Bilal S. Abuasal

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.

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.

In Silico Modeling for the Nonlinear Absorption Kinetics of UK-343,664: A P-gp and CYP3A4 Substrate

The aim of this work was to extrapolate in vitro and preclinical animal data to simulate the pharmacokinetic parameters of UK-343,664, a P-glycoprotein (P-gp) and CYP3A4 substrate, in human. In addition, we aimed to develop a simulation model to demonstrate the involvement and the controversial complex interaction of intestinal P-gp and CYP3A4 in its nonlinear absorption, first-pass extraction, and pharmacokinetics using the advanced compartmental absorption and transit (ACAT) model. Finally, we aimed to compare the results predicted from the model to the reported findings in human clinical studies. In situ perfusion, allometric scaling, PBPK Rodger mechanistic approach, in vitro metabolism, and fitting to in vivo data were used to mechanistically explain the absorption, distribution and metabolism, respectively. GastroPlus was used to build the integrated simulation model in human for UK-343,664 to mechanistically explain the observed clinical data at 30, 100, 200, 400, and 800 mg oral doses. The measured in vitro value for CYP3A4 K(m) (465 μM) in rCYPs was converted to units of μg/mL, corrected for assumed microsomal binding (17.8%) and applied to all metabolic processes. The measured in vitro values of V(max) for CYP3A4 (38.9 pmol/min/pmol), 2C8, 2C9, 2C19, and 2D6 were used along with the in vitro CYP3A4 K(m) to simulate liver first pass extraction and systemic clearance. The measured in vitro values of V(max) for CYP3A4 and 2D6 were used along with the in vitro CYP3A4 K(m) to simulate gut first pass extraction. V(max) and K(m) values for P-gp were obtained by fitting to in vivo data and used to simulate gut efflux transport activity. Investigation of the interaction mechanism of P-gp and CYP3A4 in the intestine was achieved by comparing the influence of a virtual knockout of P-gp or gut metabolism on the fraction absorbed, fraction reaching the portal vein, and fraction metabolized in the gut. Comparison between simulation and in vivo results showed that the in silico simulation provided a mechanistic explanation of the observed nonlinear absorption kinetics of UK-343,664 in human following its administration in the range of 30-800 mg as oral solutions. The simulation results of the pharmacokinetic parameters, AUC and C(max), by GastroPlus were comparable with those observed in vivo. This simulation model is one possible mechanistic explanation of the observed in vivo data and suggests that the nonlinear dose dependence could be attributed to saturation of both the efflux transport by P-gp and the intestinal metabolism. However, the concentration ranges for either protein saturation did not overlap and resulted in much greater than dose proportional increases in AUC. At low doses, producing intraenterocyte concentrations below the fitted value of K(m) for P-gp, the influence of P-gp appears to be protective and results in a lower fraction of gut 3A4 metabolism. At higher doses, as P-gp becomes saturated the fraction of gut 3A4 extraction increases, and eventually at the highest doses, where 3A4 becomes saturated, the fraction of gut 3A4 extraction again decreases. Such a complex interpretation of this in vitro-in vivo extrapolation (IVIVE) is another example of the value and insight obtained by physiologically based absorption simulation.

Enhancement of Intestinal Permeability Utilizing Solid Lipid Nanoparticles Increases γ-Tocotrienol Oral Bioavailability

Abstractγ-Tocotrienol (γ-T3), a member of the vitamin E family, has been reported to possess an anticancer activity. γ-T3 is a lipophilic compound with low oral bioavailability. Previous studies showed that γ-T3 has low intestinal permeability. Thus, we have hypothesized that enhancing γ-T3 intestinal permeability will increase its oral bioavailability. Solid lipid nanoparticles (SLN) were tested as a model formulation to enhance γ-T3 permeability and bioavailability. γ-T3 intestinal permeability was compared using in situ rat intestinal perfusion, followed by in vivo relative oral bioavailability studies. In addition, in vitro cellular uptake of γ-T3 from SLN was compared to mixed micelles (MM) in a time and concentration-dependent studies. To elucidate the uptake mechanism(s) of γ-T3 from SLN and MM the contribution of NPC1L1 carrier-mediated uptake, endocytosis and passive permeability were investigated. In situ studies demonstrated SLN has tenfold higher permeability than MM. Subsequent in vivo studies showed γ-T3 relative oral bioavailability from SLN is threefold higher. Consistent with in situ results, in vitro concentration dependent studies revealed γ-T3 uptake from SLN was twofold higher than MM. In vitro mechanistic characterization showed that while endocytosis contributes to γ-T3 uptake from both formulations, the reduced contribution of NPC1L1 to the transport of γ-T3, and passive diffusion enhancement of γ-T3 are primary explanations for its enhanced uptake from SLN. In conclusion, SLN successfully enhanced γ-T3 oral bioavailability subsequent to enhanced passive permeability.

Comparison of the intestinal absorption and bioavailability of γ‐tocotrienol and α‐tocopherol: in vitro, in situ and in vivo studies

The aim of this work was to compare the intestinal absorption kinetics and the bioavailability of γ‐tocotrienol (γ‐T3) and α‐tocopherol (α‐Tph) administered separately as oil solutions to rats in vivo. Also, to explain the significant difference in the oral bioavailability of the compounds: (1) the release profiles using the dynamic in vitro lipolysis model, (2) the intestinal permeability and (3) carrier‐mediated uptake by Niemann‐Pick C1‐like 1 (NPC1L1) transporter were examined. Absolute bioavailability studies were conducted after oral administration of γ‐T3 or α‐Tph prepared in corn oil to rats. In situ rat intestinal perfusion with ezetimibe (a NPC1L1 inhibitor) was performed to compare intestinal permeability. The in vitro interaction kinetics with NPC1L1 was examined in NPC1L1 transfected cells. While the in vitro release studies demonstrated a significantly higher release rate of γ‐T3 in the aqueous phase, the oral bioavailability of α‐Tph (36%) was significantly higher than γ‐T3 (9%). Consequent in situ studies revealed significantly higher intestinal permeability for α‐Tph compared with γ‐T3 in rats. Moreover, the NPC1L1 kinetic studies demonstrated higher Vmax and Km values for α‐Tph compared with γ‐T3. Collectively, these results indicate that intestinal permeability is the main contributing factor for the higher bioavailability of α‐Tph. Also, these results emphasize the potentially important role of intestinal permeability in the bioavailability of γ‐T3, suggesting that enhancing its permeability would increase its oral bioavailability. Copyright

Sesamin synergistically potentiates the anticancer effects of γ-tocotrienol in mammary cancer cell lines.

γ-Tocotrienol and sesamin are phytochemicals that display potent anticancer activity. Since sesamin inhibits the metabolic degradation of tocotrienols, studies were conducted to determine if combined treatment with sesamin potentiates the antiproliferative effects of γ-tocotrienol on neoplastic mouse (+SA) and human (MCF-7 and MDA-MB-231) mammary cancer cells. Results showed that treatment with γ-tocotrienol or sesamin alone induced a significant dose-responsive growth inhibition, whereas combination treatment with these agents synergistically inhibited the growth of +SA, MCF-7 and MDA-MB-231 mammary cancer cells, while similar treatment doses were found to have little or no effect on normal (mouse CL-S1 and human MCF-10A) mammary epithelial cell growth or viability. However, sesamin synergistic enhancement of γ-tocotrienol-induced anticancer effects was not found to be mediated from a reduction in γ-tocotrienol metabolism. Rather, combined treatment with subeffective doses of γ-tocotrienol and sesamin was found to induce G1 cell cycle arrest, and a corresponding decrease in cyclin D1, CDK2, CDK4, CDK6, phospho-Rb, and E2F1 levels, and increase in p27 and p16 levels. Additional studies showed that the antiproliferative effect of combination treatment did not initiate apoptosis or result in a decrease in mammary cancer cell viability. Taken together, these findings indicate that the synergistic antiproliferative action of combined γ-tocotrienol and sesamin treatment in mouse and human mammary cancer cells is cytostatic, not cytotoxic, and results from G1 cell cycle arrest.

Development and validation of a reversed-phase HPLC method for the determination of γ-tocotrienol in rat and human plasma

γ-Tocotrienol (γ-T3) is a member of the vitamin E family. Recently, γ-T3 has attracted the attention of the scientific community due to its potent anticancer activity and other therapeutic benefits. The objective of this study was to develop and validate a simple and practical reversed-phase HPLC method with satisfactory sensitivity for the routine quantification of γ-T3 in rat and human plasma. The separation of γ-T3 from the plasma components was achieved with a C(18) reversed-phase column with an isocratic elution using a mixture of methanol, ethanol and acetonitrile (85:7.5:7.5, v/v/v) with a UV detection at 295 nm. γ-T3 was extracted from rat and human plasma by liquid-liquid extraction with an average recovery of 60%. The method proved linear in the range 100-5000 ng/mL. The inter-day precision ranged from 5.8 to 12.8% and the accuracy ranged from 92.4 to 108.5%, while the intra-day precision ranged from 0.7 to 7.9% in both rat and human plasma. This data confirm that the developed method has a satisfactory sensitivity, accuracy and precision for the quantification of γ-T3 in plasma. To assess its applicability the method was successfully applied to the quantitative analysis for pharmacokinetic studies of γ-T3 in rats administered a 10 mg/kg single oral dose.

Effects of Chemically Characterized Fractions from Aerial Parts of Echinacea purpurea and E. angustifolia on Myelopoiesis in Rats

Echinacea species are used for beneficial effects on immune function, and various prevalent phytochemicals have immunomodulatory effects. Using a commercial E. purpurea (L.) Moench product, we have evaluated the myelopoietic effect on bone marrow of rats treated with various extracts and correlated this with their chemical class composition. Granulocyte/macrophage-colony forming cells (GM-CFCs) from femurs of female Sprague-Dawley rats were assessed at 24 h after 7 daily oral treatments. A 75% ethanolic extract at 50 mg dried weight (derived from 227 mg aerial parts) per kg body weight increased GM-CFCs by 70% but at 100 mg/kg was without effect. Ethanolic extracts from aerial parts of E. angustifolia DC. var. angustifolia and E. purpurea from the USDA North Central Regional Plant Introduction Station increased GM-CFCs by 3- and 2-fold, respectively, at 200 mg/kg (~1400 mg/kg plant material). Extract from another USDA E. angustifolia was inactive. Proton and APT NMR, MS, and TLC indicated alkylamides and caffeic-acid derivatives (CADs) present in ethanolic extracts of both the commercial and USDA-derived material. Cichoric and caftaric acids were prominent in both E. purpurea ethanolic extracts but absent in E. angustifolia. Aqueous extract of the commercial material exhibited polysaccharide and CAD signatures and was without effect on GM-CFCs. A methanol-CHCl3 fraction of commercial source, also inactive, was almost exclusively 1:4 nonanoic: decanoic acids, which were also abundant in commercial ethanolic extract but absent from USDA material. In conclusion, we have demonstrated an ethanolextractable myelostimulatory activity in Echinacea aerial parts that, when obtained from commercial herbal supplements, may be antagonized by medium-chain fatty acids presumably derived from a non-plant additive.

Abstract 3045: Sesamin synergistically potentiates the anticancer effects of γ-tocotrienol in mammary tumor cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Epidemiological studies have highlighted the ability of micronutrients in plants to reduce the risk of breast cancer. Phytochemicals can interfere with cell-signaling pathways that regulate cell proliferation and differentiation. γ-Tocotrienol is a natural form of vitamin E that displays a potent and selective anticancer activity against various mammary cell lines with no discernible toxicity toward normal cells. Sesamin is an abundant phytochemical in sesame seed oil which showed antiproliferative and antiangiogenic activity against human breast cancer cells. In addition, sesamin inhibits the metabolism of vitamin E isomers and increases their bioavailability and efficacy. Preliminary growth inhibition studies showed that combined treatment with subeffective doses of γ-tocotrienol and sesamin synergistically inhibited the growth of neoplastic murine +SA (ErbB2-positive) and human MCF-7 (luminal A, ER-positive) mammary epithelial cells, as determined by MTT assay and isobologram analysis. Additional studies were conducted to determine the intracellular mechanisms mediating the synergistic anticancer activity in +SA mammary epithelial cells. Flow cytometry analysis showed that combined treatment of γ-tocotrienol and sesamin increased the percentage of cells in G1 phase as compared to the vehicle-treated control group. Western blot studies revealed that blockade in cell cycle progression is associated with decreased expression of cyclin D1, CDK2, CDK4, CDK6, phospho-Rb, and E2F1 transcription factor, and corresponding increase in levels of the cyclin-dependent kinase inhibitor proteins, p27 and p16. Additional Western blot experiments showed that the combined γ-tocotrienol and sesamin treatment caused a marked decrease of ErbB2, ErbB3, and ErbB4 receptors phosphorylation. Furthermore, the combined low dose γ-tocotrienol and sesamin treatment was associated with a relatively large decrease in the intracellular levels of total or phosphorylated c-Raf, Mek1/2, Erk1/2, PI3K, PDK1, Akt, p-NF-κB, Jak1, Jak2, and Stat1 as compared to all other treatment groups. Additional studies were conducted to investigate whether the growth inhibitory effects of the combined treatment is associated with a concomitant activation of apoptotic pathways. Results showed that the combined γ-tocotrienol and sesamin treatment did not activate early or late apoptosis events in the cell indicating that the inhibitory effects of the combined treatment are cytostatic, but not cytotoxic. In summary, these findings indicate that sesamin synergistically increases the anticancer effects of γ-tocotrienol, and combined treatment with these agents may provide some benefits in the treatment of breast cancer in women. This study was supported by a grant from First Tech International Ltd., and the Malaysian Palm Oil Council. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3045. doi:1538-7445.AM2012-3045