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Martin Brandl, Gril Eide Flaten and Annette Bauer-Brandl, University of Troms, Drug Transport & Delivery Group,
Troms, Norway
doi: 10.1002/9780470048672.wecb432
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Biologic Background Physico-Chemical Approach to Passive Transport
Breivika,
This article deals with the passive transport of electrolytes, water, and small organic molecules across biologic membranes (cell membranes and membranes that conne cellular compartments). Passive transport may be classied into diffusion, facilitated diffusion, and bulk ow. The focus in the current article is on simple diffusion, a process described by the random movement of solutes that results in the net transport (ux) along a concentration gradient. This process, at rst, may be described by the rst law of Fick. More detailed models take into account the partitioning of solutes between the aqueous media and the barrier itself, as well as the ux resistance that arises from water layers adherent to the barrier, which results in the second law of Fick. The actual extent of the diffusion of molecules across biologic membranes is caused by the interaction between the composition and the structural arrangement of the membrane and by the physico-chemical characteristics of the permeant. For the sake of simplication, the primary structural element of a biomembrane, which is a phospholipid bilayer, can be considered a continuous, lipophilic phase between two aqueous compartments. Commonly used permeability screening approaches include in silico modeling, liposome-based experimental models, cell culture models, and in situ perfusion.
The permeability of compounds through cell membranes is of great interest and importance for the elucidation of many biologic cell functions. Most metabolically important substances are transported across membranes by active transport. Many other intrinsic compounds, as well as most drugs, are known to pass the membrane by passive diffusion.
Biologic Background
Biologic membranes as transport barriers
The most prominent function of biologic membranes is to control selectively the molecular transport into and out of cells. The ability of certain molecules to traverse such biologic barriers depends on their composition as well as their structural and functional features. These features are discussed in more detail on a molecular level elsewhere in this encyclopedia. In
brief, the plasma membrane of a cell, as well as any other biologic membrane, is organized basically as a bilayer structure of two sheets composed of phospholipids and neutral lipids, like cholesterol, in which membrane proteins are embedded. The polar head-group of the phospholipids is facing toward the aqueous phases, which are the cytosol on the one side and the medium around the cell on the other, whereas the lipophilic chains of the sheets face to one another to form the center of the membrane. At rst glance, such a membrane might be assumed to be a tight barrier that separates the cell interior from its surrounding or cellular compartments from each other, not allowing for any transport of compounds across this barrier. However, particular molecules need to pass through cell barriers to maintain continuously the basic functionality of the cells; they need to introduce and load substrates as well as remove and discharge toxic compounds. Furthermore, overall cell volume, osmotic pressure, intracellular pH, and ionic composition are controlled by the passage of molecules through the cell membrane (1).
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Passively absorbed compounds diffuse either through the cell itself (transcellular pathway) or in between cells (paracellular pathway). The lipid bilayers of which the mucosal and basolateral epithelial cell membranes are composed of, dene the primary transcellular diffusion resistance to solute transport across the intestinal barrier. Transcellular permeability, particularly of lipophilic solutes, depends on their partitioning between intestinal membrane and aqueous compartments (Fig. 1).
Denitions
Flux (J ) of a species is the mass (M ) (or number of molecules of this species) transported per unit time across the barrier, normalized by the cross-sectional surface area A of the barrier:
J =
dM 1 dt A
(1)
(dimension of J : g sec1 cm2 ) A plot of the mass transported versus time is linear if the donor concentration is virtually constant and the acceptor concentration is virtually zero; the ux is constant when the
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tight junction
basolateral membrane
Figure 1 Pathways of the intestinal barrier. A: paracellular passive diffusion, B: transcellular passive diffusion, CF: inux/efux facilitated transport facilitated by membrane proteins, G: transcytosis, and H: endocytosis (reprinted from Reference 2 with kind permission from Dr. Jon V aben and Dr. Roy Lysaa).
system has reached the equilibrium called steady state (i.e., the linear part in Fig. 3):
inverse of the resistance of the membrane, which is the ux normalized by the concentration in the donor compartment.
M =J At
(1a)
P = J /CD
(2)
The slope of the curve corresponds to J A. In most experimental setups, the surface area A is known and ux J can be derived. Constant ux is maintained as long as C D is much larger than C A and as long as both are practically unchanged. In biologic systems, a good example for such conditions may be the uptake of xenobiotics from the GI-tract, where the GI tract acts as a reservoir for the molecules, which are transported through the intestinal epithelium into the blood, which acts as a sink. The quantitative value of ux is widely dependent on the concentration in the donor compartment C D (guratively spoken, the pressure). Ideally, C D should not change over the period of observation; if C D is not constant, its change with time needs to be considered, which in many cases applies to real situations. However, the concentration in the acceptor compartment should be kept neglectably small (sink conditions) to make calculations easier. Fortunately, in biologic systems, this scenario is very often the case and experimental conditions in most cases can be chosen accordingly. It is handy to dene permeability P as the
Permeability, therefore, comes in the dimension of cm/sec, a self-explanatory unit. Combining Equation 1 and 2 reveals:
dM = P A CD dt
(3)
In cases where C D is not constant over time, the ux J also changes with time. Exchanging mass by concentration, M = C D V , where V is the concentration of the donor phase, reveals:
P A dCD = CD dt V
(4)
dCD =k CD
0
dt
(5)
3
CD 0
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Figure 2 Schematic illustration of concentration gradients. CD concentration in the donor compartment; CA concentration in the acceptor compartment. Scheme 2a: without a physical barrier; Scheme 2b: lipid barrier-controlled; Scheme 2c: water layer-controlled; and Scheme 2d: combined lipid- and water layer-controlled.
mass transported
time
Figure 3 Diagram illustrating mass transport versus time, lag time, and steady-state ux.
time
reveals
ln CD 0 ln CD = k t
(6)
The sigma minus plot (i.e., plotting ln C D0 ln C D vs. time) yields permeability as the slope k , where still k = P*A/V . A similar general expression also can be used under nonsink conditions. Here it is useful to describe the velocity of transport (i.e., ux J ) expressing the effect of the concentration gradient (C ) and the length of the dimension along the line (x ), as well as to introduce the diffusion coefcient as the proportionality constant (the Fick law).
J = D
4
C x
(7)
This description is particularly useful because the diffusion coefcient is reasonably well dened in aqueous solutions, it is related to molecular properties in aqueous solutions, and it can be predicted. However, in biologic systems, the observed length x for a single transport stepwill be widely unchanged. Preferably, steady state and sink conditions are studied, which simplies the Fick law and focuses on permeability as stated above. Let us now consider a barrier between the donor and the acceptor compartment, where the barrier has different properties in terms of the solvation of the traversing molecules, as a simplication of a biomembrane. Such a model is shown in Fig. 2, beginning with Scheme 2b, where a homogeneous lipid barrier between the donor and the acceptor compartment is
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introduced. The permeability also is dened in this case as described above and as indicated in the Scheme. Looking closer into the system, the diffusant will partition into the lipid barrier according to the ratio of solubility both in the aqueous phases and in the lipid phase (the partition coefcient, i.e., the ratio of chemical potentials in the two phases). The concentration ratios in the two interfaces will be equal, but not the absolute concentrations, as the concentration in the donor compartment is higher than in the acceptor compartment. The gradient between the two interfaces will make the diffusion occur (see Fig. 2). The diffusion coefcient in the aqueous phases (donor and acceptor phase) would not play a role if practically no concentration gradient exists within these compartments, in other words, if the diffusion in the aqueous compartments is not rate determining. The partition coefcient between the lipid phases and aqueous phases, K , determines whether C mD and C mA , are higher or lower than C D and C A , respectively. If the diffusion of the permeant in the lipid barrier phase is rate determining, then the thickness of the barrier has to be taken into account as well. Here, the permeability coefcient is proportional to its diffusion coefcient D in the membrane (slow diffusion in the membrane).
permeability)as the reciprocal of the overall resistanceis the sum of the resistances of all the layers involved. One should keep in mind that permeability is a material property of the system with respect to the interaction of the solute, barrier, temperature, solution composition, and other factors. Furthermore, molecules at each step would be in a solvated state. However, these solvation states cannot be directly experimentally measured, easily predicted, nor modeled. The most simple and effective mathematical approach, therefore, is to look at the entire barrier as a whole and to stick to the evaluation of the overall ux and permeability, respectively, as stated above, keeping in mind that it is the interaction on the molecular level that explains these conditions. The overall transport is described, thenas it is not physico-chemically consistentin the terminology of apparent permeability, P app . Nonsink conditions are less appropriate for experimental work on transport through barriers and, thus, are not discussed more here.
J =
D K CD dM 1 = dt A h
(8) (9)
dM D K CD A = dt h
Again, given a practically unchanged C D , the mass transport still reveals the simple form:
M =k t
(10)
Also in this example, ux J is constant during the steady state and permeability P can be revealed. However, here we have to consider a lag time t L . For a constant ux, we have to wait until the lipid barrier is saturated and shows a constant gradient of the diffusant according to the partition coefcient. The lag time that represents the intercept with the time axis is found by extrapolating the linear part of the curve (Fig. 3).
M = k (t tL )
(10a)
Lag time t L would be a measure of membrane thickness h and the diffusion coefcient (diffusion velocity) in the membrane. The barrier models can be increasingly more complex with respect to the structure of the barriers:
Adjacent water layers on both sides of the lipid barrier (Fig. 2, Schemes 2c and 2d). Nonhomogeneous barriers (as are cell membranes) Nonsymmetrical barriers (as are cell membranes)
Any of the components of the barrier may be rate determining, and in some cases, several of them are important for the diffusion process (Fig. 2, Scheme 2d; combined control). In any case, it is most straightforward to look at the overall permeability of the entire barrier. The overall permeability (apparent
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Membrane uptake of the noncharged form of a solute is favored over its charged form. As a result, solute membrane permeability versus pH curves is shifted toward the right for weak acids and toward the left for weak bases (5). This condition also explains the irregular permeability behavior of ion pairs.
readily into the lipid barrier. Biologic uids contain a variety of such charged species that originate from the dissociation of salts, acids, and bases. Another kind of transport, facilitated diffusion, is prevalent for them because their permeation via simple diffusion is rather limited because of limited partitioning into the biomembrane. Facilitated diffusion is a process where permeants diffuse across membranes with the aid of transport proteins. Water-lled pores in the membrane formed by proteins are the main pathway for ions. These pores may be gated so that they can open and close, thus regulating the ion ux. Another kind of membrane proteins, carrier proteins that change shape as the molecules are being carried through, are responsible for the facilitated transport of larger molecules like glucose and amino acids. In contrast to active transport, facilitated diffusion does not require energy and only can carry molecules or ions down a concentration gradient. The transport proteins involved are intrinsic, that is, they span the membrane. They are solute-specic, for example, they have binding sites for the specic molecule or ion that they transport. They also have a dened capacity of how many solutes they can transport. An example of facilitated diffusion is glucose that permeates through cell membranes rather slowly via conventional diffusion because it is not soluble in the membrane. However, glucose passes quickly across a cell membrane via facilitated diffusion.
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by using a chromatographic surface prepared by covalently immobilized cell membrane phospholipids. The technique is experimentally simple and can screen many compounds within a short time. The predominant factor that regulates the passive diffusion across a membrane is its ability to pass through the lipid cell layer, and the capacity factor log K s determined by this technique shows reasonable correlation with permeability across cell monolayers and partitioning into liposomes (8, 9).
ity of biologic membranes better than bulk solvents. Biologic lipid membranes are composed of charged lipids that provide a polar environment at the membrane surface, which in biologic systems, often holds a negative charge. A strong attraction of positively charged molecules, thus, is observed. Liposome partitioning, in contrast to octanol/water partitioning, can take into account hydrogen bonding and electrostatic effects. However, the partition coefcient measured in liposome systems may not reect permeation always. In some cases, it may reect more drug interactions with the membrane surface (10, 12).
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Cell-based tools
Donor chamber Phospholipid vesicle -based barrier
Filter
Acceptor chamber
Figure 5 Schematic drawing of the experimental setup used in the permeation studies with the phospholipid vesicle-based barrier (reprinted from Reference 15).
Acceptor chamber
Donor chamber
Figure 6 Schematic drawing of the PAMPA permeation cell (reprinted from Reference 15).
Single-cell studies
Cells from animal or human origin can be isolated from different tissue and used as uptake systems. Normally, the isolated cells are suspended in a buffer under O2 /CO2 in the presence of the permeant and shaken well. After a certain time period, the cells are separated from the buffer and extracted to determine the amount of permeant inside the cells. Because of the low volume of the cells, the assay is based mostly on use of radiolabeled compounds (23). Cells can be isolated from different origin, and one animal can give rise to experiments with many different compounds or conditions. However, many disadvantages exist as well, like the lack of reproducibility with large variations in viability, because of the chemical or mechanical stress during isolation. This method also only allows for uptake, not transport or permeability, to be studied, but the polarity of the uptake; whether a compound is taken up on the apical or basolateral side cannot be decided (22).
bic lter material with a mixture of lecithin and inert organic solvent creates an articial lipid membrane (17). The use of 96-well microplates coupled with the rapid analysis using a spectrophotometric plate reader makes this system compatible with automated procedures and, hence, an attractive model for screening many compounds (9). Different variants of this model have been reported in the last 10 years with different lipid compositions: n-hexadecane HDMPAMPA (18), bio-mimetic BMPAMPA (17), and Double-Sink DSPAMPA (19). A schematic drawing of the PAMPA barrier with dissolved phospholipid in an organic solvent on a lter support separating the donor and acceptor chamber is shown in Fig. 6. What is common for the PAMPA techniques and the phospholipids vesicle-based barrier is that they are much less labor intensive than many cell-based assays, they are versatile and cost efcient, and they are being claimed to be a helpful stand-in for cellular models (20, 21). They also give access to a wider pH range compared with cell-based models, are easier to automate, and give a higher throughput. The limitations of these models are that they underestimate the permeability of actively transported drugs and hydrophilic molecules with low molecular weight and that the use of UV detection excludes drug compound that do not display UV absorbance (8, 9, 20). 8
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costly (8, 9, 2225). A schematic drawing of a cell monolayer assay is shown in Fig. 7.
Apical chamber
Tissue models
It is extremely difcult to obtain viable human tissues for permeability studies on a regular basis. Because animal intestinal tissues also are made up of essentially the same kind of endothelial cells, permeability screening for drug discovery purposes routinely is carried out using various animal species. However, the viability of the existing tissues is difcult to maintain because the tissues are devoid of direct blood supply and need constant oxygenation. The everted gut technique, intestinal rings, and the Ussing chamber are the most widely used methods that use tissue for studying absorption and permeability (22). The Ussing chamber can be used to look into the permeability of many different membranes/barriers. Using this technique, the tissue architecture is preserved and closely resembles the in vivo situation and the viability of the tissue can be monitored closely by electrical parameters during the experiment. However, because these techniques also involve the determination of the active transport and efux mechanisms, we leave these topics to other parts of the encyclopedia.
Cell monolayer
Filter
Basolateral chamber
Figure 7 Schematic drawing of a cell monolayer assay with the cell monolayer on a lter; support separates the apical and basolateral chamber from each other (reprinted from Reference 15).
rapidly into conuent monolayers followed by spontaneous differentiation, which provides a good system for transport studies (22, 23). Caco2 cells have been the most extensively characterized and the most used of all cell models, especially in the eld of drug permeability and absorption; this model has been considered the gold standard used to validate other permeability assays. This cell line originates from the human colon adenocarcinoma, differentiates spontaneously, and forms polarized monolayers with a well-dened brush border on the apical side as well as tight junctions. Other commonly used culture models are: HT29, T84, MDCK, LLCPK1, and 2/4/A1. The HT29 and T84 are like the Caco2 cells, human colon cancer cell lines. MDCK, LLCPK1, and 2/4/A1 originate in animals. The rst two come from kidneys, whereas the last one is derived from fetal rat intestine. The 2/4/A1 can be used to estimate the passive permeability of the human small intestine, especially with regard to paracellular transport, because the tightness of the tight junctions better correlates with the situation in vivo; however, this cell line does not express functional active transport and efux systems (8, 12, 24, 25). The use of cell monolayers, instead of single cells or isolated membrane vesicles, provides the opportunity to investigate the transport of drugs across the intestinal epithelial instead of just looking at the uptake of the drug into the cell or vesicle. Sampling on both the apical end basolateral side of the epithelium is possible, and crossover studies can be performed. Another advantage is that the cells are of human origin, different from many tissue-based techniques where animal tissue is used. The system also is amenable to automation. The major drawback of cell monolayer models is the intrinsic variability that can be seen in permeability data. The results obtained in several laboratories can differ by more than an order of magnitude, probably because of the underlying variability in, for example, culturing techniques, genetic drift, and variable expression of transporters. Many cell lines are originations from colon cancer tissue and by that do not resemble the situation in the normal small intestine perfectly, for example, the tight junctions are tighter and thereby can result in underestimation of the permeability of hydrophilic compounds. Caco2 assays are relatively accessible and more amenable to higher throughput than tissue models, but their culture can be labor-intensive and
References
1. Ti Tien H, Ottova-Leitmannova A, Membrane biophysics as viewed from experimental bilayer lipid membranes. In: Membrane Science and Technology. 2000. Elsevier, Amsterdam. pp. 221282. 2. V aben J. Dipeptidomimetics as Pro-Moieties for hPEPT1 Targete Prodrugs. In: Department of Pharmacy, University of Troms, 2004. 3. Conradi RA, Burton PS, Borchardt RT. Physico-chemical and biological factors that inuence a drugs cellular permeability by passive diffusion: methods and principles in medicinal chemistry. Lipophil. Drug Action Toxicol. 1996;4:233252. 4. Ohki S, Sprangler RA. Passive and facilitated transport. In: The Structure of Biological Membranes. Yeagle PL, ed. 2005. CRC Press, Boca Raton, FL. pp. 329387. 5. Fleisher D, Biological transport phenomena in the gastrointestinal tract: cellular mechanisms: Review article. Drugs and the Pharmaceutical Sciences. Transport Proc. Pharmaceut. Sys. 2000;102:147184. 6. Blume A, Lipid phase transitions: water and proton permeability. Methods Enzymol. 1986;127:480486.
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7. Bresseleers GJ, Goderis HL, Tobback PP. Measurement of the glucose permeation rate across phospholipid bilayers using small unilamellar vesicles. Effect of membrane composition and temperature. Biochim. Biophys. Acta. 1984;772:374382. 8. Balimane PV, Chong S, Morrison RA. Current methodologies used for evaluation of intestinal permeability and absorption. J. Pharmacolog. and Toxicol. Methods 2000;44:301312. 9. Hamalainen MD, Frostell-Karlsson A. Predicting the intestinal absorption potential of hits and leads. Drug Disc. Today 2004;1:397405. 10. Zhang YX, Zeng CM, Li YM, Hjerten S, Lundahl P, Immobilized liposome chromatography of drugs on capillary continuous beds for model analysis of drug-membrane interactions. J. Chromatogr. A. 1996;749:1318. 11. Beigi F, Gottschalk I, Lagerquist Hagglund C, Haneskog L, Brekkan E, Zhang Y, Osterberg T, Lundahl P, Immobilized liposome and biomembrane partitioning chromatography of drugs for prediction of drug transport. Int. J. Pharm. 1998;164:129137. 12. Malkia A, Murtomaki L, Urtti A, Kontturi K, Drug permeation in biomembranes; In vitro and in silico prediction and inuence of physicochemical properties. Eur. J. Pharm. Sci. 2004;23:1347. 13. Hellwich U, Schubert R, Concentration-dependent binding of the chiral beta-blocker oxprenolol to isoelectric or negatively charged unilamellar vesicles. Biochem. Pharmacol. 1995;49:511517. 14. Kramer SD. Liposome/water partitioning: theory, techniques and applications. In: Pharmacokinetic Optimization in Drug Research. B. Testa, et al., eds. 2001. Wiley-VCH, Z urich. 15. Sugano K, Hamada H, Machida M, Ushio H, High throughput prediction of oral absorption: improvement of the composition of the lipid solution used in parallel articial membrane permeation assay. J. Biomol. Screen. 2001;6:189196. 16. Flaten GE, Dhanikula AB, Luthman K, Brandl M. Drug permeability across a phospholipid vesicle based barrier: A novel approach for studying passive diffusion. Eur. J. Pharm. Sci. 2006;27:8090. 17. Kansy M, Senner F, Gubernator K, Physicochemical high throughput screening: parallel articial membrane permeation assay in the description of passive absorption processes. J. Med. Chem. 1998;41:10071010. 18. Wohnsland F, Faller B. High-throughput permeability pH prole and high-throughput alkane/water log P with articial membranes. J Med Chem. 2001;44:923930. 19. Avdeef A. Absorption and Drug Development; Solubilty, Permeability and Charge State. 1st ed. 2003. Wiley-Interscience, NJ. pp. 116246. 20. Flaten GE. The phospholipid vesicle-based barrier; A novel method for passive drug permeability screening. In: Department of Pharmacy, University of Troms, 2007. 21. Kansy M, Avdeef A, Fischer H. Advances in screening for membrane permeability: high-resolution PAMPA for medicinal chemists: Review article. Drug Disc. Today 2004;1:349355.
22. 23.
24.
25.
Hillgren KM, Kato A, Borchardt RT. In-vitro systems for studying intestinal drug absorption. Medic. Res. Revs. 1995;15:83109. Tukker JJ. In vitro methods for the assessment of permeability: drugs and the pharmaceutical sciences. Oral Drug Absorp. 2000;106:5172. Artursson P. Cell cultures as models for drug absorption across the intestinal mucosa. Crit. Rev. Therap. Drug Carrier Sys. 1991;8:305330. Matsson P, Bergstrom CAS, Nagahara N, Tavelin S, Norinder U, Artursson P. Exploring the role of different drug transport routes in permeability screening. J. Med. Chem. 2005;48:604613.
Further Reading
Nielsen PE, Avdeef A. PAMPAa drug absorption in vitro model8. Apparent lter porosity and the unstirred water layer. Eur. J. Pharm. Sci. 2004;22:3341. Ruell JA, Tsinman KL, Avdeef A. PAMPAa drug absorption in vitro model 5. Unstirred water layer in iso-pH mapping assays and pK(a)(ux)optimized design (pOD-PAMPA. Eur. J. Pharm. Sci. 2003;20:393402. Sugano K, Hamada H, Machida M, Ushio H, Saitoh K, Terada K. Optimized conditions of bio-mimetic articial membrane permeation assay. Int. J. Pharm. 2001;228:181188. Sugano K, Takata N, Machida M, Saitoh K, Terada K. Prediction of passive intestinal absorption using bio-mimetic articial membrane permeation assay and the paracellular pathway model. Int. J. Pharm. 2002;241:241251. Ungell AL. In vitro absorption studies and their relevance to absorption from the GI tract. Drug Dev. Ind. Pharm. 1997;3:879892.
See Also
Drug Transport Ion Channels and Pores, Chemical Biology of Small Molecule Transport Transport in Cells, Mechanisms of Water Channels
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