Amal Kaddoumi

Olive-Oil-Derived Oleocanthal Enhances β-Amyloid Clearance as a Potential Neuroprotective Mechanism against Alzheimer’s Disease: In Vitro and in Vivo Studies

Oleocanthal, a phenolic component of extra-virgin olive oil, has been recently linked to reduced risk of Alzheimers disease (AD), a neurodegenerative disease that is characterized by accumulation of β-amyloid (Aβ) and tau proteins in the brain. However, the mechanism by which oleocanthal exerts its neuroprotective effect is still incompletely understood. Here, we provide in vitro and in vivo evidence for the potential of oleocanthal to enhance Aβ clearance from the brain via up-regulation of P-glycoprotein (P-gp) and LDL lipoprotein receptor related protein-1 (LRP1), major Aβ transport proteins, at the blood-brain barrier (BBB). Results from in vitro and in vivo studies demonstrated similar and consistent pattern of oleocanthal in controlling Aβ levels. In cultured mice brain endothelial cells, oleocanthal treatment increased P-gp and LRP1 expression and activity. Brain efflux index (BEI%) studies of (125)I-Aβ40 showed that administration of oleocanthal extracted from extra-virgin olive oil to C57BL/6 wild-type mice enhanced (125)I-Aβ40 clearance from the brain and increased the BEI% from 62.0 ± 3.0% for control mice to 79.9 ± 1.6% for oleocanthal treated mice. Increased P-gp and LRP1 expression in the brain microvessels and inhibition studies confirmed the role of up-regulation of these proteins in enhancing (125)I-Aβ40 clearance after oleocanthal treatment. Furthermore, our results demonstrated significant increase in (125)I-Aβ40 degradation as a result of the up-regulation of Aβ degrading enzymes following oleocanthal treatment. In conclusion, these findings provide experimental support that potential reduced risk of AD associated with extra-virgin olive oil could be mediated by enhancement of Aβ clearance from the brain.

Paclitaxel loaded PEG5000–DSPE micelles as pulmonary delivery platform: Formulation characterization, tissue distribution, plasma pharmacokinetics, and toxicological evaluation

The objective of the present study was to evaluate the potential of paclitaxel loaded micelles fabricated from PEG(5000)-DSPE as a sustained release system following pulmonary delivery. PEG(5000)-DSPE micelles containing paclitaxel were prepared by solvent evaporation technique followed by investigation of in vitro release of paclitaxel in lung simulated fluid. Tissue distribution and plasma pharmacokinetics of the PEG-lipid micelles after intratracheal and intravenous administrations were investigated in addition to intratracheally administered taxol. Finally, toxicological profile of PEG(5000)-DSPE was investigated. Paclitaxel was successfully formulated in PEG-lipid micelles with encapsulation efficiency of 95%. The PEG-lipid micelles exhibited a sustained release behavior in the simulated lung fluid. Intratracheally administered polymeric micellar paclitaxel showed highest accumulation of paclitaxel in the lungs with AUC(0-12) in lungs being 45-fold higher than intravenously administered formulation and 3-fold higher than intratracheally delivered taxol. Paclitaxel concentration in other non-targeted tissues and plasma were significantly lower as compared to other groups. Furthermore, toxicity studies showed no significant increase in levels of lung injury markers in PEG(5000)-DSPE treated group as compared to saline-treated group. PEG(5000)-DSPE micelles delivered intratracheally were able to sustain highest paclitaxel concentrations in lungs for long periods of time, thus apprehending their suitability as pulmonary drug carriers.

Enhanced Brain Amyloid-β Clearance by Rifampicin and Caffeine as a Possible Protective Mechanism Against Alzheimer’s Disease

Rifampicin and caffeine are widely used drugs with reported protective effect against Alzheimers disease (AD). However, the mechanism underlying this effect is incompletely understood. In this study, we have hypothesized that enhanced amyloid-β (Aβ) clearance from the brain across the blood-brain barrier (BBB) of wild-type mice treated with rifampicin or caffeine is caused by both drugs potential to upregulate low-density lipoprotein receptor related protein-1 (LRP1) and/or P-glycoprotein (P-gp) at the BBB. Expression studies of LRP1 and P-gp in brain endothelial cells and isolated mice brain microvessels following treatment with rifampicin or caffeine demonstrated both drugs as P-gp inducers, and only rifampicin as an LRP1 inducer. Also, brain efflux index (BEI%) studies conducted on C57BL/6 mice treated with either drug to study alterations in Aβ clearance demonstrated the BEI% of Aβ in rifampicin (82.4 ± 4.3%) and caffeine (80.4 ± 4.8%) treated mice were significantly higher than those of control mice (62.4 ± 6.1%, p < 0.01). LRP1 and P-gp inhibition studies confirmed the importance of both proteins to the clearance of Aβ, and that enhanced clearance following drugs treatment was caused by LRP1 and/or P-gp upregulation at the mouse BBB. Furthermore, our results provided evidence for the presence of a yet to be identified transporter/receptor that plays significant role in Aβ clearance and is upregulated by caffeine and rifampicin. In conclusion, our results demonstrated the upregulation of LRP1 and P-gp at the BBB by rifampicin and caffeine enhanced brain Aβ clearance, and this effect could explain, at least in part, the protective effect of rifampicin and caffeine against AD.

Role of ABC Transporters in the Pathogenesis of Alzheimer’s Disease

Alzheimers disease (AD) is a progressive neurodegenerative disorder and the most common form of age-related dementia that begins with memory loss and progresses to include severe cognitive impairment. A major pathological hallmark of AD is the accumulation of beta amyloid peptide (Aβ) in senile plaques in the brain of AD patients. The exact mechanism by which AD takes place remains unknown. However, an increasing number of studies suggests that ATP-binding cassette (ABC) transporters, which are localized on the surface of brain endothelial cells of the blood-brain barrier (BBB) and brain parenchyma, may contribute to the pathogenesis of AD. Recent studies have unraveled important roles of ABC transporters including ABCB1 (P-glycoprotein, P-gp), ABCG2 (breast cancer resistant protein, BCRP), ABCC1 (multidrug resistance protein 1, MRP1), and the cholesterol transporter ABCA1 in the pathogenesis of AD and Aβ peptides deposition inside the brain. Therefore, understanding the mechanisms by which these transporters contribute to Aβ deposition in the brain is important for the development of new therapeutic strategies against AD. This review summarizes and highlights the accumulating evidence in the literature which describe the role of altered function of various ABC transporters in the pathogenesis and progression of AD and the implications of modulating their functions for the treatment of AD.

Nilotinib potentiates anticancer drug sensitivity in murine ABCB1-, ABCG2-, and ABCC10-multidrug resistance xenograft models.

A panel of clinically used tyrosine kinase inhibitors were compared and nilotinib was found to most potently sensitize specific anticancer agents by blocking the functions of ABCB1/P-glycoprotein, ABCG2/BCRP and ABCC10/MRP7 transporters involved in multi-drug resistance. Nilotinib appreciably enhanced the antitumor response of (1) paclitaxel in the ABCB1- and novel ABCC10-xenograft models, and (2) doxorubicin in a novel ABCG2-xenograft model. With no apparent toxicity observed in the above models, nilotinib attenuated tumor growth synergistically and increased paclitaxel concentrations in ABCB1-overexpressing tumors. The beneficial actions of nilotinib warrant consideration as viable combinations in the clinic with agents that suffer from MDR-mediated insensitivity.

Mixed micelles of PEG2000-DSPE and vitamin-E TPGS for concurrent delivery of paclitaxel and parthenolide: Enhanced chemosenstization and antitumor efficacy against non-small cell lung cancer (NSCLC) cell lines

Concurrent combination of chemotherapeutic drugs is a promising alternative to single-agent therapies in cancer. In the present study, paclitaxel and parthenolide were loaded into mixed micelles and tested against taxol sensitive (A549) and resistant (A549-T24) NSCLC cell lines. Combination chemotherapy was further evaluated by isobologram analyses and combination index calculations. Drugs were loaded into micelles by the film casting method using PEG(2000)-DSPE and vitamin E-TPGS. Micelle characterization studies included the determination of particle size, encapsulation efficiency, in vitro release kinetics, as well as 1H NMR analysis. The in vitro release of both drugs was slower from the mixed micelles, which maintained an encapsulation efficiency >95% and chemical stability over a storage period of 45 days. The IC50 of paclitaxel and parthenolide determined by MTT assay were 108.6nM and 21μM, respectively, while the combination had an IC50 of 64.15nM in A549 cells. In the taxol resistant cell lines, the IC50 values of paclitaxel and parthenolide were 233nM and 32μM, respectively, while the combination had an IC(50) of 128nM. The efficacy of paclitaxel and parthenolide against both cell lines significantly increased when the drugs were coencapsulted in mixed micelles. Mixed micelles caused 79% cell death, which was significantly higher than the 46% cell death caused by the drugs in solution against taxol sensitive cell lines. In taxol resistant cell lines, the cell death caused by mixed micelles was 70% as compared to 45% cell death caused by un-encapsulated drugs. Co-encapsulation of parthenolide with paclitaxel in mixed micelles increased the anticancer activity of paclitaxel against resistant and sensitive lung cancer cell lines.

Intestinal Absorption of γ-Tocotrienol Is Mediated by Niemann-Pick C1-Like 1: In Situ Rat Intestinal Perfusion Studies

γ-Tocotrienol (γ-T3) is a member of the vitamin E family that displays potent anticancer activity and other therapeutic benefits. The objective of this study was to evaluate γ-T3 intestinal uptake and metabolism using the in situ rat intestinal perfusion model. Isolated segments of rat jejunum and ileum were perfused with γ-T3 solution, and measurements were made as a function of concentration (5–150 μM). Intestinal permeability (Peff) and metabolism were studied by measuring total compound disappearance and major metabolite, 2,7,8-trimethyl-2-(β-carboxy-ethyl)-6-hydroxychroman, appearance in the intestinal lumen. γ-T3 and metabolite levels were also determined in mesenteric blood. The Peff of γ-T3 was similar in both intestinal segments and significantly decreased at concentrations ≥25 μM in jejunum and ileum (p < 0.05), whereas metabolite formation was minimal and mesenteric blood concentrations of γ-T3 and metabolite remained very low. These results indicate that γ-T3 intestinal uptake is a saturable carrier-mediated process and metabolism is minimal. Results from subsequent in situ inhibition studies with ezetimibe, a potent and selective inhibitor of Niemann-Pick C1-like 1 (NPC1L1) transporter, suggested γ-T3 intestinal uptake is mediated by NPC1L1. Comparable findings were obtained when Madin-Darby canine kidney II cells that express endogenous NPC1L1 were incubated with increasing concentrations of γ-T3 or γ-T3 with increasing concentrations of ezetimibe. The present data show for the first time that γ-T3 intestinal absorption is partly mediated by NPC1L1.

Up-regulation of P-glycoprotein reduces intracellular accumulation of beta amyloid: investigation of P-glycoprotein as a novel therapeutic target for Alzheimer's disease

Objectives  Several studies have suggested the efflux transporter P‐glycoprotein (P‐gp) to play a role in the etiology of Alzheimers disease through the clearance of amyloid beta (Aβ) from the brain. In this study, we aimed to investigate the possibility of P‐gp as a potential therapeutic target for Alzheimers disease by examining the impact of P‐gp up‐regulation on the clearance of Aβ, a neuropathological hallmark of Alzheimers disease.

Discovery of Novel GSK-3β Inhibitors with Potent in Vitro and in Vivo Activities and Excellent Brain Permeability Using Combined Ligand- and Structure-Based Virtual Screening

Dysregulation of glycogen synthase kinase (GSK-3β) is implicated in the pathophysiology of many diseases, including type-2 diabetes, stroke, Alzheimers, and others. A multistage virtual screening strategy designed so as to overcome known caveats arising from the considerable flexibility of GSK-3β yielded, from among compounds in our in-house database and two commercial databases, new GSK-3β inhibitors with novel scaffold structures. The two most potent and selective validated hits, a 2-anilino-5-phenyl-1,3,4-oxadiazole (24) and a phenylmethylene hydantoin (28), both exhibited nanomolar affinity and selectivity over CDK2 and were potent enough for direct in vivo validation. Both were able to cause significant increases in liver glycogen accumulation in dose-dependent fashion. One also exhibited excellent blood-brain barrier permeability, the other adequate for a lead compound. Analogues of the oxadiazole 24 were synthesized to experimentally corroborate or rule out ligand-bound structures arising from docking studies. SAR results supported one docking study among a number of alternatives.

Intestinal Absorption of γ-Tocotrienol is Mediated by NPC1L1: In Situ Rat Intestinal Perfusion Studies

γ-Tocotrienol (γ-T3) is a member of the vitamin E family that displays potent anticancer activity and other therapeutic benefits. The objective of this study was to evaluate γ-T3 intestinal uptake and metabolism using the in situ rat intestinal perfusion model. Isolated segments of rat jejunum and ileum were perfused with γ-T3 solution, and measurements were made as a function of concentration (5–150 μM). Intestinal permeability (Peff) and metabolism were studied by measuring total compound disappearance and major metabolite, 2,7,8-trimethyl-2-(β-carboxy-ethyl)-6-hydroxychroman, appearance in the intestinal lumen. γ-T3 and metabolite levels were also determined in mesenteric blood. The Peff of γ-T3 was similar in both intestinal segments and significantly decreased at concentrations ≥25 μM in jejunum and ileum (p < 0.05), whereas metabolite formation was minimal and mesenteric blood concentrations of γ-T3 and metabolite remained very low. These results indicate that γ-T3 intestinal uptake is a saturable carrier-mediated process and metabolism is minimal. Results from subsequent in situ inhibition studies with ezetimibe, a potent and selective inhibitor of Niemann-Pick C1-like 1 (NPC1L1) transporter, suggested γ-T3 intestinal uptake is mediated by NPC1L1. Comparable findings were obtained when Madin-Darby canine kidney II cells that express endogenous NPC1L1 were incubated with increasing concentrations of γ-T3 or γ-T3 with increasing concentrations of ezetimibe. The present data show for the first time that γ-T3 intestinal absorption is partly mediated by NPC1L1.