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DRUG METABOLISM AND DISPOSITION
Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics
DMD 37:992–998, 2009
Vol. 37, No. 5
26286/3462750
Printed in U.S.A.
Characterization of Human Corneal Epithelial Cell Model As a
Surrogate for Corneal Permeability Assessment:
Metabolism and Transport
Cathie D. Xiang, Minerva Batugo, David C. Gale, Tao Zhang, Jingjing Ye, Chunze Li,
Sue Zhou, Ellen Y. Wu, and Eric Y. Zhang
Pharmacokinetics Dynamics and Metabolism (C.D.X., M.B., D.C.G., C.L., S.Z., E.Y.W., E.Y.Z.), and Preclinical Statistics (J.Y.),
Pfizer Global Research and Development, La Jolla, California; and College of Pharmacy, the University of Michigan, Ann Arbor,
Michigan (T.Z.)
Received December 18, 2008; accepted February 11, 2009
The recently introduced Clonetics human corneal epithelium
(cHCE) cell line is considered a promising in vitro permeability
model, replacing excised animal cornea to predict corneal permeability of topically administered compounds. The purpose of this
study was to further characterize cHCE as a corneal permeability
model from both drug metabolism and transport aspects. First,
good correlation was found in the permeability values (Papp) obtained from cHCE and rabbit corneas for various ophthalmic drugs
and permeability markers. Second, a previously established realtime quantitative polymerase chain reaction method was used to
profile mRNA expression of drug-metabolizing enzymes (major
cytochromes P450 and UDP glucuronosyltransferase 1A1) and
transporters in cHCE in comparison with human cornea. Findings
indicated that 1) the mRNA expression of most metabolizing enzymes tested was lower in cHCE than in excised human cornea, 2)
the mRNA expression of efflux transporters [multidrug resistantassociated protein (MRP) 1, MRP2, MRP3, and breast cancer resistance protein], peptide transporters (PEPT1 and PEPT2), and
organic cation transporters (OCTN1, OCTN2, OCT1, and OCT3)
could be detected in cHCE as in human cornea. However, multidrug resistance (MDR) 1 and organic anion transporting polypeptide 2B1 was not detected in cHCE; 3) cHCE was demonstrated to
possess both esterase and ketone reductase activities known to
be present in human cornea; and 4) transport studies using probe
substrates suggested that both active efflux and uptake transport
may be limited in cHCE. As the first detailed report to delineate
drug metabolism and transport characteristics of cHCE, this work
shed light on the usefulness and potential limitations of cHCE in
predicting the corneal permeability of ophthalmic drugs, including
ester prodrugs, and transporter substrates.
Topical instillation is the desired route of administration for ophthalmic drugs to treat diseases in the anterior segment of the eye
including glaucoma, inflammations, infections, and dry eye. The
primary pathway of drug permeation from the tear fluid to the anterior
chamber of the eye is via the transcorneal route. The cornea has a
multilayered structure constituted primarily of corneal epithelium,
stroma, and endothelium. However, passage through the corneal epithelium is considered to be the rate-limiting step in the transcorneal
penetration of most ophthalmic drugs (Maurice and Mishima, 1984).
In the past decade, several in vitro corneal permeability models that
can substitute for the isolated cornea to predict ocular absorption and
facilitate ophthalmic drug discovery have been developed (Reichl and
Becker, 2008). These models evolved from primary cultures using
rabbit corneas and immortalized rabbit cell lines to the immortalized
human corneal epithelial (HCE) cell lines, and even include complex
systems such as human corneal constructs (summarized in Table 1).
Given the pros and cons of each model, HCE cell lines seem to be
most appealing owing to their human origin, the relatively short
preparation time, and the good reproducibility often ensured by commercially available products. A recent comparative study suggested
that the Clonetics cHCE could be more suitable than other HCE cell
lines (i.e., HCE-T and sHCE) in predicting ocular drug permeability.
This model not only closely resembles excised human corneas in the
barrier function as measured by TEER but also seems to be able to
adequately differentiate permeability differences observed between
poorly and highly permeable compounds (Becker et al., 2008).
Not only does the cornea serve as a diffusion barrier to limit drug
penetration, it also expresses certain drug-metabolizing enzymes and
active transport systems (Duvvuri et al., 2004; Mannermaa et al.,
2006; Zhang et al., 2008). These enzymes and transporters, when
functionally expressed, could affect ocular drug absorption. For example, the corneal epithelium has robust esterase activity, which
underlies the significance of the ester prodrug approach in ophthalmic
Article, publication date, and citation information can be found at
http://dmd.aspetjournals.org.
doi:10.1124/dmd.108.026286.
ABBREVIATIONS: HCE, human cornea epithelial; TEER, transepithelial electrical resistance; PCR, polymerase chain reaction; BSS, balanced salt
solution; LC, liquid chromatography; MS/MS, tandem mass spectrometry; A, apical; B, basolateral; MRP, multidrug resistant-associated protein;
BCRP, breast cancer resistance protein; PEPT, peptide transporter; OCTN, organic cation/carnitine transporter; P-gp, P-glycoprotein; P450,
cytochrome P450; UGT, UDP glucuronosyltransferase.
992
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ABSTRACT:
993
METABOLISM AND TRANSPORT CHARACTERISTICS OF THE cHCE MODEL
TABLE 1
Summary of in vitro models used for drug corneal permeability assessment
Model
Origin and Source
1) Introduced by Kawazu et al. (1998); 2)
introduced by Chang et al. (2000)
SIRC
Established by Phillips et al. (1966); ATCC
RCE
Introduced by Araki et al. (1993); ECACC
HCE-T
Established by Araki-Sasaki et al. (1995)
cHCE
Established at Cambrex Bio Science in 2006
sHCE
Established at SkinEthic Laboratories (Nice,
France)
First described by Griffith et al. (1999)
HCC
Characteristics
Rabbit origin; TEER low (at ⬃150 ohm 䡠 cm2)
in model of Kawazu et al. (1998) and high
(at ⬎2000 ohm 䡠 cm2) in model of Chang et
al. (2000); used to study transport mechanism
Immortalized cell line from rabbit cornea;
TEER low at ⬃100 ohm 䡠 cm2; a fibroblast
phenotype; used to study permeability,
transporter and esterase presence
Immortalized cell line from albino rabbit
cornea; TEER low at ⬃150 ohm 䡠 cm2; used
to study drug permeation
Immortalized cell line by infection of human
corneal epithelial cells with a recombinant
simian virus 40-adenovirus vector; tight
multilayered with TEER ⬎400 ohm 䡠 cm2;
used to study drug transport and transporter
presence
Immortalized cell line by infection of isolated
normal human corneal cells with an
amphotropic recombinant retrovirus
containing HPV-16 ED/E7 genes; tight
multilayered with TEER ⬎400 ohm 䡠 cm2;
used to study drug permeability
Immortalized human corneal epithelia cells;
TEER low at ⬃100 ohm 䡠 cm2.
Human corneal equivalent with epithelia, stomal
and endothelial cells; tested for in vitro
permeation studies
References
Kamazu et al., 1998, 1999, 2006; Chang et al.,
2000; Dey et al., 2003; Sakanaka et al., 2006
Tak et al., 2001; Dey et al., 2003; Majumdar et al.,
2003; Becker et al., 2008; Goskonda et al.,
1999, 2000
Burgalassi et al., 2004
Toropainen et al., 2001, 2003; Ranta et al., 2003;
Becker et al., 2007, 2008
Becker et al., 2008
Becker et al., 2008
Reichl et al., 2004, 2005
RCE, rabbit corneal epithelium; SIRC, Statens Seruminstitut rabbit corneal epithelial cell line; ATCC, American Type Culture Collection; ECACC, European Cell Culture Collection; HCE, human
corneal epithelial cell line; HCC, human corneal construct; HPV, human papilloma virus.
drug design (Shirasaki, 2008). For the efflux and uptake transporters
potentially present in the cornea, the ocular absorption of their drug
substrates could be either hindered or facilitated. Because of the
restricted tissue availability of fresh human cornea, mechanistic understanding about the effect of metabolic conversion and drug-transporter interaction on ocular drug absorption is lacking. Encouragingly,
the newly introduced cHCE model could be an appropriate in vitro
system to study these mechanisms, allowing for more flexible and
in-depth experimental designs that are otherwise prohibitive with
fresh human cornea.
As described here, studies were designed to further characterize
cHCE as a corneal permeability model, particularly from drug metabolism and transport aspects. In brief, a previously established
real-time quantitative PCR approach (Zhang et al., 2008) was adopted
to profile mRNA expression of key metabolizing enzymes and transporters in cHCE in comparison with human cornea. Moreover, functional studies using probe substrates were conducted to assess drug
metabolism (i.e., esterase and ketone reductase) and active transport in
cHCE.
Materials and Methods
Materials. Acetazolamide, amantadine, ampicillin, atenolol, brimonidine,
buspirone (internal standard), cimetidine, desipramine, dexamethasone, dexamethasone 21-acetate, digoxin, diclofenac, etoposide, latanoprost, latanoprost
acid, methotrexate, metoprolol, nadolol, NADPH, pilocarpine, quinidine, and
timolol were purchased from Sigma-Aldrich (St. Louis, MO). The cHCE on
Transwell filter inserts was purchased from Lonza Walkersville, Inc. (Walkersville, MD). Fresh rabbit corneas (albino) were purchased from Pel-Freez
Biologicals (Rogers, AR), kept in Optisol-GS (Bausch & Lomb, Rochester,
NY), and used within 36 h after harvesting. BSS and BSS PLUS solution was
purchased from Alcon (Dallas, TX).
RNA Extraction, cDNA Synthesis, and Real-Time Quantitative PCR.
Human eye tissue collection, RNA extraction, cDNA synthesis, real-time PCR,
and quantitative analysis were performed as reported previously (Zhang et al.,
2008). Total RNA from cHCE cells was extracted with the QIAGEN RNeasy
kit after homogenization in a FastPrep F120 instrument (Qbiogene Inc., Irvine,
CA). PCR reactions were run in a total volume of 20 ␮l, containing cDNA
from 25 ng of total RNA. The geometric mean of human ␤-actin, glyceraldehyde-3-phosphate dehydrogenase, and peptidylpropyl isomerase A was used as
the normalization factor for the relative quantification of each gene.
Permeability Studies with the Excised Rabbit Cornea and cHCE. Excised rabbit cornea. Fresh rabbit corneas were rinsed and equilibrated in BSS
solution for 15 min. The corneas were mounted in a Ussing chamber perfusion
system (Harvard Apparatus Inc., Holliston, MA), which was placed in the
heating block (Harvard Apparatus Inc.). The exposed surface area of the
cornea is approximately 0.2 cm2/well. One milliliter of BSS or BSS PLUS
solution was added in the chamber bathing the endothelium side (the receiver
chamber), and 1 ml of test compound (10 ␮M) in BSS or BSS PLUS was
added in the chamber bathing the epithelium side (the donor chamber).
Constant mixing of the donor and receiver solutions was achieved with a gentle
gas flow of air-CO2 (95%:5%) through both chambers. The heating block was
maintained at 37°C by a circulating water bath. Serial samples of 100 ␮l were
removed from the receiver chamber every hour during a period of 4 h. Each
aliquot was replaced immediately with an equal volume of BSS. The samples,
mixed with 10 ␮l of internal standard solution, were analyzed directly by
LC-MS/MS.
cHCE. Upon the receipt of each cHCE plate, the cells were cultured in the
medium overnight and used the next day. The surface area of the membrane is
0.33 cm2 per insert. The permeability experiments were performed under a
humidified atmosphere of air-CO2 (95%:5%) at 37°C. In the A to B transport
study, 1 ml of cell medium was added in the basolateral side (the receiver side).
The study was initiated by dosing the apical compartment with 200 ␮l of cell
medium containing the test compound and permeability markers (i.e., nadolol
and metoprolol). In the B to A transport study, 200 ␮l of cell medium was
added in the apical side (the receiver side), and the study was initiated by
dosing the basolateral compartment with 1000 ␮l of cell medium containing
the test compound and permeability markers. The buffer pH in both donor and
receiver chambers was 7.4 for all of the compounds tested except for ampicillin, which was dosed in the cell medium with pH adjusted to 6.5. Samples
of 100 ␮l were collected from the receiver side every 60 min for up to 4 h.
Each 100-␮l aliquot removed was immediately replenished with an equal
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Primary RCE
994
XIANG ET AL.
-6
Papp in excised rabbit cornea, 10 cm/s
18
R2=0.95
Acetazolamide
Atenolol
Brimonidine
Chlorothiazide
Diclofenac
Dorzolamide
Ethoxyzolamide
Metoprolol
Nadolol
Pilocarpine
Timolol
16
14
12
10
8
6
4
2
0
2
4
6
10
8
12
14
16
-6
Papp in cHCE, 10 cm/s
FIG. 1. Permeability values (Papp) obtained in excised rabbit corneas and cHCE. Data are presented as the mean of triplicates.
volume of fresh cell medium. The samples, mixed with 10 ␮l of internal
standard solution, were analyzed directly by LC-MS/MS.
Permeability Data and Statistical Analysis. Apparent permeability coefficients (Papp) were calculated using the following equation: Papp ⫽ (1/A ⫻
C0) (dM/dt), where dM/dt is the flux (nanomoles per second) across the cell
layers or cornea, A (square centimeters) is the exposed surface area of the insert
membrane of cHCE or rabbit cornea, and C0 is the initial drug concentration
(micromolar) in the donor compartment at t ⫽ 0. Flux (dM/dt) across the cell
layer or cornea was determined from the slope of the linear portion of the
cumulative amount permeated versus time plot. The results are expressed as
the mean ⫾ S.D. The statistical significance ( p ⬍ 0.05) was determined by
Student’s t test.
Esterase Activity and Prodrug Permeability in cHCE. Esterase activity
of cHCE cells was monitored by the conversion of ester prodrug, latanoprost
(10 ␮M), to the acid form. The permeability value of latanoprost was calculated according to the concentrations of either latanoprost or its acid in the
receiver chamber of the cHCE system. In addition, the Papp value of latanoprost acid was calculated when the acid itself was dosed in the donor chamber.
Ketone Reductase Activity in cHCE. Ketone reductase activity was monitored by LC-mass spectrometry detection of dihydrolevobunolol in the receiver side after levobunolol was dosed at 10 ␮M in the donor chamber.
LC-MS/MS. The analytical system comprised a Sciex API 4000 mass
spectrometer (Applied Biosystems, Foster City, CA), a CTC HTS PAL autosampler (LEAP Technologies, Carrboro, NC), and Shimadzu LC-10ADvp
pumps (Shimadzu, Kyoto, Japan). The samples were analyzed using reversephase chromatography (Zorbax SB-Phenyl, 50 mm ⫻ 2.1 mm, 5 ␮m; Agilent
Technologies, Santa Clara, CA). A 4-min linear gradient from 5 to 90% of
mobile phase I (acetonitrile-water 98:2, containing 0.1% formic acid) was used
at the flow of 0.6 ml/min, and mobile phase II was water-acetonitrile (98:2)
containing 0.1% formic acid. Multiple reaction monitoring was used to
monitor drug levels in the various experiments. The parent/fragment information is as follows: acetazolamide (223/181), amantadine (152/135), ampicillin
(350/106), atenolol (267/145), brimonidine (292/212), buspirone (386/122),
cimetidine (253/159), desipramine (267/208), dexamethasone (393/373), dexamethasone 21-acetate(435/415), digoxin (781/651), diclofenac (296/215), dihydrolevobunolol (294/220), dorzolamide (325/199), etoposide (589/229),
ethoxyzolamide (259/178), indinavir (614/421), latanoprost (433/397), latanoprost acid (391/355), levobunolol (292/236), methotrexate (455/308), metoprolol (268/116), nadolol (310/254), pilocarpine (209/163), quinidine (325/
307), timolol (317/261), and verapamil (455/165).
Results
Transcorneal Permeability in Excised Rabbits Corneas and
cHCE. The permeability of eight ophthalmic drugs and three ␤-blockers (atenolol, metoprolol, and nadolol) was assessed using either
excised rabbit corneas or cHCE. The Papp values for the 11 compounds track well between values for the two models with a correlation coefficient (R2) of 0.95 (Fig. 1). In addition, Spearman’s rank
correlation coefficient was calculated to be 0.92, suggesting the similar rank order of permeability data obtained from the two models.
mRNA Expression of Drug-Metabolizing Enzymes in Human
Cornea and cHCE. Consistent with the previous report (Zhang et al.,
2008), the mRNA level of each detectable metabolizing enzyme in
human cornea was less than 1% of the corresponding hepatic level.
CYP1A2 and CYP2B6 mRNAs were not detected in either cHCE or
human cornea. All of the enzymes tested except CYP2A6 were lower
in cHCE than in human cornea (Fig. 2).
mRNA Expression of Drug Transporter in Human Cornea and
cHCE. Similar to that in human cornea, the mRNA expression of
efflux transporters (MRP1, MRP2, MRP3, and BCRP), peptide transporters (PEPT1 and PEPT2), and organic cation transporters (OCTN1,
OCTN2, OCT1, and OCT3) could be detected in cHCE. Their expression levels were more or less comparable between human cornea
and cHCE. In addition, the mRNA expression of MDR1 and organic
anion-transporting protein 2B1, which was detected in human cornea
but quantified as very low (Zhang et al., 2008) was not detected in
cHCE (Fig. 3).
Esterase Activity in cHCE and Rabbit Cornea. Esterase activity
was evident in both cHCE and rabbit cornea as latanoprost acid
was readily detected in the receiver chambers after latanoprost was
dosed in the donor chamber. Permeability values of latanoprost
(calculated based on the concentrations of either prodrug or acid in
the receiver chambers) and latanoprost acid are shown in Table 2.
In both models, latanoprost acid had low and similar permeability.
Latanaprost exhibited poor permeability when measured as the
parent ester itself but had a much higher permeability when measured as latanoprost acid.
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0
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METABOLISM AND TRANSPORT CHARACTERISTICS OF THE cHCE MODEL
UGTIA1
CYP3A5
CYP3A4
CYP2E1
CYP2D6
CYP2C19
CYP2C9
CYP2C8
CYP2B6
CYP1A2
Human Cornea
HCE
*
0.00
0.05
0.10
Relative expression of each gene
FIG. 2. Relative mRNA expression level of major P450s and UGT1A1 in human cornea and cHCE. Data are presented as the mean of triplicates with the S.D. as the error
bar. ⴱ, not detected.
B
A
Cornea
cHCE
BCRP
Cornea
cHCE
OCTN2
OCTN1
MRP3
OCT3
OCT1
MRP2
OATP2B1
*
MRP1
PEPT2
MDR1
*
PEPT1
Relative expression of each gene
Relative expression of each gene
FIG. 3. Relative mRNA expression level of selected (A) drug efflux and (B) uptake transporter genes in human cornea and cHCE. Data are presented as the mean of
triplicates with the S.D. as the error bar. ⴱ, not detected.
Ketone Reductase Activity in cHCE. Dihydrolevobunolol was
readily detected in the receiver side after levobunolol was dosed in the
donor side of the cHCE model (reaction shown in Fig. 4). As a
nonselective ␤-adrenergic antagonist, levobunolol is used in the treatment of glaucoma. It has been shown that the metabolic reduction of
the cyclohexanone functional group to form dihydrolevobunolol is
mediated by NADPH-dependent ketone reductase in ocular tissues
including cornea (Lee et al., 1988).
Permeability of Probe Substrates for Efflux Transporters in
cHCE. The permeability of digoxin (P-gp substrate) and indinavir
(substrate for P-gp, MRP1, and MRP2) in the cHCE model was
measured at both A to B and B to A directions under various
concentrations (Fig. 5). When measured in either direction, Papp
values of either digoxin or indinavir did not seem to vary significantly
with the increase in concentration. In addition, the flux ratios did not
appear to significantly deviate from unity (i.e., in the range from 0.6
to 1.5) for either compound under different concentrations. The permeability of two MRP3 substrates, etoposide and methotrexate, was
also tested but was found to be too low in both directions to derive a
Papp ratio (data not shown).
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*
CYP2A6
996
XIANG ET AL.
TABLE 2
Ester conversion and permeability of latanoprost in cHCE and excised rabbit
cornea (dosed as either ester or acid)
Data are presented as the mean of triplicates ⫾ S.D.
Papp
Compound Dosed
Measured as the Ester
Measured as the Acid
10⫺6 cm/s
Latanoprost
Latanoprost
Latanoprost
Latanoprost
in cHCE
acid in cHCE
in cornea
acid in cornea
0.8 ⫾ 0.3
N.A.
0a
N.A.
18.5 ⫾ 2.3
0.4 ⫾ 0.2
13.8 ⫾ 2.7
0.4 ⫾ 0.1
N.A., not applicable.
a
Latanoprost was not detected in the receiver chamber.
O
Ketone reductase
O
OH
OH
NH
NH
Dihydrolevobunolol
Levobunolol
FIG. 4. Ketone reductase activity in cHCE as evidenced by the formation of
dihydrolevobunolol from levobunolol.
Permeability of Probe Substrates for Uptake Transporters in
cHCE. The permeabilities of quinidine and verapamil (substrates for
OCTN1 and OCTN2), amantadine and desipramine (substrates
for OCT1), cimetidine (substrate for OCT3), and ampicillin (substrate for PEPT1 and PEPT2) in the cHCE model were measured in
both A to B and B to A directions at 10 ␮M (Table 3). At this
concentration, the Papp_A to B of quinidine, verapamil, amantadine,
desipramine, and cimetidine appeared to be similar to the Papp_B to A,
suggesting the lack of active uptake of these compounds in cHCE
cells. Papp values of ampicillin were found to be poor in both directions with an apparent higher Papp_B to A (0.56 ⫾ 0.1 ⫻ 10⫺6 cm/s)
than Papp_A to B (0.12 ⫾ 0.07 ⫻ 10⫺6 cm/s). It should be noted that the
codosed low-permeability marker (i.e., nadolol) also appeared to be
more permeable in the B to A direction (Papp_B to A 0.60 ⫾ 0.15 ⫻
10⫺6 cm/s versus Papp_A to B 0.36 ⫾ 0.15 ⫻ 10⫺6 cm/s). Nevertheless,
the data suggested that there may also be little active uptake of
ampicillin in cHCE cells.
Discussion
Assessing the transcorneal permeability of drug candidates is essential
in ophthalmic drug discovery for front-of-eye delivery. This assessment
is often achieved with excised animal (i.e., rabbit) cornea using a diffusion chamber setup, which offers reasonable predictability for human
transcorneal permeability. However, such experiments are labor-intensive with low throughput and involve the sacrifice of animals. These
disadvantages may be alleviated by cell-based models derived from
corneal tissues (Table 1). In this work, we focused on the newly introduced Clonetics cHCE model, which is regarded as a promising in vitro
model to minimize the use of animal corneas in transcorneal permeability
testing (Beck et al., 2008). Permeability values for 11 compounds were
obtained from excised rabbit cornea and cHCE models (Fig. 1). Good
correlation between the two models suggests that the cHCE model could
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O
OH
indeed be substituted for excised rabbit cornea as a surrogate permeability
model in ocular drug discovery.
Human cornea is not simply a physical barrier that limits the
diffusion of xenobiotics into the eye. It is equipped with sophisticated
metabolic and potential active transport systems that should be taken
into consideration for ophthalmic drug delivery and disposition (Duvvuri et al., 2004; Mannermaa et al., 2006). In previous work (Zhang
et al., 2008), the mRNA expression of major P450s and drug transporters was profiled for human ocular tissues, including cornea. A
similar real-time PCR approach was adopted in this present work to
compare the mRNA expression of major metabolizing enzymes and
drug transporters between human cornea and the cHCE. Furthermore,
functional studies using probe substrates were explored to characterize cHCE as a permeability model from both metabolism and transport aspects.
Drug Metabolism in cHCE. The mRNA expression of most P450s
and UGT1A1 tested was shown to be lower in cHCE than in human
cornea, which itself possesses limited mRNA expression of these
enzymes in comparison with the liver. There was little metabolic
turnover when P450s and UGT1A1 probe substrates were incubated
in rabbit corneal homogenates (data not shown). Taken together, the
data suggest that 1) the human cornea probably possesses minimal
metabolic capacity mediated by P450 enzymes and UGT1A1 and 2)
although the cHCE cells may contain even lesser amounts of these
enzymes than human cornea, the cHCE cells may still be a relevant
model to evaluate the transcorneal permeability of substrates for these
enzymes.
The cHCE cells were shown to have evident esterase activity to
convert latanoprost (Xalatan) to its pharmacologically active metabolite (latanoprost acid), as well as ketone reductase activity to convert
levobunolol (Betagan) to its equipotent ocular metabolite, dihydrolevobunolol. It should be noted that although these results confirmed that the esterases and ketone reductase possessed by human
cornea are also functional in cHCE cells, it did not provide any
quantitative comparison on the rate and extent of these enzyme
activities between cHCE cells and human cornea.
Similar permeability data for latanoprost and latanoprost acid were
obtained in both cHCE and excised rabbit cornea models (Table 2).
Latanoprost acid itself had low permeability, whereas its ester prodrug
(latanoprost, quantified in the form of the acid) showed much improved permeability in both cHCE (⬎40 fold) and excised rabbit
corneas (⬎30 fold). As the prodrug design is a proven approach in
ophthalmic drug design (Shirasaki, 2008), our data suggest that the
cHCE model may be used to confirm metabolic conversion and
evaluate transcorneal permeability of ester prodrug candidates. Further experiments are needed to quantitatively compare esterase activity between cHCE and human cornea in terms of esterase composition, specificity, and kinetics.
Drug Transport in cHCE. The mRNA expression and potential
functional presence of drug transporters in human cornea have been
documented in the literature (Duvvuri et al., 2004; Mannermaa et al.,
2006; Karla et al., 2007; Zhang et al., 2008). In the present work, five
efflux and seven uptake transporters, which were found to have
quantifiable mRNA expression in human cornea (Zhang et al., 2008),
were studied for their mRNA expression (Fig. 3) and functional
relevance in cHCE cells (Table 3; Fig. 5). The different probe substrates for the different transporters were based on transporter substrate
recommendations found in the U.S. Food and Drug Administration
transporter database (http://www.fda.gov/cder/drug/drugInteractions/
tableSubstrates.htm#major).
Except for MDR1 (not detectable in cHCE, but detected at a very
low level in human cornea), the mRNA expression of other efflux
997
METABOLISM AND TRANSPORT CHARACTERISTICS OF THE cHCE MODEL
3.0
2.5
3.0
A>B
B>A
Flux ratio
2.0
1.5
1.5
1.0
1.0
0.5
0.5
0.0
0.0
100 µM
200 µM
600 µM
A>B
B>A
Flux ratio
4.5
4.0
4.5
3.5
3.5
3.0
2.5
2.5
2.0
1.5
1.5
Flux ratio (B>A/A>B)
Papp of Indinavir in cHCE, ×10 -6 cm/s
5.0
1.0
0.5
0.5
0.0
1 µM
10 µM
100 µM
500 µM
FIG. 5. Permeability parameters of digoxin (A) and indinavir (B) obtained in cHCE. Data are presented as the mean of triplicates with the S.D. as the error bar.
transporters including MRP1, MRP2, MRP3, and BCRP was detected in cHCE cells. Consistent with the lack of MDR1 mRNA
expression in cHCE cells, there was no significant unidirectional
flux of digoxin (a substrate for P-gp) at different concentrations
(Fig. 5). These data are consistent with a study that showed no
significant difference in rhodamine 123 (a substrate for P-gp) flux
in either direction in excised human cornea or cHCE (Becker et al.,
2007, 2008). Although mRNAs of MRP1, MRP2, and BCRP could
be detected in cHCE cells, their activities did not appear to
manifest in these cells because no significant unidirectional flux of
indinavir (a common substrate for these transporters) was observed
under different concentrations (Fig. 5).
Despite the detectable mRNA expression of uptake transporters
(PEPT1, PEPT2, OCT1, OCT3, OCTN1, and OCTN2) in cHCE cells,
the mRNA presence does not seem to translate to a net increase in the
absorptive flux of their substrates (Table 3), suggesting that the active
transport function of these uptake transporters also may be minimal in
cHCE cells. It is important to note that the similarities or differences
in mRNA expression (Fig. 3) between cHCE and human cornea of
these tested efflux and uptake transporters do not necessarily correlate
to protein expression and transport activity. Therefore, it is still
difficult to conclude whether the cHCE model is predictive or not for
human transcorneal permeability of these transporter substrates and
whether further studies (i.e., permeability data obtained from freshly
excised human cornea) would be needed.
In summary, this work further validated the fact that the cHCE
model can be adopted as a surrogate permeability model to substitute
for the excised rabbit cornea model in ophthalmic drug discovery and
research. The detailed characterization of cHCE as a permeability
model from both drug metabolism and transport aspects sheds light on
the potential similarities and differences between cHCE and human
cornea. These characteristics should be taken into account when
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 3, 2017
2.0
10 µM
B
2.5
Flux ratio (B>A/A>B)
Papp of Digoxin in cHCE , ×10 -6 cm/s
A
998
XIANG ET AL.
TABLE 3
Papp values and Papp_ A to B /Papp_ B to A ratios of probe substrates (10 ␮M) of uptake transporters in cHCE
Data are presented as the mean of triplicates ⫾ S.D.
Transporter
Probes
Papp_ A to B
Papp_ B to A
Papp_ A to B/Papp_ B to A
8.41 ⫾ 1.6
10.5 ⫾ 1.0
13.2 ⫾ 0.1
3.4 ⫾ 0.7
1.5 ⫾ 0.1
1.5
1.0
1.1
1.2
0.7
10⫺6 cm/s
OCTN1 and OCTN2
OCTN1 and OCTN2
OCT1
OCT1
OCT3
Quinidine
Verapamil
Amantadine
Desipramine
Cimetidine
12.4 ⫾ 0.9
10.8 ⫾ 0.5
15.1 ⫾ 0.8
3.9 ⫾ 0.4
1.0 ⫾ 0.2
cHCE is used to predict transcorneal permeability of drug candidates,
including prodrug and transporter substrates.
Acknowledgments. We thank Ping Kang and Deepak Davie for
insightful scientific discussions and critical reading of the manuscript.
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Address correspondence to: Dr. Eric Y. Zhang, 10724 Science Center Drive,
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