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Experimental estimation of the effective unstirred water layer thickness in the human jejunum, and its importance in oral drug absorption
iii
EUROPEAN
ELSEVIER
European Journal of Pharmaceutical Sciences 3 (1995) 247-253
JOURNAL
OF
PIIARMACEUTICAL SCIENCES
Leading Article
Experimental estimation of the effective unstirred water layer thickness in the human jejunum, and its importance in oral drug absorption Urban Fagerholm, Hans LennernS.s* Division of Biopharmaceutics and Pharmacokinetics, Department of Pharmacy, University of Uppsala, P.O. Box 580. S-751 23 Uppsala, Sweden Received 6 January 1995; accepted 26 May 1995
Abstract
Diffusion through the unstirred water layer (UWL) has previously been considered to be the rate limiting step in the intestinal uptake of highly permeable drugs. In order to evaluate the thickness and importance of the U W L , we used previously published effective intestinal permeability ( P , , ) data of two high permeability compounds, D-glucose and antipyrine, obtained in single-pass perfusions of the human jejunum. These two compounds are actively and passively transported, respectively, across the jejunal mucosa. The hydrodynamics within the segment is turbulent (well-mixed), and higher perfusion rates are assumed to lead to a more efficient luminal stirring, which results in a decreased thickness of the U W L (6), and consequently, to an enhanced P , of rapidly absorbed solutes. Previous published P , estimates obtained at a physiological range of perfusion flow rates between 1.5 and 6.0 ml min -~ were reanalysed, and the overall median 6 was estimated to 83 and 188 ,am for D-glucose and antipyrine, respectively. Our main conclusion is that human intestinal effective permeability in vivo of rapidly absorbed compounds is mainly determined by the membrane permeability and not the aqueous permeability. This statement is based on the following observations; (a) no significant differences in either P~, or 6 between the lowest and the highest flow rates of these solutes were observed, (b) despite equal diffusion coefficients in buffer of these two compounds, D-glucose was about 2 - 3 times more rapidly absorbed than antipyrine, (c) good agreement with recently reported animal and in vitro data. The P ~ estimates of D-glucose (carrier-mediated absorption) and antipyrine (passively absorbed) were highly correlated (r: = 0.79; p = 0.0001), which then suggests that the apical membrane of the intestinal mucosa is the main diffusion barrier for both passively and actively absorbed solutes. Furthermore, the membrane area is variable due to nonspecific regulation of the villi and microvilli, which might have a marked influence on the absorption rate. Our results indicate that the clinical relevance of the U W L as a factor responsible for considerable variability in the oral absorption of highly permeable drugs seems unlikely. Keywords: Aqueous permeability; D-Glucose absorption; Intestinal perfusion; Intestinal permeability; Oral drug absorption; Pre-epithelial diffusion barrier; Unstirred water layer
1. Introduction Solute absorption from the intestinal lumen to blood implicates molecular diffusion through the unstirred water layer (UWL) (Fig. 1), across the epithelial cell, through the interstitial fluid, and *Corresponding author. Tel. (+46-18) (+46-18) 174 003).
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into the blood capillary. The U W L is a more or less stagnant layer of water, mucus and glycocalyx adjacent to the intestinal wall, and is created because it is virtually impossible to stir the luminal contents so that complete mixing occurs right up to the intestinal mucosal surface (Thomson and Dietchy, 1984). Whether the UWL has a major or minor impact on the uptake of a drug from the lumen is thought to depend on
U. Fagerholm. H. Lennernds / European Journal of Pharrnaceutical Sciences 3 (1995) 247-25.;
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Fig. 1. Cross-section of the lumen of the human small intestine. the ability of the drug molecule to permeate the cell membrane (Thomson and Dietchy, 1984). The rate-limiting step in the transmucosal uptake of a low permeability compound is the transport across the apical membrane, rather than the diffusion through the UWL. Hence, the UWL can be considered as a negligible barrier to the uptake of slowly absorbed drugs (Thomson and Dietchy, 1984). For a rapidly permeating solute (effective intestinal permeability value, Pelf ~> 2 x 10 -4 cm s - ' ) (Lennernfis, 1994), the UWL is suggested to contribute to the major resistance to intestinal absorption (Levitt et al., 1984; Thomson and Dietchy, 1984; Winne, 1987). Since absorbed drug is slowly replaced by new molecules from the bulk solution due to slower diffusion across the UWL, a concentration gradient is created between the exterior side of the UWL and the intestinal wall. By definition, the effective thickness of the U W L (6) is determined by this concentration difference (Winne, 1984). The 6 is actually not a true value of the layer thickness, rather it is an operational value of the distance to the apical membrane from the point where the linear extrapolation of the concentration gradient reaches the homogenous concentration in the well-stirred bulk phase. Thus, compounds that have high membrane permeabilities will have the same P~ff-values if the diffusion coefficients across the UWL are the same. The most common way to evaluate the thickness and importance of the .UWL on solute absorption has been to determine the Pelf or
intestinal absorption rates at different perfusion flow rates for highly permeable compounds (Winne, 1979; Levitt et al., 1987). A majority of these reports have been carried out in the perfused animal small intestine during anaesthesia. estimates have earlier been approximated to be about 300-800 ~ m based on values from highly permeable solutes (Read et al., 1977; H6gerle and Winne, 1983; Levitt et al., 1984; Westergaard et al., 1986; Winne, 1987). However, recently reevaluated absorption data in humans and as well in vivo studies in unanaesthetized animals obtained estimations of the UWL-thickness of about 30-100 ~ m . These new values are suggested to be a reflection of a more effective gut motility, and thereby higher stirring in the intestinal lumen during the conscious in vivo state (Andersson et al., 1988; Levitt et al., 1990; Levitt et al., 1992a; Levitt et al., 1992b). A human perfusion technique has been validated for absorption of drugs and nutrients (Lennernfis et al., 1992, Lennern~is, 1994; Fagerholm et al., 1995). From those studies we have obtained a database of Pelf-values of two rapidly absorbed compounds, D-glucose and antipyrine, at different perfusion flow rates between 1.5 and 6.0 ml rain -1 (Lennernhs et al., 1992; Lennernfis et al., 1993; Lennern/is et al., 1994; Nilsson et al., 1994; Fagerholm et al., 1995). In this report, these P~f-values were used to estimate the thickness of the U W L in the human jejunum, and to investigate the importance of the UWL as a factor for both inter-and intraindividual variability in the absorption rate of high permeability compounds in vivo in man.
2. Materials and methods
The perfusion instrument (Loc-I-Gut ®" Synectics AB, Sweden) is a 175 cm long and sterile polyvinyl tube (external diameter 5.3 mm) intended for intestinal perfusions in humans (Knutson et al., 1989; Lennern~is et al., 1992). It has six inner channels and is distally provided with two elongated latex balloons, placed 10 cm apart. The tube is inserted and positioned in the human proximal jejunum under the guidance of a fluoro-
U. Fagerholrn, H. Lennerniis / European Journal of Pharmaceutical Sciences 3 (1995) 247-253
scopic technique (Philips 21-S). The balloons are inflated with air via two of the smaller channels, and a closed segment allowing perfusion is created. Two wider channels in the center of the tube are used for infusion and aspiration of the perfusate. A more detailed description of the perfusion technique can be found elsewhere (Knutson et al., 1989; Lennern/is et al., 1992). In these studies, jejunal perfusions with Dglucose (I0 raM; n = 5 3 ) and antipyrine (0.510.5 raM; n = 7 5 ) were carried out in healthy subjects in the morning after a 10 h overnight fast. The P~f~. for passively absorbed solutes, as for example antipyrine, is independent on the concentration entering the intestinal segment (Lennernfis et al., 1992; Lennern/is et al., 1993; Lennernfis et al., 1994; Nilsson et al., 1994: Fagerholm et al., 1995). The perfusion flow rates at which perfusions were performed were 1.5, 2.0, 3.0 and 6.0 ml rain -~ The number of subjects participating at each flow rate is presented in Table 1. All perfusion syringes and perfusate samples were weighed, and the samples were quantitatively collected on ice in 10 min intervals, immediately frozen and stored at -20°C until analysis. The permeability measurements were performed during isotonic conditions (about 270-300 mOsm l -s) in order to obtain a minimal net fluid flux across the jejunal epithelium. More details of the composition of the perfusion solution are available in each paper (Lennernfis et al., 1992; Lennernfis et al., 1993; Lennernfis et al., 1994; Nilsson et al., 1994;
249
Fagerholm et al., 1995). Approval for these studies was given by the Ethics Committee of the Medical Faculty, Uppsala University.
3. Calculations
Residence time analysis has shown that the hydrodynamics of the solution within the intestinal segment can best be described by a wellstirred model (Lee et al., 1993). Hence. the P~f~ was calculated according to the following equation (Amidon et al., 1980): O(C n - Coui)/c,,u,
P'~ff =
2"rrRL
(1)
where Q is the perfusion flow rate through the intestinal segment, and C m and C,,u~ are the inlet and fluid flux-corrected outlet solute concentrations, respectively. The mass transfer surface area (2~rrL) within the intestinal segment is assumed to be the cylinder area with the length (L) of 10 cm and a radius (r) of 1.75 cm. The reciprocal of P~,f (I/P~f) is an estimate for the total effective resistance (R~ff) to drug absorption, and is a combination of two diffusional resistances in series, namely the UWL (1/P,,q) and the cell membrane (l/P,,) (Johnson and Amidon, 1988): R~t t = l / P ~ l t. = 1/P:~q +
1/P,,
(2)
According to the widely used assumption that rapidly absorbed solutes have negligible resist-
Table l Median ( 2 5 - 7 5 % percentiles) steady-state effective permeability (Pe,f) and effective unstirred water layer thickness (0) for antipyrine (0.5-10.5 mM) and D-glucose (10 mM) in the human j e j u n u m Perfusion flow rate (ml min - ' )
P~t~ ( x 10 -4 cm s
a ( p-m)
1)
number of experiments antipyrine / D-glucose
antipyrine 1.5 2.0 3.0 6.0 Total median
D-glucose
antipyrine
3.5
274
(-)
(-)
3.3 (2.4-5.5) 5. l (3.4-9.0) 6./1 (3.1-18) 4.8
6,2 (3.5-14) 1l (7.3-18) 9.3 (3.4-44) 9.6
280 (17/I-396) 177 (101-268) 152 (68-41)2) 188
D-glucose 2/128 (57-231) 74 (43-11/)) 96 (41-3/)3) 83
16/15 49/30 8/8 75/53
250
U. Fagerholm, H. Lennernds / European Journal of Pharmaceutical Sciences 3 (1995) 247-253
ance to passage through the cell membrane (Pro >> P,q) (Winne, 1978), Eq. 2 can be simplified: (3)
Pelt ~ P~q
The effective UWL thickness (6) can then be estimated by the use of the following equation (Winne, 1978): 6 "~ D/Pet. f (4) where D is the diffusion coefficient of antipyrine (0.91 × 10 -5 cmz s -~) and D-glucose (0.80 × 10 -5 c m 2 S-j) in buffer solution o r water at 37°C (Wilke and Chang, 1955; Levitt et al., 1992b). The estimation of P~ff and 6 from perfusion experiments is based upon several assumptions, e.g., (a) the intestine has an effective absorptive area of a smooth cylinder, (b) the diffusion rates obtained in plain saline buffer equals that in the UWL, (c) the intestinal segment is totally filled with perfusate, and (d) the enterocyte membrane is considered to be a negligible barrier to intestinal absorption of high P~f~ solutes. Results are presented as median and percentiles (25% and 75%) due to the large interindividual variability in the data.
3. Results and discussion
In the present report, human effective permeabilities (P~,) obtained at different physiologi40'
cal perfusion rates, were used to estimate the importance and the thickness (6) of the UWL. These data are presented in Table 1 and Figs. 2, 3 and 4. No significant differences in either Pelf or fi between the lowest and the highest flow rates of these solutes were observed. The overall median 6-values estimated from Pef:values of antipyrine and o-glucose were 188 and 83 /zm, respectively. Our results do not agree with previous reported values where an increased perfusion flow rate gave a significant reduction of the 6, and consequently, an increased P~ff for rapidly absorbed solutes (Winne, 1979; Levitt et al., 1987). For instance, in one study it was demonstrated that by increasing the perfusion flow rate through the rat jejunum from 0.1 to 0.5 ml min -~, the 6 of antipyrine was reduced from 540 to 330 /.Lm (Winne, 1979). Furthermore, in another rat experiment of Levitt et al., 1987, an increase of the perfusion flow rate from 0.19 to 7.35 ml min -t lead to a reduction of the 6 from 720 to 180/xm (calculated from permeability of carbon oxide). In both of these studies the animals were under anaesthesia (Winne, 1979; Levitt, et al., 1987). Our human experimental median 6 data from all flow rates were approximately 74-280 p,m (Table 1), which also agrees with in vivo data from animal studies, in vitro studies in cell culture (Caco-2 cells) and reanalysed human data (Andersson et al., 1988; Levitt et al., 1990; Levitt et -" Itq
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(b)
Fig. 2. Individual effective intestinal permeability data (P~ff) of antipyrine (left) and D-glucose (right) obtained at different perfusion flow rates in the human jejunum: ( - - ) and (--) represent median, and 25% and 75% percentiles, respectively.
U. Fagerholm, H. Lennerntis / European Journal of Pharmaceutical Sciences 3 (1995) 247-253
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(b) Fig. 3. [ndividual unstirred water layer thickness data (~) calculated from Pcff estimates of antipyrine (left) and o-glucose (right) at different perfusion flow rates in the human jejunum: (--) and (--) represent median, and 25% and 75% percentiles, respectively. (a)
al., 1992a; Levitt et al., 1992b, Karlsson and Artursson, 1992). This is probably a reflection of a more effective luminal mixing during the conscious in vivo state, produced by the macromotility and morphology of the intestinal tissue, and the movement of the villi (Andersson et al., 1988; Levitt et al., 1990; Levitt et al., 1992a; Levitt et al., 1992b). Our in vivo calculated U W L thickness also agrees well with the measured mucus gel thickness (162 /~m) obtained in biopsy samples from the human duodenum (Sarosiek et al., 1991). Estimating the Pe, and the /5 requires a measure of the available surface area. In our calculations it is assumed that the intestinal area equals that of a smooth cylinder, but the true anatomical area is, of course, larger than this. However, the general opinion is that the absorption of highly permeable solutes occurs at the tips of the villi (Chang and Rao, 1994; Strocchi and Levitt, 1993). Strocchi and Levitt, 1993, suggested that solutes are absorbed within 5% of the tips of the villi, and that less than 2% of the cells in the intervillous spaces contribute to a major fraction of the intestinal absorption of glucose. Thus, for compounds with high Peff, the cylinder area is probably a good prediction of the available membrane area for absorption. Compounds with low membrane permeability might however diffuse deeper down into the intervillous spaces.
In this report, we present in vivo human data that demonstrate that the U W L is neglible compared to the intestinal cell membrane as the rate-limiting barrier to intestinal uptake of highly permeable solutes. Instead, a reappraisal of the importance of this immobilized fluid layer suggests that the apical membrane of the intestinal mucosa is the main diffusion barrier for both slowly and rapidly absorbed solutes in humans regardless of absorption mechanism. There are several indications in our present data that support this suggestion; (a) the low overall median/5 estimates of U W L in vivo between 83-188 ~ m , (b) no significant differences in either Peff or /5 between the lowest and the highest flow rates of these solutes were observed, (c) despite equal diffusion coefficients in buffer solution of these two compounds, D-glucose was about 2-3 fold more rapidly absorbed than antipyrine (Fig. 4 and Table 1). Support is given by other researchers (Levitt et al., 1990; Chiou, 1994). Levitt et al., 1990, stated that the minimal contribution of the epithelial resistance to intestinal drug absorption in man must be at least 40%. Implied in Chiou (1994), a 15 of 40 /~m in the human intestine would only impair the transmucosal uptake rate of solutes with absorption half-lives shorter than 6 min. Furthermore, the high correlation between the Peff of D-glucose (carrier-mediated absorption) and antipyrine (passively ab-
252
U. Fagerholrn, H. Lennernds / European Journal of Pharmaceutical Sciences 3 (1995) 247-253
_---, Itn
100" y = -3.5 ÷ 2.6X
variability in the intestinal absorption of high permeability solutes is due to other factors than a variable thickness of the UWL, such as available surface area and lipid composition in the membrane.
r^~ = 0.79 P = 0 . 0 0 0 1 / v
so.
~
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~.
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References
/~ ° ° a O/
10
] II 2ml-min-1 I I o3ml.min-i ]
20
AnflpyrineeffectivepermeabiUty
30
4O
(E-04 cm s - I)
Fig. 4. Correlation between PC,. estimates of antipyrine and o-glucose obtained after simultaneous administration of the compounds at different perfusion flow rates (2.0, 3.0 and 6.0 ml rain -~) in the human jejunum (n =53).
sorbed) (rZ=0.79; p =0.0001; n =53) suggests that the available surface of the apical membrane of the intestinal mucosa is the main diffusion barrier for both actively and passively absorbed solutes (Fig. 4) (Lennernfis et al., 1992). Our variable surface area theory might be explained by nonspecific regulation of the intestinal mucosa by hypertrophy and changes in microvillous and villous dimensions (Karasov and Diamond, 1983; Diamond, 1991). This might be a rapid and dynamic mechanism explained by the villous contractility that have the potential to alter the functional absorptive area (Strocchi and Levitt, 1993). In conclusion, we suggest that the resistance of the UWL to the intestinal absorption of highly permeable solutes in vivo in humans have earlier been markedly overestimated. Instead we consider the intestinal absorption in humans to be membrane controlled for both low and high permeability compounds, irrespective of the transport mechanism. The minor importance of the thickness of the UWL as a factor for absorption rate is probably due to a gastrointestinal motility in vivo that produces a highly efficient luminal stirring. We also suggest that the available surface of the apical membrane of the intestinal mucosa is the main diffusion barrier for both passively and actively absorbed compounds. Finally, we suggest that inter- and intrasubjectal
Amidon, G.L., Kou, J., Elliot, R.L. and Lightfoot, E.N. (1980) Analysis of models for determining intestinal wall permeabilities. J. Pharm. Sci. 69, 1369-1373. Andersson, B.W., Levine, A.S., Levitt, D.G., Kneip, J.M. and Levitt, M.D. (1988) Physiological measurement of luminal stirring in perfused rat jejunum. Am. J. Physiol. 254, G843-848. Chang, E.B. and Rao, M.C. (1994) Intestinal water and electrolyte transport: mechanisms of physiological and adaptive responses. In: L.R. Johnson (editor) Physiology of the Gastrointestinal Tract. Raven Press, New York, pp. 2027-2081. Chiou, W.L. (1994) Effect of "unstirred'" water layer in the intestine of the rate and extent of absorption after oral administration. Biopharm&Drug Disp. 15, 709-717. Diamond, J.M. (1991) Evolutionary design of intestinal nutrient absorption: Enough but not too much. News Physiol. Sci. 6, 92-96. Fagerholm, U., Borgstr6m, L., Ahrenstedt, O. and Lennernfis, H. (1995) The lack of effect of induced net fluid absorption on the in vivo permeability of terbutaline in the human jejunum. J. Drug Targeting (in press). H6gerle, M.L. and Winne, D. (1983) Drug absorption by the rat jejunum perfused in situ. Naunyn-Schmiedeberg's Arch. Pharmaco[. 322, 249-355. Johnson, D.A. and Amidon, G.L. (1991) Determination of intrinsic membrane transport parameters from perfused intestine experiments: a boundary layer approach to estimating the aqueous and unbiased membrane permeabilities. J. Theor. Biol. 131, 93-106. Karasov, W.H. and Diamond, J.M. (1983) Adaptive regulation of sugar and amino acid transport by vertebrate intestine. Am. J. Physiol. 245:G443-462. Karlsson, J. and Artursson, P. (1992) A new diffusion chamber system for the determination of drug permeability coefficients across the human intestinal epithelium that are independent of the unstirred water layer. Biochim. Biophys. Acta 1111, 204-210. Knutson, L., Odlind, B. and Hfillgren, R. (1989) A new technique for segmental jejunal perfusion in man. Am. J. Gastroenterol. 84, 1278-1284. Lee, I-D., Fagerholm, U., Lennernfis, H. and Amidon, G. (1993) A study of hydrodynamic characteristics in human intestine applying residence time distribution analysis. In: A. Urtti (editor) Methods to Overcome Biological Barriers in Drug Delivery, Syrup. 26-28 August. Acta Pharm. Sci. 10, Kuopio University, 73 pp.
U. Fagerholm. H. Lennern?is / European Journal of Pharmaceutical Sciences 3 (1995) 247-253
Lennern/is, H., Ahrenstedt, 6., H/illgren, R., Knutson, L., Ryde, M. and Paalzow, L. (1992) Regional jejunal perfusion, a new in vivo approach to study oral drug absorption in man. Pharm. Res. 9, 1243-1251. LennernS.s, H., Nilsson, D., Aquilonius, S-M., Ahrenstedt, (3., Knutson, L. and Paalzow, L. (1993) The effect of L-leucine on the absorption of levodopa, studied by regional jejunual perfusion in man. Br. J. Clin. Pharmacol. 35, 243-250. Lennern~is, H. (1994) Gastrointestinal absorption mechanisms: a comparison between animal and human models. Eur. J. Pharm. Sci. 2, 39-43. Lennern/is, H., Ahrenstedt, O. and Ungell, A-L. (1994) Intestinal drug absorption during induced net water absorption in man: a mechanistic study using antipyrine, atenolol and enalaprilat. Br. J. Clin. Pharmacol. 37,589596. Levitt, M.D., Aufderheide, T., Fetzer, C.A., Bond, J.H. and Levitt, D.G. (1984) Use of carbon monoxide to measure luminal stirring in the rat gut. J. Clin. Invest. 74, 2O56-2O64. Levitt, M.D., Fetzer, C.A., Kneip, J.M., Bond, J.H. and Levitt, D.G. (1987) Quantitative assessment of luminal stirring in the perfused small intestine of the rat. Am. J. Physiol. 252, G325-332. Levitt, M.D., Furne, J.K., Strocchi, A., Anderson, B.W. and Levitt, D.G. (1990) Physiological measurements of luminal stirring in the dog and human small bowel. J. Clin. Invest. 86, 1540-1547. Levitt, M.D., Strocchi, A. anti Levitt, D.G. (1992a) Human jejunal unstirred layer: evidence for extremely efficient luminal stirring. Am. J. Physiol. 262, G593-596. Levitt, M.D., Furne, J.K. and Levitt, D.G. (1992b) Shaking of the intact rat and intestinal angulation diminish the jejunal unstirred layer. Am. J. Gastroenterol. 103, 14601466. Nilsson, D., Fagerholm, U. and Lennern~is, H. (1994) The influence of net water absorption on the permeability of antipyrine and levodopa in the human jejunum. Pharm. Res. 11, 1541-1545.
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Read, N.W., Barber, D.C., Levin, R.J. and Holdsworth, C.D. (1977) Unstirred layer and kinetics of electrogenic glucose absorption in the human jejunum in situ. Gut lS, 865-876. Sarosiek, J., Marshall, B.J., Peura, D.A., Hoffman, R.N., Feng, T. and McCallum, R.W. (1991) Gastroduodenal mucus gel thickness in patients with Helicobacter pylori: a method for assessment of biopsy specimens. Am. J. Gastroenterol. 86, 729-734. Strocchi A. and Levitt M.D. (1993) Role of villous surface area in absorption. Dig. Dis.&Sci. 38, 385-387. Thomson, A.B.R and Dietchy, J.M. (1984) The role of the unstirred water layer in intestinal permeation. Chapter 21 in: T.Z. Csaky (editor) Pharmacology of Intestinal Permeation II. Springer, Berlin. Westergaard, H., Holtermiiller, K.H. and Dietschy, J.M. (1986) Measurement of resistance of barriers to solute transport in vivo in rat jejunum. Am. J. Physiol. 250, G727-735. Wilke, C.R. and Chang, P. (1955) Correlation of diffusion coefficients in dilute solutions. Am. Inst. Chem. Eng. J. 1, 264-27/). Winne, D. (1978) Dependence of intestinal absorption in vivo on the unstirred layer. Naunyn-Schmiedeberg's Arch. Pharmacol. 304, 175-181. Winne, D. (1979) Rat jejunum perfused in situ: effect of perfusion rate and intraluminat radius on absorption rate and effective unstirred layer thickness. NaunynSchmiedeberg's Arch. Pharmacol. 307, 265-274. Winne, D. (1984) In: Unstirred layer as a diffusion barrier in vitro and in vivo. E. Skadhauge and K. Heintze (editors) Intestinal Absorption and Secretion. MTP Press, Lancaster, pp. 21-38. Winne, D (1987) Closed rat jejunal segment in situ: role of pre-epithelial diffusion resistance (unstirred layer) in the absorption process and model analysis. NaunvnSchmiedeberg's Arch. Pharmacol. 335, 204-215.
EUROPEAN
ELSEVIER
European Journal of Pharmaceutical Sciences 3 (1995) 247-253
JOURNAL
OF
PIIARMACEUTICAL SCIENCES
Leading Article
Experimental estimation of the effective unstirred water layer thickness in the human jejunum, and its importance in oral drug absorption Urban Fagerholm, Hans LennernS.s* Division of Biopharmaceutics and Pharmacokinetics, Department of Pharmacy, University of Uppsala, P.O. Box 580. S-751 23 Uppsala, Sweden Received 6 January 1995; accepted 26 May 1995
Abstract
Diffusion through the unstirred water layer (UWL) has previously been considered to be the rate limiting step in the intestinal uptake of highly permeable drugs. In order to evaluate the thickness and importance of the U W L , we used previously published effective intestinal permeability ( P , , ) data of two high permeability compounds, D-glucose and antipyrine, obtained in single-pass perfusions of the human jejunum. These two compounds are actively and passively transported, respectively, across the jejunal mucosa. The hydrodynamics within the segment is turbulent (well-mixed), and higher perfusion rates are assumed to lead to a more efficient luminal stirring, which results in a decreased thickness of the U W L (6), and consequently, to an enhanced P , of rapidly absorbed solutes. Previous published P , estimates obtained at a physiological range of perfusion flow rates between 1.5 and 6.0 ml min -~ were reanalysed, and the overall median 6 was estimated to 83 and 188 ,am for D-glucose and antipyrine, respectively. Our main conclusion is that human intestinal effective permeability in vivo of rapidly absorbed compounds is mainly determined by the membrane permeability and not the aqueous permeability. This statement is based on the following observations; (a) no significant differences in either P~, or 6 between the lowest and the highest flow rates of these solutes were observed, (b) despite equal diffusion coefficients in buffer of these two compounds, D-glucose was about 2 - 3 times more rapidly absorbed than antipyrine, (c) good agreement with recently reported animal and in vitro data. The P ~ estimates of D-glucose (carrier-mediated absorption) and antipyrine (passively absorbed) were highly correlated (r: = 0.79; p = 0.0001), which then suggests that the apical membrane of the intestinal mucosa is the main diffusion barrier for both passively and actively absorbed solutes. Furthermore, the membrane area is variable due to nonspecific regulation of the villi and microvilli, which might have a marked influence on the absorption rate. Our results indicate that the clinical relevance of the U W L as a factor responsible for considerable variability in the oral absorption of highly permeable drugs seems unlikely. Keywords: Aqueous permeability; D-Glucose absorption; Intestinal perfusion; Intestinal permeability; Oral drug absorption; Pre-epithelial diffusion barrier; Unstirred water layer
1. Introduction Solute absorption from the intestinal lumen to blood implicates molecular diffusion through the unstirred water layer (UWL) (Fig. 1), across the epithelial cell, through the interstitial fluid, and *Corresponding author. Tel. (+46-18) (+46-18) 174 003).
174 317; Fax
0928-0987/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved S S D I 0928-0987(95)00027-5
into the blood capillary. The U W L is a more or less stagnant layer of water, mucus and glycocalyx adjacent to the intestinal wall, and is created because it is virtually impossible to stir the luminal contents so that complete mixing occurs right up to the intestinal mucosal surface (Thomson and Dietchy, 1984). Whether the UWL has a major or minor impact on the uptake of a drug from the lumen is thought to depend on
U. Fagerholm. H. Lennernds / European Journal of Pharrnaceutical Sciences 3 (1995) 247-25.;
248
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- 3.$ ©m
I
Fig. 1. Cross-section of the lumen of the human small intestine. the ability of the drug molecule to permeate the cell membrane (Thomson and Dietchy, 1984). The rate-limiting step in the transmucosal uptake of a low permeability compound is the transport across the apical membrane, rather than the diffusion through the UWL. Hence, the UWL can be considered as a negligible barrier to the uptake of slowly absorbed drugs (Thomson and Dietchy, 1984). For a rapidly permeating solute (effective intestinal permeability value, Pelf ~> 2 x 10 -4 cm s - ' ) (Lennernfis, 1994), the UWL is suggested to contribute to the major resistance to intestinal absorption (Levitt et al., 1984; Thomson and Dietchy, 1984; Winne, 1987). Since absorbed drug is slowly replaced by new molecules from the bulk solution due to slower diffusion across the UWL, a concentration gradient is created between the exterior side of the UWL and the intestinal wall. By definition, the effective thickness of the U W L (6) is determined by this concentration difference (Winne, 1984). The 6 is actually not a true value of the layer thickness, rather it is an operational value of the distance to the apical membrane from the point where the linear extrapolation of the concentration gradient reaches the homogenous concentration in the well-stirred bulk phase. Thus, compounds that have high membrane permeabilities will have the same P~ff-values if the diffusion coefficients across the UWL are the same. The most common way to evaluate the thickness and importance of the .UWL on solute absorption has been to determine the Pelf or
intestinal absorption rates at different perfusion flow rates for highly permeable compounds (Winne, 1979; Levitt et al., 1987). A majority of these reports have been carried out in the perfused animal small intestine during anaesthesia. estimates have earlier been approximated to be about 300-800 ~ m based on values from highly permeable solutes (Read et al., 1977; H6gerle and Winne, 1983; Levitt et al., 1984; Westergaard et al., 1986; Winne, 1987). However, recently reevaluated absorption data in humans and as well in vivo studies in unanaesthetized animals obtained estimations of the UWL-thickness of about 30-100 ~ m . These new values are suggested to be a reflection of a more effective gut motility, and thereby higher stirring in the intestinal lumen during the conscious in vivo state (Andersson et al., 1988; Levitt et al., 1990; Levitt et al., 1992a; Levitt et al., 1992b). A human perfusion technique has been validated for absorption of drugs and nutrients (Lennernfis et al., 1992, Lennern~is, 1994; Fagerholm et al., 1995). From those studies we have obtained a database of Pelf-values of two rapidly absorbed compounds, D-glucose and antipyrine, at different perfusion flow rates between 1.5 and 6.0 ml rain -1 (Lennernhs et al., 1992; Lennernfis et al., 1993; Lennern/is et al., 1994; Nilsson et al., 1994; Fagerholm et al., 1995). In this report, these P~f-values were used to estimate the thickness of the U W L in the human jejunum, and to investigate the importance of the UWL as a factor for both inter-and intraindividual variability in the absorption rate of high permeability compounds in vivo in man.
2. Materials and methods
The perfusion instrument (Loc-I-Gut ®" Synectics AB, Sweden) is a 175 cm long and sterile polyvinyl tube (external diameter 5.3 mm) intended for intestinal perfusions in humans (Knutson et al., 1989; Lennern~is et al., 1992). It has six inner channels and is distally provided with two elongated latex balloons, placed 10 cm apart. The tube is inserted and positioned in the human proximal jejunum under the guidance of a fluoro-
U. Fagerholrn, H. Lennerniis / European Journal of Pharmaceutical Sciences 3 (1995) 247-253
scopic technique (Philips 21-S). The balloons are inflated with air via two of the smaller channels, and a closed segment allowing perfusion is created. Two wider channels in the center of the tube are used for infusion and aspiration of the perfusate. A more detailed description of the perfusion technique can be found elsewhere (Knutson et al., 1989; Lennern/is et al., 1992). In these studies, jejunal perfusions with Dglucose (I0 raM; n = 5 3 ) and antipyrine (0.510.5 raM; n = 7 5 ) were carried out in healthy subjects in the morning after a 10 h overnight fast. The P~f~. for passively absorbed solutes, as for example antipyrine, is independent on the concentration entering the intestinal segment (Lennernfis et al., 1992; Lennern/is et al., 1993; Lennernfis et al., 1994; Nilsson et al., 1994: Fagerholm et al., 1995). The perfusion flow rates at which perfusions were performed were 1.5, 2.0, 3.0 and 6.0 ml rain -~ The number of subjects participating at each flow rate is presented in Table 1. All perfusion syringes and perfusate samples were weighed, and the samples were quantitatively collected on ice in 10 min intervals, immediately frozen and stored at -20°C until analysis. The permeability measurements were performed during isotonic conditions (about 270-300 mOsm l -s) in order to obtain a minimal net fluid flux across the jejunal epithelium. More details of the composition of the perfusion solution are available in each paper (Lennernfis et al., 1992; Lennernfis et al., 1993; Lennernfis et al., 1994; Nilsson et al., 1994;
249
Fagerholm et al., 1995). Approval for these studies was given by the Ethics Committee of the Medical Faculty, Uppsala University.
3. Calculations
Residence time analysis has shown that the hydrodynamics of the solution within the intestinal segment can best be described by a wellstirred model (Lee et al., 1993). Hence. the P~f~ was calculated according to the following equation (Amidon et al., 1980): O(C n - Coui)/c,,u,
P'~ff =
2"rrRL
(1)
where Q is the perfusion flow rate through the intestinal segment, and C m and C,,u~ are the inlet and fluid flux-corrected outlet solute concentrations, respectively. The mass transfer surface area (2~rrL) within the intestinal segment is assumed to be the cylinder area with the length (L) of 10 cm and a radius (r) of 1.75 cm. The reciprocal of P~,f (I/P~f) is an estimate for the total effective resistance (R~ff) to drug absorption, and is a combination of two diffusional resistances in series, namely the UWL (1/P,,q) and the cell membrane (l/P,,) (Johnson and Amidon, 1988): R~t t = l / P ~ l t. = 1/P:~q +
1/P,,
(2)
According to the widely used assumption that rapidly absorbed solutes have negligible resist-
Table l Median ( 2 5 - 7 5 % percentiles) steady-state effective permeability (Pe,f) and effective unstirred water layer thickness (0) for antipyrine (0.5-10.5 mM) and D-glucose (10 mM) in the human j e j u n u m Perfusion flow rate (ml min - ' )
P~t~ ( x 10 -4 cm s
a ( p-m)
1)
number of experiments antipyrine / D-glucose
antipyrine 1.5 2.0 3.0 6.0 Total median
D-glucose
antipyrine
3.5
274
(-)
(-)
3.3 (2.4-5.5) 5. l (3.4-9.0) 6./1 (3.1-18) 4.8
6,2 (3.5-14) 1l (7.3-18) 9.3 (3.4-44) 9.6
280 (17/I-396) 177 (101-268) 152 (68-41)2) 188
D-glucose 2/128 (57-231) 74 (43-11/)) 96 (41-3/)3) 83
16/15 49/30 8/8 75/53
250
U. Fagerholm, H. Lennernds / European Journal of Pharmaceutical Sciences 3 (1995) 247-253
ance to passage through the cell membrane (Pro >> P,q) (Winne, 1978), Eq. 2 can be simplified: (3)
Pelt ~ P~q
The effective UWL thickness (6) can then be estimated by the use of the following equation (Winne, 1978): 6 "~ D/Pet. f (4) where D is the diffusion coefficient of antipyrine (0.91 × 10 -5 cmz s -~) and D-glucose (0.80 × 10 -5 c m 2 S-j) in buffer solution o r water at 37°C (Wilke and Chang, 1955; Levitt et al., 1992b). The estimation of P~ff and 6 from perfusion experiments is based upon several assumptions, e.g., (a) the intestine has an effective absorptive area of a smooth cylinder, (b) the diffusion rates obtained in plain saline buffer equals that in the UWL, (c) the intestinal segment is totally filled with perfusate, and (d) the enterocyte membrane is considered to be a negligible barrier to intestinal absorption of high P~f~ solutes. Results are presented as median and percentiles (25% and 75%) due to the large interindividual variability in the data.
3. Results and discussion
In the present report, human effective permeabilities (P~,) obtained at different physiologi40'
cal perfusion rates, were used to estimate the importance and the thickness (6) of the UWL. These data are presented in Table 1 and Figs. 2, 3 and 4. No significant differences in either Pelf or fi between the lowest and the highest flow rates of these solutes were observed. The overall median 6-values estimated from Pef:values of antipyrine and o-glucose were 188 and 83 /zm, respectively. Our results do not agree with previous reported values where an increased perfusion flow rate gave a significant reduction of the 6, and consequently, an increased P~ff for rapidly absorbed solutes (Winne, 1979; Levitt et al., 1987). For instance, in one study it was demonstrated that by increasing the perfusion flow rate through the rat jejunum from 0.1 to 0.5 ml min -~, the 6 of antipyrine was reduced from 540 to 330 /.Lm (Winne, 1979). Furthermore, in another rat experiment of Levitt et al., 1987, an increase of the perfusion flow rate from 0.19 to 7.35 ml min -t lead to a reduction of the 6 from 720 to 180/xm (calculated from permeability of carbon oxide). In both of these studies the animals were under anaesthesia (Winne, 1979; Levitt, et al., 1987). Our human experimental median 6 data from all flow rates were approximately 74-280 p,m (Table 1), which also agrees with in vivo data from animal studies, in vitro studies in cell culture (Caco-2 cells) and reanalysed human data (Andersson et al., 1988; Levitt et al., 1990; Levitt et -" Itq
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Perfusion flow rate (ml m i n - I )
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Fig. 2. Individual effective intestinal permeability data (P~ff) of antipyrine (left) and D-glucose (right) obtained at different perfusion flow rates in the human jejunum: ( - - ) and (--) represent median, and 25% and 75% percentiles, respectively.
U. Fagerholm, H. Lennerntis / European Journal of Pharmaceutical Sciences 3 (1995) 247-253
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(b) Fig. 3. [ndividual unstirred water layer thickness data (~) calculated from Pcff estimates of antipyrine (left) and o-glucose (right) at different perfusion flow rates in the human jejunum: (--) and (--) represent median, and 25% and 75% percentiles, respectively. (a)
al., 1992a; Levitt et al., 1992b, Karlsson and Artursson, 1992). This is probably a reflection of a more effective luminal mixing during the conscious in vivo state, produced by the macromotility and morphology of the intestinal tissue, and the movement of the villi (Andersson et al., 1988; Levitt et al., 1990; Levitt et al., 1992a; Levitt et al., 1992b). Our in vivo calculated U W L thickness also agrees well with the measured mucus gel thickness (162 /~m) obtained in biopsy samples from the human duodenum (Sarosiek et al., 1991). Estimating the Pe, and the /5 requires a measure of the available surface area. In our calculations it is assumed that the intestinal area equals that of a smooth cylinder, but the true anatomical area is, of course, larger than this. However, the general opinion is that the absorption of highly permeable solutes occurs at the tips of the villi (Chang and Rao, 1994; Strocchi and Levitt, 1993). Strocchi and Levitt, 1993, suggested that solutes are absorbed within 5% of the tips of the villi, and that less than 2% of the cells in the intervillous spaces contribute to a major fraction of the intestinal absorption of glucose. Thus, for compounds with high Peff, the cylinder area is probably a good prediction of the available membrane area for absorption. Compounds with low membrane permeability might however diffuse deeper down into the intervillous spaces.
In this report, we present in vivo human data that demonstrate that the U W L is neglible compared to the intestinal cell membrane as the rate-limiting barrier to intestinal uptake of highly permeable solutes. Instead, a reappraisal of the importance of this immobilized fluid layer suggests that the apical membrane of the intestinal mucosa is the main diffusion barrier for both slowly and rapidly absorbed solutes in humans regardless of absorption mechanism. There are several indications in our present data that support this suggestion; (a) the low overall median/5 estimates of U W L in vivo between 83-188 ~ m , (b) no significant differences in either Peff or /5 between the lowest and the highest flow rates of these solutes were observed, (c) despite equal diffusion coefficients in buffer solution of these two compounds, D-glucose was about 2-3 fold more rapidly absorbed than antipyrine (Fig. 4 and Table 1). Support is given by other researchers (Levitt et al., 1990; Chiou, 1994). Levitt et al., 1990, stated that the minimal contribution of the epithelial resistance to intestinal drug absorption in man must be at least 40%. Implied in Chiou (1994), a 15 of 40 /~m in the human intestine would only impair the transmucosal uptake rate of solutes with absorption half-lives shorter than 6 min. Furthermore, the high correlation between the Peff of D-glucose (carrier-mediated absorption) and antipyrine (passively ab-
252
U. Fagerholrn, H. Lennernds / European Journal of Pharmaceutical Sciences 3 (1995) 247-253
_---, Itn
100" y = -3.5 ÷ 2.6X
variability in the intestinal absorption of high permeability solutes is due to other factors than a variable thickness of the UWL, such as available surface area and lipid composition in the membrane.
r^~ = 0.79 P = 0 . 0 0 0 1 / v
so.
~
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~.
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References
/~ ° ° a O/
10
] II 2ml-min-1 I I o3ml.min-i ]
20
AnflpyrineeffectivepermeabiUty
30
4O
(E-04 cm s - I)
Fig. 4. Correlation between PC,. estimates of antipyrine and o-glucose obtained after simultaneous administration of the compounds at different perfusion flow rates (2.0, 3.0 and 6.0 ml rain -~) in the human jejunum (n =53).
sorbed) (rZ=0.79; p =0.0001; n =53) suggests that the available surface of the apical membrane of the intestinal mucosa is the main diffusion barrier for both actively and passively absorbed solutes (Fig. 4) (Lennernfis et al., 1992). Our variable surface area theory might be explained by nonspecific regulation of the intestinal mucosa by hypertrophy and changes in microvillous and villous dimensions (Karasov and Diamond, 1983; Diamond, 1991). This might be a rapid and dynamic mechanism explained by the villous contractility that have the potential to alter the functional absorptive area (Strocchi and Levitt, 1993). In conclusion, we suggest that the resistance of the UWL to the intestinal absorption of highly permeable solutes in vivo in humans have earlier been markedly overestimated. Instead we consider the intestinal absorption in humans to be membrane controlled for both low and high permeability compounds, irrespective of the transport mechanism. The minor importance of the thickness of the UWL as a factor for absorption rate is probably due to a gastrointestinal motility in vivo that produces a highly efficient luminal stirring. We also suggest that the available surface of the apical membrane of the intestinal mucosa is the main diffusion barrier for both passively and actively absorbed compounds. Finally, we suggest that inter- and intrasubjectal
Amidon, G.L., Kou, J., Elliot, R.L. and Lightfoot, E.N. (1980) Analysis of models for determining intestinal wall permeabilities. J. Pharm. Sci. 69, 1369-1373. Andersson, B.W., Levine, A.S., Levitt, D.G., Kneip, J.M. and Levitt, M.D. (1988) Physiological measurement of luminal stirring in perfused rat jejunum. Am. J. Physiol. 254, G843-848. Chang, E.B. and Rao, M.C. (1994) Intestinal water and electrolyte transport: mechanisms of physiological and adaptive responses. In: L.R. Johnson (editor) Physiology of the Gastrointestinal Tract. Raven Press, New York, pp. 2027-2081. Chiou, W.L. (1994) Effect of "unstirred'" water layer in the intestine of the rate and extent of absorption after oral administration. Biopharm&Drug Disp. 15, 709-717. Diamond, J.M. (1991) Evolutionary design of intestinal nutrient absorption: Enough but not too much. News Physiol. Sci. 6, 92-96. Fagerholm, U., Borgstr6m, L., Ahrenstedt, O. and Lennernfis, H. (1995) The lack of effect of induced net fluid absorption on the in vivo permeability of terbutaline in the human jejunum. J. Drug Targeting (in press). H6gerle, M.L. and Winne, D. (1983) Drug absorption by the rat jejunum perfused in situ. Naunyn-Schmiedeberg's Arch. Pharmaco[. 322, 249-355. Johnson, D.A. and Amidon, G.L. (1991) Determination of intrinsic membrane transport parameters from perfused intestine experiments: a boundary layer approach to estimating the aqueous and unbiased membrane permeabilities. J. Theor. Biol. 131, 93-106. Karasov, W.H. and Diamond, J.M. (1983) Adaptive regulation of sugar and amino acid transport by vertebrate intestine. Am. J. Physiol. 245:G443-462. Karlsson, J. and Artursson, P. (1992) A new diffusion chamber system for the determination of drug permeability coefficients across the human intestinal epithelium that are independent of the unstirred water layer. Biochim. Biophys. Acta 1111, 204-210. Knutson, L., Odlind, B. and Hfillgren, R. (1989) A new technique for segmental jejunal perfusion in man. Am. J. Gastroenterol. 84, 1278-1284. Lee, I-D., Fagerholm, U., Lennernfis, H. and Amidon, G. (1993) A study of hydrodynamic characteristics in human intestine applying residence time distribution analysis. In: A. Urtti (editor) Methods to Overcome Biological Barriers in Drug Delivery, Syrup. 26-28 August. Acta Pharm. Sci. 10, Kuopio University, 73 pp.
U. Fagerholm. H. Lennern?is / European Journal of Pharmaceutical Sciences 3 (1995) 247-253
Lennern/is, H., Ahrenstedt, 6., H/illgren, R., Knutson, L., Ryde, M. and Paalzow, L. (1992) Regional jejunal perfusion, a new in vivo approach to study oral drug absorption in man. Pharm. Res. 9, 1243-1251. LennernS.s, H., Nilsson, D., Aquilonius, S-M., Ahrenstedt, (3., Knutson, L. and Paalzow, L. (1993) The effect of L-leucine on the absorption of levodopa, studied by regional jejunual perfusion in man. Br. J. Clin. Pharmacol. 35, 243-250. Lennern~is, H. (1994) Gastrointestinal absorption mechanisms: a comparison between animal and human models. Eur. J. Pharm. Sci. 2, 39-43. Lennern/is, H., Ahrenstedt, O. and Ungell, A-L. (1994) Intestinal drug absorption during induced net water absorption in man: a mechanistic study using antipyrine, atenolol and enalaprilat. Br. J. Clin. Pharmacol. 37,589596. Levitt, M.D., Aufderheide, T., Fetzer, C.A., Bond, J.H. and Levitt, D.G. (1984) Use of carbon monoxide to measure luminal stirring in the rat gut. J. Clin. Invest. 74, 2O56-2O64. Levitt, M.D., Fetzer, C.A., Kneip, J.M., Bond, J.H. and Levitt, D.G. (1987) Quantitative assessment of luminal stirring in the perfused small intestine of the rat. Am. J. Physiol. 252, G325-332. Levitt, M.D., Furne, J.K., Strocchi, A., Anderson, B.W. and Levitt, D.G. (1990) Physiological measurements of luminal stirring in the dog and human small bowel. J. Clin. Invest. 86, 1540-1547. Levitt, M.D., Strocchi, A. anti Levitt, D.G. (1992a) Human jejunal unstirred layer: evidence for extremely efficient luminal stirring. Am. J. Physiol. 262, G593-596. Levitt, M.D., Furne, J.K. and Levitt, D.G. (1992b) Shaking of the intact rat and intestinal angulation diminish the jejunal unstirred layer. Am. J. Gastroenterol. 103, 14601466. Nilsson, D., Fagerholm, U. and Lennern~is, H. (1994) The influence of net water absorption on the permeability of antipyrine and levodopa in the human jejunum. Pharm. Res. 11, 1541-1545.
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Read, N.W., Barber, D.C., Levin, R.J. and Holdsworth, C.D. (1977) Unstirred layer and kinetics of electrogenic glucose absorption in the human jejunum in situ. Gut lS, 865-876. Sarosiek, J., Marshall, B.J., Peura, D.A., Hoffman, R.N., Feng, T. and McCallum, R.W. (1991) Gastroduodenal mucus gel thickness in patients with Helicobacter pylori: a method for assessment of biopsy specimens. Am. J. Gastroenterol. 86, 729-734. Strocchi A. and Levitt M.D. (1993) Role of villous surface area in absorption. Dig. Dis.&Sci. 38, 385-387. Thomson, A.B.R and Dietchy, J.M. (1984) The role of the unstirred water layer in intestinal permeation. Chapter 21 in: T.Z. Csaky (editor) Pharmacology of Intestinal Permeation II. Springer, Berlin. Westergaard, H., Holtermiiller, K.H. and Dietschy, J.M. (1986) Measurement of resistance of barriers to solute transport in vivo in rat jejunum. Am. J. Physiol. 250, G727-735. Wilke, C.R. and Chang, P. (1955) Correlation of diffusion coefficients in dilute solutions. Am. Inst. Chem. Eng. J. 1, 264-27/). Winne, D. (1978) Dependence of intestinal absorption in vivo on the unstirred layer. Naunyn-Schmiedeberg's Arch. Pharmacol. 304, 175-181. Winne, D. (1979) Rat jejunum perfused in situ: effect of perfusion rate and intraluminat radius on absorption rate and effective unstirred layer thickness. NaunynSchmiedeberg's Arch. Pharmacol. 307, 265-274. Winne, D. (1984) In: Unstirred layer as a diffusion barrier in vitro and in vivo. E. Skadhauge and K. Heintze (editors) Intestinal Absorption and Secretion. MTP Press, Lancaster, pp. 21-38. Winne, D (1987) Closed rat jejunal segment in situ: role of pre-epithelial diffusion resistance (unstirred layer) in the absorption process and model analysis. NaunvnSchmiedeberg's Arch. Pharmacol. 335, 204-215.