Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2000-03-17
2004-08-24
Weber, Jon (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S370000, C435S366000, C435S325000
Reexamination Certificate
active
06780580
ABSTRACT:
TECHNICAL FIELD
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
AUC
area under the curve
BSEP
bile salt export pump
Cl
B
biliary clearance
Cl
in
intrinsic clearance
cMOAT
canalicular multispecific organic anion transporter
CFDA
carboxyfluorescein diacetate
DMEM
Dulbecco's modified Eagle's medium
EDTA
ethylenediamine tetraacetate
HP
Hewlett Packard
HPLC
high performance liquid chromatography
hr
hour
i.v.
intravenous
i.p.
intraperitoneal
K
m
Michaelus-Menten constant for enzyme-substrate reaction
LC/MS
liquid chromatography/mass spectrometry
mg pr.
milligrams protein
min
minute
MDR2
multidrug resistance protein 2
MRP2
multidrug resistance associated protein 2
Ntcp
Na
+
/taurocholate cotransporting polypeptide
OATP1
organic ion anion transporting polypeptide 1
OATP2
organic ion anion transporting polypeptide 2
P-gp
P-glycoprotein
SD
standard deviation
UV
ultraviolet
UV/VIS
ultraviolet/visible
V
max
maximum velocity of enzyme-catalyzed reaction
BACKGROUND ART
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.
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.
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.
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.
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 Sci.
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. Heptaology
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.
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
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.
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.
It is a further object of the present invention to provide an in vitro method of screening candidate compounds for susceptibility to biliary excretion.
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.
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.
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.
REFERENCES:
patent: 5602026 (1997-02-01), Dunn et al.
patent: WO 94/12662 (1994-06-01), None
patent: WO 96/01426 (1996-01-01), None
LeCruse et al. Am. J. Physiol. 266 (Cell Physiol.35):C1764-C1774, 1994.*
Poole et al. Archives of Toxicology. (1990) 64:474-481.*
Liu et al., “Partial Maintenance of Taurocholate Uptake by Adult Rat Hepatocytes Cultured in a Collagen Sandwich Configuration,” Pharmaceutical Research, vol. 15, No. 10, 1998. (Journal Article).
Liu et al., “Biliary Excretion of Taurocholate (TC) in Rat Hepatocytes Cultured in a Collagen Sandwich Configuration (SC)”, Hepatology 24:370A (973), 1996,
Brouwer Kim L. R.
LeCluyse Edward L.
Liu Xingrong
Afremova Vera
Jenkins & Wilson & Taylor, P.A.
The University of North Carolina at Chapel Hill
Weber Jon
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