Department of Internal Medicine, University of Michigan Medical
Center, Ann Arbor, Michigan 48109 (P.S.-R., K.S.L., P.B.W.), and
Department of Pharmaceutics, University of Washington, Seattle,
Washington 98195 (K.E.T., J.M.F., M.F.P.)
The human colon carcinoma cell line, Caco-2, is widely used as a model
for oral absorption of xenobiotics. The usefulness of Caco-2 cells has
been limited, however, because they do not express appreciable
quantities of CYP3A4, the principle cytochrome P450 present in human
small bowel epithelial cells. We report that treatment of Caco-2 cells
with 1
,25-dihydroxyvitamin D3, beginning at confluence,
results in a dose- and duration-dependent increase in CYP3A4 mRNA and
protein, with little apparent effect on the expression of CYP3A5 or
CYP3A7. This treatment also results in increases in NADPH cytochrome
P450 reductase and P-glycoprotein (the MDR1 gene product)
but has no detectable effect on expression of CYP1A1, CYP2D6,
cytochrome b5, liver or intestinal fatty
acid binding proteins, or villin. Maximal expression of CYP3A4 requires an extracellular matrix on a permeable support and the presence of
serum. In the treated cells, the intrinsic formation clearance of
1
-hydroxymidazolam (a reaction characteristically catalyzed by CYP3A
enzymes) was estimated to be somewhat lower than that of human jejunal
mucosa (1.14 and 3.67 ml/min/g of cells, respectively). The
1
-OH-midazolam/4-OH-midazolam product ratio produced by the cells
(~5.3) is comparable to, but somewhat lower than, that observed in
human jejunal microsomes (7.4-15.4), which may reflect the presence of
CYP3A7 in the Caco-2 cells. 25-Hydroxyvitamin D3 is less
efficacious but reproduces the effects of the dihydroxy compound, whereas unhydroxylated vitamin D is without appreciable effect. These
observations, together with the time course of response, suggest that
the vitamin D receptor may be involved in CYP3A4 regulation. The
culture model we describe should prove useful in defining the role of
CYP3A4 in limiting the oral bioavailability of many xenobiotics.
 |
Introduction |
CYP3A4, the principal cytochrome
P450 present in human liver (1) and small intestinal epithelial cells
(enterocytes) (2, 3), has been implicated in the metabolic elimination
of many drugs (4). It has been proposed that first-pass metabolism by
intestinal CYP3A4 contributes to the poor oral bioavailability of some
of these drugs (5-9). An in vitro model capable of
predicting the oral bioavailability of CYP3A4 substrates could be very
useful during drug development. It has also been proposed that drug
interactions involving induction or inhibition of CYP3A4 may be largely
occurring at the level of the intestine (3, 6, 7) and not exclusively within the liver, as originally thought. Therefore, an in
vitro model suitable for studying the role of intestinal CYP3A4 in
drug/drug interactions would also be quite valuable.
Caco-2 cells (10), when grown as monolayers on permeable supports, have
proved to be useful as a model for studying intestinal permeability
(11, 12) and several transport functions, including the transport of
some drugs (13-16). However, this human colon carcinoma cell line has
thus far fallen short as an ideal model for predicting oral
bioavailability or studying drug/drug interactions, in part because of
its failure to express substantial amounts of CYP3A4. Although a Caco-2
cell clone was recently shown to express a protein that was detected by
an anti-CYP3A antibody (17), the protein did not comigrate with CYP3A4
on polyacrylamide gels. Subsequently, others found that cultures of the
parent Caco-2 cell line were capable of metabolizing cyclosporin A to
one of the major metabolites known to be produced by CYP3A4; however, two other metabolites of cyclosporin A characteristically produced by
CYP3A4 were not detected (18). The rate of nifedipine oxidation, another CYP3A4-catalyzed reaction, has also been reported to be low in
Caco-2 cells (19).
In the current study, in which we added to the culture medium a variety
of compounds reported to influence cellular differentiation, we have
found that 1
,25-dihydroxyvitamin D3 results in a dose- and duration-dependent increase in the expression of metabolically active CYP3A4 by confluent Caco-2 cells. This expression is further enhanced when the cells are grown on certain extracellular matrices. The system described should prove useful as a model for assessing the
roles of intestinal CYP3A4 in limiting oral bioavailability and in
participating in drug/drug interactions.
 |
Experimental Procedures |
Materials.
Caco-2 cells (American Type Culture Collection
HTB37) were obtained from American Type Culture Collection, Rockville,
MD. DMEM, other media and supplements, and HBSS were obtained from GIBCO (Grand Island, NY). FBS was obtained from Hyclone (Logan, UT).
Millicell CM and PCF culture inserts and the Millicell ERS device were
obtained from Millipore (Bedford, MA). Uncoated and commercially coated
[unpolymerized collagen type I (200 µg/cm2), fibrillar
collagen (polymerized type I collagen; 200 µg/cm2),
collagen type IV (15 µg/cm2), laminin (25 µg/cm2), and Matrigel (2.86 mg/cm2)]
track-etched PET inserts, Matrigel, Growth Factor-Reduced Matrigel (used at 2.86 mg/cm2), Dispase, ITS, and ITS+ were obtained
from Collaborative Biomedical Products (Bedford, MA).
1
,25-(OH)2-D3, 25-OH-D3, and
unhydroxylated D3 were obtained from Calbiochem (La Jolla,
CA).
N-Methyl-N-(t-butyl-dimethylsilyl)trifluoroacetamide was purchased from Pierce Chemical (Rockford, IL).
-Glucuronidase (G-7770) and other chemicals were obtained from Sigma Chemical (St.
Louis, MO) and were of tissue culture or molecular biology grades where
appropriate. MDZ, 15N3-MDZ, 4-OH-MDZ,
1
-OH-MDZ, and 1
-[2H2]1
-OH-MDZ were gifts
from Dr. Bruce Mico (Roche Laboratories, Nutley, NJ). Additional stable
isotope-labeled internal standards (15N3-1
-OH-MDZ and
15N3-4-OH-MDZ) were generated from an
incubation of 15N3-MDZ with human liver
microsomes. Briefly, human liver microsomes, containing 6 nmol of total
cytochrome P450, were incubated with 100 µg of
15N3-MDZ and 12 mg of NADPH in 100 mM potassium phosphate buffer, pH 7.4, for 10 min at 37°.
The reaction (8 ml final volume) was stopped by the addition of 8 ml of
100 mM Na2CO3, pH 12.5. 15N3-labeled hydroxy products were extracted
twice with 20 ml of ethyl acetate. Solvent was removed under nitrogen.
The remaining solid was redissolved in 20 ml of methanol and stored in
aliquots at
20°.
Stock solutions of 1
,25-(OH)2-D3,
25-(OH)-D3, and D3 were made in absolute
ethanol or dimethylsulfoxide; test and control cultures contained
0.1-0.2% ethanol or 0.2% dimethylsulfoxide. The midazolam stock
solution was 4 mM in dimethylsulfoxide.
Cell culture.
All cultures were maintained in a humidified
37° incubator with a 5% carbon dioxide in air atmosphere. Caco-2
cells were obtained at passage 19 and grown in plastic tissue culture
dishes in a medium consisting of DMEM containing 25 mM
glucose and 2 mM L-glutamine and supplemented
with 0.1 mM nonessential amino acids, 45 nM
DL-
-tocopherol, 100 units/ml sodium penicillin G, and
100 µg/ml streptomycin. Complete growth medium was then prepared by
the addition of 20% heat-inactivated FBS. When the cells reached 80%
confluence, they were removed using 0.1% trypsin/0.537 mM
EDTA, diluted 1:3, and reseeded onto fresh tissue culture dishes.
Clones were prepared from the parent cell line at passage 27 by
limiting dilution (i.e., a dilute suspension of passage 27 Caco-2 cells
was prepared and distributed into the wells of microtiter plates such
that each well was expected to receive 0.5-1 cell; these wells were
observed microscopically, and the cells from wells that seemed to
contain only a single colony were propagated further in culture). For
the experiments described below, the clones were used at passages 9- 16, whereas the parent cell line was used at passage 24 or 27.
For our experiments, the cells were seeded at 6 × 105
cells/cm2 onto the membranes of culture inserts using
complete growth medium. The following day, the membranes were washed
three times, and fresh medium was added. Subsequent medium changes were
at 2-3-day intervals. In all experiments, protein analysis and
metabolic activity assay were done from a single 30-mm insert per time
point or per culture condition variation. RNA analysis was done from a
separate 12- or 10-mm insert. Millicell CM (Teflon membrane; 0.4 µm
pore size) culture inserts, which require an extracellular matrix
coating for cell adherence, were used primarily. All CM inserts were
coated with a dried film of Matrigel according to manufacturer's
(Millipore) instructions. Matrigel was thawed on ice, diluted with
ice-cold sterile water to 1.35 mg/ml, and dispensed (225 µg/cm2) into precooled inserts using precooled pipette
tips. The inserts were then allowed to dry overnight in a laminar flow
hood.
In our initial studies, we found that Caco-2 cells demonstrated a
marked tendency to pull away from the perimeter of Matrigel-coated inserts [as previously reported by others using Madin Darby Canine Kidney cells (20)] and form complex structures rather than maintaining a confluent monolayer pattern of growth, thereby making many of the
cultures unusable. This was worsened by increasing the amount of
Matrigel/cm2 of surface area of the inserts or using it as
a gel instead of a dried film. In a small trial to compare various
extracellular matrices, we used commercially coated PET inserts (1 µm
pore size). Caco-2 cells grown on Growth Factor-Reduced Matrigel,
laminin, collagen type IV, collagen type I, and polymerized collagen
type I (fibrillar collagen) demonstrated a monolayer pattern of growth and a more rapid achievement of confluence than did cells grown on
Matrigel. On the inserts commercially coated with unpolymerized collagen type I and, to a lesser degree, on the inserts coated with
collagen type IV, the Caco-2 cells demonstrated dome formation, indicating that there was impairment of water and electrolyte movement
across the membrane. This alone renders the inserts commercially coated
with unpolymerized collagen type I and collagen type IV unusable for
studies of drug absorption. In an experiment to study the effect of the
presence or absence of extracellular matrix, Millicell PCF inserts
(polycarbonate membrane; 3.0 µm pore size), which do not require a
matrix coating for cell adherence, were used with and without a
Matrigel coating.
On achieving confluence (transepithelial resistance of
250
·cm2 (21) as measured using a Millicell ERS device),
the basic medium was additionally supplemented (to the indicated final
concentrations) with sodium selenite (0.1 µM), zinc
sulfate (3 µM), and ferrous sulfate (5 µM).
Complete differentiation medium was then prepared by the addition of
5% heat-inactivated FBS. Where indicated, the medium of some cultures
was additionally supplemented with retinol acetate, menadione sodium
bisulfite,
-aminolevulinic acid, sodium pyruvate, sodium butyrate,
vitamin D2, 1
,25-(OH)2-D3,
25-(OH)-D3, or D3. In another experiment,
serum-free differentiation media were compared with the 5%
FBS-containing medium. The cells from all experiments were maintained
in their respective differentiation media for 2 weeks postconfluence
and then used for MDZ metabolism studies and/or harvested for analysis
of protein or mRNA.
A separate comparison was made using several different cell culture
media as the basic component of the 5% FBS-containing differentiation
medium used during the 2 weeks postconfluence. All of the complete
differentiation media in this trial were formulated to contain equal
amounts of glucose, L-glutamine, selenium, zinc, and
vitamin E, and all were supplemented with 0.25 µM
1
,25-(OH)2-D3. Results showed that CYP3A
catalytic activity was greater in cultures grown in DMEM, DMEM/F12
(1:1), or Williams' E based medium than in cultures grown in
Iscove's, Iscove's/F12/NCTC135 (5:5:1), RPMI 1640, McCoy's 5A,
Waymouth 752/1, Waymouth with 0.1 mM nonessential amino
acids, Medium 199, CMRL 1066, or CMRL/F12 (4:1) based medium (not
shown). However, the differences in catalytic activity among cells
grown in the various media were < 4-fold. Review of the media
components did not reveal any single factor shared by DMEM and
Williams' E media that is not also present in one or more of the other
media that did not perform as well.
An experiment to determine whether the duration of exposure to
1
,25-(OH)2-D3 could be shortened without
diminishing the expression of CYP3A was conducted in such a manner that
all cultures were of the same age (2 weeks postconfluence) at the time
of harvesting. On reaching confluence, the medium in all cultures was
changed from growth medium containing 20% FBS to differentiation
medium containing 5% FBS, but 0.25 µM
1
,25-(OH)2-D3 was added at different times
postconfluence and continued until the cells of each particular culture
were 2 weeks postconfluence. Each time point was represented by
duplicate cultures: one for mRNA analysis and another for immunoblots and catalytic activity assay.
For MDZ metabolism studies, the medium was aspirated, and the inserts
were washed three times with HBSS. Then, 1.5 ml of fresh complete
differentiation medium was placed on each side of the insert, with 4 µM MDZ present in the medium placed on only one side of
the monolayer. The incubation media did not contain vitamin D (except
that contributed by the 5% FBS).
Preparation for transmission electron microscopy.
The medium
was aspirated, and the inserts were washed three times with HBSS. The
monolayers were fixed in 2% glutaraldehyde/4% tannic acid in
cacodylate buffer, postfixed in 1% osmium tetroxide in cacodylate
buffer, dehydrated in a graded series of ethanol, and embedded in EPON
resin. Sections (70 nm thick) were prepared, poststained sequentially
with 4% uranyl acetate and Reynolds lead citrate, and examined with a
transmission electron microscope (model CM-10; Philips Electronics
Instruments).
Harvesting for immunoblots.
The medium was aspirated, and
the inserts were washed three times with HBSS containing 15 mM HEPES. Cells grown on Matrigel or Growth Factor-Reduced
Matrigel-coated inserts were removed from the membranes using Dispase
according to the manufacturer's directions. Then, 5 mM
EDTA in normal saline containing 1 mM PMSF and 1 mM benzamidine was added to inhibit further digestion. The cell suspension was transferred to a microcentrifuge tube and pelleted
by centrifugation at 325 × g. The pellets were washed three times with HBSS containing 15 mM HEPES, 1 mM PMSF, and 1 mM benzamidine. The final pellet
was resuspended in a solution of 20% v/v glycerol, 100 mM
Tris·HCl, pH 7.4, 10 mM EDTA, 1 mM dithiothreitol, 1 mM PMSF, 1 mM benzamidine,
and 100 µg/ml aprotinin and homogenized in a conical ground glass
tissue grinder with 20 passes of a ground-glass pestle. The homogenate
was subjected to 10 seconds of sonication and was then kept at
80°
until analysis.
Cells grown on uncoated inserts or inserts coated with non-Matrigel
matrices were harvested by scraping into HBSS containing HEPES and
protease inhibitors, washed three times, and then homogenized, sonicated, and frozen as above.
Immunoblots.
The protein concentrations of the cell
sonicates were measured according to the method of Bradford (22) using
bovine serum albumin standards. The sonicates were electrophoresed in
polyacrylamide gels containing 0.1% sodium dodecyl sulfate, and the
separated proteins were electrophoretically transferred to
nitrocellulose. Immunoblot development was performed as previously
described (23). CYP3A proteins were detected using a mouse monoclonal
antibody named 13-7-10 (24) (a gift from Dr. Pierre Kremers,
Université de Liège, Liège, Belgium). This antibody
detects all known forms of human CYP3A. CYP3A5 was detected using an
immunoadsorbed polyclonal rabbit antibody (25) received as a gift from
Dr. Steven A. Wrighton (Eli Lilly, Indianapolis, IN). CYP2D6 was
detected using a polyclonal rabbit antibody developed against unique
antigenic peptides (26) and obtained as a gift from Dr. Alastair Cribb
(Merck Research Laboratories, West Point, PA). CYP1A1 was detected
using a polyclonal goat antibody (Gentest, Woburn, MA) developed
against rat CYP1A1 and CYP1A2 that has been shown to also detect the
human forms of these proteins. Reductase and cytochrome
b5 were detected using polyclonal rabbit
antibodies developed against the specific rat proteins that were found
to also recognize the human forms of these proteins. Briefly, NADPH
cytochrome P450 reductase (reductase) was purified from
phenobarbital-induced adult rat liver microsomes as previously
described (27). Cytochrome b5 was purified from adult rat liver according to a method identical to that previously described for the purification of rabbit cytochrome
b5 (28). Rabbit polyclonal antibody was produced
against each antigen in female New Zealand White rabbits. Animals
received an initial subcutaneous injection (250 µg of reductase or
200 µg of cytochrome b5) in Freund's complete
adjuvant followed by an identical booster dose in Freund's incomplete
adjuvant 3 weeks later. Peripheral blood was collected over the next 20 weeks, and serum IgG was isolated as previously described (29). Pgp was
detected using a polyclonal rabbit antibody (Oncogene Science,
Uniondale, NY). Villin was detected using a mouse monoclonal antibody
(Chemicon International, Temecula, CA). IFABP and LFABP were detected
using polyclonal rabbit antibodies developed against rat proteins that have been shown to detect the human forms of these proteins (30); these
antibodies were received as gifts from Dr. Jeffrey Gordon (Washington
University School of Medicine, St. Louis, MO). Secondary antibodies of
appropriate specificities conjugated with horseradish peroxidase were
obtained commercially (rabbit anti-mouse IgG and goat anti-rabbit
IgG/A/M from Zymed Laboratories, San Francisco, CA; mouse anti-goat
IgG/A/M from Pierce, Rockford, IL). Binding of secondary antibodies was
detected using the ECL reagents and film from Amersham (Arlington
Heights, IL).
In some cases, immunoblot protein concentrations were determined by
computer-aided densitometry. Optical densities were performed on a
Macintosh computer using the public-domain NIH Image
program.1 Individual exposures were scanned
into binary images with a ScanJetIIc color scanner (Hewlett Packard,
Greeley, CO). An absorbance-concentration standard curve was prepared
using the bands from second loaded serial dilutions of purified CYP3A4
protein, and the absorbances of unknowns were converted to quantitative
numbers by comparison to this curve. This allowed for correction of the
nonlinearity of ECL light output and ECL Hyperfilm.
Harvesting for mRNA analyses; RNA isolation.
The medium was
aspirated, and the culture inserts were washed three times with HBSS
containing 15 mM HEPES. Chomczynski and Sacchi's
denaturing solution (31) was applied to the monolayer (333 µl/cm2 insert membrane surface area). After 5 min, the
cell lysate was transferred to a microcentrifuge tube and stored at
80°. The lysate was later thawed quickly at 65°, and total RNA
was isolated according to the published protocol (31), adjusting
reagent volumes proportionately as appropriate for the initial lysate volume.
mRNA analyses.
cDNA was prepared from the total RNA as
previously described (32). The polymerase chain reaction was performed
on 1 µl of a 1:10 dilution in water of a prepared cDNA. Primer
sequences for amplification of CYP3A3, CYP3A4, CYP3A5, and CYP3A7
(product sizes of 619, 382, 350, and 469 bp, respectively) cDNAs were
as previously described (33), with the following exceptions: the antisense primer for CYP3A5 was 5
-TTC TGG TTG AAG AAG TCC TTG CGT
GTC-3
, and the first sense primer (33) for CYP3A3 was paired with the
second antisense primer (33). The primers for mdr-1 cDNA
(34) amplification were 5
-GTC ATT GTG GAG AAA GGA AAT CAT G-3
and
5
-ATT CCA AGG GCT AGA AAC AAT AGT G-3
, product size of 478 bp. The
primers for IFABP cDNA (35) amplification were 5
-AGG AAG CTT GCA GCT
CAT GAC AAT TTG AAG-3
and 5
-AGT ATT CAG TTC GTT TCC ATT GTC TGT
CCG-3
, product size of 231 bp. The primers used for villin cDNA (36,
37) amplification were 5
-CAG CTA GTG AAC AAG CCT GTA GAG GAG CTC-3
and 5
-GCC ACA GAA GTT TGT GCT CAT AGG CAC ATC-3
, product size of 303 bp. If CYP3A5 and CYP3A3 are organized in the
same fashion as CYP3A4 (38) and CYP3A7 (39), then
all synthetic oligonucleotide primer pairs used spanned at least one
intron. PCR mixtures were as previously described (32). Each thermal
cycle consisted of 95° for 1 min and then 65° for 1 min 15 sec;
after completing all cycles, a 10-min extension step at 65° was
performed. PCR products were electrophoresed on agarose gels and
stained with ethidium bromide. A reagent control, included in each PCR
run, was confirmed to be negative when run on the gel. PCR products
were judged to be of the size appropriate for amplification of the
specific cDNA by comparison with molecular weight standards included on
each gel. The sequence of a PCR product produced from Caco-2 cell cDNA by each primer pair was determined by the method of Sanger et al. (40) using the plasmid pCR II (InVitrogen, San Diego, CA) and
was confirmed to be consistent with the published specific cDNA
sequence.
An internal standard for quantitative competitive PCR (41, 42) of
CYP3A4 cDNA was prepared by inserting a 30-bp segment into the cDNA
sequence to be amplified. PCR mixtures were prepared by combining
serial dilutions of a known amount of the standard cDNA and constant
amounts of the unknown cDNA. The two cDNAs were amplified together, and
the products were separated on agarose gels. After staining with
ethidium bromide, the two bands were quantified densitometrically. An
equivalence point (at which the densities of the standard and unknown
bands are of equal absorbance) was determined; the concentration of the
unknown is equal to that of the standard at the point of equivalence.
Evidence supports the assumption that the insertion of the 30-bp
segment into the standard does not significantly alter amplification
efficiency.2
MDZ, 1
-hydroxymidazolam, and 4-hydroxymidazolam assays.
Measurement of the rate and extent of MDZ 1
-hydroxylation was used as
the principal means to assess CYP3A catalytic activity in Caco-2 cell
cultures. All culture samples were analyzed in duplicate, and the mean
of the two measurements is reported. For quantification of 1
-OH-MDZ,
0.2-1.0 ml of medium removed from the apical or basolateral
compartment of a Caco-2 cell culture was diluted with water to a volume
of 1 ml. To this was added 1 ml of 50 mM
Na2CO3, pH 12.5, and 20 ng of
1
-[2H2]1
-OH-MDZ internal standard. For the
analysis of MDZ, 0.02 ml of culture medium was diluted to a volume of 1 ml. To this was added 1 ml of 50 mM
Na2CO3, pH 12.5, and 40 ng of
15N3-MDZ internal standard. Standards were also
prepared in duplicate. Known amounts of MDZ and 1
-OH-MDZ (1-50 ng)
were combined with 100 mM potassium phosphate buffer, pH
7.4. To this was added 1 ml of 50 mM
Na2CO3, pH 12.5, and the appropriate amount of
stable isotope-labeled internal standard.
Unknowns and standards were extracted with 5 ml of ethyl acetate, and
the upper organic layer was transferred to clean tubes. The solvent was
evaporated to dryness under a stream of nitrogen. The remaining solid
was reconstituted in 100 µl of derivatizing reagent [10%
N-methyl-N-(t-butyldimethylsilyl)trifluoroacetamide in acetonitrile]. Samples were transferred to glass autoinjector vials, sealed, and heated at 80° for 2 hr before analysis by gas chromatography-selective ion mass spectrometry (9).
To determine whether the glucuronide conjugate of 1
-OH-MDZ was formed
during Caco-2 cell incubations, 0.25-ml aliquots of apical and
basolateral culture medium from cells incubated for 2-24 hr with 4 µM MDZ (administered apically) were diluted to a volume
of 0.5 ml and added to 0.5 ml of 100 mM sodium acetate, pH
5.0, containing 200 units of
-glucuronidase. Samples were spiked
with 20 ng of 1
-[2H2]1
-OH-MDZ and incubated
at 37° for 24 hr. Each of a complete set of parallel 0.25-ml samples
was diluted to a volume of 1 ml and spiked with 20 ng of
1
-[2H2]1
-OH-MDZ (no glucuronidase
treatment). All samples were then alkalinized and processed as above,
and the 1
-OH-MDZ concentrations were quantified by mass spectrometry.
The effect of verapamil on MDZ apical/basolateral distribution and
1
-OH-MDZ formation and distribution in Caco-2 cell cultures was also
examined. Verapamil (100 µM) was added to the apical medium of 0.25 µM
1
,25-(OH)2-D3 treated 2 weeks postconfluent Caco-2 cells simultaneously with apical or basolateral administration of 4 µM MDZ. After incubations of 0-24 hr, both apical
and basolateral compartments were sampled for measurement of MDZ and
1
-OH-MDZ concentrations.
In selected cultures, the minor CYP3A metabolite, 4-OH-MDZ, was
quantified along with 1
-OH-MDZ after incubations with 4 µM MDZ added to the apical medium. For the assay,
base-treated culture medium was spiked with
15N3-1
-OH-MDZ and
15N3-4-OH-MDZ (~50 ng/10 ng fixed ratio) and
processed as described above. Standard curves for both 1
-OH-MDZ (1-50
ng) and 4-OH-MDZ (0.5-25 ng) were also prepared. The only
modifications to the mass spectrometric assay involved monitoring
molecular ions with m/z 455 and 460 (for unlabeled and
15N3-labeled 37Cl isotope of
1
-OH-MDZ, respectively) and base peak fragment ions
([M-t-Bu(CH3)2SiOH]
)
with m/z 323 and 328 (for unlabeled and
15N3-labeled 37Cl isotope of
4-OH-MDZ, respectively). 4-OH-MDZ exhibited a slightly shorter gas
chromatography column retention time than 1
-OH-MDZ (12.7 versus 14.0 min), under previously defined gas chromatographic conditions (9).
For comparison, 1
-OH/4-OH-MDZ product ratios were also measured in
incubations of human jejunal microsomes. Microsomes were prepared from
12 different organ donors, as previously described (8) and stored at
70°. Protein concentrations were determined according to the method
of Lowry et al. (43) using bovine serum albumin standards.
Each preparation was analyzed by immunoblotting for CYP3A4 and CYP3A5
content (29). Six were preselected for determination of the MDZ
metabolite ratio, based on the detection of both CYP3A4 and CYP3A5
(n = 3) or CYP3A4 only (n = 3).
Microsomal incubations were carried out in duplicate as previously
described (8) using 100-200 µg of microsomal protein, with the final MDZ concentration set at 0, 0.25, 1, 4, or 8 µM.
Alkalinized incubation samples were spiked with ~50 ng
15N3-1
-OH-MDZ and ~10 ng
15N3-4-OH-MDZ (fixed ratio) and processed for
mass spectrometric analysis as described above.
Determination of 1
-hydroxymidazolam intrinsic formation
clearance.
An intrinsic 1
-OH-MDZ formation clearance was
determined from the 24-hr time course data for 1
-OH-MDZ formation in
confluent cultures dosed apically with 4 µM MDZ. Parent
drug and metabolite concentrations measured at 1 and 2 hr after MDZ
administration were used for computation. The amount of 1
-OH-MDZ
formed during this interval was determined as the sum of metabolite in
the apical and basolateral compartments at 2 hr minus the total amount
measured at 1 hr. The rate of product formation was normalized for the total mass of Caco-2 cells in each culture insert (an average of 65 mg)
measured at the end of the MDZ incubation period (after Dispase
digestion, washing, and centrifugation of the cells).
Because the incubation medium contained significant amounts of FBS, the
fraction of MDZ bound to culture medium proteins was measured by
equilibrium dialysis (8). The unbound concentration of MDZ in the
apical and basolateral compartments during cell culture was calculated
as the product of the experimentally determined MDZ free fraction and
total MDZ concentration. Because MDZ concentrations in the apical and
basolateral compartments were not at equilibrium during the 1-2-hr MDZ
incubation interval, the intracellular reaction was assumed to be
driven by a MDZ concentration that was more closely aligned with the
concentration in the basolateral compartment. Thus, the intrinsic
1
-OH-MDZ formation clearance (assuming subsaturating MDZ
concentrations) was calculated as the ratio of the 1
-OH-MDZ formation
rate and the average (1-2 hr) unbound MDZ concentration in the
basolateral compartment. This calculation assumes that any potential
MDZ transport mechanism is located in the apical membrane.
For comparison, an intrinsic 1
-OH-MDZ formation clearance was also
determined for eight paired human duodenal and jejunal mucosae. The
mucosal mass was removed from ~1-foot sections of duodenum and
jejunum and total homogenate and microsomes were prepared, as described
above. Protein concentrations for homogenate and microsomes were
measured according to the method of Lowry et al. (43). The
specific CYP3A4 content (pmol/mg of protein) in homogenate and
microsomes was measured by immunoblot analysis (29). The total amount
of microsomal protein per gram of mucosa was determined from the
following relationship:
where
This calculation assumes that CYP3A4 found in mucosal homogenate
resides exclusively within the microsomal fraction. The intrinsic
1
-OH-MDZ formation clearance/g of mucosa was calculated as the product
of the microsomal intrinsic formation clearance and the computed amount
of microsomal protein/g of mucosa.
 |
Results |
To obtain genetic homogeneity among the cells to be used in our
experiments, five clones were prepared from the parent Caco-2 cell line
at passage 27. As judged by relative band intensities of RT-PCR
products on ethidium-stained gels, all five of the clones seemed to
express greater levels of CYP3A4 mRNA than did the parent cell line
(not shown). The clone (no. 5) that seemed to have the highest level of
expression of CYP3A4 mRNA was found to have poor growth
characteristics. A clone (no. 7) with an intermediate level of CYP3A4
mRNA expression was therefore selected for use in the experiments
described below.
To define culture conditions that favored expression of CYP3A4 by
Caco-2 cells, RT-PCR was used to assess relative levels of CYP3A4 mRNA
in 2-week postconfluent cells that had been grown with or without
Matrigel or had been exposed to test compounds added to the
differentiation medium beginning at the time of confluence. The
achievement of confluence was delayed on the Matrigel-coated inserts
(average, ~12-25 days) compared with uncoated polycarbonate inserts
(~7 days). However, the levels of CYP3A4 mRNA (as well as villin and
IFABP mRNAs) generally seemed to be higher when using Matrigel-coated
inserts (not shown), and therefore Matrigel was used in subsequent
cultures. Vitamin A (retinol acetate, 0.3 µM), vitamin K
(menadione sodium bisulfite, 145 nM), vitamin
D2 (ergocalciferol, 0.63 µM),
-aminolevulinic acid (50-100 µM), sodium butyrate (2 mM, in the presence or absence of vitamin D2), or sodium pyruvate (1 mM) did not seem to enhance CYP3A4
mRNA expression. However, the addition of
1
,25-(OH)2-D3 (1
,
25-dihydroxycholecalciferol, 0.63 µM) to the medium was
found to result in a dramatic increase in the expression of CYP3A4
mRNA, and this effect was studied further.
A dose-response experiment was performed with concentrations of
1
,25-(OH)2-D3 that spanned the original 0.63 µM concentration for 2 weeks, administered beginning at
the time of confluence. 1
,25-(OH)2-D3
resulted in increased CYP3A4 mRNA expression at all concentrations used
(0.05-1.00 µM) (Fig. 1). An attempt was made to quantify the fold increase in CYP3A4 mRNA using serial dilutions of an internal standard for competitive amplification and
titration (see Experimental Procedures). Due to the very low levels of
CYP3A4 mRNA present in untreated cells, precise quantification of the
fold induction was difficult, but it appeared that a 40-80-fold increase had occurred. Expression of the closely related CYP3A7 and
CYP3A5 mRNAs (44) also seemed to increase in response to 1
,25-(OH)2-D3, but the increase in CYP3A5
mRNA seemed to be of much lesser magnitude (Fig. 1). The effect of
1
,25-(OH)2-D3 was not a general effect on
all mRNAs, however, because the levels of mdr-1, IFABP, and
villin mRNAs did not seem to increase (Fig. 1). CYP3A3 mRNA was not
detected in treated or untreated Caco-2 cells by
RT-PCR.3

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Fig. 1.
Changes in Caco-2 cell levels of specific mRNAs in
response to treatment with varying doses of
1 ,25-(OH)2-D3. Caco-2 cells grown on
Matrigel-coated Teflon culture inserts were treated with varying
concentrations of 1 ,25-(OH)2-D3 for 2 weeks
beginning at the time of confluence. Total RNA was then prepared and
subjected to RT-PCR using synthetic oligonucleotide primer pairs
designed to selectively amplify fragments of the indicated cDNAs. One
culture was used for each concentration of
1 ,25-(OH)2-D3 (number of PCR cycles: CYP3A4,
36; CYP3A5, 33; CYP3A7, 39; mdr-1, 34; IFABP, 32;
villin, 27). PCR was done twice with each pair of primers, and the
results shown are representative.
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The effects of equal concentrations of
1
,25-(OH)2-D3, 25-(OH)-D3, and
D3 were then compared in parallel cultures. The CYP3A substrate MDZ (45) (4 µM), was administered apically
after 2 weeks of treatment with the vitamin D analogs (i.e., at 2 weeks postconfluence). Treatment with
1
,25-(OH)2-D3 resulted in a dramatic increase in the extent of MDZ metabolism (Fig. 2A;
apical concentrations shown). Increases in CYP3A catalytic activity
(Fig. 2A) and immunoreactive protein (Fig. 2C) were evident at 0.05 µM 1
,25-(OH)2-D3, and both had
plateaued at 0.25-0.5 µM
1
,25-(OH)2-D3. Untreated Caco-2 cells
contained ~7.9 pmol of total CYP3A/mg of protein, whereas the maximal
expression (at 0.5 µM 1
,
25-(OH)2-D3) corresponded to 20.6 pmol of total
CYP3A/mg of cell protein. For comparison, the mean ± standard
deviation CYP3A4 content in mucosal homogenates prepared from human
duodenum and jejunum was found to be 9.2 ± 6.2 and 8.4 ± 5.4 pmol/mg of protein, respectively (Table 1).

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Fig. 2.
Changes in Caco-2 cell levels of CYP3A catalytic
activity and immunoreactive protein in response to treatment with
varying doses of 1 ,25-(OH)2-D3,
25-(OH)-D3, and D3. Caco-2 cells grown on
Matrigel-coated Teflon culture inserts were treated with varying concentrations of 1 ,25-(OH)2-D3,
25-(OH)-D3, or D3 for 2 weeks beginning at the
time of confluence. The medium was then replaced with medium not
supplemented with vitamin D. MDZ (4 µM) was added to the
apical medium. After 6 hr, the apical and basolateral media were
removed separately and analyzed for 1 -OH-MDZ concentration. A and B,
Data from the apical media. C, Results of immunoblots of cell sonicates
(10 µg of protein/lane) developed with a monoclonal antibody that
recognizes all known human forms of CYP3A. Each point on
the graphs represents one culture and corresponds to one
lane on the immunoblots. A composite blot from two sets
of cultures is shown for 25-(OH)-D3, which required higher
doses than did 1 ,25-(OH)2-D3 to achieve a
maximal response.
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TABLE 1
CYP3A content and MDZ hydroxylation activity in human duodenum and
jejunum
Total homogenate and microsomes were prepared from mucosal scrapings of
paired human duodenum and jejunum from eight different donors. Protein
concentration and specific CYP3A content for each tissue fraction was
measured as described in Experimental Procedures. Two of eight
preparations contained relatively low amounts of CYP3A5 in addition to
CYP3A4, which were resolved from one another by SDS-polyacrylamide gel
electrophoresis. Microsomes were incubated with varying concentrations
of MDZ for determination of Km and Vmax parameters for 1 -hydroxylation. The
microsomal intrinsic formation clearance was calculated as the ratio of
Vmax to Km. The mucosal
intrinsic formation clearance was calculated from the product of
microsomal intrinsic clearance and an experimentally determined
concentration of microsomal protein in the total mucosal mass (see
text).
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In the initial dose-response experiment using 25-(OH)-D3,
CYP3A catalytic activity and immunoreactive protein began to increase at 0.25 µM 25-(OH)-D3 but the effect had not
yet plateaued by 1.0 µM 25-(OH)-D3, and
therefore a dose-response experiment extending to higher concentrations
of this agent was performed. CYP3A catalytic activity (Fig. 2B) was
maximal at 5 µM 25-(OH)-D3 and had
substantially declined by 15 µM, declining still further
at 20 µM 25-(OH)-D3. Changes in CYP3A
immunoreactive protein followed a similar pattern (Fig. 2C). The
maximal CYP3A catalytic activity achieved with 25-(OH)-D3
was not as great as that achieved with
1
,25-(OH)2-D3. Unhydroxylated D3
had no effect on immunoreactive CYP3A protein or MDZ metabolism, but
concentrations of >1.0 µM were not examined.
Immunoblots showed little change in the level of CYP3A5 in response to
1
,25-(OH)2-D3 (Fig. 3) or to
25-(OH)-D3 (not shown). Levels of CYP3A7 could not be
assessed because a specific antibody is not available. Levels of CYP1A1
and CYP2D6 did not seem to change in response to
1
,25-(OH)2-D3 (Fig. 3) or
25-(OH)-D3 (not shown). The level of reductase seemed to
increase in response to concentrations of
1
,25-(OH)2-D3 of
0.05 µM
(Fig. 3) and concentrations of 25-(OH)-D3 of
0.5
µM (not shown). An antibody to cytochrome b5 recognized a protein on immunoblots whose
level of expression did not seem to change in response to either
1
,25-(OH)2-D3 (Fig. 3) or
25-(OH)-D3 (not shown). The cytochrome
b5 immunoreactive protein in the Caco-2 cells
did not comigrate with the major immunoreactive protein present in
adult small bowel and liver but did comigrate with a smaller
immunoreactive protein present in duodenal homogenate but not detected
in human liver microsomes (Fig. 3).

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Fig. 3.
Caco-2 cell levels of a variety of proteins in
response to varying doses of 1 ,25-(OH)2-D3.
Caco-2 cells grown on Matrigel-coated Teflon culture inserts were
treated with varying concentrations of
1 ,25-(OH)2-D3 for 2 weeks beginning at the
time of confluence. The results of immunoblots of cell sonicates,
developed with several antibodies as described in Experimental
Procedures, are shown. Each 1 ,25-(OH)2-D3
concentration represents one culture. These are the same
1 ,25-(OH)2-D3 treated cultures as were used
for catalytic activity and immunoblotting in Fig. 2. Caco-2 protein loads were 10 µg for CYP3A, CYP3A5, Pgp, and villin; 25 µg for CYP1A1 and CYP2D6; 40 µg for IFABP and LFABP; and 80 µg for
reductase and cytochrome b5. Human duodenal
biopsy homogenate protein loads were 25 µg for CYP3A, Pgp, and
villin; 40 µg for CYP1A1; 50 µg for CYP2D6; 60 µg for IFABP and
LFABP; and 80 µg for cytochrome b5. Human
liver protein loads were 3 µg for CYP1A2 and CYP2D6, 10 µg for
cytochrome b5, 12 µg for CYP3A5, 25 µg
for reductase, and 40 µg for LFABP. Note that liver microsomes were
used except for LFABP, for which liver homogenate was used.
ND, not done. Quantification of the CYP3A immunoreactive
bands (top) by computer-aided densitometry, using second
loaded serial dilutions of purified CYP3A4 protein, revealed that the
band from untreated cells (lane 1) corresponded to 7.9 pmol of CYP3A/mg of total protein, whereas the band from cells treated
with 0.5 µM 1 ,25-(OH)2-D3
(lane 6) corresponded to 20.6 pmol of CYP3A/mg of total
protein. Thus, visual interpretation of immunoblots developed with ECL
may overestimate fold increases in levels of expression.
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The effect of 1
,25-(OH)2-D3 and
25-(OH)-D3 on the expression of Pgp was examined because
many substrates of this transport protein are also CYP3A4 substrates
(46, 47). 1
,25-(OH)2-D3 (Fig. 3) and
25-(OH)-D3 (not shown) resulted in a modest increase in
levels of immunoreactive Pgp at concentrations of
0.5
µM and
2.5 µM, respectively.
As control proteins, the responses of IFABP, LFABP, and villin
expression to 1
,25-(OH)2-D3 and
25-(OH)-D3 were examined. Neither IFABP nor LFABP showed
changes in levels of expression on immunoblots in response to
1
,25-(OH)2-D3 (Fig. 3) or
25-(OH)-D3 (not shown). Levels of villin immunoreactive
protein seemed to decrease slightly in response to 1 µM
1
,25-(OH)2-D3 (Fig. 3) or concentrations of
25-(OH)-D3 of
10 µM (not shown).
To try to elucidate whether the conditions we had defined for optimal
expression of CYP3A4 resulted in a general increase in differentiation,
electron micrographs of Caco-2 cells grown in the presence or absence
of Matrigel and with or without a 2-week exposure to 0.25 µM 1
,25-(OH)2-D3 were
obtained. Ultrastructural differences to indicate differing degrees of
differentiation were not detected by an experienced observer (W. O. Dobbins III) who was blinded to the treatments. Measurements of cell
height (~16.5 µm) and microvillar height (~1.02 µm) were
not clearly affected by the presence or absence of vitamin D or
Matrigel. These measurements are similar to those obtained by Wilson
et al. (17.2 and 0.94 µm), who used uncoated
nitrocellulose filters (12), but less than those obtained by Hidalgo
et al. (29.6 and 1.19 µm), who used collagen type I-coated
polycarbonate filters (11). The microvillar height was also less than
that reported by Halline et al. (1.52 µm) for Caco-2 cells
grown on plastic and treated with 0.1 µM
1
,25-(OH)2-D3 in medium containing 20% FBS
(48). A representative Caco-2 cell grown on a Matrigel-coated
polycarbonate membrane and treated with
1
,25-(OH)2-D3 is shown in Fig.
4.

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Fig. 4.
Electron micrographs of Caco-2 cells grown on a
Matrigel-coated polycarbonate culture insert and treated with 0.25 µM 1 ,25-(OH)2-D3 for 2 weeks
beginning at the time of confluence. Magnification, 10,905×.
A, apical compartment; ECM, Matrigel
extracellular matrix; M, insert membrane;
P, pore; B, basolateral compartment.
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Because 1
,25-(OH)2-D3 is quite expensive, an
experiment was undertaken to determine whether the duration of exposure
to 1
,25-(OH)2-D3 could be shortened without
diminishing the expression of CYP3A. The experiment was conducted so
that all cultures were of the same age (2 weeks postconfluence) at the
time of harvesting. An increase in CYP3A4 mRNA was evident after 3-4
hr of exposure to 0.25 µM
1
,25-(OH)2-D3 and, by computer aided
densitometry, seemed to plateau at 120 hr (5 days) of exposure to
1
,25-(OH)2-D3 (Fig. 5A).
Increased CYP3A protein expression became evident on the immunoblots at
18 hr and continued to rise throughout the remainder of the 2-week
experiment (Fig. 5A). MDZ metabolism showed a similar pattern, first
becoming detectable in cultures exposed to
1
,25-(OH)2-D3 for 12 hr and then continuing
to rise throughout the remainder of the 2-week experiment (Fig. 5B).
Immunoreactive reductase did not seem to increase until 168 hr (7 days)
of exposure to 1
,25-(OH)2-D3 (Fig. 5A).
Immunoreactive Pgp seemed to increase beginning at ~72 hr of exposure
to 1
,25-(OH)2-D3 (Fig. 5A). The levels of villin mRNA and immunoreactive protein did not seem to change over the
2-week course of the experiment (Fig. 5A).

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Fig. 5.
Duration of exposure to
1 ,25-(OH)2-D3 necessary to achieve increased
expression of (A) CYP3A4 mRNA and CYP3A4, reductase, and Pgp
immunoreactive proteins and (B) CYP3A catalytic activity by Caco-2
cells. Each time point represents two different cultures: one used for
mRNA analysis by RT-PCR, and another used for immunoblots of cell
sonicates and catalytic activity. Caco-2 cells were grown on
Matrigel-coated Teflon culture inserts and were treated with 0.25 µM 1 ,25-(OH)2-D3 for varying
periods. The experiment was conducted such that all cultures were of
the same age (2 weeks postconfluence) at the time of harvesting (i.e.,
on reaching confluence, the medium in all cultures was changed from
growth medium containing 20% FBS to differentiation medium containing
5% FBS, but 0.25 µM
1 ,25-(OH)2-D3 was added at different times
postconfluence and continued until the cells of each particular culture
were 2 weeks postconfluence). The medium was then replaced with medium not supplemented with vitamin D. MDZ (4 µM) was added to
the apical medium. After 6 hr, the amount of 1 -OH-MDZ was measured in
the combined luminal plus basolateral medium from each culture, and the
rate of formation was calculated (numbers of PCR cycles: villin, 27;
CYP3A4, 36; immunoblot protein loads: 5 µg for CYP3A, Pgp, villin; 40 µg for reductase).
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We next undertook time course experiments in which 4 µM
MDZ was administered for varying lengths of time to either the apical or basolateral medium of Caco-2 cells cultured on Matrigel and exposed
to 1
,25-(OH)2-D3 for 2 weeks postconfluence.
1
-OH-MDZ (Fig. 6A) became detectable by 1 hr after MDZ
administration to either compartment. The rate of 1
-OH-MDZ formation
seemed to slow slightly over time but remained roughly constant after 6 hr. At every time point, the concentration of 1
-OH-MDZ was greater in
the apical medium than in the basolateral medium (Fig. 6A), even when
MDZ had been administered basolaterally. The apical concentration of
parent compound after apical administration (Fig. 6B) declined over the
first 12 hr, but a stable ~1.4-fold apical to basolateral
concentration gradient was then maintained for the remainder of the 24 hr. By 12 hr, the parent compound had also been distributed
preferentially to the apical medium after basolateral administration
(Fig. 6B).

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Fig. 6.
Time course of MDZ metabolism by Caco-2 cells
treated with 1 ,25-(OH)2-D3 and the effect of
verapamil. Caco-2 cells were grown on Matrigel-coated Teflon culture
inserts and treated with 0.25 µM
1 ,25-(OH)2-D3 for 2 weeks beginning at the
time of confluence. The medium was then replaced with medium not
supplemented with vitamin D. MDZ (4 µM) was added to the
apical or basolateral medium. C and D, In selected cultures, 100 µM verapamil was applied apically. After incubation, the
apical and basolateral media were removed separately and analyzed for
concentrations of 1 -OH-MDZ and MDZ. Each point (A and
C) represents metabolite data from a separate culture, with
corresponding parent compound data (B and D). Note the different scales
for A and C. , Apical medium after apical administration of MDZ.
, Basolateral medium after apical administration of MDZ. , Apical
medium after basolateral administration of MDZ. , Basolateral medium
after basolateral administration of MDZ.
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Aliquots of the apical and basolateral media at various time points
after apical administration of MDZ were subjected to treatment with
glucuronidase before being assayed for 1
-OH-MDZ. There were no
differences between the 1
-OH-MDZ concentrations obtained after glucuronidase treatment (not shown) and the concentrations obtained without hydrolysis, indicating that glucuronidation of 1
-OH-MDZ had
not occurred in the Caco-2 cells.
We next examined the effect of adding verapamil (100 µM)
to the apical compartment during MDZ metabolism time courses (Fig. 6, C
and D). This resulted in a ~90% inhibition of 1
-OH-MDZ formation (Fig. 6C versus 6A), but the apical-to-basolateral concentration gradients of both parent MDZ (Fig. 6D) and 1
-OH metabolite (Fig. 6C)
were similar to those seen in the absence of verapamil (Fig. 6, B and
A, respectively).
An intrinsic 1
-OH-MDZ formation clearance was calculated, as outlined
in Experimental Procedures, from the time course data obtained in the
cultures dosed apically with MDZ in the absence of verapamil. The rate
of 1
-OH-MDZ formation during the 1-2-hr interval was found to be 75.9 pmol/min/g of cells (Table 2). Under equilibrium
dialysis conditions, ~89.8% of MDZ was found to be bound to medium
proteins, yielding an unbound fraction of 10.2%. Thus, the total and
unbound MDZ concentrations in the basolateral compartment were
estimated to be 650 and 66.3 nM, respectively. The
intrinsic 1
-OH-MDZ formation clearance under nonsaturating conditions
[Km ~3.4
µM (8)] was estimated to be 1.14 ml/min/g of
cells. The mean intrinsic 1
-OH-MDZ formation clearance for eight
different human duodenal and jejunal microsomes was found to be 133.8 and 118.3 µl/min/mg of protein, respectively (Table 1). Values for
each section of intestine were quite variable, with an interindividual
variation of 15- and 20-fold for duodenum and jejunum, respectively.
The average microsomal protein content (± standard deviation) in
duodenal and jejunal mucosa was found to be 25.4 ± 14.4 and
27.7 ± 18.7 mg of protein/g of mucosa, respectively. Taking the
product of the microsomal intrinsic 1
-OH-MDZ formation clearance and
microsomal protein content in duodenal and jejunal mucosa, we arrived
at intrinsic clearance values of 3.83 ± 3.62 and 3.67 ± 3.81 ml/min/g of mucosa, respectively (Table 1). Thus, the intrinsic
1
-OH-MDZ formation clearance for
1
,25-(OH)2-D3-pretreated Caco-2 cells
compared quite respectably with that for human intestinal mucosa,
representing 30% and 31% of the mean intrinsic formation clearance
determined for human duodenal and jejunal mucosa, respectively.
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TABLE 2
MDZ 1 -hydroxylation kinetics in Caco-2 cell cultures at the 1-2-hr
incubation interval
MDZ (4 µM) was added at time zero to the medium in the
apical compartment of replicate Caco-2 cell cultures that had been treated with 0.25 µM
1 ,25-(OH)2-D3 for 2 weeks after confluence. 1 ,25-(OH)2-D3 was not present in the MDZ
incubation medium. Both apical and basolateral media were sampled 1 and
2 hr later (from separate cultures) for measurement of MDZ and
1 -OH-MDZ. The unbound fraction of MDZ in culture medium, measured by
equilibrium dialysis, was found to be 10.2%. Calculations were
performed as described in Experimental Procedures.
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All CYP3A isoforms catalyze both the 1
- and 4-hydroxylation of MDZ.
Different 1
-OH-MDZ/4-OH-MDZ product ratios are produced by the three
known human CYP3A isoforms (CYP3A4, CYP3A5, and CYP3A7) (49). For
CYP3A4, metabolism of MDZ at the 1
-position predominates, although
hydroxylation at the 4-position is increasingly favored with increasing
MDZ concentrations (49). The 1
-OH-MDZ/4-OH-MDZ product formation ratio
was measured in selected Caco-2 cell cultures (Table 3)
as part of the CYP3A isoform characterization. There was an increase in
the product ratio from 3.8 to 5.3 with an increase in duration of
treatment (12 versus 336 hr) with
1
,25-(OH)2-D3. Clone 5, which maximally
expressed CYP3A immunoreactive protein, had sufficient metabolic
activity for measurements to be made in both the
1
,25-(OH)2-D3-treated and untreated states.
In the 1
,25-(OH)2-D3-treated culture, the
product ratios were 5.4 and 5.2 for apical and basolateral
compartments, respectively, whereas in the untreated culture, the
product ratios were slightly lower at 4.5 (apical) and 3.6 (basolateral). In cultures of clone 7, which was used for all of the
other experiments described here, a product formation ratio of >5 was
obtained in 1
,25-(OH)2-D3-treated cells
grown on either laminin or Matrigel. In general, product formation
ratios determined for intestinal microsomes (Table 4) were higher than those found in Caco-2 cell cultures. For those intestines that contained only CYP3A4, the 1
-OH-MDZ/4-OH-MDZ ratios
varied from 7.4 to 9.0 for incubations with 0.25 µM MDZ and from 5.6 to 5.8 for incubations with 8 µM MDZ. As
expected, because CYP3A5 gives a higher product ratio than does CYP3A4
(49), higher ratios were generated in incubations with intestinal
microsomes containing both CYP3A4 and CYP3A5. Values of 11.1 to 15.4 and 6.7 to 9.7 were measured at 0.25 and 8 µM MDZ,
respectively. Thus, product ratios from Caco-2 cell cultures were most
similar to those from intestinal microsomes that contained only CYP3A4,
although lower than those from intestinal microsome incubations with
the most comparable concentrations (0.25-1.0 µM) of MDZ
(predicted unbound intracellular concentrations in the Caco-2 cells
were ~0.07 µM).
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TABLE 3
1 -OH-MDZ/4-hydroxymidazolam formation ratios from incubations of MDZ
with cultured Caco-2 cells
Clone 5 is the clone that maximally expresses CYP3A immunoreactive
protein, whether treated or untreated with
1 ,25-(OH)2-D3. Clone 7 is the clone that was
used for all of the other experiments described.
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TABLE 4
1 -OH-MDZ/4-OH-MDZ metabolite concentration ratios from incubations of
MDZ with human jejunal microsomes
Microsomes prepared from the jejunum of each of six subjects were
selected based on the results of prescreening for the presence of
CYP3A4 only or CYP3A4 and CYP3A5. The microsomes from each of these
subjects were incubated with varying concentrations of MDZ. Metabolite
formation was quantitated as described in Experimental Procedures. The
ratios of 1 -OH-MDZ/4-OH-MDZ for all permutations are presented.
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Several other experiments were performed to further characterize our
Caco-2 culture model. The use of a particular clone may not be a
necessary component to the model because all five of our clones as well
as the parent cell line showed increased levels of CYP3A immunoreactive
protein after 2 weeks of treatment with 0.25 µM
1
,25-(OH)2-D3 (Fig. 7B). The
MDZ 1
-hydroxylation activity of each cell line with and without
1
,25-(OH)2-D3 treatment (Fig. 7A) correlated
quite closely with the level of CYP3A immunoreactive protein
(p = 0.0003, r = 0.987). There
was little variability of reductase expression among the clones and
parent cell line when untreated, but there was variable responsiveness
to 1
,25-(OH)2-D3; clone 7 had a response
similar to that of the parent cell line but greater than that of clone
5 (Fig. 7B). There was considerable variability among the clones with
respect to levels of expression of immunoreactive LFABP, with clone 7 being a relatively high expressor (not shown). There was slight
variability of Pgp and villin expression among the clones, with clone 7 being a relatively high expressor of both immunoreactive proteins (Fig.
7B). Levels of expression of CYP3A5 (Fig. 7B) and cytochrome
b5, CYP1A1, CYP2D6, and IFABP (not shown)
immunoreactive proteins were similar among the clones and the parent
cell line.

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Fig. 7.
Comparison of levels of selected immunoreactive
proteins and CYP3A catalytic activity in Caco-2 cell clones and the
parent cell line and their responses to
1 ,25-(OH)2-D3. All cells were grown on
Matrigel-coated Teflon culture inserts. One set of cultures was treated
with 0.25 µM 1 ,25-(OH)2-D3 for
2 weeks beginning at the time of confluence (+) while a duplicate set
was left untreated ( ). The medium was then replaced with medium not
supplemented with vitamin D. A, MDZ (4 µM) was added to
the apical compartment. After 7.5 hr, the apical and basolateral media
were collected separately. The rates of 1 -OH-MDZ accumulation in the
apical medium are shown. blq, below limits of
quantification. B, Immunoblots of sonicates of the cells were developed
with antibodies of the indicated specificities. With longer exposures
of the film, a CYP3A band was seen with each of the untreated cells
(protein loads: 5 µg for CYP3A, CYP3A5, Pgp, villin; 60 µg for
reductase). P, parent cell line.
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Matrigel (225 µg/cm2) alone did not result in increased
expression of CYP3A immunoreactive protein. Nevertheless, although 1
,25-(OH)2-D3 resulted in increased CYP3A
protein and catalytic activity in the absence of Matrigel (Fig.
8A), the increase in CYP3A expression in response to
1
,25-(OH)2-D3 was clearly enhanced when the
Caco-2 cells were grown on Matrigel. The rate of 1
-OH-MDZ formation by
Caco-2 cells grown on uncoated culture inserts and treated with 0.25 µM 1
,25-(OH)2-D3 was only 17%
of that of treated cells grown on Matrigel-coated inserts (Fig. 8A).
All extracellular matrices were not equal with respect to expression of
CYP3A immunoreactive protein and catalytic activity: using commercially
coated inserts, unpolymerized collagen type I was associated with
relatively low levels of expression and activity, whereas fibrillar
collagen (polymerized type I collagen), laminin, Growth Factor-Reduced Matrigel (2.86 mg/cm2), and (to a lesser extent) collagen
type IV were associated with levels of expression and activity similar
to that seen with Matrigel (2.86 mg/cm2) (Fig. 8B).

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Fig. 8.
Effects of extracellular matrices and FBS on the
increase in CYP3A in response to
1 ,25-(OH)2-D3. At 2 weeks postconfluence, 1 ,25-(OH)2-D3 was removed, if present, and
the cells were incubated with 4 µM MDZ added to the
apical medium. After incubations of 4.25-7 hr (uniform duration within
each group), the apical and basolateral media were collected.
Top, rates of 1 -OH-MDZ accumulation in the apical
medium are shown. ND, not done. Bottom, immunoblots are
of 5-µg loads of cell sonicates developed with antibodies of the
indicated specificities. A, Contribution of Matrigel. Caco-2 cells were
grown on polycarbonate culture inserts (+M) with or ( M) without Matrigel. Selected cultures
(+D) were treated with 0.25 µM
1 ,25-(OH)2-D3 for 2 weeks beginning at the
time of confluence while duplicate cultures were left untreated
( D). (Note that, in the case of Pgp, the first pair of
lanes is from a different gel than the second pair of lanes, and
therefore direct comparisons can only be made within each pair and not
between the pairs of immunoreactive bands.) B, Comparison of
extracellular matrices. Caco-2 cells were grown on PET culture inserts
commercially coated with several different extracellular matrices
[CI, unpolymerized collagen type I; FC,
fibrillar collagen (polymerized type I collagen); CIV,
collagen type IV; L, laminin; M,
Matrigel] as well as inserts that we coated with Growth Factor-Reduced
Matrigel (GFR) (see Experimental Procedures for matrix
application densities). Note that the growth factor-reduced matrix
formed a 1-2-mm-thick gel. All cultures were treated with 0.25 µM 1 ,25-(OH)2-D3 for 2 weeks beginning at the time of confluence. C, Contribution of FBS. Caco-2 cells were grown on Matrigel-coated Teflon culture inserts and treated
with 0.25 µM 1 ,25-(OH)2-D3 in
the presence or absence of 5% FBS for 2 weeks beginning at the time of
confluence. The serum-free medium was supplemented with ITS (final
concentrations: 5 µg/liter selenous acid, 5 mg/liter insulin and
transferrin), ITS+ (final concentrations: 6.2 µg/liter selenous acid,
6.2 mg/liter insulin and transferrin, 1.25 g/liter bovine serum
albumin, 5.35 mg/liter linoleic acid), or ITS+ with 100 nM
dexamethasone (Dex).
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It also seemed that Caco-2 cell expression of immunoreactive Pgp,
although slightly greater with Matrigel than in the absence of a
substratum (Fig. 8A), was greater with single component substrata (collagen type I, collagen type IV, or laminin) than with
multicomponent substrata (Matrigel or Growth Factor-Reduced Matrigel)
(Fig. 8B). Polymerized collagen type I was associated with a level of
expression similar to that seen with the multicomponent substrata.
These results are similar to those reported for Pgp expression in
cultured rat hepatocytes (50).
A Matrigel substratum seemed