Selective transport of an anti-transferrin receptor antibody through the blood-brain barrier in vivo

The brain capillary endothelium, which makes up the blood-brain barrier (BBB) in vivo, expresses high concentrations of transferrin receptor, and recent studies show that an antitransferrin receptor monoclonal antibody may function as a BBB drug transport vector. The present report examines the pharmacokinetics of clearance of radiolabeled antitransferrin receptor monoclonal antibody from the bloodstream in rats in vivo, and also assesses the extent to which brain selectively extracts the antibody from the blood compared to other peripheral organs such as liver, kidney, myocardium, or lung. [125I]Mouse immunoglobulin G2a control antibody was cleared monoexponentially with a half-time of 9.8 +/- 2.3 h. The clearance of the [3H]OX-26 antitransferrin receptor antibody from blood was biexponential with half-times of 2.2 +/- 0.8 min (61 +/- 10% of clearance) and 3.9 +/- 0.2 h (39 +/- 4% of clearance). The OX-26 antibody was rapidly taken up by liver during the first 60 min after injection, but this uptake reached rapid saturation, and hepatic OX-26 content actually declined subsequent to the first hour after injection. In contrast, brain continuously extracted the OX-26 antibody from the bloodstream, and the brain volume of distribution of OX-26 reached a value 18-fold greater than the volume of distribution of the mouse immunoglobulin G2a at 5 h after injection. There was no specific uptake of the OX-26 by myocardium or lung, and minor uptake by kidney was observed that also reached saturation within the first 60 min after injection.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo evidence for carrier-mediated uptake of beta-lactam antibiotics through organic anion transport systems in rat kidney and liver

The transport mechanisms of beta-lactam antibiotics in the rat kidney and liver were studied with an in vivo tissue-sampling single-injection technique using [3H]benzylpenicillin [( 3H]PCG) as a substrate. Concentration-dependent uptake of [3H]PCG was observed in the kidney, and the in vivo kinetic parameters were estimated as follows: the maximum uptake rate (Jmax) was 6.88 mumol/min/g of kidney, MIchaelis constant (Kt) was 1.39 mM and nonsaturable first-order rate constant (kd) was 0.414 ml/min/g of kidney. The uptake of [3H]PCG was inhibited by organic anions but not by organic cations. Several beta-lactam antibiotics also reduced the uptake of [3H]PCG. Furthermore, the organic anion, probenecid, and beta-lactam antibiotic, cefpiramide, showed a dose-dependent inhibitory effect. These results suggest participation of an organic anion transport system in uptake of beta-lactam antibiotics across the renal plasma membrane. Saturable uptake of [3H]PCG was also observed in the liver and Jmax, Kt and Kd were estimated to be 3.62 mumol/min/g of liver, 3.59 mM and 0.223 ml/min/g of liver, respectively. The in vivo influx rate calculated from Jmax/Kt in the liver was 1.01 ml/min/g of liver and was close to the in vitro value, 1.54 ml/min/g of liver, estimated previously from isolated hepatocytes. Although dipeptides and organic cations showed no effect on the hepatic uptake of [3H]PCG, probenecid significantly reduced its uptake. Several beta-lactam antibiotics also reduced the uptake of [3H]PCG by the liver. These features of the hepatic uptake of beta-lactam antibiotics through an organic anion transport system are in agreement with the previous results obtained in isolated hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of liver single photon emission computed tomography in a case of fulminant hepatic failure

Fulminant hepatic failure is associated with a high mortality rate. Thus, accurate assessment of hepatic functional reserve and hepatic regeneration is important. We describe a 67-year-old woman who survived subacute hepatic failure. We had an opportunity to monitor the clinical course of the patient using single photon emission computed tomography (SPECT) with 99mTc-galactosyl-human serum albumin (99mTc-GSA) and frequent hematological examinations. On admission, prothrombin time was remarkably prolonged (23.1% of control). The liver uptake of 99mTc-GSA was also considerably low. She responded well to treatment. Four weeks after admission, SPECT analysis showed a dramatic increase in liver uptake of 99mTc-GSA, suggesting promotion of hepatic regeneration. Moreover, functional liver volume calculated from the SPECT data showed a marked increase at 4 weeks after admission, whereas CT scan showed no change at that point. This indicated that SPECT with 99mTc-GSA reflected functional hepatocytes more accurately than liver volume determined by CT scan, which cannot exclude nonfunctional hepatocytes. The patient's condition improved in parallel with the improvements in the indices measured by SPECT and hematological examinations. SPECT analysis is practically useful for the prompt assessment of improvement in patients with fulminant hepatic failure.     

Abnormal renal and hepatic glucose metabolism in type 2 diabetes mellitus.

Release of glucose by liver and kidney are both increased in diabetic animals. Although the overall release of glucose into the circulation is increased in humans with diabetes, excessive release of glucose by either their liver or kidney has not as yet been demonstrated. The present experiments were therefore undertaken to assess the relative contributions of hepatic and renal glucose release to the excessive glucose release found in type 2 diabetes. Using a combination of isotopic and balance techniques to determine total systemic glucose release and renal glucose release in postabsorptive type 2 diabetic subjects and age-weight-matched nondiabetic volunteers, their hepatic glucose release was then calculated as the difference between total systemic glucose release and renal glucose release. Renal glucose release was increased nearly 300% in diabetic subjects (321+/-36 vs. 125+/-15 micromol/min, P < 0.001). Hepatic glucose release was increased approximately 30% (P = 0.03), but increments in hepatic and renal glucose release were comparable (2.60+/-0.70 vs. 2.21+/-0.32, micromol.kg-1.min-1, respectively, P = 0.26). Renal glucose uptake was markedly increased in diabetic subjects (353+/-48 vs. 103+/-10 micromol/min, P < 0.001), resulting in net renal glucose uptake in the diabetic subjects (92+/-50 micromol/ min) versus a net output in the nondiabetic subjects (21+/-14 micromol/min, P = 0.043). Renal glucose uptake was inversely correlated with renal FFA uptake (r = -0.51, P < 0.01), which was reduced by approximately 60% in diabetic subjects (10. 9+/-2.7 vs. 27.0+/-3.3 micromol/min, P < 0.002). We conclude that in type 2 diabetes, both liver and kidney contribute to glucose overproduction and that renal glucose uptake is markedly increased. The latter may suppress renal FFA uptake via a glucose-fatty acid cycle and explain the accumulation of glycogen commonly found in the diabetic kidney.     

Cellular targets and receptors for interleukin-6 I. In vivo and in vitro uptake of IL-6 in liver and hepatocytes

Interleukin-6 (IL-6) is a potent stimulator of the hepatic synthesis of acute-phase proteins. 125I-labelled IL-6 disappeared from the blood of rats with an overall half-time of about 1.5 min; 41% of the injected tracer dose was recovered in the liver by 15 min. The clearance was biphasic. The simultaneous injection of tracer and an excess of unlabelled IL-6 eliminated the initial rapid phase, and reduced the hepatic uptake to 14%. Light microscopic autoradiography showed 5% of the grains over non-hepatocytes, and 80% over hepatocytes, accumulating in areas around the bile canaliculi. Thereafter, degradation products accumulated in the bile. At 4 degrees C, isolated rat hepatocytes bound IL-6 with an apparent Kd of 39 pmol l-1 to a uniform class of 4500 receptors per cell with an apparent molar mass of 115-120 kg mol-1. The HepG2 human hepatocellular cell line bound IL-6 with an apparent Kd of 21 pmol l-1 to a uniform class of 1200 receptors per cell with an apparent molar mass of 155-160 kg mol-1. At 37 degrees C, both cell types endocytosed the bound ligand slowly, and degradation products appeared in the medium after a relatively long lag period (40 min in hepatocytes and 1 h in HepG2 cells).     

In vivo tumor transfection mediated by polyplexes based on biodegradable poly(DMAEA)-phosphazene.

In recent years, increasing interest is being paid to the design of transfectants based on non-toxic and biodegradable polymers for gene therapy purposes. We recently reported on a novel, biodegradable polymer, poly(2-dimethylamino ethylamino)phosphazene (p(DMAEA)-ppz) for use in non-viral gene delivery. In this study, the biodistribution and in vivo transfection efficiency of polyplexes composed of plasmid DNA and p(DMAEA)-ppz were investigated after intravenous administration in tumor bearing mice. Data were compared with those of polyplexes based on the non-biodegradable polyethylenimine (PEI 22kDa). Both polyplex systems were rapidly cleared from the circulation (<7% ID, at 60 min after administration) and showed considerable disposition in the liver and the lung, all in line with earlier work on cationic polyplex systems. The lung disposition is attributed to aggregates formed by interaction of the polyplexes with blood constituents. Redistribution of the polyplexes from the lung was observed for both polyplex formulations. Importantly, both polyplex systems showed a substantial tumor accumulation of 5% and 8% ID/g for p(DMAEA)-ppz and PEI22 polyplexes, respectively, at 240 min after administration. The tumor disposition of the p(DMAEA)-ppz and PEI22 polyplexes was associated with considerable expression levels of the reporter gene. In contrast to PEI22 polyplexes, p(DMAEA)-ppz polyplexes did not display substantial gene expression in the lung or other organs (organ gene expression<1/100 of tumor gene expression). The observed preferential tumor gene expression mediated by the p(DMAEA)-ppz polyplexes enables the application of this polymer to deliver therapeutic genes to tumors.     

Altered hepatic calcium homeostasis in guinea pigs with halothane-induced hepatotoxicity

Exposure of guinea pigs to 1% halothane in air for 4 hr resulted in extensive centrizonal hepatic necrosis in 70% of animals examined 2 to 3 days later. In contrast, confluent hepatic necrotic lesions were not present in animals studied 24 hr after halothane exposure; only microvascular fatty change of hepatocytes with occasional necrotic cells was observed at that time (in 84% of animals). This delayed onset of lesion development afforded the opportunity to study microsomal membrane composition and indices of Ca++ homeostasis before and after the onset of halothane-induced hepatic necrosis. Hepatic microsomal cytochrome P-450 levels were unaltered 24 hr after halothane exposure, but fell to approximately 50% of control values at 72 hr. This indicates that such changes were most likely the result of hepatocellular necrosis. Microsomal lipid composition, including the relative proportions of individual phospholipids, was unaltered during halothane-induced hepatotoxicity. In contrast, microsomal Ca++ sequestration, as assessed by 45Ca uptake, was reduced 24 hr after halothane exposure compared with controls (2.76 +/- 1.32 nmol/mg of protein per min, vs. 6.63 +/- 2.88 nmol/mg of protein per min, P less than .001). This early change in microsomal Ca++ uptake was associated with a 10-fold increase in total hepatic Ca++ content at 24 hr. Subsequent changes in hepatic Ca++ content were proportionate to the severity of liver necrosis. The observation that abnormalities of hepatic Ca++ homeostasis antedate the presence of liver cell necrosis is consistent with a role for altered Ca++ fluxes in the mechanism of halothane-induced liver injury.     

Vitamin D3 uptake by the isolated perfused rat liver from lipoprotein fractions is separate from cholesterol and triglyceride uptake

Using the isolated perfused rat liver we examined the uptake of [14C] or [3H] vitamin D3 and [14C] triglycerides or [3H] cholesterol by the liver of normal rats, from different lipoprotein fractions, as measured by disappearance from the perfusate. When incorporated into chylomicrons only 43% of the vitamin D3 remained in the perfusate at 60 min (i.e. 57% uptake) as compared to 68% of the triglycerides (i.e. 32% uptake). When added on very low density lipoproteins (VLDL) at 60 min 37 +/- 3% (n = 6) of the vitamin D3, 38 +/- 2% of the cholesterol (n = 3) (P NS), and 83 +/- 4% of the triglycerides (n = 3) remained in the perfusate (P less than 0.0005 for cholesterol:triglycerides and vitamin D3:triglycerides). For high density lipoprotein fraction (HDL) isolated perfused livers were studied with and without albumin present in the perfusate. Without albumin 19 +/- 8% (n = 3) of the vitamin D3 and 43 +/- 8% (n = 3) of the cholesterol remained in the perfusate at 60 min. The results with albumin present were 40 +/- 1% (n = 5) of the vitamin D3 and 63 +/- 4% (n = 5) of the cholesterol at 60 min (P less than 0.0005). The cholesterol:cholesterol ester ratio of the VLDL fraction was 8.8:1 and of the HDL fraction 1:1.4. There was no metabolism of vitamin D3 during the 1 h perfusion. These results suggest that vitamin D3 is in equilibrium with the lipoprotein surface, and the hepatic uptake of vitamin D is a surface phenomenon independent of lipoprotein metabolism.     

Carrier-mediated delivery of 9-(2-phosphonylmethoxyethyl)adenine to parenchymal liver cells: a novel therapeutic approach for hepatitis B

Our aim is to selectively deliver 9-(2-phosphonylmethoxyethyl)adenine (PMEA) to parenchymal liver cells, the primary site of hepatitis B virus (HBV) infection. Selective delivery is necessary because PMEA, which is effective against HBV in vitro, is hardly taken up by the liver in vivo. Lactosylated reconstituted high-density lipoprotein (LacNeoHDL), a lipid particle that is specifically internalized by parenchymal liver cells via the asialoglycoprotein receptor, was used as the carrier. PMEA could be incorporated into the lipid moiety of LacNeoHDL by attaching, via an acid-labile bond, lithocholic acid-3alpha-oleate to the drug. The uptake of the lipophilic prodrug (PMEA-LO) by the liver was substantially increased after incorporation into LacNeoHDL. Thirty minutes after injection of [(3)H]PMEA-LO-loaded LacNeoHDL into rats, the liver contained 68.9% +/- 7.7% of the dose (free [(3)H]PMEA, <5%). Concomitantly, the uptake by the kidney was reduced to <2% of the dose (free [(3)H]PMEA, >45%). The hepatic uptake of PMEA-LO-loaded LacNeoHDL occurred mainly by parenchymal cells (88.5% +/- 8.2% of the hepatic uptake). Moreover, asialofetuin inhibited the liver association by >75%, indicating uptake via the asialoglycoprotein receptor. The acid-labile linkage in PMEA-LO, designed to release PMEA during lysosomal processing of the prodrug-loaded carrier, was stable at physiological pH but was hydrolyzed at lysosomal pH (half-life, 60 to 70 min). Finally, subcellular fractionation indicates that the released PMEA is translocated to the cytosol, where it is converted into its active diphosphorylated metabolite. In conclusion, lipophilic modification and incorporation of PMEA into LacNeoHDL improves the biological fate of the drug and may lead to an enhanced therapeutic efficacy against chronic hepatitis B.     

Improved regional selectivity of hepatic arterial mitomycin by starch microspheres

Biodegradable starch microspheres, 40 microns in diameter, were administered through hepatic arterial catheters in 16 subjects with primary and metastatic liver tumors. These microspheres temporarily obstruct blood flow at the precapillary arteriole (microcirculation) level. Our study was undertaken to determine whether such occlusion would enhance hepatic deposition of, and thereby decrease systemic exposure to, simultaneously administered hepatic arterial mitomycin C (mito). When mito (10 mg/m2 over 1 min) was given with 90 X 10(6) microspheres (10 subjects), there was a 17% to 70% reduction in systemic mito exposure. When mito (10 mg/m2 over 1 min) was given with 36 X 10(6) microspheres (six subjects), there was a 15% to 60% reduction in systemic exposure, which may correlate with dose-dependent shunting (8% to 29%) through the liver to the lung (and hence to the systemic circulation), attributed to the starch microspheres. No life-threatening myelosuppression was noted; hepatic toxicity consisted of transient pain and elevation of liver enzymes.     

Uptake of L-carnitine and its short-chain ester propionyl-L-carnitine in the isolated perfused rat liver

Hepatic uptake of propionyl-L-carnitine (PLC) and L-carnitine (LC) was assessed with the impulse-response technique in the single-pass perfused rat liver. The experiments involved a rapid injection (impulse) of a mixture of the radiolabeled test compound (PLC or LC) and a reference compound (sucrose) into portal vein inflow and collection and radiochemical analysis (response) of the venous outflowing perfusate samples. The impulse injection was made in the presence of increasing unlabeled background concentrations of PLC (0-50 microM) or LC (50-500 microM) perfusing the liver. The hepatic uptake was minimal or negligible for LC, whereas the hepatic influx clearance was found to be low (0.095 ml/s equivalent to 5.7 ml/min) for PLC relative to the perfusate flow rate (30 ml/min). When background concentrations of PLC were increased (from 1-50 microM), the influx clearance was reduced in a concentration-dependent behavior, indicating partial saturation of the entry of compound into hepatocytes. PLC was taken up into hepatocytes via a unidirectional transport process with negligible efflux. The hepatic uptake of PLC was significantly reduced in the presence of unlabeled LC (500 microM), indicating an inhibition of the sinusoidal membrane transport of PLC by LC. The study showed the sinusoidal membrane is a permeability barrier to the entry of PLC and LC into hepatocytes, and it is the site of a common carrier-mediated transporter for both compounds.     

New, simple models to evaluate zone-specific damage due to hypoxia in the perfused rat liver: time course and effect of nutritional state

Models were developed to study zone-specific damage in periportal and pericentral regions of the liver lobule due to hypoxia produced in the perfused liver by ischemia, nitrogen or perfusion with low flow followed by reflow. Damage was assessed by lactate dehydrogenase release and trypan blue uptake in specific regions. Perfusion for up to 120 min under the conditions employed in all models failed to damage liver from well fed rats. In contrast, perfusion of livers from fasted rats for 30 min with N2-saturated buffer produced dye uptake of 37% and 66% in periportal and pericentral regions, respectively. Damage tended to be greater in this model when calcium was omitted from the perfusate (69% and 88% staining of periportal and pericentral regions, respectively). Release of lactate dehydrogenase correlated well with the percentage of cells stained with dye. In livers from fasted rats, 90 min of low flow (ca. 1 ml/g/min) followed by 30 min of reflow at normal flow rates (ca. 4 ml/g/min) produced damage exclusively to pericental regions of the liver lobule. On the average, about 40% of hepatocytes were stained with the dye under these conditions. Sixty minutes of ischemia followed by 13 min of reflow produced damage in 12% of periportal and 32% of pericentral regions of the liver lobule. When perfusion was in the retrograde direction (60 min low flow, 30 min reflow), periportal areas were damaged but pericentral regions were spared. Thus, models have been developed to study zone-specific damage due to hypoxia in the perfused liver. The data indicate that nutritional status is an important determinant of damage to hepatocytes due to hypoxia.     

Hepatic oxygen and glucose metabolism in the fetal lamb. Response to hypoxia.

Although the fetal liver is an active metabolic organ, its oxygen and glucose requirements have not previously been described. We measured hepatic blood flows and the oxygen and glucose differences across the liver in 12 late gestation fetal lambs in utero. Four animals were studied at least 1 wk postsurgically and again 2-5 d later to assess daily variations in hepatic blood flow and metabolism (group I). A second group of eight animals was studied 3-5 d postsurgically during a control period and during acute fetal hypoxia (group II). Under control conditions total hepatic blood flow averaged 400 ml/min per 100 g in both groups, and 75-80% was of umbilical origin. Liver blood flow and oxygen consumption were usually similar during repeated measurements, but in one animal varied considerably. During periods of normoxia, oxygen consumption for both the right and left lobes of liver was 4 ml/min per 100 g. Oxygen consumption of the whole liver accounted for 20% of total fetal oxygen consumption. This was achieved with oxygen extraction of 10-15%, so that hepatic venous blood was well oxygenated and provided an important source of oxygen for other fetal tissues. Under control conditions we could demonstrate no net hepatic uptake or release of glucose suggesting that the liver ultimately utilizes another carbon source to support its oxidative metabolism. During acute hypoxia total liver blood flow and its umbilical venous contribution both fell by 20%. Blood flow to the right lobe of the liver fell twice as much as that to the left lobe. Hepatic oxygen consumption was linearly related to oxygen delivery during the control and hypoxic periods. Consequently, right hepatic oxygen uptake fell by 45% whereas left hepatic oxygen uptake was unchanged, suggesting a functional difference between the lobes. During hypoxia glucose was released from both liver lobes; 6 mg/min per 100 g for the right lobe and 9 mg/min per 100 g for the left lobe. Total hepatic release of glucose was estimated to nearly equal umbilical uptake, so that 45% of the glucose available to fetal tissues was of hepatic origin. We conclude that the fetal liver responds to acute hypoxia by reducing its own oxygen consumption and releasing glucose to facilitate anaerobic metabolism.     

Mechanism of hepatotoxicity to periportal regions of the liver lobule due to allyl alcohol: role of oxygen and lipid peroxidation

The objective of this study was to evaluate the role of local oxygen tension in the zone-specific hepatotoxicity due to allyl alcohol. Infusion of allyl alcohol (350 microM) for 60 min into livers from normal fed rats perfused in the anterograde direction damaged virtually all cells in periportal areas of the liver lobule as indexed by trypan blue uptake (half-maximal uptake at 38 min). Under these conditions, oxygen uptake was inhibited only in periportal hepatocytes. Increasing the time of infusion of allyl alcohol to 90 min, however, caused dye uptake in virtually all cells across the liver lobule, with half-maximal staining in pericentral regions occurring at 70 min, indicating that hepatocytes in pericentral areas are not immune to damage by allyl alcohol. In livers from diethylmaleate-treated rats, the half-time for staining of periportal and pericentral regions was 27 and 39 min, respectively. Perfusion in the retrograde direction reverses the oxygen gradient in the liver. When allyl alcohol was infused in the retrograde direction for 60 min, cells in pericentral regions took up trypan blue whereas those in periportal areas were not damaged. Concomitantly, oxygen uptake was decreased only in pericentral regions. Infusion of allyl alcohol in oxygen-saturated perfusate in either direction caused release of lactate dehydrogenase and malondialdehyde. If oxygen tension was decreased by lowering the flow rate or perfusing with air-saturated buffer in the anterograde direction, however, malondialdehyde release and dye uptake due to allyl alcohol was reduced markedly.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tissue and hepatic subcellular distribution of liposomes containing bleomycin after intravenous administration to patients with neoplasms.

1. Liposomes containing 111In-labelled bleomycin were injected intravenously into two patients. One patient had a hepatoma and the other had secondary adenocarcinomatous deposits in the liver. 2. The tissue distribution of 111In was determined by whole-body scanning and by measurement of the radioactivity in organs at autopsy. 3. Scans in vivo and post-mortem measurement of radioactivity indicated that liposomes accumulate predominantly in the liver, but that there is no selective uptake of liposomes by the malignant tissue. 4. The subcellular distribution of radioactivity in the liver was measured 90 min after injection by fractionation of percutaneous liver biopsies on sucrose density gradients. 5. Radioactivity within the liver was concentrated in lysosomes. 6. Electron microscopy of tissue obtained before the administration of liposomes revealed particles morphologically indistinguishable from liposomes in hepatoma cells and hepatocytes.     

Contribution of OATP2 (OATP1B1) and OATP8 (OATP1B3) to the hepatic uptake of pitavastatin in humans

Pitavastatin, a novel potent 3-hydroxymethylglutaryl-CoA reductase inhibitor, is selectively distributed to the liver in rats. However, the hepatic uptake mechanism of pitavastatin has not been clarified yet. In the present study, we investigated the contribution of organic anion transporting polypeptide 2 (OATP2/OATP1B1) and OATP8 (OATP1B3) to pitavastatin uptake using transporter-expressing HEK293 cells and human cryopreserved hepatocytes. Uptake studies using OATP2- and OATP8-expressing cells revealed a saturable and Na(+)-independent uptake, with K(m) values of 3.0 and 3.3 microM for OATP2 and OATP8, respectively. To determine which transporter is more important for its hepatic uptake, we proposed a methodology for estimating their quantitative contribution to the overall hepatic uptake by comparing the uptake clearance of pitavastatin with that of reference compounds (a selective substrate for OATP2 (estrone-3-sulfate) and OATP8 (cholecystokinin octapeptide) in expression systems and human hepatocytes. The concept of this method is similar to the so-called relative activity factor method often used in estimating the contribution of each cytochrome P450 isoform to the overall metabolism. Applying this method to pitavastatin, the observed uptake clearance in human hepatocytes could be almost completely accounted for by OATP2 and OATP8, and about 90% of the total hepatic clearance could be accounted for by OATP2. This result was also supported by estimating the relative expression level of each transporter in expression systems and hepatocytes by Western blot analysis. These results suggest that OATP2 is the most important transporter for the hepatic uptake of pitavastatin in humans.     

Quantitation of HSV mass distribution in a rodent brain tumor model

A number of different viral vectors have been used for gene therapy of tumors, with many more under construction, ultimately designed to improve tumor targeting and transduction efficiency. It has become apparent that insufficient viral delivery can be a key limitation to treatment efficacy. We have studied the in vivo mass distribution of a herpes simplex virus type 1 (HSV) vector, hrR3, by radiolabeling it with 111In-oxine. The virus was administered to intracerebral 9L glioma bearing Fisher (F-344) rats by intracarotid and intratumoral injection. The blood half-life of the virus was 1 min (fast component, 10% contribution) and 180 min (slow component, 90% contribution). Approximately 20% of activity had been excreted by 24 h. With intracarotid injection, the total amount of virus that accumulated in tumor was 0.10+/-0.07% of the injected dose (ID)/g at 1 h and 0.19+/-0.01% ID/g at 24 h. By comparison, co-injection of RMP-7, a synthetic bradykinin analog, with the virus, resulted in slightly increased tumor delivery of 0.17+/-0.10% ID/g (P 0.05) at 1 h. The 1 h organ distribution after intra-arterial injection (%ID/organ) was as follows: liver 273+/-2.86%, lung 2.10+/-0.68% and kidney 1.78+/-1.60% with lesser amounts in other organs. When virus was injected directly into the tumor, 71% of virus remained in tumor at 24 h (590+/-212 %ID/g, consistent with the small tumor mass containing most of the virus) with the following distribution regions: tumor > border zone > normal brain (99:40: 1). These studies are the first quantitative mass distribution studies of HSV vectors in an experimental brain tumor model. Localization and quantitation of viral accumulation in vivo will enable detailed analysis of viral and organ interactions critical for advancing the therapeutic use of vectors.     

Renal selective N-acetyl-L-gamma-glutamyl prodrugs. III. N-acetyl-L-gamma-glutamyl-4'-aminowarfarin is not targeted to the kidney but is selectively excreted into the bile

The pharmacokinetics of N-acetyl-L-gamma-glutamyl-4'-aminowarfarin (AGAW) was studied in the rat. The aim of this prodrug was to cause a renal-specific inhibition of the vitamin K cycle as a result of renal-specific release of the active drug 4'-aminowarfarin (AW). In vitro, it was found that kidney and liver homogenates and cytosol were able to convert the prodrug. In vivo, plasma concentrations of AW rose only slowly after a dose of 10 mg/kg AGAW i.v. to give a maximum concentration of about 3 micrograms AW/ml at t = 14 to 24 h. The tissue distribution of AGAW and AW was measured after 10 mg/kg AGAW i.v. It was found that AGAW did not accumulate in the kidney (9.7 micrograms/g in the kidney; 83 micrograms/ml in plasma at t = 60 min). AW concentrations were very low (0.1 microgram/ml or mg at t = 60 min). These results suggest that AGAW is not transported via a carrier into the kidney. The uptake of AGAW in vitro by rat kidney slices was investigated. It was found that AGAW did not accumulate in the slices. Neither did AGAW influence the accumulation of N-acetyl-gamma-glutamyl sulfamethoxazole in kidney slices. A second explanation for the lack of selectivity of AGAW in vivo could be its high (approximately 90%) plasma protein binding. Instead of being targeted to the kidney, however, AGAW was found to be excreted via a carrier-mediated mechanism into the bile: 50% of the dose was recovered unchanged in the bile within 3 hr.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for the involvement of a pulmonary first-pass effect via carboxylesterase in the disposition of a propranolol ester derivative after intravenous administration

The disposition kinetics of O-butyryl propranolol (butyryl-PL), a model compound containing an ester moiety, after intravenous administration was compared with that of PL in rats and beagle dogs. Rats showed only 30% conversion of butyryl-PL to PL up to 2 h after dosing, whereas dogs showed nearly complete conversion within 10 min after administration. The CL(total) of butyryl-PL in rats was 5.8 l/h/kg and that in dogs was 65.6 +/- 18.6 l/h/kg, both of which were greater than hepatic blood flow. The in vivo conversion from butyryl-PL to PL in the rat could be explained on the basis of the hydrolysis characteristics in the liver and blood. The in vitro hydrolysis data and the in vivo data after intra-arterial administration clearly demonstrated that the extremely high CL(total) of butyryl-PL in dogs was dependent on first-pass hydrolysis in the lung in addition to hydrolysis at a blood flow-limited rate in the liver and kidney. The availability of butyryl-PL after passage through the lung was 50%. Furthermore, the isoform of carboxylesterase involved in the pulmonary hydrolysis of butyryl-PL in the dog was identified as D1, a CES-1 group enzyme. However, butyryl-PL was not recognized as a substrate by CES-1 family carboxylesterases, which are present at high levels in the rat lung (RH-1) and kidney (RL-1). These findings indicate that extrahepatic metabolism, especially in the lung, is important in the disposition of drugs containing an ester moiety after intravenous administration and that the substrate specificity of carboxylesterase isozyme distinguishes from others.     

How organic anions accumulate in hepatocytes lacking Mrp2: evidence in rat liver

In the liver, the accumulation of hepatobiliary contrast agents is a crucial issue to understand the images of liver scintigraphy or magnetic resonance (MR) imaging. Thus, depending on the regulation of uptake and exit membrane systems in normal and injured hepatocytes, these contrast agents will accumulate differently within cells. Gadobenate dimeglumine (Gd-BOPTA) is a hepatobiliary MR contrast agent that distributes to the extracellular space and enters into rat hepatocytes through the sinusoidal transporters, organic anion-transporting polypeptides. Gd-BOPTA is not metabolized during its transport to the canalicular membrane where it is excreted into bile through multiple resistance protein-2 (Mrp2). It is not well known how Gd-BOPTA accumulates in normal livers and in livers lacking Mrp2. We perfused livers from normal rats and from rats lacking Mrp2 with (153)Gd-BOPTA at increasing concentrations and assessed the hepatic accumulation of this agent using a gamma probe placed above the livers. By use of a pharmacokinetic model that best described the amounts of Gd-BOPTA in perfusate, bile, and hepatic tissue over time, we showed how increasing concentrations and the absence of Mrp2 modify the hepatic accumulation of the contrast agent. It is noteworthy that despite the absence of Gd-BOPTA bile excretion and a similar efflux back to sinusoids in livers lacking Mrp2, the maximal hepatic accumulation of contrast agent was similar to normal rats. We also showed how hepatic accumulation relies on the concomitant entry into and exit from hepatocytes. Such information improves our understanding of liver imaging associated with the perfusion of hepatobiliary contrast agents, which was recently introduced in clinical practice.