Pharmacokinetics, antitumor and cardioprotective effects of liposome-encapsulated phenylaminoethyl selenide in human prostate cancer rodent models.

PURPOSE: Cardiotoxicity associated with the use of doxorubicin (DOX), and other chemotherapeutics, limits their clinical potential. This study determined the pharmacokinetics and antitumor and cardioprotective activity of free and liposome encapsulated phenyl-2-aminoethyl-selenide (PAESe). METHODS: The pharmacokinetics of free PAESe and PAESe encapsulated in liposomes (SSL-PAESe) were determined in rats using liquid chromatography tandem mass-spectrometry. The antitumor and cardioprotective effects were determined in a mouse xenograft model of human prostate (PC-3) cancer and cardiomyocytes (H9C2). RESULTS: The encapsulation of PAESe in liposomes increased the circulation half-life and area under the drug concentration time profile, and decreased total systemic clearance significantly compared to free PAESe. Free- and SSL-PAESe improved survival, decreased weight-loss and prevented cardiac hypertrophy significantly in tumor bearing and healthy mice following treatment with DOX at 5 and 12.5 mg/kg. In vitro studies revealed PAESe treatment altered formation of reactive oxygen species (ROS), cardiac hypertrophy and gene expression, i.e., atrial natriuretic peptide and myosin heavy chain complex beta, in H9C2 cells. CONCLUSIONS: Treatment with free and SSL-PAESe exhibited antitumor activity in a prostate xenograft model and mitigated DOX-mediated cardiotoxicity.

Inhibition of JNK and p38 MAPK phosphorylation by 5-(acetylamino)-4-oxo-6-phenyl-2-hexenoic acid methyl ester and 4-phenyl-butenoic acid decreases substance P-induced TNF-α upregulation in macrophages.

The interactions between the immune and nervous systems play an important role in immune and inflammatory conditions. Substance P (SP), the undecapeptide RPKPQQFFGLM-NH2, is known to upregulate the production of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α. We report here that 5-(acetylamino)-4-oxo-6-phenyl-2-hexenoic acid methyl ester (AOPHA-Me) and 4-phenyl-3-butenoic acid (PBA), two anti-inflammatory compounds developed in our laboratory, reduce SP-stimulated TNF-α expression in RAW 264.7 macrophages. We also show that AOPHA-Me and PBA both inhibit SP-stimulated phosphorylation of JNK and p38 MAPK. Furthermore, molecular modeling studies indicate that both AOPHA-Me and PBA dock at the ATP binding site of apoptosis signal-regulating kinase 1 (ASK1), a member of the MAPKs upstream of both JNK and p38 MAPK, with predicted interaction energies of -7.0 kcal/mol and -5.9 kcal/mol, respectively; this binding overlaps with that of staurosporine, a known inhibitor of ASK1. Taken together, these findings suggest that AOPHA-Me and PBA inhibition of TNF-α expression in SP-stimulated RAW 264.7 macrophages is a consequence of the inhibition of JNK and p38 MAPK phosphorylation. We have previously shown that AOPHA-Me and PBA inhibit the amidative bioactivation of SP, which also would be expected to decrease formation of pro-inflammatory cytokines. It is conceivable that this dual action of inhibiting amidation and MAPK phosphorylation may be of some advantage in enhancing the anti-inflammatory activity of a therapeutic molecule.

Cellular antioxidant activity of phenylaminoethyl selenides as monitored by chemiluminescence of peroxalate nanoparticles and by reduction of lipopolysaccharide-induced oxidative stress.

Hydrogen peroxide (H2O2), produced in living cells by oxidases and by other biochemical reactions, plays an important role in cellular processes such as signaling and cell cycle progression. Nevertheless, H2O2 and other reactive oxygen species are capable of inducing damage to DNA and other cellular components, and oxidative stress caused by overproduction of cellular oxidants has been linked to pathologies such as inflammatory diseases and cancer. Therefore, new approaches for reducing the accumulation of cellular oxidants are of considerable interest from both a biotechnological and a therapeutic perspective. Recognizing that selenium is an essential component of the active sites of several antioxidant enzymes, we have developed a family of novel phenylaminoethyl selenide compounds that are readily taken up into cells and have low toxicity in vivo. We now report chemiluminescent imaging of hydrogen peroxide consumption by phenylaminoethyl selenides, via the use of peroxalate nanoparticle methodology. Further, we demonstrate the ability of phenylaminoethyl selenides to decrease lipopolysaccharide-induced oxidative stress in human embryonic kidney cells. We also report the successful encapsulation of a phenylaminoethyl selenide within poly(lactide-co-glycolide) nanoparticles, and we show that these selenide-loaded nanoparticles exhibit antioxidant activity in cells. Taken together, these results significantly enhance the attractiveness of phenylaminoethyl selenides as potential agents for supplementing cellular defenses against reactive oxygen species.

Myo-inositol hexakisphosphate, isolated from female gametophyte tissue of loblolly pine, inhibits growth of early-stage somatic embryos.

• Myo-inositol hexakisphosphate (InsP(6)), abundant in animals and plants, is well known for its anticancer activity. However, many aspects of InsP(6) function in plants remain undefined. We now report the first evidence that InsP(6) can inhibit cellular proliferation in plants under growth conditions where phosphorus is not limited. • A highly anionic molecule inhibitory to early-stage somatic embryo growth of loblolly pine (LP) was purified chromatographically from late-stage LP female gametophytes (FGs), and then characterized structurally using mass spectrometry (MS) and nuclear magnetic resonance (NMR) analyses. • Exact mass and mass spectrometry-mass spectrometry (MS-MS) fragmentation identified the bioactive molecule as an inositol hexakisphosphate. It was then identified as the myo-isomer (i.e. InsP(6)) on the basis of (1)H-, (31)P- and (13)C-NMR, (1)H-(1)H correlation spectroscopy (COSY), (1)H-(31)P heteronuclear single quantum correlation (HSQC) and (1)H-(13)C HSQC. Topical application of InsP(6) to early-stage somatic embryos indeed inhibits embryonic growth. • Recently evidence has begun to emerge that InsP(6) may also play a regulatory role in plant cells. We anticipate that our findings will help to stimulate additional investigations aimed at elucidating the roles of inositol phosphates in cellular growth and development in plants.

The antioxidant phenylaminoethyl selenide reduces doxorubicin-induced cardiotoxicity in a xenograft model of human prostate cancer.

Anthracyclines are potent anticancer agents, but cardiotoxicity mediated by free radical generation limits their clinical use. This study evaluated the anticancer activity of phenyl-2-aminoethyl selenide (PAESe) and its potential to reduce doxorubicin (DOX)-induced cardiotoxicity. Growth inhibitory effects of PAESe with DOX, and vincristine, clinically used anticancer agents, and tert-butylhydroperoxide (TBHP), a known oxidant, on the growth of human prostate carcinoma (PC-3) cells was determined. PAESe (≤1µm) did not alter the growth of PC-3 cells, however, concomitant use of PAESe decreased the oxidative-mediated cytotoxicity of TBHP, but had limited effect on vincristine or DOX activity. Further, PAESe decreased the formation of intracellular reactive oxygen species from TBHP and DOX. The effect of PAESe on the activity of DOX was determined using a tumor (PC-3) xenograft model in mice. PAESe did not alter DOX antitumor activity and showed evidence of direct antitumor activity relative to controls. DOX treatment decreased mice body weight significantly, whereas concomitant administration of PAESe and DOX was similar to controls. Most importantly, PAESe decreased DOX-mediated infiltration of neutrophil and macrophages into the myocardium. These data suggest PAESe had in vivo antitumor activity and in combination with DOX decreased early signs of cardiotoxicity while preserving its antitumor activity.

Identification of a thioselenurane intermediate in the reaction between phenylaminoalkyl selenoxides and glutathione.

Selenium has a long history of association with human health and disease, and a low concentration of selenium in plasma has been identified in epidemiological studies as a risk factor for several disorders associated with oxidative stress. This association suggests that organoselenium compounds capable of propagating a selenium redox cycle might supplement natural cellular defenses against oxidants, such as peroxynitrite and hydrogen peroxide. While several such organoselenium compounds are under active investigation as potential therapeutic agents, chemical characterization of reaction intermediates involved in their redox cycling has been problematical. We now report evidence that the reaction between phenylaminoalkyl selenoxides and glutathione (GSH) proceeds through the intermediacy of a thioselenurane species. The results of stopped-flow kinetic experiments were consistent with a rapid and stoichiometric initial reaction of GSH with selenoxide to generate a kinetically-detectable intermediate, followed by a slower reaction of this intermediate with a second molecule of GSH to produce the final selenide and GSSG products. Flow injection ESI-MS and ESI-MS/MS experiments confirmed that the reaction intermediate is indeed a thioselenurane. Final structural characterization of the thioselenurane intermediate was obtained from analysis of the daughter ions produced in flow injection ESI-MS/MS experiments. These results help to elucidate the chemical nature of the redox cycling of phenylaminoalkyl selenides, and represent, to our knowledge, the first evidence for the intermediacy of a thioselenurane species in the reaction of thiols with selenoxides.

L-Phenylalanine concentration in blood of phenylketonuria patients: a modified enzyme colorimetric assay compared with amino acid analysis, tandem mass spectrometry, and HPLC methods.

BACKGROUND: Phenylketonuria (PKU) is an autosomal recessive disorder caused by an impaired conversion of L-phenylalanine (Phe) to L-tyrosine, typically resulting from a deficiency in activity of a hepatic and renal enzyme L-phenylalanine hydroxylase. The disease is characterized by an increased concentration of Phe and its metabolites in body fluids. METHODS: A modified assay based on an enzymatic-colorimetric methodology was developed for measuring blood Phe levels in PKU patients; this method is designed for use with undeproteinized samples and avoids the use of solvents or amphiphilic agents. Thus, the method could be suitable for incorporation into a simple home-monitoring device. RESULTS: We report here on a comparison of blood Phe concentrations in PKU patients measured in undeproteinized plasma using this enzyme colorimetric assay (ECA), with values determined by amino acid analysis (AAA) of deproteinized samples, and HPLC and tandem mass spectrometry (MS/MS) analyses of dried blood spot (DBS) eluates. Pearson correlation coefficients of 0.951, 0.976 and 0.988 were obtained when AAA-measured Phe concentrations were compared with the ECA-, HPLC- or MS/MS-measured values, respectively. A Bland-Altman analysis revealed that mean Phe concentrations determined using AAA were on average 65 µmol/L lower than values measured by our ECA. These results may be the result of minimizing the manipulations performed on the patient sample compared with AAA, HPLC, and MS/MS methods, which involve plasma deproteinization or DBS elution and derivatization. CONCLUSIONS: The results reported here confirm that Phe concentrations determined by our ECA method are comparable to those determined by other widely used methods for a broad range of plasma Phe concentrations.

Isolation and characterization of a molecule stimulatory to growth of somatic embryos from early stage female gametophyte tissue of loblolly pine.

Loblolly pine (LP, Pinus taeda) is the primary commercial species in southern forests of the US. Somatic embryogenesis (SE) is an effective technique to implement clonal tree production of high-value genotypes from breeding and genetic engineering programs. Unlike angiosperm embryos with attached cotyledons as seed storage organs, the diploid conifer embryo is surrounded by the unattached haploid female gametophyte (FG). The FG is not present in culture. This presents a dilemma if the FG produces necessary or regulatory compounds for embryo growth, since in culture these important compounds would be missing and would have to be added as supplements. We report here the direct evidence that extracts from early-stage FG indeed stimulate early-stage somatic embryo (SME) growth and multiplication, whereas extracts from late-stage FG inhibit early-stage SME growth. Furthermore, we have now isolated this stimulatory substance from early-stage FG tissue, and identified this substance as citric acid on the basis of NMR and mass spectrometry. We then demonstrated that topical application of citric acid to SMEs stimulates embryo colony growth at P = 0.05. Moreover, we find that there is a good correlation between the amount of citric acid isolated from FG tissue (65 nmoles per stage 2-3 FG) and the amount of citric acid that stimulates colony growth (25-50 nmoles) when applied topically to SMEs. This approach of isolating and characterizing a molecule from plant tissue, and investigating its role on SE processes can provide valuable information leading to further applications of these molecules to improve LP SE protocols.