The Antioxidant Tempamine:  In Vitro Antitumor and Neuroprotective Effects and Optimization of Liposomal Encapsulation and Release

Abstract

The piperidine nitroxide tempamine (TMN) is a cell-permeable, stable radical having antioxidant, anticancer, and proapoptotic and/or pronecrotic activities, as was demonstrated by us in cell cultures. We also demonstrated synergism between TMN and doxorubicin in doxorubicin-sensitive and doxorubicin-resistant cell lines. Treatment of the C26 mouse colon carcinoma model in vivo also demonstrated synergism between TMN and doxorubicin in sterically stabilized liposomes (SSLs) containing TMN (SSL-TMN) and those containing doxorubicin. The above effects of TMN and SSL-TMN motivated us to develop and optimize the SSL-TMN formulation so that it will be able to reach the disease site with a sufficiently high TMN level and a release rate needed to achieve a therapeutic effect. Because TMN is an amphipathic weak base, it was remote loaded by an intraliposome high/extraliposome low transmembrane ammonium sulfate gradient. The kinetics and level of TMN loading were monitored by cyclic voltammetry (CV) and electron paramagnetic resonance (EPR); the latter also indicates TMN precipitation in the intraliposomal aqueous phase. The regeneration of the original CV and EPR signals by the ionophore nigericin indicates that TMN remained fully intact during loading and release. The cardinal role of the transmembrane ammonium ion gradient in the loading process was proven by the use of the selective ionophores nonactin (for NH4+) and nigericin (for H+). The anion of the ammonium salts affects loading stability and the rate of TMN release, both mediated through the TMN state of aggregation in the intraliposomal aqueous phase. The greater the TMN salt precipitation, the slower the TMN release rate. This was supported by measurement of osmolality, which is inversely related to TMN salt precipitate. Precipitation is in the order SO4(-2)>Cl-1>glucuronate-1. Liposome lipid composition, magnitude of the transmembrane ammonium ion gradient, and type of anion of the ammonium salt determine the amount of TMN loaded and its release rate.

Synthesis and effects of some novel tetrahydronaphthalene derivatives on proliferation and nitric oxide production in lipopolysaccharide activated Raw 264.7 macrophages

“…Cyclic stable nitroxide free radical 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) and its derivative tempamine possess anticancer and antioxidant properties due to its antitumor activities and protective effects against oxidative damage [21,44,45]. New TEMPO-labeled retinoid derivative 27 displayed 18.2% and 59.2% nitrite inhibition at 5 mM and 50 mM concentrations, respectively and therefore seemed to be the most active compound in this series with 59.2% nitrite inhibition.…”

“…A different substitution pattern exists in compounds 16e32 in which the second position of the tetrahydronaphthalene ring bears the substituents instead of the methyl group as seen in the first set of compounds. One of these linked amine compounds is a cyclic, stable nitroxide free radical tempamine which is known to possess antioxidant properties [21]. Thus, the first group of compounds indicate that the NHeCO moiety binds to tetrahydronaphthalene ring at 3rd position whereas the second substitution pattern occurred with the reverse amide formation (COeNH) at the 2nd position of the ring.…”

“…In the present study, we aim to reveal more active and less toxic novel retinoid analogues compared to ATRA. We have prepared a series of novel retinoid compounds consisting of a tetrahydronaphthalene ring system which is integrated with several moieties including alpha-lipoic acid, caffeic acid, ferulic acid, trolox and tempamine residues which have been reported to possess free radical scavenging activities [20,21]. We investigated the effects of these retinoid analogues in vitro on proliferation and NO production in LPS-activated RAW 264.7 macrophages with comparison to known antioxidant compounds.…”

Tailorable nanocarrier emulsion for drug delivery

“…Using such a method, various drugs had been successfully loaded into liposome based on pH (Nichols and Deamer, 1976, Qiu et al, 2008, Dos Santos et al, 2004, Swenson et al, 2001 or ion gradient (Lasic et al, 1995, Wasserman et al, 2007, Clerc and Barenholz, 1998. Intravenously injected liposomes are varied in size, but most of them are smaller than 400 nm to facilitate extravasation into the interstitial space from the bloodstream.…”

“…Drugs can be loaded into liposomes through: i) conventional methods such as thin-film rehydration and the freezing-and-thawing method, where liposomes were formed in the aqueous phase saturated with soluble drugs (Szoka and Papahadjopoulos, 1980, Lasic, 1988, Woodle and Papahadjopoulos, 1989 or ii) the more widely used remote or active loading approach, where drugs are loaded into a preformed liposome by a driving force caused by the trans-membrane gradient (Clerc and Barenholz, 1995, Clerc and Barenholz, 1998, Haran et al, 1993. Using such a method, various drugs had been successfully loaded into liposome based on pH (Nichols and Deamer, 1976, Qiu et al, 2008, Dos Santos et al, 2004, Swenson et al, 2001 or ion gradient (Lasic et al, 1995, Wasserman et al, 2007, Clerc and Barenholz, 1998. Intravenously injected liposomes are varied in size, but most of them are smaller than 400 nm to facilitate extravasation into the interstitial space from the bloodstream.…”