Increased Plasma Clearance Research Articles

Pharmacokinetic and Pharmacodynamic Interactions of Ethanol and Propofol in Rabbits

Propofol is a short-acting intravenous anesthetic which is rapidly metabolized by glucuronidation and ring hydroxylation catalyzed by cytochrome P450. In clinical trials, it was found that patients receiving both ethanol and propofol injection in the surgery awoke significantly earlier than those who were injected propofol alone. To investigate pharmacokinetic and pharmacodynamic interactions of propofol and ethanol, propofol was injected alone and in combination with ethanol to rabbits. Propofol was injected intravenously at a dose rate of 15 mg kg−1. Ethanol was injected intraperitoneally at a dose rate of 2.5 mg kg−1. An LC assay with native fluorescence detection was developed and validated for determining the concentration of propofol in rabbit plasma and the effect of co-injected ethanol on the plasma pharmacokinetics of propofol in rabbits. Co-injection of ethanol decreased the maximum plasma concentration (Cmax) and area under the plasma concentration–time curve (AUC0–∞) of propofol by 53.28 and 53.42%, respectively, as compared to the control (P < 0.001). Concomitant ethanol also caused a 66.67% increase in plasma clearance (CL) (P < 0.001). These may partially explain the finding that co-injected ethanol reduced the anesthetic effect of propofol by introducing hepatic microsomal drug-metabolizing enzymes activity in rabbits.

Pharmacokinetics and Tumor Disposition of PEGylated, Methotrexate Conjugated Poly-l-lysine Dendrimers

Dendrimers have potential for delivering chemotherapeutic drugs to solid tumors via the enhanced permeation and retention (EPR) effect. The impact of conjugation of hydrophobic anticancer drugs to hydrophilic PEGylated dendrimer surfaces, however, has not been fully investigated. The current study has therefore characterized the effect on dendrimer disposition of conjugating alpha-carboxyl protected methotrexate (MTX) to a series of PEGylated (3)H-labeled poly-l-lysine dendrimers ranging in size from generation 3 (G3) to 5 (G5) in rats. Dendrimers contained 50% surface PEG and 50% surface MTX. Conjugation of MTX generally increased plasma clearance when compared to conjugation with PEG alone. Conversely, increasing generation reduced clearance, increased metabolic stability and reduced renal elimination of the administered radiolabel. For constructs with molecular weights >20 kDa increasing the molecular weight of conjugated PEG also reduced clearance and enhanced metabolic stability but had only a minimal effect on renal elimination. Tissue distribution studies revealed retention of MTX conjugated smaller (G3-G4) PEG(570) dendrimers (or their metabolic products) in the kidneys. In contrast, the larger G5 dendrimer was concentrated more in the liver and spleen. The G5 PEG(1100) dendrimer was also shown to accumulate in solid Walker 256 and HT1080 tumors, and comparative disposition data in both rats (1 to 2% dose/g in tumor) and mice (11% dose/g in tumor) are presented. The results of this study further illustrate the potential utility of biodegradable PEGylated poly-l-lysine dendrimers as long-circulating vectors for the delivery and tumor-targeting of hydrophobic drugs.

Hepatobiliary Disposition of Thyroid Hormone in Mrp2-Deficient TR− Rats: Reduced Biliary Excretion of Thyroxine Glucuronide Does Not Prevent Xenobiotic-Induced Hypothyroidism

The hepatobiliary disposition of thyroxine (T4) was evaluated in Groningen Yellow transport deficient (TR(-)) rats lacking functional multidrug resistance-associated protein 2 (Mrp2; Abcc2). Male Wistar and TR(-) rats were dosed orally (4 days) with phenobarbital (PB; 100 mg/kg) or DMP 904 (200 mg/kg), after which T4 homeostasis and hepatic cytochromes P450, UDP-glucuronosyltransferase, xenobiotic transporters, and T4 glucuronidation were determined. Serum concentrations of T4 were approximately 50% higher in control TR(-) rats than Wistars. PB and DMP 904 increased hepatic levels of P450s and T4-glucuronidation (T4-G), and these changes were associated with decreased serum T4 levels in both strains. In Wistar but not TR(-) rats, DMP 904 increased thyroid stimulating hormone levels twofold. Hepatobiliary clearance of T4 was determined after intravenous infusion of [(125)I]T4 to rats dosed with PB and DMP 904 (4 days). PB and DMP 904 increased plasma clearance and hepatic uptake of [(125)I]T4 equivalents in Wistar but not TR(-) rats. Total biliary clearance (Cl(bile)) was approximately 0.85 and 0.2 ml/h in Wistar and TR(-) rats, respectively, with virtually no T4-G excreted in bile in TR(-) rats. Biliary clearance of unconjugated T4 was also lower in control TR(-) rats than in Wistars, although DMP 904 increased its biliary clearance in both strains. These results suggest that Mrp2 is likely to be responsible for biliary excretion of T4-G and contributes in part to excretion of T4. Decreased biliary clearance of T4 and metabolites in TR(-) rats mitigated but did not prevent drug-induced changes in serum T4, suggesting that other factors contribute to changes in T4 homeostasis in these rats.

Low-Intensity Exercise Exerts Beneficial Effects on Plasma Lipids via PPARγ

An important mechanism by which physical activity reduces the risk of cardiovascular disease is through regulating plasma lipids. We investigated whether low-intensity exercise modulates lipid metabolism and the transcription factors peroxisome proliferator-activated receptor gamma (PPARgamma) and liver X receptor alpha (LXRalpha) responsible for controlling reverse cholesterol transport (RCT). Thirty-four sedentary adults, mean age 45.6 +/- 11.1 yr, participated in an 8-wk low-intensity exercise program consisting of walking 10,000 steps, three times a week. Subjects were randomly allocated to either an exercise group or a sedentary control group, and serum lipid or lipoprotein concentrations were determined. Compared with controls, there was a significant decrease in total cholesterol (preexercise, 5.73 +/- 1.39 mmol x L; postexercise, 5.32 +/- 1.28 mmol x L) and a significant increase in HDL (preexercise, 1.46 +/- 0.47 mmol x L; postexercise, 1.56 +/- 0.50 mmol x L) after the exercise program. There was a significant increase in serum oxidized LDL (oxLDL) concentrations in the exercise group before and after exercise (0 wk, 554 +/- 107 ng x mL; 4 wk, 698 +/- 134 ng x mL; 8 wk, 588 +/- 145 ng x mL). A significant increase in leukocyte mRNA expression for PPARgamma (4 wk, 1.8 +/- 0.9-fold; 8 wk, 4.3 +/- 1.9-fold) was observed, which was reinforced by increased PPARgamma DNA-binding activity postexercise (preexercise, 0.22 +/- 0.09 OD units; postexercise, 1.13 +/- 0.29 OD units). A significant increase in gene expression was observed for the oxLDL scavenger receptor CD36 (4 wk, 3.8 +/- 0.6-fold; 8 wk, 2.7 +/- 0.5-fold) and LXRalpha (8 wk, 3.5 +/- 0.8-fold). Two LXRalpha-regulated genes involved in RCT, namely, ATP-binding cassette transporters A1 and GI (ABCA1 and ABCG1, respectively), were significantly up-regulated postexercise (8 wk: ABCA1, 3.46 +/- 0.56-fold; ABCG1, 3.06 +/- 0.47-fold). We propose that the net effect of these changes may be to increase oxLDL uptake, to stimulate RCT, and thus to promote clearance of proatherogenic lipids from the vasculature, ultimately contributing to the cardiovascular benefits of low-intensity aerobic exercise.

Metabolism of amyloid proteins.

Metabolic processing of amyloid precursor proteins is an important factor in the genesis of practically all forms of amyloidosis. Of the three major forms of systemic amyloidosis, reactive amyloid (amyloid A protein; AA) formation shows the most consistent role of partial proteolysis of serum amyloid A (SAA) to AA proteins which form fibrils. Immunoglobulin amyloidosis is also usually associated with C-terminal degradation of the fibril precursor light chain protein. Although it is commonly thought that transthyretin amyloidosis is associated with fibril formation from the tetrameric circulating plasma transthyretin, chemical analyses of transthyretin fibril deposits show significant fragmentation of the fibril protein constituents. In addition, it has been documented that proteolytic fragments are the fibril subunit proteins in gelsolin, cystatin C. Alzheimer's beta-amyloid precursor protein and apolipoprotein AI (apoAI) amyloidoses. Notable exceptions to the role of proteolysis in amyloid fibril formation would appear to be the lysozyme and beta 2-microglobulin amyloidoses. Few studies have examined the metabolism of amyloid-forming proteins. Perhaps the best data are on apoAI, which show decreased plasma residence time for the amyloidogenic Gly26Arg apoAI (1.8 d vs. normal 4.5 d). Similarly, preliminary data show increased clearance of Val30Met transthyretin when compared with the wild-type protein (18 h vs. 26 h). Also, biosynthetically 35S-labelled SAA proteins reconstituted with HDL show increased plasma clearance of murine SAA2, the amyloid fibril subunit protein, when compared with murine SAA1. Few data are available on metabolism of amyloid immunoglobulin light chain proteins, but it has been shown that radiolabelled Bence-Jones proteins are cleared very rapidly from the circulation. A better understanding of the metabolism of precursor proteins in each of the amyloid deposition diseases will give insight into the mechanisms of fibril formation and pathogenesis of amyloidosis.

LINEZOLID-RESISTANT STAPHYLOCOCCUS AUREUS IN TWO PEDIATRIC PATIENTS RECEIVING LOW-DOSE LINEZOLID THERAPY

We report 2 sisters with hyper-IgE syndrome treated with daily suppressive dosages of linezolid (LZD) who developed LZD-resistant Staphylococcus aureus carrying the G2576T mutation in the 23S rRNA gene. Molecular typing suggested transmission of the resistant strain from one sister to the other. LZD-susceptible S. aureus was isolated 2 months after LZD discontinuation. LZD-resistant S. aureus remains rare but may occur while receiving suppressive therapy.

Quantitation of Binding of Ricinus communis Agglutinin I to Von Willebrand Factor (VWF): Investigation of Relationship with Plasma Clearance of VWF in Type 1 Von Willebrand Disease.

Type 1 von Willebrand disease (VWD) is a heterogeneous bleeding disorder manifest by a partial quantitative deficiency of the sialoglycoprotein von Willebrand factor (VWF). Glycosylation of VWF results in the addition of 12 N-linked and 10 O-linked oligosaccharide chains and modification of these structures has been shown to result in increased plasma clearance of VWF. Exposed galactose residues are required for the binding of VWF to the hepatic asialoglycoprotein receptor. The lectin Ricinus communis agglutinin I (RCA I) has a high affinity for the terminal β-linked galactose residues on VWF. Recent findings have reported increased binding of RCA-I to VWF in a proportion of patients with a partial quantitative deficiency of VWF. We have evaluated the binding of RCA-I to VWF in patients with type 1 VWD and normal controls and investigated its relationship with the plasma clearance of VWF antigen (VWF:Ag) in patients with type 1 VWD. An ELISA was developed to assess binding of biotinylated RCA-I to VWF in 30 patients with type 1 VWD and 17 normal controls. Plasma samples were analysed at baseline in both groups and following desmopressin (DDAVP) infusion in VWD group. VWF:Ag levels were assessed in parallel and RCA-I binding to VWF was expressed as a ratio of RCA-I/VWF:Ag. In the VWD patients VWF:Ag levels were quantitated over 6 hours following the administration of intravenous DDAVP. The half-life of VWF (VWF:Ag t1/2) was calculated by determining the first-order rate constant for the elimination phase of VWF:Ag.1 Median VWF:Ag levels in the VWD patients and normal controls were 36 IU/dL (range, 4–50), and 103 IU/dL (64–197) respectively. At baseline, RCA-I lectin was found to bind to VWF with a median RCA-I/VWF:Ag ratio of 0.98 (0.48–1.74) in VWD group as compared to 0.85 (0.69–1.28) in the controls. This represents a significant difference in RCA-I/VWF:Ag ratios between the two groups, p<0.05. Quantitation of RCA-I binding to VWF secreted post DDAVP in VWD group is pending. Median VWF:Ag t1/2 in the type 1 VWD patients was 4.1 hours (0.92–8.7) representing significantly increased plasma clearance of VWF:Ag in these patients.2 There was significant correlation between baseline RCA/VWF:Ag ratio and VWF:Ag t1/2 in the type 1 VWD patients, r=0.48, p<0.01. This represents significant inverse correlation between RCA-I/VWF binding and plasma clearance of VWF:Ag. There was no correlation between baseline RCA/VWF:Ag ratio and VWF:Ag in either the patient or control groups, p>0.1. The finding of increased binding of RCA-I to VWF in type 1 VWD patients at baseline is suggestive of variation in glycosylation in a proportion of these patients. This could be causative of or contributory towards increased VWF clearance, be otherwise related to the type 1 VWD disease phenotype or may represent normal variability. The inverse correlation found between baseline RCA-I/VWF binding and plasma clearance of VWF:Ag may reflect differences between VWF secreted post-DDAVP and baseline VWF: investigation is ongoing.

Von Willebrand Factor Propeptide: Response to 1-Deamino-8-D-Arginine Vasopressin and Investigation of Relationship with Plasma Clearance of Von Willebrand Factor Antigen in Type 1 Von Willebrand Disease.

The propeptide of von Willebrand factor (VWFpp) has been demonstrated to be critical for multimerization and intracellular storage of the mature von Willebrand factor (VWF) protein. Although VWFpp and VWF have been shown to circulate at a distinct ratio in plasma, they are stored in endothelial cells in equimolar amounts and stimulation of the endothelium results in acute simultaneous release of these polypeptides in equimolar concentrations. This includes following DDAVP administration to healthy individuals. An increased ratio of plasma VWFpp to VWF antigen (VWF:Ag) may reflect increased clearance of VWF:Ag or reduced clearance of VWFpp. We have quantitated plasma VWFpp levels and the VWFpp to VWF:Ag ratio in patients with type 1 VWD and examined their relationship with the plasma clearance of VWF:Ag and VWFpp. We have also examined the endothelial release of VWFpp and VWF:Ag following DDAVP stimulation in these patients. Plasma molar VWF:Ag and VWFpp concentrations were determined by immunosorbent assay1 in 27 patients with type 1 VWD and 21 normal controls. In the VWD patients VWF:Ag and VWFpp levels were quantitated over 6 hours following the administration of intravenous DDAVP. The half-lives of VWF:Ag (VWF:Ag t1/2) and VWFpp (VWFpp t1/2) were calculated by determining the first-order rate constant for the elimination phase of the respective proteins.2 To determine the molar ratio of VWFpp and VWF:Ag released following DDAVP, values at 30 mins were extrapolated from semi-log plots of VWFpp and VWF:Ag against time. Median concentrations of VWFpp and VWF:Ag were 2.9nmol/L (range, 1.4–10.9) and 24.5nmol/L (2.5–42) pre-DDAVP in VWD group and 9.7nmol/L (5.8–18.1) and 51.5nmol/L (32–98.5) at baseline in normal subjects. The median VWFpp/VWF:Ag ratio was 0.13 (0.06–1.5) in the VWD group as compared to 0.23 (0.08–0.34) in the normals. Median VWF:Ag t1/2 and VWFpp t1/2 values in the VWD patients were 4 hours (0.9–8.7) and 2.5 hours (1.7–8) respectively. VWF:Ag normally has a half-life of 8–12 hours in plasma,3 while that of VWFpp is 2–3 hours.1 In the patient group, inverse correlation was found between VWFpp/VWF:Ag ratio and VWF:Ag t1/2, r=−0.48, p=0.01. There was also significant inverse correlation between VWFpp and VWF:Ag t1/2, r=−0.53, p<0.005. In contrast, there was no correlation between VWFpp/VWF:Ag ratio and VWFpp t1/2, r=−0.29, p>0.1. The values for VWFpp and VWF:Ag extrapolated 30 mins post DDAVP were corrected for baseline values and plotted to give a slope of 0.48, representing release of two moles of VWF per mole VWFpp in the patient group. The range of the plasma VWFpp/VWF:Ag ratio is wide in type 1 VWD patients and this may reflect heterogeneity in the pathophysiological processes, including post secretion events such as increased clearance of VWF:Ag, that contribute towards disease phenotype. The finding of inverse correlation between VWF:Ag t1/2 and the ratio of VWFpp to VWF:Ag indicates that increased plasma clearance of VWF:Ag is not associated with an increased VWFpp clearance in some patients with type 1 VWD. In addition, we have not demonstrated equimolar release of VWFpp and mature VWF in patients with type 1 VWD.

Neuropsychology/Language/Behavior: All Ages

Neuropsychology/Language/Behavior: All Ages

Oligosaccharide structures of von Willebrand factor and their potential role in von Willebrand disease

Oligosaccharides make up approximately 20% of the mass of VWF and although their structures are well established, their functional role remains unclear. Modification of the VWF oligosaccharide structures has been shown to result in increased plasma clearance of the protein. A mutation which alters cell type-specific expression of the Galgt2 glycosyltransferase gene in the RIIIS/J mouse results in an autosomal dominant partial quantitative deficiency of VWF. Increased plasma clearance of VWF has been demonstrated in some individuals with a partial quantitative deficiency of the protein and it is possible that variation in VWF glycosylation may contribute towards this. ABH antigens occur within the oligosaccharide component of VWF and may account for the variation in plasma VWF:Ag levels observed between individuals of different ABO blood groups. The structures and functional roles of the oligosaccharide side chains of VWF and possible pathogenetic mechanisms by which they may contribute towards VWD are reviewed in this article.

Influence of trauma on plasma elimination of exogenous fat and on lipoprotein lipase activity and mass

Trauma is followed by an increased plasma clearance and oxidation of exogenous fat but the underlying mechanism is not fully understood. To examine the influence of a surgical trauma on the plasma elimination of exogenous triglycerides (TG) and its relationship with lipoprotein lipase (LPL) activity and LPL mass. Nine patients underwent a hypertriglyceridaemic clamp and a lipolytic capacity test before and after open abdominal surgery. The infusion rate was adjusted to maintain a stable TG concentration of 4 mmol x l(-1) during 180 min. The lipolytic capacity was determined as the change in LPL activity and mass following a bolus dose of 100 IU x kg BW(-1) heparin sodium. Postoperatively, the plasma elimination rate of fat was 2.6 times higher (P<0.001). Infusion of lipids in the postoperative state was followed by a smaller rise in free fatty acids (P<0.05) in comparison with the preoperative situation. The postoperative basal fasting LPL activity was half of that in the preoperative state and the LPL activity rose almost two-fold during the clamp. The heparin-induced rises in LPL activity and LPL mass were similar (n.s.) before and after surgery. A moderate surgical trauma is accompanied by a greater than two-fold rise in plasma elimination rate of exogenous fat despite a lower basal LPL activity and a virtually unchanged LPL pattern during infusion of lipids. Our study demonstrates that although trauma may substantially enhance the fat elimination capacity a significant proportion of the infused fat is not utilized for metabolic purposes.

Efficacy of PLD-118, a novel inhibitor of candida isoleucyl-tRNA synthetase, against experimental oropharyngeal and esophageal candidiasis caused by fluconazole-resistant C. albicans.

PLD-118, formerly BAY 10-8888, is a synthetic antifungal derivative of the naturally occurring beta-amino acid cispentacin. We studied the activity of PLD-118 in escalating dosages against experimental oropharyngeal and esophageal candidiasis (OPEC) caused by fluconazole (FLC)-resistant Candida albicans in immunocompromised rabbits. Infection was established by fluconazole-resistant (MIC > 64 microg/ml) clinical isolates from patients with refractory esophageal candidiasis. Antifungal therapy was administered for 7 days. Study groups consisted of untreated controls; animals receiving PLD-118 at 4, 10, 25, or 50 mg/kg of body weight/day via intravenous (i.v.) twice daily (BID) injections; animals receiving FLC at 2 mg/kg/day via i.v. BID injections; and animals receiving desoxycholate amphotericin B (DAMB) i.v. at 0.5 mg/kg/day. PLD-118- and DAMB-treated animals showed a significant dosage-dependent clearance of C. albicans from the tongue, oropharynx, and esophagus in comparison to untreated controls (P </= 0.05, P </= 0.01, P </= 0.001, respectively), while FLC had no significant activity. PLD-118 demonstrated nonlinear plasma pharmacokinetics across the investigated dosage range, as was evident from a dose-dependent increase in plasma clearance and a dose-dependent decrease in the area under the plasma concentration-time curve. The biochemical safety profile was similar to that of FLC. In summary, PLD-118 demonstrated dosage-dependent antifungal activity and nonlinear plasma pharmacokinetics in treatment of experimental FLC-resistant oropharyngeal and esophageal candidiasis.

Pharmacokinetic models for the saturable absorption of cefuroxime axetil and saturable elimination of cefuroxime

Since oligopeptidic drugs such as beta-lactam antibiotics share the same carriers in humans and animals, the absorption and elimination kinetics of cefuroxime (C) were investigated in rats. Plasma C concentrations were measured by liquid chromatography. Pharmacokinetics and bioavailability of C in the rat were examined after intravenous (i.v.) administration at three doses (1.78, 8.9 and 17.8mg) of cefuroxime sodium and oral administration at two doses (2.02 and 8.9mg) of cefuroxime axetil (CA). Preliminary fits using data from intravenous administration of C showed that the drug disposition kinetics were clearly nonlinear, with an increase in plasma clearance as the intravenous dose increased. After oral administration of CA, normalized C(max) was higher for smaller dose than for the largest dose. The population pharmacokinetic parameters were obtained by means of nonlinear mixed effect modelling approach according to a nonlinear elimination and nonlinear absorption two-compartment model. The nonlinear elimination could be attributed to a saturable renal tubular reabsorption of the antibiotic and nonlinear intestinal absorption of CA mediated by carrier system. The oral bioavailability of C, calculated by numeric integration of an amount of CA drug absorbed was 22 and 17% for 2.02 and 8.9mg of prodrug administered orally.

Type 1 diabetic mice are protected from acetaminophen hepatotoxicity.

Streptozotocin (STZ)-induced diabetic (DB) mice challenged with single ordinarily lethal doses of acetaminophen (APAP), carbon tetrachloride (CCl4), or bromobenzene (BB) were resistant to all three hepatotoxicants. Mechanisms of protection against APAP hepatotoxicity were investigated. Plasma alanine aminotransferase, aspartate aminotransferase, and liver histopathology revealed significantly lower hepatic injury in DB mice after APAP administration. HPLC analysis of plasma and urine revealed lower plasma t1/2, increased volume of distribution (Vd), and increased plasma clearance (CLp) of APAP in the DB mice and no difference in APAP-glucuronide, a major metabolite in mice. Interestingly, covalent binding of 14C-labeled APAP to liver target proteins; arylation of APAP to 58, 56, and 44 kDa acetaminophen binding proteins (ABPs); and glutathione (GSH) depletion in the liver did not differ between nondiabetic (non-DB) and DB mice in spite of downregulated hepatic microsomal CYP2E1 and 1A2 proteins in the DB mice, known to be involved in bioactivation of APAP. Compensatory cell division measured via 3H-thymidine pulse labeling and immunohistochemical staining for proliferating cell nuclear antigen (PCNA) indicated earlier onset of S-phase in the DB mice after exposure to APAP. Antimitotic intervention of liver cell division by colchicine (CLC) after administration of APAP led to significantly higher mortality in the DB mice suggesting a pivotal role of liver cell division and tissue repair in the protection afforded by diabetes. In conclusion, the resistance of DB mice against hepatotoxic and lethal effects of APAP appears to be mediated by a combination of enhanced APAP clearance and robust compensatory tissue repair.

Differential regulation of adrenal corticosteroids after restriction-induced drinking in rats.

Water-restricted rats exhibit a rapid decrease in plasma corticosterone after drinking. The present study examined the effect of restriction-induced drinking on plasma aldosterone and plasma clearance of corticosterone. Rats were water restricted for 6-7 days and then killed before or 15 min after water administration; plasma and adrenal hormones were assayed. Plasma and adrenal corticosterone decreased after drinking without a change in plasma corticosteroid-binding globulin; plasma ACTH decreased or did not change. In contrast, plasma aldosterone did not change or increased after drinking; plasma renin activity was elevated by water restriction and increased further after drinking. In another experiment, rats were adrenalectomized, and corticosterone and aldosterone were replaced with pellets and osmotic minipumps, respectively. Rats were water restricted and killed. There was a small decrease in plasma corticosterone but no change in aldosterone after drinking in adrenalectomized animals. These data suggest that changes in plasma steroids after restriction-induced drinking result from zone-specific responses of the adrenal to known secretagogues, with minimal contribution from increased plasma clearance.

Hepatic Endosomal Trafficking of Lipoprotein-Bound Endotoxin in Rats

Triglyceride-rich lipoproteins (chylomicrons (CM), VLDL) can bind and protect against endotoxin (LPS)-induced shock and mortality in rodents. The protective effect of lipoproteins is in part due to the increased plasma clearance and biliary excretion of LPS. Specifically, CM–LPS complexes are principally removed from the circulation by the liver with a rapid plasma half-life approximating that for CM alone. Thus, we hypothesized that hepatocytes clear CM-bound LPS via known lipoprotein receptors and traffic the toxic macromolecule through the same endosomal pathway employed for the catabolism of triglyceride-rich lipoproteins. To examine the endosomal uptake and biliary excretion of LPS, we isolated early and late hepatic endosomal fractions and hepatic bile from rats following the injection of radiolabeled CM-bound LPS. The uptake of 125I-LPS was compared in animals that overexpressed either the LDL receptor or the LDL receptor-related protein (LRP) versus untreated control with normal lipoprotein levels. Herein we present data indicating that both the LDL receptor and the LRP participate in the rapid internalization of CM-bound LPS by hepatocytes. Upregulation of the LDL receptor increased the accumulation of 125I-LPS in late endosomes (P < 0.03). In contrast, increased levels of the LRP were associated with negligible movement of LPS into late endosomes but a trend toward the increased biliary excretion of the radiolabeled macromolecule. Taken together these data further elucidate the role of the liver in the host innate immune response to infection and potentially implicate distinct roles for the LDL receptor and LRP in the catabolism of CM-bound LPS.

Endothelin-1: increased plasma clearance, pulmonary affinity and renal vasoconstriction in young smokers.

Pulmonary and renal haemodynamics and elimination of endothelin-1 (ET-1) were studied in six young smokers in response to 20 min intravenous infusion of ET-1 (4 pmol kg(-1) min(-1)) after smoking. At 20 min of ET-1 infusion fractional ET-1 extractions in the lungs and kidneys were 60 +/- 2 and 60 +/- 7%, respectively. Cardiac output and renal blood flow (RBF) fell by 18 +/- 4% (P<0.05) and 34 +/- 5% (P<0.01). Mean systemic arterial pressure increased (P<0.05) whereas pulmonary pressures were unchanged. Compared with previously published data in non-smokers (Weitzberg et al., 1991, 1993) basal arterial ET-1 and ET-1-values during ET-1 infusion were lower with a more rapid return to basal value. Smokers had higher pulmonary extraction of ET-1 at the same pulmonary arterial concentration (P<0.05). RBF reduction was more pronounced (P<0.05). Systemic vascular resistance increased while pulmonary vascular resistance did not increase as in non-smokers. Increased plasma clearance and more efficient pulmonary elimination of ET-1 lowers the arterial level in young smokers. In addition ET-1 evokes more pronounced renal vasoconstriction in these individuals.

Cryopreservation enables long-term storage of 9-(2-phosphonylmethoxyethyl)adenine prodrug-loaded reconstituted lactosylated high-density lipoprotein.

Chronic hepatitis B is caused by an infection of parenchymal liver cells with hepatitis B virus (HBV). The nucleoside analog 9-(2-phosphonylmethoxyethyl)adenine (PMEA) is a promising candidate drug for the treatment of HBV infections. PMEA effectively inhibits the replication HBV in cell culture (1,2), and the orally available bis(POM) derivative has entered clinical trials (3). Although initial results are promising, the disposition of PMEA remains a matter of concern. Most of the drug is cleared by the kidneys, whereas only a small fraction taken up by parenchymal liver cells (4). To enhance the effectiveness of PMEA against HBV, we developed a carrier-based strategy for selective delivery of PMEA to parenchymal liver cells. PMEA was conjugated to lithocholic acid-3a-oleate (LO), yielding the lipophilic prodrug PMEA-LO (5). The prodrug was incorporated into reconstituted lactosylated high-density lipoprotein (LacNeoHDL), a lipidic carrier that is selectively taken up by parenchymal liver cells via the asialoglycoprotein receptor (6,7). LacNeoHDLassociated PMEA-LO was delivered rapidly to parenchymal liver cells (8). Once internalized, PMEA was released from the PMEA-LO prodrug and phosphorylated to its active metabolite (8). The selective delivery of PMEA to parenchymal liver cells is expected to result in an enhanced therapeutic efficacy against HBV. An important prerequisite for clinical application is that PMEA-LO-loaded LacNeoHDL remains stable during storage and transport. An attractive option for storage of PMEALO-loaded LacNeoHDL is cryopreservation. Freezing may, however, seriously damage the particles. Studies on cryopreservation of lipoproteins focus on low-density lipoprotein (LDL). Freezing and thawing of LDL results in aggregation of the particles (9,10). The aggregation leads to non-specific binding of LDL to cultured human fibroblasts (9), and to increased plasma clearance and enhanced spleen uptake, suggesting phagocytosis by cells of the reticulo-endothelial system (11). However, cryoprotectants like sucrose protect LDL from freezing-induced damage (9,12). In this study we investigated the effect of cryopreservation of PMEA-LO-loaded LacNeoHDL on the stability of the PMEA-LO prodrug and on the physical and biological properties of the prodrug-loaded particles. As sucrose is beneficial for LDL during freezing, we examined the ability of sucrose to stabilize the prodrug-loaded carrier during cryopreservation

Drug Targeting Efficacy to Underlying Muscle Following Topical Application. I. Evaluation Based on a Physiological Pharmacokinetic Model.

A physiological pharmacokinetic model describing the absorption and disposition of topically applied drugs was proposed, and the effect of various pharmacokinetic and physiological parameters on the drug delivery into the targeted muscle was simulated. The proposed model consists of vehicle, and stratum corneum, viable epidermis and muscle below the application and reference sites, and plasma, each joined with transfer clearance and plasma flow. Indomethacin concentrations in tissues and plasma after topical application to rats could be explained by the model. Most indomethacin delivered into the underlying muscle was via direct penetration. The model simulation showed that the increase in plasma clearance and clearance between viable skin and muscle, and the decrease in application area and plasma flow rate into viable skin and muscle would promote the targeting efficacy of topically applied drugs to the underlying muscle.

Increased Clearance Explains Lower Plasma Levels of Tissue-Type Plasminogen Activator by Estradiol: Evidence for Potently Enhanced Mannose Receptor Expression in Mice

Several clinical studies have demonstrated an inverse relationship between circulating levels of estrogen and tissue-type plasminogen activator (t-PA). The present study was designed to test the hypothesis that estrogens lower plasma levels of t-PA by increasing its clearance from the bloodstream. 17-Ethinyl estradiol (EE) treatment resulted in a significant increase in the clearance rate of recombinant human t-PA in mice (0.46 mL/min in treated mice v 0.32 mL/min in controls; P &lt; .01). The clearance of endogenous, bradykinin-released t-PA in rats was also significantly increased after EE treatment (area under the curve [AUC], 24.9 ng/mL · min in treated animals v 31.9 ng/mL · min in controls; P &lt; .05). Two distinct t-PA clearance systems exist in vivo: the low-density lipoprotein receptor-related protein (LRP) on liver parenchymal cells and the mannose receptor on mainly liver endothelial cells. Inhibition of LRP by intravenous injection of receptor-associated protein (RAP) as a recombinant fusion protein with Salmonella japonicum glutathione S-transferase (GST) significantly retarded t-PA clearance in control mice (from 0.41 to 0.25 mL/min; n = 5, P &lt; .001) and EE-treated mice (from 0.66 to 0.35 mL/min; n = 5, P &lt; .005), but did not eliminate the difference in clearance capacity between the 2 experimental groups. Similar results were obtained in mice in which LRP was inhibited via overexpression of the RAP gene in liver by adenoviral gene transduction. In contrast, administration of mannan, a mannose receptor antagonist, resulted in identical clearances (0.22 mL/min in controls and 0.24 mL/min in EE-treated mice). Northern blot analysis showed a 6-fold increase in mannose receptor mRNA expression in the nonparenchymal liver cells of EE-treated mice, whereas the parenchymal LRP mRNA levels remained unchanged. These findings were confirmed at the protein level by ligand blotting and Western blotting analysis. Our results demonstrate that EE treatment results in increased plasma clearance rate of t-PA via induction of the mannose receptor and could explain for the inverse relationship between estrogen status and plasma t-PA concentrations as observed in humans.