Human airway epithelial cell lines for in vitro drug transport and metabolism studies

1461-5347/00/$ – see front matter ©2000 Elsevier Science Ltd. All rights reserved. PII: S1461-5347(99)00231-X

▼ Inhalation to the lung is an increasingly im-portant route for drug delivery. Pharmaceuticalaerosols are a well established means of localizeddrug delivery for the treatment of lung diseases.The most frequently used aerosols are smallamounts of bronchodilator or steroid delivereddirectly to the lung to treat obstructive airwaydiseases. Direct targeting of the lung results in animmediate onset of drug action and reduced sideeffects. The permeability of the lung to a varietyof drugs, including peptides and proteins, hasalso been recognized for some time1,2. Althoughthis review will focus on the airway rather thanthe alveolar epithelium, recent improvements indelivery device technology have created muchoptimism over using inhalation to the deep lungfor the delivery of drugs with poor oral absorp-tion to the systemic circulation3,4. Gene therapyis another rapidly emerging area, and several in-herited and acquired lung diseases are potentiallytreatable through the delivery of therapeuticgenes to the respiratory epithelium5.

Despite the popularity of inhalation as a drugdelivery route, assessing the fate of inhaled com-pounds is difficult because of the inaccessibility,delicate nature and complex structure of the lung.The interpretation of results obtained in animaland tissue models is complicated by inter-speciesvariation, imprecise delivery of drug to the lungand concerns over preparation viability. The useof epithelial cell culture models has many advan-tages (Box 1). In particular, airway cell lines aremore convenient models in which to assess ab-sorption mechanisms, compared with intact lungmodels which are not usually capable of discern-ing such properties. The value of epithelial cellculture is best illustrated by reference to the wellcharacterized Caco-2 cell line, the foremost cellculture model of intestinal drug absorption6,7.Caco-2 cell monolayers are used to study drugtransport mechanisms, assess absorption en-hancement strategies, and predict oral bioavail-ability8–11. Caco-2 monolayers are also used inhigh throughput screening programmes, and asummary of their applications illustrates the valueof cell culture models (Box 2). The Caco-2 cellculture model is widely used to generate data forregulatory purposes, and this has recently led theUnited States Pharmacopoeial Division of Stan-dards Development to attempt to introducestandard methods for drug transport experimentsutilizing Caco-2 cells7.

Compared with the development of intestinalcell culture drug-absorption models, interest inthe use of cell lines to model the respiratory epi-thelium is comparatively recent. The respiratorytract is usually considered as two distinct regions;the central conducting airways and peripheral al-veolar regions (Table 1). Deposition of pharma-ceutical aerosols occurs primarily via impactionat airway bifurcations, gravitational sedimen-tation within the airway tubules and diffusion

Human airway epithelial cell lines forin vitro drug transport and metabolismstudies Ben Forbes

Ben ForbesDepartment of PharmacyFranklin-Wilkins Building

King’s College London150 Stamford Street

LondonUK SE1 8WA

tel: +44 20 7848 4823fax: +44 20 7848 4800

e-mail: [email protected]

reviews research focus

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PSTT Vol. 3, No. 1 January 2000

The pharmaceutical industry relies on appropriate in vitro models for

the evaluation of drug absorption and metabolism. Despite increas-

ing interest in drug delivery via the lung, there is currently no widely

accepted cell culture model of the airway epithelium. This review

considers the airway epithelium, the culture of airway epithelial cells

and the need for cell lines which can model the airway epithelium.

Three of the most promising human bronchial cell lines,

16HBE14o2, Calu-3 and BEAS-2B, are reviewed, with emphasis on

their recent application for the study of drug transport, drug metab-

olism and gene delivery. Current limitations and future directions

for the development of these cell lines as models of the airway

epithelium are discussed.

within the alveoli. The airway epithelium is pseudostratified,features leakier tight junctions and a smaller surface area thanthe alveolar epithelium, and is protected by a mucociliary clear-ance mechanism.The alveolar epithelium is extremely thin andhas an extensive surface area which is patrolled by alveolarmacrophages. In 1998, more than 20% of the drug deliveryproducts sold worldwide were aimed at the central regions ofthe lung, for the treatment of asthma, chronic obstructive pul-monary disease and other bronchial-related disease.

Pharmaceuticals in development for delivery to the periph-eral lung are primarily for systemic absorption rather than localdelivery.Although delivery to the deep lung is technically morechallenging than delivery to the airways, the alveolar regionsare an attractive site for absorption into the systemic circu-lation, and may present the opportunity to achieve prolongedresidence times using sophisticated aerosol formulations12. Pri-mary cultures of type II alveolar epithelial cells, which formtype I cell-like monolayers in culture, are the most widely usedcell model for the study of drug transport across the alveolarepithelium. The A549 cell line exhibits many features of al-veolar type II cells, and has been used to study alveolar drug ab-sorption13 and metabolism14. However, there are questions overthe ability of A549 cells to form functional tight junctions15

and to detect chemically induced alveolar toxicity16. The suit-ability of A549 cells as a drug absorption model is also debat-

able because – despite the greater number of type II cells pres-ent in the alveoli – approximately 95% of the alveolar epithelialsurface area is composed of type I alveolar epithelial cells.Thereis currently no satisfactory human cell line to model the al-veolar epithelium, and this region of the lung will not be con-sidered further in this review.

The major site of deposition in the lung from current phar-maceutical aerosol devices is the airways where the epitheliumis both the principal barrier to drug absorption and the firstcellular metabolic barrier encountered by inhaled compounds.To usefully predict the fate of compounds delivered to the lung,a model of the airway epithelium should reflect the drug trans-port characteristics and metabolic activity encountered in vivo.Airway cell lines derived from human airway epithelium arebeginning to find use as drug transport models17–22.Three air-way-derived cell lines in particular have shown promise as invitro models of the airway epithelium; 16HBE14o2 and Calu-3cell lines as absorption models and the BEAS-2B cell line tostudy drug metabolism. The origins of these cell lines, theirproperties and current use in drug delivery research are re-viewed in this article.The transport and metabolism of salbuta-mol and formoterol by these bronchial epithelial cell lines areused to illustrate the utility of these cell lines as tools for evalu-ating the fate of inhaled compounds.

Airway epitheliumThe human airway epithelium consists of a pseudostratified layercomposed of at least six distinct epithelial cell types: ciliatedcells, mucous goblet cells, Clara cells, serous cells, basal cells, anddense core-granulated cells23,24.The cellular composition of the

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PSTT Vol. 3, No. 1 January 2000 reviews research focus

Box 1. Summary of the advantages andlimitations of cell culture models for in vitrodrug transport and metabolism studies

Advantages• Small amounts of compounds are required for experiments• Easier and more economical than in vivo experiments and

reduces animal usage• Rapid, with a high throughput capacity• Provides mechanistic information about epithelial transport• Environmental conditions, such as temperature and pH, can

be controlled• Drug analysis is simplified by the use of aqueous buffer

solutions

Limitations• Models are usually based on a single cell type and the cells

are monoclonal in nature• Tight junctions may not be representative of the target

tissue• Non-representative cell cycles affect transport and

metabolism mechanisms• Cell lines derived from adenocarcinomas often have

untypical phenotypes

Box 2. Summary of the applications of cellculture modelsa in the design and development of drugs

• Estimation of permeability characteristics of drugcandidates

• Deduction of drug transport pathways• Determination of structure–activity relationships for active

transport and efflux processes• Determination of optimal physicochemical characteristics

for passive absorption• Assessment of chemical strategies for absorption

enhancement• Elucidation of drug metabolism• Rapid assessment of potential toxic effects of drug

candidates or formulations

aCell culture models provide systems in which the biochemical and physical

barriers posed by absorptive epithelia can be studied.

epithelium varies substantially between species and at differentlevels of the lung between the trachea and the terminal bron-chioles23. Ciliated cells, mucous goblet cells and Clara cellsmake up almost the entire surface of the epithelium. Basal cellsand dense core-granulated cells do not form part of the epi-thelial surface under normal conditions, and the other celltypes, including serous cells, represent ,5% of cells24.

The human airway epithelial surface principally comprisesciliated cells, which are the most abundant cell at all levels ofthe airways24,25. In the higher airways, the ciliated cells are interspersed by secretory cells, mainly mucus-secreting gobletcells.At lower levels the ciliated cells are interspersed mainly byClara cells. Considering this distribution of epithelial cells, theciliated cell is clearly the most appropriate single cell-type formodelling of the airway epithelium. Diagrammatic represen-tations of the typical epithelium in the higher airways, at the

bronchiolar level, and in the alveolar region are illustrated inFig. 1.

Primary cultures of mixed populations of human airway epi-thelial cells provide the closest in vitro representation of the air-way epithelium. However, the lack of availability of normalhuman airway tissue, the limited amount of cells generated byprimary cell culture, and donor variation are major limitations.Despite the optimization of culture conditions, the mucociliarydifferentiation of primary cultures becomes significantly im-paired after two to three passages26. Major factors that promotethe differentiation of airway epithelial cells in culture are theextracellular matrix (such as collagen gel), the composition of

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PSTT Vol. 3, No. 1 January 2000reviews research focus

Table 1. The airways and alveolar region of the lung

Airways Alveolar region

Tracheobronchial tubes Acinus

Structure ofthe lung

Airway generationis shown in brackets

Primary Transport of air to the Gas exchangefunction gas-exchange regions

Mechanisms Impaction and Sedimentation andof aerosol sedimentation diffusiondeposition (particles 3–10 mm) (particles <3 mm)

Epithelium Pseudostratified, ciliated, Squamous epitheliummucus-secreting epithelium

The lung is usually considered as two distinct regions, the airways (central lung) and the

alveolar regions (peripheral or deep lung). The anatomical and histological differences

between the regions are related to their physiological function. These features determine

the deposition and absorption of inhaled drugs. In the classic model of the lung, the

airways are regarded as 16 generations of serial dichotomous airway branchings and the

alveolar regions are regarded as generation 17–23 [Weibel, E.R. (1963) Morphometry of

the human lung, Springer Verlag, Berlin, Germany].

Trachea (0)

Small bronchi and bronchioles(4–16)

Large bronchi(1–3)

Respiratorybronchioles(17–19)

Alveolar ducts(20–22)

Alveolar sacs(23)

Figure 1. Typical lung epithelia showing the major cell types. (a) Thebronchial epithelium showing the pseudostratified nature of thecolumnar epithelium, principally comprised of ciliated cells (c),interspersed with goblet cells (g), and the basal cells (b). (b) Thebronchiolar epithelium showing the cuboidal nature of the epithelium,principally comprised of ciliated cells (c), and interspersed with Claracells (cl). (c) The alveolar epithelium showing the squamous nature ofthe epithelium, comprised of the extremely thin type I cell (I), whichaccounts for approximately 95% of the epithelial surface, and thecuboidal type II cell (II).

(a)

(b)

(c)

I II

c

c

b

g

cl

Pharmaceutical Science and Technology Today

the growth medium (such as inclusion of retinoic acid) andculture at an air–liquid interface24,27,28.

Human bronchial epithelial cell linesCell lines have the advantage of providing a much more con-venient, reproducible model than primary cell cultures. Earlystudies concentrated on the ability of cell lines to model thephysiological processes of the airway epithelium, particularlyion transport, and characterization of their cell biology27.Therehas also been considerable interest in their use for pathophysio-logical studies, such as the response of the epithelium to injuryand inflammatory mediators. In the 1990s the potential ofhuman bronchial epithelial cell lines for use as drug absorptionmodels was recognized.The ability of cells to form tight junc-tions is critical to their use as a drug absorption model.16HBE14o2 and Calu-3 cell lines both form polarized cell lay-ers and have been identified as two of the better differentiatedcell lines24, with potential as drug absorption models20.Pulmonary epithelial cell lines were recently compared to estab-lish which could be used to study the effects of house dust-miteproteinases on epithelial permeability15. 16HBE14o2 and Calu-3cells were shown to express the proteins of the major intercellu-lar junctions (functional tight junctions, desmosomes and zonu-lae adherentes), but A549 cells were functionally deficient intight junctions. BEAS-2B cell lines do not form tight junctionsas readily as 16HBE14o2 and Calu-3 cells. Therefore, despitebeing one of the most widely used cell lines for the study of cellbiology, pathologic processes and drug metabolism, BEAS-2Bcells have limited potential in modelling drug absorption.

The 16HBE14o2 cell line was developed by transformationof cultured bronchial-surface epithelial cells from a one-year-old male heart–lung patient29.The Department of Pharmacy atKing’s College London obtained the cells as a gift from DieterGruenert at the Cardiovascular Research Institute at Universityof California, San Francisco, USA. When grown on collagen-coated supports at an air–liquid interface, the cells retain im-portant properties of differentiated airway epithelial cells,including the formation of tight junctions, regulated ion trans-port and morphological features, including apical microvilliand cilia29. Freeze-fracture electron microscopy of cultured16HBE14o2 cells has revealed extensive and well-formed tightjunctional belts (Fig. 2)30.The cell line was developed to studythe chloride channel activity of the cystic fibrosis transmem-brane conductance regulator (CFTR) in normal airway epi-thelial cells29,31,32. Recently, polarized 16HBE14o2 cell layershave been used to study drug transport18,19,21 and gene deliv-ery33–35.

The Calu-3 cell line is derived from a bronchial adenocarci-noma in a 25-year-old Caucasian male. Calu-3 cells are availablefrom the American Type Culture Collection (ATCC) for research

purposes, but commercial interests must be negotiated withMemorial Sloan-Kettering Cancer Center in New York, USA.CFTR levels are higher in Calu-3 cells than in 16HBE14o2

cells31, although CFTR is the major chloride ion channel inboth cell lines and is similarly regulated32. Calu-3 cells have alsobeen reported to produce much greater amounts of secretorycomponent than 16HBE14o2 cells36.These features, plus otherproperties of the Calu-3 cell, are consistent with those of theserous cell of the tracheobronchial gland37, although the cellsalso contain secretory granules24 and express mucus genesmore typical of goblet cells or mucous gland cells24,38,39. Calu-3 and 16HBE14o2 cells have been used to study the barrierfunction of the airway epithelium following radiation damage,using bovine serum albumin, sodium fluorescein and vitaminB12 as markers of epithelial integrity40. Polarized Calu-3 layerswith transepithelial electrical resistance (TER) .300 Ohmscm2 have been used to study drug transport17,22,41.

The BEAS-2B cell line was derived from normal human epi-thelial cells immortalized using the adenovirus 12-simian virus40 hybrid virus42, and is available from the ATCC provided usagedoes not infringe US patent claim US4885238A (immortalizedhuman bronchial epithelial and mesothelial cell lines).The cellshave been popular in studies of airway epithelial cell structureand function, including phenotyping and cytokine regu-lation43,44, regulation of glucocorticoid receptors45 and responseto challenges such as tobacco smoke46, particles47 and hyper-oxia48,49. However, it is not easy to generate TER greater than100 Ohms cm2 using BEAS-2B cells of intermediate passage50,51.Higher TER has been induced using high calcium culturemedium, but only using cells of higher passage (passage 84 to124)51. BEAS-2B cells have been used to study the expressionand activity of drug metabolizing enzymes52,53.

Drug transportThe major requirement of an airway drug absorption model isthat it accurately represents the barrier properties of thebronchial epithelium in vivo for both passively absorbed and ac-tively transported compounds. Ultimately, the model shouldbe capable of predicting drug transport across the respiratoryepithelium and bioavailability from the lung. Cell culture drugabsorption models of the airway are based on epithelial cellscultured as confluent cell layers grown on semi-permeable cellculture supports. A variety of commercial and customized dif-fusion chamber systems have been used to accommodate thecell layers, but all operate on the principle that the polarizedcells separate an apical chamber from a basolateral chamber(Fig. 3). Drug compound is applied to the donor chamber (ei-ther the apical or basolateral chamber) and the appearance ofdrug is measured in the contralateral receiver chamber. The‘tightness’ of the cell layer is determined by the tight junctions

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between cells, and can be assessed by measurement of TER orthe permeability of compounds which are transported acrossthe cell layers exclusively by the paracellular route. The trans-port of compounds across cell culture models is expressed asthe percentage transported (%/cm2/h) or apparent permeabil-ity coefficient (Papp). Papp (nm sec21) is calculated using Papp 5 (dq/dt)/(A.Co), where dq/dt is the linear transport rateof the compound,A is the surface area of the cell layer and Co isthe initial compound concentration in the donor chamber.

Calu-3 cells with TER of 300–400 Ohms cm2 have been usedas models of the bronchial epithelium for the study of drugtransport. The first reported use of Calu-3 cells as a drug ab-sorption model in a diffusion chamber system was a study of

diltiazem and insulin transport41. Diltiazem transport was simi-lar to the good permeability observed in Caco-2 monolayers,whereas insulin transport was low. In a more detailed report,the transport of ciprofloxacin has been shown to be primarilytranscellular and dominated by simple diffusion-solubilityprocesses17.Transepithelial transport of mannitol, ciprofloxacin,digoxin and vinblastine was proportional to log D in Calu-3cells and there was no net secretion of any compound in theapical to basolateral or basolateral to apical direction17.

The transport of salbutamol and formoterol across16HBE14o2 cells illustrates the value of cell lines for determin-ing transport mechanisms and pathways. Equivalent apical tobasolateral and basolateral to apical transport rates point to passive

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Figure 2. Examples of tight junctional belts found in 16HBE14o2 cells visualized by freeze-fracture electron microscopy. Samples were rapidly frozen inliquid nitrogen slush, and platinum or carbon-shadowed replicas were produced. (a) An extensive tight junctional belt is shown ranging from approximatelyeight-junctional elements (right side of micrograph) to three-junctional elements (left side of micrograph). P-face fracture view 3 40,000 (M 5 microvilli).(b) and (c) Further examples of tight junctional belts. Predominantly P-face fractures 3 40,000 (M 5 microvilli). Micrographs kindly supplied by RobGodfrey, Lung Pathology Unit, Royal Brompton Hospital, UK.

diffusional mechanisms of transport for these compounds. Fur-ther analysis of apical to basolateral transport allows the pathwayof absorption to be elucidated18. The flux of salbutamol acrossindividual 16HBE14o2 cell layers correlates strongly with fluxof the paracellular marker mannitol, and the near-zero interceptcalculated by linear regression suggests exclusive paracellulartransport (Fig. 4a). The permeability of formoterol is lessstrongly correlated with the permeability of mannitol, and thepositive y-intercept calculated by linear regression indicates atranscellular component to formoterol transport (Fig. 4b).

The effect of formulation on drug absorption is well demon-strated by the transport of salbutamol and formoterol across16HBE14o2 monolayers (Fig. 5)19.The transport of salbutamol,which is almost fully ionized between pH 6 and 8, is not af-fected by altering the pH in the apical chamber. In terms of for-moterol, as the proportion of the unionized species increasesfrom 3% at pH 6 to 69% at pH 8, the extent of transcellulartransport increases and the Papp of formoterol is positively cor-related with the pH in the apical chamber.The threefold differ-ence in Papp produced by a small difference in pH demonstratesthe potential importance of formulation on drug absorption.The use of formulation approaches has been proposed to opti-mize the delivery of macromolecules and modify the pharma-cokinetic profiles of drugs delivered to the lung54.The small vol-ume of fluid lining the airways makes the lung susceptible tohigh localized concentrations of deposited aerosol formulations.The effects of delivery systems on drug absorption and res-piratory epithelial integrity are likely to become an importantissue, and the availability of a well characterized model in whichto assess airway epithelial permeability will be valuable.

It has been shown in Caco-2 cell cultures that the expressionof transporters (including efflux mechanisms) is important ifcultured monolayers are to accurately model the epithelium invivo55. Formoterol is subject to an efflux mechanism in Caco-2monolayers, but not in 16HBE14o2 cells19, and ciprofloxacin,

23


Figure 3. The diffusion chamber system for drug transport studies.Polarized bronchial epithelial cells are cultured at an air–liquidinterface on a semi-permeable support. For transport experiments,drug solution is added to the donor chamber (apical or basolateral) andthe appearance of drug is monitored in the receptor chamber.

Bronchial cells

Semi-permeable support

Culture mediumBasolateral

Apical


Figure 4. Co-transport across the 16HBE14o2 drug absorption modelof the fluid-phase marker mannitol 2 mM and (a) salbutamol 100 mM,(b) formoterol 100 mM. The apparent permeability (Papp) of thecompounds across individual cell layers was compared. Salbutamolpermeability showed a strong correlation with the permeability ofmannitol, and a zero-intercept following regression analysis indicatesexclusive paracellular transport. The weaker correlation of formoterolpermeability with mannitol permeability and the positive interceptfollowing regression analysis indicates both paracellular andtranscellular components to the transport of formoterol.

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digoxin and vinblastine are effluxed by Caco-2 cells but notCalu-3 cells17.There is currently limited information about theexpression of transporters in the airway epithelium when com-pared with the much better characterized intestinal epithelium.Although non-specific epithelial models have shown reasonablecorrelation with human oral bioavailability, such asMadin–Darby Canine Kidney cells to model intestinal absorp-tion56, the use of organ-specific human cell culture models re-mains desirable for many applications.

Drug metabolismIn order to predict the metabolic fate of a xenobiotic deliveredto the lung, it is important to consider the region of the res-piratory tract to which it will be targeted.The regional and cel-lular location of enzymes will be important, as will the effectof genetic, physiological and environmental factors on enzymeactivity. A broad range of enzymes is expressed by the lung,but, in general, their cellular location has been poorly charac-terized57. Studies that use homogenates of the whole lung areof limited value for modelling the metabolic fate of inhaledcompounds following selective regional deposition. Even dataobtained from intact lung models is highly influenced by ex-perimental methodology such as the species used and sam-pling procedures. The surface of the airway epithelium is theprimary metabolic barrier to inhaled foreign compounds.Therefore, the expression and activity of xenobiotic metaboliz-ing enzymes in cultured human airway epithelial cells is highlyrelevant to the fate of inhaled xenobiotics.

Xenobiotic metabolizing enzymes of the airway epithelialcells have been identified using reverse transcriptase-polym-erase chain reaction to assess primary human bronchial epi-thelial cell cultures58. Cytochrome P450 enzymes 1A1, 1B1,2B7, 2E1, 2F1 and 4B1 and microsomal epoxide hydrolasewere shown to be in bronchial epithelial cells, but not alveolarmacrophages, whereas several phase II enzymes were detectedin both bronchial epithelial cells and alveolar macrophages.In a similar study, the levels of phase I enzymes were lower inprimary cultured bronchial epithelial cells compared withbronchial mucosal samples59.

Data from studies utilizing continuous cell lines is minimal,particularly for the Calu-3 and 16HBE14o2 cell lines. BEAS-2Bcells have been shown to express peptidase activity in the formof neutral endopeptidase 24.11 and aminopeptidase M, butcarboxypeptidase and dipeptidylpeptidase IV activity was notdetected53. Peptidase expression has implications for both theregulation of biologically active endogenous peptides inhealthy and diseased epithelium and for the pulmonary deliv-ery of peptide-based therapeutic agents. Studies using BEAS-2Bcells and lung homogenates have shown similar stereoselectivesulphation of the active R(-) enantiomer of salbutamol to thatseen in the intestine52,60. The metabolism of inhaled pharma-ceuticals, for example esterified b2-agonists and cortico-steroids, is an important determinant of their pharmacokinet-ics, and bronchial epithelial cells should be an appropriatemodel for predicting the potential for presystemic metabolism.

Gene deliveryThe respiratory epithelium is a target for therapeutic gene de-livery. The transfer of genes in vivo is inefficient and polarizedairway epithelial cell cultures provide a convenient in vitro sys-tem in which to test gene delivery systems. Such studies haveshown that epithelial cell polarization is an important determi-nant of gene transfer efficiency to respiratory epithelial cells,both for virus-mediated gene transfer to primary airway cul-tures61,62 and 16HBE14o2 cells34 and for non-viral methodsof gene transfer to primary cultures63. Other studies utilizingbronchial cell lines to study gene delivery have shown cytokineproduction by BEAS-2B cells and primary airway epithelial cul-tures following exposure to viral gene delivery systems64. Theability of polycation-DNA complexes to transfect 16HBE14o2

cells has also been studied33,65 and a reduction in the ability oflipid systems to transfect 16HBE14o2 cells in the presence ofexogenous sputum33 and surfactant66 has been reported.

Future directionsIt is important that the biological properties of bronchial epi-thelial cell lines mirror those of airway epithelial cells in vivo.Culture conditions are critical to the differentiation of bronchial

24


Figure 5. The effect of apical chamber pH on the apparentpermeability coefficient (Papp) of salbutamol (100 mM) and formoterol(100 mM) across the 16HBE14o2 drug absorption model. The increasein formoterol transport reflects an increasing proportion of unionizedspecies available for transcellular transport (3% formoterol isunionized at pH 6, increasing to 69% unionized at pH 8). In contrast,there is no pH-dependant change in the transport of salbutamol,which is 100% ionized at pH 6–8 (data represent mean 6 standarderror, n 5 3). Salbutamol (white) and formoterol (black).

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epithelial cells in vitro. Further characterization of markers of cel-lular differentiation in vivo would provide additional features, bywhich the status of cultured cells can be assessed.This wouldaid further optimization of culture methods to promote cellulardifferentiation and improve the expression of normal bronchialairway epithelial phenotype by cells in culture. Both cell cultureconditions and time in culture are likely to affect the expressionof drug-metabolizing enzymes and active transport mecha-nisms. The active transport mechanisms that exist in the lungare not well characterized at present.Although not flawless, pri-mary cultured human airway epithelial cultures that show ahigh degree of mucociliary differentiation provide a goodmodel in which to look for such mechanisms, provided thatculture conditions favour the expression of active transportmechanisms native to the airway epithelium.

For acceptance as in vitro drug absorption models, airway cellmodels must establish robust correlation between data obtainedin vitro and pulmonary drug absorption in vivo. Differences inmethodology, particularly dosing methods and species vari-ation, present difficulties when using drug absorption datafrom the literature. However, the collated data of Schanker andcoworkers have been used as a measure of in vivo pulmonaryavailability and correlate well with data obtained using Xenopuspulmonary membranes67.

A limitation of the 16HBE14o2 absorption model is that itdoes not incorporate the protective covering of mucus that pro-tects the epithelium in vivo.To allow for an evaluation of the ef-fect of the mucus layer on drug transport, an absorption modelbased on the rat tracheal goblet cell line, SPOC1, has been de-veloped in the Department of Pharmacy at King’s College London68. Mucus secretion in this model is under purinergiccontrol, allowing drug absorption to be correlated with theamount of mucus coating the cells. The absorption of testos-terone clearly shows the extent to which mucus may affect theabsorption of certain drug compounds (Fig. 6)69.

In further experiments an approximate doubling of thethickness of the mucus layer has been found to reduce thetransport of certain compounds (benzylpenicillin, salicylicacid, urea and inulin transport were reduced by 55%, 33%,21% and 57%, respectively), but had no effect on the transportof antipyrine, mannitol or sucrose70. More research will be re-quired to discern any relationship between the physicochemi-cal properties of a compound and the absorption barrier posedby mucus. If mucus can reduce the absorption of some com-pounds from simple solutions, then it can be imagined thatthere will be even greater potential for mucus interaction withmore complex inhaled formulations.

In conclusion, 16HBE14o2 and Calu-3 cells are the mostpromising cell lines for use as in vitro models of airway epithelialpermeability. However, much work is still required to evaluate

these models and define their strengths and limitations beforeit will be possible to take advantage of the opportunities theyoffer for the rapid assessment of pulmonary transport, metab-olism and gene delivery. It is also worth noting that althoughencouraging progress is being made in the development of celllines to model the airways, there has been far less developmentor success in generating cell lines that can be cultured as robustmodels of the alveolar regions.

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Figure 6. Reduction in testosterone transport following the stimulationof mucus secretion by adenyl imidodiphosphate (100 mM). Mucussecretion in the apical chamber of individual Transwells (area 1 cm2)was inversely correlated with testosterone transport. The meanapparent permeability coefficient (Papp) of testosterone across theSPOC1 drug absorption model was reduced from 228 6 98 ms s21 to82 6 10 nm s21 (n 5 6, mean 6 SD). Stimulated mucus secretion (filledsquares), baseline mucus secretion (clear squares). Data from Ref. 69.

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In brief…

Guilford Pharmaceutical and Amgen (ThousandOaks, CA, USA) have announced that thecompanies have initiated the first clinical testing ofneuroimmunophilins. A novel class of smallmolecule neurotrophic agents,neuroimmunophilins may represent a newapproach to the treatment of neurodegenerativedisorders.

Preclinical studies, which included models ofParkinson’s disease, saw neuroimmunophilinsdemonstrate potential for the promotion of nerveregeneration and repair. The initial study is aPhase 1 safety, tolerability and pharmacokineticstudy in healthy subjects and is being conductedin Europe. The initial disease target will beParkinson’s disease.

According to Craig R. Smith, president and chiefexecutive officer of Guilford, the companies‘…have reached an important milestone in thedevelopment of neuroimmunophilins. We willundoubtedly learn a great deal aboutneuroimmunophilins over the next few months,and, assuming a successful outcome of the phase1 clinical program, we look forward to the firstclinical trials in patients with Parkinson’s disease.’

Human airway epithelial cell lines for in vitro drug transport and metabolism studies

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