* Department of Drug Safety Evaluation, Pfizer Global Research and Development, Ann Arbor, Michigan 48105;
University of Pittsburgh Medical Center, Department of Pathology, Pittsburgh, Pennsylvania 15261;
Departments of Pharmokinetics, Dynamics, and Metabolism and
Pharmaceutical Sciences, Prizer Global Research and Development, Ann Arbor, Michigan 48105;
¶ Veterans Administration Medical Center, White River Junction, Vermont 05009; and
|| Departments of Biochemistry and Pharmacology/Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03756
Received July 2, 2003; accepted August 6, 2003
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ABSTRACT |
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Key Words: cholestasis; preclinical; clinical; toxicity; hepatocytes; bile acids; macrolides; transport.
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INTRODUCTION |
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The common characteristic of the drugs used in this study is the preferential elimination from the body via the biliary pathway, which accounts for at least 50% of total drug clearance. We and others have hypothesized that hepatotoxicity in humans taking these drugs may be associated with drug-mediated inhibition of active canalicular transport of bile components, including, but not limited to, bile acids (Kostrubsky et al., 2001; Stieger et al., 2000
). These drugs are likely substrates for active liver transporter-mediated uptake and efflux into the bile canaliculi. The transporters that participate in biliary drug elimination also transport endogenous bile components. Therefore, there is a potential for mutual inhibition of drug and bile acid efflux from the liver, resulting in an increase in drug and bile acids retained in the liver over time. We hypothesized that compounds showing greater inhibitory potency for bile acid transport will have a greater risk of being hepatotoxic. To address this issue, we have used an in vitro model of cultured human hepatocytes with an extensive canalicular network. We developed a competition assay between the drug and radioactively labeled bile acid to test whether canalicular efflux of taurocholate can be inhibited in a concentration-dependent manner, and whether this inhibition would correlate with clinical hepatotoxicity. For the current study, we have used six macrolide antibiotics with both high and low incidences of clinical hepatotoxicity. Our data indicate that drugs with greater hepatotoxic risk are stronger inhibitors of taurocholate transport in cultured human hepatocytes.
Previously, Fattinger et al.(2001) observed an increase in rat serum bile acids after treatment with drugs that cause hepatotoxicity in clinic. These authors found a dose-dependent increase in rat total bile acids after drugs were administered, either alone or in combination. We investigated whether inhibitors of taurocholate transport in cultured hepatocytes would also cause an increase in rat serum bile acids. Since bile acids are increased in humans taking these drugs, and associate with clinical hepatotoxicity, the increase in rat serum bile acids might be predictive of hepatotoxicity in humans.
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MATERIALS AND METHODS |
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Hepatocyte culture.
Human hepatocytes were prepared from livers not used for whole organ transplant within 24 h of procurement. Hepatocytes were isolated by a three-step collagenase perfusion technique as described previously (Strom et al., 1996) and plated at a cell density of 2 x 106 cells per well in 6-well plates previously coated with 0.2 mg/ml type I collagen. The isolated hepatocytes (approximately 92% purity) were maintained in HMM medium supplemented with 10-7 M dexamethasone, 10-7 M insulin, 100 units/ml penicillin G, 100 µg/ml streptomycin, and 10% bovine calf serum, and were kept at 37°C in a humidified incubator with 95% air/5% CO2. Cells were allowed to attach for 46 h. At this time the medium was replaced with serum-free medium and changed on a daily basis thereafter. Following 2448 h in culture, medium was removed and the cells overlaid with a neutralized preparation of collagen (0.1 ml/well) at a final concentration of 1.5 mg/ml, as described previously, with modifications (LeCluyse et al., 1994
; Liu et al., 1999a
,b
). Specifically, collagen stock solution on ice was diluted with cold HMM culture medium, and pH was adjusted to 7.4 using cold, sterile 0.2 N NaOH. The plate was tilted to spread collagen, and excess collagen on the sides of wells was pipetted to fill uncovered spots. After 60 min in a humidified incubator at 37°C, culture medium was added to the plates and changed every 24 h thereafter. Some plated cells were not overlaid for the duration of the experiment, for comparison of transport with sandwiched cells.
Inhibition of bile acid transport.
After 96120 h in culture, to allow for canaliculi to develop, the culture medium was replaced with regular Hanks balanced salt solution (HBSS). Following 10 min incubation, 1 µM [3H]taurocholic acid or 5 µM CLF, with or without test compound, at concentrations indicated in the figure legends, was added in standard HBSS buffer, and plates were incubated for 15 min at 37°C. Transport was stopped by removing buffer and washing cells three times with 2 ml of cold standard HBSS. Taurocholate efflux from canalicular spaces was initiated by adding, in a time-dependent sequence, 1 ml of standard HBSS or Ca/Mg2+-free HBSS and incubating at 37°C (Liu et al., 1999c). Removal of Ca/Mg2+ from the incubation buffer opens the tight junctions and releases the bile acid that has accumulated therein (City, 1992
). Aliquots of media (100 ml) were harvested at the indicated time points and counted in a liquid scintillation counter. Cells were washed once with regular HBSS and harvested in 1 ml of 0.2 N NaOH/0.1% SDS. Aliquots of cell lysate were counted in a liquid scintillation counter to determine the amount of taurocholate retained by the cells. All transport activities were normalized per milligram of total protein (Lowry et al., 1951
).
The quantity of bile acid that accumulated in canaliculi was defined by the difference in efflux of radioactive taurocholate in incubation buffers, in the absence and presence of Ca/Mg2+, and calculated at 10 min after addition of the corresponding buffer. The difference in amount of radioactivity between the two buffer conditions in the absence of inhibitor corresponded to a 100% taurocholic acid efflux in canaliculi. In the presence of an inhibitor, this difference became smaller and was used to calculate the percent inhibition of canalicular taurocholic acid efflux.
Inhibition of taurocholate cellular uptake was calculated based on the amount of radioactivity recovered from cell lysates, in the presence or absence of inhibitors incubated in standard HBSS buffer.
The results represent the data from seven separate hepatocyte cultures prepared from different donors. We observed that taurocholate transport was strongly present and produced little variation from culture to culture when plated hepatocytes formed a confluent monolayer with microscopically distinguishable bile canaliculi on the day of the experiment. In contrast, absolute values of taurocholate transport were not sufficient to study the effect of inhibitors if the cells were subconfluent and formed separate aggregates of cells.
Fluorescent images of canalicular-accumulated CLF were taken immediately after washing cells and adding regular or Ca/Mg2+-free buffer.
Inhibition of bile acid transport in intact rats.
In vivo experiments were conducted in jugular vein precannulated Sprague-Dawley rats weighing approximately 300 g. Animals received powdered rodent chow (Purina certified chow; Purina, St. Louis, MO) and tap water by bottle ad libitum. All procedures involving animals were conducted in accordance with Guide for the Care and Use of Laboratory Animals and under a protocol approved by the Institutional Animal Care and Use Committee. Animals were fasted overnight prior to experiment. The drugs were administered, via the tail vein, to restrained animals, as follows: a single iv dose of vehicle-control glyburide (25 mg/kg), CI-1034 (25 mg/kg), or their combination, at 25 mg/kg each. The vehicle was a mixture of N,N-dimethylacetamide (DMA) and a 40% ß-cyclodextrin sulfobutyl ether sodium salt (SBECD) solution (w/v) prepared in 50 mM Tris(hydroxymethyl)aminomethane such that the total vehicle volume was composed of 5% DMA and 95% SBECD solution. Blood samples were collected from the jugular vein at indicated time-points, and serum was analyzed for total bile acids using a Hitachi 911 analyzer and Sigma reagents.
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RESULTS |
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The overall effect of different compounds on canalicular efflux, measured in two hepatocyte cultures, is shown in Figures 6A and 6B
. Efflux at 100% is shown by an arrow in the control cells. A decrease in difference between the two buffer conditions, in the presence and absence of potential inhibitors, represents the percent inhibition compared to untreated hepatocytes, as summarized in Figure 6B
. At the concentrations tested, salicylic acid did not inhibit transport of taurocholic acid. In contrast, CyA, CI-1034, bosentan, glyburide, and TAO, drugs that are eliminated preferentially via bile and clinically known to cause liver toxicity, inhibited taurocholate efflux. CyA, CI-1034, and glyburide caused a concentration-dependent inhibition of taurocholate efflux. CyA at 10 µM completely inhibited taurocholate efflux, since no difference in taurocholate efflux was observed between the two buffers.
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DISCUSSION |
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The rationale for the present study comes from the understanding that some drugs are substrates for hepatic active biliary transport and are eliminated via the bile. Clinical adverse liver effects, including cholestasis, are associated with inhibition of biliary transport. Compounds with molecular structures that make them likely candidates for biliary elimination can inhibit bile acid transport and be potentially hepatotoxic. Drugs, including CI-1034, bosentan, TAO, glyburide, and erythromycin estolate, are known to cause hepatic dysfunction in humans. Bosentan causes an increase in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in 1011% of patients at levels at least 3 times the upper limit of normal, and in 4% of patients at levels greater than 8 times the upper limit of normal (NDA, 2001). In addition, Fattinger et al. (2001)
reported that bosentan-induced cholestatic liver injury developed within the first 4 weeks of treatment, with increased serum bile acid levels preceding increased ALT levels by 5 to 7 days. The same group reported administration of glyburide as a risk factor, because concomitant bosentan and glyburide treatment increased the incidence of liver injury to 29%. They also found an additive response on rat serum bile acids when both drugs were coadministered intravenously. We found a similar response with CI-1034 when it was combined with glyburide (Fig. 9
). In a clinical Phase 1 study, CI-1034 was administered only to a small number of patients before being discontinued from development. Similarly to bosentan, CI-1034 increased ALT, AST, and serum bile acids at between 2 and 7 weeks of treatment.
TAO and erythromycin estolate are drugs with known hepatotoxic potential in humans. Serum aminotransferase elevations with erythromycin estolate have been reported in up to 38% of patients, with another study reporting an increase in about 10% of patients and a cholestatic pattern of injury developing two weeks after the beginning of therapy (reviewed in Stricker and Spoelstra 1985). Changes appear in most cases within 5 to 15 days after starting treatment. In agreement with many clinical reports of cholestasis, in our study, erythromycin estolate was found to be a potent inhibitor of taurocholate efflux (Fig. 8
). In addition, erythromycin estolate has been shown to reduce bile acid concentrations and bile flow in isolated rat liver, effects that were not observed to the same extent with erythromycin base (Gaeta et al., 1985
). We also found that erythromycin base, in contrast to erythromycin estolate, was ineffective in inhibiting taurocholate transport. It also does not have the same reported clinical hepatotoxicity as erythromycin estolate. Similar to erythromycin estolate, TAO has a high reported rate of liver toxicity increasing serum aminotransferase in 30% and producing jaundice in 4% of patients, typically after about two weeks of treatment (Ticktin and Zimmerman, 1962
). TAO followed erythromycin estolate in inhibiting taurocholate efflux in our system (Fig. 8
). In contrast to the high frequency of reported clinical hepatotoxicity after treatment with erythromycin estolate and TAO, new-generation macrolides are less hepatotoxic. Although roxithromycin, spiramycin, and telithromycin have clinical records of cholestatic hepatitis (Denie et al., 1992
; Easton-Carter et al., 2001
; U.S. FDA, 2003
; Zuazo et al., 1997
), they have less frequent incidences of liver injury. Similarly, in sandwiched human hepatocytes, roxithromycin did not inhibit efflux of taurocholate, and it increased liver aminotransferase in <1% of patients (product information). Both telithromycin and spiramycin inhibited taurocholate efflux only at 100 µM. Recent clinical data on telithromycin indicates that it increased ALT > 3 times the upper limit of normal in approximately 1% of patients at any post-therapy time point (FDA AIDAC Meeting, 2003).
The inhibitory potencies of individual compounds within a given therapeutic area need to be evaluated in conjunction with the projected human therapeutic plasma concentration. Higher plasma concentrations will likely be associated with greater levels of the drug in liver. When the drug has a high potency of inhibition of bile-acid transport in the described systems, this compound will be expected to have a greater probability of clinically adverse liver effects. For bosentan, CI-1034, TAO, and all tested macrolides, the human plasma concentrations are above 1 µg/ml (112 µg/ml). This is similar to the serum concentration of bile acids, which are concentrated in bile at 1000 times the concentration detected in serum. In contrast, the plasma concentration for glyburide is about 0.1 µg/ml and is associated with a low incidence of hepatic injury, despite the potent inhibition of bile-acid transport. Yet, a few reports have demonstrated cholestatic hepatitis with glyburide, an effect that may be attributed to the ability of glyburide to accumulate in liver and to be a potent inhibitor of bile-acid transport (Kelner et al., 1969; Stricker and Spoelstra, 1985
; Wongpaitoon et al., 1981
). Similarly, high hepatic concentrations (150 times greater than in the serum) were also reported in rats for erythromycin (Lee et al., 1953
), and roxithromycin, an oxime derivative of erythromycin that has an elimination half-life of about six times that of erythromycin, also reached high tissue concentrations (Puri and Lassman, 1987
). CyA is a potent inhibitor of Bsep (Stieger et al., 2000
), as well as of taurocholate transport in our system (Fig. 6
). In agreement with in vitro data, treatment of transplant patients with CyA resulted in 2- to 3-fold increases in total serum bile acids and this correlated well with blood levels of CyA (Tripodi et al., 2002
). At the same time, CyA has a lower incidence of hepatotoxicity in comparison to nephrotoxicity (Klintmalm et al., 1981
). CyA is pharmacologically active at plasma concentrations less than 1 µg/ml (0.10.4 µg/ml) and has a low hepatic extraction ratio, likely maintaining a low level of drug in the liver, a factor contributing to the low frequency of hepatotoxicity.
The fact that both glyburide and CI-1034 increased rat serum total bile acids after iv administration suggests their cholestatic potential by acting on the hepatobiliary transporters. Fattinger et al., 2001, observed a similar response to glyburide and bosentan. These authors found a dose-dependent increase in rat total bile acids after drugs were administered, either alone or in combination. Since bile acids are increased in humans taking these drugs and associate with clinical hepatotoxicity, the increase in rat total bile acids might be predictive of hepatotoxicity in humans.
In summary, cultured human hepatocytes transport bile acid into and out of cells. This transport can be inhibited, in a dose-dependent fashion, by compounds that are substrates for biliary elimination. The potency of this inhibition by a drug is likely to be a reflection of its in vivo inhibitory effect on bile-acid transport and could therefore be associated with adverse clinical liver effects. Even though rats may be resistant to liver toxicity induced by some drugs, a transient increase in serum bile acids combined with inhibitory effects on bile-acid efflux in sandwich cultured human hepatocytes may be better predictors of the cholestatic potential of compounds in humans. The evaluation of compounds from a given therapeutic area, using our proposed strategy, will help rank compounds according to their hepatotoxic potential.
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ACKNOWLEDGMENTS |
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NOTES |
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