Hepatic Disposition Characteristics of 111In-Labeled Lactosaminated Bovine Serum Albumin in Rats

The hepatic disposition of lactosaminated bovine serum albumin (Lac-BSA) in rats was studied at the whole body, isolated liver, and isolated parenchymal cell levels. After intravenous injection, ¹¹¹In-Lac-BSA (1 mg/kg) was rapidly eliminated from the plasma due to extensive uptake by liver parenchymal cells; however, a significant decrease in hepatic clearance was observed at high dose (50 mg/kg). In a single-pass, constant infusion experiment in the isolated liver, ¹¹¹In-Lac-BSA was continuously extracted. The extraction ratio at steady state (E_ss) for ¹¹¹In-Lac-BSA was significantly decreased by coadministrating galactose, NH₄Cl, or chloroquine, and at low temperature, suggesting that hepatic uptake of Lac-BSA proceeds via receptor-mediated endocytosis for asialoglycoprotein. Kinetic analysis of ¹¹¹In-Lac-BSA binding with isolated parenchymal cells at 4°C yielded a dissociation constant (K_d) of 2.5 ×10^–8M and a value of 3.5 × 10⁵ maximal binding sites/cell (B_max). The internalization rate constant (k_int) for ¹¹¹In-Lac-BSA was calculated to be 0.46 min^–l in liver perfusion experiments using the EDTA-wash method.     

Pharmacokinetic analysis of scavenger receptor-mediated uptake of anionized proteins in the isolated perfused rat liver

The hepatic uptake characteristics of ¹¹¹In-labeled succinylated lysozyme (Suc-LZM), superoxide dismutase (Suc-SOD), bovine serum albumin (Suc-BSA), catalase (Suc-CAT), and maleylated SOD (Mal-SOD), BSA (Mal-BSA) were studied in rat liver perfusion experiments. During a single-pass constant infusion mode, [¹¹¹In]Suc-BSA, [¹¹¹In]Suc-CAT, and [¹¹¹In]Mal-BSA were significantly extracted while extraction of [¹¹¹In]Suc-LZM, [¹¹¹In]Suc-SOD, and [¹¹¹In]Mal-SOD were small, suggesting the importance of molecular weight or total number of anionic charges per one protein molecule for the hepatic uptake of anionized proteins. The extraction ratio at steady state (E_ss) for [¹¹¹In]Suc-BSA was significantly decreased by co-administration of Mal-BSA or dextran sulfate, which is known to be taken up via scavenger receptor, and NH₄Cl, dinitrophenol, or cytochalasin B, suggesting that hepatic uptake of [¹¹¹In]Suc-BSA proceeds via receptor-mediated endocytosis. The internalization rate constant (k_int) for [¹¹¹In]Suc-BSA was calculated to be 0.27 min⁻¹ in liver perfusion experiments using the acid-wash method. The outflow patterns of [¹¹¹In]Suc-BSA at various inflow concentrations were simultaneously fitted to a physiological one-organ pharmacokinetic model, in which the hepatic uptake was represented by division into the processes of binding to the cell surface and internalization, by the use of the MULTI (RUNGE) program. The obtained pharmacokinetic parameters (maximum binding amount X_∞, binding constant K, and internalization rate constant k_int for [¹¹¹In]Suc-BSA clearly characterized the difference in their hepatic uptake mechanisms compared with lactosylated and cationized BSA. The present study has demonstrated that large succinylated and maleylated proteins should be useful as a carrier for the intracellular delivery of drugs specifically into the liver endothelial cells.     

Nanoparticle Surface Charges Alter Blood–Brain Barrier Integrity and Permeability

Purpose: The blood–brain barrier (BBB) presents both a physical and electrostatic barrier to limit brain permeation of therapeutics. Previous work has demonstrated that nanoparticles (NPs) overcome the physical barrier, but there is little known regarding the effect of NP surface charge on BBB function. Therefore, this work evaluated: (1) effect of neutral, anionic and cationic charged NPs on BBB integrity and (2) NP brain permeability.

Methods: Emulsifying wax NPs were prepared from warm oil-in-water microemulsion precursors using neutral, anionic or cationic surfactants to provide the corresponding NP surface charge. NPs were characterized by particle size and zeta potential. BBB integrity and NP brain permeability were evaluated by in situ rat brain perfusion.

Results: Neutral NPs and low concentrations of anionic NPs were found to have no effect on BBB integrity, whereas, high concentrations of anionic NPs and cationic NPs disrupted the BBB. The brain uptake rates of anionic NPs at lower concentrations were superior to neutral or cationic formulations at the same concentrations.

Conclusions: (1) Neutral NPs and low concentration anionic NPs can be utilized as colloidal drug carriers to brain, (2) cationic NPs have an immediate toxic effect at the BBB and (3) NP surface charges must be considered for toxicity and brain distribution profiles.     

The influence of hepatic transport on the distribution volumes and mean residence time of drug in the body and the accuracy of estimating these parameters by the traditional pharmacokinetic calculations

The influence of hepatic uptake and efflux, which includes passive diffusion and transporter‐mediated component, on drug distribution volumes [steady‐state volume of distribution (V_ss) and terminal volume of distribution (V_β)], mean residence time (MRT), clearance, and terminal half‐life is considered using a simplified physiologically based pharmacokinetic model. To account for hepatic uptake, liver is treated as two‐compartmental unit with drug transfer from extracellular water into hepatocytes. The exactly calculated distribution volumes and MRT are compared with that obtained by the traditional equations based on the assumption of central elimination. It was found that V_ss may increase more than 10‐fold and V_β more than 100‐fold due to the contribution of transporter‐mediated uptake. The terminal half‐life may be substantially shortened (more than 100‐fold) due to transporters. It may also decrease significantly due to the increase of intrinsic hepatic clearance (CL_int), whereas hepatic clearance has already reached saturation (and stays close to the possible maximum value). It is shown that in case of transporter‐mediated uptake of compound into hepatocytes, in the absence of efflux and passive diffusion (unidirectional uptake), hepatic clearance is independent of CL_int and is determined by hepatic blood flow and uptake rate constant. The effects of transporter‐mediated uptake are mostly pronounced for hydrophilic acidic compounds and moderately lipophilic neutral compounds. For basic compounds and lipophilic neutral compounds the change of distribution volumes due to transporters is rather unlikely. It was found that the traditional equations provide very accurate values of V_ss, V_β, and MRT in the absence of transporter action even for very low rates of passive diffusion. On the other hand, the traditional equations fail to provide the correct values of these parameters when the increase of distribution volumes due to transporters takes place, and actually yield the values substantially smaller than the true ones (up to an order of magnitude for V_ss and MRT, and three orders of magnitude for V_β). © 2011 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:5031–5047, 2011     

Disposition of a polymeric prodrug of mitomycin C, mitomycin C-dextran conjugate, in the perfused rat liver

The disposition of a polymeric prodrug of mitomycin C (MMC), mitomycin C-dextran conjugate (MMC-D), was studied in the single-pass perfused rat liver in order to clarify the effect of physico-chemical properties, such as molecular weight and electric charge, on the hepatic uptake of MMC-D. Six types of MMC-D were used: both cationic MMC-D (MMC-Dcat) and anionic MMC-D (MMC-Dan) conjugated with dextran with molecular weights of 10,000, 70,000, and 500,000. Outflow curves were analyzed using statistical moment theory. Remarkable hepatic uptake of MMC-Dcat was observed and the uptake amount increased with an increase in molecular weight (i.e., approximately 80% of the dose was taken up by the liver during a single passage of the conjugate with a molecular weight of 500,000). Intrinsic clearance (CLint,i) and apparent distribution volume (Vi) also increased as the molecular weight increased. On the other hand, almost 100% of applied MMC-Dan was recovered in the outflow regardless of molecular weight, with almost the same moment parameters as those of the vascular reference substance (VRS), 131I-labeled human serum albumin (HSA). In a repeated application, the uptake of MMC-Dcat decreased in a stepwise manner, suggesting a saturation in the hepatic uptake of MMC-Dcat, while the uptake of MMC-Dan was unchanged. The MMC-Dcat pretreatment also affected the uptake of Evans blue (EB) bound to bovine serum albumin (BSA). These results demonstrate that molecular weight and electric charge determine the hepatic disposition of macromolecular prodrugs.     

Transdermal Macromolecular Delivery: Real-Time Visualisation of Iontophoretic and Chemically Enhanced Transport Using Two-Photon Excitation Microscopy

Purpose. To investigate the transdermal delivery of a modelmacromolecule by passive and iontophoretic means following pretreatment withC₁₂-penetration enhancers and to visualise transport across humanstratum corneum (SC) in real time. Methods. Transport studies of dextran, labelled with fluorescentCascade Blue® (D-CB; M_R = 3 kDa) across human stratum corneum,were conducted during passive and iontophoretic modes of deliveryfollowing pretreatment with either dodecyltrimethylammoniumbromide (DTAB), sodium dodecyl sulphate (SDS) or Azone®.Size-exclusion chromatography was used to assess maintenance of dextranstructural integrity throughout experimental lifetime. Two-photonexcitation microscopy was employed to visualise real-time dextran transportduring current application. Results. The positively charged C₁₂-enhancer DTAB elevated passiveD-CB steady-state flux (J_ss) and was the only enhancer to do soabove control during iontophoresis. The negatively charged SDS had theleast effect during both stages. On-line macromolecular transport wasvisualised, indicating both inter- and intra-cellular pathways across SCduring current application. No transport was visible across untreatedSC during passive transport. Conclusions. Use of a positively charged enhancer may improve J_ssof anionic macromolecular penetrants during passive and iontophoreticdelivery. On-line visualisation of iontophoresis across SC was possibleand can provide mechanistic insight into SC transport pathways.     

Pharmacokinetic evaluation of high pulmonary disposition of clarithromycin after systemic administration

1. The distribution characteristics of clarithromycin to the lung were investigated in vivo and in isolated lung perfusion experiments. The in-vivo integration plot analysis showed that the pulmonary uptake and extracellular distribution in the lung were significantly higher for clarithromycin than for erythromycin. 2. In the rat lung single-pass perfusion study, the pulmonary extraction ratio (E_ss) of clarithromycin at steady-state was significantly higher than that of erythromycin, and the E_ss of clarithromycin tended to decrease as the inflow concentration increased, suggesting the involvement of carrier-mediated transport in the pulmonary disposition of clarithromycin. 3. The outflow patterns of clarithromycin or erythromycin at various inflow concentrations were simultaneously analysed based on a pharmacokinetic model, which consists of the non-specific binding site, the specific binding site and the subsequent uptake process. The parameters obtained suggested that clarithromycin would have the higher affinity and higher capacity for the specific binding site, and the higher equilibrium constant for the non-specific binding site than erythromycin. 4. The simulation study using those parameters demonstrated that clarithromycin could be bound to the specific binding site and subsequently taken up more extensively than erythromycin. 5. A multiple-indicator dilution study also indicated that clarithromycin was more readily associated and extracted with the lung than with erythromycin. In the inhibition study, it was suggested that the pulmonary uptake of clarithromycin could be ascribed not only to the non-specific binding depending on its lipophilic nature, but also in part to some specialized mechanisms such as organic cation transporters.     

以叶酸受体为靶向的阳离子脂质体的制备与性质考察

为了研制一种能通过叶酸受体途径靶向肿瘤细胞的叶酸受体靶向脂质体，将叶酸(folate，folic acid，F)、 聚乙二醇二胺(polyoxyethylene-bis-amine，NH₂-PEG-NH₂)、 琥珀酸酐(succinic anhydride，SUC)和二硬脂酰磷脂酰乙醇胺(distearoylphosphatidylethanolamine，DSPE)按序共价连接， 并使用薄层色谱和飞行时间质谱确证合成产物为叶酸-聚乙二醇-二硬脂酰磷脂酰乙醇胺(folate-polyethyleneglycol-distearoylphosphatidylethanolamine，F-PEG-DSPE)。膜材选用二棕榈酰磷脂酰胆碱(dipalmitoylphosphatidylcholine，DPPC)， 3β-［N-(N′,N′-二甲基胺乙基)胺基甲酰基］胆固醇(3β-［N(N′,N′-dimethylaminoethane) carbamoyl］ cholesterol，DC-Chol)和F-PEG-DSPE，以10∶10∶0.75(摩尔比)的配比，以荧光素标记的阴离子葡聚糖(dextran fluorescein anionic，DFA)为模型，用薄膜分散法制备含DFA的叶酸受体靶向脂质体，其包封率较高(>55%)、稳定性好，平均粒径为144 nm，体外释放慢。MTT法考察其对细胞的毒性结果表明该阳离子脂质体具有一定的细胞毒性，在低浓度时(0.012 5～0.1 μmol·L^{-1)脂质体的细胞毒性与DC-chol浓度成正比。流式细胞技术检测KB细胞和HepG2细胞对DFA脂质体的摄取，结果表明叶酸受体靶向的长循环阳离子脂质体能提高细胞对脂质体的摄取。该研究为进一步研究叶酸受体靶向阳离子脂质体在肿瘤基因治疗中的应用提供了理论基础。}     

Predominant Contribution of Rat Organic Anion Transporting Polypeptide-2 (Oatp2) to Hepatic Uptake of β-Lactam Antibiotics

Purpose To identify the rat hepatic basolateral transporters involved in the hepatic uptake of β-lactam antibiotics using nafcillin as a model β-lactam antibiotic that undergoes extensive biliary excretion. Materials and Methods Uptake by isolated rat hepatocytes and Xenopus laevis oocytes expressing organic anion transporting peptides (Oatp1, 2, and 4) and organic anion transporter (OAT2) was evaluated. Results Nafcillin uptake by isolated rat hepatocytes was saturable with the K _m of 210 μM and was significantly inhibited by anionic compounds (estrone-3-sulfate and sulfobromophthalein), but not by cationic compounds (tetraethylammonium and 1-methyl-4-phenylpyridinium). In an in vitro uptake study by Xenopus oocytes expressing hepatic basolateral membrane transporters, nafcillin was transported by multiple Oatps with K _m values of 4120 μM (Oatp1/Oatp1a1), 198 μM (Oatp2/Oatp1a4), and 1,570 μM (Oatp4/Oatp1b2), though it was not transported by hOAT2. Comparison of affinity and analysis by the relative activity factor method indicated that Oatp2 is the predominant contributor to the hepatic uptake of nafcillin. Cefadroxil, cefazolin, cefmetazole, cefoperazone, cefsulodin, and cephalexin, though not cefotaxime or ceftriaxone, were also substrates of Oatp2. Conclusion These findings suggest that Oatp2 plays a key role in the hepatic uptake of nafcillin and most other β-lactam antibiotics in rats.     

Pharmacokinetic analysis of uptake process of lactosaminated albumin in rat liver constant infusion experiments

The hepatic uptake of ¹¹¹In-labeled lactosaminated bovine serum albumin (Lac-BSA) was studied in comparison with ¹¹¹In-labeled cationized albumin (Cat-BSA) in rat liver perfusion experiments. During a single-pass constant infusion mode, [¹¹¹In]Lac-BSA (0.7–10 μg/ml) was continuously extracted by the liver and its exctaction ratio at steady-state (E_ss) decreased as the inflow concentration increased. The outflow pattern of [¹¹¹In]Lac-BSA was significantly different from that of [¹¹¹In]Cat-BSA. The outflow patterns of [¹¹¹In]Lac-BSA at various inflow concentrations were simultaneously fitted to a physiological one-organ model, in which the hepatic uptake was represented by division into the processes of binding to the cell surface and internalization, by the use of the MULTI(RUNGE) program. The obtained kinetic parameters (maximum binding amount X_∞, binding constant K and internalization rate constant k_int) correlated closely with the experimental data, suggesting the validity of this model. The kinetic parameters for [¹¹¹In]Lac-BSA and [¹¹¹In]Cat-BSA clearly characterized the difference in their hepatic transport mechanisms and the in vivo targeting characteristics of these albumin derivatives were discussed based on computer simulation.     

A physiologically based pharmacokinetic model to predict the pharmacokinetics of highly protein‐bound drugs and the impact of errors in plasma protein binding

Predicting the pharmacokinetics of highly protein‐bound drugs is difficult. Also, since historical plasma protein binding data were often collected using unbuffered plasma, the resulting inaccurate binding data could contribute to incorrect predictions. This study uses a generic physiologically based pharmacokinetic (PBPK) model to predict human plasma concentration–time profiles for 22 highly protein‐bound drugs. Tissue distribution was estimated from in vitro drug lipophilicity data, plasma protein binding and the blood: plasma ratio. Clearance was predicted with a well‐stirred liver model. Underestimated hepatic clearance for acidic and neutral compounds was corrected by an empirical scaling factor. Predicted values (pharmacokinetic parameters, plasma concentration–time profile) were compared with observed data to evaluate the model accuracy. Of the 22 drugs, less than a 2‐fold error was obtained for the terminal elimination half‐life (t_1/2, 100% of drugs), peak plasma concentration (C_max, 100%), area under the plasma concentration–time curve (AUC_0‐t, 95.4%), clearance (CL_h, 95.4%), mean residence time (MRT, 95.4%) and steady state volume (V_ss, 90.9%). The impact of f_up errors on CL_h and V_ss prediction was evaluated. Errors in f_up resulted in proportional errors in clearance prediction for low‐clearance compounds, and in V_ss prediction for high‐volume neutral drugs. For high‐volume basic drugs, errors in f_up did not propagate to errors in V_ss prediction. This is due to the cancellation of errors in the calculations for tissue partitioning of basic drugs. Overall, plasma profiles were well simulated with the present PBPK model. Copyright © 2016 John Wiley & Sons, Ltd.     

Effect of cationic drugs on the transporting activity of human and rat OCT/Oct 1–3 in vitro and implications for drug–drug interactions

The inhibitory effects of cationic drugs (β-adrenoreceptor antagonists, calcium (Ca)-channel blocker, I_f channel inhibitor, antiarrhythmic drugs, and antibacterial drugs) that inhibit 1-methyl-4-phenylpyridinium (MPP) and/or metformin uptake into hOCT1–3/rOct1–3-expressing cells and human/rat hepatocytes were investigated in this study. The drug–drug interaction (DDI) potential of these drugs for the hOCT/rOct-mediated hepatic/renal uptake process was also assessed. The IC₅₀ values of cardiovascular drugs, including an I_f channel inhibitor with a new mechanism of action, were greater for hOCT2/rOct2 than those for hOCT1/rOct1 or hOCT3/rOct3. No species differences in these values were observed between hOCTs and rOcts. As for hOCT2-mediated uptake, the IC₅₀ values of quinidine and the I_f channel inhibitor for metformin uptake were lower than those for MPP uptake. However, previous clinical studies found that the IC₅₀ values of these drugs for hOCT1/rOct1 and hOCT2/rOct2 were much greater than their unbound plasma concentrations, which suggests that the DDIs of these cationic compounds may not be related to hOCT/rOct-mediated hepatic/renal uptake pathways. In addition, investigation of the luminal transporters of cationic compounds in the kidney, as well as the in vitro DDI potential of their inhibitors, is important for the clarification of cationic compound DDIs in humans.     

Exposure to High Environmental Temperature in the Sauna Does not Change Plasma Indocyanine Green (ICG) Clearance in Healthy Subjects

Abstract: Indocyanine green (ICG) was given intravenously (0.5 mg kg^-1) to seven healthy male volunteers in random order during a control session and a session in the sauna bath. The sauna bathing session consisted of three 10 min. stays in the sauna (temperature 85-95°, relative humidity 25-30%), separated by two 5-min. periods of resting at 22°. Blood samples were taken for 60 min. in order to calculate ICG plasma clearance (CI), volume of distribution (V_ss) and elimination half-life (t_1/2β). The mean±S.E.M. values of ICG plasma clearance, V_ss and t_1/2β for the control session and the sauna bathing session were 0.47±0.08 1 min.^-1 versus 0.39±0.04 1 min.^-1, 2.4±0.4 1 versus 2.3±0.2 1 and 3.9±0.3 min. versus 4.4±0.3 min., respectively. No statistically significant differences in the CI, t_1/2β or V_ss of ICG were detected between the control and sauna bathing sessions. The results suggest that short-term exposure to high ambient temperatures during sauna bathing does not affect hepatic blood flow. Consequently, short-term hyperthermia and associated changes in hepatic blood flow are assumed to have little, if any, effect on the hepatic clearance of drugs.     

Comparison of predictability for human pharmacokinetics parameters among monkeys,rats, and chimeric mice with humanised liver

1.?The aim of the present study was to evaluate the usefulness of chimeric mice with humanised liver (PXB mice) for the prediction of clearance (CL_t) and volume of distribution at steady state (Vd_ss), in comparison with monkeys, which have been reported as a reliable model for human pharmacokinetics (PK) prediction, and with rats, as a conventional PK model.

2.?CL_t and Vd_ss values in PXB mice, monkeys and rats were determined following intravenous administration of 30 compounds known to be mainly eliminated in humans via the hepatic metabolism by various drug-metabolising enzymes. Using single-species allometric scaling, human CL_t and Vd_ss values were predicted from the three animal models.

3.?Predicted CL_t values from PXB mice exhibited the highest predictability: 25 for PXB mice, 21 for monkeys and 14 for rats were predicted within a three-fold range of actual values among 30 compounds. For predicted human Vd_ss values, the number of compounds falling within a three-fold range was 23 for PXB mice, 24 for monkeys, and 16 for rats among 29 compounds. PXB mice indicated a higher predictability for CL_t and Vd_ss values than the other animal models.

4.?These results demonstrate the utility of PXB mice in predicting human PK parameters.     

Similarity or Discrepancy in Pharmacokinetic Parameter Estimation Between Bolus and Infusion Studies

A recent study by Heatherington and Rowland showing discrepancies in steady-state volume of distribution (V_ss) estimation of two barbiturates between bolus and infusion studies in rat hindlimb preparations was reviewed. Their rationale is that increasing the duration of administration may increase the accessibility for tissue distribution and thus increase V_ss for compounds showing slow tissue uptake. Such a dosing-duration-dependent distribution concept is, however, inconsistent with the principle in linear kinetics that the fate or disposition function of any drug molecules is independent of time of administration and presence of other molecules. When their well-designed bolus studies were reanalyzed by including extrapolated outflow data from the last sampling time to infinity, the V_ss values for the two barbiturates were found to be very similar to those obtained by the infusion method. Our analysis seems to validate a theoretical concept that parameter estimation is independent of the duration of administration in linear kinetics. A potential complication of using the bolus method to study V_ss is presented.     

Verofylline pharmacokinetics in dietary-induced obese rats: Role of fat mass and protein binding in determining volume of distribution

1. Verofylline, a lipophilic polysubstituted methylxanthine, was utilized to examine how severely altered body composition in obesity affects drug disposition; the role of fat-free mass, fat mass and protein binding in determining the volume of distribution (V_ss) was investigated. Obesity was induced by feeding Sprague-Dawley rats for 8 months with a calorie-dense diet; the obese rats showed increases of 50% in total body mass and 150% in body fat.

2. Both the absolute V_ss and the clearance (Cl) in the obese rats increased 2-fold over control. Since Cl and V_ss increased similarly, the half-life of verofylline in obese rats did not change.

3. The increase of Cl in obese rats can be accounted for by metabolic function related to fat-free mass and decreased serum binding. Similarly, an increase in fraction unbound and in total body mass accounted for the increase in V_ss.

4. Based on in vitro measurements of muscle and fat tissue uptake of verofylline, and the assumed body spaces (from tritium dilution method), the predicted values for V_ss closely approximated those of observed values. The semi-physiological model proposed here appears adequate to relate changes of body composition and serum protein binding in obesity to V_ss.     

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.     

Significant Role of Liver Sinusoidal Endothelial Cells in Hepatic Uptake and Degradation of Naked Plasmid DNA After Intravenous Injection

Purpose. Uptake and degradation of naked plasmid DNA (pDNA) by liver sinusoidal endothelial cells (LSECs) were investigated. Methods. Tissue distribution and intrahepatic localization were determined after an intravenous injection of ¹¹¹In- or ³²P-labeled pDNA into rats. Cellular uptake and degradation of fluorescein- or ³²P-labeled pDNA were evaluated using primary cultures of rat LSECs. Results. Following intravenous injection, pDNA was rapidly eliminated from the circulation and taken up by the liver. Fractionation of liver-constituting cells by centrifugal elutriation revealed a major contribution of LSECs to the overall hepatic uptake of pDNA. Confocal microscopic study confirmed intracellular uptake of pDNA in cultured LSECs. Apparent cellular association of pDNA was similar at 37°C and 4°C. However, trichloroacetic acid (TCA) precipitation experiments showed the TCA-soluble radioactivity in the culture medium increased in an accumulative manner at 37°C. Involvement of a specific mechanism was demonstrated, as the uptake of pDNA was significantly inhibited by excess unlabeled pDNA and some polyanions (polyinosinic acid, dextran sulfate, heparin) but not by others (polycytidylic acid, dextran). These inhibitors also reduced the amount of TCA-soluble radioactivity in the culture medium. Conclusion. These results suggest that LSECs efficiently ingested and rapidly degraded naked pDNA in vivo and in vitro and released the degradation products into the extracellular space.     

No effect of diltiazem on the hepatic clearance of indocyanine green in the rats

In order to investigate the effect of the pretreatment with various doses of diltiazem (DTZ) on the pharmacokinetics of indocyanine green (ICG) at steady state, especially the hepatic blood clearance due to the change of hepatic blood flow, the following experiments were carried out with ICG, a hepatic function test marker, not metabolized in liver and only excreted in bile. The intravenous bolus injection (3,780 μg/kg) and the constant-rate infusion (10, 100 μg/kg/hr) or ICG into the left femoral vein were made in order to check the steady-state plasma concentration (C _ss of 10 μg/ml) of ICG at 20, 25 and 30 min. Following a 90-min washout period, the intravenous bolus injection (108, 430, 860 and 1,720 μg/kg) and the constant-rate infusion (108, 433, 866 and 1,730 μg/kg/hr) of DTZ into the right femoral vein were made and the achievement of the steady-state plasma levels (C _ss of 50, 200, 400 and 800 ng/ml) of DTZ were conformed at 60, 70 and 80 min. During the steady state of DTZ, the intravenous bolus injection (3,780 μg/kg) and the constant-rate infusion (10,200 μg/kg/hr) of ICG into the left femoral vein were made and also the steady-state plasma concentration of ICG was checked at 20, 25 and 30 min. The plasma concentrations of DTZ and ICG were determined using a high performance liquid chromatographic technique. At the steady state, the hepatic blood clearance of ICG was obtained from the plasma concentration and blood-to-plasma concentration ratio (R _b ) of ICG. The pretreatment with various doses of DTZ did not influence the plasma concentrations,R _B and plasma free fraction (f _p ) of ICG. So the hepatic blood clearance of ICG was independent of concentration of DTZ. The hepatic blood clearance of ICG could be affected by both hepatic blood flow and hepatic intrinsic clearance. But there was no change of the hepatic blood clearance of ICG between the control and the DTZ-pretreated rats in this study. So it may be suggested that DTZ does not influence hepatic blood flow.     

The Fate of Plasmid DNA After Intravenous Injection in Mice: Involvement of Scavenger Receptors in Its Hepatic Uptake

Purpose. We examined the stability and disposition characteristics of a naked plasmid DNA pCAT as a model gene after intravenous injection in mice to construct the strategy of in vivo gene delivery systems. Methods. After the injection of pCAT to the mice, stability, tissue distribution, hepatic cellular localization, and effect of some polyanions on the hepatic uptake were studied. Results. The in vitro study demonstrated that the pCAT was rapidly degraded in mouse whole blood with a half-life of approximately 10 min at a concentration of 100 µg/ml. After intravenous injection, pCAT was degraded at a significantly faster rate than that observed in the whole blood, suggesting that pCAT in vivo was also degraded in other compartments. Following intravenous injection of [³²P] pCAT, radioactivity was rapidly eliminated from the plasma due to extensive uptake by the liver. Hepatic accumulation occurred preferentially in the non-parenchymal cells. The hepatic uptake of radioactivity derived from [³²P] pCAT was inhibited by preceding administration of polyanions such as polyinosinic acid, dextran sulfate, maleylated and succinylated bovine serum albumin but not by polycytidylic acid. These findings indicate that pCAT is taken up by the liver via scavenger receptors on the non-parenchymal cells. Pharmacokinetic analysis revealed that the apparent hepatic uptake clearance was fairly close to the liver plasma flow. Conclusions. These findings provide useful information for the development of delivery systems for in vivo gene therapy.