Stephen D. Lee

Impact of lipoproteins on the biological activity and disposition of hydrophobic drugs: implications for drug discovery

In contrast to many traditional pharmaceutical agents that exhibit a high degree of aqueous solubility, new drug candidates are frequently highly lipophilic compounds. The aqueous environment of the blood provides a thermodynamically unfavourable environment for the disposition of such hydrophobic drugs. However, this limitation can be overcome by association with circulating lipoproteins. Elucidation of the mechanisms that dictate drug–lipoprotein association and blood-to-tissue partitioning of lipoprotein encapsulated drugs might yield valuable insight into the factors governing the pharmacological activity and potential toxicity of these compounds. This Review discusses the impact of hydrophobic drug–lipoprotein interactions on pharmacokinetics, drug metabolism, tissue distribution and biological activity of various hydrophobic compounds, and outlines how best to use this information in drug discovery and development programmes.

The influence of hyperlipoproteinemia on in vitro distribution of amiodarone and desethylamiodarone in human and rat plasma.

PurposeTo study the effect of hyperlipoproteinemia on in vitro distribution of amiodarone (AM) and its prevalent metabolite desethylamiodarone (DEA) in human and rat plasma.Materials and MethodsHuman and rat normolipidemic (NL) and hyperlipidemic (HL) plasma were spiked with AM and DEA. The fractions (high and low density lipoproteins, triglyceride rich lipoproteins and lipoprotein deficient plasma) were separated using ultracentrifugation.ResultsHuman and rat displayed similar patterns in terms of association of AM and DEA in NL plasma, in which the highest and lowest associations were observed in lipoprotein deficient (LPDP) and triglyceride (TRL) rich plasma fractions, respectively. In HL a substantial shift was observed in partitioning of AM and DEA mostly to TRL. The shift of AM and DEA into TRL fraction of HL plasma was more drastic for rat than human. In HL, association of AM with rat LPDP and HDL fractions were 10 and 26-fold lower than in the corresponding human fractions, respectively. The DEA:AM ratio in rat, but not human, was significantly affected by HL.ConclusionHL caused a major shift of AM and DEA to TRL fraction in both species. The findings were consistent with the higher AM concentrations previously noted in HL rats given the drug.

Effect of dietary fat on hepatic liver X receptor expression in P-glycoprotein deficient mice: implications for cholesterol metabolism

Pgp (P-glycoprotein, MDR1, ABCB1) is an energy-dependent drug efflux pump that is a member of the ATP-binding cassette (ABC) family of proteins. Preliminary studies have reported that nonspecific inhibitors of Pgp affect synthesis and esterification of cholesterol, putatively by blocking trafficking of cholesterol from the plasma membrane to the endoplasmic reticulum, and that relative increases in Pgp within a given cell type are associated with increased accumulation of cholesterol. Several key efflux proteins involved in the cholesterol metabolic pathway are transcriptionally regulated by the nuclear hormone liver X receptor (LXR). Therefore, to examine the interplay between P-glycoprotein and the cholesterol metabolic pathway, we utilized a high fat, normal cholesterol diet to upregulate LXRα without affecting dietary cholesterol. Our research has demonstrated that mice lacking in P-glycoprotein do not exhibit alterations in hepatic total cholesterol storage, circulating plasma total cholesterol levels, or total cholesterol concentration in the bile when compared to control animals on either a normal (25% calories from dietary fat) or high fat (45% calories from dietary fat) diet. However, p-glycoprotein deficient mice (Mdr1a-/-/1b-/-) exhibit increased hepatic LXRα protein expression and an elevation in fecal cholesterol concentration when compared to controls.

Dose-Dependent Targeted Suppression of P‑glycoprotein Expression and Function in Caco‑2 Cells

The efflux transporter P-glycoprotein (Pgp), encoded by the ABCB1 gene, decreases the bioavailability of a wide range of orally administered drugs. Drug permeability studies using the in vitro Caco-2 cell model commonly rely on small molecule modulators to estimate the contribution of Pgp to drug efflux. The use of such modulators may be limited by their interactions with other membrane transporters. RNA interference, a tool allowing for the specific degradation of a target gene’s mRNA, has emerged as a technique to study gene expression and function. This manuscript describes the use of chemically modified small interfering RNA (siRNA) for a dose-dependent suppression of ABCB1 in Caco-2 cells and the subsequent drug permeability assay. We transfected Caco-2 cells while in suspension with chemically modified synthetic siRNA–lipid complexes and then seeded the cells on polycarbonate semipermeable supports. Once the monolayer of Caco-2 cells formed tight junctions and expressed brush border enzymes, we determined the dose-dependent suppression of the ABCB1 gene using RT-qPCR. We measured the duration of silencing at the optimal siRNA dose by Western blot for Pgp protein. The utility of this in vitro model was determined by performing bidirectional transport studies using a well-established substrate for Pgp, rhodamine 123. A single 4 h transfection of the Caco-2 cells with ≥100 nM siRNA reduced the expression of ABCB1 mRNA by >85% at day five in culture. The time-course study revealed that the single transfection reduces Pgp protein levels for 9 days in culture. This magnitude of silencing was sufficient to reduce the efflux of rhodamine 123 as measured by the apparent permeability coefficient and intracellular accumulation. In this study, we demonstrate the dose-dependent, targeted degradation of Pgp in Caco-2 cells as a new model for assessing drug efflux from enterocytes. The dose-dependent nature of the Pgp silencing in this study offers significant improvements over other approaches to creating a Caco-2 model with suppressed ABCB1 expression. We envision that this technique, in conjunction with better small molecule inhibitors, will provide a useful tool for future drug permeability studies.

Inhibition of Cholesterol Absorption: Targeting the Intestine

ABSTRACTAtherosclerosis, the gradual formation of a lipid-rich plaque in the arterial wall is the primary cause of Coronary Artery Disease (CAD), the leading cause of mortality worldwide. Hypercholesterolemia, elevated circulating cholesterol, was identified as a key risk factor for CAD in epidemiological studies. Since the approval of Mevacor in 1987, the primary therapeutic intervention for hypercholesterolemia has been statins, drugs that inhibit the biosynthesis of cholesterol. With improved understanding of the risks associated with elevated cholesterol levels, health agencies are recommending reductions in cholesterol that are not achievable in every patient with statins alone, underlying the need for improved combination therapies. The whole body cholesterol pool is derived from two sources, biosynthesis and diet. Although statins are effective at reducing the biosynthesis of cholesterol, they do not inhibit the absorption of cholesterol, making this an attractive target for adjunct therapies. This report summarizes the efforts to target the gastrointestinal absorption of cholesterol, with emphasis on specifically targeting the gastrointestinal tract to avoid the off-target effects sometimes associated with systemic exposure.

Evaluation of the Contribution of the ATP Binding Cassette Transporter, P-glycoprotein, to in Vivo Cholesterol Homeostasis

P-glycoprotein (Pgp, encoded by ABCB1, commonly known as MDR1), an ATP-dependent transporter with a broad range of hydrophobic drug substrates, has been associated with the in vitro intracellular transport of cholesterol; however, these findings have not been confirmed in vivo. In this manuscript we tested the contributions of Pgp to in vivo cholesterol homeostasis by comparing the cholesterol phenotype of wild type mice with mice lacking both murine isoforms of Pgp (Abcb1a(-/-)/1b(-/-)) by measuring cholesterol absorption, circulating cholesterol, and lipoprotein cholesterol profiles. The mice were fed diets containing normal or high levels of dietary fat (25% vs 45% kcal from fat) and cholesterol (0.02% vs 0.20% w/w) for 8 weeks to challenge their capacity to maintain homeostasis. There were no significant differences in cholesterol absorption, circulating cholesterol levels, and lipoprotein profiles between Pgp knockout and wild type mice fed matching diets. Compensatory shifts were observed in the activation of two key transcription factors involved in maintaining cholesterol balance, the Liver X Receptor and SREBP-2, which may have maintained the wild type phenotype in the knockout mice. Deletion of Pgp affected the molar composition of gallbladder bile, when the mice were fed diets containing high levels of dietary fat, cholesterol, or both. The mole fraction of bile salts was reduced in the gallbladder bile of Pgp knockout mice, while the mole fraction of cholesterol was increased. In this paper, we provide evidence that Pgp knockout mice maintain cholesterol homeostasis, even when challenged with high cholesterol diets. We suggest that the specific shifts in cholesterol regulatory networks identified in the jejunum and liver of the knockout mice may have compensated for the lack of Pgp. Our finding that Pgp knockout mice were unable to maintain gallbladder bile composition when challenged with high dietary fat and/or cholesterol compliments recent reports that Pgp may be a secondary bile salt export pump.

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.

Abstract 3532: P-glycoprotein downregulation using RNAi decreases cholesterol efflux from human renal cancer cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: Renal cancer is responsible for an estimated 55,000 new cases and 13,000 deaths per year in the United States (2008). Human renal cancer tissue contains eight times more cholesterol than adjacent normal kidney tissue indicating that cholesterol may be important for cancer. Increased cholesterol levels have also been found to protect kidneys from ischemia. Interestingly, a p-glycoprotein (Pgp) knockout mouse model has been found to be protective against ischemic renal injury suggesting a link between Pgp and increased renal cholesterol. Our objectives were to investigate cholesterol accumulation, biosynthesis and efflux in 3 models: a) a human embryonic kidney cell line (293T) where Pgp expression is reduced by RNA interference (RNAi), b) kidneys from a murine Pgp knockout model and c) a canine kidney cell line (MDCKII) overexpressing human Pgp. Methods: All cell culture studies were performed in DMEM. The 293T cells were transfected with 3 Pgp specific RNAi oligos and compared to a scrambled RNAi control; protein analysis was performed 72h post-transfection. Cellular cholesterol concentrations were determined using the Amplex Red cholesterol assay. Cholesterol synthesis was evaluated by immunoblotting of HMG CoA reductase, the rate limiting enzyme in the biosynthesis pathway. Cholesterol efflux to high density lipoprotein (HDL) acceptors over 6h was measured after a 24h incubation with 3H-cholesterol. Male Pgp knockout and FVB control (wild type) mice were maintained 12 weeks on a controlled chow diet (25% of calories coming from fat and 0.02% cholesterol). Cells were lysed in RIPA buffer with protease inhibitors and analyzed for HMG CoA reductase and actin expression by immunoblotting. Lipids from kidney tissues were extracted by Folch method and cholesterol levels were determined using an enzymatic assay. Results: Pgp protein expression was downregulated 80-90% with this RNAi system. Despite reduced HMG CoA reductase levels in both Pgp knockdown cells (29%) and the kidneys from the Pgp knockout (35%) there was no change in cholesterol levels suggesting either uptake was increased or efflux was reduced in these samples. When cholesterol efflux was analyzed in the Pgp knockdown cells, we found a 31-46% reduction in cholesterol efflux to HDL. In the case of the MDR1-MDCKII cells overexpressing Pgp, we found a 100% increase in HMG CoA reductase expression with no changes in cholesterol cellular levels, further suggesting that cholesterol efflux is increased in these cells. Conclusions: The reduced cholesterol efflux in Pgp knockdown cells suggests a role for Pgp in cholesterol efflux. This finding is supported by the observation that Pgp over-expression causes compensatory changes to the levels of the cholesterol synthesis enzyme, HMG CoA reductase. ACKNOWLEDGEMENTS: Funding for this project was provided by the Canadian Institutes of Health Research (CIHR). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3532.