Research PaperPEGylated γ-tocotrienol isomer of vitamin E: Synthesis, characterization, in vitro cytotoxicity, and oral bioavailability
Graphical abstract
Introduction
Vitamin E from palm oil refers to a family of eight related, lipid soluble, compounds that are divided into two subfamilies known as tocopherols (T) and tocotrienols (T3). Each subgroup has α, β, γ, and δ isomers that differ in the number of methyl substitutions on the chromane moiety and the degree of saturation in their phytyl side chain [1].
Vitamin E was initially discovered as an essential factor for reproduction [2]. The antioxidant properties of this compound were later identified [3]. The poor water solubility of vitamin E, however, limited its clinical use [4]. To overcome this limitation, a PEG conjugate of the α-tocopherol isomer of vitamin E, known as d-alpha tocopheryl polyethylene glycol 1000 succinate or simply vitamin E TPGS was synthesized [5]. This water soluble derivative of vitamin E (1 g/10 mL [6]), was successfully used to treat children with low birthweight, and patients with cholestasis [7]. The tocotrienol isomers of vitamin E were only discovered during the 1960s [8], [9], [10]. Remarkably, it was only during the 1990s that the anticancer activities of tocotrienols were identified [11], which established a distinction in the health and therapeutic benefits between the tocotrienol and tocopherol isomers of vitamin E [12].
The γ tocotrienol isomer of vitamin E (γ-T3) was found to have the most promising anticancer activity [11]. Several studies have reported that γ-T3 isomer is the most abundant and active as anticancer agent [1], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22]. Unfortunately, it has also been reported that it is difficult to obtain therapeutic levels of γ-T3 in the blood and target tissues by simple oral administration due to its poor aqueous solubility and limited absorption and oral bioavailability [4], [23], [24], [25], [26]. To overcome these limitations, our laboratory was engaged over the past decade in developing lipid-based delivery systems for the oral and parenteral administration of γ-T3 for use in breast cancer prevention and therapy [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34]. Preliminary results demonstrated that incorporating γ-T3 into lipid-based delivery systems enhances its oral bioavailability and potentiates its anticancer activity [24], [26], [32], [34], [35]. The enhancement in the oral bioavailability of γ-T3, nonetheless, was limited and was found to be as low as 5.6% at a dose of 1 mg/kg of body weight [25]. In addition to its low aqueous solubility, recent in situ permeability studies have revealed that the intestinal permeability of γ-T3 is dose dependent when tested at 1, 2.5, 10, 25 and 50 mg/kg dose intervals [4], [24], [25], [26]. The intestinal absorption of γ -T3 is a saturable process that was found to be mediated by the Niemann-Pick C1-like 1 (NPC1L1) transporter [24], [25], [26]. Moreover, it was found that surfactants (i.e. Cremophor EL (BASF, Mount Olive, NJ) and Labrasol (Gattefossé, Paramus, NJ)) used in lipid-based formulations for the solubilization and oral delivery of γ-T3 caused a reduction in its cellular uptake due to the surfactant-induced inhibition of the NPC1L1 transporter protein resulting in nonlinear absorption kinetics [25], [26]. In addition to passive diffusion and NPC1L1 mediated transport of γ-T3, vitamin E intestinal absorption was also found to be, at least in part, mediated by the scavenger receptor class B type I (SR-B1) protein [4], [23], [24].
In light of the aforementioned issues, we aimed to improve the oral bioavailability of γ-T3 by synthesizing water soluble derivatives. We hypothesized that chemically conjugating γ-T3 to a polyethylene glycol (PEG) moiety via a succinate linker would enhance the solubility, oral bioavailability, and therapeutic activity of γ-T3. PEGylation, the process of covalent attachment of PEG, has already been successfully used to enhance the activity of therapeutic proteins and small organic molecules, such as naloxegol (MOVANTIK™), a PEGylated derivative of naloxone, which was recently approved by the FDA [36], [37]. While the oral bioavailability and pharmaceutical applications of vitamin E TPGS are well documented [5], the bioavailability and therapeutic activity of the PEG conjugates of the tocotrienol isomers have not been previously reported in the literature. Therefore, the objectives of the current study were to (a) synthesize and characterize two PEGylated γ-T3 variants with m-PEG molecular weights of approximately 350 (γ-T3PGS 350) and 1000 (γ-T3PGS 1000); (b) examine the biological activity of the PEGylated γ-T3 by evaluating their in vitro cytotoxic activity against two human breast cancer cell lines (MCF-7 and MDA-MB-231) and (c) determine the oral bioavailability of the PEGylated γ-T3 in rats. To the best of our knowledge this marks the first report on the biological evaluation of PEGylated tocotrienol isomers of vitamin E and their self-assembly in aqueous media.
Section snippets
Materials
Methoxy polyethylene glycols (mPEG 1000 and 350) were provided by INEOS Oxide (Antwerp, Belgium). Tocotrol™ L50P, a tocotrienol rich fraction of palm oil was purchased from Fuji Health Science, Inc. (Burlington, NJ). Triethylamine and succinic anhydride were from Alfa Aesar (Ward Hill, MA). Toluene and Chloroform-d (CDCL3) were from Acros (Bridgewater, NJ). p-Toluenesulfonic acid monohydrate (p-TsOH), hexane, sodium sulfate (Na2SO4) and sodium bicarbonate (NaHCO3) were from Avantor (Center
Extraction of γ-T3
γ-T3 was extracted as a pale yellow viscous oil from Tocotrol™ L50P in multigram quantities (∼25 g). The purity of γ-T3 in the extract was ⩾90% as confirmed by TLC and HPLC analysis (data not shown). The compound 1H NMR spectrum was in agreement with the literature values for the chemical shift of the phytyl chain of γ-T3 at 1.5–2.2 and the aromatic proton at 7–8 ppm [46].
Synthesis and characterization of γ-T3PGS 350 and γ-T3PGS 1000
Two γ-T3PGS variants with mPEG molecular weights of approximately 350 and 1000 were synthesized via a two-step route outlined
Discussion
In the present study we reported on the synthesis and conjugation of PEG to the γ-T3 isomers of vitamin E using a succinate linker. As illustrated in Fig. 1, the existing route to γ-T3PGS 350 and γ-T3PGS 1000 synthesis is straightforward owing to the simple single ring-opening step of half acid ester precursor, followed by controlled coupling to the terminally methylated PEG. Succination and subsequent PEGylation of γ-T3 were confirmed by HPLC, MS, and 1H-NMR studies. For example, the
Conclusion
In the present study the synthesis, physical properties, and biological activity of PEGylated γ-T3 were reported. The succination of γ-T3 and subsequent conjugation to mPEG 350 and 1000 were confirmed by 1H NMR, mass spectroscopy, and HPLC. PEGylation of γ-T3 was found to significantly enhance the oral bioavailability and plasma concentration of γ-T3 and to retain the cytotoxic activity of γ-T3 against breast cancer cells. Overall, PEGylated γ-T3 were similar to vitamin E TPGS in their physical
Acknowledgments
Drs. Patrick O’neal and Pratik Adhikari from the Institute for Micromanufacturing and Biomedical Engineering at Louisiana Tech University are acknowledged for their assistance with fluorescence spectroscopy. Acknowledgment is also extended to Dr. Terje Dokland and Ms. Cynthia Rodenburg for cryo-TEM imaging. The EM work was carried out in the Cryo-EM core facility, Department of Microbiology, at the University of Alabama at Birmingham.
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