Biomedical Engineering Reference
In-Depth Information
23.3. IN VITRO SYSTEMS
Questions are often posed as to how to interrelate in vitro data to in vivo data: namely,
how to model the data in order to answer questions mechanistically and make pre-
dictions. Vital information is provided from in vitro systems to arrive at transport
data to predict events in vivo. The in vitro systems for the study of drug transport
include use of cell lines transfected with transporter genes
44
,
45
and hepatocyte uptake
studies.
1
,
46
,
47
Transfected cells and freshly prepared isolated hepatocytes are often
used to determine, within the linear uptake period, the extent of cellular accumula-
tion to yield the initial velocity of uptake (amount/time). Beyond these time points,
efflux and metabolism become dominant and drug accumulation is no longer pro-
portional to time. The velocity data derived may be fitted to the Michaelis-Menten
equation or similar relationships to provide the maximum transport capacity (
V
max
)
and affinity constant (Michaelis-Menten constant
K
m
or concentration of drug at
half
V
max
). Because of the time-involvement and lack of availability of human livers
for the preparation of fresh hepatocytes, cryopreservation has been used to retain
and preserve functions of the hepatocyte.
48
,
49
Zonal hepatocytes, prepared accord-
ing to the digitonin-collagenase method of Lindros and Pentılla,
50
have been used
for the study of acinar drug uptake. Vesicles from either basolateral or canalicu-
lar membranes may be used for study. The transmembrane transport often aims at
identification of the driving forces of uptake.
51
,
52
Inside-out vesicles are needed for
examination of canalicular transport, especially for drugs that are too polar to traverse
the basolateral membrane. With molecular cloning, in vitro gene expression systems
in
Xenopus laevis
oocytes or mammalian cells have been applied extensively to drug
transport studies, in either transiently or stably transfected systems.
44
,
45
,
53
,
54
Dou-
ble transfection systems that express rat transporters such as Ntcp/Bsep
55
have been
developed. Moreover, double-transfected MDCK II or LLC-PK1 cells expressing
human OATP2/MRP2,
57
,
58
OATP2/MDR1, OATP2/BCRP,
59
or OATP8/MRP2,
60
,
61
or even quadruple-transfected cells
62
have been utilized to study vectorial transport
of solutes in cell monolayers. Transgenic animals bearing a human transporter gene
have succeeded to show that the Eisai hyperbilirubinemic rat that is deficient in Mrp2
could regain its ability to excrete Mrp2-substrates into bile.
63
Analogously, many systems exist at the subcellular level: the S9 fraction, cytosol,
and microsomes purified enzyme systems
64
,
65
for the assessment of metabolic ac-
tivities. In other systems, transgenic animals,
66
−
68
antibodies,
69
and reconstituted or
cDNA-expressed systems
70
are used routinely to identify the metabolic system as
well as the kinetic parameters (
K
m
and
V
max
). However, one drawback of these in
vitro studies is that the cofactors or cosubstrates are usually added in excess, and this
type of study rarely divulges the rate-limiting role of cofactors or cosubstrates that
may be depleted in vivo.
15
,
16
In other instances, the in vitro system is not able to fully
reflect the activity of the enzyme in vivo.
71
,
72
The isolated hepatocyte that retains the intact cellular membrane houses both trans-
porters and enzymes and encompasses the pertinent information for both transporter
and enzymes.
11
,
21
Similar types of information may be obtained in the sandwich-
cultured hepatocytes
73
−
75
and bioreactors composed of cultured hepatocytes.
76
,
77
However, caution is needed with cultured systems since enzyme levels and regulators
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