PERMEABILITY- invitro, ex vivo, in silico

This presentation discusses various permeability studies and invitro, ex vivo, insilico permeability models used in healthcare research

1. PERMEABILITY in vitro, in vivo, Ex vivo, in situ, in silico Presenter V. Sravani

2. Invitro In vivo Ex vivo In situ In silico Permeation studies

3. In vitro methods Cell based methods Non cell based methods

4. Non biological methods

5. Parallel Artificial Membrane Permeability Assay (PAMPA) Filter infused with phospholipids in an organic solvent to mimic the lipid composition of the intestinal membrane The extent of permeation through the membrane is measured and compared to known extent of drug absorption in humans Hexadecane membrane-PAMPA, biomimetic-PAMPA, dioleoyl phosphatidylcholine-PAMPA , Double-Sink TM PAMPA Double artificial membrane permeation assay (DAMPA) containing an intracellular compartment to mimic the intracellular space between membranes improved accuracy of predictions of drug intestinal transport, to a certain extent

6. PVPA (Vesicle based permeation study) The membrane comprised phospholipid-made liposomes (mainly composed of phosphatidylcholine) deposited into the pores and onto the surface of a nitrocellulose filter support. liposome-based membrane by using negatively charged liposomes that more closely mimic the lipid composition of intestinal cells. The model was supplemented with an additional layer of porcine mucus on top of the liposome-based membrane Passive transport and transmucosal delivery

7. Permea Pad Sandwich-like structure consisting of a layer of dry phospholipids (soybean phosphatidylcholine S-100) wrapped in two support layers (cellulose-hydrate membranes). Sandwich-like structure prevents the erosion of the wrapped lipid layer and the leakage of lipids into the aqueous environment. PermeaPad barrier exhibits stronger resistance to pH changes and aggressive additives (e.g., cosolvents) compared to the PAMPA and PVPA models. Study transcellular transport.

8. Cell based methods Mono culture models Co culture models Triple culture models 3D cells models

9. Skin models A number of culture-derived skin equivalents such as living skin equivalent models (LSEs) and human reconstructed epidermis (HRE) have been used to measure percutaneous absorption Episkin ( Episkin SNC, Chaponost , France), EpiDerm ( MatTek Corp., Ashland, MA, USA) SkinEthic ( Laboratoire SkinEthic , Nice, France). EpiSkin comprises a human collagen (types III and I) matrix, representing the dermis, covered with a film of type IV human collagen, on which stratified differentiated epidermis derived from human keratinocytes is grown. Reconstructed human skin models are able to mimic human skin to a large extent, contrasting with the limitations of classical cell monolayers

10. Ocular models Cornea: The human corneal epithelium HCE-T cell model Conjuctiva : The IOBA-NHC cell line, a non-transfected, spontaneously immortalized epithelial cell line derived from human conjunctiva Chang conjunctiva epithelial cell and primary human conjunctiva-derived epithelial cell (HCDEC) models were used for studying the efficiency of a novel carrier system, bacterial ghosts Retina: Monolayers of ARPE-19 cells are a well-established in vitro model of the outer blood--retinal barrier (BRB). This model has been used for the development of targeted drug delivery systems to the posterior segment of the eye (regulation of gene expression, polarized distribution and secretion of proteins, delivery of genes and antisense oligonucleotides, for toxicity studies, and as models of retinal disease)

11. Pulmonary and nasal models Air pathway : Human bronchial epithelial cell lines, Calu3 and 16HBE14o- cell monolayers. This in vitro system allowed studying both the deposition and permeation of particles, two of the critical parameters in the efficacy of an inhalable formulation. Alveolar region: A549 cells have been used as model for studying the absorption of peptides and proteins 3D systems based on human-derived tracheal/bronchial epithelial cells ( EpiAirway , MatTek Corp., USA) Recently, co-cultures have been developed for nasal permeation studies composed by a collagen matrix with embedded human nasal fibroblasts covered by a RPMI 2650 epithelial cell layer which resembles nasal mucosa.

12. Vaginal and Rectal models The use of in vitro cell models for studying rectal absorption of drugs has relied mainly on the Caco-2 monolayer model. CaSki endocervical cell line was shown to form cell monolayers or bi/ trilayers , depending on cell seeding density, and has been shown useful for studying transport mechanisms Cell-covered filters are typically mounted on Ussing chambers in order to proceed with permeation experiments commercially available vaginal--ectocervical tissue-like model – EpiVaginal . The model is based on a 3D culture of non-transformed human vaginal--ectocervical epithelial cells grown on polycarbonate cell culture tissue inserts.

13. Intestinal models

14. BBB models Static models Dynamic models Monolayer models BMECs are co-cultured with other CNS cells that directly contribute to the barrier properties of BBB-(BMECs with astrocytes or pericytes human cerebral microvascular endothelial cell line ( hCMEC /D3) and immortalized human cerebral endothelial cells Co culture models Stem cell based models Micro fluid based models

15. Dynamic in vitro BBB model BMECs and astrocytes are implanted in the inner (luminal) and outer (abluminal) sides of the porous hollow fibers, respectively. The culture medium is pushed into the system through a variable-speed pump to produce shear stress equivalent to that of physiological conditions in vivo (5–23 dynes/cm2 ). To maintain the stable microenvironment, a gas-permeable tubing system was used for the exchange of O2 and CO2. This dynamic in vitro BBB model has been used to study the pathophysiology of various CNS diseases, including ischemia-reperfusion-induced injury and epilepsy.52,53 Recently, an updated model with hollow fibers

16. Brush border membrane vesicles BBMV models are based on isolated apical membranes from different parts of the GIT, thus allowing evaluation of apical membrane transport without the influence of the basolateral membrane or regional differences in the GIT. The purified fraction of apical membranes is extracted from a homogenate of frozen inverted intestine BBMVs have been successfully extracted from different sources, including the human Drug uptake is evaluated by quantifying the amount of drug in both vesicles and the medium Incomplete information about the absorption process both for movement into and out of cells via the apical and basolateral membranes. Difficult to study transcellular mechaanism

17. Microfluidics-based systems Gut -on-a-chip and human-microbial cross talk ( HuMiX ), have emerged as cell-culture models for studying drug transport across intestine barriers These models utilize microfluidic technology for in vitro cell culture, thus reproducing the 3D topology, dynamic environment and gut microbiome observed in the human intestine gut-on-a-chip microdevices consists of a porous membrane that supports a monolayer of intestinal epithelium cells and separates two compartments, simulating the intestinal lumen and blood circulation

18. Ex vivo models InTESTineTM Intestinal rings Everted Intesinal sac Diffusion chambers GI tissue robotic interface system

19. Ussing Chamber First developed by Ussing and Zerahn (1951) to study transepithelial ion transport across frog skin This method involves the isolation of the intestinal tissues, cutting it into strips of appropriate size, clamping it on a suitable device. It consists of an opened intestinal segment supported, creating two isolated compartments: one corresponding to the luminal side, where the molecule of interest is placed, and the serosal side, corresponding to the basolateral region. This system also controls and maintains temperature and continuously gasses the chamber. Two voltage-sensing electrodes and two current-passing electrodes are placed on each side, which monitor and provide current. It enables measurement of transepithelial electrical resistance (TEER) short-circuit current across the membrane

20. An intestinal section is reverted, and both ends are tied, after filling it with an appropriate buffer solution. Then, this intestinal section is incubated with the chosen buffer and the compound of interest. The incubation usually involves a controlled temperature and aeration or stirring. Adv: Possibility to test distinct intestinal regions The sample volume on the serosal side is relatively small and drugs accumulate faster Everted sac technique

21. InTESTineTM Commercially available physiologically relevant intestinal tissue model developed by TNO Uses freshly isolated healthy porcine intestinal tissue from different regions of the animal’s GIT. The model promises to be a cost-effective way to study absorption, metabolism and the complex physiology of the intestine. The system is a medium-throughput system that is available in 24- to 96-well plate format

22. Intestinal Rings and Segments It involves exposing intestinal segments or rings, to the compound of interest, dissolved in a buffer, to assess its uptake by enterocytes and subsequent metabolism. An intestinal segment is isolated and, after washing, is cut either into small rings or segments and submerged in a proper oxygenated medium. The intestinal rings might also be incubated in an everted position, as in the evert sac technique. The main advantages of these methods are their practicality and simplicity, the ability to choose distinct intestinal regions and their high throughput

23. Circulation techniques In this method, small intestine may or may not be everted. This involves isolating either the entire small intestine of small lab animal or a segment and circulating oxygenated buffer containing the drug through the lumen. Drug free buffer is circulated on the serosal side of the intestinal membrane and oxygenated. Absorption rate from the lumen to the outer solution are determined by sampling both the fluid circulating through the lumen and outside.

24. Franz diffusion cells Temperature-controlled donor and acceptor compartments separated by the tissue. The test sample is introduced in the donor compartment, and sampling is performed from the acceptor compartment at different time intervals. The receptor chamber is continuously mixed using a magnetic stirrer, which significantly reduces the unstirred water layer Skin permeation studies

25. Animal models Blank urine or blood sample is taken from the test animal before the experiment. The test dosage form is administered to the animal and at appropriate intervals of time the blood or urine sample are collected and assayed for the drug content. The experimental animal chosen should bear some resemble to man. It is reported that pigs most closely resemble to man but are not used due to the handling problems. The other animal that can be used are dogs, rabbits and rats In Vivo models

26. In situ methods Perfusion models The gut loop model The isolated intestinal perfusion model

27. In silico studies Quantitative structure-permeability relationship(QSPR) models. Utilize statistical relationshipsextracted from experimental permeability measurementscombined with the physiochemical properties of a set oftraining compounds Molecular dynamics model simulate the molecular process of diffusion at the atomic level GROMACS4.5.5 (membrane builder) AutomatedTopology Builder server (topology generation) ChemAxon /Chemicalize server ( pKa ) CLogP miLogP Chemicalize LogP MOE SLogP

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