Introduction

Abstract Introduction Results Discussion References

Recently, a variety of small peptidessomatostatin analogs (1-3), endothelin antogonists (4, 5), and renin inhibitors (6)have been developed as therapeutic agents. Despite of their stability to enzymatic degradation in the gastrointestinal tract and liver, the short half-lives in plasma hinder their development as therapeutic agents. This short half-life was the result of extensive hepatic uptake and biliary excretion (7-9). Bile acids and organic anions are known ligands taken up efficiently by the liver from blood. It has been shown that active transport is involved in their hepatic uptake. Organic anions such as DBSP¹ (10), benzylpenicillin (11), and HMG-CoA reductase inhibitor (pravastatin) (12) are transported by a Na⁺-independent multispecific organic anion transporter (13); whereas bile acids are transported both by a Na⁺-dependent bile acid transporter and by a Na⁺-independent organic anion transporter (14). Organic cations are taken up by a different system from that of organic anions (15). In addition, previous reports on isolated hepatocytes have demonstrated carrier-mediated transport in the hepatic uptake of small peptides, such as the somatostatin analog, octreotide (16-18), renin inhibitors (19-22), and endothelin antagonists (23). The hepatic uptake of these ligands is also inhibited by bile acids and organic anions. These results suggest that peptides may be transported by common transport systems for organic anions and bile acids (17, 19, 24). However, analysis of the tissue distribution of peptides in vivo has received little detailed attention. The tissue distribution of drugs is governed by many biological and physiological factors, such as tissue volume, blood flow, protein binding in plasma and tissue, specific transport mechanisms, etc. In particular, there is a specific transport mechanism for organic anions in liver and kidney, organs involved in the metabolism and excretion of exogenous compounds, such as DBSP (liver) and PAH (kidney) (25). A transport system for organic cations is also known to exist in both organs (15).

In this study, analysis of the tissue distribution of the cyclooctapeptide, octreotide, was conducted using it as a model compound for peptides. More than 70% of an intravenous dose of octreotide is excreted into the bile in unchanged form, and 30% of the dose is excreted into urine (26). To investigate the initial tissue distribution of octreotide, the time profiles of [¹⁴C]octreotide in plasma and several tissues were determined to calculate the tissue uptake clearance of octreotide in vivo in several tissues, including the liver and kidney. The uptake clearances obtained were compared among the tissues. To determine whether specific transport exists in the tissue distribution of octreotide, the effects of organic anions (DBSP and PAH), bile acid (TCA), and unlabeled octreotide on the tissue uptake of [¹⁴C]octreotide were examined. The characteristics of octreotide uptake and the magnitude of the uptake and inhibition by another compound were compared in vivo and in vitro using isolated rat hepatocytes and primary cultured hepatocytes. In addition, biliary excretion was determined in EHBRs in which the ATP-driven canalicular transport system (cMOAT) for organic anions such as DBSP and dinitrophenylglutathione is genetically defective, whereas the biliary excretion system for bile acids is conserved (27).

Materials and Methods

Chemicals and Reagents. Unlabeled octreotide and [¹⁴C]octreotide (specific activity: 46.2 mCi/mmol) were supplied by Sandoz Pharma Ltd. (Basel, Switzerland). [³H]Inulin was purchased from New England Nuclear (Boston, MA). DBSP was from the Société d'Etudes et de Recherches Biologiques (Paris, France). All other chemical and reagents were commercial products of analytical grade.

Analysis of the Time Profiles of the Plasma Disappearance and the Biliary Excretion of [¹⁴C]Octreotide. Male SDRs (Nisseizai, Tokyo, Japan) and EHBRs (Eisai Laboratories, Gifu, Japan) weighing ~220 g were used throughout the experiments. Under light ether anesthesia, both femoral arteries and veins were cannulated with polyethylene catheters (PE-50) for blood sampling and the bolus injection of octreotide and infusion of DBSP or TCA, respectively. The bile duct was cannulated with polyethylene catheters (PE-10) for bile collection.

Determination of Kinetic Parameters. Plasma concentration profiles were fitted with the following biexponential equation using the nonlinear iterative least squares method (28).

(1)

AUC at t was calculated by integrating eq.1 from 0 to t.

(2)

(3)

(0-)

Analysis of Tissue Uptake after Intravenous Administration of [¹⁴C]Octreotide in SDRs and EHBRs. After intravenous bolus administration of [¹⁴C]octreotide (8 µCi/kg) via the femoral vein, blood was collected from the femoral artery. At time 1, 3, or 5 min after administration of [¹⁴C]octreotide, rats were killed, and liver, kidney, intestine, pancreas, lung, muscle, spleen, and cerebrum were excised immediately, and a portion of each tissue was weighed and radioactivity counted. The tissue uptake rate can be described by the following differential equation during the relatively early phase when the efflux of [¹⁴C]octreotide is minimal:

(4)

where X(t) is the amount of octreotide in the tissue at time t, CL_up is the tissue uptake clearance, and Cp(t) is the plasma concentration of octreotide. Integration of eq. 4 gives:

(5)

where AUC_(0-t) represents the area under the plasma concentration-time curve from 0 to t.

(6)

Inhibition of Octreotide Uptake by DBSP, TCA, PAH, and octreotide. Thirty minutes before administration of the mixture of [¹⁴C]octreotide and [³H]inulin, DBSP (2.5 µmol/min/kg and 25 µmol/kg as loading dose), TCA (10 µmol/min/kg), PAH (23 µmol/min/kg and 700 µmol/kg as loading dose), or octreotide (250 nmol/min/kg) infusions were started and continued throughout the experiment. As a control experiment, an equivalent volume of saline was infused.

Uptake of Octreotide by Isolated Hepatocytes. Hepatocytes were isolated from male SDRs (250-280 g) by the procedure of Baur et al. (31). After isolation, hepatocytes were suspended (2 × 10⁶ cells/ml) at 0°C in Krebs-Henseleit buffer supplemented with 12.5 mM HEPES (pH 7.4) containing 2% BSA. Cell viability was checked by the trypan blue (0.4%) exclusion test.

Uptake of Octreotide by Primary Cultured Hepatocytes. Hepatocytes were prepared from male SDRs (220-280 g) by the procedure of Kato and Sugiyama (33). After isolation, hepatocytes were suspended (5 × 10⁵ cells/well) in a 12-well plate (Corning, Corning, NY) in 5% calf serum, 1 nM insulin, and 1 nM dexamethasone containing William's medium E. Cells were cultured for 4.5 hr at 37°C and 5% CO₂. Cell viability was checked by the trypan blue (0.4%) exclusion test.

Estimation of In Vivo Uptake Clearance from In Vitro Data. The kinetic parameters for octreotide uptake were estimated from the following equation:

(7)

where V_o is the initial uptake rate of octreotide (pmol/min/10⁶ cells), S is the octreotide concentration in the medium (µM), K_M is the Michaelis constant (µM), V_max is the maximum uptake rate (pmol/min/10⁶ cells), and P_diff is the nonspecific uptake clearance (µl/min/10⁶ cells). The aforementioned equation was fitted to the uptake data sets by an iterative nonlinear least squares method using a MULTI program (28) to obtain estimates of kinetic parameters. Input data were weighted as reciprocals of the square of the observed values, and the algorithm used for fitting was the Damping Gauss Newton Method.

(8)

(9)

	Results

Abstract Introduction Results Discussion References

Effect of DBSP and TCA on Plasma Elimination and Biliary Excretion of [¹⁴C]Octreotide. The effects of an organic anion, DBSP, and a bile acid, TCA, on the plasma elimination and biliary excretion of [¹⁴C]octreotide were examined (fig. 1). Intravenous infusion of DBSP and TCA into rats was conducted at two infusion rates before and after bolus administration of [¹⁴C]octreotide. Biliary excretion rates of TCA after infusion at a rate of 4 or 10 µmol/min/kg were 3.9 or 7.1 µmol/min/kg from 20 to 30 min after initiation of infusion, respectively, and the plasma concentration after 30 min was 100 or 350 µM, respectively. Biliary excretion rates of DBSP after infusion at a rate of 1 or 2.5 µmol/min/kg were 0.9 or 1.7 µmol/min/kg, and plasma concentration was 60 and 300 µM, respectively. Biliary excretion of DBSP and TCA exhibited minimal increase by further increasing the infusion rates. Biliary excretion rates at the infusion rates of 10 µmol/min/kg for TCA and 2.5 µmol/min/kg for DBSP were then estimated as transport maximum (T_max). At 30 min after starting of infusion of TCA or DBSP, [¹⁴C]octreotide was administered by intravenous bolus injection, and plasma and biliary excretion profiles were examined. Plasma elimination and biliary excretion of [¹⁴C]octreotide were inhibited by both TCA and DBSP in a dose-dependent manner and the biliary excretion of [¹⁴C]octreotide was reduced to <10% of that of controls at the T_max of each compound. Moreover, the plasma elimination of [¹⁴C]octreotide was markedly delayed at T_max of TCA and DBSP. The biliary excretion clearance (CL_bile,p) defined with respect to the octreotide concentration in plasma was reduced to 0.5% by TCA and to 6.6% by DBSP, compared with controls (table 1).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Effect of TCA and DBSP on plasma elimination (A) and biliary excretion (B) of octreotide. Thirty minutes before intravenous administration of [14C]octreotide (40 µCi/kg), DBSP (, 1 µmol/min/kg; , 2.5 µmol/min/kg) or TCA (, 4 µmol/min/kg; , 10 µmol/min/kg) infusion was started and continued throughout the experiment. As a control (), an equivalent volume of saline was infused. Each point and vertical bar represents the mean ± SEM of three rats.

Comparison of Plasma Elimination and Biliary Excretion of [¹⁴C]Octreotide between SDRs and EHBRs. Plasma elimination and biliary excretion of [¹⁴C]octreotide in EHBR after intravenous bolus administration were compared with SDRs (fig. 2). Plasma elimination was delayed, compared with SDRs and the cumulative biliary excretion over 120 min was reduced to 40% of that in SDRs. The CL_bile,p in SDRs and EHBRs was 7.4 ml/min/kg and 1.7 ml/min/kg, respectively, and the CL_bile,p in EHBRs was reduced to 23% of that in SDRs (table 1).

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Plasma elimination and biliary excretion of [14C]octreotide in SDRs and EHBRs. After intravenous administering of [14C]octreotide (40 µCi/kg) to SDRs () and EHBRs (), plasma elimination (A) and cumulative biliary excretion (B) were monitored. Each point and vertical bar represents the mean ± SEM of three rats.

Analysis of Early-Phase Tissue Distribution of [¹⁴C]Octreotide in SDRs. The early-phase tissue distribution of [¹⁴C]octreotide was examined (fig. 3). The time profiles of [¹⁴C]octreotide concentrations in plasma and several tissues 1, 3, and 5 min after intravenous bolus injection were determined to calculate CL_up of octreotide in vivo. The CL_up values in the liver and kidney were 0.2 ml/min/g and 0.58 ml/min/g, respectively, and were much higher than other organs, such as spleen, pancreas, lung, muscle, intestine, and brain. K_p of [₁₄C]octreotide was similar to that of [³H]inulin as an extracellular space marker in muscle, intestine, and brain, whereas slightly higher K_p values were observed in the spleen, lung, and pancreas (table 2). To determine the contribution of glomerular filtration to the uptake of octreotide by the kidney, the CL_up of [³H]inulin at 5 min was determined to be 1.02 ± 0.16 ml/min/g kidney. The glomerular filtration clearance of octreotide in kidney estimated using the unbound fraction of octreotide in plasma (0.41) and the CL_up of inulin (1.02 ml/min/g kidney) was 0.42 ml/min/g kidney, as indicated by a broken line in fig. 3B. This value was similar to the CL_up of octreotide (0.58 ml/min/g) in the kidney. These results suggest that most of the apparent uptake of octreotide by the kidney is mainly due to the glomerular filtration (fig. 3B).

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Early-phase tissue uptake of [14C]octreotide (8 µCi/kg) after intravenous administration to rats (integration plot). [14C]Octreotide plasma concentration-time profiles and uptake by each tissue after 1, 3, and 5 min were measured and data were expressed as integration plots. Initial slope represents the CLup for each tissue. The broken line represents nonspecific uptake of [14C]octreotide by glomerular filtration (fp multiplied by GFR) in the kidney. (A) , liver; , lung; , pancreas; , cerebrum. (B) , kidney.

Inhibition of [¹⁴C]Octreotide Hepatic Uptake by Taurocholic Acid and Organic Anions in SDRs. The effects of coinfusion of TCA, DBSP, PAH, and unlabeled octreotide on the initial hepatic uptake clearance of [¹⁴C]octreotide were examined 5 min after intravenous bolus injection of [¹⁴C]octreotide (fig. 4, table 2). The CL_up was reduced to 10% of controls by coinfusion of TCA and DBSP, but PAH had no effect. In addition, the CL_up was reduced to 40% of controls by coinfusion of excess unlabeled octreotide, when the estimated octreotide concentration in plasma was 30 µM. A decrease in K_p value at 5 min after administration, which reflects the early-phase distribution to each tissue, was observed not only in liver but also in the lung and intestine with coinfusion of TCA and DBSP. However, the reduction in the K_p value in the lung and intestine was <15%, compared with that in liver.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Effect of DBSP, TCA, PAH, and octreotide on the hepatic uptake of [14C]octreotide in SDRs and hepatic uptake in EHBRs. Thirty minutes before administration of the mixture of [14C]octreotide and [3H]inulin, DBSP (2.5 µmol/min/kg and 25 µmol/kg as a loading dose), TCA (10 µmol/min/kg), PAH (23 µmol/min/kg and 700 µmol/kg as a loading dose), or octreotide (250 nmol/min/kg) infusion was started and continued throughout the experiment. As a control, an equivalent volume of saline was infused. Plasma concentration-time profile and the amount in each tissue were determined at 1 and 5 min after administration of [14C]octreotide. Each value and bar represents the mean ± SEM of three rats.

Comparison of [¹⁴C]Octreotide Hepatic Uptake between SDR and EHBRs. The CL_up of [¹⁴C]octreotide observed in EHBRs under the same condition was 0.05 ml/min/g liver (fig. 4). The CL_up in EHBRs thus decreased to 25% of SDRs. The K_p value in the liver 5 min after administration of octreotide in EHBRs also decreased to one-third that in SDRs (table 2). A change of K_p value in other tissues was minimal (table 2).

In Vitro Uptake Study. The concentration dependence of octreotide by isolated hepatocytes was investigated (fig. 5 and table 3). A marked concentration dependence was observed and the estimated parameters (K_M, V_max, and P_diff) were 108 ± 37 µM, 223 ± 75 pmol/min/10⁶ cells, and 0.51 ± 0.14 µl/min/10⁶ cells, respectively. Similar experiments were also conducted using primary cultured hepatocytes, and a similar concentration dependence was observed. The estimated parameters (K_M, V_max, and P_diff) were 97 ± 8 µM, 185 ± 11 pmol/min/10⁶ cells, and 0.55 ± 0.04 µl/min/10⁶ cells, respectively. The PS product for hepatic uptake (PS_influx), which represents the membrane permeability clearancecalculated from the equation, V_max/(K_M + P_diff) was 2.57 µl/min/10⁶ cells for isolated hepatocytes and 2.46 µl/min/10⁶ cells for primary cultured hepatocytes.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Concentration dependency of octreotide uptake by isolated hepatocytes and primary cultured hepatocytes. Uptake of [14C]octreotide (1-1000 µM) by isolated hepatocytes prepared from SDRs () was initiated by adding the ligand to the preincubated (37°C for 3 min) cell suspension (2 × 106 cells/ml). At 0.5 or 2 min, reaction was terminated by separating cells from the medium using a centrifugal filtration technique. Initial uptake velocity Vo) was calculated by linear regression of points taken at 30 and 120 sec. Each point represents the mean ± SEM of six determinations. The primary hepatocytes () cultured for 4.5 hr were preincubated at 37°C for 10 min with 600 µl of Krebs-Henseleit buffer. Initial uptake velocity (Vo) was calculated by linear regression of points taken at 1 and 5 min. Each point and bar represent mean ± SEM of three determinations.

Effect of Plasma Prepared from SDRs and EHBRs on [¹⁴C]Octreotide Uptake by Primary Cultured Hepatocytes. To examine the cause of the reduced CL_up EHBRs in vivo, the effects of plasma obtained from SDRs and EHBRs on the uptake of [¹⁴C]octreotide were investigated using primary cultured hepatocytes prepared from SDRs (fig. 6). The addition of EHBR plasma reduced the uptake clearance to 75% that prepared from SDRs. Part of the decrease of CL_up observed in EHBRs is thus supposed to be due to an endogenous substance (not identified yet) in the plasma of EHBRs.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Uptake of [14C]octreotide by primary cultured rat hepatocytes in the presence of plasma prepared from SDRs and EHBRs. Cultured cells were preincubated at 37°C for 10 min with 2% BSA containing buffer (), 90% (v/v) SDR plasma (), or 90% (v/v) EHBR plasma (). Uptake was started by the addition of [14C]octreotide to give a final concentration of 6 µM. Each point and vertical bar represent mean ± SEM of three experiments.

	Discussion

Abstract Introduction Results Discussion References

Recently, cyclic and linear small peptides consisting of 5 to 10 amino acids have been developed as therapeutic agents: the somatostatin analog octreotide (17), endothelin antagonists (4, 5), and renin inhibitors (10). Efficient hepatic uptake followed by rapid biliary excretion of unchanged compound is a common characteristic of these small peptides. This short plasma half-life caused by them has hindered their development as therapeutic agents. Several recent studies have demonstrated that a specific active transport system for the uptake of peptide exists on the sinusoidal hepatocyte membrane using isolated hepatocytes in vitro (17-21). We have already examined the hepatic uptake of an anionic pentapeptide, the endothelin antagonist BQ-123 (23). Pharmacokinetic and biochemical analyses have demonstrated that there is carrier-mediated transport in the hepatic uptake of peptides, and this transport consists of both Na⁺-dependent and -independent uptake. The former is the same transport system for bile acids and latter shares the uptake system with the multispecific anion transporter (23). Moreover, we have demonstrated that the biliary excretion of BQ-123 from liver to bile is by primary active transport and BQ-123 is excreted by cMOAT (37). Decreased biliary excretion of BQ-123 was demonstrated in EHBRs, which have a defect of cMOAT (27, 38-40).

In this study, the distribution of a peptide into organs, including liver and kidney, was examined in vivo using the cationic octapeptide, octreotide, in contrast to the anionic BQ-123. Moreover, the uptake of octreotide was examined using isolated hepatocytes and primary cultured hepatocytes in vitro, and uptake clearance observed was compared with that obtained in vivo. Previous studies have reported that the uptake of octreotide is by carrier-mediated transport and is competitively inhibited by bile acids and organic anions (16, 17). In this study, the effects of organic anions and bile acids on the hepatic uptake of octreotide were examined, and saturation of the uptake of octreotide was also investigated in vivo. Both the biliary excretion and CL_bile,p of octreotide were reduced to <10% of that of controls by TCA and DBSP at their T_max (fig. 1 and table 1). From a pharmacokinetic point of view, decreased CL_bile,p is brought by the decrease in the hepatic uptake from blood to liver and/or the decrease in biliary excretion from liver to bile. Thus, to determine how far the hepatic uptake, as the first process of hepatobiliary transport, is influenced by TCA and DBSP, the early-phase tissue distribution of octreotide was determined in vivo (figs. 3 and 4). A high CL_up was observed in the liver and kidney (0.2 ml/min/g liver and 0.58 ml/min/g kidney), compared with other organs (fig. 3). The apparent CL_up in the kidney involves amount of octreotide remaining in the lumen after being excreted by glomerular filtration. Thus, to obtain the exact uptake clearance for kidney via the basolateral membrane, a correction was made using the values of the unbound fraction of octreotide in plasma [0.41 (26)] and GFR (1.02 ml/min/g) estimated from the renal uptake clearance of [³H]inulin (fig. 3B). The glomerular filtration clearance of octreotide, estimated at 0.42 ml/min/g, demonstrated that 70% of the apparent uptake of octreotide by kidney was accounted for by glomerular filtration. This result demonstrates that octreotide is specifically taken up mostly by the liver during the early distribution phase. The hepatic uptake thus evaluated was reduced to <15% of controls by TCA and DBSP (fig. 4). On the other hand, PAH had no effect on the uptake of octreotide by the liver. Uptake of [¹⁴C]octreotide was reduced to 40% of controls by 30 µM octreotide in plasma resulting from infusion of unlabeled octreotide, thus indicating saturation of hepatic uptake of octreotide in vivo. Inhibition by TCA and DBSP in vivo confirmed the previous study (17) that reported inhibition of octreotide uptake by TCA and bromosulfophthalein using isolated hepatocytes. Although a significant but slight decrease in K_p at 5 min was observed in the intestine and lung after DBSP and TCA administration (table 2), the mechanism of the inhibitory effect is not known yet. The K_p value of octreotide at 5 min in the kidney was significantly reduced to 3.8 by PAH, compared with 5.3 in controls. However, the K_p value of inulin in the kidney reflecting glomerular filtration showed a similar decrease from 9.2 to 6.9. Considering that glomerular filtration contributes 70% of the uptake clearance of octreotide in the kidney, the decrease in uptake clearance of octreotide in kidney after administration of excess PAH may not be due to inhibition of the uptake process via basolateral membrane.

To compare the hepatic uptake clearance in vitro and in vivo, the uptake clearance of octreotide was also measured using isolated hepatocytes and primary cultured hepatocytes (fig. 5 and table 3). Similar results were obtained in both experimental systems. The estimated parameters (K_M, V_max, and P_diff) were ~100 µM, 200 pmol/min/10⁶ cells, and 0.5 µl/min/10⁶ cells. A similar uptake clearance under a linear condition (V_max/K_M + P_diff) was obtained in both systems: 2.57 µl/min/10⁶ cells (isolated hepatocytes) and 2.46 µl/min/10⁶ cells (primary cultured cell). The uptake clearance for octreotide in vivo was estimated from eq. 8 (venous equilibrium model) and eq. 9 (sinusoidal perfusion model). The uptake clearance in vivo was calculated from in vitro data to be 0.23 ~ 0.26 ml/min/g liver (isolated hepatocytes) and 0.22 ~ 0.26 ml/min/g liver (primary cultured hepatocytes). These values were comparable with the uptake clearance (0.20 ml/min/g) obtained in vivo (table 3). Thus, the carrier-mediated transport system detected in vitro using isolated hepatocytes and primary cultured hepatocytes is considered to be largely responsible for the hepatic uptake in vivo.

We have already compared hepatobiliary transport of octreotide in SDRs with that in EHBRs (41). In this study, the steady-state concentration in plasma and liver and the biliary excretion rate were monitored after intravenous infusion. Comparing SDRs and EHBRs, differences were observed in CL_bile,p, but not in biliary excretion clearance defined in respect to the concentration in liver. In addition, the liver-to-plasma concentration ratio (K_p) in EHBRs (1.15) was half that of SDRs (2.20) (41). This result suggests that the decrease in CL_bile,p is not caused by a decrease in biliary excretion via the bile canalicular membrane, but rather a decrease in hepatic uptake via the sinusoidal membrane. In fact, we have already determined the primary active ATP-dependent uptake of octreotide by bile canalicular membrane vesicles, which is maintained in EHBRs (41), as well as in SDRs. To determine the mechanism of the reduced K_p in EHBRs, the hepatic uptake clearance in EHBRs was compared with that in SDRs in vivo using integration plot analysis. As shown in fig. 4, the hepatic uptake clearance in EHBRs was ~25% of that in SDR. However, considering that only the transporter (cMOAT) on the bile canalicular membrane has been believed to be deficient in EHBRs, it is unlikely that the transport carrier on the sinusoidal membrane is also deficient in EHBRs. To examine the mechanism for the decrease in the hepatic uptake in EHBRs, the uptake of octreotide by primary cultured hepatocytes from SDRs was monitored in the presence of plasma prepared from SDRs and EHBRs, and compared with that in the absence of plasma (fig. 6). The uptake clearance in the presence of plasma prepared from EHBRs was reduced to 75% that from SDRs. The bilirubin glucuronide concentration was reported to increase in EHBR plasma to 49 µM from 1 µM in the normal plasma (38). This suggested that an endogenous substance, such as bilirubin glucuronide, elevated in EHBR plasma may play a role in part of the decreased hepatic uptake in EHBRs. Thus, we should be careful not to conclude easily from the observation of reduced biliary excretion of ligands in EHBRs that the ligands are excreted into bile by cMOAT, which is deficient in EHBRs.

In conclusion, the existence of a carrier-mediated transport for the hepatic uptake of octreotide and its inhibition by organic anions and bile acids were investigated in vivo. Kinetic analysis demonstrated that the decreased biliary excretion of octreotide observed in EHBR and the marked decreased biliary excretion caused by bile acids and organic anions were due to reduced hepatic uptake rather than biliary excretion.