Selective Effect of Adjuvant Arthritis on the Disposition of Propranolol Enantiomers in Rats Detected Using a Stereospecific HPLC Assay

Nonstereospecific studies have indicated that the pharmacokinetics of propranolol (PR) are altered in inflammatory conditions such as arthritis. However, as the kinetics and dynamics of PR are stereoselective, we examined the effect of adjuvant arthritis (AA) on the disposition of the individual enantiomers. A novel normal-phase stereospecific HPLC assay for PR was developed involving chiral derivatization with S-(naphthyl)ethyl isocyanate and fluorescence detection. Oral and iv doses of racemic PR were administered to control and AA rats (n = 6). AA had no significant effect on either clearance or S:R ratio after iv doses. On the other hand, after oral doses, clearance was significantly decreased in AA. Although significant for both enantiomers, this effect was more pronounced on the less active R-enantiomer. The AUC R:S ratio was, therefore, significantly altered (AA, 14 ± 3.0; control, 4.3 ± 1.2). Increased total (S + R) plasma concentrations of PR in AA, possibly due to a reduced intrinsic clearance, therefore, reflect mainly increased concentrations of the less active R-enantiomer.     

Efficacy assessment of sustained intraperitoneal paclitaxel therapy in a murine model of ovarian cancer using bioluminescent imaging

We evaluated the pre-clinical efficacy of a novel intraperitoneal (i.p.) sustained-release paclitaxel formulation (PTX_ePC) using bioluminescent imaging (BLI) in the treatment of ovarian cancer. Human ovarian carcinoma cells stably expressing the firefly luciferase gene (SKOV3^Luc) were injected i.p. into SCID mice. Tumour growth was evaluated during sustained or intermittent courses of i.p. treatment with paclitaxel (PTX). In vitro bioluminescence strongly correlated with cell survival and cytotoxicity. Bioluminescent imaging detected tumours before their macroscopic appearance and strongly correlated with tumour weight and survival. As compared with intermittent therapy with Taxol®, sustained PTX_ePC therapy resulted in significant reduction of tumour proliferation, weight and BLI signal intensity, enhanced apoptosis and increased survival times. Our results demonstrate that BLI is a useful tool in the pre-clinical evaluation of therapeutic interventions for ovarian cancer. Moreover, these results provide evidence of enhanced therapeutic efficacy with the sustained PTX_ePC implant system, which could potentially translate into successful clinical outcomes.     

The involvement of the pregnane X receptor in hepatic gene regulation during inflammation in mice

Inflammation and proinflammatory cytokines suppress the expression of several hepatic transporters and metabolic enzymes, often resulting in cholestatic liver disease. However, mechanism(s) of this down-regulation have not been fully elucidated. As the pregnane X receptor (PXR) is involved in inducing many of these hepatic proteins, it is possible that PXR is also involved in their down-regulation during inflammation. Thus, we compared the effect of inflammation on hepatic gene regulation in wild-type (PXR(+/+)) versus PXR-null (PXR(-/-)) mice. Treatment of PXR(+/+) but not PXR(-/-) mice with the PXR activators 5-pregnen-3beta-ol-20-one-16alpha-carbonitrile (PCN) or 17beta-hydroxy-11beta-[4-dimethylamino phenyl]-17alpha-[1-propynyl] estra-4,9-dien-3-one (RU486) resulted in increased mRNA levels of bsep, mdr1a, mrp2, mrp3, oatp2, and cyp3a11, indicating involvement of PXR in their regulation. Significantly lower mRNA levels of bsep, mdr2, mrp2, mrp3, ntcp, oatp2, and cyp3a11 were found in endotoxin-treated PXR(+/+) mice. In endotoxin-treated PXR(-/-) mice, the extent of mrp2 suppression was significantly diminished. Changes in MRP2 expression were supported by Western blot analysis. Although interleukin (IL)-6 imposed significant decreases in the expression of bsep, mrp2, and cyp3a11 in PXR(+/+) mice, this was not observed in PXR(-/-) mice. Of note, significantly lower levels of PXR mRNA and protein were detected in endotoxin- and IL-6-treated PXR(+/+) mice. In addition, endotoxin and IL-6 were also able to suppress PCN-mediated induction of bsep, mrp2, cyp3a11, and PXR. Taken together, our results suggest that PXR plays a role in the down-regulation of several hepatic proteins during inflammation.     

Pharmacokinetics of acebutolol enantiomers in humans.

The chiral beta-blocker acebutolol (AC) is marketed as a racemic mixture. Both AC and its major metabolite, diacetotol (DC), are chiral, the S-enantiomer possessing beta-blocking activity. The pharmacokinetics of AC and DC enantiomers was determined in 12 healthy subjects following oral administration of 200 mg of AC. Plasma and urine were collected over a 24-hr period and both AC and DC enantiomers were measured utilizing a stereospecific HPLC assay. Concentrations of S-AC were predominant in both plasma and urine [AUC S:R, 1.20 +/- 0.1; cumulative urinary excretion (sigma Xu) S:R, 1.17 +/- 0.05), which corresponded to a significantly greater oral clearance of R-AC (106 +/- 30 L/h) than S-AC (87 +/- 22 L/h). The Cmax of R-DC was significantly greater than for S-DC (S/R 0.7 +/- 0.1). The half-life (t1/2) of R-DC (6.4 +/- 1.6 h) was significantly shorter than that of S-DC (8.8 +/- 2.4 h). The observed AUC values for R- and S-DC were not significantly different. Renal clearance of R-DC (70 +/- 34 mL/min) was significantly greater than that of S-DC (53 +/- 29 mL/min). The data suggest that the first-pass metabolism of R-AC to R-DC is stereoselective. This metabolism, coupled with the stereoselective renal excretion of R-DC is likely a major contributor to the observed stereoselective disposition of AC and its major metabolite in humans.     

Influence of molecular organization and interactions on drug release for an injectable polymer-lipid blend

An injectable blend composed of a water soluble chitosan (WSC) derivative, egg phosphatidylcholine (ePC), and fatty acid chlorides (FACl) was explored for localized delivery of anticancer agents. The composition–property relationships of the injectable WSC–FACl–ePC blend were determined by investigating the physico-chemical and performance properties of the blend as a function of the ratio of the components, as well as the acyl chain length of the FACl (C10–C16) employed. Thermal and rheological measurements revealed that the melting transitions and viscosities of the blends increased as a function of FACl acyl chain length. FTIR analysis demonstrated that the stability of the blends was attributed to the specific interactions among the molecules. In addition, confocal laser scanning microscopy revealed that the incorporation of C10–C16 FACl altered the molecular organization of ePC and WSC within the blends, which resulted in distinct physico-chemical properties. Specifically, the formation of micro-domains within the blends increased the stability, as well as delayed the release of paclitaxel from the formulation under physiologically relevant conditions. Overall, the interactions identified among the components, and the relationships established between the composition and properties of the blend can be used as a tool to develop advanced injectable drug delivery systems for pharmaceutical applications.     

Characterization of Guanidine Transport in Human Renal Brush Border Membranes

Purpose. Organic cation transporters in the renal proximal tubule are important in the secretion of many clinically used drugs and their metabolites. The goal of this study was to determine the mechanisms of guanidine transport in human kidney. Methods. Brush-border membrane vesicles were prepared from donor human kidneys deemed unsuitable for renal transplantation. Results. Uptake of [¹⁴C]-guanidine (50 M) in the vesicles, as determined by rapid filtration, was significantly greater in the presence of an outwardly-directed proton gradient, at all early time points, than in the absence of the gradient. Proton-stimulated uptake of [¹⁴C]-guanidine at 30 sec (32.0 ± 1.24 pmol/mg protein) was significantly inhibited by a number of organic cations including 5 mM unlabeled guanidine (14.8 ± 1.84 pmol/mg protein) and 5 mM MIBA (9.14 ± 3.80 pmol/ mg protein), but not by 5 mM TEA (28.4 ± 5.67 pmol/mg protein). Guanidine, but not TEA, trans-stimulated [¹⁴C]-guanidine uptake. Conversely, TEA, but not guanidine, trans-stimulated [¹⁴C]-TEA uptake in the vesicles. The proton-dependent transport of guanidine was characterized by a K_m of 3.52 ± 0.42 mM (SE) and a V_max of 34.6 ± 8.64 pmol/mg protein/sec (SE). Conclusions. These results demonstrate that guanidine transport in human renal brush border membrane vesicles is stimulated by a proton gradient. Evidence was obtained suggesting that the transporter for guanidine is distinct from the previously described organic cation proton antiporter for TEA.     

Effect of adjuvant arthritis on the disposition of acebutolol enantiomers in rats

Disease states such as arthritis may interact with the kinetics of -blockers. Acebutolol (AC) is a chiral -blocker which is available as a racemate. The beneficial properties of AC, however, is attributed mainly to theS-(+)-enantiomer. The disposition of AC enantiomers and their active, chiral metabolites, diacetolol (DC) were examined after oral administration to healthy and adjuvant-induced arthritic (AA) female Sprague-Dawley rats. Arthritis was induced by tail base injection ofMycobacterium butyricum. Swelling of hind and forepaws were apparent in 10–16 days in AA but not controls. Control and AA rats were sacrificed at 0.5, 1, 2, 4, 6 and 8 h after a 25 mg/kg oral AC dose and blood was collected (n=6). Significant three to tenfold increases in the initial plasma concentrations (0.5–2h) of AC were observed in AA. Enantiomers were equally affected, thus ACSR ratio was not changed. Higher plasma concentrations of the metabolite were only significant at 2h. The ratio of DCAC, however, was unaffected by AA. The DCSR ratio was significantly decreased at 0.5 and 1 h in AA. The limited protein binding of AC (10%) was neither stereoselective nor affected by AA. Reduced intrinsic clearance in AA may be responsible for these observations.