U.S. patent application number 12/728858 was filed with the patent office on 2010-07-08 for method of screening candidate compounds for susceptibility to biliary excretion.
Invention is credited to Kim L.R. Brouwer, Edward L. LeCluyse, Xingrong Liu.
Application Number | 20100173333 12/728858 |
Document ID | / |
Family ID | 22416897 |
Filed Date | 2010-07-08 |
United States Patent
Application |
20100173333 |
Kind Code |
A1 |
LeCluyse; Edward L. ; et
al. |
July 8, 2010 |
METHOD OF SCREENING CANDIDATE COMPOUNDS FOR SUSCEPTIBILITY TO
BILIARY EXCRETION
Abstract
A method of screening a candidate compound for susceptibility to
biliary excretion. The method includes the steps of providing a
culture of hepatocytes, the culture having at least one bile
canaliculus; exposing a candidate compound to the culture; and
determining an amount of candidate compound in the at least one
bile canaliculus, the amount of candidate compound in the at least
one bile canaliculus indicating the susceptibility of the candidate
compound to biliary excretion. Optionally, the culture of
hepatocytes is a long-term culture in a sandwich configuration. The
method is particularly applicable to the screening of multiple
candidate compounds in a single effort.
Inventors: |
LeCluyse; Edward L.; (Chapel
Hill, NC) ; Brouwer; Kim L.R.; (Chapel Hill, NC)
; Liu; Xingrong; (East Lyme, CT) |
Correspondence
Address: |
JENKINS, WILSON, TAYLOR & HUNT, P. A.
Suite 1200 UNIVERSITY TOWER, 3100 TOWER BLVD.,
DURHAM
NC
27707
US
|
Family ID: |
22416897 |
Appl. No.: |
12/728858 |
Filed: |
March 22, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10855085 |
May 27, 2004 |
7682781 |
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12728858 |
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09527352 |
Mar 17, 2000 |
6780580 |
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10855085 |
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60124810 |
Mar 17, 1999 |
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Current U.S.
Class: |
435/7.21 |
Current CPC
Class: |
G01N 33/5067
20130101 |
Class at
Publication: |
435/7.21 |
International
Class: |
G01N 33/567 20060101
G01N033/567 |
Goverment Interests
GRANT STATEMENT
[0002] This invention was made in part from government support
under Grant No. GM41935 from the National. Institute of Health.
Thus, the U.S. Government has certain rights in the invention.
Claims
1. A method of screening a candidate compound for susceptibility to
biliary excretion, the method comprising the steps of: (a)
providing a culture of hepatocytes, the culture of hepatocytes
comprising at least one bile canaliculus; (b) exposing a candidate
compound to the culture; and (c) determining an amount of candidate
compound in the at least one bile canaliculus, the amount of the
candidate compound in the at least one bile canaliculus indicating
the susceptibility of the candidate compound to biliary
excretion.
2. The method of claim 1, wherein the hepatocytes are isolated from
a source selected from the group consisting of rat, human, monkey,
ape, cat, dog, pig, hog, cattle, oxen, sheep, horses, turkeys,
chickens, ducks and geese.
3. The method of claim 1, wherein the culture of hepatocytes
further comprises a long-term culture of hepatocytes.
4. The method of claim 1, wherein the culture of hepatocytes
further comprises a canalicular network.
5. The method of claim 1, wherein the culture of hepatocytes is
further characterized as having a configuration selected from the
group consisting of clusters of hepatocytes, aggregates of
hepatocytes, at least one layer of hepatocytes, and combinations
thereof.
6. The method of claim 5, wherein the hepatocytes are embedded in a
matrix.
7. The method of claim 5, wherein the culture of hepatocytes
further comprises a sandwich culture of hepatocytes, the sandwich
culture comprising at least one layer of hepatocytes and at least
one bile canaliculus with the at least one layer of
hepatocytes.
8. The method of claim 7, wherein the sandwich culture of
hepatocytes further comprises a long-term sandwich culture of
hepatocytes.
9. The method of claim 7, wherein the at least one layer of
hepatocytes is sandwiched between two layers of matrix.
10. The method of claim 9, wherein the matrix is selected from the
group consisting of a biological matrix medium, a synthetic matrix
medium, and combinations thereof.
11. The method of claim 10, wherein the biological matrix medium is
selected from the group consisting of collagens, laminins, basement
membrane-derived complexes, derivatives thereof and combinations
thereof.
12. The method of claim 1, wherein the amount of the candidate
compound in the at least one bile canaliculus is determined by
calculating a biliary clearance value for the culture, the
calculated biliary clearance value indicating the susceptibility of
the candidate compound to biliary excretion.
13-64. (canceled)
Description
RELATED APPLICATION INFORMATION
[0001] This application claims the benefit of and priority to U.S.
Provisional Patent Application 60/124,810, filed Mar. 17, 1999, the
disclosure of which is incorporated herein by reference in its
entirety.
TECHNICAL FIELD
[0003] The present invention relates to a method of screening
compounds which are candidates primarily for use as therapeutic
agents for susceptibility to biliary excretion. More particularly,
the present invention relates to an in vitro method of screening
candidate compounds for susceptibility to biliary excretion.
Compounds can be chosen for use as therapeutic agents for
administration to humans and other warm-blooded vertebrates.
TABLE OF ABBREVIATIONS
[0004] AUC--area under the curve [0005] BSEP--bile salt export pump
[0006] Cl.sub.B--biliary clearance [0007] Cl.sub.in--intrinsic
clearance [0008] cMOAT--canalicular multispecific organic anion
transporter [0009] CFDA--carboxyfluorescein diacetate [0010]
DMEM--Dulbecco's modified Eagle's medium [0011]
EDTA--ethylenediamine tetraacetate [0012] HP--Hewlett Packard
[0013] HPLC--high performance liquid chromatography [0014] hr--hour
[0015] i.v.--intravenous [0016] i.p.--intraperitoneal [0017]
K.sub.m--Michaelis-Menten constant for enzyme-substrate reaction
[0018] LC/MS--liquid chromatography/mass spectrometry [0019] mg
pr.--milligrams protein [0020] min--minute [0021] MDR2--multidrug
resistance protein 2 [0022] MRP2--multidrug resistance associated
protein 2 [0023] Ntcp--Na.sup.+/taurocholate cotransporting
polypeptide [0024] OATP1--organic ion anion transporting
polypeptide 1 [0025] OATP2--organic ion anion transporting
polypeptide 2 [0026] P-gp--P-glycoprotein [0027] SD--standard
deviation [0028] UV--ultraviolet [0029] UV/VIS--ultraviolet/visible
[0030] V.sub.max--maximum velocity of enzyme-catalyzed reaction
BACKGROUND ART
[0031] First-pass metabolism pertains to the absorption of
therapeutic agents, drugs or other compounds into the portal blood
supply that leads to the liver. When a drug is swallowed, the
stomach and small intestine absorb it, with subsequent flow in the
blood to the portal vein entry to the liver. The liver may then in
turn rapidly absorb and metabolize the drug at high concentrations
through the liver blood supply. Thus, large amounts of the drug may
never be seen by the systemic circulation or drug effect site.
Additionally, rapid metabolism via the first-pass metabolism route
can lead to the formation of high plasma concentrations of unwanted
metabolites.
[0032] Thus, in the liver, therapeutic compositions are often
undesirably removed from an animal's circulatory system in that
they are taken up by hepatocytes (liver cells) and excreted in bile
via the bile canaliculi. Uptake into the hepatocytes is mediated by
transport systems endogenous to hepatocytes, including Ntcp and
cMOAT. Such transporters move xenobiotics like therapeutic
compositions as well as endogenous compounds across the sinusoidal
membrane of the hepatocytes. Bile canaliculi are structures within
liver tissue that receive excreted components from the hepatocytes
and transport the bile to a common bile duct for removal from the
animal. Biliary excretion of substrates is thus a complex process
involving translocation across the sinusoidal membrane, movement
through the cytoplasm, and transport across the canalicular
membrane.
[0033] The advent of combinatorial chemistry techniques has enabled
the identification of extremely high numbers of compounds that have
potential as therapeutic agents. However, assays for susceptibility
to biliary excretion that can rapidly identify those candidate
compounds that have a lower potential for uptake by hepatocytes and
excretion through bile canaliculi have lagged behind the pace of
synthesis and screening of pharmacological activities. Numerous in
vivo (e.g. bile duct cannulated animals) and in vitro preparations
(e.g. isolated perfused livers, isolated hepatocytes, hepatocyte
couplets, liver plasma membrane vesicles and expressed transport
proteins) have been used to investigate biliary excretion
processes. See e.g. Oude Elferink et al., Biochim. Biophys. Acta
1241:215-268, 1995.
[0034] Additionally, short-term (3-8 hour) cultured hepatocyte
couplets have been employed to examine directly the biliary
excretion of fluorescent compounds utilizing fluorescence
microscopy, as described by Graf and Boyer, J. Hepatol. 10:387-394,
1990. However, the application of cultured hepatocyte couplets to
study biliary excretion of xenobiotics is limited in that the
substrate must contain a fluorescent chromophore.
[0035] Long-term (typically more than 24 hour) cultured hepatocytes
have been reported to restore polarity with canalicular-like
structures. See e.g., Barth and Schwarz, Proc. Natl. Acad. Sci.
79:4985-4987, 1982; Maurice et al., J. Cell 90:79-92, 1988;
Talamini et al., Hepatology 25:167-172, 1997. Although primary
hepatocytes maintained under conventional culture conditions have
been used to study drug metabolism and hepatotoxicity, long-term
cultures of hepatocytes have not been a suitable model for studying
hepatobiliary transport. Particularly, as described by Groothuis
and Meijer, J. Hepatology 24(Suppl. 1):3-28, 1996 and LeCluyse et
al., Adv. Drug Del. Rev. 22:133-186, 1996, rapid loss of
liver-specific function; including hepatic transport properties,
and failure to establish normal bile canalicular networks and to
maintain normal hepatocyte morphology have been observed in such
cultures.
[0036] Existing methods have not been demonstrated to be widely
applicable to investigate human biliary excretion. In addition,
existing approaches cannot be used to examine efficiently biliary
excretion processes for a large number of drug candidates. Thus,
there is a long-felt need for an assay to assess susceptibility of
candidate compounds for hepatic uptake and biliary excretion. Such
an assay would facilitate elimination of those compounds with an
undesirably high susceptibility for biliary excretion from further
evaluation as therapeutic agents early in the evaluation process.
Correspondingly, there is a long-felt need for the rapid
identification of suitable candidate compounds (i.e., compounds
that are not susceptible to biliary excretion) for further testing
as therapeutic agents.
SUMMARY OF THE INVENTION
[0037] A method of screening a candidate compound for
susceptibility to biliary excretion is disclosed herein. The method
comprises the steps of providing a culture of hepatocytes, the
culture comprising at least one bile canaliculus having a
canalicular space; exposing a candidate compound to the culture;
and determining an amount of the candidate compound in the
canalicular space of the at least one bile canaliculus, the amount
of the candidate compound in the canalicular space of the at least
one bile canaliculus indicating the susceptibility of the candidate
compound to biliary excretion. The culture of hepatocytes
preferably comprises a long-term culture in a sandwich
configuration.
[0038] Accordingly, it is an object of the present invention to
provide a rapid and inexpensive method of screening of candidate
compounds for susceptibility to biliary excretion.
[0039] It is a further object of the present invention to provide
an in vitro method of screening candidate compounds for
susceptibility to biliary excretion.
[0040] It is yet a further object of the present invention to
provide a method of screening candidate compounds for
susceptibility to biliary excretion which facilitates the screening
of many candidate compounds in a single effort.
[0041] It is still a further object of the present invention to
provide a high throughput method of screening of candidate
compounds for susceptibility to biliary excretion.
[0042] Some of the objects of the invention having been stated
herein above, other objects will become evident as the description
proceeds, when taken in connection with the accompanying Laboratory
Examples and Drawings as best described herein below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0043] FIG. 1A is a graph depicting cumulative uptake of
[.sup.3H]inulin (1 .mu.M) standard buffer (closed symbols) and
Ca.sup.++-free buffer (open symbols) in hepatocyte monolayers
cultured for 3 hr;
[0044] FIG. 1B is a graph depicting cumulative uptake of
[.sup.3H]inulin (1 .mu.M) in standard buffer (closed symbols) and
Ca.sup.++-free buffer (open symbols) in hepatocytes cultured in a
sandwich configuration for 96 hr;
[0045] FIG. 2A is a graph depicting cumulative uptake of
[.sup.14C]salicylate (1 .mu.M) in standard buffer (closed symbols)
and Ca.sup.++-free buffer (open symbols) in hepatocyte monolayers
cultured for 3 hr;
[0046] FIG. 2B is a graph depicting cumulative uptake of
[.sup.14C]salicylate (1 .mu.M) in standard buffer (closed symbols)
and Ca.sup.++-free buffer (open symbols) in hepatocytes cultured in
a sandwich configuration for 96 hr;
[0047] FIG. 3A is a graph depicting cumulative uptake of
[.sup.3H]methotrexate (1 .mu.M) in standard buffer (closed symbols)
and Ca.sup.++-free buffer (open symbols) in hepatocyte monolayers
cultured for 3 hr;
[0048] FIG. 3B is a graph depicting cumulative uptake of
[.sup.3H]methotrexate (1 .mu.M) in standard buffer (closed symbols)
and Ca.sup.++-free buffer (open symbols) in hepatocytes cultured in
a sandwich configuration for 96 hr;
[0049] FIG. 4A is a graph depicting cumulative uptake of
[.sup.3][D-pen.sup.2,5]enkephalin (15 .mu.M) in standard buffer
(closed symbols) and Ca.sup.++-free buffer (open symbols) in
hepatocyte monolayers cultured for 3 hr;
[0050] FIG. 4B is graph depicting cumulative uptake of
[.sup.3H][D-pen.sup.2,5]enkephalin (15 .mu.M) in standard buffer
(closed symbols) and Ca.sup.++-free buffer (open symbols) in
hepatocytes cultured in a sandwich configuration for 96 hr;
[0051] FIG. 5A is a graph depicting cumulative uptake of
[.sup.3]taurocholate (1 .mu.M) in standard buffer (closed symbols)
and Ca.sup.++-free buffer (open symbols) in hepatocyte monolayers
cultured for 3 hr;
[0052] FIG. 5B is a graph depicting cumulative uptake of
[.sup.3H]taurocholate (1 .mu.M) in standard buffer (closed symbols)
and Ca.sup.++-free buffer (open symbols) in hepatocytes cultured in
a sandwich configuration for 96 hr;
[0053] FIG. 6A is a graph depicting the relationship between the
percentage of the dose excreted in rat bile in vivo and the Biliary
Excretion Index in 96-hr sandwich cultured hepatocytes for the
following model substrates: inulin (.quadrature.), salicylate
(.diamond-solid.), methotrexate (.largecircle.),
[D-pen.sup.2,5]enkephalin (.tangle-solidup.), and taurocholate ( ).
The Biliary Excretion Index was calculated from the 10-min
cumulative uptake data (FIGS. 1A-5B) based on Equation 3. The
broken line is the fit of a linear regression equation to the
data;
[0054] FIG. 6B is a graph depicting the relationship between the
percentage of the dose excreted in rat bile in vivo and in vivo
intrinsic biliary clearance and in vitro biliary clearance in 96-hr
sandwich cultured hepatocytes for the following model substrates:
inulin (.quadrature.), salicylate (.diamond-solid.), methotrexate
(.largecircle.), [D-pen.sup.2,5]enkephalin (.tangle-solidup.), and
taurocholate ( ). The in viva intrinsic biliary clearance was
calculated from Equation 2 based on in vivo biliary clearance
values from the literature. The in vitro biliary clearance was
calculated from Equation 4. The broken line is the fit of a linear
regression equation to the data;
[0055] FIG. 7A is a graph depicting cumulative uptake of
[.sup.3H]264W94 (3 .mu.M) in standard buffer (closed symbols) and
Ca.sup.++-free buffer (open symbols) in hepatocyte monolayers
cultured for 3 hr;
[0056] FIG. 7B is a graph depicting cumulative uptake of
[.sup.3H]264W94 (3 .mu.M) in standard buffer (closed symbols) and
Ca.sup.++-free buffer (open symbols) in hepatocytes cultured in a
sandwich configuration for 96 hr;
[0057] FIG. 8A is a graph depicting cumulative uptake of
[.sup.3H]2169W94 (3 .mu.M) in standard buffer (closed symbols) and
Ca.sup.++-free buffer (open symbols in hepatocyte monolayers
cultured for 3 hr; and
[0058] FIG. 8B is a graph depicting cumulative uptake of
[.sup.3H]2169W94 (3 .mu.M) in standard buffer (closed symbols) and
Ca.sup.++-free buffer (open symbols) in hepatocytes cultured in a
sandwich configuration for 96 hr;
[0059] FIG. 9A presents the chemical structures of the compound
264W94, wherein the asterisk sign indicates the position of
.sup.14C incorporated uniformly; and
[0060] FIG. 9B presents the chemical structures of the compound
2169W94, wherein the asterisk sign indicates the position of
.sup.14C incorporated uniformly.
DETAILED DESCRIPTION OF THE INVENTION
[0061] In accordance with the present invention, a method is
provided for the screening of a candidate compound or substrate for
susceptibility to biliary excretion. The method comprises the steps
of providing a culture of hepatocytes, the culture comprising at
least one bile canaliculus having a canalicular space; exposing a
candidate compound to the culture; and determining an amount of the
candidate compound in the canalicular space of the at least one
bile canaliculus, the amount of the candidate compound in the
canalicular space of the at least one bile canaliculus indicating
the susceptibility of the candidate compound to biliary
excretion.
[0062] As would be appreciated by one of ordinary skill in the art,
in vivo biliary excretion of substrates involves translocation
across the sinusoidal membrane, movement through the cytoplasm, and
transport across the canalicular membrane. Thus, in a preferred
hepatocyte culture of the present invention, functional properties
displayed by hepatocytes in vivo are established. For example, the
establishment of hepatic transport systems, such as sinusoidal or
canalicular transport systems, or both sinusoidal and canalicular
transport system's is particularly contemplated in accordance with
the present invention. Exemplary transport systems include, but are
not limited to, Ntcp, cMOAT, OATP1, OATP2, MRP2, P-gp, BSEP and
MDR2.
[0063] Additionally, the establishment of at least one bile
canaliculus and the establishment of normal hepatocyte morphology
in the hepatocyte cultures are also contemplated in accordance with
the present invention. Preferably, the culture comprises a
plurality of bile canaliculi. More preferably, the plurality of
bile canaliculi comprise a canalicular network. The amount of
candidate compound, as discussed in detail below, in the
canalicular space of the at least one bile canaliculus indicates
the susceptibility of the candidate compound to biliary
excretion.
[0064] While the following terms are believed to be well understood
by one of ordinary skill in the art, the following definitions are
set forth to facilitate explanation of the invention.
[0065] The term "candidate compound" or "candidate substrate" is
meant to refer to any compound wherein the characterization of the
compound's susceptibility to biliary excretion is desirable.
Exemplary candidate compounds or substrates include xenobiotics
such as drugs and other therapeutic agents, carcinogens and
environmental pollutants, as well as endobiotics such as steroids,
fatty acids and prostaglandins.
[0066] The candidate drugs and other therapeutic agents screened in
accordance with the method of the present invention are
contemplated to be useful in the treatment of warm-blooded
vertebrates. Therefore, the invention concerns mammals and
birds.
[0067] Contemplated is the treatment of mammals such as humans, as
well as those mammals of importance due to being endangered (such
as Siberian tigers), of economical importance (animals raised on
farms for consumption by humans) and/or social importance (animals
kept as pets or in zoos) to humans, for instance, carnivores other
than humans (such as cats and dogs), swine (pigs, hogs, and wild
boars), ruminants (such as cattle, oxen, sheep, giraffes, deer,
goats, bison, and camels), and horses. Also contemplated is the
treatment of birds, including the treatment of those kinds of birds
that are endangered, kept in zoos, as well as fowl, and more
particularly domesticated fowl, i.e., poultry, such as turkeys,
chickens, ducks, geese, guinea fowl, and the like, as they are also
of economical importance to humans. Thus, contemplated is the
treatment of livestock, including, but not limited to, domesticated
swine (pigs and hogs), ruminants, horses, poultry, and the
like.
[0068] The term "biliary excretion" is meant to refer to a
biological process wherein substances are removed from an animal's
circulatory system by being taken up by hepatocytes (liver cells)
and excreted in bile via the bile canaliculi. Uptake into the
hepatocytes is mediated by transport systems endogenous to
hepatocytes, including, but not limited to, Ntcp and OATP1. Bile
canaliculi are structures within liver tissue which receive
excreted components from the hepatocytes and transport the bile to
a bile duct for removal from the animal.
[0069] By the phrase "an amount of candidate compound" and/or the
phrase "determining an amount of candidate compound in the at least
one bile canaliculus", as used herein and in the claims, it is
meant to refer to any amount of candidate compound that is taken up
by hepatocytes and excreted into the at least one bile canaliculus
in accordance with the assay of the present invention. For example,
"an amount" can refer to substantially no candidate compound
residing in the at least one bile canaliculus after exposure of a
candidate compound to a culture in accordance with the present
invention. Alternatively, "an amount" can refer to substantially
all of the candidate compound residing in the at least one bile
canaliculus after exposure of a candidate compound to a culture in
accordance with the present invention. Thus, the phrase "an amount
of candidate compound in the at least one bile canaliculus" can be
used to describe candidate compounds that are not highly excreted,
extensively excreted, and extensively and rapidly excreted.
[0070] The phrase "determining an amount of candidate compound in
the at least one bile canaliculus" is also meant to refer to the
use of a biliary excretion index calculation and a biliary
clearance calculation as described herein below. The phrase
"determining an amount of a candidate compound in the at least one
bile canaliculus" may also refer to the detection of a reduced
amount of a marker compound due to uptake of candidate compound
into the at least one bile canaliculus as described in the high
throughput embodiment of the assay of the present invention
described herein below. Thus, quantitative and qualitative
determinations of "an amount of candidate compound in the at least
one bile canaliculus" are contemplated to be within the scope of
the present invention.
[0071] The phrase "an amount of candidate compound" and/or the
phrase "determining an amount of candidate compound in the at least
one bile canaliculus" are also meant to refer to the screening of,
for example, a class or series of candidate compounds and then
establishing a ranking of susceptibility to biliary excretion of
the candidate compounds within the class or series. It is thus
contemplated in accordance with a preferred embodiment of the
present invention that the candidate compound or compounds wherein
lesser or lower susceptibility to excretion is observed according
to such a ranking may be chosen for further experimentation or
development as a therapeutic agent, while compounds wherein higher
or greater susceptibility to excretion is observed according to
such a ranking may be excluded from further experimentation or
development as a therapeutic agent.
[0072] However, as would be readily apparent to one of ordinary
skill in the art, the characteristic that a compound is susceptible
to biliary excretion does not necessarily preclude further
development of the compound as a therapeutic agent. Indeed, the
decision of whether to proceed with the development of a particular
candidate compound as a therapeutic agent is based on many factors,
including, but not limited to, the biological activity of the
candidate compound. While susceptibility to biliary excretion is an
important factor, it is not the only factor that is typically
considered by one of ordinary skill in the art. Characterization of
susceptibility to biliary excretion in accordance with the method
of the present invention thus provides data that is desirable for
use by one of ordinary skill in the art in evaluating whether to
proceed with the development of a candidate compound as a
therapeutic agent.
[0073] The term "marker compound" is meant to refer to a chemical
compound that is readily detectable using a standard detection
technique, such as fluorescence or chemiluminescence
spectrophotometry, scintillation spectroscopy, chromatography,
liquid chromatography/mass spectroscopy (LC/MS), colorimetry, and
the like. Exemplary marker compounds thus include, but are not
limited to, fluorogenic or fluorescent compounds, chemiluminescent
compounds, colorimetric compounds, UV/VIS absorbing compounds,
radionuclides and combinations thereof.
[0074] Therapeutic compositions that are taken up and excreted
extensively though the biliary excretion processes described herein
typically have a minimal chance of imparting therapeutic effects in
a subject. It is thus very desirable to establish an in vitro test
for a compound's susceptibility to hepatocyte uptake and biliary
excretion so as to facilitate elimination of a compound with an
undesirably high susceptibility from further evaluation as a
therapeutic agent early in the evaluation process. The biliary
excretion assay of the present invention provides such a test.
[0075] Rat hepatocytes are preferred in a culture for use in the
method of the present invention; but, any suitable source of
hepatocytes as would be apparent to one of ordinary skill in the
art is contemplated to be within the scope of the present
invention. Exemplary sources include the warm-blooded vertebrates
listed above. In particular, exemplary sources include, but are not
limited to, human beings, monkeys, apes, cats, dogs, pigs, hogs,
cattle, oxen, sheep, horses, turkeys, chickens, ducks and
geese.
[0076] The biliary excretion assay method of the present invention
may optionally comprise establishing a sandwich culture of
hepatocytes wherein at least one hepatocyte layer is formed between
two layers of matrix. While configuration as a sandwich culture is
the preferred configuration for the culture, any suitable
configuration as would be apparent to one of ordinary skill in the
art is contemplated to be within the scope of the present
invention. For example, clusters, aggregates or other associations
or groupings of hepatocytes in a culture wherein at least one bile
canaliculus is formed and wherein functional properties of
hepatocytes are established are contemplated to fall within the
scope of the present invention. Preferably, the culture
configuration facilitates the formation of a plurality of bile
canaliculi. More preferably, the culture configuration facilitates
the formation of a canalicular network. The amount of candidate
compound, as discussed in detail herein, in the canalicular space
of the bile canaliculi indicates the susceptibility of the
candidate compound to biliary excretion.
[0077] Additionally, in the preferred sandwich configuration,
hepatocytes are cultured in monolayers between two layers of matrix
or scaffolding. But, the hepatocytes can also be embedded in the
matrix or can extend non-uniformly through the matrix vertically,
horizontally, diagonally, or in any combination thereof, such that
one-dimensional, two-dimensional and three-dimensional hepatocytes
aggregates are formed. In accordance with the present invention, it
is thus contemplated that the hepatocyte cultures can be formed by
mixing hepatocyte cells with an appropriate matrix and inserting
the mixture into a suitable culture container, such as a multi-well
plate.
[0078] While collagen is a preferred substrate or scaffolding for
the culture of hepatocytes, any suitable substrate or scaffolding
whether natural, synthetic or combinations thereof as would be
apparent to one of ordinary skill in the art is contemplated to be
within the scope of the present invention. For example, other
biological substrates, including but not limited to laminin and the
basement membrane derived biological cell culture substrate sold
under the registered trademark MATRIGEL.RTM. by Collaborative
Biomedical Products, Inc. of Bedford, Mass., are contemplated to
comprise suitable substrate or scaffolding material. Synthetic
matrix materials, substrate materials or scaffolding materials,
which are typically made from a variety of materials such as
polymers, are also contemplated to fall within the scope of the
present invention. The variation of component materials with a
particular matrix for use in culturing hepatocytes is also
contemplated in accordance with the method of the present
invention.
[0079] The cultured hepatocytes are preferably cultured as a
"long-term culture". By "long-term culture" it is meant to refer to
hepatocytes that have been cultured for at least about 0.12 hours.
More preferably, by "long-term culture" it is meant to refer to
hepatocytes that have been cultured for at least about 24 hours,
for at least about 48 hours, or for at least about 72 hours. Even
more preferably, by "long-term culture" it is meant to refer to
hepatocytes that have been cultured for at least about 96 hours.
Long-term culturing facilitates the formation of bile canaliculi
and the establishment of functional properties within the
hepatocytes.
[0080] Following long-standing patent law convention, the terms "a"
and "an" mean "one or more" when used in this application,
including the claims.
Side-by-Side Embodiment
[0081] In accordance with one embodiment of the present invention,
replicate hepatocyte cultures are established, preferably in
sandwich configuration. A first culture is exposed to a standard
buffer and a second culture is exposed to a Ca.sup.++-free buffer.
Exposure to the Ca.sup.++-free buffer disrupts the bile canaliculi
within the hepatocyte monolayers by breaking down adhesional
processes or junctional complexes in the monolayer of hepatocytes.
While exposure to the Ca.sup.++-free buffer is a preferred method
of breaking down the adhesional processes or junctional complexes
to substantially disrupt the bile canaliculi, any suitable
technique for breaking down the adhesional processes or junctional
complexes to promote substantial disruption of the bile canaliculi
as would be apparent to one of ordinary skill in the art is
contemplated to be within the scope of the present invention.
Exemplary techniques include, but are not limited to, the
administration to the culture of peptides which interact with
cell-to-cell binding sites to thereby prevent neighboring cells
from binding.
[0082] A candidate compound or compounds is/are then added to each
culture. The candidate compound(s) cannot be retained within the
bile canaliculi in the culture that was treated with Ca.sup.++-free
buffer. Thus, in this culture, candidate compound(s) may be taken
up into the hepatocytes and retained within the cytoplasm of the
hepatocytes. However, any amount of the candidate compound(s) that
is excreted by the hepatocytes across the canalicular membrane will
flow into the buffer medium and will be removed when the buffer
medium is removed. In contrast, when candidate compound(s) is/are
administered to the hepatocyte sandwich culture in which the bile
canaliculi are intact, any candidate compound(s) that is/are taken
up by the cells and excreted by the cells is/are maintained both in
the cytoplasm of the hepatocytes and in the bile canaliculi.
[0083] It is then desirable to obtain a measurement of the amount
of candidate compound present within the intact bile canaliculi.
The buffer media is removed from the cultures and the cultures are
washed and lysed. As described in the Laboratory Examples presented
herein below, the lysing of the cells within the cultures may be
accomplished by addition of a suitable lysis buffer coupled with
agitation of the culture. A preferred lysis buffer includes a
detergent. The desired measurement is obtained by comparing the
amount of candidate substance present in the lysate from the
culture which has disrupted bile canaliculi (such as by exposure to
Ca.sup.++-free medium) as compared to the lysate from the culture
with intact bile canaliculi. Two particular calculations have been
utilized to compare the cultures and to determine an amount of the
candidate compound residing in the intact bile canaliculi. As
described above, the amount of candidate compound in the intact
bile canaliculi indicates the candidate compound's susceptibility
to biliary excretion.
[0084] One calculation is described as a biliary excretion index,
which is a calculation of the uptake and excretion of the candidate
compound as follows: 100%.times.{(uptake in the culture with intact
bile canaliculi minus uptake within hepatocytes only in the
Ca.sup.++-free culture) divided by (uptake in the culture with
intact bile canaliculi)}. The other calculation is a biliary
clearance calculation, which is performed as follows: (uptake in
the culture with intact bile canaliculi minus uptake within
hepatocytes only in the Ca.sup.++-free culture) divided by (time of
incubation multiplied by the concentration of the candidate
compound in the buffer medium).
[0085] Upon comparison of the in vitro assay of the present
invention to a standard in vivo assay for biliary excretion as
described in the Laboratory Examples presented herein below, it was
determined that biliary clearance provided a more accurate and
desirable evaluation of excretion. Particularly, the in vitro
biliary clearance calculation adequately differentiated among
candidate substances that are: (1) not highly excreted; (2)
extensively excreted; and (3) extensively and rapidly excreted.
Thus, the use of the biliary clearance calculation comprises an
important aspect of the invention.
Metabolite Assay Embodiment
[0086] In the hepatocytes of the method of the present invention
certain metabolic activities (called Phase I activities) may be
substantially reduced. The substantial reduction in metabolic
activity coupled with maintenance of biliary transport represents
an advantage of the in vitro biliary excretion assay of the present
invention in that a differentiation can be made between biliary
excretion of a parent candidate compound versus a metabolite or
metabolites of the parent candidate compound. This feature
comprises an important aspect of the present invention.
[0087] In accordance with a preferred embodiment of the metabolite
assay of the present invention, the method comprises establishing a
first set and second set of two cultures of hepatocytes, with each
culture preferably comprising at least one layer of hepatocytes
sandwiched between two layers of collagen and at least one bile
canaliculus formed within at least one layer of hepatocytes. The
first set of cultures includes intact bile canaliculi and the
second set of cultures includes disrupted bile canaliculi.
[0088] Metabolic enzyme activity and/or transport systems are then
induced in the hepatocytes of one of the cultures within each of
the first set and second set of cultures in accordance with
art-recognized techniques using inducers which are known to
up-regulate Phase I hepatic enzyme activity, such as phenobarbital
and .beta.-naphthoflavone. Exemplary inducers and techniques
associated with the same are described by Parkinson, A. (1996)
Biotransformation of Xenobiotics in Casarett and Doull's
Toxicology. The Basic Science of Poisons., 5.sup.th Ed. (Klaassen,
C. D. ed.) pp. 113-186, McGraw Hill, New York, and by LeCluyse et
al., (1996) Cultured rat hepatocytes, in Models for Assessing Drug
Absorption and Metabolism (Borchard et al. eds), pp 121-160, Plenum
Press, New York, the contents of each of which are herein
incorporated by reference.
[0089] A candidate parent compound is exposed to the first and
second sets of cultures for a time sufficient to allow uptake of
the candidate parent compound. Each set of cultures is washed and
then lysed. The amount of candidate parent compound present in the
lysate obtained from the culture in each set of cultures having
inactive metabolic enzymes is determined. The amount of metabolite
of the candidate parent compound present in the lysate obtained
from the culture in each set of cultures having active metabolic
enzymes is also determined.
[0090] A biliary clearance value for the cultures having inactive
metabolic enzymes is calculated using the amount of candidate
parent compound in the culture lysate. The calculated biliary
clearance value is then used to determine the susceptibility of the
candidate parent compound to biliary excretion, as described above.
A biliary clearance value for the cultures having active metabolic
enzymes is calculated using the amount of metabolite of the
candidate parent compound in the culture lysate. The calculated
biliary clearance value is then used to determine the
susceptibility of the metabolite to binary excretion, as described
above. This information is contemplated to be useful, for example,
in evaluating whether or not to administer a therapeutic
composition in a pro-drug form.
High Throughput Assay Embodiment
[0091] An additional alternative embodiment of the present
invention pertains to a high throughput hepatic uptake and biliary
excretion assay. Such an assay preferably involves the use of
cultured hepatocytes as described above, in conjunction with a
marker compound that is a substrate for endogenous sinusoidal or
canalicular transport systems, or both sinusoidal and canalicular
transport systems. Exemplary transport systems include, but are not
limited to, Ntcp, cMOAT, OATP1, OATP2, MRP2, P-gp, BSEP and MDR2.
Particularly, a candidate compound is administered to a hepatocyte
culture in conjunction with a marker compound in accordance with
the cell culture and compound administration techniques described
in the Laboratory Examples presented below.
[0092] Uptake and excretion competition between a candidate
compound and the marker compound is then evaluated. That is, a
significant drop in the amount of marker compound (e.g. measured or
detected signal from the marker compound) within bile canaliculi in
a culture may indicate that the candidate compound (as opposed to
the marker compound) is taken up and excreted extensively.
[0093] A ranking of susceptibility to hepatic uptake and biliary
excretion of the candidate compounds is then established. It is
thus contemplated in accordance with a preferred embodiment of the
high throughput assay of the present invention that the candidate
compound or compounds wherein lesser or lower susceptibility to
excretion is observed according to such a ranking may be chosen for
further experimentation or development as a therapeutic agent,
while compounds wherein higher or greater susceptibility to
excretion is observed according to such a ranking may be excluded
from further experimentation or development as a therapeutic
agent.
[0094] An exemplary marker compound comprises the fluorescent
cMOAT/MRP2 substrate, carboxydichlorofluorescein. Preferably,
carboxydichloroflorescein diacetate, Which exhibits only a weak
fluorescence, is utilized as a fluorogenic precursor due to its
rapid penetration into the hepatocyte plasma membrane.
Carboxydichlorofluorescein diacetate is hydrolyzed readily in the
cytoplasm of hepatocytes by intracellular esterases to a highly
fluorescent product, carboxydichlorofluorescein as described in
Haugland, Molecular Probes: Handbook of Fluorescent Probes and
Research Chemicals (1992-1994), p. 134, Molecular Probes, Inc.,
1992.
[0095] The fluorescence of carboxydichlorofluorescein is sensitive
to pH and thus any assay based on the intensity of
carboxydichlorofluorescence should consider the effects of pH.
However, it has been observed that less than a 0.3 pH unit
difference has been found between cytosol and bile canaliculi in
hepatocyte couplets. Although carboxydichlorofluorescein has been
used for pH determinations in acidic organelles, its fluorescence
intensity is not altered markedly between pH 7.1 and pH 7.4. The
fluorescence of carboxydichlorofluorescein at pH 7.4 is only about
10-20% higher than at pH 7.1 at maximum emission wavelength.
Inasmuch as the fluorescence of carboxydichlorofluorescein is used
as a qualitative probe to localize carboxydichlorofluorescein
cellular distribution, the slight pH gradient between cytosol and
the canaliculi do not affect the application of the high throughput
assay of the present invention.
[0096] Additional marker compounds include, but are not limited to,
fluorescein-labeled taurocholate, a bile acid that is rapidly and
extensively taken up by hepatocytes and excreted into the bile
canaliculi as described in the Laboratory Examples presented herein
below; cholyiglycylamido fluorescein, another fluorescent bile acid
described by Boyer and Soroka, Gastroenterology 109:1600-1611
(1995); rhodamine 123 for MDR2 and P-gp; and carboxyfluorescein
diacetate (CFDA).
[0097] It is contemplated that the method of the present invention
may be performed within standard multi-well assay plates as are
well known in the art, such as the 96-well micro-titer plates that
are available from ICN Pharmaceuticals, Inc. (Costa Mesa, Calif.).
Thus, a plurality of candidate compounds can be simultaneously
screened for susceptibility to biliary excretion within multiple
wells of a multi-well plate.
[0098] The following Laboratory Examples have been included to
illustrate preferred modes of the invention. Certain aspects of the
following Laboratory Examples are described in terms of techniques
and procedures found or contemplated by the present inventors to
work well in the practice of the invention. These Laboratory
Examples are exemplified through the use of standard laboratory
practices of the inventors. In light of the present disclosure and
the general level of skill in the art, those of skill will
appreciate that the following Laboratory Examples are intended to
be exemplary only and that numerous changes, modifications and
alterations can be employed without departing from the spirit and
scope of the invention.
Laboratory Examples
[0099] The following Laboratory Examples pertain to the
establishment of a correlation of biliary excretion in
sandwich-cultured rat hepatocytes (present method) and in vivo in
rats (standard). Five model substrates representing a diverse
spectrum of biliary excretion properties were selected to examine
the relationship between the percentage of the dose excreted in
bile in vivo in rats and in vitro using sandwich-cultured
hepatocytes in accordance with the methods of the present
invention. The five model substrates included inulin, salicylate,
methotrexate, [D-pen.sup.2,5]enkephalin and taurocholate.
[0100] Additionally, a comparison of in vivo and in vitro biliary
excretion of 264W94 and its metabolites is set forth in Example 4.
Compound 2169W94 is the O-demethylated metabolite of 264W94 in rats
and humans, which can undergo further conjugation with
urindine-5'-diphosphoflucuronic acid to form a glucuronide
conjugate (Silver et al., ISSX Proceedings, (San Diego, Calif. USA)
pp. 387, 1996). The structural formulas of compounds 264W94 and
2169W94 are presented in FIG. 9.
Materials and Methods Used in the Examples
[0101] Chemicals. [.sup.3H]Taurocholate (3.4 Ci/mmol;
purity>97%), [.sup.14C]salicylate (55.5 mCi/mmol;
purity>99%), and [.sup.3H][D-pen.sup.2,5]enkephalin (36 Ci/mmol;
purity>97% 0 were obtained from Dupont New England Nuclear
(Boston, Mass.). [.sup.3H]Methotrexate (13.7 Ci/mmol;
purity>99%) and [.sup.3H]inulin (1.3 Ci/mmol; purity 97%) were
obtained from Amersham international plc (Buckinghamshire,
England). Compounds [.sup.14C]264W94
((3R,5R)-3-butyl-3-ethyl-2,3,4,5-tetrahydro-7,8-dimethoxy-5-phenyl-1,4-be-
nzothiazepine-1,1-dioxide; 45.5 mCi/mmol; purity>99%) and
[.sup.14C]2169W94
((3R,5R)-3-ethyl-2,3,4,5-tetrahydro-7-methoxy-8-hydroxy-5-phenyl-1,4-benz-
othiazepine-1,1-dioxide; 43.7 mCi/mmol; purity>99%) were
obtained from Glaxo Wellcome, Inc. (Research Triangle Park, North
Carolina). Collagenase (type I, class I) was obtained from
Worthington Biochemical Corp. (Freehold, N.J.). Dulbecco's modified
Eagles's medium (DMEM), fetal bovine serum and insulin were
pumhased from Gibco (Grand Island, New York). Rat tail collagen
(type I) was obtained from Collaborative Biomedical Research
(Bedford, Mass.). All other chemicals and reagents were of
analytical grade and were readily available from commercial
sources.
[0102] Animals. Male Wistar rats (250-280 g) from Charles River
Laboratory (Raleigh, N.C.) were used as liver donors. Rats were
housed individually in stainless-steel cages in a constant
alternating 12-hr light and dark cycle at least 1 week before the
study was performed, and were fed ad libitum until use. Bile duct
cannulated rats (200-250 g) were obtained from Charles River
(Raleigh, N.C.). All procedures were approved by the Institutional
Animal Care and Use Committee at the University of North Carolina
at Chapel Hill, Chapel Hill, N.C.
[0103] Preparation of Culture Dishes. Plastic culture dishes (60
mm) were precoated with rat tail collagen at least 1 day prior to
preparing the hepatocyte cultures. To obtain a gelled collagen
substratum, ice-cold neutralized collagen solution (0.1 ml, 1.5
mg/ml, pH 7.4) was spread onto each culture dish. Freshly coated
dishes were placed at 37.degree. C. in a humidified incubator for
approximately 1 hr to allow the matrix material to gel, followed by
addition of 3 ml DMEM to each dish and storage in a humidified
incubator.
[0104] Culture of Rat Hepatocytes. Hepatocytes were isolated with a
two-step perfusion method. Briefly, rats were anesthetized with
ketamine and xylazine (60 and 12 mg/kg i.p., respectively) prior to
portal vein cannulation. The liver was perfused in situ with
oxygenated Ca.sup.2+-free Krebs-Henseleit bicarbonate buffer
containing collagenase type I (0.5 mg/ml) for 10 min. The hepatic
capsule was removed with forceps. The hepatocytes were released by
shaking the liver gently in 100 ml DMEM.
[0105] The released cells were filtered through a sterile nylon
mesh (70-.mu.m). The hepatocyte suspensions were centrifuged at
50.times.g for 3 min. The cell pellet was resuspended in 25 ml DMEM
and an equal volume of 90% isotonic polyvinylpyrrolidone-coated
silica colloid centrifugation medium (pH 7.4) sold under the
registered trademark PERCOLL.RTM. by Pharmacia, Inc. of Piscataway,
N.J. The resulting cell suspension was centrifuged at about 70 to
about 150.times.g for 5 min. The pellet was resuspended in 50 ml
DMEM and the cell suspensions were combined into one tube followed
by centrifugation at 50.times.g for 3 min. Hepatocyte viability was
determined by trypan blue exclusion. Only those hepatocyte
preparations with viability greater than 90% were utilized for
further studies.
[0106] Hepatocyte suspensions were prepared with DMEM containing 5%
fetal calf serum, 1 .mu.M dexamethasone and 4 mg/L insulin.
Hepatocyte suspensions were added to the precoated dishes at a
density of about 2-3.times.10.sup.6 cells/60-mm dish. Approximately
1 hr after plating the cells, the medium was aspirated and 3-ml
fresh DMEM was added. For transport studies, hepatocytes that had
been seeded for 3-5 hr without collagen overlay were defined as
3-hr or short-term cultured hepatocytes.
[0107] To prepare sandwich-cultured hepatocytes, neutralized
collagen solution (0.1 ml, about 1.5 to about 3.0 mg/ml, pH 7.4)
was added to the monolayers 24 hr after the cells were seeded.
Cultures with collagen overlay were incubated for 45 min at
37.degree. C. in a humidified incubator to allow the collagen to
gel before addition of DMEM. Medium was changed on a daily basis
until the fourth day after the cells were seeded. These hepatocytes
were referred to as 96-hr or long-term cultured hepatocytes.
[0108] Cumulative Uptake Studies in Sandwich-Cultured Hepatocytes.
Hepatocytes cultured in a collagen-sandwich configuration were
incubated in 3 ml standard buffer or Ca.sup.2+-free buffer at
37.degree. C. for 10 min. After removing the incubation buffer,
uptake was initiated by addition of 3 ml standard buffer containing
substrate to each dish. After incubation for designated times,
cumulative uptake was terminated by aspirating the incubation
solution and rinsing 4 times with 3 ml ice-cold standard buffer to
remove extracellular substrate. After washing, 2 ml of 1% Triton
X-100 solution was added to culture dishes, and the cells were
lysed by shaking the dish on a shaker for 20 min at room
temperature. An aliquot (1 ml) of lysate was analyzed by liquid
scintillation spectrometry. Bio-rad DC Protein Assay Kit (Bio-Rad
Laboratories, Hercules, Calif.) was used to determine the protein
concentration in the culture extracts using bovine serum albumin as
standard. Triton X-100 (1%) did not interfere with the assay. All
values for substrate uptake into cell monolayers were corrected for
nonspecific binding to the collagen by subtracting the substrate
uptake determined in the appropriate control dishes in the absence
of cells as described previously.
[0109] Biliary Excretion in Rats after Intravenous Administration
of 264W94 and Oral Administration of 2169W94. [.sup.14C]264W94 was
formulated as a solution in a mixture of sterile
water/polypropylene glycol 400/ethanol (2:1:1 v/v/v) at a
concentration of 0.125 mg/mL. Following collection of pre-dose
bile, [.sup.14C]1264W94 solution was administrated by caudal vein
injection (0.1 mg/kg). For the 2169W94 studies, [.sup.14C]2169W94
was prepared as a suspension at a concentration of 0.1 mg/mL in
0.5% (w/v) methylcellulose in water. Following collection of
pre-dose bile, [.sup.14C]2169W94 suspension was administrated by
gavage (1.0 mg/kg). All rats were placed into individual plastic
metabolism cages that allowed the rats unrestrained movement. Bile
was collected into polypropylene containers surrounded by ice. For
the 264W94 studies, the bile container was changed at 8 and 24
hours after the dose. Previous studies indicated that samples were
stable on ice for 24 hours. Bile samples were stored at -20.degree.
C. until analysis.
[0110] Analytical Procedure. Aliquots of cell lysate or bile
samples containing 264W94 or 2169W94 were mixed with 2-fold volumes
of ice-chilled acetonitrile, and centrifuged to remove precipitated
proteins. The supernatant was evaporated under nitrogen at room
temperature, and reconstituted in 100 .mu.L of a 70/30 mixture of
50 mM ammonium acetate/acetonitrile/trifluoroacetic acid (95:5:0.1
v:v:v) and acetonitrile. The sample extracts were injected onto a
WATERS.TM. SYMMETRY.TM. C18 column (3.9.times.150 mm) and eluted by
a 85/15 mixture of 50 mM ammonium acetate (pH 4.0) and
acetonitrile; the percentage of acetonitrile was increased by a
WATERS.TM. 600E System Controller to 55% over a period of 20
minutes, and then to 100% during the next 10 minutes.
[0111] Radiocarbon that eluted from the HPLC was quantified with an
on-line radioactivity detector (RADIOMATIC FLO-ONE/BETA.TM.
Radio-Chromatography Detector Series 500 TR Series, Packard
Instrument Co.). The peaks of 264W94, 2169W94, and 2169W94
glucuronide were identified by comparing with purified standard
compound. Under these conditions, baseline separation of these
three components was achieved. The concentration of the three
components was determined by normalizing the eluted radioactivity
in each peak to the total injected radioactivity.
[0112] Data Analysis. Uptake data were normalized to the protein
content and expressed as mean.+-.SD from 3-4 separate preparations
of hepatocytes. Statistical differences between mean values for the
10-min cumulative substrate uptake in the presence and absence of
Ca.sup.2+ were determined by the use of the well-known Student's
t-test. A P value of <0.05 was considered significant.
[0113] In vivo biliary clearance, Cl.sub.B (ml/min/kg body weight),
was calculated according to Equation 1:
Cl B = Amount bile ( 0 - T ) AUC 0 - T Equation 1 ##EQU00001##
where Amount.sub.bile(O-T) represents the amount of parent drug
recovered in bile from 0 to time T when most drug was eliminated
from the systemic circulation, and AUC.sub.0-T represents the area
under the plasma concentration-time curve from 0 to time T (in
minutes).
[0114] The in vivo intrinsic biliary clearance (Cl.sub.Bin,
ml/min/kg body weight) was estimated according to Equation 2 based
on the well-stirred model of hepatic disposition assuming biliary
excretion is the predominant elimination pathway (Pang et al., J.
Pharmacokinet. Biopharm. 5:625-653, 1977).
Cl Bin = Q Cl B Q - Cl B Equation 2 ##EQU00002##
where Q represents rat hepatic plasma flow, 40 ml/min/kg of body
weight {(blood flow.times.(1-hematocrit)}; Pollack et al., J.
Pharmacol. Exp. Ther. 18:197-202, (1989), and Cl.sub.B represents
billiary clearance for model compounds reported in the literature
or calculated from Equation 1.
[0115] Biliary excretion of substrates in the monolayers was
quantitatively assessed by the Biliary Excretion Index based on
Equation 3:
BiliaryExcretionIndex = Uptake standard - Uptake ca ++ - free
Uptake standard 100 % Equation 3 ##EQU00003##
where Uptake.sub.standard and Uptake.sub.Ca++-free represent the
cumulative uptake of substrate over a 10-min interval in the
hepatocyte monolayers pre-incubated in standard buffer and in
Ca.sup.++-free buffer, respectively.
[0116] Biliary clearance in the sandwich-cultured hepatocytes,
Cl.sub.B(culture) (ml/min/kg per body weight), was calculated
according to Equation 4:
Cl B ( culture ) = Uptake standard - Uptake Ca ++ - free Time
incubation Concentration medium Equation 4 ##EQU00004##
where Time.sub.incubation was 10 min and Concentration.sub.medium
represented the initial substrate concentration in the incubation
medium. Rat liver weight and protein content in liver tissue were
assumed to be 40 g/kg of body weight and 0.20 .mu.g of liver weight
(Seglen et al, Methods in Cell Biology (13.sup.th Ed., Prescott D.
M. Eds.) pp. 30-78, Academic Press, New York, 1976), respectively,
in all calculations.
Summary of the Results of the Examples
[0117] Biliary excretion of the five model substrates in long-term
sandwich-cultured hepatocytes in accordance with the present
invention was consistent with their in vivo biliary excretion
properties. Quantification of biliary excretion in the cultured
hepatocytes utilizing the biliary excretion index calculation is
described hereinabove. Briefly, the biliary excretion index
represents the percentage of retained substrate in the bile
canaliculi. The results of the Laboratory Examples indicate that
compounds undergoing negligible biliary excretion in vivo based on
the percentage of dose excreted in bile (e.g., inulin, salicylate)
have a low biliary excretion index (approximately zero). Compounds
that are more extensively excreted in bile in vivo (e.g.,
methotrexate, [D-pen.sup.2,5]enkephalin, and taurocholate) have a
high biliary excretion index (approximately 50%).
[0118] The relationship between the biliary excretion index and the
percentage of the dose excreted in bile in vivo only reveals a
categorical correlation. Methotrexate and [D-pen.sup.2,5]enkephalin
represent compounds that are "highly" excreted in bile
(approximately 60% and 70% of the i.v. dose was recovered in bile
in 1 hr, respectively). In contrast, taurocholate is "rapidly and
extensively" excreted in that almost all of the i.v. dose was
excreted in bile in less than 1 hr. The biliary excretion index can
thus differentiate between compounds that undergo extensive versus
negligible or low biliary excretion.
[0119] However, the biliary excretion index appears unable to
differentiate between compounds that are highly excreted in bile,
like methotrexate (biliary excretion index: approximately 55%) or
[D-pen.sup.2,5]enkephalin (biliary excretion index: approximately
42%), and compounds that are "rapidly and extensively" excreted in
bile, like taurocholate (biliary excretion index: approximately
56%). This limitation in the biliary excretion index may be due to
the fact that this index is determined predominantly by the
canalicular excretory functions. The percentage of
i.v.-administered substrate excreted into the bile in vivo is
determined by sinusoidal uptake activity, canalicular excretory
activity, as well as other competitive elimination processes.
[0120] Biliary clearance represents a more effective parameter for
comparison of the relationship between in vivo and in vitro biliary
excretion. The in vivo biliary clearance was calculated in the
Laboratory Examples as the ratio of the amount excreted into bile
at time T and the plasma AUC between time 0 and time T. Because
most of the administered dose was eliminated at time T, the biliary
clearance approximates the biliary clearance calculated from time 0
to time infinity. Biliary clearance calculated in this matter is a
function of intrinsic biliary clearance and the hepatic plasma flow
rate. To eliminate the effects of plasma flow, the intrinsic
biliary clearance was calculated based on the "well stirred" model
of hepatic disposition described by Pang and Rollan in J.
Pharmacokinet. Biopharm. 5:625-653, 1977. Likewise, in vitro
biliary clearance was calculated as the ratio of the amount
excreted in the canalicular networks in the hepatocyte monolayers
and the AUC in the incubation medium.
[0121] In the sandwich-cultured hepatocytes, the incubation medium
was accessible to all hepatocytes in the dish at the same time.
Thus, the calculated in vitro biliary clearance should represent
the intrinsic biliary clearance. However, since biliary excretion
involves two processes, uptake across the sinusoidal membrane and
excretion across the canalicular membrane, the true intrinsic
biliary clearance should be determined by transport across the
canalicular membrane and calculated based on intracellular
substrate concentrations. Therefore, the in vivo and in vitro
"intrinsic" clearance values calculated in the Laboratory Examples
may be referred to as an "apparent" intrinsic biliary clearance
value, which would be rate limited by the slowest step in the
process, either sinusoidal uptake or canalicular excretion.
[0122] The correlation between in vitro biliary clearance and in
vivo intrinsic biliary clearance was high (r.sup.2=0.9865) for the
five model substrates. According to the in vivo intrinsic biliary
clearance, the five model substrates can be classified into three
groups: compounds that are not excreted in bile (inulin and
salicylate; approximately 0 ml/min/kg), compounds that are highly
excreted in bile (methotrexate and [D-pen.sup.2,5]enkephalin,
approximately 17.3 ml/min/kg and approximately 34.4 ml/min/kg,
respectively); and compounds that are rapidly and extensively
excreted in bile (taurocholate, approximately 116.9 ml/min/kg). The
estimated in vitro biliary clearance adequately differentiated
between these three groups of compounds (approximately 0, 4-13, and
56 ml/min/kg, respectively). These results suggest that the binary
clearance more accurately characterizes the relationship between in
viva and in vitro biliary excretion as compared to the biliary
excretion index.
Example 1
Cumulative Uptake in Cultured Hepatocytes
[0123] The cumulative uptake of inulin was negligible (less than
0.01% of initial added substrate) at all incubation times in either
short-term or long-term cultured hepatocytes (FIGS. 1A and 1B). In
the 3-hr cultured hepatocytes, the cumulative uptake of salicylate,
methotrexate and [D-pen.sup.2,5]enkephalin was not significantly
different in standard buffer and in Ca.sup.2+-free buffer (FIGS.
2A, 3A, and 4A; p>0.05). However, slightly higher cumulative
uptake of taurocholate in standard buffer compared to
Ca.sup.2+-free buffer was observed (FIG. 5A); at 10 min, the
cumulative uptake in standard buffer was approximately 10% higher
than in Ca.sup.2+-free buffer (p=0.0352). In 96-hr cultured
hepatocytes, extracellular Ca.sup.2+ had no effect on the
cumulative uptake of salicylate (FIG. 2B, p>0.05). However, the
uptake of methotrexate, [D-pen.sup.2,5]enkephalin, and taurocholate
in long-term cultured hepatocytes in standard buffer was
significantly higher than in Ca.sup.2+-free buffer (FIGS. 3B, 4B,
and 5B; p<0.05).
Example 2
Relationship Between the Percentage of Dose Excreted in Bile in
Rats and Biliary Excretion Index in Cultured Hepatocytes
[0124] The five model substrates representing a diverse spectrum of
biliary excretion properties were selected to examine the
relationship between the percentage of the dose excreted in bile in
vivo in rats and the Biliary Excretion Index in sandwich-cultured
hepatocytes. Information regarding the percentage of the dose
excreted in rat bile after i.v. administration was obtained from
the literature. The extent of inulin and salicylate secretion into
bile was negligible (Eriksson et al., Acta. Physiol. Scand. 95:1-5,
1975; Laznicekand et al., Eur. J. Drug Met. Pharmacokinet.
19:21-26, 1994). Approximately 50-60% of a 22 .mu.mol/kg
methotrexate dose (Bremnes et al., Cancer Res. 49:2460-2464, 1989;
Masuda et al., Cancer Res. 57:3506-10, 1997) and 70% of a 14.5
.mu.mol/kg [D-pen.sup.2,5]enkephalin dose (Chen et al.; Pharm. Res.
14:345-350, 1997) were excreted into rat bile as unchanged drug in
1 hr. Taurocholate biliary excretion was more rapid and extensive
than methotrexate and [D-pen.sup.2,5]enkephalin. In 1 hr, virtually
100% of the dose (8.0 .mu.mol/kg) was recovered in rat bile (Inoue
et al., Biochim. Biophys. Acta. 833:211-216, 1985).
[0125] Biliary excretion in the sandwich-cultured hepatocytes can
be expressed quantitatively as the Biliary Excretion Index
calculated from Equation 3 based on the 10-min cumulative uptake
data in FIGS. 3B-5B. The Biliary Excretion Index of inulin and
salicylate was assumed to be negligible because no statistically
significant differences in the cumulative uptake of inulin or
salicylate were observed between standard buffer and Ca.sup.2+-free
buffer (p>0.05). The Biliary Excretion Index of methotrexate,
[D-pen.sup.2,5]enkephalin and taurocholate was 55.4.+-.18.3%,
42.4.+-.6.5% and 56.4.+-.5.2%, respectively. The relationship
between the percentage of the dose excreted in rat bile in vivo and
the Biliary Excretion Index measured in the in vitro system is
depicted in FIG. 6A. The Biliary Excretion Index was very low for
compounds undergoing negligible biliary excretion in vivo (e.g.,
inulin and salicylate). In contrast, the Biliary Excretion Index
was moderately high for compounds that are excreted in bile in vivo
(e.g., methotrexate, [D-pen.sup.2,5]enkephalin, and
taurochloate).
Example 3
Correlation of In Vitro and In Vivo Biliary Clearance
[0126] The in vivo biliary clearance (ml/min per kg body weight) of
inulin, salicylate, methotrexate and taurocholate was 0.035 (Utesch
et al., Vitro Cell. Dev. Biol. 27A:858-863, 1991), .about.0
(Laznicekand et al., Eur. J. Drug Met. Pharmacokinet. 19:21-26,
1994), 12.1 (Masuda et al., Cancer Res. 57:3506-10, 1997), and 29.8
(Inoue et al., Biochim. Biophys. Acta. 833:211-216, 1985),
respectively. In vivo biliary clearance of
[D-pen.sup.2,5]enkephalin, 18.5 ml/min/kg, was calculated based on
Equation 1 from the data reported by Chen and Pollack (Chen and
Pollack, Pharm. Res. 14:345-350, 1997). Based on these in vivo
biliary clearance values, the intrinsic biliary clearance of
inulin, salicylate, methotrexate, [D-pen.sup.2,5]enkephalin and
taurocholate was calculated from Equation 2 (0.04, 0. 17.3, 34.4,
and 116.9 ml/min/kg, respectively).
[0127] The in vitro biliary clearance of inulin, salicylate,
methotrexate, [D-pen.sup.2,5]enkephalin and taurocholate,
calculated from Equation 4 based on the 10-min cumulative uptake
data (FIGS. 1B-5B) was -0, -0, 4.1.+-.1.0, 12.6.+-.2.2, and
56.2.+-.6.0 ml/min/kg, respectively. The in vivo intrinsic biliary
clearance correlated well with the in vitro biliary clearance
(r.sup.2=0.9865) for the five model compounds (FIG. 6B).
Example 4
Comparison of In Vivo and In Vitro Biliary Excretion of 264W94 and
its Metabolites
[0128] The structural formulas of compounds 264W94 and 2169W94 are
presented in FIG. 9. Compound 2169W94 is the O-demethylated
metabolite of 264W94 in rats and humans, which can undergo further
conjugation with urindine-5'-diphosphoflucuronic acid to form a
glucuronide conjugate (Silver et al., ISSX Proceedings, (San Diego,
Calif. USA) pp. 387, 1996).
[0129] After i.v. administration of [.sup.14C]264W94 to rats (0.24
.mu.mol/kg), neither 264W94 nor 2169W94 was detected in bile in 24
hr. However, 35.4% (n=2) of the total administered radioactivity
was recovered in bile in the first hour. Approximately, 30.0% of
the radioactivity recovered in bile was the 2169W94 glucuronide;
the remaining 70% of radioactivity in bile represented unidentified
metabolites. After oral administration of [.sup.14C]264W94 to rats
(2.4 .mu.mol/kg), 2169W94 was not detected in the bile in 24 hr.
However, 66.4% (n=2) of the total administered radioactivity was
recovered in bile in 8 hr. Approximately, 88.7% of the
radioactivity in bile was in the form of the 2169W94 glucuronide
conjugate. These in vivo results demonstrate that 264W94 and its
O-demethylated product, 2169W94, undergo negligible biliary
excretion, but the glucuronide conjugate of 2169W94 undergoes
extensive biliary excretion in rats.
[0130] To determine the biliary excretion of 264W94 and metabolites
in 3-hr and 96-hr cultured hepatocytes, hepatocyte monolayers were
incubated in standard or Ca.sup.2+-free buffer before cumulative
uptake was conducted in standard buffer containing 3 .mu.M of
[.sup.14C]264W94 or [.sup.14C]2169W94 (FIGS. 7 and 8). In 3-hr
cultured hepatocytes, the cumulative uptake measured by total
radioactivity of 264W94 or 2169W94 was similar in the hepatocytes
pre-incubated in standard buffer or Ca.sup.++-free buffer
(p>0.05), suggesting that the uptake of 264W94 and 2169W94 in
short-term cultured hepatocytes was not affected by pre-incubation
of the monolayers in Ca.sup.++-free buffer. In 96-hr cultured
hepatocytes, the 10-min cumulative uptake of 264W94 measured by
total radioactivity was not significantly different in the
monolayers pre-incubated in standard buffer or Ca.sup.++-free
buffer (p>0.05).
[0131] HPLC analysis of the cell lysate at 10 min revealed that
73.0% of the total radioactivity was in the form of 264W94 and 3.3%
was the 2169W94 glucuronide conjugate; 2169W94 was not detected in
the lysate. In 96-hr sandwich-cultured hepatocytes, 10-min
cumulative uptake of 2169W94 was approximately 70% greater in the
presence of Ca.sup.2+ than in the absence of Ca.sup.2+ (p>0.05).
In the 10-min cell lysate, approximately 16.7% of total
radioactivity was in the form of 2169W94, and approximately 58.5
was the 2169W94 glucuronide conjugate. Compound 2169W94 forms the
glucuronide conjugate which is excreted into bile canalicular
networks in long-term cultured hepatocytes.
[0132] To further characterize the utility of the in vitro biliary
excretion assay of the present invention to predict in vivo biliary
excretion of drug metabolites, the in vitro and in vivo biliary
excretion of 264W94, and its O-demethylated metabolites 269W694 and
2169W94 glucuronide were examined. Previous in vitro studies
conducted with rat and human liver microsomes, precision cut liver
slices, and cDNA expressed hepatic cytochrome p450 isozymes
indicated that 264W94 formed an O-demethylated metabolite at the
8-methoxy position. Among the several cytochrome p450 isozymes
examined, CYP3A4 was the isozyme primarily involved in the
metabolism of 264W94 (Silver et al., ISSX Proceedings (San Diego,
Calif. USA) p. 387, 1996).
[0133] In vivo disposition studies demonstrated that neither 264W94
nor its O-demethylated metabolite, 2169W94, was excreted in the
bile. But, the 2169W94 glucuronide conjugate, along with other
unidentified metabolites, were extensively excreted in bile. The
lack of biliary excretion of 264W94 in long-term sandwich-cultured
hepatocytes was consistent with negligible in vivo biliary
excretion of 264W94.
[0134] In vivo, approximately 35% of 264W94 equivalent was excreted
in bile as metabolites in 1 hr after i.v. administration of 264W94.
In cultured hepatocytes, however, the biliary excretion of 264W94
metabolites was negligible (FIG. 7B). This apparent discrepancy
between the in vivo and in vitro biliary excretion for metabolites
of 264W94 may be explained by differences in metabolic activities.
In vivo, 264W94 undergoes O-demethylation to form 2169W94; and
subsequently, 2169W94 is conjugated with
uridine-5'-diphosphoglucuronic acid to form 2169W94 glucuronide.
This glucuronide conjugate accounts for 30% of the total amount
excreted in bile. In the lysate of long-term sandwich-cultured
hepatocytes incubated with 264W94, only approximately 3% of the
total amount incubated was detected as the 2169W94 glucuronide
conjugate. These results indicated that the long-term cultured
hepatocytes were not capable of the O-demethylation reaction.
Consequently, negligible glucuronide conjugate was formed and
excreted in the bile.
[0135] However, after incubation of the monolayers with 2169W94,
the O-demethylated metabolite of 264W94, 58.5% of 2169W94 was
converted to glucuronide conjugates and significant biliary
excretion was observed in the cultured hepatocytes (FIG. 8B).
Evidently, phase I metabolic activities such as O-demethylation
deteriorate significantly, while the phase II metabolic activities
such as glucuronide conjugation are maintained, at least in part,
in the long-term sandwich-cultured hepatocytes used in accordance
with the present invention. Thus, this Laboratory Example further
indicates that the assay of the present invention can be employed
to predict in vivo biliary excretion of a substrate in its parent
form. Indeed, the application of the present in vitro assay method
to study and to predict in vivo biliary excretion of metabolites
requires consideration of the status of metabolic activities in the
monolayers.
REFERENCES
[0136] The references listed below as well as all references cited
in the specification are incorporated herein by reference to the
extent that they supplement, explain, provide a background for or
teach methodology, techniques and/or compositions employed herein.
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[0138] Bremnes et al., Cancer Res. 49:2460-2464, 1989. [0139] Chen
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[0160] It will be understood that various details of the invention
may be changed without departing from the scope of the invention.
Furthermore, the foregoing description is for the purpose of
illustration only, and not for the purpose of limitation--the
invention being defined by the claims.
* * * * *