Manisha Ramaswamy

Differences in Lipoprotein Lipid Concentration and Composition Modify the Plasma Distribution of Cyclosporine

AbstractPurpose. The purpose of this study was to define the relationship between lipoprotein (LP) lipid concentration and composition and the distribution of cyclosporine (CSA) in human plasma. Methods. 3H-CSA LP distribution was determined in normolipidemic human plasma that had been separated into different LP and lipoprotein-deficient plasma (LPDP) fractions by either affinity chromatography coupled with ultracentrifugation, density gradient ultracentrifugation or fast protein liquid chromatography. 3H-CSA LP distribution (at a concentration of 1000 ng/ml) was also determined in patient plasma samples with defined dyslipidemias. Furthermore, 3H-CSA LP distribution was determined in patient plasma samples of varying LP lipid concentrations. Following incubation, the plasma samples were separated into their LP and LPDP fractions by sequential phosphotungistic acid precipitation in the dyslipidemia studies and by density gradient ultracentrifugation in the specific lipid profile studies and assayed for CSA by radioactivity. Total plasma and lipoprotein cholesterol (TC), triglyceride (TG) and protein (TP) concentrations in each sample were determined by enzymatic assays. Results. When the LP distribution of CSA was determined using three different LP separation techniques, the percent of CSA recovered in the LP-rich fraction was greater than 90% and the LP binding profiles were similar with most of the drug bound to plasma high-density (HDL) and low-density (LDL) lipoproteins. When 3H-CSA was incubated in dyslipidemic human plasma or specific patient plasma of varying LP lipid concentrations the following relationships were observed. As the very low-density (VLDL) and LDL cholesterol and triglyceride concentrations increased, the percent of CSA recovered within the VLDL and LDL fractions increased. The percent of CSA recovered within the HDL fraction significantly decreased as HDL triglyceride concentrations increased. The percent of CSA recovered in the LPDP fraction remained constant except in hypercholesterolemic/hypertriglyceridemic plasma where the percent of CSA recovered decreased. Furthermore, increases in VLDL and HDL TG/TC ratio resulted in a greater percentage of CSA recovered in VLDL but less in HDL. Conclusions. These findings suggest that changes in the total and plasma LP lipid concentration and composition influence the LP binding of CSA and may explain differences in the pharmacological activity and toxicity of CSA when administered to patients with different lipid profiles.

Amphotericin B Lipid Complex or Amphotericin B Multiple-Dose Administration to Rabbits with Elevated Plasma Cholesterol Levels: Pharmacokinetics in Plasma and Blood, Plasma Lipoprotein Levels, Distribution in Tissues, and Renal Toxicities

ABSTRACT The purpose of the present study was to determine if a relationship exists between the plasma cholesterol concentration, the severity of amphotericin B (AmpB)-induced renal toxicity, and the pharmacokinetics of AmpB in plasma in hypercholesterolemic rabbits administered multiple doses of amphotericin B (AmB) deoxycholate (Doc-AmB) and AmB lipid complex (ABLC). After 7 days of administration of a cholesterol-enriched diet (0.50% [wt/vol]) or a regular rabbit diet, each rabbit was administered a single intravenous bolus of Doc-AmB (n = 8) or ABLC (n = 10) (1.0 mg/kg of body weight) daily for 7 consecutive days (a total of eight doses). Blood samples were obtained daily before and 24 h after the administration of each dose and serially thereafter following the administration of the last dose for the assessment of pharmacokinetics in plasma, kidney toxicity, plasma lipoprotein levels, and drug distribution in tissue. The pharmacokinetics of AmB in blood following the administration of ABLC were also determined in rabbits fed cholesterol-enriched and regular diets (n = 3 each group). Before drug treatment, cholesterol-fed rabbits demonstrated marked increases in total, low-density lipoprotein (LDL), and triglyceride-rich lipoprotein (TRL) cholesterol levels in plasma compared with the levels in rabbits on a regular diet. No significant differences in total plasma triglyceride levels were observed. Significant increases in plasma creatinine levels were observed in rabbits fed a cholesterol-enriched diet (P < 0.05) and rabbits fed a regular diet (P < 0.05) when administered AmB. However, the magnitude of this increase was twofold greater in rabbits fed a regular diet than in rabbits fed a cholesterol-enriched diet. An increase in plasma creatinine levels was observed only in rabbits on a cholesterol-enriched diet administered ABLC. The pharmacokinetics of AmB were significantly altered in rabbits on a cholesterol-enriched diet administered Doc-AmB or ABLC compared to those in rabbits on a regular diet administered each of these compounds. The pharmacokinetics of AmB in blood were significantly different following ABLC administration but not following Doc-AmB administration in both rabbits fed cholesterol-enriched diets and rabbits fed regular diets compared to their corresponding pharmacokinetics in plasma. An increased percentage of AmB was recovered in the TRL fraction when Doc-AmB was administered to rabbits fed a cholesterol-enriched diet than when it was administered to rabbits fed a regular diet. Furthermore, an increased percentage of AmB was recovered in the LDL and TRL fractions when ABLC was administered to rabbits fed a cholesterol-enriched diet rabbits fed a regular diet. These findings suggest that an increase in plasma cholesterol levels modifies the pharmacokinetics of AmB and renal toxicity following the administration of multiple intravenous doses of Doc-AmB and ABLC.

The stereoselective distribution of halofantrine enantiomers within human, dog, and rat plasma lipoproteins.

Purpose. To study the in vitro distribution of the enantiomers of theantimalarial drug halofantrine in human, dog and rat plasmalipoprotein-fractions.Methods. Plasma was spiked with racemic halofantrine (1000 ng/ml)and incubated for 1 h at 37°C. The fractions (high and low densitylipoproteins, triglyceride-rich lipoproteins and lipoprotein deficientplasma) were separated using density gradient ultracentrifugation.Fractions were assayed for halofantrine enantiomer using stereospecifichigh performance liquid chromatography.Results. The (−) enantiomer of halofantrine displayed higher affinityfor the lipoprotein-deficient fraction than the (+) enantiomer in allthree species. The (+) enantiomer was predominately located in thelipoprotein rich fractions of dog and human plasma (the (+):(−) ratioranging from 1.2–9.6). In contrast, the (+):(−) ratio was consistently<1 in lipoprotein-deficient fractions. Dog displayed a large magnitudeof stereoselectivity in halofantrine distribution to the plasma fractionstested. There were substantial interspecies differences in the pattern ofdistribution of halofantrine enantiomers within the different fractions. Asignificant positive relationship was observed between halofantrineuptake into lipoprotein-rich fractions and the percent of apolar corelipid in those fractions. There was also a strong negative correlationbetween total protein concentration and the enantiomeric ratio in thelipoprotein-deficient plasma fraction.Conclusion. Distribution of halofantrine enantiomer to plasma lipoprotein-fractions is stereoselective and species specific. This differentialbinding of halofantrine enantiomers to lipoproteins may need to beconsidered in viewing pharmacokinetic and pharmacodynamic datainvolving the drug.

Heat Treatment of Amphotericin B Modifies Its Serum Pharmacokinetics, Tissue Distribution, and Renal Toxicity following Administration of a Single Intravenous Dose to Rabbits

ABSTRACT The purpose of this investigation was to determine the serum pharmacokinetics, tissue distribution, and renal toxicity of amphotericin B (AmpB) following administration of a single intravenous dose (1 mg/kg of body weight) of Fungizone (FZ) and a heat-treated form of FZ (HFZ) to New Zealand White female rabbits. FZ solutions were heated at 70°C for 20 min to produce HFZ. Blood samples were obtained before drug administration and serially thereafter. After collection of the 48-h blood sample, each rabbit was humanely sacrificed and the right kidney, spleen, lungs, liver, and heart were harvested for AmpB analysis. Serum creatinine levels were measured before and 10 h after drug administration. AmpB concentrations in the serum and tissues were analyzed using high-performance liquid chromatography. FZ administration to rabbits resulted in a greater-than-50% increase in serum creatinine concentrations compared to baseline. However, HFZ administration resulted in no difference in serum creatinine concentrations compared to baseline. The AmpB area under the concentration-time curve (AUC) after HFZ administration was significantly lower than the AmpB AUC in rabbits administered FZ. However, AmpB systemic total body clearance was significantly greater in rabbits administered HFZ than in rabbits administered FZ without any differences in volume of distribution at steady state. Kidney tissue AmpB concentrations, although not significantly different, were greater in rabbits administered FZ than in rabbits administered HFZ. Likewise, lung and spleen AmpB concentrations, although not significantly different, were greater in rabbits administered FZ than in rabbits administered HFZ. However, liver AmpB concentrations were significantly lower in rabbits administered FZ than in rabbits administered HFZ. No significant differences in heart AmpB concentration between rabbits administered FZ and those given HFZ were found. These findings suggest that the pharmacokinetics, tissue distribution, and renal toxicity of AmpB are modified following administration of HFZ. HFZ could be an improved low-cost AmpB drug delivery system that has a potentially higher therapeutic index than FZ.

Role of plasma lipoproteins in modifying the toxic effects of water-insoluble drugs: Studies with cyclosporine A

Lipoproteins are a heterogeneous population of macromolecular aggregates of lipids and proteins that are responsible for the transport of lipids through the vascular and extravascular fluids from their site of synthesis or absorption to peripheral tissues. Lipoproteins are involved in other biological processes as well, including coagulation and tissue repair, and serve as carriers of a number of hydrophobic compounds within the systemic circulation. It has been well documented that disease states (eg, AIDS, diabetes, cancer) significantly influence circulating lipoprotein content and composition. Therefore, it appears possible that changes in the lipoprotein profile would affect not only the ability of a compound to associate with lipoproteins but also the distribution of the compound within the lipoprotein subclasses. Such an effect could alter the pharmacokinetics and pharmacological action of the drug. This paper reviews the factors that influence the interaction of one model hydrophobic compound, cyclosporine A, with lipoproteins and the implications of altered plasma lipoprotein concentrations on the pharmacological behavior of this compound.

Cyclosporine Transfer from Low- and High-Density Lipoproteins Is Partially Influenced by Lipid Transfer Protein I Triglyceride Transfer Activity

AbstractPurpose. The purpose of this study was to determine if lipid transfer protein (LTP I) facilitated triglyceride (TG) transfer activity regulates the plasma lipoprotein distribution of cyclosporine (CSA). Methods. To assess the influence of drug concentration and incubation time on the plasma lipoprotein distribution of CSA, 3H-CSA (50 to 1000 ng/ml) was incubated in human plasma for 5 to 120 minutes at 37°C. To determine if LTP I facilitated TG transfer activity regulates the plasma lipoprotein distribution of CSA, 3H-Triolein (TG)- or 3H-CSA-enriched high-density lipoproteins (HDL) or low-density lipoproteins (LDL) were incubated in T150 buffer (50 mM Tris-HCl, 150 mM NaCl, 0.02% Sodium Azide, 0.01% Disodium EDTA), pH 7.4 which contained a 3H-Triolein (TG) or 3H-CSA-free lipoprotein counterpart ± exogenous LTP I (1.0 μg protein/ml) or in delipidated human plasma which contained 1.0 μg protein/ml of endogenous LTP I for 90 minutes at 37°C. These experiments were repeated in the presence of a monoclonal antibody TP1 (15 μg protein/ml) directed against LTP I. Results. No differences in CSA lipoprotein distribution were observed following incubation of the drug at varying concentrations and incubation times in human plasma. The percent transfer of TG from HDL to LDL and LDL to HDL was greater in T150 buffer than in human plasma. However, the percent transfer of CSA from only LDL to HDL was greater in T150 buffer than in human plasma. Furthermore, undetectable 3H-CSA transfer from HDL to LDL in T150 buffer containing purified LTP I was observed. In addition, when the percent transfer of TG and CSA were determined in the presence of TP1, the percent transfer of TG and CSA from only LDL to HDL were significantly decreased in T150 buffer and human plasma compared to controls. Conclusions. These findings suggest that the transfer of CSA between different lipoprotein particles is only partially influenced by LTP I facilitated TG transfer activity.

Rat and Rabbit Plasma Distribution of Free and Chylomicron-Associated BIRT 377, a Novel Small Molecule Antagonist of LFA-1-Mediated Cell Adhesion

AbstractPurpose. The objectives of this study are to determine the plasma distribution of free and chylomicron-associated BIRT 377 within rats and rabbits. Methods. For the rat studies free and chylomicron-associated BIRT 377 was incubated in plasma from CD 1 non-fasted rats for 60 minutes at 37°C. Following incubation the plasma was separated into its lipoprotein and lipoprotein-deficient plasma (LPDP) fractions by three different methods and analyzed for BIRT 377 content by HPLC. For the rabbit studies New Zealand fasted white rabbits (3 kg; n = 4) were administered an intravenous dose of free BIRT 377 (1 mg/kg). Following administration, serial blood samples were obtained and the plasma was analyzed for BIRT 377. The plasma collected at the 0.083-h time point was separated into each of its lipoprotein fractions and analyzed for BIRT 377. Results. 37.8 ± 1.2% of the original drug amount incubated in rat plasma was recovered within the lipoprotein-rich fraction. 41.5 ± 0.4% of the original chylomicron-associated drug concentration incubated was recovered within the lipoprotein-rich fraction. The percentage of drug recovered within the TRL fraction was significantly greater following the incubation of chylomicron-associated BIRT 377 compared to free BIRT 377. In addition, BIRT 377 apparently follows a two-compartment pharmacokinetic model following single intravenous dose administration to rabbits. Conclusions. These findings suggest that plasma lipoprotein binding of BIRT 377 is evident and may be a factor in evaluating the pharmacological fate of this drug when administered to patients that exhibit changes in their plasma lipoprotein lipid.

Differences in the method by which plasma is separated from whole blood influences amphotericin B plasma recovery and distribution following amphotericin B lipid complex incubation within whole blood.

A previous investigation suggested that the use of plasma as the biological fluid for measurement of amphotericin B (AmpB) concentrations greatly underestimates the concentrations of AmpB in the total blood circulation following amphotericin B lipid complex (ABLC) administration to humans. The purpose of this study was to determine if differences in the method used to obtain plasma from whole blood influences the percentage of AmpB recovered in plasma following ABLC incubation in whole blood. ABLC (5 μg AmpB/ml; peak blood concentration observed in rabbits following intravenous bolus of ABLC at a dose of 1 mg/kg) was incubated in whole blood for 5 min at 25°C. These conditions were used to mimic the sample retrieval conditions used when blood is obtained from animals and human patients. Following incubation, plasma was obtained from whole blood using five different methods: (A) Whole blood was centrifuged for 5 min at 23°C, and the plasma was separated; (B) whole blood was stored at 4°C for 18 h, and the plasma was separated by gravity; (C) whole blood was stored at 23°C for 18 h, and the plasma was separated by gravity; (D) whole blood was stored at 37°C for 18 h in a water bath, and the plasma was separated by gravity; and (E) whole blood was stored at 30°C for 18 h in a water bath, and the plasma was separated by gravity. All samples were protected from light throughout the duration of the experiment. AmpB concentration in each plasma sample was determined by high-performance liquid chromatography (HPLC) using an external calibration curve. The whole blood : plasma Amp B concentration ratio and the percentage of AmpB partitioned into plasma following incubation of ABLC in whole blood for each plasma separation procedure was as follows: (A) 6.5 : 1 blood : plasma AmpB concentration ratio, 15.4% ± 1.6% AmpB in plasma; (B) 2.98 : 1 blood : plasma AmpB concentration ratio, 33.6% ± 7.7% AmpB in plasma; (C) 1.5 : 1 blood : plasma AmpB concentration ratio, 67.6% ± 10.3% AmpB in plasma; (D) 1.5 : 1 blood : plasma concentration ratio, 68.1% ± 1.1% AmpB in plasma; and (E) 1.2 : 1 blood : plasma AmpB concentration ratio; 83.4% ± 5.5% AmpB in plasma. These findings suggest that when measurement of AmpB in plasma is required following ABLC administration, incubation of whole blood at 30°C for 18 h appears to be the most effective method.