Hepatic Uptake Kinetics Research Articles

Transporter Roles in the Pharmacokinetics and Tissue Distribution of Voxilaprevir, a Pan‐genotypic HCV NS3/4A Protease Inhibitor

IntroductionVoxilaprevir (VOX; GS‐9857) is a pan‐genotypic hepatitis C virus nonstructural (NS) protein 3/4A protease inhibitor (HCV‐PI) with potent antiviral activity against HCV genotypes 1 to 6 and an enhanced resistance profile over previous HCV‐PIs1. VOX is a component of Vosevi™, a fixed‐dose combination of three agents (SOF/VEL/VOX) and is approved for treatment of HCV. In vitro and in vivo assessments were conducted to determine the roles of drug transporters in the pharmacokinetics (PK) and preferential tissue distribution of VOX.MethodsVoxilaprevir metabolic stability was assessed in vitro in hepatic microsomal fraction and cryopreserved hepatocytes from the rat, dog, monkey and human. Permeability and efflux transport were characterized in Caco‐2 cells and Pgp‐ or BCRP‐overexpressing MDCKII cells. The uptake kinetics of VOX was characterized in rat and human hepatocytes. VOX PK and distribution into liver was determined following a single intravenous (IV) administration in rat, dog, and monkey. VOX elimination was evaluated in bile‐duct cannulated rat following a single IV dose. The distribution of VOX in select tissues (liver, kidney, & lung) was assessed with and without rifampicin pretreatment following IV administration in rat. PK following single ascending oral administration of VOX was also evaluated in rat.ResultsVOX was metabolically stable in hepatic microsomal fractions across all species tested, except for the monkey. In the rat, VOX was mainly eliminated via biliary excretion as unchanged parent drug (70% of dose), and to a lesser extent as its oxidated metabolites. Liver uptake of VOX was observed across the nonclinical species tested ‐ liver to plasma concentration ratio was 970, 34, and 41 in the rat, dog, and monkey, respectively. VOX was found to be a substrate of efflux transporters P‐gp and BCRP, and also a substrate of hepatic uptake transporters (OATPs). VOX displayed a non‐linear and saturable OATP‐mediated hepatic uptake kinetics in vitro. In the rat, VOX shown substantially higher liver concentration than reference tissues (kidney and lung) with and without rifampicin pretreatment. Non‐linear PK was also observed following ascending oral doses of VOX in rat.ConclusionsTaken together, these studies demonstrate the contribution of drug transporters in the PK and preferential liver distribution of VOX; which was also corroborated in its clinical PK and efficacy studies.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sodium fluorescein is a probe substrate for hepatic drug transport mediated by OATP1B1 and OATP1B3

The aim of this study was to characterize the in vitro hepatic uptake kinetics of sodium fluorescein (NaFluo) and identify the transporters involved. NaFluo exhibited saturable uptake kinetics in suspended rat and human hepatocytes as reflected by Km values of 22.5 and 14.1 µM, and Vmax values of 98.3 and 5.8 pmol/(million cells • min), respectively. Coincubation with known inhibitors (e.g. rifampicin) of organic anion transporting polypeptide (OATP/Oatp; SLCO gene family) significantly decreased NaFluo uptake in hepatocytes. In contrast, neither inhibitors/substrates of the organic cation transporter or organic anion transporter family nor depletion of extracellular sodium resulted in significant inhibition of NaFluo uptake. To explore the contribution of individual uptake transporters, NaFluo uptake was determined in Chinese hamster ovary cells transfected with OATP1B1, OATP1B3, and OATP2B1. Transporter‐mediated uptake of NaFluo was observed in OATP1B1‐ and OATP1B3‐transfected cells (Km = 4.2 and 10.9 µM; Vmax = 30.9 and 135 [pmol/(mg protein • min)], respectively). NaFluo can be used as a probe substrate to study Oatp/OATP1B‐mediated drug interactions in fluorescence‐based in vitro transport assays of rat and human liver. Labeling of drugs or bile salts with a fluorescein moiety can be expected to result in fluorescent conjugates with substantially altered hepatic uptake characteristics as compared with the unconjugated compounds. © 2011 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:5018–5030, 2011

Uptake of phosphatidylserine-containing liposomes by liver sinusoidal endothelial cells in the serum-free perfused rat liver

We studied the kinetics of hepatic uptake of liposomes during serum-free recirculating perfusion of rat livers. Liposomes consisted of phosphatidylcholine, cholesterol and phosphatidylserine in a 6:4:0 or a 3:4:3 molar ratio and were radiolabelled with [ 3H]cholesteryl oleyl ether. The negatively charged liposomes were taken up to a 10-fold higher extent than the neutral ones. Hepatic uptake of fluorescently labelled liposomes was examined by fluorescence microscopy. The neutral liposomes displayed a typical Kupffer cell distribution pattern, in addition to weak diffuse staining of the parenchyma, while the negatively charged liposomes showed a characteristic sinusoidal lining pattern, consistent with an endothelial localization. In addition, scattered Kupffer cell staining was distinguished as well as diffuse parenchymal fluorescence. The mainly endothelial localisation of the negatively charged liposomes was confirmed by determining radioactivity in endothelial and Kupffer cells isolated following a 1-h perfusion. Perfusion in the presence of polyinosinic acid, an inhibitor of scavenger receptor activity, reduced the rate of uptake of the negatively charged liposomes twofold, indicating the involvement of this receptor in the elimination mechanism. These results are compatible with earlier in vitro studies on liposome uptake by isolated endothelial cells and Kupffer cells, which showed that in the absence of serum also endothelial cells in situ are able to take up massive amounts of negatively charged liposomes. The present results emphasize that the high in vitro endothelial cell uptake in the absence of serum from earlier observations was not an artifact induced by the cell isolation procedure.

Consideration on moments of outflow profile in liver perfusion system with change in perfusate flow rate using oxacillin as model drug.

The effect of perfusate flow rate on hepatic structure and hepatic uptake kinetics was investigated using oxacillin as a model drug and bovine serum albumin (BSA) as a reference substance in the liver perfusion system from the standpoint of a dispersion model and moment characteristics. The estimated recovery ratio (FH) of oxacillin was about 40% which was independent of the change in perfusate flow rate. The mean transit time (tH) of oxacillin decreased with an increase in flow rate, while the relative variance (sigma 2/t2H) of oxacillin was independent of the flow rate. The tH of BSA decreased with an increase in the flow rate to the same extent as that of oxacillin, while sigma 2/t2H of BSA was independent of flow rate. When the dispersion model is adopted as a model system to analyze hepatic perfusion data following the pulse input, the moment characteristics (FH, tH and sigma 2/t2H) are given in complicated equations. It is demonstrated by the present investigation that these moment equations can be extensively simplified for a drug with a medium extraction ratio (FH > 50%), i.e., FH is independent of the distribution, both FH and tH are independent of the dispersion process in the hepatic blood space, and both tH and sigma 2/t2H are independent of the elimination. Thus, it is shown that FH and tH are exactly the indices of elimination and distribution, respectively, and sigma 2/t2H is the index of dispersion in the blood space plus nonequilibrium in the hepatic distribution.

Pharmacokinetic Studies on the Drug Delivery System Using Liposomes.

This review describes our recent investigation on the pharmacokinetic analysis of liposome disposition, especially hepatic uptake of liposomes. We have examined the saturable uptake processes of liposomes by the liver and postulated a new kinetic model “Manpuku Model (Satiated Model)” which explains AUC-dependent saturation kinetics of liposome uptake by the liver. This model assumes that the hepatic uptake clearance (CLh) is described by the maximum hepatic uptake clearance (CLh, max), maximum hepatic uptake (Xmax) and the hepatic uptake (X) as CLh=CLh, max (1-X/Xmax). This model explains the saturation kinetics of hepatic uptake of liposomes well and can be applicable to characterize the saturation kinetics of other kinds of liposome uptake.The mechanism of hepatic uptake of liposomes was investigated using isolated perfused liver and hepatic uptake of liposomes was shown to be enhanced by preincubation with serum. This opsonic activity was shown to be mediated via complement receptor mediated phagocytosis. The activation of complement system dependended on the size of liposomes. There are two kinds of uptake pathways, one via size dependent, complement receptor mediated pathway, the other is size independent, nonspecific pathway.The intracellular disposition of liposomes was further examined using 125I-BSA as a degradable, aqueous phase marker in the isolated peritoneal macrophages. The intracellular degradation of liposomes was shown to be biphasic. The degradation process of liposomes was also examined in intact liver and biphasic pattern was confirmed. These results indicate heterogeneous intracellular disposition (both transport and degradation) of liposomes.The rational strategies for the drug delivery system using liposomes will be developed based on both quantitative evaluation of liposome movement in blood circulation, hepatic uptake and intracellular disposition and clarification of the mechanisms of liposome uptake and intracellular disposition of liposomes.

Hepatic blood flow: accuracy of estimation from infusions of indocyanine green in anaesthetized cats.

Experiments were performed to determine the accuracy of the estimation of hepatic blood flow from infusions of indocyanine green (ICG) in anaesthetized cats. The estimated flows were compared to hepatic blood flows measured directly in a hepatic venous long-circuit preparation. This preparation allowed direct measurement and alteration of hepatic blood flows, and collection of arterial and mixed hepatic venous blood samples without depletion of blood volume in the animal. Mean hepatic plasma flows estimated during infusions of 3.22 and 6.44 nmol kg-1 min-1 were reliable indicators of true hepatic flow at three different flow levels, provided that a sufficiently long time (greater than 30 min) was allowed for distribution equilibrium and that data from several animals were pooled to reduce random variability. Variability arose through subtraction of plasma arterial and hepatic venous levels to obtain the arteriovenous difference. Estimations of hepatic plasma flow by intravenous infusions of ICG were more accurate and reliable than estimations from bolus injections of ICG, or intravenous infusions of galactose studied previously. The kinetics of hepatic uptake of ICG are complex. Extraction and clearance of ICG fell steadily with time during the infusions and constant plasma ICG levels were not attained during 150 min infusions. This is attributed to the effects of accumulation of ICG within the liver cells since hepatic uptake substantially exceeded biliary excretion rate. Total ICG concentrations in sinusoid and liver cells increased in parallel. The concentration in the liver cell was 88 (60-115) times the concentration in the sinusoid but we have no data on whether or not the free concentrations in plasma and cell were in equilibrium.

Lactosylation of albumin reduces uptake rate of dibromosulfophthalein in perfused rat liver and dissociation rate from albumin in vitro.

Two types of models have recently been proposed to describe hepatic uptake kinetics of protein bound drugs: a model in which dissociation from plasma protein is rate limiting the process, and a model in which an interaction between protein and hepatocyte surface is thought to promote dissociation and uptake of the drug. This study was designed to investigate several aspects of both models, using lactosylated albumin as a binding protein that can interact with the Ashwell receptor abundantly present on the hepatocyte. Dibromosulfophthalein clearance was studied in rat liver in the presence of 150 microM (1%) albumin or 150 microM lactosylated albumin. Initial disappearance rate from perfusate in the presence of lactosylated albumin indicated a 2-fold decrease in hepatic uptake rate compared with native albumin. This was confirmed by compartmental analysis, showing a similar decrease in hepatic uptake rate constant. Protein binding of dibromosulfophthalein to lactosylated albumin was only marginally different from normal albumin. Consequently, modification of the protein retarded uptake of the organic anion at an essentially unchanged unbound concentration. Fluorescence spectroscopy of lactosylated albumin showed a blue-shifted tryptophan emission spectrum compared with albumin, indicating increased hydrophobicity of the neoglycoprotein. We therefore considered a change in off-and-on rate for binding of dibromosulfophthalein in lactosylated albumin. Rapid filtration experiments indicated that the dissociation rate constant of dibromosulfophthalein from lactosylated albumin was half that of controls. We conclude that the decreased off-rate from lactosylated albumin can explain the retarding influence on hepatic uptake rate of dibromosulfophthalein. This observation argues for the concept of dissociation-limited uptake in the hepatic clearance of the organic anion.

Effects of liver blood flow on hepatic uptake kinetics of galactose in anesthetized cats: parallel tube model.

Hepatic galactose uptake in cats anesthetized with pentobarbital was determined during (i) steady-state infusions at several doses, (ii) rapidly increasing infusion rates at different blood flows, and (iii) prolonged infusion of a single dose at different blood flows. The hepatic venous long-circuit technique was used to allow frequent sampling of arterial, portal, and hepatic venous blood without depletion of the animal's blood volume and to allow measurement and alteration of total hepatic blood flow. Uptake was shown to follow Michaelis-Menten kinetics and was consistent with the "parallel tube model." The kinetic parameters Vmax and Km could be determined under steady-state and nonsteady-state conditions and were independent of hepatic blood flow over the range 60-150% of control flow. Mean Vmax was 80 mumol/(min X 100 g liver) and mean Km was 215 microM. Vmax declined by 50% when flow was reduced to half normal. It is concluded that the parallel tube model can be used to describe and predict hepatic galactose kinetics in anesthetized cats, although other models may fit the data equally well.

The kinetics of unmatched and HLA-matched 111in-labelled homologous platelets in recipients with chronic marrow hypoplasia and anti-platelet immunity.

The kinetics of homologous platelets, labelled in plasma with 111In-tropolonate, have been studied in five recipients with chronic marrow hypoplasia and severe thrombocytopenia, who were refractory to platelet transfusions as a result of alloimmunization. Mean platelet life span (MPLS), recovery, plasma 111In level and splenic and hepatic uptake kinetics were studied on two occasions, one using HLA-matched platelets and the other unmatched platelets. In each case, recovery of labelled platelets at 1 h post-injection and MPLS improved with HLA matching, although this improvement was highly variable. Only two of the five subjects would have derived any significant benefit from HLA-matched as compared with unmatched platelet transfusions. It was concluded that the need exists for additional cross-matching procedures, possibly related to platelet specific antigens, in patients who remain refractory to platelet transfusion.

Difference in hepatic uptake kinetics of aspirin and salicylamide in rats

Immediately after intraportal administration to rats, the ratio of liver to plasma concentrations for total aspirin was close to unity, whereas that for total salicylamide ranged from about 3 to 7. The hepatic accumulation of salicylamide appeared to be capacity-limited because of the ratio decreased with increases in the dose. In vitro experiments with isolated hepatocytes indicated that aspirin was slowly transported into the hepatocytes by an apparently linear process only, while salicylamide was taken up very rapidly by both saturable and apparently linear transport processes. The cell to medium concentration ratio estimated for the initially net transported component of the unchanged drug was significantly larger with salicylamide, which give ratios from 3.5 to 19, than with aspirin which gave an almost constant value lower than 2 despite wide variations in the initial concentration. For the capacity-limited uptake process of salicylamide; the kinetic parameters were estimated as V max = 0.325 nmole · (g cellular protein) −1. sec −1 and K m = 201 μM. Among various metabolic inhibitors, 2,4-dinitropherol (50μM) inhibited the uptake of salicylamide most extensively. The present comparison of the in vivo and in vitro data for aspirin with those for salicylamide confirmed the previously reported difference in the hepatic first-pass effect of these two drugs.

The role of conjugation reactions in enhancing biliary secretion of bile acids.

Shortening the five-carbon carboxylic acid side chain of cholic acid by one methylene group gave rise to a bile acid (norcholate) that was not a substrate for the bile acid-conjugating enzymes. The metabolism and biliary secretion of norcholate in intact liver was examined in the isolated perfused rat liver system. When rat livers were perfused with 14-20 microM solutions of norcholate for 10 min, norcholate was found in the unconjugated form in liver, venous effluent and bile. Neither tauronorcholate nor glyconorcholate was detectable by high-pressure liquid chromatography or fast-atom-bombardment mass spectrometry. The kinetics of hepatic uptake and biliary secretion of norcholate was compared with that for cholate, taurocholate and chemically synthesized tauronorcholate. The latter three bile acids were completely cleared from the perfusate and efficiently secreted into the bile. However, norcholate was incompletely extracted from the perfusate, and this was shown to be at least partially due to its relatively lower rate of hepatic uptake. Furthermore, the rate of norcholate secretion into bile was greatly reduced relative to the secretion of cholate or chemically synthesized tauronorcholate, even though the concentration of norcholate in the liver was comparatively high. These data demonstrate that the conjugation of bile acids greatly facilitates their secretion into bile.

Hepatic uptake and degradation of unilamellar sphingomyelin/cholesterol liposomes: a kinetic study.

The kinetics of hepatic uptake and degradation of sphingomyelin/cholesterol (2:1, M/M) small unilamellar liposomes were investigated in a BALB/c mouse. The tissue distribution of liposomes was determined by scintillation spectrometry. The percentage of intact liposomes in tissues was estimated by the technique of gamma-ray perturbed angular correlation. A kinetic model was developed to analyze the above data. A remarkable agreement was noted between the experimental data and the corresponding theoretical values. Our results indicate that the sphingomyelin/cholesterol unilamellar liposomes had an unusually long half-life of 16.5 hr in the circulation after intravenous administration to mice. The hepatic degradation of the liposomes in vitro at 37 degrees C followed first-order kinetics, with a half-life of 3.5 +/- 0.2 (SEM) hr. Furthermore, the rate of the in vivo degradation of liposomes in the liver was found to be quite similar to that in vitro, with a half-life of 3.6 +/- 0.4 hr. The rate of release of the liposome-encapsulated agent, indium-111, in the liver was not constant, and reached a maximum at about 8 hr after the administration of liposomes. The approach developed in the present study is general and can be applied to the investigation of factors that may control the release of pharmacologically active agents in any tissue.

Uptake of bile acids by perfused rat liver.

The uptake of 14C-labeled cholic, taurocholic, and chenodeoxycholic acid by the perfused rat liver was studied to characterize the mechanism responsible for hepatic uptake of bile acids. A rapid-injection multiple indicator-dilution technique and the three-compartment model of Goresky were employed. The kinetics of hepatic uptake of the three bile acids could be described by the Michaelis-Menten equation. The maximal uptake velocities (Vmax) were 24.9 +/- 2.2 (mean +/- SD), 20.8 +/- 1.2, 1.2, and 11.4 +/- 0.9 nmol/s-g liver for cholic, taurocholic, and chenodeoxycholic acid, respectively. The corresponding apparent half-saturation constants (Km) were 526 +/- 125, 258 +/- 43, and 236 +/- 48 nmol/g liver. Competitive inhibition could be demonstrated between cholate and taurocholate as well as between cholate and chenodeoxycholate. Substitution of 94% of the Na+ in the perfusion medium decreased the Vmax and the apparent Km of taurocholate uptake by 68 and 55%, respectively. These findings are consistent with the hypothesis that bile acids are taken up into the hepatocyte by Na+-dependent carrier-mediated transport.

Kinetics of hepatic uptake of unconjugated bilirubin.

1. The uptake of bilirubin was studied in the perfused rat liver by a multiple-indicator dilution technique employing the three-compartment model of Goresky. 2. The kinetics of hepatic bilirubin uptake could be described by the Michaelis-Menten equation. 3. The maximal uptake velocity (V max.) and the apparent half-saturation constant (Km) were 4-4 +/- 0-5 nmol s-1 g-1 of liver and 58 +/- 16 nmol/g of liver respectively, indicating that the hepatic uptake system for bilirubin under normal conditions is operating far below saturation. 4. Sodium taurocholate did not compete with bilirubin for hepatic uptake. 5. These findings are consistent with the concept that carrier-mediated transport is responsible for hepatocellular uptake of bilirubin and that bilirubin and bile acids enter the hepatocyte via separate pathways.

Kinetics of hepatic dye absorption for indocyanine green. Influence of bilirubin and sodium glycocholate

Kinetics of hepatic uptake of indocyanine green, a dye which is used for evaluation of liver function, were studied in the rat. The results indicate that the relationship between ICG-dose and initial hepatic dye uptake obeys Michaelis-Menten kinetics suggesting an interaction of the dye with a carrier or fixed site in the liver cell. Thus it was possible to calculate maximum ICG-uptake (vmax) and the Michaelis constant (Km) of this transport system from several submaximal values.vmax was 7.65 (6-06-9.65)22 mg per 100 g liver/min and Km 0.56 (0.31–0.81)22. Under the influence of substances which inhibit the elimination of dyes by the liver the parametersvmax and Km showed changes which allowed characterization of the type of inhibition. While sodium glycocholate had no influence on maximum hepatic ICG-uptake and the Michaelis constant bilirubin caused a significant increase of Km to 1.29 (0.68–1.90)22 without significantly changingvmax. These data suggest that bilirubin interferes with hepatic uptake of indocyanine green by competitive inhibition and that uptake of bile acids is dependent on a different mechanism.