the use of the intestinal epithelial cell culture model, caco-2, in pharmaceutical development

advanced

drug delivery reviews

ELSEVTER* Advanced Drug Delivery Reviews 22 (1996) 85-103

The use of the intestinal epithelial cell culture model, Caco-2, in pharmaceutical development

Carole A. Bailey*, Piotr Bryla’, A. Waseem Malick

Pharmaceutical Research and Development, Hqffmann La-Roche Inc., Nutley, New Jersey 07110, USA

Abstract

The need for more innovative and readily orally bioavailable therapeutics that can be developed faster has driven the development of tools and strategies that will have a major impact on those needs. The application of the Caco-2 human epithelial cell line for use as a tissue culture model for permeability measurements that can be used to predict oral absorption effectively demonstrates this strategy. This article discusses some of the strategic applications of the Caco-2 tissue culture model in pharmaceutical development. The development of an experimental program to characterize the endogenous gut peptide transport system is described, as well as its application to the understanding of the oral absorption of cephalosporin molecules. Other studies describe the use of permeability studies for a chemical series of drug candidates to predict their oral absorption in vivo. Finally. the application of the model to aid in the development of formulation strategies is discussed.

Keywords: Caco-2: Permeability; Partition coefficient; Peptide transporters; Formulation development: Tissue culture

Contents

Introduction ..........................................................................................................................................................................

1.1. General introduction and scope of the review ...............................................................................................................

1.2. Tissue culture systems in the hierarchy of experimental models.. ..................................................................................

1.3. Application of the cell culture model in the development process ................................................................................

Characteristics of the model system.. ....................................................................................................................................

2.1. General considerations ..................................................................................................................................................

2.2. Experimental conditions.. ..............................................................................................................................................

2.3. Growth of cells on filters in transport wells ...................................................................................................................

2.4. Correlation between absorption and permeability coefficients.. ....................................................................................

Representative applications ..................................................................................................................................................

3.1. Endogenous transport systems: the peptide transporter(s) ............................................................................................

3.2. Permeability within a chemical series ............................................................................................................................

3.3. LJse of the tissue culture model for formulation development.. .....................................................................................

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*Corresponding author.

‘Current address: Bayer Corporation, West Haven, CT 06516, USA.

Ahhreviations: DMA, dimethyl amiloride; DIDS, diisothiocyanatostilbene-disulfonic acid: FCCP, carbonyl cyanide p-trifluoro-

methoxyphenyl hydrazone; HBSS, Hank’s balanced salt solution; HEPES. N-2-hydroxyethyl piperazine-N’-2.ethane sulfonate:

MEM. Dulbecco’s minimal essential medium; MES, 2-(N-morpholino) ethanesulfonic acid; MMS, methyl methanethiosulfonate;

NEM, n-ethyl maleimide: SEDDS, self-emulsifying drug delivery system; TEER, trans-epithelial electrical resistance

0169-409X/96/$32.00 @ 1996 Elsevier Science B.V. Ail rights reserved

PI/ SO169-409X(96)00416-4

4. Concluding remarks _..__..,,,,......_..........,,.,............................

Acknowledgments ._....__.___..,,,.,...............,,,,................,,...........

References ..___.........................................................................

1. Introduction

1.1. General introduction and scope C$ the review

The objective of this review is to discuss applications of the human intestinal epithelial

cell culture model. Caco-2, to the drug develop-

ment process in the pharmaceutical industry. The general approach will be to address the use of

the cell culture model as a pragmatic working model. with emphasis on Caco-2 cells. While other cell models have been described in the literature (reviewed in [l] and references there-

in), this model is currently the best characterized

and most commonly used cell system for absorption studies. Caco-2 cells are human in origin,

and can be manipulated in culture so that they exhibit many characteristics of the human small intestinal epithelium. Thus, these cells readily

lend themselves to application as an in vitro model to study Gl absorption in a human cell

model. However. it should be noted that the cells originate from a human cola-rectal adenocar-

cinema, and have been reported in some cases to retain some of the characteristics of colonic epithelium [2-5,321. Other reviews in this volume

will address more theoretical issues of transport

and permeability in cell culture models, in vitro- in vivo correlations. and drug transport and efflux systems in epithelia. and these issues will

not be dealt with in depth here (see related reviews in this issue).

Historically. the pharmaceutical industry has

employed different experimental designs. analytical techniques and animal models to study the absorption process. and frequently these differences have made direct comparison of results difficult. With respect to in vivo studies, no single animal species has emerged as a suitable model for human studies. In addition, there is currently a concerted effort to decrease the use of animals in experimental studies. Cell culture models for the study of absorption and transport across

,.. loo

endogenous barriers naturally emerge as a viable

alternative in this area. Use of these cellular

models can decrease the number of animals

needed for experimental studies, and offer ease of set up and handling as well as some level of

cost control, once the initial tissue culture facility is established. An additional advantage of the

cell culture model is that multiple studies can be performed with a relatively small amount of test

compound. which is often a key factor early in the development of a drug candidate.

1.2. Tissue culture systems in the hierarchy of experimental models

Since the overall goal of the pharmaceutical

industry is the delivery of the therapeutic candidate in humans, the use of cell culture models must be classified as a reductionist approach to

this challenge. In fact, cell culture models occur approximately midway on the organization chart of the most complex human systems to the highly

simplistic molecular models that are used early in the drug discovery process. which are frequently generated by recombinant techniques. Therefore, the nature of the cell culture model allows for

the design of experiments which probe a certain subset of experimental questions at the cellular

and molecular level, but are precluded from gaining information that might result from the interplay of multiple complex biological func-

tions. In its most useful application, the process of absorption or permeation, which is represented via passage across the entire intact intestinal epithelial barrier from the luminal (apical) surface to the abluminal (basolateral) side. is readily addressed in the Caco-2 cell model.

Because of the reductionist nature of the C’aco-2 cell culture model, conclusions must be interpreted with caution on more complex issues that are the result of the interplay between different cell types, tissues, and biological signals in the whole organism. If. for example, the test molecule is subject to strong interaction with the

C.A. Bailey et al. I Advanced Drug Delivery Reviews 22 (1996) 8_%10_3 87

mucous or unstirred boundary layer in vivo, or suffers rapid clearance and/or a high degree of metabolism in the intact animal, the correlations drawn from the tissue culture model necessarily become less predictive of the situation in humans. In these cases, the highest predictive efficiency is obtained from intact animal studies, but it is nonetheless useful to compare the tissue culture data with pharmacokinetic data generated in vivo early in the development process. The propensity for a molecule or class of molecules to undergo abnormal metabolism or large clearance effects can then be factored into the pool of data that will impact on the overall development scheme for the product. In such cases, the tissue culture model can still be used to obtain useful information on the absorption step in the overall process.

Other models most commonly employed for the study of oral absorption are brush border membrane vesicles, everted intestinal rings, and single pass intestinal perfusion. As might be expected, each of these absorption models has inherent advantages and disadvantages. For example. vesicular studies (brush border membrane vesicles) measure only the uptake step into the vesicle interior. Further, establishing that the carrier systems have retained the correct directionality, and have not undergone a scrambling in orientation during the process of preparation of the vesicles is important, and experimentally challenging. in the vesicular system. In contrast, the correct carrier system orientation is intrinsic to a controlled, well characterized cell monolayer system in culture, as well as to the animal tissue and organ models. A second commonly used system is the isolated, everted intestinal ring, most frequently prepared from intestinal segments isolated from rodents or small animals such as the rabbit. This experimental model usually maintains some semblance of the endogenous mucous layer, in addition to the epithelial cell barrier. In this model, the accumulation of drug into the intestinal tissue is measured in vitro, and tissue uptake is equated with absorption. This is a relatively rapid and simple system which requires minimal maintenance facilities. However, the method usually requires radiolabelled test compound, as the extraction and

analysis of non-labelled test substance from tissue samples can be tedious and time consuming at best. A third approach is the single pass intestinal perfusion. In this in situ model, steady state disappearance of the test compound from a circulating pool presented to the luminal side of the isolated intestinal segment is generally equated with transport across the barrier. While disappearance from the lumen may equate with transport of the test compound across the entire barrier, it should be noted that other factors might also impact this measurement, such as entrapment of test compound in the mucous layer, adsorption in the experimental chamber and/or the tissue itself, or uptake into the cells without final release of compound out to the basal side. All of these experimental conditions will present as a disappearance from the circulating perfusate, and may not accurately represent the entire transport process across the epithelial barrier. As with the Caco-2 cell culture system, analysis is usually from a relatively simple buffer system, and is generally not problematic. The single pass perfusion is the most complex model for the study of absorption as circulation, enerva- tion, metabolism and clearance function all re- main intact; by extension, it is also probably closest to the actual in vivo situation. However, the model requires a large number of samples to obtain good statistical data, due to the physiolog- ical complexity and variability inherent in the system. In addition, these studies require the maintenance of a substantial animal facility.

Multiple studies comparing these absorption models have been described, most recently in a comprehensive report by Stewart et al. [6]. The general conclusion from these multiple studies is that the Caco-2 cell culture model is a positive predictor of drug absorption in the intestinal tract, and by extension, of bioavailability in vivo.

1.3. Application of the cell culture model in the development process

The Caco-2 intestinal epithelial model is generally employed as support for drug discovery, and in development of formulation strategies to address the oral absorption step early in the

xx C.A. Builq et al. / Advmced Drug Delivcyy Reviews 22 (19%) X5- IO_+’

pharmaceutical development process. While

there are usually several physico-chemical properties of a drug molecule that aid in the predic-

tion of potential oral absorption such as solubility, electrostatic charge, partition coefficient, pK and interaction with artificial membranes. no

one of these completely mimics the interplay

between the test molecule and an intact bio-

logical barrier such as the epithelial cell system.

In the intact biological system, the drug molecule

can take the paracellular route where diffusion and water solubility play a key role, or it can partition across the membranes of the cell, where

lipophilicity, solvation state, and electrostatic charge all can impact on the process. These functions can be easily addressed in the cell

culture model, where permeability across the monolayer is readily characterized experimentally. The resultant permeability coefficients can

then be applied to predict absorption characteris-

tics for the test molecule. An additional application of the Caco-2 model

has been in the delineation of endogenous carrier systems present in the gut. which may be enlisted for the delivery of drug candidates across the

epithelial barrier. The rationale here is to use the endogenous carrier systems that have evolved naturally for the absorbtion of proteins. carbohy- drates, lipids and vitamins to deliver drug mole-

cules via the oral route. Probably the best example of this approach is described by studies of the

peptide transporter in Caco-2 cells that have been reported in the literature over the last

several years [7-131. Other studies using the Caco-2 model to study endogenous transport systems have described amino acid transport [ 14- 171. a hexose transporter [ 181, nucleotide trans-

port [19], bile acid transporters [3,20,31]. vitamin carriers [21,22], and a system for the uptake of phosphate 1231.

In order to maximize the potential of these approaches, a detailed characterization of the carrier system must be obtained. The Caco-2 model lends itself readily to this kind of study at the molecular and cellular level. It should be noted. however, that some investigators have reported differences in the expression of carrier systems in the cell culture model depending on the conditions used for culture and/or the pas-

sage number of the cells in culture [3.24,25,30,31]. C are must be exercised, therefore, when using the cell culture model to study

carrier systems, to ensure that experiments are

performed over a broad range of experimental conditions and cell culture passages. This will

establish whether there are changes in the ex-

pression of the transporter system under inves- tigation for which a subset of experimental

conditions must be carefully controlled.

2. Characteristics of the model system

2.1. General considerations

It is of prime importance that the cell culture system to be employed be carefully characterized

and experimentally controlled. The rationale for this careful definition of the model cell culture

system is to be able to make direct comparisons

between measured permeability values for known compounds and unknown or test drug molecules which are under development. The

absolute value of the apparent permeability coefficient may vary somewhat among laboratories because of differences in experimental con-

ditions and techniques employed during the determination of these values. Accurate definition of a model system and of the window of

permeability values for drugs with known oral absorption characteristics and bioavailability

under a carefully standardized set of experimen-

tal culture and transport conditions allows for the use of the cell culture system to obtain predictive information for new compounds under development.

While other models, such as HT-29 cells. have been described [1,27], Caco-2 cells in culture are

the best characterized gut epithelial monolayer system available at the present time. They exhibit the correct morphology and express many of the brush border hydrolases, ion transport properties and carrier systems typical of human gut epithelium upon differentiation in culture ([3,26-291. Ba’l 1 ey. unpublished observations). In addition, mature, intact cell monolayers are characterized by a discreet transepithelial electrical

C.A. Bailey et al. I Advanced Drug Delivery Reviews 22 (1996) 8%10-3 89

resistance (TEER), which is a function of the presence of tight junctional complexes between neighboring cells. The magnitude of the measured TEER is variable, and has been described as being closer to the resistance in the human colon, i.e. the resistance in the Caco-2 culture system may be higher than is actually present in the small intestine in vivo [2-41.

are best characterized using short transport times so that saturation kinetics apply, and compounds which exhibit low permeabilities are more readily detected in the larger transport units due to the greater surface area of the monolayer available for transport. A summary list of variable parameters is presented in Table 1.

2.2. Experimental conditions

There are several experimental parameters that can be varied in describing the typical Caco- 2 cell culture model system. Among these are the source of cells and their passage number, the incubation medium employed for cell growth and maintenance, the diameter of the filters in the transport unit, which impacts on the experimental volumes and amount of test compound needed, the nature of the filter support and its porosity, the buffering system used during cell culture experiments, variability in time and temperature, and/or the use of mixing or stirring apparatus during the experiment. It is recom- mended that this list of parameters be surveyed and considered in the context of the type of studies to be conducted, and that choices be made that best fit the nature and objectives of a particular study or program. For example, experimental compounds in short supply are more readily studied in small transport units to con- serve compounds, enzyme and carrier systems

Once the experimental parameters of the model system have been decided upon, a careful characterization of that system must be performed. Morphological studies should be conducted using electron microscopy to follow the development of intact microvilli on the apical surface of the monolayer, and to establish evidence of the presence of mature tight junctions throughout the monolayer. Development of transepithelial electrical resistance (TEER) should be monitored to determine the maturity and intactness of the monolayer barrier. This is readily performed using a volt-ohm meter that has been equipped with electrodes which are offset for ease of placement in the upper and lower chambers of the transport unit. In addition, the permeability of a series of markers, of increasing size or molecular weight, should be characterized once the culture or transport system has been defined. In general, the intact monolayer should exclude the passage of marker molecules from the apical to the basal chamber of the transport unit under the desired experimental conditions. By contrast, the filter unit itself should allow the markers to equilibrate

Table 1

Factors potentially influencing the measure of permeability in the tissue culture model

Factor Parameter affected

Source of cells, passage number

Incubation medium for cell growth and maintenance

Type of transport unit

Composition and porosity of filter in transport unit

LJse of extracellular matrix coating on filter

Length of time cells grown on filters

Size of transport unit filter (area in cm2)

Buffer volume in apical and basolateral chambers

Intactness of monolayer. expression of endogenous transporters

Time to confluency, expression of endogenous transporters

Cost. amount of test compound needed. adsorption

Adsorption, free diffusion of solutes

Characteristics of monolayer and matrix secreted by cells

Maturity of monolayer, tight junctions

Number of cells needed, cost of unit. amount of test compound

needed, amount of transport measured

Amount of test compound needed. dilution factor

which impacts analytics

Composition of transport buffer Cost, efficiency, ease of use, analysis, experimental flexibility pH of transport buffer Experimental flexibility Time and temperature Experimental flexibility

Mixing or stirring apparatus Unstirred boundarv laver effects

readily into both chambers within the time frame

of the experiment. That is. the transport unit must not present a limiting barrier to transport

under the experimental conditions of the study. The choice of markers is limited to molecules

which cannot enlist the use of one of the endogenous transport systems as a mechanism of

transport across the monolayer, so that any experimentally measured transport represents

the exclusion or barrier function of the system. Typical marker molecules are mannitol. lucifer

yellow. PEG-4000. and dextrans of defined mo-

lecular weight (e.g. 20 000 and/or 70 000 Da). These molecules bracket the reasonable size

range that might be expected of candidate drug

molecules. Further. they are readily available either with a radiolabelled or fluorescent tag,

allowing for ease of analytical detection. Once the culture system to be used for trans-

port studies has been well characterized and found to be reproducible. permeability coefti- cients can be determined for a series of com-

pounds with known oral absorption characteristics in vivo. This approach will control for vari-

ability that may occur in absolute values for

permeability coefficients among laboratories, due to differences either in the transport unit being

employed, the cells, and/or the conditions under which the measurements are made. Once a range

of apparent permeability coefficients has been defined in the chosen experimental test system. direct comparisons between measured values for known compounds and unknown or new drug

molecules which are under development can bc performed. allowing for the prediction of the absorption characteristics of the unknown com-

pound in vivo.

There are numerous conditions that can be employed to conduct transport or permeability

studies using the cell culture model. Following is a typical set of conditions employed in our laboratory for transport studies using the Caco-2 cell model. For routine use. Caco-2 cells are grown on 6.5mm diameter inserts in 24-well cluster plates. In some studies, transport units containing wells of 25mm diameter (six wells per

plate) have been used. The insert filter consists of a tissue culture treated polycarbonate membrane

with a 3.O-P_m pore size. Filters are precoated with 45 I_r,g/cm” of rat tail Type I collagen, and

Caco-2 cells are seeded onto the pre-equilibrated

filters at a cell density of 60 000 cells per square centimeter. Maintenance medium consists of Dulbecco’s Modified Eagle Medium (D-MEM) supplemented with 2 mM t_-glutamine. 1% non-

essential amino acids, lo-25 mM N-2-hydroxyethyl piperazine-N’-2-ethane sulfonate buffer

(HEPES) and 10% fetal or newborn bovine

serum. In experiments conducted at more acidic

conditions, the HEPES is replaced with 2-(N- morpholino) ethanesulfonic acid (MES). Incuba- tion conditions for cell growth are maintained at

37°C. in a 5% CO, atmosphere and U-90%

relative humidity. Cells growing on filters are fed by careful aspiration of spent media from both compartments of the transport unit, followed by replacement with fresh media that has been prewarmed to 37°C. usually every other day. Cells

are routinely used in transport experiments after approximately 21 days growth on filters, at which

time the confluent monolayers exclude the pas-

sage of the small molecular weight marker mannitol, and express a TEER of approximately 400 Ohm-cm’. Fig. 1 shows the development of a

tight epithelial barrier, characterized by the exclusion. with time in culture, of molecular weight markers of increasing size. It can be seen from

these data that the largest marker, dextran 70 000, was the first to be excluded from passage across the barrier. followed by exclusion of the

PEG 4000 marker. The last marker to be excluded was mannitol, which was the smallest

molecule tested. Routine measurements in this system indicate that mannitol passage across the mature monolayer is f 0.5% /h at 37°C at pH 7.4. after 21 days in culture. This graded size

exclusion correlates with the development of correct cellular morphology as evidenced by well delined microvilli and tight junctions throughout the monolayer as seen by transmission electron microscopy ( [26-291, Bailey. data not shown). In our laboratory. measurements of trans-epithelial electrical resistance indicate that it has reached a maximum by approximately 15 days. after which it is maintained up to at least 30 days in culture.

C.A. Bailey et al. I Advanced Drug Delivery Review3 22 (1996) 8.5-103

Fig. I. Exclusion of molecular markers from passage across

the monolayer as a function of molecular weight and maturity

of cell monolayer. [ ‘Hlmannitol, [ ‘JC]polyethylene glycol

(m.w. = 4000) or fhroresceinated dextran (m.w. = 70 000)

were introduced into the apical compartment of the transport

unit, and the hasolateral chamber was analyzed for appear-

ance of the marker molecule after 60 min incubation at 37°C.

Experimental values represent the average of a minimum of

triplicate determinations: standard errors were less than 10%.

Data supplied by C. Bailey, R. Festen and P. Lynch. Hoff-

mann-La Rochc. Inc.. Nutley, NJ.

2.4. Correlution between absorption and permeability coyfficients

Prior to the use of transport studies using the Caco-2 human cell line as a predictive model to study oral absorption, the in vitro cell culture model should be characterized using several different compounds of known oral absorption in humans. Fig. 2 shows just such a correlation for eight compounds whose oral absorption characteristics in humans have been previously described, ranging from O-100%. The permeability coefficients (cm/s) were measured in the Caco-2 model at both pH 6.1 and 7.4. A plot of oral absorption vs. permeability data displays as a sigmoid curve, with compounds that are poorly absorbed exhibiting apparent permeabilities (P,,,,,) 5 0.5 X 10 -’ cm/s, while those compounds that are most efficiently absorbed (90-100%) have permeabilities 2 4 X 10eh cm/s. Permeabili- ty coefficients measured at both pH 7.4 and 6.1 did not differ greatly from each other for this set of compounds, with the exception of salicylic

91

Fig. 2. Correlation between permeability coefficients in cell

culture and absorption in humans. Apparent permeability

coefficients were measured as a function of time at 37°C in

the Caco-2 tissue culture model as described in the text. and

values were plotted against data available in the literature for

absorption in humans. The permeability values were mea-

sured in Hank’s balanced salt solution adjusted to pH 7.4

with HEPES (open squares) or to pH 6.1 with MES (closed

squares). Test compounds were as follows: 1. vasopressin; 2.

sulfasalazine: 3, PEG-4000; 4. mannitol: 5. atenolol: 6, dexa-

methasone; 7. salicylic acid, and 8. propranolol. Data supplied

by C. Bailey, P. Bryla and P. Lynch. Hoffmann-La Roche

Inc.. Nutley, NJ.

acid, which had a significantly lower value for measured permeability at pH 6.1. It should be noted that, despite the difference in absolute values, the apparent permeabilities measured at both pH’s predicted the correct absorption characteristics for the molecule.

These values were determined in a model system similar to that described in Section 2.3, above. The transport filters were 6.5 mm in diameter, the buffer was Hank’s Balanced Salt Solution (HBSS), supplemented with 25 mM glucose and buffered to pH 7.4 with 10 mM HEPES, or to pH 6.1 with 10 mM MES. Cells were prepared by gentle aspiration of spent media, followed by gently washing the monolayers with prewarmed assay buffer. An aliquot of buffer was then introduced into the basolateral chamber, and the cell insert replaced into the well. The test compound was dissolved in the appropriate buffer, prewarmed to 37°C and then carefully layered over the monolayer in the apical chamber. After 60 min incubation at 37°C during which time the plates were gently stirred on a mixing platform, the cell insert was removed from the culture well, and the amount of com-

pound transported across the monolayer to the basolateral chamber was determined. Analytical methods used radiolabelled compound with quantitation via scintillation techniques, or unlabelled compound determined by UV-visible spectrophotometry. or reversed-phase HPLC.

When these experimentally determined permeability coefficients are compared across different laboratories, the effect of experimental conditions on the absolute value of these coefficients becomes apparent in the different numerical ranges that are reported for a similar set of test compounds (cf. Fig. 1 with [2,4]). However, the overall correlation with absorption in humans is conserved in these studies with the Caco-2 cell culture model, confirming its utility in obtaining useful predictive information on oral absorption. These results also emphasize the importance of determining permeability coefficients for a set of known compounds using the experimental model selected in a particular laboratory, prior to the use of the cell culture experimental model for predicting the absorption of unknown test compounds in vivo.

3. Representative applications

In the following section, applications that illustrate the use of the Caco-2 cell culture model will be described for the characterization of the endogenous peptide transport system in the gut epithelium, the use of the cell culture system to predict absorption for a chemical series of compounds, and finally a discussion on the use of the cell culture system in formulation development.

3.1. Endogenous transport systems: the peptide transporter(s)

These studies were conducted using the Caco-2 cell culture model with the following experimental goals: to study the transcellular transport system in the gut by directly employing a dipeptide substrate: to separate, functionally, uptake into the cells from transport across the monolayer: and to characterize the effect of representative cephalosporins on these processes. These experiments were performed at short

incubation times. so that kinetic parameters could be measured that would accurately characterize the transporter systems, and minimize the effects of non-saturable passive functions. The study conditions employed radiolabelled dipeptides as substrates for direct uptake and transport studies. and a series of dipeptides and cephalosporins in competition and inhibitor studies.

The Caco-2 cell culture system readily allowed for preliminary experiments that optimized the directionality for the transport of dipeptide substrates, the time course of both uptake and transport, and confirmation of the previously described acidic pH dependence of the transporter(s) [7- 131. Based on these preliminary data, all studies were performed at pH 6.1 in the apical to basolateral direction, and transport and uptake were measured after 3 min incubation at 37°C. The 3-min time point was chosen to allow enough transport across the monolayer so that measurable and reproducible experimental values could be obtained. During this time period. uptake into the cells was approaching saturation, but still in the linear portion of the saturable kinetic function. In contrast. the transport across the cells was in an early phase at this time, with only low levels present in the basolateral chamber. Therefore, the ability to accurately measure the levels of substrate present in the basolateral chamber of the transport unit strongly impacted on the experimental time points that were chosen. Further, it was established by HPLC analysis that loss of the peptide substrates to proteolysis in the cell system was in the range of S-IO% after 5 min at 37°C leaving greater than 90% of the substrate available for transport. Longer time periods resulted in significantly greater losses to proteolytic degradation (Bailey and Bryla, data not shown).

Results of these studies confirm the presence of at least two saturable carrier systems for the transport of dipeptides, and an apparently passive. non-saturable function. A first carrier system had high affinity (K,,‘s in the micromolar range) for the dipeptide substrate. while the second system exhibited lower substrate affinity (K,,,‘s in the millimolar range). The capacities of these systems were also different, as exhibited by

C.A. Bailey et al. I Advanced Drug Delivery Reviews 22 (1996) 85-103 93

the approximately five- to sevenfold higher Mich- aelis constant for V,,, in the low affinity system. In addition, the studies indicated that the cells transported significantly less substrate across the monolayer than was taken up into the cells. These data are summarized in Table 2. The data also indicate that the Caco-2 cells were able to switch from high affinity to low affinity carrier systems. and finally to non-saturable transport function as the concentration of substrate is progressively increased from micromolar to millimolar concentrations. This effect is depicted graphically in Fig. 3. Taken together, these studies provide evidence that there may be a family of transporters in the gut designed to absorb peptide and peptide-like substrates under different conditions. Further, the data indicate that passive transport can contribute significantly to overall transport at substrate concentrations in

Table 2 Kinetic parameters for the peptide carrier function deter-

mined in the Caco-2 model

Function K,(mmol) V_,,(nmol/min/mg)

High affinity carrier Uptake 0.13 0.77

Transport 0.15 0.09

Low affinity carrier Uptake 3.70 3.30

Transport 3.80 0.31

The dependence of initial rates of [“‘Iltyrosyl-glycine uptake

and transport was studied at pH 6.1 and 37°C at the 3-min

time point, with increasing amounts of tyrosyl-glycine sub-

strate. Kinetic parameters (apparent K, and V,,,) were

calculated by linear regression analysis of Eadie-Hofstee

plots (v vs. v/[S]), after subtracting out the non-saturable

portion of the transport function. The contribution of the

non-saturable portion of the transport function (K,) was

calculated from the slope of the straight line generated from

transport rates at higher substrate concentrations. All values

were normalized for protein concentration of the cell sample,

determined by bicinchoninic acid assay. A wash in ice cold

buffer containing fivefold molar excess of unlabelled tyrosyl-

glycine was used to stop the uptake/transport function, and

to correct for any adsorption or binding of the radiolabelled

substrate to the external membranes of the cell monolayer.

Uptake is defined as the amount of substrate taken up into

the cells in the monolayer growing on the filter, and transport

is defined as the substrate that is transported completely

across the monolayer barrier into the basolateral chamber of

the transport unit. Data supplied by C. Bailey and P. Bryla,

Hoffmann-La Roche Inc., Nutley, NJ.

100

80-

i 60-

2 l- 1 40-

E 8 20-

O-

5 15 25 SO 100 250 500 750 1000

Concentration (uM)

Fig. 3. Ability of transporter function to switch between

carrier systems to transport dipeptide substrate across mono-

layer. Total transport across the Caco-2 monolayer was

measured using increasing amounts of tyrosyl-glycine as

substrate, at concentrations up to 1 mM. Radiolabelled

substrate (“‘I) was used for analytical detection. The clear

bars represent the saturable portion of the transport function

and the non-saturable function in this substrate concentration

range is represented by the black bars. Initial transport rates

measured experimentally were separated into two compo-

nents, a saturable function and an apparently non-saturable

first-order process, which represents the switch over to the

second carrier system as substrate concentration is increased.

The initial rate of substrate transport can be expressed by the

following equation: v = {V,,,,,[S]/(K,, + [S])) + KJS], where [S] is the substrate concentration, v represents the

initial transport rate, K,,, is the Michaelis constant, and K, is

the apparent first-order rate constant for the nonsaturable

process. Data provided by C. Bailey and P. Bryla.

the millimolar range, even when an active transport mechanism is present. This is especially evident as concentrations rise above the levels of the K, for the low affinity dipeptide transporter.

Competition studies in the Caco-2 model were performed using a series of dipeptide substrates and cephalosporin molecules. Both the uptake and transport functions of a glycine-tyrosine substrate were inhibited by a series of dipeptide substrates with differing electrostatic charge and hydrogen bonding characteristics, indicating that the native uptake and transport systems will accept a variety of peptide substrates (Fig. 4). In contrast, when a series of cephalosporins was tested, inhibition constants indicated that these molecules are not efficient substrates for the high

94 C.A. Bailey et al. I Advunced Drug Delivery Reviews 22 (196,) 85-103

Cefaclor 0

la,,i-fl-t

Cl N \

S 2 H H

Cefuroxime Axetil H,C yy;

N \ y;;$

OL . 2 s

Cefuroxime Sodium

Fig. 4. Chemical structures of cephalosporin molecules re-

ferred to in text and Table 3.

affinity carrier system, as evidenced by inhibition constants that were an order of magnitude greater than the Michaelis affinity constants for the dipeptide substrate. However, these cephalosporins do possess varying abilities to inhibit the low affinity transport function, based on the observation that some of the inhibition constants are in the same range as the Km’s for the lower affinity dipeptide carrier system. These data are summarized in Table 3.

Unlike other models available for transport studies, the cell culture model is unique in that it lends itself readily to studies that separate the overall transport process into the individual uptake and efflux steps. For example, it could be experimentally determined in peptide transport studies that the uptake and transport functions in the epithelial cell exhibited differing sensitivities

Table 3

Inhibition constants for uptake and transport function mea-

sured in the Caco-2 model for a series of dipeptide substrates

and cephalosporin molecules

Competitor Uptake WI .,,p) Transport (K, .+,,p)

Gly-tyr

*rg-gly Asp-gly

Gly-gly

Cefaclor

Cefixime

Cefuroxime axetil

Cefuroxime sodium

0.11

0.30

0.45

0.73

9.7

4.1

2.3

5.5

0.09 0.16

0.18

0.24

13.2

7.3

4.5

40.9

The affinity of the uptake and transport carrier proteins in

the Caco-2 cell monolayer for a series of dipeptides and

cephalosporins was determined using [“‘I]tyrosyl-glycine in

the presence of the competing substrates. An apparent K, in

the presence of the competitor was determined by the Eadie-

Hofstee method as described in Table 2. The apparent K, for

the competing substrate was then calculated using the follow-

ing equation: v={~,,+~[S]/K,[I + ([I]lK,)]+[S]} + K,[S].

where [I] and K, represent the competitor concentration and

inhibition constant. respectively. The apparent Km (K,,, ,,,,,,) is

a function of [I] and K, based on the relationship K,,,( I + [f]/

K,): the affinity constant for the competing substrate is l/K,.

Thus, the lower the absolute value for K,, the greater the

affinity of the competing substrate for the carrier. K, ~,pp is

derived from the carrier-mediated component after correc-

tion for the nonsaturable uptake and transport. as described

in Table 2. Dipeptide substratc abbreviations are as follows:

ply. glycine: tyr, tyrosine; arg, arginine; asp. aspartic acid.

Data supplied by C. Bailey and P. Bryla, Hoffmann-La Roche

Inc.. Nutley. NJ.

to inhibition by different cephalosporins. These data suggest that there are different carrier molecules in the apical and basolateral membranes of the Caco-2 cell, and that the individual cephalosporins vary in their ability to recruit these carriers for transport across the gut epithelium. This is especially evident in the case of the cephalosporin cefuroxime sodium, which is available only as a parenteral formulation, due to a failure to be orally absorbed. Transport studies in the Caco-2 cell culture model indicate that this molecule efficiently enters the cells via the dipeptide uptake function, but cannot exit out across the basolateral membrane of the cells. In contrast, the orally active combination, cefuroxime axetyl, is able to compete with a dipeptide substrate for exit from the cell. This experimental observation that cefuroxime axetyl can trans-

C.A. Bailey et al. I Advanced Drug Delivery Reviews 22 (1996) 8_5-103 95

port across the Caco-2 monolayer is in good agreement with the ability of this cephalosporin to be orally absorbed, while cefuroxime sodium is not (Table 3).

Another advantage of the use of the Caco-2 cell culture model is exhibited by the ability to use pharmacological agents to probe the mech- anistics of uptake and transport functions at the molecular level. For example, it could be determined that both uptake and transport were sensitive to the protonophore carbonyl cyanide p-(trifluoromethoxy)phenyl-hydrazone (FCCP), confirming that hydrogen ions are actively involved in the dipeptide transport process. This is in good agreement with the acidic dependence of dipeptide transport described above. In contrast, inhibitors of the various cellular sodium transport systems, or the replacement of sodium ions with choline + ’ ions in the transport system had minimal effects on peptide transport, indicating that neither sodium ions, nor Na+/H’ exchange are the best descriptors of the co-transport process. Inhibition of transport by bumetanide, an inhibitor of the Na / K /2Cl co-transporter, sug- gested the possible involvement of this carrier system or family of ions was greater on the exit of dipeptides out of the cell than on the process of uptake from the apical surface. Other studies indicated that neither the uptake nor the trans-

port functions were greatly affected by ver- apamil, indicating that the multi-drug resistance transporter and/or calcium channels do not play a major role in the dipeptide transport system. Colchicine was employed to test for the role of the cytoskeleton in the peptide transport function, and results suggest that the cytoskeleton probably is not directly involved. It should be noted that all of the agents tested caused changes in the uptake and transport functions in the Caco-2 cell system. However, only a few were able to reduce the uptake/transport system to the level of the passive function. These data suggest that most of the pharmacologic agents tested may have been acting more generally on overall cellular function, rather than specifically on the peptide transport system. Notable excep- tions were the protonophore FCCP, and, to a lesser extent, bumetanide (Na/K/2Cl co-transport). Table 4 summarizes the use of pharmacological inhibitors to characterize the dipeptide transport system at the molecular level.

Basic molecular questions, such as the nature of the carriers in the apical and basolateral membrane domains of the epithelial cell, are also readily addressed in the cell culture model. This type of information is not accessible in the other in vitro transport models. In our laboratory, sensitivity to sulfhydryl reagents was employed

Table 4

Effect of pharmacologic agents on uptake and transport of dipeptide substrate by mature Caco-2 monolayers

Inhibitor Affected parameter Inhibitor cont. % Inhibition uptake % Inhibition transport

Ouabain Na/K ATPase (Na’) 1 mM 35.3 32.1

DMA NaiH antiporter (Na’ ) IO pM 11.1 ( + ) 10.3” Choline buffer Na ’ Complete substitution for all Na’ 37.3 6.4

Sodium azide Metabolic poison 0.5 mM 40.7 32.2

FCCP H’ 40 )LM 77,s 80.2 Valinomycin K’ 1 PM ( + ) 6.9” ( + ) 37.4” DIDS CIIHCO, ions 0.1 mM 8.6 23.6 NEM Sulfhydryl groups 5 mM 33.3 SO.8 MMS Sulthydryl groups I mM 79.9% @ 0.66 mM 72.9% @ 0.15 mM Colchicine Cytoskeleton 0.3 mM 34.6 43.1 Vcrapamil MDR, Ca ions 0.1 mM 33.1 51.4 Bumetanide NalKI2 Cl transport 0.1 mM 38.3 60.2

Mature Caco-2 monolayers were incubated in the presence or absence (control) of the indicated concentrations of pharmacologic

agents for 1.5 min at 37°C. The initial rates of [“‘I]tyrosyl glycine uptake and transport were then measured as described in the

legend to Table 2. Percent inhibition was calculated as the decrease in the amount of uptake or transport function measured

compared lo control values. in which cells were exposed to Hank’s buffer at pH 6.1. Data provided by C. Bailey and P. Bryla,

Hoffmann-La Roche, Nutley, NJ.

“Represents stimulation of indicated function, rather than inhibition.

to probe whether the carriers in the apical and basolateral membranes of the Caco-2 cell are the same protein. The permeable and non-specific sulfhydryl reagent, N-methyl maleimide (NEM), was used to ascertain whether the uptake and transport functions were sensitive to sulfhydryl inhibition in initial experiments. Having determined that the dipeptide function was sensitive to sulfhydryl inhibition, a second, impermeant reagent, methyl methanethiosulfonate (MMS), was employed to test for differential sensitivity in the apical and basolateral membrane domains of the cell. In these studies, the MMS was present either in the apical or basolateral chamber of the transport unit during the uptake and transport experiment. Different inhibition sensitivities from the vectorial application of increasing amounts of MMS would suggest that the transport protein(s) were not the same protein in the apical and basolateral membranes of the Caco-2 cells. Results of these studies indicate that the transport function is approximately fourfold more sensitive than the uptake function to inhibition by MMS (50% inhibition at 0.15 mM for transport vs. 0.66 mM for uptake), suggesting that the protein(s) responsible for transport out of the cell on the basolateral side are not the same as those responsible for uptake into the cell on the apical surface. This is in good agreement with the cephalosporin inhibition data described above for the cefuroxime sodium and cefuroxime axetyl molecules. in which each possessed differing abilities to enter and exit the epithelial cells during the transport process.

In summary, these studies exemplify the man- ner in which the Caco-2 tissue culture model lends itself readily to the study of endogenous transport systems at the cellular and molecular level. In addition to the ability to character& kinetic parameters of the system, it is possible to obtain mechanistic information on co-factors that may be involved in the transport and uptake functions, and the ability of drug molecules to efficiently use the endogenous system(s) to cross the epithelial barrier. These types of studies can be extended to the other carrier systems present in the gut. The information gained will extend our knowledge of endogenous transport systems. and also obtain information on the ability of

specitic drug molecules or classes of compounds to use these systems to cross endogenous epithelial barriers in the gut, and by extension, to be orally absorbed.

3.2. Permeability within a chemical series

The oral route of delivery is always the most desirable mode of administration for new pharmaceutical candidates, as well as for line exten- sions for previously marketed products. It represents the ‘gold standard’ for all drug delivery routes, and because of this preferred status, there is much research and development work focused in this area. A great deal of effort has been expended in searching for correlations between experimental systems in vitro and oral absorption in humans, that might be used as efficient predic- tors. These have included the small animal GI models, cell culture models such as the Caco-2 system. octanol-water partition coefficients, measures of hydrogen bonding and desolvation energies. immobilized artificial membranes, and re- tention time on reversed phase HPLC columns. In general, small animal GI studies and the Caco-2 tissue culture model have shown the best correlation with oral absorption in vivo. and are most commonly used in the industry at the present time, although other models are continu- ing to be studied.

The Caco-2 cell culture model allows for the rapid screening of a series of compounds, usually with less than 50 mg of compound. Indeed, it is possible to conduct a minimum transport experiment with from 5-10 mg of test drug substance, provided a sensitive analytical method is available for detecting transported compound in the hasolateral chamber of the transport unit. Data from Caco-2 transport studies of two separate chemical series follow. Both groups of compounds, representing a chemical series of enzyme inhibitors designed for different therapeutic areas, were at an early phase in the discovery program at the time of the screening for possible absorption characteristics. At this point in the development of a drug molecule, chemical syn- thesis of alternate structures is still a viable strategy to optimize both the activity at the target site and the absorption characteristics of

C.A. Bailey et al. I Advanced Drug Delivery Reviews 22 (1996) 85-103 91

the candidate molecule. Both of these challenges must be satisfied in order for the molecule to be developed into a marketable candidate.

A summary of physico-chemical properties, as well as the biological properties of permeability in the Caco-2 tissue culture model and bioavailability in a rat model, for the two groups of test compounds is shown in Table 5. Both groups contained eight different molecules of a chemical series, although not all measures were performed on all molecules in each series. It can be seen from the physico-chemical and biological data that there are similarities in molecular weight, permeability and bioavailability characteristics between the two molecular series. There are, however, major differences in solubility (Group II is more soluble as a class) and in octanol/ water partition coefficients. Here, Group I is represented by a narrower range of lipophilicities compared to the compounds in Group II.

The correlation(s) among permeability in the Caco-2 cell model, log D (octanol/water), and bioavailability are explored in Fig. 5A-D. It can be seen from these data that there is excellent correlation within each individual group between the Caco-2 permeability coefficients and the percent bioavailability measured in rats (cf. Fig. 5A and C).

When the apparent permeability in tissue culture is compared directly to the octanol/water partition coefficient, it can be seen that there is a good experimental fit for both Groups I and II,

Table 5

Physico-chemical characteristics of compounds in the chemi-

cal series identified in the text as Groups I and II

Group I Group II

Molecular weight 450-650 300-500

Solubility (mgiml) 0.003-0.327 0.8- > 100

Partition coefficient“ 1.20-2.45 (()0.35-( +)l.s

Apparent permeabilityh 0.001-9.1 0.0001-10.9

Bioavailability’ S-23% 1.3-31%

Apparent permeability constant data provided by C. Bailey.

P. Bryla and E. Spence, Hoffmann-La Roche Inc.. Nutley, NJ.

USA. Physical constants and bioavailability data courtesy of

Medicinal Chemistry and Pharmacology Divisions of Hof-

fman-La Roche Inc., Welwyn Garden, UK.

“As log D, determined in octanol/water.

hem/s, X 10mh. determined in Caco-2 cell monolayers.

‘Determined in rats.

when each group of compounds is considered individually (cf. Fig. 5B and D). However, when Group I is now directly compared to Group II, it is apparent that while the shape of the two curves is similar, there is a major difference in the y-intercept for the two curves. That is, the entire curve for the compounds in Group II is shifted down significantly on the Y-axis of the plot, which represents the log D (octanol/water) values. These data suggest that there are parameters which are impacting on the permeability and absorption of the test compounds (e.g. solubility, solvation energies, hydrogen bonding, hydropho- bicity), which are not impacting on the octanol/ water partition coefficient measurement, and that these factors play a role in the transport and absorption process.

This observation is readily apparent when the combined data from both Groups I and II are plotted on the same graph (Fig. 6A and B) In Fig. 6A, the excellent correlation between bioavailability in vivo and permeability in the Caco- 2 tissue culture model is again apparent. One could predict from these data that permeabilities in the range of 2-10 x 10eh cm/s might represent viable candidates for oral delivery for compounds from either both Groups I and/or II, with higher values representing greater absorption. If these data were used to predict what partition properties would be desirable to meet the needs for transport or absorption, the data would predict that a log D greater than 1.5 would be needed for the compounds in Group I, while a value of greater than 0.2 would be needed for the compounds in Group II. Thus, a general correlation between octanol/water partition properties and either transport or bioavailability is tenuous at best. The lack of correlation between bioavailability and log D (octanollwater) data is readily seen in a plot of the combined data from Groups I and II, which is shown in Fig. 6B.

In summary, the studies above that use the Caco-2 tissue culture model to measure permeabilities, indicate that the apparent permeability as measured in the tissue culture system is a reliable predictor of oral absorption for a broad cross section of compounds. Further, these data indicate that the octanol/water partition coefficient is not an efficient model for the

50

40

z E 30

._ z b . 20

9

10

0

I I 3 I I

5C 2.5 - 5D

.

.

.

Fig. 5. Correlation between pcrmcahility, hioavailabilitv and partition coefticient for compounds comprising Groups I and II.

Apparent permeability (cm/s. X 10 “) in the Caco-2 model was measured for the test compounds in Groups I and II at 37°C and

pH 7.4. Transport was measured in a 0.33 cm’ filter unit: cells wcrc grown a minimum of 3 weeks in the transport unit prior to the

experiment. All data rcprcscnt the avcragc ol a minimum of triplicate determinations: standard errors were less that 10%.

Permeability data provided by C. Bailey, P. Bryla and E. Spence. Hoffmann-La Rocbe Inc.. Nutley, NJ. Partition coefficients

(octanoliwater) and % bioavailability (in the rat) were determined by Medicinal Chemistry and Pharmacology Departments.

Hoffmann La-Roche Inc.. Welwyn Garden. IJK. (A) Group I: Correlation between apparent permeability measured in the Caco-2

model and bioavailability. (B) Group I: Correlation bctwccn partition cocflicicnt and apparent permeability measured in the

Caco-2 model. (C) Group II: Correlation between apparent permeability mcasurcd in the Caco-2 model and bioavailability. (D)

Group II: Correlation between partition coefficient and apparent permeability mcasurcd in the (‘nco-7 model.

prediction of oral absorption; there is signiti- cantly lower correlation between log D values and bioavailability overall. It should be noted

that the compounds described by these data are representative of test compounds which exhibit relatively low oral absorption characteristics. which were being characterized in an effort to increase oral absorption strategies in their development profile. Thus. they all fall on the lower portion of the sigmoid curve which was described earlier (Fig. 2). and are not representative of the entire oral absorption range.

A further advantage of the permeability mea-

surement in the tissue culture model is that only small amounts of test compound are required,

the studies can be performed with non-radio labelled compound, and the studies can take place prior to the availability of plasma assays. Although the data were not presented here, we have also observed in our laboratory that the propensity for some metabolism and preliminary solution stability of the drug candidates can be obtained from the studies in the Caco-2 model relatively early in the discovery and development process. However, as noted above, the tissue culture model. because of its reductionist nature.

C.A. Bailey et al. I Advanced Drug Delivery Reviews 22 (1996) 85- 103 99

50

6A

10

l . l l *

0. 1 -0.5 0 0.5 1 1.5 2 2.5

log D (octanol/wster)

Fig. 6. Correlation between permeability, bioavailability and

partition coefficient for the combined data for compounds

from Groups I and II. (A) Correlation between permeability

measured in the Caco-2 model and bioavailability for the

pool of compounds in Groups I and II. (B) Correlation

between partition coefficient determined in octanoliwater

and bioavailabihty for the pool of compounds in Groups I

and II. See Fig. 5 for details of experimental conditions.

does not measure factors such as clearance, stability and/or toxicity, all of which may have an impact in vivo for a given test molecule.

3.3. Use of the tissue culture model for formulation development

It is not unusual during development in the pharmaceutical industry to be faced with a drug candidate which must be delivered by the oral route, but which has little or no oral activity as a neat molecule. In this case, it is incumbent on the formulator to develop a strategy that will result in a formulation that will allow the test molecule to be absorbed via the GI tract, from a formulation which exhibits good manufacturability and stability, and is at the same time safe and cost effective. The availability of a high throughput

screen that allows for the rapid screening of a large number of permutations in formulation candidates is a distinct advantage in dealing with this often formidable challenge. The Caco-2 tissue culture model can be used for this purpose, once a few simple caveats are taken into consid- eration.

It should be noted that the strategies employed by the formulator to obtain oral bioavailability via formulation manipulation generally occur in a developmental time frame at which resynthesis of a new structure, or a new salt form, are no longer viable alternatives for various reasons. For example, the test molecule may have simply resisted any alterations in structure that did not result in drastic loss of activity, or alternately may not have lent itself well to the formation of a soluble, stable, and acceptable salt form. Ideal- ly, it should be routine that the absorption of test molecules be checked very early in the discovery process. This policy allows either for an informed choice of the best all around candidate to be made, and/or for synthetic changes to be made to difficult molecules whenever possible. As described above, the Caco-2 model is especially efficient in this kind of study. However, there are cases where an entire class of drug candidates resists changes that would increase solubility, and by extension, oral absorption, due to losses in activity at the target site that are unacceptable to the program. In these cases, a formulation strategy may be able to compensate for the lack of intrinsic absorption parameters inherent in the molecule. The Caco-2 tissue culture model is useful for these studies, because a large number of permutations in formulation can be checked for ability to enhance or increase the permeability of the drug candidate across the epithelial barrier. Caution must be used, however, in inter- preting the results of the permeability studies because of the simplistic nature of the cell culture model compared to the situation in vivo. For example, dissolution and dilution in the acidic milieu of the stomach are not addressed by the cell culture model, and the model therefore cannot be used to measure the effects of this step on the overall absorption of the drug molecule. In particular, the effects of dilution play a key role in the interpretation of data from the tissue

100 C.A. Builey et al. I Advanced Drug Delivery Reviews 22 (1996) 8.5- IO.3

culture model, because the dilution in vivo is difficult to control or to be predicted, being affected by such complex parameters as the fed state, activity levels of the subject, chronobiolo- gy, etc. Nevertheless, it is possible to obtain useful information from the tissue culture model to feed back to the formulator, primarily as it applies to the absorption step across the epithelial barrier.

We have studied the effect of the commercially available formulations on the orally available cephalosporins using the Caco-2 epithelial barrier model. Our data indicate that a sampling of the formulations available on the market cause anywhere from a four- to eightfold increase in transport across the Caco-2 monolayer, when compared to the transport of the unformulated cephalosporins (i.e. compounds dissolved in buffer and then presented to the cells for transport). The cephalosporin formulations have an effect on the epithelial cell barrier manifested as a decrease in trans-epithelial resistance, and a change in permeability of the barrier which allows the transient passage of small molecular weight markers such as mannitol. Larger molecule weight markers, such as PEG-4000 and dextrans of molecular weight 20 000 and 70 000 Da are excluded from passage by the effects of the formulations on the monolayer. There was no measurable effect on the viability of the cells exposed to the formulations compared to control cells, as measured by neutral red assay. These studies characterized and quantified experimental evidence in vitro for the positive role that formulation can play in the oral absorption of drug candidates [33].

In other studies, we have employed the cell culture model to screen excipients to be used in a self-emulsifying drug delivery system (SEDDS). While these studies allowed for the rapid screening of approximately 80 permutations in formulation, using only a small amount of active drug substance and small experimental test formulations, it must be noted that the results should be interpreted with caution. It is useful to study the effects of these formulations as a function of dilution, presenting at least two different dilu- tions of the formulation to the cells for permeability measurements as a function of time.

Our routine procedure is to test the formulations at a 1 / 10 and a l/50 dilution in transport buffer. In this way, the effects of dilution on the ability of formulation excipients to impact on absorption. or to maintain the test molecule in solution can be ascertained. If the transport function is strongly dependent on the concentration of ex- cipient, then a recommendation is made for formulation as an enteric coated capsule, allowing for bypass of the dilution that may occur in the stomach.

Other factors that must be considered in this area are possible effects of the excipients on biological conditions or functions which are not represented by the Caco-2 epithelial cell model. Thus, excipients that are designed to route compounds to biliary absorption or to the lymphatics might not be seen as effective in the Caco-2 model, but may in fact be successful in the in vivo situation. It is therefore strongly recom- mended that studies in this area be conducted with full understanding of the excipients involved. and the potential impact that may be obtained in vivo, particularly if the effect is not targeted to the transport step across the gut epithelium.

4. Concluding remarks

In the current atmosphere of shorter drug development times, with the focus on innovative and readily bioavailable therapeutics, tools and strategies that lead to faster development and efficient oral delivery of candidates will have a major impact on the success of those candidates. Oral bioavailability is extremely important to the success of many of these new drug candidates. The more the processes that impact on oral absorption are understood, the better and safer will be the pharmaceutical products that result. Further, there is a need for rapid and efficient in vitro systems that are indeed predictive of the situation in vivo, as those systems will impact positively on shortening the time to market.

The Caco-2 epithelial cell culture model in fact satisfies both of these criteria. It is an excellent model for probing the absorption step at the cellular and molecular level. Increasing our

C.A. Bailey et al. I Advanced Drug Delivery Reviews 22 (lYY6) 8.5-103 101

knowledge in this area will most certainly lead to more effective and efficient new drug candidates that can be delivered via the oral route. This in vitro system embodies the all important characteristics of simplicity and ease of manipulation, allowing for the study of molecular and cellular questions. Studies of this nature should be useful in piecing together more of the ‘big picture’ that describes absorption across the epithelial barrier in the gut. Continued exploration of the endogenous transport systems, the routes across the epithelial barrier (e.g. diffusion, endocytosis, transcytosis) and the role and activity of the tight junctions in the paracellular transport pathway are readily addressed in the Caco-2 model. By employing models such as this in more mechanistic studies, we may come to understand better the function of the tight junctions, and begin to probe some of the important questions on the use and safety of the so called enhancer molecules. More understanding in this area might also eventually be applied to better delivery of peptide and protein molecules across other endogenous biological barriers, such as the pulmonary and nasal systems.

From a more pragmatic point of view, the Caco-2 tissue culture model can serve as an important part of the drug discovery support function in pre-clinical pharmaceutical development. Apparent permeability values obtained in the Caco-2 model should be a key part of the pre-formulation package, meeting the needs of biological predictive data that complement the physico-chemical profile which is collected on new drug candidates early in the discoverylde- velopment process. The correlation between permeability across the Caco-2 monolayer and oral absorption in vivo is now well established for a large number of compounds, in multiple laboratories. It is the simplest and fastest in vitro method for predictive permeability studies. In addition, the analytical and test compound needs are minimized in this tissue culture model. The other major predictive model in this arena is the small animal gut permeability model. As described above, this model requires the maintenance of a substantial animal colony and the availability of radiolabelled compound, or of larger amounts of cold compound and a plasma assay for the drug substance.

It is clear that the cellular models are rapidly increasing our knowledge of the endogenous processes by allowing a large number of studies to take place in vitro. Parallel to the large increase in our knowledge base on penetration across endogenous biological barriers which has been made possible by tissue culture models, inroads are being made in the understanding of liver enzyme metabolism, cellular toxicity studies, and some of the functions of the lymphatic system. It is envisioned that in the future a ‘package’ of in vitro cell culture models will be available to obtain the maximum amount of information on each new drug candidate in vitro, early in the discovery/development process. At the same time, the move toward cellular models will reduce the number of experimental animals needed for drug discovery and development. This approach should make the entire process more efficient, effective and rapid, which in turn should impact positively on the delivery of new therapeutics to the patient.

Acknowledgments

The authors would like to acknowledge the continued visionary support of Dr S. Udenfriend regarding the use of tissue culture models in the pharmaceutical industry. The ready and open sharing of information by Dr Ken Audus on the development of new approaches to the use of tissue culture models, such as Caco-2, is grateful- ly appreciated. Finally, we would like to thank Drs L. Bailey and R. Tarantino for their helpful discussions and critical reading of the document.

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the use of the intestinal epithelial cell culture model, caco-2, in pharmaceutical development

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