Novel astaxanthin prodrug (CDX-085) attenuates thrombosis in a mouse model.

BACKGROUND: Cardiovascular disease remains the leading cause of morbidity and premature mortality in most industrialized countries as well as in developing nations. A pro-oxidative state appears to promote and/or exacerbate vascular disease complications. Furthermore, a state of low-grade chronic inflammation can promote increased oxidative stress and lead to endothelial cell and platelet dysfunction ultimately contributing to thrombogenesis. OBJECTIVES: In this study, the effect of a proprietary astaxanthin prodrug (CDX-085) on thrombus formation was investigated using a mouse model of arterial thrombosis. The influence of free astaxanthin, the active drug of CDX-085, on human endothelial cells and rat platelets was evaluated to investigate potential mechanisms of action. METHODS AND RESULTS: Oral administration of CDX-085 (0.4% in chow, approximately 500 mg/kg/day) to 6-8 week old C57BL/6 male mice for 14 days resulted in significant levels of free astaxanthin in the plasma, liver, heart and platelets. When compared to control mice, the CDX-085 fed group exhibited significant increases in basal arterial blood flow and significant delays in occlusive thrombus formation following the onset of vascular endothelial injury. Primary human umbilical vein endothelial cells (HUVECs) and platelets isolated from Wistar-Kyoto rats treated with free astaxanthin demonstrated significantly increased levels of released nitric oxide (NO) and significantly decreased peroxynitrite (ONOO-) levels. CONCLUSION: Observations of increased NO and decreased ONOO- levels in endothelial cells and platelets support a potential mechanism of action for astaxanthin (CDX-085 active drug). These studies support the potential of CDX-085 and its metabolite astaxanthin in the treatment or prevention of thrombotic cardiovascular complications.

Hydrophilic carotenoids: surface properties and aggregation of an astaxanthin-lysine conjugate, a rigid, long-chain, highly unsaturated and highly water-soluble tetracationic bolaamphiphile.

The surface and aggregation properties of a synthetic, highly water-soluble carotenoid, the tetracationic astaxanthin-lysine conjugate (Asly), have been examined through measurements of surface tension, optical absorption and dynamic light scattering. The following parameters were determined: critical aggregation concentration c(M), surface concentration Gamma, molecular area a(m), free energy of adsorption and aggregation (DeltaG(ad) degrees and DeltaG(M) degrees , respectively), and the aggregate size r(H). The compound forms true monomolecular solutions in water below c(M); aggregates emerge only at rather high concentrations (> or =2.18 mM).

Electron- and energy-transfer properties of hydrophilic carotenoids.

The antioxidant activities-expressed as the electron-donating properties-of five hydrophilic carotenoids (carotenoid surfactants) and three related hydrophobic carotenoids were investigated by flash photolysis. The electron-transfer rates of the carotenoids to the triplet state of the sensitizer 2-nitronaphthalene and the energy transfer rates of triplet 2-nitronaphthalene to the carotenoids were determined. The results demonstrate that the electron-donating effects of the hydrophilic and hydrophobic carotenoids were comparable when evaluated in acetonitrile. In the presence of water, however, electron transfer (i.e., antioxidant efficiency) was enhanced by a factor of four for the hydrophilic carotenoids. The increased hydrophilicity of carotenoids, therefore, could expand their antioxidant properties, thus facilitating their use as aqueous-phase radical scavengers. At the same time, it was shown that supramolecular assembly ("aggregation") of the amphiphilic carotenoids prevented electron transfer, thus deactivating the antioxidant function. Modulation of the biophysical properties of carotenoids through synthetic modification is capable of increasing the biological and medical utility of this natural class of predominantly hydrophobic antioxidant compound.

Differential effects of carotenoids on lipid peroxidation due to membrane interactions: X-ray diffraction analysis.

The biological benefits of certain carotenoids may be due to their potent antioxidant properties attributed to specific physico-chemical interactions with membranes. To test this hypothesis, we measured the effects of various carotenoids on rates of lipid peroxidation and correlated these findings with their membrane interactions, as determined by small angle X-ray diffraction approaches. The effects of the homochiral carotenoids (astaxanthin, zeaxanthin, lutein, beta-carotene, lycopene) on lipid hydroperoxide (LOOH) generation were evaluated in membranes enriched with polyunsaturated fatty acids. Apolar carotenoids, such as lycopene and beta-carotene, disordered the membrane bilayer and showed a potent pro-oxidant effect (>85% increase in LOOH levels) while astaxanthin preserved membrane structure and exhibited significant antioxidant activity (40% decrease in LOOH levels). These findings indicate distinct effects of carotenoids on lipid peroxidation due to membrane structure changes. These contrasting effects of carotenoids on lipid peroxidation may explain differences in their biological activity.

Retrometabolic syntheses of astaxanthin (3,3'-dihydroxy-beta,beta-carotene-4,4'-dione) conjugates: a novel approach to oral and parenteral cardio-protection.

Disodium disuccinate astaxanthin has potent cardioprotective effects in animals, with demonstrated preclinical efficacy in the rat, rabbit, and canine models of experimental infarction. It has been effective in subchronic and acute dosing regimens after parenteral administration, and recently published data in rats demonstrate that oral cardioprotection is also readily achieved. Myocardial salvage in the canine can reach 100% with a 4-day subchronic dosing regimen; single-dose I.V. cardioprotection, when given 2 hours before experimental coronary occlusion, is on average two-thirds of that achieved with the subchronic regimen in dogs. In conscious animals, no effects on hemodynamic parameters have been observed. Recently, the beneficial properties of this prototypical astaxanthin conjugate have been extended to include second- and third-generation compounds with improved pharmacokinetic and/or potency profiles. The primary mechanism of cardioprotection appears to be antioxidant activity: potent direct scavenging of the lynchpin radical in ischemia-reperfusion injury, superoxide anion, has been documented in appropriate model systems. In addition, modulation of serum complement activity, reduction of the levels of deposition of C-reactive protein (CRP) and the membrane attack complex (MAC) in infarcted tissue, and reduction in oxidative stress markers from the arachidonic acid and linoleic acid pathways also suggest a significant anti-inflammatory component to the mechanism of cardioprotection. Favorable plasma protein binding has been demonstrated in vitro for several astaxanthin conjugates; this binding capacity overcomes the supramolecular assembly of the compounds that occurs in aqueous solution, which in itself improves the stability and shelf-life of aqueous formulations. Astaxanthin readily populates cardiac tissue after metabolic hydrolysis of both oral and parenteral administration of the astaxanthin ester derivates, providing a reservoir of cardioprotective agent with a significant half-life due to favorable ADME in mammals. Due to the well-documented safety profile of astaxanthin in humans, disodium disuccinate astaxanthin may well find clinical utility in cardiovascular applications in humans following successful completion of preclinical and clinical pharmacology and toxicology studies in animals and humans, respectively.

Hydrophilic carotenoid amphiphiles: methods of synthesis and biological applications.

Carotenoid bioactivity, namely the quenching of reactive oxygen species (ROS) and other excited-state oxidants, suggests significant clinical potential for these natural products. However, a thorough therapeutic evaluation of the carotenoids has been hampered by limited water-solubility and/or water-dispersibility ("hydrophilicity") as well as poor bioavailability. Hydrophilic carotenoid derivatives have been designed and prepared for parenteral administration. Synthetic methods, selected physical characteristics, and potential biological applications of these novel therapeutics will be discussed.

Molecular modeling of the non-covalent binding of the dietary tomato carotenoids lycopene and lycophyll, and selected oxidative metabolites with 5-lipoxygenase.

Numerous studies on human prostate cancer cell lines indicate a role for arachidonic acid (AA) and its oxidative metabolites in prostate cancer proliferation. The metabolism of AA by either the cyclooxygenase (COX) or the lipoxygenase (LOX) pathways generates eicosanoids involved in tumor promotion, progression, and metastasis. In particular, products of the 5-LOX pathway (including 5-HETE and 5-oxo-EET) have been implicated as potential 'survival factors' that may confer escape after androgen withdrawal therapy through fatty-acid (i.e., AA) drive. Potent natural dietary antioxidant compounds such as lycopene and lycophyll, with tissue tropism for human prostate, have been shown to be effective in ameliorating generalized oxidative stress at the DNA level. Suppressing the 5-LOX axis pharmacologically is also a promising avenue for intervention in human patients. The recently recognized direct interaction of the astaxanthin-based soft-drug Cardax to human 5-LOX with molecular modeling, and the downregulation of both 5-HETE and 5-oxo-EET in vivo in a murine peritonitis model, suggest that other important dietary carotenoids may share this enzyme regulatory feature. In the current study, the acyclic tomato carotene lycopene (in all-trans and 5-cis isomeric configurations) and its natural dihydroxy analog lycophyll (also present in tomato fruit) were subjected to molecular modeling calculations in order to investigate their predicted binding interaction(s) with human 5-LOX. Two bioactive oxidative metabolites of lycopene (4-methyl-8-oxo-2,4,6-nonatrienal and 2,7,11-trimethyl-tetradecahexaene-1,14-dial) were also investigated. A homology model of 5-LOX was constructed using 8-LOX and 15-LOX structures as templates. The model was validated by calculating the binding energy of Cardax to 5-LOX, which was demonstrated to be in good agreement with the published experimental data. Blind docking calculations were carried out in order to explore the possible binding sites of the carotenoids on 5-LOX, followed by focused docking to more accurately calculate the predicted energy of binding. Lycopene and lycophyll were predicted to bind with high affinity in the superficial cleft at the interface of the beta-barrel and the catalytic domain of 5-LOX (the 'cleavage site'). Carotenoid binding at this cleavage site provides the structural rationale by which polyenic compounds could modify the 5-LOX enzymatic function via an allosteric mechanism, or by radical scavenging in proximity to the active center. In addition, the two bioactive metabolites of lycopene were predicted to bind to the catalytic site with high affinity--therefore suggesting potential direct competitive inhibition of 5-LOX activity that should be shared by both lycopene and lycophyll after in vivo supplementation, particularly in the case of the dial metabolite.

Rofecoxib increases susceptibility of human LDL and membrane lipids to oxidative damage: a mechanism of cardiotoxicity.

Clinical investigations have demonstrated a relationship between the extended use of rofecoxib and the increased risk for atherothrombotic events. This has led to the removal of rofecoxib from the market and concern over the cardiovascular safety of other cyclooxygenase (COX)-2 selective agents. Experimental findings from independent laboratories now indicate that the cardiotoxicity of rofecoxib may not be a class effect but because of its intrinsic chemical properties. Specifically, rofecoxib has been shown to increase the susceptibility of human low-density lipoprotein and cellular membrane lipids to oxidative modification, a contributing factor to plaque instability and thrombus formation. Independently of COX-2 inhibition, rofecoxib also promoted the nonenzymatic formation of isoprostanes and reactive aldehydes from biologic lipids. The basis for these observations is that rofecoxib alters lipid structure and readily forms a reactive maleic anhydride in the presence of oxygen. By contrast, other selective (celecoxib, valdecoxib) and nonselective (naproxen, diclofenac) inhibitors did not influence rates of low-density lipoprotein and membrane lipid oxidation. We have now further confirmed these findings by demonstrating that the prooxidant activity of rofecoxib can be blocked by the potent antioxidant astaxanthin in homochiral form (all-trans 3S, 3'S). These findings provide a mechanistic rationale for differences in cardiovascular risk among COX-selective inhibitors because of their intrinsic physicochemical properties.

Association studies of aggregated aqueous lutein diphosphate with human serum albumin and alpha1-acid glycoprotein in vitro: evidence from circular dichroism and electronic absorption spectroscopy.

Water-dispersible C40 carotenoid derivatives, with increased utility in mammalian therapeutic applications, include natural stereoisomer-based (3R,3'R,6'R)-lutein (beta,epsilon-carotene-3,3'-diol) derivatives. Esterification with inorganic phosphate and conversion to the sodium salt produced compounds (lutein diphosphate sodium salt; 'LdP') capable of forming red-orange aqueous suspensions after addition to USP-purified water. The aqueous dispersibility of this diphosphate salt reached 29 mg/mL without the addition of heat, detergents, co-solvents, or other additives, and was a potent direct scavenger of superoxide anion (by EPR spectroscopy) in an isolated human neutrophil assay. In the current study, preliminary evidence of the aqueous aggregation of this compound in EPR studies was confirmed using circular dichroism (CD) and electronic absorption (UV-vis) spectroscopy. Evidence for H-type ('card-pack') and J-type ('head-to-tail') self-assemblies was obtained. In vitro analysis of the potential binding interaction between LdP and human serum albumin (HSA) and alpha1-acid glycoprotein (AGP) revealed only non-specific binding with HSA (and none with AGP), contrasting with previous reports of direct interaction between astaxanthin-based soft drugs and the major plasma protein albumin. The rapid in vivo cleavage of this phosphodiester by promiscuous mammalian phosphatases may overcome the aqueous aggregation of the formulated compound. This difference in potential plasma protein interaction with prior reports reflects the subtle structural differences inherent in either the parent carotenoid scaffolds and/or the esterifying moieties.

Molecular modeling of non-covalent binding of homochiral (3S,3'S)-astaxanthin to matrix metalloproteinase-13 (MMP-13).

Inhibitors for matrix metalloproteinases (MMPs) are under investigation for the treatment of various important chronic illnesses, including cancer, arthritis, and cardiovascular disease (CVD). In particular, MMP-13 is currently being probed as a potential key target in CVD and malignant disease due to its documented effects on extracellular matrix (ECM) remodeling, important in the pathophysiology of these diseases. Within the family of related mammalian MMP enzymes, MMP-13 possesses a large hydrophobic binding pocket relative to that of other MMPs. Homochiral astaxanthin (3S,3'S-AST; 3S,3'S-dihydroxy-beta,beta-carotene-4,4'-dione), an important antioxidant and anti-inflammatory xanthophyll carotenoid, is an active metabolite of several novel soft drugs in clinical development; it is also extensively used and tested as a human nutraceutical. In the current study, the prediction of the geometry and energetics of its binding to human MMP-13 was conducted with molecular modeling. The method used was found to predict the energy of binding of known ligands of MMP-13 with great precision. Blind docking using the whole protein target was then used in order to identify the possible binding site(s) of AST. AST was predicted to bind at several sites in close proximity to the active center. Subsequent analyses focused on the binding site at the atomic (i.e., amino acid sequence) level suggested that AST can bind to MMP-13 with high affinity and favorable energetics. Therefore, the modeling study predicts potential direct enzyme-inhibitory activity of AST against MMP-13, a behavior that may be exploited in mammalian systems in which pathological upregulation of MMP activity is paramount.

Purification of synthetic all-E lycophyll (psi,psi-carotene-16,16'-diol).

An efficient purification of synthetic all-trans (all-E) lycophyll is described. The synthetic preparation of the rare xanthophyll lycophyll produces a mixture of geometric isomers. Purification by HPLC using reverse-phase C30 silica affords milligram quantities of the desired all-trans isomer in >95% purity, as confirmed by (1)H NMR and LC/MS. Most recently, a facile work-up of the geometric mixture formed during total synthesis was found to provide multigrams of the targeted all-E geometric isomer of lycophyll. The acquisition of modest quantities of this specific lycopene analog allows its therapeutic potential to be explored.

The effects of oral Cardax (disodium disuccinate astaxanthin) on multiple independent oxidative stress markers in a mouse peritoneal inflammation model: influence on 5-lipoxygenase in vitro and in vivo.

Disodium disuccinate astaxanthin ('rac'-dAST; Cardax) is a water-dispersible C40 carotenoid derivative under development for oral and parenteral administration for cardioprotection of the at-risk ischemic cardiovascular patient. In experimental infarction models in animals (rats, rabbits, and dogs), significant myocardial salvage has been obtained, up to 100% at the appropriate dose in dogs. The documented mechanism of action in vitro includes direct scavenging of biologically produced superoxide anion; in vivo in rabbits, modulation of the complement activity of serum has also been shown. A direct correlation between administration of the test compound in animals and reductions of multiple, independent markers of oxidative stress in serum was recently obtained in a rat experimental infarction model. For the current study, it was hypothesized that oral Cardax administration would inhibit oxidative damage of multiple relevant biological targets in a representative, well-characterized murine peritoneal inflammation model. A previously developed mass spectrometry-based (LC/ESI/MS/MS) approach was used to interrogate multiple distinct pathways of oxidation in a black mouse (C57/BL6) model system. In vivo markers of oxidant stress from peritoneal lavage samples (supernatants) were evaluated in mice on day eight (8) after treatment with either Cardax or vehicle (lipophilic emulsion without drug) orally by gavage at 500 mg/kg once per day for seven (7) days at five (5) time points: (1) baseline prior to treatment (t=0); (2) 16 h following intraperitoneal (i.p.) injection with thioglycollate to elicit a neutrophilic infiltrate; (3) 4 h following i.p. injection of yeast cell wall (zymosan; t=16 h/4 h thioglycollate+zymosan); (4) 72 h following i.p. injection with thioglycollate to elicit monocyte/macrophage infiltration; and (5) 72 h/4 h thioglycollate+zymosan. A statistically significant sparing effect on the arachidonic acid (AA) and linoleic acid (LA) substrates was observed at time points two and five. When normalized to the concentration of the oxidative substrates, statistically significant reductions of 8-isoprostane-F(2alpha) (8-iso-F(2alpha)) at time point three (maximal neutrophil recruitment/activation), and 5-HETE, 5-oxo-EET, 11-HETE, 9-HODE, and PGF(2alpha) at time point five (maximal monocyte/macrophage recruitment/activation) were observed. Subsequently, the direct interaction of the optically inactive stereoisomer of Cardax (meso-dAST) with human 5-lipoxygenase (5-LOX) was evaluated in vitro with circular dichroism (CD) and electronic absorption (UV/Vis) spectroscopy, and subsequent molecular docking calculations were made using mammalian 15-LOX as a surrogate (for which XRC data has been reported). The results suggested that the meso-compound was capable of interaction with, and binding to, the solvent-exposed surface of the enzyme. These preliminary studies provide the foundation for more detailed evaluation of the therapeutic effects of this compound on the 5-LOX enzyme, important in chronic diseases such as atherosclerosis, asthma, and prostate cancer in humans.

Seven day oral supplementation with Cardax (disodium disuccinate astaxanthin) provides significant cardioprotection and reduces oxidative stress in rats.

In the current study, the improved oral bioavailability of a synthetic astaxanthin derivative (Cardax; disodium disuccinate astaxanthin) was utilized to evaluate its potential effects as a cardioprotective agent after 7-day subchronic oral administration as a feed supplement to Sprague-Dawley rats. Animals received one of two concentrations of Cardax in feed (0.1 and 0.4%; approximately 125 and 500 mg/kg/day, respectively) or control feed without drug for 7 days prior to the infarct study carried out on day 8. Thirty minutes of occlusion of the left anterior descending (LAD) coronary artery was followed by 2 h of reperfusion prior to sacrifice, a regimen which resulted in a mean infarct size (IS) as a percentage (%) of the area at risk (AAR; IS/AAR,%) of 61 +/- 1.8%. The AAR was quantified by Patent blue dye injection, and IS was determined by triphenyltetrazolium chloride (TTC) staining. Cardax at 0.1 and 0.4% in feed for 7 days resulted in a significant mean reduction in IS/AAR,% to 45 +/- 2.0% (26% salvage) and 39 +/- 1.5% (36% salvage), respectively. Myocardial levels of free astaxanthin achieved after 7-day supplementation at each of the two concentrations (400 +/- 65 nM and 1634 +/- 90 nM, respectively) demonstrated excellent solid-tissue target organ loading after oral supplementation. Parallel trends in reduction of plasma levels of multiple lipid peroxidation products with disodium disuccinate astaxanthin supplementation were observed, consistent with the documented in vitro antioxidant mechanism of action. These results extend the potential utility of this compound for cardioprotection to the elective human cardiovascular patient population, for which 7-day oral pre-treatment (as with statins) provides significant reductions in induced periprocedural infarct size.

The synthesis and aqueous superoxide anion scavenging of water-dispersible lutein esters.

Xanthophyll carotenoids of the C40 series, which includes commercially important compounds such as lutein, zeaxanthin, and astaxanthin, have poor aqueous solubility in the native state. Hawaii Biotech, Inc. (HBI) and others have shown that the aqueous dispersibility of derivatized carotenoids can be increased by varying the chemical structure of the esterified moieties. In the current study, the published series of novel, highly water-dispersible C40 carotenoid derivatives has been extended to include (3R,3'R,6'R)-lutein (beta,epsilon-carotene-3,3'-diol) derivatives. Two novel derivatives were synthesized by esterification with inorganic phosphate and succinic acid, respectively, and subsequently converted to the sodium salts. Red-orange, clear, aqueous suspensions were obtained after addition of these novel derivatives to USP-purified water. Aqueous dispersibility of the disuccinate sodium salt of lutein was 2.85 mg/mL; the diphosphate salt demonstrated a >10-fold increase in dispersibility at 29.27 mg/mL. As reported previously, these aqueous suspensions were obtained without the addition of heat, detergents, co-solvents, or other additives. The direct aqueous superoxide scavenging abilities of these novel derivatives were subsequently evaluated by electron paramagnetic resonance (EPR) spectroscopy in a well-characterized in vitro isolated human neutrophil assay. The novel derivatives were nearly identical aqueous-phase scavengers, demonstrating dose-dependent suppression of the superoxide anion signal (as detected by spin-trap adducts of DEPMPO) in the millimolar range. These lutein-based soft drugs will likely find utility in those commercial and clinical applications for which aqueous-phase singlet oxygen quenching and direct radical scavenging may be required.

Disodium disuccinate astaxanthin (Cardax): antioxidant and antiinflammatory cardioprotection.

Disodium disuccinate astaxanthin (Cardax), DDA) has cardioprotective effects in the rat, rabbit, and canine models of experimental infarction. It is highly effective by parenteral administration in subchronic and acute dosing regimens. Unpublished data in rats suggest that oral cardioprotection is also readily achievable. DDA-induced myocardial salvage in the canine can reach 100% with a 4-day subchronic dosing regimen. At a single i.v. dose DDA is cardioprotective, when given 2 h before experimental coronary occlusion, but the protection is on the average two-thirds of that achieved with the subchronic regimen in dogs. In conscious animals DDA has no effects on hemodynamic parameters. The primary mechanism of cardioprotection appears to be antioxidant activity involving direct scavenging of superoxide anion, the lynchpin radical in ischemia-reperfusion injury. In addition, modulation of serum complement activity, as well as the reduction in the levels of C-reactive protein (CRP) and the membrane attack complex (MAC) in infarcted tissue suggest a significant antiinflammatory component in the mechanism of cardioprotective action of DDA. Stoichiometric binding of the meso-form of the compound to human serum albumin (HSA) has been demonstrated in vitro. This binding capacity overcomes the supramolecular assembly of the compound in aqueous solution, which by itself improves the stability and shelf life of aqueous formulations. Non-esterified astaxanthin readily enters cardiac tissue after either oral or parenteral administration, providing a reservoir of a cardioprotective agent with a significant half-life due to favorable ADME in mammals. Due to the well-documented safety profile of non-esterified astaxanthin in humans, disodium disuccinate astaxanthin may well find clinical utility in cardiovascular indications in humans following successful completion of preclinical and clinical pharmacology and toxicology studies.

Circular dichroism and absorption spectroscopic data reveal binding of the natural cis-carotenoid bixin to human alpha1-acid glycoprotein.

Using circular dichroism (CD) and electronic absorption spectroscopy techniques, interaction of the natural dietary cis-carotenoid bixin with an important human plasma protein in vitro was demonstrated for the first time. The induced CD spectra of bixin obtained under physiological conditions (pH 7.4, 37 degrees C) revealed its binding to the serum acute-phase reactant alpha(1)-acid glycoprotein (AGP), a member of the lipocalin protein family. Spectral features of the extrinsic Cotton effects of bixin suggested the inclusion of a single, chirally distorted ligand molecule into the asymmetric protein environment. Compared with the absorption spectra obtained in ethanol and benzene, the strong red shift of the main absorption peak of AGP-bound bixin indicated that the proposed binding site was rich in aromatic residues, and also suggested that hydrophobic interactions were involved in the binding. Using the data obtained from the CD titration experiments, the association constant (Ka=4.5x10(5)M-1) and stoichiometry of the binding (0.15) were calculated. The low value of the stoichiometry was attributed to the structural polymorphism of AGP. To the authors' knowledge, the current study represents the first human lipocalin protein for which carotenoid binding affinity has been explored in vitro with these techniques.

In vitro binding of leukotriene B4 (LTB4) to human serum albumin: evidence from spectroscopic, molecular modeling, and competitive displacement studies.

Circular dichroism (CD) and UV absorption spectroscopy were utilized for the first time to investigate the interaction between leukotriene B4 (LTB4) and human serum albumin (HSA) in vitro. The weak intrinsic CD signal of LTB4 was enhanced fivefold in the presence of HSA. The red-shifted, hypochromic, and reduced vibrational fine structure of the ligand/protein UV absorption spectrum indicated complexation of the two molecules in solution. Results obtained from CD titration experiments were subjected to non-linear regression analysis to estimate the binding parameters (Ka = 6.7 x 10(4) M(-1), n = 1). Palmitic acid strongly decreased the induced CD signal of the LTB4/HSA complex, suggesting the role of a high-affinity fatty acid HSA binding site in the leukotriene complexation. Molecular modeling calculations based on the crystal structure of HSA predicted that the long-chain fatty acid site that overlaps with drug binding site II in subdomain IIIA was the most likely binding location for LTB4. Using the drug site II-specific marker ligand rac-ibuprofen, this prediction was confirmed with induced-CD displacement measurements. To the authors' knowledge, the current study represents the first demonstration of binding of LTB4 to HSA in vitro and has implications for the biological transport of this important pro-inflammatory mediator in vivo.

Acute and chronic administration of disodium disuccinate astaxanthin (Cardax) produces marked cardioprotection in dog hearts.

Previous results from our laboratory have shown that a novel carotenoid derivative (disodium disuccinate astaxanthin; Cardax) produced dose-related reductions in myocardial infarct size (IS) in Sprague-Dawley rats when it was administered at any of three doses (25, 50 and 75 mg/kg, iv) on four consecutive days, followed by the acute infarct size study on day 5. Maximum salvage occurred at the highest dose (75 mg/kg) tested, and was shown as a 56% reduction in IS. In the present follow-up study, we used a more relevant large animal model, the dog, and looked at the effect of administering Cardax iv either acutely 2 h prior to occlusion (N = 8) or for 4 days at 50 mg/kg iv as previously done in the rat model (N = 6). The results were compared to a saline vehicle-treated group (N = 10). In all groups, dogs were subjected to 60 min of left anterior descending (LAD) coronary artery occlusion and 3 h of reperfusion. IS was determined using a triphenyltetrazolium chloride (TTZ) histochemical stain and was expressed as a percent of the area at risk (IS/AAR). IS/AAR was 20.9 +/- 1.6 % (mean +/- S.E.M.) in controls and was reduced to 11.0 +/- 1.7% (47.3% salvage; p < 0.01) in dogs treated only once iv at 2 h prior to occlusion, and 6.6 +/- 2.8% (68.4% salvage; p < 0.001) in dogs treated for 4 days. In the chronic treatment group, two of the three dogs with plasma concentrations of non-esterified astaxanthin above 1 microM had 0% IS/AAR (100% cardioprotection). These results suggest that Cardax has marked cardioprotective properties in both rodents and canines. Thus, Cardax may be a novel and powerful new means to prevent myocardial injury and/or necrosis associated with elective and/or urgent cardiac surgical interventions such as coronary angioplasty and stenting, as well as coronary artery bypass surgery (CABG).

Hydrophilic carotenoids: surface properties and aqueous aggregation of a rigid, long-chain, highly unsaturated dianionic bolaamphiphile with a carotenoid spacer.

The water dispersibility of astaxanthin was greatly enhanced by converting it to a disodium disuccinate salt. This carotenoid salt behaved as a bolaamphiphile in water; dynamic light scattering (DLS) revealed the formation of stable aggregates with an average hydrodynamic radius close to 1 microm. Larger aggregates were observed in solutions of increased osmolarity. Absorption spectra demonstrated that the aggregates could withstand the addition of 20% acetonitrile before disintegrating to monomers. The physicochemical properties of this astaxanthin derivative in solution were comprehensively studied by measuring surface tension, critical aggregate concentration, surface concentration, molecule area, free energy of adsorption and micellation, adsorption-aggregate energy relationship, and equilibrium constants, and then compared with similar compounds reported previously in the literature.

Inhibition of chemically-induced neoplastic transformation by a novel tetrasodium diphosphate astaxanthin derivative.

Carotenoids have been implicated in numerous epidemiological studies as being protective against cancer at many sites, and their chemopreventive properties have been confirmed in laboratory studies. Astaxanthin (AST), primarily a carotenoid of marine origin, responsible for the pink coloration of salmon, shrimp and lobster, has received relatively little attention. As with other carotenoids, its highly lipophilic properties complicate delivery to model systems. To overcome this issue we have synthesized a novel tetrasodium diphosphate astaxanthin (pAST) derivative with aqueous dispersibility of 25.21 mg/ml. pAST was delivered to C3H/10T1/2 cells in an aqueous/ethanol solution and compared with non-esterified AST dissolved in tetrahydrofuran. We show pAST to (i) upregulate connexin 43 (Cx43) protein expression; (ii) increase the formation of Cx43 immunoreactive plaques; (iii) upregulate gap junctional intercellular communication (GJIC); and (iv) cause 100% inhibition of methylcholanthrene-induced neoplastic transformation at 10(-6) M. In all these assays, pAST was superior to non-esterified AST itself; in fact, pAST exceeded the potency of all other previously tested carotenoids in this model system. Cleavage of pAST to non-esterified (free) AST and uptake into cells was also verified by HPLC; however, levels of free AST were approximately 100-fold lower than in cells treated with AST itself, suggesting that pAST possesses intrinsic activity. The dual properties of water dispersibility (enabling parenteral administration in vivo) and increased potency should prove extremely useful in the future development of cancer chemopreventive agents.