Verica Risovic

Effects of Lipid-Based Oral Formulations on Plasma and Tissue Amphotericin B Concentrations and Renal Toxicity in Male Rats

ABSTRACT The purpose of this study was to determine the effects of various lipid and mixed-micelle formulations on the oral absorption and renal toxicity of amphotericin B (AMB) in rats. The maximum concentration of AMB in plasma and the area under the concentration-time curve for 0 to 24 h for AMB were elevated in rats administered triglyceride (TG)-rich AMB formulations in comparison to those in rats given (i) AMB preformulated as a micelle containing sodium deoxycholate with sodium phosphate as a buffer (DOC-AMB), (ii) an AMB-lipid complex suspension, or (iii) AMB solubilized in methanol. Furthermore, our findings suggest that AMB incorporated into TG-based oral formulations has less renal toxicity than DOC-AMB.

Potential Mechanisms by Which Peceol® Increases the Gastrointestinal Absorption of Amphotericin B

Purpose, The purpose of this study was to ascertain how the incorporation of AmpB into a glyceride‐rich excipient Peceol® significantly increased Amphotericin Bs (AmpB) gastrointestinal absorption in white male Sprague‐Dawley rats. Based on preliminary studies, our working hypothesis was that incorporation of AmpB into mixed micelles composed of Peceol® would significantly enhance gastro‐intestinal (GI) tract absorption by increasing lymphatic drug transport and decreasing P‐glycoprotein (PGP) mediated drug efflux. Methods. I. Lymphatic Transport Studies: Following an overnight fast (12–16 hr) and 48 hr postsurgery, rats were divided into two treatment groups and received a single‐dose oral gavage (1 mL total volume) at 0700 h of either desoxycholates (DOC)–AmpB (5 mg AmpB/kg; n = 6 at each time point) or AmpB incorporated into 100% Peceol (Peceol®–AmpB; 5 mg AmpB/kg; n = 6 at each time point). Mesenteric lymph samples were obtained prior to and at 0–4‐hr, 4–6‐hr, and 6–8‐hr intervals post oral gavage. An equal volume of normal saline (1 mL) was administered intravenously to the animal following each blood draw to prevent fluid depletion throughout the duration of the study. Lymph was immediately harvested by centrifugation and analyzed for drug by high‐performance liquid chromatography (HPLC). II. Multidrug Resistance 1 (mdr‐1) Studies: Caco‐2 cells were seeded at 10,000 cells/cm2 in T‐75 flasks. When the cells reached 80% confluency, they were treated for 1 day and 7 days with 0.1% to 1.0% (v/v) Peceol® or media alone (control). Following treatment, total RNA was is olated using TRIzol® reagent, followed by reverse transcription into single‐stranded cDNA. Polymerase chain reactions (PCR) were performed with specific primers for mdr‐1. The PGP protein was determined by Western Blot Analysis. Results: Mean weight of rats was not significantly different prior to and following drug administration. Similarly, kidney, liver, lung, spleen, and heart weights were not different between DOC–AmpB and Peceol®–AmpB treatment group. A significantly greater amount of AmpB was transported through the mesenteric lymph duct for all the time intervals used following the administration of Peceol®‐AmpB treatment group compared to the administration of DOC–AmpB (suspension). A significant lower mdr‐1 mRNA and PGP protein expression within Caco‐2 cells was observed following 1 and 7 days treatment with Peceol® 0.1% to 1.0% (v/v) compared to nontreated controls. Conclusions, Taken together, these findings suggest that Peceol® increases the gastrointestinal absorption of AmpB by increasing the amount of drug that is transported through the mesenteric lymph duct and by decreasing mdr‐1 mRNA and PGP protein expression, resulting in lower PGP‐mediated AmpB efflux.

Assessing the Antifungal Activity of a New Oral Lipid-Based Amphotericin B Formulation Following Administration to Rats Infected with Aspergillus Fumigatus

ABSTRACT The purpose of this study was to assess the antifungal activity of a new oral amphotericin B (AmpB) lipid-based formulation following administration to rats infected with Aspergillus fumigatus. Aspergillus fumigatus inoculum (2.1–2.5 × 107 colony forming units [CFU]) were injected via the jugular vein; 48h later male albino Sprague-Dawley rats (350–400 g) were administered either a single oral dose of AmpB incorporated into Peceol (50 mg AmpB/kg), physiologic saline (nontreated controls) or Peceol alone (vehicle control) once daily for 4 days. To assess antifungal activity Brain, Lung, Heart, Liver, Spleen and Kidney sections were homogenized with normal saline (1 mL/g of tissue) and a 0.1-mL aliquot was spread plated onto a Sabourand dextrose agar plate. The plates were incubated for 48 hr at 37°C, at which time the number of fungal CFU were determined and corrected for tissue weight. In addition, plasma galactomannan antigen concentrations were determined. Data was reported as mean ± standard error of the mean. The AmpB-Peceol oral formulation significantly decreased total fungal CFU concentrations recovered in all the organs added together, brain CFU concentrations, spleen CFU concentrations and plasma galactomannan antigen concentrations compared to baseline. No significant differences in lung, heart, liver and kidney CFU concentrations between treatment and control groups were observed. Peceol vehicle control did not exhibit any antifungal activity. These findings suggest that a new oral lipid-based formulation of AmpB incorporated into Peceol can significantly decrease brain and spleen CFU concentrations and plasma galactomannan antigen concentrations compared to non-treated controls.

Assessing lipid lowering and plasma cholesteryl ester transfer protein activity of simvastatin following administration to rabbits fed a high fat/cholesterol diet.

ABSTRACT Purpose: The purpose of this study was to assess the lipid lowering and plasma cholesteryl ester transfer protein (CETP) activity following administration of simvastatin to rabbits fed a high fat/cholesterol diet. Methods: Male New Zealand white rabbits were housed in individual cages and fed a standard diet for 7 days. After 7 days, animals were fed 10 g of a regular chow diet plus 100 g of the same diet supplemented with 0.5% (w/v) cholesterol and 14.0% (w/v) coconut oil for 28 days. Following 28 days on this diet, the animals were randomized based on plasma cholesterol and triglyceride levels, into a group of control animals and a group (n = 6) of animals fed 100 g of cholesterol/coconut diet plus 10 g regular chow diet containing simvastatin (3 mg/kg/day) for an additional 28 days. Blood samples were taken from the marginal ear vein prior to and 28 days after the initiation of drug treatment. Plasma was harvested and stored at 4°C prior to lipid analysis. Plasma total cholesterol and triglyceride levels were quantified using enzymatic kits. HDL (high-density lipoproteins) cholesterol levels were determined using the dextran sulfate-Mg2+ precipitation method. ApoB cholesterol levels were determined by subtracting total cholesterol from HDL cholesterol. Cholesteryl ester transfer protein (CETP) activity was determined by standard assay methods. Results: We observed that simvastatin significantly reduced total plasma cholesterol, triglyceride, and apoB cholesterol compared to non-treated controls. Simvastatin treatment did not alter serum CETP activity compared to non-treated controls. Conclusions: These findings suggest that decreasing plasma lipid levels by treatment with simvastatin is not due to changes in serum CETP activity in rabbits fed a high fat/cholesterol diet.