in rats. It can offer us an idea of which part of the small intestine the medication will be absorbed, making this technique beneficial for drugs with windows absorption. This approach involves using two tubes: the first tube for sample perfusion introduced in the proximal part of the intestine and a second tube for drainage inserted in the distal part, as in figure 6. The drug solution must be pumped from a perfusion tube, passed through the intestinal segment, and collected after passing through the drainage tube to determine the medication's absorption rate41. The absorption measurement is based on the variations between the medication solution's intake and outlet42.


Figure 6: Show the in situ intestinal permeation technique adapted from41


Because this technique has a complete blood supply and intestinal nerve 20, the challenge of intestinal tissue viability is overcome in comparison with chamber technique.  However, the disadvantage, is more expensive and need more numbers of an animal; it was also shown that surgical intervention of the intestine along with anesthesia resulted in a considerable alteration in blood flow to the intestine and had a significant impact on the pace of absorption 37, this model used for assessment the permeation of berberine hydrochloride for oral administration43.




C.   Rat gut sac technique:

This technique has two types, everted and non-everted techniques. The everted type was created by extracting the small intestine from an anaesthetized rat, then placing it over a glass rode and everting it 41; this everted intestine was connected at both ends with surgical sutures before being immersed in an oxygenated ringer solution containing the medication to be tested as present in figure 7. Then, to assess the rate of drug permeation through the everted intestine's membrane, a specified volume of this solution must be drawn at specific time intervals44. The advantage of the everted model is that it allows us to estimate the effect of efflux transporters on drug and excipients absorption. Also it can study the effect of cyclodextrin and poloxamer on oral absorption of boswellic acid45, as well as the occurrence of a mucous layer and a high surface area to simulate the in vivo human situation44. The change in gut morphology is the main drawback of this model as a result of the eversion, as well as maintaining the integrity of intestinal tissue during the experiment37.


Figure 7: Diagram for everted rat gut sac technique adapted      from 44


The non-everted methodology is easier and fast method to evaluate intestinal absorption in vivo. The excised intestinal sac was not everted in the non-everted rat gut sac technique, but all other procedures were identical to the everted model 36. Compared to the everted approach, the amount of medication used in the study is comparatively tiny. The everting procedure causes morphological destruction of the tissue, so this method is an alternative method to the everted gut sac model for assessing drug permeability 47. This technique was applied to evaluate the oral absorption of curcumin 36 and the effect of glidant peptides as permeation enhancers for drug delivery 48.


Advanced non-everted gut sac technique:

The procedure was conducted through several steps, which include as follows: the abdomen of the anaesthetized rat was opened; the small intestine was excised and washed with glucose saline, then kept in Tyrode's solution. After that, a 5cm segment was isolated from the fresh small intestine, especially from the jejunum site. The bottom of this segment end was securely tied with a surgical suture, while the top end was secured with a suture that was long enough to link the segment to the transducer. The intestinal segment carefully was immersed in a tissue chamber which fills with Tyrode's solution with tiny bubbles created from a flowing mixture of 95% O2 and 5% CO2 at 37°C and buffer pH 7.4.


The segment should not touch the inside of the tissue bath, and the upper suture thread should be vertical. A chromatogram is recorded by DT-475 Displacement Transducer from this system to assess the tension of the intestinal segment. After that, a small amount of drug solution was injected into the segment from the upper end to analyze drug permeability at a specific time interval49. This method's advantage is that the drug's permeation and intestinal motility can be assessed in the same experiment. Intestinal smooth muscle contraction represents by intestinal motility controlled by the enteric nervous system that has a substantial effect on the absorption of luminal material into the systemic circulation50. Also, membrane depolarization triggers the activation of voltage-gated calcium channels, releasing the intracellular storage of calcium then smooth muscle contraction51,52, the same occur in intestinal smooth muscle53. The advancement in this non-everted model is reflected by using the I Worx system (DT-475), as shown in figure 8, which acts as a recorder for intestinal motility by chromatogram that explains intestinal contraction, as seen in the research effect of ranitidine and neostigmine54 effect of Azadirachta indica extract55, and effect of Asperidine B46 on intestinal motility.


The ex vivonon-everted model can be a simple, quick, and low-cost approach. The integrity of the intestinal membrane could be maintained throughout the experiment57. Also, it gives an assessment for intestinal motility, which affect the diffusion of drug through the intestinal membrane and then absorption into the systemic circulation because Segmental motions and constant mixing of the luminal substance in the intestine assist keep the concentration at the mucosal surface at a maximum, this is important for permeation process because there is a linear relationship between the concentration of the substance and passive diffusion through the intestinal membrane. Also, the pressures created during segmentation may favor a modest amount of filtering57.


Cut segment from rat small intestine


Insert segment in tissue chamber


Figure 8: diagram for assessment of drug permeation with intestinal motility



In recent decades, many efforts have been made to develop permeability evaluation models for studying the oral absorption of different medicines. This review overviews various methods for measuring intestinal permeability, including in vitro and ex vivo models. There are eight approaches in this review, which have been explained in detail (in vitro cell-free and cell culture, ex vivo rat gut sac, Using chamber, cell culture; in situ: intestinal perfusion). The benefits and drawbacks of each model were explained, and the best applications for each were identified. There are problems associated with cost and laboratory variation connected with the cell culture process in cell-free and cell-based models. In contrast, the tissue-based model shows the low cost and low laboratory heterogenicity.


In addition, the development of the non-everted rat gut method was also discussed. This approach is relatively easy and inexpensive compared to other methods; this technique can also be used to record intestinal tension as a specific chromatogram provided by the iworx system DT-457, which indicates intestinal segment motility and then continuing the diffusion process during the experiment. As a result, this drug permeation study can be made with high robustness using this technique.



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Received on 05.08.2022            Modified on 07.09.2022

Accepted on 30.09.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(5):2492-2498.

DOI: 10.52711/0974-360X.2023.00410