Kristina Sachs-Barrable

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.

Effects of Monoglycerides on P-Glycoprotein: Modulation of the Activity and Expression in Caco-2 Cell Monolayers

The purpose of this study was to analyze the effects of two common monoglyceride components of lipid excipients, 1-monoolein and 1-monostearin, on the activity and expression of P-glycoprotein (P-gp) in Caco-2 cells. Non-cytotoxic concentrations of 1-monoolein and 1-monostearin were determined by assessing membrane permeability and mitochondrial activity in Caco-2 cells, a human colon adenocarcinoma cell line. Concentrations of 500 and 100 microM were used to evaluate P-gp activity through Rh123 accumulation and bifunctional transport studies. The P-gp protein expression levels were quantified through the use of immunoblots. The changes in cell membrane fluidity and nuclear membrane integrity upon the addition of monoglycerides were analyzed by fluorescence anisotropy using DPH and TMA-DPH as the fluorescent labels and by using increasing salt concentrations to release the nuclear contents, respectively. The absorptive flux (apical to basolateral) in the bifunctional transport studies was not found to be statistically significant for the non-cytotoxic concentrations of 1-monoolein and 1-monostearin. However, treatments of 500 and 100 microM of 1-monoolein or 1-monostearin displayed statistically lowered efflux (basolaterial to apical, P < 0.05) compared to the controls (7.9 +/- 0.8, 12.9 +/- 2.6 x 10 (6) cm/s for 1-monoolein or 11.1 +/- 2.0, 11.4 +/- 2.3 x 10 (6) cm/s for 1-monostearin, respectively, compared to the untreated control, 21.1 +/- 2.9 x 10 (6) cm/s, n = 5). Rh123 accumulation was also found to be enhanced upon 24 h incubation with both concentrations of the monoglycerides; however, only concentrations of 500 muM of the monoglycerides were shown to significantly reduce the P-gp protein expression. The results from this study suggest that these two monoglycerides, common components in various lipid excipients, are inhibitors of P-gp.

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.

Cyclosporine A and Rapamycin induce in vitro cholesteryl ester transfer protein activity, and suppress lipoprotein lipase activity in human plasma

PURPOSE Cyclosporine A (CsA), Rapamycin (RAPA), Tacrolimus (FK-506) and Mycophenolate mofetil (MMF) are immunosuppressants that are widely used in solid organ transplant patients. However, some of these drugs have been reported to cause dyslipidemia in patients. Our aim was to determine the effects of these drugs on in vitro cholesteryl ester transfer protein (CETP), hepatic lipase (HL) and lipoprotein lipase (LPL) activity within human plasma. METHODS We measured CETP activity in human normolipidemic plasma with and without drug treatment, by measuring the incorporation of labeled cholesteryl ester into lipoproteins. To further confirm the result, we also measured recombinant CETP (rCETP) activity with and without drug treatment. We measured HL and LPL activity in post-heparin normal human plasma in the presence and absence of the drugs by measuring the release of fatty acids from radiolabeled triolein. RESULTS We found an increase in CETP activity in human normolipidemic plasma and rCETP treated with CsA and RAPA. By contrast, CETP activity was not altered significantly in the presence of FK-506 and MMF. LPL activity in post-heparin normal human plasma was suppressed following the co-incubation with CsA, RAPA, FK-506 or MMF whereas HL activity remained unaffected. CONCLUSIONS The increase in CETP activity and suppression in LPL activity following CsA and RAPA treatment observed in the present study may be associated with elevated LDL cholesterol levels and hypertriglyceridemia seen in patients administered these drugs.

Tacrolimus-Induced Elevation in Plasma Triglyceride Concentrations After Administration to Renal Transplant Patients Is Partially Due to a Decrease in Lipoprotein Lipase Activity and Plasma Concentrations

Background. Hyperlipidemia is a frequent and persistent complication in solid organ transplant recipients, leading to the high occurrence of cardiovascular disease in this patient population. Lipid abnormalities including increased total cholesterol, triglycerides (TG), and low-density lipoprotein-cholesterol have been reported frequently in transplantation patients and a variety of immunosuppressive therapies seem to be one of the main factors that influence posttransplant lipidemic profiles. For many years, tacrolimus (TAC) has been used as an immunosuppressive drug in transplantation. The aim of our investigation was to determine the effect of TAC administration on the plasma lipid profile and some key regulatory proteins of plasma lipid metabolism including cholesterol ester transfer protein, hepatic lipase and lipoprotein lipase (LPL) within renal transplant patients. Methods. Twenty-five renal transplant patients were recruited and received TAC therapy, of which nine of these patients were treated with statin therapy for dyslipidemia. The effects of TAC on plasma total cholesterol, TG, HDL-C, low-density lipoprotein-cholesterol, cholesterol ester transfer protein, hepatic lipase and LPL concentration and activity were determined from patients plasma samples collected before the transplant surgery (baseline), and weekly for four consecutive weeks after surgery and TAC administration. Results. We observed that TAC significantly increases plasma TG concentrations and reduces LPL plasma concentration and activity in renal transplant patients, independent of any lipid lowering drug treatment patients received. Conclusions. Taken together, these findings suggest that the reduction in LPL activity, partly due to the decrease of plasma LPL concentration after TAC administration may be an explanation for hypertriglyceridemia observed in patients administered TAC.

The use of the United States FDA programs as a strategy to advance the development of drug products for neglected tropical diseases

Abstract Neglected tropical diseases (NTDs) are infections which are endemic in poor populations in lower- and middle-income countries (LMIC). Approximately one billion people have now or are at risk of getting an NTD and yet less than 5% of research dollars are focused on providing treatments and prevention of these highly debilitating and deadly conditions. The United States Food and Drug Administration (FDA) Orphan Drug Designation program (ODDP) provides orphan status to drugs and biologics, defined as those intended for the safe and effective treatment, diagnosis or prevention of rare diseases and/or disorders that affect fewer than 200 000 people in the United States, or that affect more than 200 000 persons but are not expected to recover the costs of developing and marketing a treatment drug. These regulations have led to the translation of rare disease knowledge into innovative rare disease therapies. The FDA Guidance for Industry on developing drugs for the treatment and prevention of NTDs describes the following regulatory strategies: Orphan Product Designation, Fast Track Designation, Priority Review Designation, Accelerated Approval and Tropical Disease Priority Review Voucher. This paper will discuss how these regulations and especially the ODDP can improve the clinical development and accessibility of drug products for NTDs.

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.

Does p-glycoprotein play a role in gastrointestinal absorption and cellular transport of dietary cholesterol?

ABSTRACT This commentary discusses the potential role of p-glycoprotein (Pgp) on the gastrointestinal absorption and cellular transport of dietary cholesterol. This is currently a controversial issue due to the conflicting evidence about the role of this ABC transporter in cholesterol transport. During the preparation of this commentary, several key publications on this topic arguing for and against this mechanism have been published. If true, this mechanism of Pgp could represent a novel role for Pgp and provide a potentially new molecular target for drug design and development.

The effect of two novel cholesterol-lowering agents, disodium ascorbyl phytostanol phosphate (DAPP) and nanostructured aluminosilicate (NSAS) on the expression and activity of P-glycoprotein within Caco-2 cells

BackgroundMany drugs are substrates for P-glycoprotein (P-gp) and interactions involving P-gp may be relevant to clinical practice. Co-administration with P-gp inhibitors or inducers changes the absorption profile as well as the risk for drug toxicity, therefore it is important to evaluate possible P-gp alterations. The purpose of this study was to investigate the effect of two novel cholesterol-lowering agents, disodium ascorbyl phytostanol phosphate (DAPP) and nanostructured aluminium silicate (NSAS), a protonated montmorillonite clay, on mdr-1 gene expression and its protein, P-glycoprotein (P-gp) within Caco-2 cells.MethodsThe effects of DAPP and NSAS on the regulation of mdr-1 gene, P-gp protein expression and activity within Caco-2 cells, were determined using cell viability and cytotoxicity tests, RT-PCR, Western Blot analysis and bi-directional transport studies.ResultsWe observed a significant down-regulation of mdr-1 mRNA (e.g. 38.5 ± 17% decrease vs. control at 5 μM DAPP and 61.2 ± 25% versus control at 10 μM DAPP; n = 6, P* < 0.05) within Caco-2 cells. Western Blot analysis of P-gp expression showed that changes in mdr-1 gene expression lead to correlating changes in P-gp protein expression. This down-regulation of P-glycoprotein also resulted in decreased activity of P-glycoprotein compared to untreated control. In contrast, when Caco-2 cells were treated with NSAS, no changes in mdr-1 gene expression, P-gp protein expression nor P-gp activity were observed.ConclusionsDAPP but not NSAS decreases P-gp mediated drug efflux through decreased mdr-1 gene expression and consequently decreased P-gp protein expression. These findings have to be taken into consideration when DAPP is concurrently given with other drugs that are substrates for P-gp since drug-drug interactions harbour a safety issue and alter bioavailability profiles.NSAS does not have any P-gp altering properties and therefore might not affect drug-drug interactions. We conclude from this study that NSAS might make a safer drug candidate compared to DAPP for lowering LDL-cholesterol.

Hepatic lipase deficiency in a Middle-Eastern-Arabic male

Hepatic lipase (HL) deficiency is a rare genetic disorder that has been associated with premature atherosclerosis despite high plasma high-density lipoprotein (HDL) cholesterol concentrations in the affected individuals. The authors describe the clinical and biochemical features of HL deficiency in a young male of Middle-Eastern-Arabic origin. This is the first report of cholesterol ester transfer protein (CETP) activity and mass in HL deficiency in a patient from this ethnic group. While the CETP mass was high, its activity was low, a discrepancy likely due to the abnormal composition of patients HDL particles.