Hepatocytes take up a variety of ligands via receptor-mediated endocytosis, yet little is known regarding either the volume of fluid or the amount of membrane internalized via endocytosis in liver cells. In these studies, we have utilized radiolabeled inulin to characterize fluid phase endocytosis by rat hepatocytes in primary culture and perfused rat liver. Uptake of inulin by cultured hepatocytes was nonlinear with time, occurring most rapidly during the first 2 min. Inulin uptake and efflux in cultured hepatocytes and inulin uptake by perfused rat liver were kinetically compatible with the entry of inulin into a rapidly (t1/2, 1-2 min) turning-over (presumably endosomal) compartment that exchanged contents with the extracellular space and comprised -3% of hepatocyte volume, as well as entry into and concentration of inulin within slowly (t1/2, >1 hr) turning-over storage compartments. Based on inulin uptake, it is estimated that cultured hepatocytes endocytosed the equivalent of 20% or more of their volume and 5 or more times their plasma membrane surface area each hour. Neither chloroquine (1 mM) nor taurocholate (200 ,uM) affected inulin handling by cultured cells, whereas colchicine (10 ,uM) inhibited transfer to storage compartments by >50%. In conjunction with our previous observations, the present findings suggest that inulin endocytosed across the basolateral membrane is largely (Q80%) regurgitated back into plasma, with smaller amounts transported to intracellular storage compartments ("18%) or to bile (-2%). Transport of inulin via these pathways is unaffected by taurocholate and does not require vesicle acidification, whereas intact microtubular function is required for transfer to storage compartments or biliary secretion.The ability to internalize extracellular material appears to be a property shared by most, if not all, cells. Hepatocytes are actively endocytic and internalize a variety of ligands by receptor-mediated endocytosis (1, 2), yet relatively little is known regarding the overall rate at which hepatocytes internalize extracellular fluid or fluid phase markers (3, 4).We have recently reported evidence that the intact perfused rat liver transports a variety of fluid phase markers from perfusate to bile via a transcellular vesicular mechanism (5). In the present study, we have extended these observations in perfused liver and also characterized fluid phase endocytosis by rat hepatocytes in primary culture. In addition to measuring the rate at which hepatocytes endocytose extracellular fluid, our principal objectives were as follows. First, several recent reports indicate that fluid phase markers are internalized initially into a compartment that exchanges with extracellular fluid (6-8); however, the kinetics of this process, which presumably reflect membrane recycling, remain incompletely characterized. We therefore sought evidence of such a process in mammalian liver and characterized it kinetically. Our second objective was to integrate observations in perfused liver and culture...

In these studies, we have used several approaches to systematically explore the contribution of transcellular vesicular transport (transcytosis) to the blood-to-bile movement of inert fluid-phase markers of widely varying molecular weight. First, under steady-state conditions, the perfused rat liver secreted even large markers in appreciable amounts. The bile-to-plasma (B/P) ratio of these different markers, including microperoxidase (B/P ratio = 0.06; mol wt = 1,879), inulin (B/P ratio = 0.09, mol wt = 5,000), horseradish peroxidase (B/P ratio = 0.04, mol wt = 40,000), and dextran (B/P ratio = 0.09, mol wt = 70,000), exhibited no clear ordering based on size alone, and when dextrans of two different sizes (40,000 and 70,000 mol wt) were studied simultaneously, the relative amounts of the two dextran species in bile were the same as in perfusate. Taurocholate administration produced a 71% increase in bile flow but little or no (0-20%) increase in the output of horseradish peroxidase, microperoxidase, inulin, and dextran. Second, under nonsteady-state conditions in which the appearance in or disappearance from bile of selected markers was studied after their abrupt addition to or removal from perfusate, erythritol reached a B/P ratio of 1 within 2 min. Microperoxidase and dextran appeared in bile only after a lag period of 12 min and then slowly approached maximal values, whereas sucrose exhibited kinetically intermediate behavior. A similar pattern was observed after removal of >95% of the marker from the perfusate. Erythritol rapidly reapproached a B/P ratio of 1, whereas the B/P ratio for sucrose, dextran, and microperoxidase fell much more slowly and exceeded 1 for a full 30 min after perfusate washout. Finally, electron microscopy and fluorescence microscopy of cultured hepatocytes demonstrated the presence of horseradish peroxidase and fluoresceindextran, respectively, in intracellular vesicles, and fractionation of perfused liver homogenates revealed that at least 35-50% of sucrose, inulin, and dextran was associated with subcellular organelles.Collectively, these observations are most compatible with a transcytosis pathway that contributes minimally to the secretion of erythritol, but accounts for a substantial fraction of sucrose secretion and virtually all (>95%) of the blood-to-bile

Pulmonary delivery of aqueous bolus aerosols of insulin in healthy subjects resulted in rapid absorption with an associated hypoglycemic effect quicker than is achieved after subcutaneous dosing of regular insulin. Inhaled insulin pharmacokinetics and pharmacodynamics were independent of formulation variables (pH, concentration) but affected by certain respiratory maneuvers.

Poly(lactic-co-glycolic) acid (PLGA) bioresorbable microspheres are used for controlled-release drug delivery and are particularly promising for ocular indications. The objective of the current study was to evaluate the pharmacokinetics and safety of a recombinant human monoclonal antibody (rhuMAb HER2) in rabbits after bolus intravitreal administration of a solution or a PLGA-microsphere formulation. On Day 0, forty-eight male New Zealand white rabbits (2.3-2.6 kg) were immobilized with intramuscular ketamine/xylazine, and the test materials were injected directly into the vitreous compartment. Group 1 animals received rhuMAb HER2 in 50:50 lactide: glycolide PLGA microspheres; Group 2 animals received rhuMAb HER2 in solution (n = 24/group). The dose for each eye was 25 microg (50 microl). After dosing, animals were sacrificed at 2 min, and on 1, 2, 4, 7, 14, 23, 29, 37, 44, 50, and 56 days (n = 2/timepoint/group). Safety assessment included direct ophthalmoscopy, clinical observations, body weight, and hematology and clinical chemistry panels. At necropsy, vitreous and plasma were collected for pharmacokinetics and analysis for antibodies to rhuMAb HER2, and the vitreal pellet (Group 1) was prepared for histologic evaluation. All animals completed the study per protocol-both treatments were well tolerated, and no suppurative or mixed inflammatory cell reaction was observed in the vitreal samples (Group 1) at any of the time points examined. Antibodies to rhuMAb HER2 were detected in plasma samples by Day 7 in both treatment groups, but infrequently in vitreous samples. There were no safety implications associated with this immune response. The in vitro characterization of the PLGA microspheres provided reasonable projections of the in vivo rhuMAb HER2 release kinetics (Group 1). The total amount of antibody that was released was similar in vitro (25.9%) and in vivo (32.4%). RhuMAb HER2 (Group 2) was cleared slowly from the vitreous compartment, with initial and terminal half-lives of 0.9 and 5.6 days, respectively. The volume of distribution approximated the vitreous volume in a rabbit eye.

The development of a neovascular supply (angiogenesis) is a major aspect of tumorigenesis. Recent work has indicated that vascular endothelial growth factor (VEGF) is a major regulator of angiogenesis. In vitro and in vivo studies have demonstrated that an anti-VEGF antibody is capable of suppressing the growth of human tumor cell lines. The following study was conducted in tumor-bearing nude mice to evaluate the concentration-response relationship of murine anti-VEGF monoclonal antibody (muMAb VEGF) so that an efficacious plasma concentration of the recombinant humanized form (rhuMAb VEGF) in cancer patients could be estimated. (This study was included in our Investigational New Drug application to support the clinical dosing regimen and projected human safety factors for the toxicology program.) Additionally, the growth dynamics of the tumors were evaluated as a function of dose to explore whether a mechanismic interpretation of tumor growth inhibition by muMAb VEGF is possible. On day 1, A673 human rhabdomyosarcoma cells (2 x 10(6) cells/mouse) were injected subcutaneously in 188 beige nude mice (16-24 g). Treatment with muMAb VEGF (0.05-5.0 mg/kg; n = 24/group), phosphate-buffered saline (n = 10), or anti-gp120 isotype-matched control antibody (5.0 mg/kg; n = 10) began 24 hr later. Each animal received intraperitoneal injections of test material twice weekly for 4 wk. Immediately prior to each dose, 2 mice from each muMAb VEGF group were selected randomly, and plasma was collected for pharmacokinetic evaluation; at the end of the study, samples were collected from all animals for pharmacokinetic evaluation. Tumor dimensions were recorded weekly, and at the end of the study, tumor weight and dimensions were recorded. Satisfactory tumor suppression in nude mice was achieved at muMAb VEGF doses of > or =2.5 mg/kg, where the average trough muMAb VEGF plasma concentration was 30 microg/ml (concentrations in individual animals >10 microg/ml). Assuming the pharmacokinetics of rhuMAb VEGF in patients will resemble the pharmacokinetics of a similar humanized anticancer monoclonal antibodies, a clinical dosing regimen was designed to maintain the rhuMAb VEGF plasma concentration in this efficacious range. This study shows an approach that can be used to estimate a human dosing regimen from preclinical pharmacokinetic/pharmacodynamic data. Because we have just initiated clinical trials with rhuMAb VEGF we cannot judge clinical outcome in relation to these preclinical predictions; nonetheless, it is hoped that by sharing our approach and thought processes with other investigators we can assist the discovery and development of anticancer therapeutics.

Cholecystokinin (CCK) was conjugated to 125I-Bolton-Hunter reagent (125I-BH-CCK), and the binding of this ligand to CCK receptors in isolated mouse pancreatic acini was correlated with the regulation by CCK of both amylase release and the transport of 2-deoxyglucose and alpha-aminoisobutyric acid. Stimulation of amylase release by CCK was biphasic. At low CCK concentrations (less than 200 pM), amylase release was progressively stimulated, whereas at higher CCK concentrations (greater than 200 pM), amylase release was progressively reduced. In contrast, stimulation of 2-[3H]deoxyglucose transport and inhibition of alpha-[3H]aminoisobutyric acid transport were monophasic, being one-half maximal at 0.85 and 0.44 nM, respectively. Under incubation conditions identical to those employed for measuring biological functions, the binding of 125I-BH-CCK to receptors in acini was rapid and reversible. Competition-inhibition curves and Scatchard plots of equilibrium binding were compatible with two orders of binding sites. Employing a computer program for analysis of multiple binding sites, a high-affinity, low-capacity binding component having a Kd of 26 pM and a lower-affinity, higher-capacity binding component having a component Kd of 2.2 nM were resolved. Regulation of 2-[3H]deoxyglucose and alpha-[3H]aminoisobutyric acid uptake appeared, therefore, to be the result of fractional occupancy of the lower-affinity CCK receptors. Regulation of amylase released was more complex and appeared to be due to the concomitant occupancy of both the high- and low-affinity CCK receptors.

Male volunteer subjects smoked one marijuana cigarette containing 100, 200, or 250 micrograms/kg delta-9-tetrahydrocannabinol (THC) and were tested on three perceptual-motor performance measures related to driving. Performance was measured and blood samples were collected for 24 h after smoking. The covariation between pharmacodynamics of performance and pharmacokinetics of THC in plasma was investigated for decrement in performance as the response to smoking a single marijuana cigarette. A significant linear correlation was found between tracking errors under divided attention and THC plasma levels over 5-25 ng/ml for approximately 2 h after smoking. A sigmoid relation was found between critical tracking breakpoint and log THC plasma levels over 2-25 ng/ml for approximately 7 h after smoking.

We determined a rate of protein egress from the pleural cavity into thoracic duct lymph in seven anesthetized sheep in whom we had made 10-ml/kg hydrothoraces containing 1% plasma protein labeled with 125I-albumin. The labeled protein left the pleural space at an average rate of 0.02 ml X kg-1 X h-1. In 25 unanesthetized sheep we injected labeled protein intravenously and collected pleural fluid and plasma at intervals up to 48 h. The radioactivity in the sheeps' plasma fell twice as rapidly as the rate observed in humans. The half time for equilibration between plasma and pleural fluid protein specific activity was approximately 6 h. In seven sheep we gave a continuous infusion of tracer protein. In six of these seven sheep we collected superficial cervical lymph. The protein concentration in the lymph was three times that of pleural fluid. The half time for protein specific activity equilibration between plasma and pleural fluid was 5 h and 3-4 h between plasma and superficial cervical lymph. The rate of protein turnover in the pleural space appears to be much slower than previously reported.