Computer simulation of sulfobromophthalein kinetics in the rat using flow-limited models with extrapolation to man

A linear, flow-limited mathematical model of drug kinetics was used to simulate total sulfobromophthalein (BSP) kinetics in normal anesthetized rats during intravenous infusions and following rapid intravenous injections. Four parameters were used to characterize the distribution and biliary and urinary excretion of BSP: liver- to- plasma concentration ratio, extrahepatic tissue- to- plasma concentration ratio, liver clearance rate constant, and renal plasma clearance rate constant. The same parameters appear to characterize the kinetics of BSP in man through the successful application of scale- up techniques utilizing data from experiments in rats. Plasma levels of BSP corresponding to intravenous infusions and rapid intravenous injections in man are approximated by computer simulation.The notation used is that of Bischoff et al. (2) with a few additions C _p BSP concentration in plasma - C _l BSP concentration in liver - C _bi BSP concentration in bile compartmenti, i = 1,2,3 - C _m BSP concentration in extrahepatic tissue - V _p volume of plasma - V _l volume of liver - V _b volume of bile - V _m volume of extrahepatic tissue - Q _l plasma flow rate through liver - Q _m plasma flow rate through extrahepatic tissue - Q _b bile flow rate through biliary tract - R _l hepatic tissue-to-hepatic venous plasma concentration ratio of BSP - R _m extrahepatic tissue-to-extrahepatic venous plasma concentration ratio of BSP - k _l hepatic tissue elimination rate constant for BSP - k _k renal plasma clearance rate constant for BSP - f(t) infusion rate function - f _i volume of bile in the bile duct compartment as a fraction of the total bile volume V_b     

Transport of organic anions into liver cells and bile

The distribution of [¹⁴C]para-acetylaminohippuric acid ([¹⁴C]PAAH) and [³⁵S]sulfobromophthalein ([³⁵S]BSP) among plasma, liver cells and bile of rats was measured under steady-state conditions. The electrical potential difference (PD) was measured across the sinusoidal membrane of the liver cell and between plasma and bile in the common duct. A PD of 40.3 ± 1.0 mV (mean ± S.E.M.) was recorded across the sinusoidal membrane (cell negative with respect to plasma). The PD between plasma and bile was 3.9 ± 0.5 mV (bile negative with respect to plasma). The electrical potentials, considered together with the concentrations of PAAH and BSP in plasma, liver cells and bile, indicate that these organic anions are actively transported into liver cells across the sinusoidal membrane. The concentrations of PAAH and BSP in liver cells and bile suggest that they are transferred across the canalicular membrane by active transport processes. When the plasma concentration of PAAH was increased, the bile/liver cell ratio of PAAH reached an asymptote, suggesting that the transport of PAAH from liver cells into bile is a carrier-mediated system which can be saturated. An infusion of sodium taurocholate, which stimulated bile flow and increased the excretion of BSP in bile, did not influence the biliary excretion of PAAH. The transport of PAAH from liver cells into bile appears to act as a ‘sink’, thereby maintaining a low liver cell PAAH concentration. The membrane transport system for the uptake and excretion of PAAH may be relevant to the biliary excretion of other exogenous and endogenous organic anions. The results of this study also demonstrate the importance of considering electrical potentials when studying the distribution of a charged compound in the hepato-biliary system.     

Targeting the sodium-dependent multivitamin transporter (SMVT) for improving the oral absorption properties of a retro-inverso Tat nonapeptide

Purpose. To investigate the potential for delivering large peptides orally by altering their absorptive transport pathways and improving intestinal permeability. The absorptive transport of retro-inverso (R.I.-) K-Tat9 and R.I.-K(biotin)-Tat9, novel peptidic inhibitors of the Tat protein of HIV-1, and their interactions with human SMVT (hSMVT), a high affinity, low capacity transporter, were investigated using Caco-2 and transfected CHO cells. Methods. Following synthesis on a PAL resin using Fmoc chemistry, the transport of R.I.-K-Tat9 (0.01-25 M) and R.I.-K(biotin)-Tat9 (0.1-25 M) was evaluated across Caco-2 cells. The transport and kinetics of biotin, biocytin and desthiobiotin (positive controls for SMVT) were also determined. Uptake of R.I.-K-Tat9 and R.I.-K(biotin)-Tat9 (both 0.1-10 M) was determined in CHO/hSMVT and CHO/pSPORT (control) cells. Results. The absorptive transport of R.I.-K-Tat9 was passive, low (P_m1 × 10^–6 cm/sec) and not concentration dependent. R.I.-K(biotin)-Tat9 permeability was 3.2-fold higher than R.I.-K-Tat9 demonstrating active (E_a = 9.1 kcal/mole), concentration dependent and saturable transport (K_m = 3.3 M). R.I.-K(biotin)-Tat9 uptake in CHO/hSMVT cells (K_m = 1.0 M) was 500-fold greater than R.I.-K-Tat9 (at 10 M). R.I.-K(biotin)-Tat9 transport in Caco-2 and CHO/hSMVT cells was significantly inhibited by known substrates of SMVT including biotin, biocytin, and desthiobiotin. Passive uptake of R.I.-K(biotin)-Tat9 was significantly greater than R.I.-K-Tat9 uptake in CHO/pSPORT cells. Conclusions. These results demonstrate that the structural modification of R.I.-K-Tat9 to R.I.-K(biotin)-Tat9 altered its intestinal transport pathway resulting in a significant improvement in its absorptive permeability by enhancing nonspecific passive and carrier-mediated uptake by means of SMVT. The specific interactions between R.I.-K(biotin)-Tat9 and SMVT suggest that targeting approaches utilizing transporters such as SMVT may substantially improve the oral delivery of large peptides.     

Organic Anion Transporter oatp2-Mediated Interaction between Digoxin and Amiodarone in the Rat Liver

Purpose. The interaction between amiodarone and digoxin has been known to increase serum concentrations of digoxin in humans and rats. In this study, we assessed the molecular mechanism(s) of that drug interaction, focusing on digoxin transport mediated by P-glycoprotein (Pgp) and by rat liver organic anion transporter (oatp2). Methods. Digoxin transport by Pgp and oatp2 was assessed using Pgp-overexpressing transfectant LLC-GA5-COL150 monolayers and oatp2-expressing Xenopus oocytes, respectively. The digoxin uptake into the isolated rat hepatocytes was also examined. Results. Amiodarone (10 M) inhibited slightly the transcellular transport of digoxin in LLC-GA5-COL150 monolayers, whereas itraconazole (10 M), a potent Pgp inhibitor, markedly blocked the transport. The digoxin uptake by the isolated rat hepatocytes and by the oatp2-expressing Xenopus oocytes was decreased markedly in the presence of amiodarone but not in the presence of itraconazole. In addition, amiodarone inhibited the oatp2-mediated digoxin uptake in a competitive manner with an apparent inhibition constant value of 1.8 M. Conclusion. These findings suggest that rat oatp2 rather than Pgp may be one of the interaction sites for digoxin and amiodarone in the liver.     

The inhibition of the sarcoplasmic calcium pump by prenylamine,reserpine, chlorpromazine and imipramine

Summary 1. The calcium induced increase in ATPase and the rate of calcium uptake of the vesicular fragments of sarcoplasmic reticulum isolated from rabbit skeletal muscle are reduced by reserpine, prenylamine, chlorpromazine and imipramine. 3×10^–5M reserpine, prenylamine, chlorpromazine and 3×10^–4M imipramine are required to produce 50% inhibition of both activities.2. In the presence of the drugs the rate of calcium exchange at the cessation of calcium uptake is reduced to the same extent as the initial rate of calcium uptake.3. Neither the calcium storing capacity nor the ability to concentrate calcium are impaired by the drugs.4. The drugs do not affect the calcium dependent phosphate exchange between ATP and ADP and the calcium dependent formation of phosphoprotein.This work was presented at the 30. Tagung der Deutschen Pharmakologischen Gesellschaft 1966 in Kiel (Balzer et al., 1967).     

Intracellular Drug Delivery Using Low-Frequency Ultrasound: Quantification of Molecular Uptake and Cell Viability

Purpose. To determine the dependence on acoustic parameters of molecular uptake and viability of cells exposed to low-frequency ultrasound. Methods. DU145 prostate cancer cells bathed in a solution of calcein were exposed to ultrasound at 24 kHz over a range of different acoustic pressures, exposure times, pulse lengths, and duty cycles. Flow cytometry was employed to quantify the number of calcein molecules delivered into each cell and levels of cell viability. Results. Both molecular uptake and cell viability showed a strong dependence on acoustic pressure and exposure time, weak dependence on pulse length, and no significant dependence on duty cycle. When all of the data were pooled together, they exhibited good correlation with acoustic energy exposure. Although molecular uptake showed large cell-to-cell heterogeneity, up to 15% of cells achieved an intracellular calcein concentration approximately equal to its extracellular concentration. Conclusions. Large numbers of molecules can be delivered intracellularly using low-frequency ultrasound. Both uptake and viability correlate with acoustic energy, which is useful for design and control of ultrasound protocols.     

Pravastatin,an HMG-CoA Reductase Inhibitor,Is Transported by Rat Organic Anion Transporting Polypeptide,oatp2

Purpose. We previously demonstrated the HMG-CoA reductase inhibitor, pravastatin, is actively taken up into isolated rat hepatocytes through multispecific organic anion transporters. The present study examined whether a newly cloned organic anion transporting polypeptide (oatp2) transports pravastatin. Methods. We investigated functional expression of oatp2 in Xenopus laevis oocytes, to examine [¹⁴C] pravastatin uptake. Results. [¹⁴C] Pravastatin (30 M) uptake into oatp2 cRNA-injected oocytes was 40 times higher than that of water-injected control oocytes. The oatp2-mediated pravastatin uptake was Na⁺-independent and saturable. The Michaelis-Menten constant was 37.5 ± 9.9 M, a level comparable to that obtained in isolated rat hepatocytes in our previous study. As is the case with rat hepatocytes, the uptake of pravastatin (30 M) was inhibited by 300 M concentrations of taurocholate, cholate, bromosulfophthalein, estradiol-17-glucuronide, and simvastatin acid, but not by para-aminohippurate. On the other hand, [¹⁴C] simvastatin acid (30 M) uptake of oatp2 cRNA-injected oocytes was not significantly different from that of water-injected oocytes. Conclusions. The cloned oatp2 was identified as the transporter responsible for the active hepatocellular pravastatin uptake.     

Hepatobiliary Transport of a Nonpeptidic Endothelin Antagonist, (+)-(5S,6R, 7R)-2-Butyl-7-[2((2S)-2-Carboxypropyl)-4-Methoxyphenyl]-5-(3,4-Methylenedioxyphenyl) Cyclopentenol[1,2-b]Pyridine-6-Carboxylic Acid: Uptake by Isolated Rat Hepatocytes and Canalicular Membrane Vesicles

Purpose. Hepatobiliary excretions of drugs from the blood to the bile include two essential transmembrane processes: uptake into hepatocytes and secretion from hepatocytes. The purpose of this study was to clarify the transport mechanisms underlying these processes for a new non-peptide endothelin antagonist, (+)-(5S,6R,7R)-2-butyl-7-[2((2S)-2-carboxypropyl)-4-methoxyphenyl]-5-(3,4-methylenedioxy- phenyl)cyclopentenol[1,2-b]pyridine-6-carboxylic acid (J-104132). Methods. Biliary excretion of J-104132 was assessed in rats after intravenous injection. To evaluate the hepatic uptake process, J-104132 was incubated with freshly isolated rat hepatocytes and the uptake of J-104132 was calculated. To evaluate the biliary secretion process, the uptake of J-104132 into rat canalicular membrane vesicles that were isolated from normal Sprague-Dawley rats or Eisai hyperbilirubinemic rats was measured. Results. After intravenous injection, J-104132 was recovered from the bile quantitatively (99.7 ± 1.3%) as its intact form. J-104132 was taken up by isolated rat hepatocytes in a time- and temperature-dependent manner. The uptake was saturable with K _m and V _max of 5.7 M and 564 pmol/min/10⁶ cells, respectively. The uptake was Na⁺ independent and was reduced in the presence of ATP depleters (rotenone and carbonyl cyanide-p-(trifluoromethoxy)-phenylhydra- zone), organic anions (dibromosulfophthalein, indocyanine green, BQ-123, and pravastatin), and bile acids (taurecholate and cholate). In Sprague-Dawley rats, J-104132 was taken up by canalicular membrane vesicle ATP-dependently with K_m and V_max values of 6.1 M and 552 pmol/min/mg protein, respectively. However, ATP-dependent uptake disappeared in Eisai hyperbilirubinemic rats. Conclusions. These data suggest that energy-dependent and carrier-mediated transport systems play important roles in hepatobiliary excretion of J-104132 (both uptake and secretion processes), which is the main excretion route in rats. As for the secretion process of J-104132, an involvement of mrp2 was demonstrated.     

PEPT2-Mediated Uptake of Neuropeptides in Rat Choroid Plexus

Purpose. The peptide transporter PEPT2 was recently shown to be functionally active in rat choroid plexus, suggesting that it may play a role in neuropeptide homeostasis in the cerebrospinal fluid. This study, therefore, examined the role of PEPT2 in mediating neuropeptide uptake into choroid plexus. Methods. Whole-tissue rat choroid plexus uptake studies were performed on GlySar in the absence and presence of neuropeptides and on carnosine. Results. The neuropeptides NAAG, CysGly, GlyGln, kyotorphin, and carnosine inhibited the uptake of radiolabeled GlySar at 1.0 mM concentrations. In contrast, TRH, [D-Arg²]-kyotorphin, glutathione, and homocarnosine did not inhibit GlySar uptake. Kyotorphin, an analgesic, was a competitive inhibitor of GlySar with a Ki of 8.0 M. The direct uptake of carnosine was also shown to be mediated by PEPT2 in isolated choroid plexus (Km = 39.3 M; Vmax = 73.9 pmol/mg/min). Radiolabeled carnosine uptake was inhibited by 1.0 mM concentrations of GlySar or carnosine but not homocarnosine, L-histidine, or -alanine. Conclusions. These findings indicate that PEPT2 mediates the uptake of a diverse group of neuropeptides in choroid plexus, and suggests a role for PEPT2 in the regulation of neuropeptides, peptide fragments, and peptidomimetics in cerebrospinal fluid.     

Hepatic Disposition of Fexofenadine: Influence of the Transport Inhibitors Erythromycin and Dibromosulphothalein

Purpose. To examine the disposition of fexofenadine in the isolated perfused rat liver and the influence of erythromycin and dibromosulphthalein (DBSP) on the hepatic uptake and biliary excretion of fexofenadine. Methods. Livers from four groups of rats were perfused in a recirculatory manner with fexofenadine HCl added as a bolus (125, 250, 500, or 1000 g) to perfusate. Livers from another three groups of rats were perfused with 250 g of fexofenadine HCl. With one group as control, erythromycin (4.0 g/ml) or DBSP (136 g/ml) was added to the perfusate of the other groups. In all experiments, perfusate and bile were collected for 60 min; in addition, livers from the second experiment were retained for assay. Fexofenadine was determined in perfusate, bile, and homogenized liver by HPLC. Results. The area under the curve (AUC) of fexofenadine was linearly related to concentration. It was unchanged from control (12,800 ± 200 ng·h/ml) by erythromycin (14,400 ± 2000 ng·h/ml), but was increased 95% by DBSP (25,000 ± 2600 ng·h/ml, P <0.001). The ratios of the concentrations of fexofenadine in liver/perfusate were decreased significantly by DBSP; those for bile/liver were increased by erythromycin. Conclusions. Erythromycin reduced the canalicular transport of fexofenadine into bile, whereas DBSP reduced uptake across the sinusoidal membrane.     

Sinusoidal Efflux of Taurocholate Is Enhanced in Mrp2-Deficient Rat Liver

Purpose. It has been shown that plasma concentration and urinary excretion of bile acids is elevated under the cholestatic/hyperbilirubinemic conditions. Previously, it was demonstrated that the plasma concentration of bile acids was elevated in the multidrug resistance-associated protein 2 (Mrp2)-deficient rats. The purpose of the present study was to compare the sinusoidal efflux clearance of taurocholate (TC) between Mrp2-deficient Eisai hyperbilirubinemic rats (EHBR) and normal rats. Method. Hepatic disposition of the [³H]TC was examined in the perfused liver. Apparent efflux clearance (PS_{net, eff}) of [³H]TC from hepatocytes to outflow across the sinusoidal membrane was defined as the amount of [³H]TC excreted into the outflow from the liver divided by hepatic AUC of [³H]TC. Additionally, influx clearance (PS_inf) was also determined by multiple indicator dilution method because PS_{net, eff} is also affected by PS_inf. Results. PS_{net, eff} was significantly higher in EHBR than that in Sprague-Dawley (SD) rats (16.6 ±1.7 vs. 6.1 ± 1.3 L/min/g liver, P <0.01). In contrast, PS_inf was comparable between SD rats and EHBR. Kinetic analysis suggested that the intrinsic clearance for the efflux of [³H]TC across the sinusoidal membrane in EHBR was higher than that in SD rats (10.4 ± 1.0 v.s. 23.3 ± 1.7 L/min/g liver, P <0.01). Conclusions. Enhanced sinusoidal efflux of TC in EHBR may be related to the altered disposition of bile acids in the mutant rats. Because Mrp3 transports TC and its expression is induced on the basolateral membrane of Mrp2-deficient rats, the enhanced sinusoidal efflux of TC in EHBR may be accounted for, at least partially, by the increased expression of Mrp3.     

Blood-Brain Barrier Transport of Reduced Folic Acid

Purpose. The brain is relatively resistant to folic acid deficiency, indicating specialized transport systems may exist for this vitamin localized within the brain capillary endothelial wall, which makes up the blood-brain barrier (BBB) in vivo. The present studies quantify the BBB transport of [³H]-methyltetrahydrofolic acid (MTFA) in vivo and in isolated human brain capillaries in vitro. Methods. BBB transport of [³H]-MTFA was compared to that of [¹⁴C]-sucrose, a plasma volume marker, following either intravenous injection or intracarotid perfusion in anesthetized rats. Competition by 10 M MTFA or 10 M folic acid was examined to determine whether folic acid is also transported by the MTFA uptake system. Results. The BBB permeability-surface area (PS) product of [³H]-MTFA, 1.1± 0.3 L/min/g, was 6-fold greater than that of [¹⁴C]-sucrose following intravenous injection. The BBB PS product determined by intracarotid arterial perfusion was not significantly different from the BBB PS product calculated following intravenous injection. A time- and temperature- dependent uptake of [³H]-MTFA in human brain capillaries was observed. The uptake of [³H]-MTFA by either rat brain in vivo or by human brain capillaries in vitro was equally inhibited by 10 M concentrations of either unlabeled MTFA or unlabeled folic acid. Conclusions. (1) A saturable transport system exists at the BBB for folic acid derivatives and since this transport is equally inhibited by either folic acid or MTFA, it is inferred that this transport system is the folic acid receptor, and not the reduced folic acid carrier. (2) The presence of a folate transport system at the BBB may offer an endogenous transport system for brain drug delivery of conjugates of folates and drugs that do not normally cross the BBB in vivo.     

The Effect of RPR 102341 on Theophylline Metabolism and Phenacetin O-Deethylase Activity in Human Liver Microsomes

Purpose. RPR 102341 is structurally similar to the fluoroquinolone class of antibiotics. Because some fluoroquinolones have been shown to inhibit theophylline metabolism, concomitant administration may increase plasma levels of theophylline resulting in serious adverse effects. The purpose of this study was to determine if RPR 102341 affects theophylline metabolism in vitro and, thus, predict whether a clinically significant drug interaction is likely to occur. In addition, the effect of RPR 102341 on phenacetin O-deethylase activity was determined to address the enzymatic basis of a potential drug interaction. Methods. The in vitro theophylline metabolism assay was conducted according to a modification of a published procedure. The phenacetin O-deethylase assay was conducted according to a modification of a published procedure. Results. The rate of conversion of theophylline to 3-methylxanthine in human liver microsomes in the presence of 100 M and 500 M RPR 102341 was 93.6 and 106 percent of the control reactions, respectively. The formation of 1-methylxanthine was 97.6 and 100 percent of the control, and 1,3-dimethyluric acid formation was 88.9 and 95.2 percent of control at 100 M and 500 M RPR 102341, respectively. In agreement, RPR 102341 caused no inhibition of human liver CYP1A2—catalyzed phenacetin O-deethylase activity. Finally, no inhibition was observed when RPR 102341 was incubated with human liver microsomes and an NADPH regenerating system prior to the addition of theophylline. Conclusions. Based on these studies, RPR 102341 is not expected to cause significant drug interactions with theophylline.     

Atorvastatin Transport in the Caco-2 Cell Model: Contributions of P-Glycoprotein and the Proton-Monocarboxylic Acid Co-Transporter

Purpose. The purpose of this study was to elucidate the mechanismsby which an HMG-CoA reductase inhibitor, atorvastatin (an organicacid with a pKa of 4.46), was transported in the secretory and absorptivedirections across Caco-2 cell monolayers. Methods. Caco-2 cells were grown on polycarbonate membrane insertsin 6-well Snapwell plates (Costar). The permeability of radiolabeledcompounds across Caco-2 cell monolayers was determined using aside-by-side diffusion apparatus (NaviCyte) and an automated liquidhandler (Hamilton Microlab 2200). The apical uptake of¹⁴C-atorvastatin was also determined in Caco-2 cells. Cyclosporin A (20 M) waspresent in the uptake media to block potential P-glycoprotein-mediatedatorvastatin efflux. Results. Polarized permeation of atorvastatin was observed with thebasolateral-to-apical (B-to-A) permeability being 7-fold greater thanthe A-to-B permeability (35.6 × 10^–6 and 4.9 × 10^–6 cm/s,respectively). The secretion of atorvastatin was a saturable process with anapparent K_m of 115 M. The B-to-A permeability of atorvastatin wassignificantly reduced by cyclosporin A (10 M), verapamil (100 M),and a P-glycoprotein specific monoclonal antibody, UIC2(10 g/ml)(43%, 25%, and 13%, respectively). Furthermore, both CsA andverapamil significantly increased the A-to-B permeability of atorvastatinby 60% however, UIC2 did not affect the A-to-B permeability ofatorvastatin. CsA uncompetitively inhibited the B-to-A flux ofatorvastatin with a K_i of 5 M. In addition, atorvastatin (100 M) significantlyinhibited the B-to-A permeability of vinblastine by 61%. The apicaluptake of atorvastatin increased 10.5-fold when the apical pH decreasedfrom pH 7.4 to pH 5.5 while the pH in the basolateral side wasfixed at pH 7.4. A proton ionophore, carbonylcyanidep-trifluoro-methoxyphenylhydrazone (FCCP) significantly decreased atorvastatinuptake. In addition, atorvastatin uptake was significantly inhibited bybenzoic acid, nicotinic acid, and acetic acid each at 20 mM (65%,14%, and 40%, respectively). Benzoic acid competitively inhibitedatorvastatin uptake with a K_i of 14 mM. Similarly, benzoic acid,nicotinic acid, and acetic acid significantly, inhibited the A-to-Bpermeability of atorvastatin by 71%, 21%, and 66%, respectively. Conclusion. This study demonstrated that atorvastatin was secretedacross the apical surface of Caco-2 cell monolayers viaP-glycoprotein-mediated efflux and transported across the apical membrane in theabsorptive direction via a H⁺-monocarboxylic acid cotransporter(MCT). In addition, this study provided the first evidence thatnegatively charged compounds, such as atorvastatin, can be a substrate forP-glycoprotein.     

Dipeptide Uptake and Transport Characteristics in Rabbit Tracheal Epithelial Cell Layers Cultured at an Air Interface

Purpose. To determine the functional presence of a H⁺/peptide cotransport process in rabbit tracheal epithelial cell layers cultured at an air-interface and its contribution to transepithelial dipeptide transport. Methods. Rabbit tracheocytes were isolated, plated on Transwells, and cultured at an air-interface. After 5 or 6 days in culture, uptake and transepithelial transport of carnosine were examined. Results. Carnosine uptake by tracheocytes was pH-dependent and was saturable with a Michaelis-Menten constant of 170 M. Moreover, carnosine uptake was inhibited 94% by Gly-L-Phe, 28% by (-Ala-Gly, but not at all by Gly-D-Phe or by the amino acids -Ala and L-His. Unexpectedly, transepithelial carnosine transport at pH 7.4 (i.e., in the absence of a transepithelial pH gradient) was similar in both the apical-to-basolateral (ab) and basolateral-to-apical (ba) directions. Lowering the apical fluid pH to 6.5 reduced abtransport 1.6 times without affecting ba transport, consistent with predominantly paracellular diffusion of carnosine under an electrochemical potential gradient. Conclusions. The kinetic behavior of carnosine uptake into cultured tracheal epithelial cell layers is characteristic of a H⁺-coupled dipeptide transport process known to exist in the small intestine and the kidney. Such a process does not appear to be rate-limiting in the transport of carnosine across the tracheal epithelial barrier.     

Massive and selective delivery of lipid-coated cationic lipoplexes of oligonucleotides targeted in vivo to hepatic endothelial cells

Purpose. Previously we reported on massive uptake of liposomes surface-modified with negatively charged aconitylated albumin (Aco-HSA) by liver sinusoidal endothelial cells (EC) in vivo. In the present work we applied this principle for the in vivo delivery of antisense oligonucleotides (ODN) to these cells. Methods. Anti ICAM-1 ODN was complexed with the cationic lipid DOTAP and the complex was coated by an excess of neutral lipids including a lipid-anchored poly(ethylene glycol). Aco-HSA was coupled to the coated cationic lipoplexes (CCLs). Plasma disappearance, organ and intrahepatic distribution of Aco-HSA modified CCLs were determined in rats, using [³H]-cholesteryl oleyl ether and ³²P-labeled ODN as markers. Results. The Aco-HSA coupled CCLs were <160 nm in size, contained 1.03 ± 0.35 nmol ODN and 54 ± 18 g Aco-HSA per mol total lipid. These CCLs were rapidly eliminated from plasma, about 60% the injected dose of ³H- or ³²P-label being recovered in the liver after 30 min. Within the liver, the EC accounted for two thirds of total liver uptake. Control non-targeted CCLs were eliminated very slowly: after 30 min still >90% of the particles was in the blood. Conclusions. Our results demonstrate efficient targeting of antisense ODN to EC in vivo, employing plasma-stable coated cationic lipoplexes, surface modified with negatively charged albumin. 40% of the injected ODN was delivered to the target cells within 30 min.     

Triterpenoid saponins from cultural plants of Stenocereus stellatus (Cactaceae)

A new triterpenoid saponin, named stellatoside A (1), and a known saponin (2), were isolated from cultural plants of the cactus Stenocereus stellatus (Ludwig G. K. Pfeiffer) V. Riccobono. The structure of 1 was determined to be a stellatogenin 3-O--l-rhamnopyranosyl(14)--l-rhamnopyranosyl(12)--d-glucuronopyranoside by spectroscopic methods. This is the first study to examine the substances of the cultural plants of cacti.     

Is ligandin relevant for the uptake and storage of phallotoxins in liver cell?

Summary To exclude an involvement of ligandin in the uptake and storage of phalloidin in hepatocytes equilibriumdialysis studies were made with phalloidin, cholic acid and bromosulfophthalein (BSP). Binding studies with isolated ligandin indicated that the affinity of ligandin for phalloidin is low (K _D=0.8×10^–3 M). Phalloidin neither displaced BSP (K _D=1.3×10^–7M) or cholic acid (K _D=7.6×10^–5 M) from ligandin, when preloaded with these substrates.Hepatocytes prepared from rats after daily treatment with phenobarbital during 5 days contained 3–4-fold concentrations of ligandin and bound greater amounts of BSP than controls. Nevertheless the velocity of the uptake both of [³H]-demethylphalloin ([³H]-DMP) and of [³⁵S]-BSP was not augmented. Also the sensitivity of liver cells to phalloidin was not drastically modified after induction with phenobarbital and agrees with earlier findings in vivo. We conclude that ligandin plays a negligible role in the uptake and a minor role in the storage of phallotoxins in liver cells.This work was supported by the Deutsche Forschungsgemeinschaft     

Uptake of FITC-chitosan nanoparticles by A549 cells

Purpose. The objective of this study was to evaluate the extent and mechanism of uptake of fluorescent chitosan nanoparticles by the A549 cells, a human cell line derived from the respiratory epithelium. Methods. Covalent conjugation with fluorescein-5-isothiocyanate yielded stably labeled chitosan molecules, which were successfully formulated into nanoparticles by ionotropic gelation. Uptake of fluorescein-5-isothiocyanate-chitosan nanoparticles and chitosan molecules by confluent A549 cells was quantified by fluorometry. Results. Cellular uptake of chitosan nanoparticles was concentration and temperature dependent, having K_m and V_max of 3.84 M and 58.14 g/mg protein/h, respectively. Uptake of chitosan nanoparticles was up to 1.8-fold higher than that of chitosan molecules alone and was not inhibited by excess unlabeled chitosan molecules. Hyperosmolarity, chlorpromazine and K⁺ depletion inhibited by 65, 34, and 54%, respectively, the uptake of chitosan nanoparticles at 37°C, but filipin had no influence on the uptake. Confocal imaging confirmed the internalization of the chitosan nanoparticles by the A549 cells at 37°C. Conclusions. Formulation of chitosan into nanoparticles significantly improved its uptake by the A549 cells. Internalization of chitosan nanoparticles by the cells seems to occur predominantly by adsorptive endocytosis initiated by nonspecific interactions between nanoparticles and cell membranes, and was in part mediated by clathrin-mediated process.