George M. Grass

In Vitro Measurement of Gastrointestinal Tissue Permeability Using a New Diffusion Cell

A new diffusion cell, derived from the Ussing chamber, was developed for the measurement of tissue permeability. This cell incorporates the attributes of using a single material and laminar flow across the tissue surface. In addition, the design allows the cell to be manufactured in a wide range of sizes to allow optimization of surface area to volume for a variety of tissues. The apparatus is applicable to the evaluation of transport of compounds through mucosal/epithelial barriers, i.e., gastrointestinal tissue. Active transport, permeability enhancers, enzymatic degradation, and absorption in various tissue sections can be explored. Preliminary data are consistent with the expected effects of molecular size and partition coefficient of a transported molecule on permeability in epithelial tissue. In addition, active transport of D-glucose and inhibition by phloridzin and ouabain can be demonstrated.

Comparison of the permeability characteristics of a human colonic epithelial (Caco-2) cell line to colon of rabbit, monkey, and dog intestine and human drug absorption.

The in vitro permeabilities of Caco-2 monolayers and permeabilities in tissue sections from colon of monkey, rabbit, and dog were compared using a series of compounds. The selected compounds differed in their physicochemical properties, such as octanol/water partition coefficient, water solubility, and molecular weight. Their structure included steroids, carboxylic acids, xanthins, alcohols, and polyethylene glycols. A linear permeability relationship was established between Caco-2 and colon tissue from both rabbit and monkey. The results suggest that Caco-2 is twice as permeable as rabbit and five times as permeable as monkey colon. However, no clear relationship could be established between Caco-2 monolayers and dog colon permeability. A relationship between permeability in Caco-2 monolayers and human absorption was found. The results suggest that within certain limits, permeability of Caco-2 monolayers may be used as a predictive tool to estimate human drug absorption.

CHARACTERIZATION OF THE UNSTIRRED WATER LAYER IN CACO-2 CELL MONOLAYERS USING A NOVEL DIFFUSION APPARATUS

Caco-2 monolayers grown on Transwell polycarbonate membranes have been characterized as a valuable tool in drug transport studies. Despite the clear advantages of this system, the lack of stirring may create an unstirred water layer (UWL) whose resistance may limit the transcellular transport of lipophilic molecules. The objective of this study was to evaluate a novel diffusion cell where the transport buffer is mixed by gas lift and to determine the mixing flow rate needed to reduce the thickness (h) of the UWL adjacent to cell monolayers. The transport of the leakage marker, mannitol, remained at least 15-fold lower than the flux of testosterone, indicating that the stirring flow rates used did not affect the integrity of the monolayers. The permeability (P) of testosterone (log PC 3.13) across monolayers mounted on this diffusion cell was 4.07, 10.90, and 14.18 × 10−5 cm/sec at flow rates of 0, 15, and 40 ml/min, respectively, and the apparent UWLs were calculated to be 1966, 733, and 564µm. P and h in the stagnant Transwell were 3.08 × 10−5 cm/sec and 2597 µm, respectively. On the other hand, h was significantly smaller in the unstirred, cell-free membranes than in their cell-containing counterparts. P was correlated with lipophilicity and, in the case of the more lipophilic compounds, with the mixing flow rate.

Permeability Characteristics of Various Intestinal Regions of Rabbit, Dog, and Monkey

The in vitro permeability of a series of both hydrophilic and lipophilic compounds, as defined by the octanol/water partition coefficient, was measured in four segments of rabbit, monkey, and dog intestine using a side-by-side diffusion cell. A linear relationship was established for tissue resistance to hydrophilic compound diffusion in jejunum and colon among rabbit, monkey, and dog. The results suggest that rabbit jejunum is twice as permeable as monkey and dog jejunum. The colonic tissues of monkey, rabbit, and dog demonstrate similar permeabilities. Measuring the permeabilities of different tissues with compounds of similar physicochemical properties allows comparison of tissue restriction to transport. Thus, in vitro permeability measurements may be used to investigate physiological differences of various intestinal tissue segments that influence tissue permeability. Investigating the permeability of different intestinal segments from various species could allow the identification of an appropriate in vitro intestinal permeability model that will lead to the prediction of intestinal absorption in humans, eliminating the need for extensive and often misleading in vivo animal testing.

Evidence for Site-Specific Absorption of a Novel ACE Inhibitor

Moexipril {2-[(l-ethoxycarbonyl)-3-phenylpropyl]amino-l-oxopropyl]-6,7-dimethoxy-1,2,3,4-tetra-hydroisoquinoline-3-carboxylic acid (S,S,S)}, an ester prodrug of an ACE inhibitor, was formulated in controlled-release preparations with a range of in vitro release rates, to provide a prolonged input of drug in vivo. However, pharmacokinetic studies with the controlled-release dosage forms in humans produced plasma profiles with the same characteristics and time to peak as an immediate-release capsule. In vitro dissolution data from the controlled-release dosage form, as well as the known characteristics of the polymer used to control drug release from the dosage form, suggest no reason to suspect an abrupt halt to the in vivo release of the drug after 1–2 hr. The lack of sustained blood levels is, therefore, most likely due to failure of the GI tract to absorb the drug beyond some location in the upper small intestine, i.e., site-specific absorption. This theory is supported by a series of computer simulations involving moexipril and the active moiety, moexipril diacid. Possible mechanisms include poor drug permeability, a pH effect whereby the zwitterionic form of the drug is more rapidly absorbed, and esterase cleavage of moexipril to the poorly absorbed moexipril diacid.

A Correlation of Permeabilities for Passively Transported Compounds in Monkey and Rabbit Jejunum

Permeability measurements were conducted for a series of compounds using in vitro tissue sections from monkey and rabbit jejunum. Jejunal segments were stripped of serosal musculature and mounted in a diffusion-cell system, using previously described methods and equipment. Permeability determinations of radiolabeled compounds ranging over two orders of magnitude in molecular weight were conducted. For the compounds examined, the permeability of the rabbit jejunum was approximately twice that of the monkey. This was in contrast to the relationship implied by the stripped tissue thickness measurements of 0.92 and 0.83 mm for rabbit and monkey, respectively. An investigation of the size of the paracellular space in the jejunum was undertaken to account for this apparent discrepancy in tissue permeability. Scanning electron micrographs of intestinal sections revealed a similar packing density of cells between species; however, a difference was noted in the shape and number of villi per unit area. Comparative measurements of the paracellular volume in both species using mannitol and methoxyinulin as extracellular space markers further suggests that the paracellular junctions are similar in size but more numerous per unit area of rabbit jejunum than that of the monkey. In contrast to passively transported compounds, the active transport of D-glucose was greater in monkey jejunum compared to rabbit tissue segments. When active transport was inhibited by blockade of the sodium pump with ouabain, the passive component of D-glucose transport for both rabbit and monkey tissue was in agreement with the relationship demonstrated above for compounds which are solely transported by passive processes.

A new side-by-side diffusion cell for studying transport across epithelial cell monolayers

Dear Editor: Frequently, transport studies with cell monolayers are performed under stagnant conditions (1-3,7,8), where the absence of stirring could result in the formation of a large and potentially non-physiological unstirred water layer (UWL). In the absence of stirring, the transepithelial fluxes of lipophihc compounds will be controlled by the UWL. When transport is hmited by the UWL, it becomes very difficult to predict accurately the permeability coefficient of compounds that are absorbed via passive diffusion (4) and to calculate the transport parameters (i.e., K m and V ~ ) of compounds absorbed by active transport (4,12-14). The diffusion apparatus described in the present study was developed to provide a system where the transport across cell monolayers grown on microporous membranes can be studied under wellstirred conditions so as to minimize the influence of the UWL surrounding the cell monolayer. The main advantage of this diffusion cell is that it does not involve potentially damaging manipulation of the cell monolayers. In addition, the system uses a very low profile (3/8 inch) well to mount the monolayer in the diffusion cell (Fig. 1). Flow within the diffusion cell can be accurately controlled by an associated set of valves. Caco-2 cell monolayers were utilized in the study because of their recent characterization as a model transport cell culture system of polarized intestinal epithelium (5,8) and because of the importance of the small intestinal epithelial barrier in the transport of drugs and nutrients. Because the flow rates utilized in this study are higher than those normally encountered in the intestinal lumen (11), the effect that the stirring flow rates (SFR) used in this study had on the barrier function of the monolayer or on cell morphology was assessed carefully. The fluxes of the lipophilic compounds testosterone and N-butanol at different stirring rates were utilized to determine the extent of transeellular permeability. In addition, the concomitant paracellular diffusion of the hydrophilic compound mannitol was used as an indicator of monolayer leakage. The effect of stirring flow on the barrier properties of the monolayers was also monitored by microscopic examination of cell morphology. Caco-2 ceils (America n Type Culture Collection, Rockville, MD) were grown for 15-20 days on short inserts (0.3 cm height, 6.5 mm diameter, 0.4 #m pore size) (Costar, Bedford, MA) which were placed inside standard Transwell~ inserts (24.5 mm dia., Costar) that contained 2.6 ml culture media outside and 0.5 ml inside. The culture medium, which consisted of Dulbeccos modified Eagles medium supplemented with 10% heat-inactivated fetal bovine serum, 1% non-essential amino acids, 100 U/ml penicillin and 100 #g/ml streptomycin (all from Hazleton Research, Lenexa, Kansas), was changed every other day. For transport studies, the small inserts containing cell monolayers were mounted in the new diffusion cell (Precision Instrument

Mechanism of Dextran Transport Across Rabbit Intesi Tissue and a Human Colon Cell-Line (CACO-2)

The in vitro permeabilities of 14C labeled dextrans (10, 40, and 70 kD) were calculated from mass transport across Peyers patches and non-patch tissues derived from rabbit jejunum, and a human colon cell line (Caco-2) grown as a monolayer on polycarbonate filters. Size distribution of dextrans did not change upon transport as judged from size exclusion chromatography. Permeabilities decreased in a size-dependent manner. Ranking of permeabilities for dextran 10 and 40 kD were: Caco-2 > non-patch tissue > Peyers patches; while dextran 70 kD demonstrated no difference among the barriers. Tissue resistance, expressed as 1/(permeability.tissue thickness) was virtually the same in Peyers patches and non-patch tissue, suggesting that tissue thickness and not interaction determines the difference in permeability. ATP depletion with ouabain, Na(+)-azide and 2-deoxy-D-glucose, and low temperature (4 degrees C) did not result in reduced permeabilities suggesting passive transport. The results suggest that the investigated intestinal barriers transport dextrans in a similar fashion independent of their source. However, comparison of the ratios dextran 10 kD/mannitol and PEG 900/mannitol between rabbit tissue and Caco-2 monolayers suggests Caco-2 monolayers may serve as a model to study absorption potential of potentially harmful compounds in coeliac disease, gastroenteritis, and colon carcinoma.

Evaluation of the performance of controlled release dosage forms of ticlopidine using in vitro intestinal permeability and computer simulations.

Prototype controlled release formulations of ticlopidine hydrochloride were developed, but when administered to humans, these formulations significantly reduced the bioavailability of intact drug in plasma. In order to examine the intestinal permeability characteristics and gastrointestinal metabolism of 14C-ticlopidine, we employed an in vitro diffusion cell system to directly measure the permeation of ticlopidine across various segments of monkey and rabbit intestine. High pressure liquid chromatography was used to determine the amount of intact ticlopidine on both the mucosal and serosal sides of the intestinal tissue. Simulations based upon the known pharmacokinetics of ticlopidine were conducted using STELLA, a modeling program, to provide insight as to the nature of the decreased bioavailability of these ticlopidine CR dosage forms. These simulations indicate that the absorption of intact ticlopidine is a non-linear phenomena, with inordinately large increases in absorbed intact drug with increases in dose. Conversely, decreases in drug available for immediate absorption, as with the controlled release dosage forms, lead to non-linear decreases in bioavailability. Such a finding is very consistent with the extensive first-pass metabolism suggested from the tissue permeability studies.

The effects of enprostil and RS-86505-007 on in-vitro intestinal permeability of rabbit and monkey.

Abstract— Enprostil is a prostaglandin E2 analogue characterized as a racemic mixture of four stereoisomers. Enprostil and a single isomer, RS‐86505‐007, were evaluated for their effects on the permeability of actively and passively transported compounds in segments of small intestine from rabbits and monkeys. Consistent with human in‐vivo studies, which have demonstrated decreases in absorption of D‐xylose, both compounds inhibited D‐glucose transport. The passively transported compounds mannitol and progesterone were also less permeable in this model in the presence of enprostil or RS‐86505‐007. In contrast to the concentration‐dependent inhibition displayed by ouabain, RS‐86505‐007 had no effect on purifed Na+K+‐ATPase. It is suggested that an effect of a general nature, possibly an increase in the barrier properties at the intestinal surface, may explain the transport inhibition. Of two other enprostil isomers, RS‐86812‐007 inhibited D‐glucose transport in rabbit small intestine, while RS‐86505‐008 had no effect. The prostaglandin E1 analogue misoprostol was ineffective in monkey and poorly effective in rabbit. This suggests that the inhibition of D‐glucose transport by enprostil and its active stereoisomers is mediated through some structurally specific receptor interaction.