Anethole Dithiolethione Research Articles

Manganese Porphyrin-Containing Polymeric Micelles: A Novel Approach for Intracellular Catalytic Formation of Per/Polysulfide Species from a Hydrogen Sulfide Donor.

Per/polysulfide species that are generated from endogenously produced hydrogen sulfide have critical regulatory roles in a wide range of cellular processes. However, the lack of delivery systems that enable controlled and sustained release of these unstable species in biological systems hinders the advancement of sulfide biology research, as well as the translation of knowledge to therapeutic applications. Here, a novel approach is developed to generate per/polysulfide species in cells by combining an H2 S donor and manganese porphyrin-containing polymeric micelles (MnPMCs) that catalyze oxidization of H2 S to per/polysulfide species. MnPMCs serve as a catalyst for H2 S oxidation in aerobic phosphate buffer. HPLC-MS/MS analysis reveals that H2 S oxidation by MnPMCs in the presence of glutathione results in the formation of glutathione-SnH (n = 2 and 3). Furthermore, co-treatment of human umbilical vein endothelial cells with the H2 S donor anethole dithiolethione and MnPMCs increases intracellular per/polysulfide levels and induces a proangiogenic response. Co-delivery of MnPMCs and an H2 S donor is a promising approach for controlled delivery of polysulfides for therapeutic applications.

Anethole Dithiolethione Increases Glutathione in Kidney by Inhibiting γ-Glutamyltranspeptidase: Biochemical Interpretation and Pharmacological Consequences.

Aims Anethole dithiolethione (ADT) is a marketed drug to treat xerostomia. Its mechanism of action is still unknown, but several preclinical studies indicate that it is able to increase intracellular glutathione (GSH) and protect against oxidative stress. Here, we investigated the molecular mechanisms behind these effects. Results Oral treatment of rats confirmed the GSH enhancing properties of ADT; among the different organs examined in this study, only the kidney showed a significant GSH increase that was already observed at low-dose treatments. The increase in GSH correlated with a decrease in γ-glutamyltranspeptidase (γ-GT) activity of the different tissues. In vitro and ex vivo experiments with tubular renal cells and isolated perfused rat kidney showed that the cellular uptake of intact GSH was correlated with the extracellular concentrations of GSH. Conclusions. The prominent in vivopharmacological effect of ADT was a marked increase of GSH concentration in the kidney and a decrease of some systemic and renal biomarkers of oxidative stress. In particular, by inhibition of γ-GT activity, it decreased the production cysteinylglycine, a thiol that has prooxidant effects as the consequence of its autooxidation. The activity of ADT as GSH enhancer in both the circulation and the kidney was long-lasting. All these characteristics make ADT a promising drug to protect the kidney, and in particular proximal tubule cells, from xenobiotic-induced damage.

Slow-Release H2S Donor Anethole Dithiolethione Protects Liver From Lipotoxicity by Improving Fatty Acid Metabolism.

“Lipotoxicity” induced by free fatty acids (FAs) plays a central role in the pathogenesis of many metabolic diseases, with few treatment options available today. Hydrogen sulfide (H2S), a novel gaseous signaling molecule, has been reported to have a variety of pharmacological properties, but its effect on FAs metabolism remains unclear. The purpose of this study was to investigate the effect and mechanisms of anethole dithiolethione (ADT, a sustained-release H2S donor) on hepatic FAs metabolism. ADT was administered daily for 4 weeks in male Syrian golden hamsters fed a high fat diet (HFD), and FAs profiles of liver tissues were analyzed using GC-MS. The results showed that in HFD-fed hamsters, ADT treatment significantly reduced the accumulation of toxic saturated and monounsaturated fatty acids (C16:0, C18:0, C16:1, and C18:1n9), while increased the content of n-6 and n-3 series polyunsaturated fatty acids (C20:3n6, C20:4n6, and C22:6n3). Mechanistically, ADT obviously inhibited the overexpression of acetyl-CoA carboxylase1 (ACC1), fatty acid synthase (FAS), and stearoyl-CoA desaturase1 (SCD1), and up-regulated the levels of fatty acid transport proteins (FATPs), liver fatty acid binding protein (L-FABP), carnitine palmitoyltransferase 1α (CPT1α), fatty acid desaturase (FADS)1 and FADS2. Notably, ADT administration significantly promoted Mitofusin1-mediated mitochondrial fusion and fatty acid β-oxidation. These findings suggest that ADT plays a beneficial role by regulating the synthesis, desaturation, β-oxidation, uptake, binding/isolation, and transport of FAs. In conclusion, ADT is effective in improving FAs metabolic disorders and liver injuries caused by HFD, which renders ADT a candidate drug for lipotoxicity-induced diseases.

Hydrogen sulfide-releasing micelles for promoting angiogenesis

Hydrogen sulfide (H2S), an important gaseous signalling molecule in the human body, has been shown to be involved in many physiological processes such as angiogenesis. Since the biological activities of H2S are known to be significantly affected by the dose and exposure duration, the development of H2S delivery systems that enable control of H2S release is critical for exploring its therapeutic potential. Here, we prepared polymeric micelles with different H2S release profiles, which were prepared from amphiphilic block copolymers consisting of a hydrophilic poly(N-acryloyl morpholine) segment and a hydrophobic segment containing H2S-releasing anethole dithiolethione (ADT) groups. The thermodynamic stability of the micelles was modulated by altering the ADT content of the polymers. The micelles with higher thermodynamic stability showed significantly slower H2S release. Furthermore, the sustained H2S release from the micelles enhanced migration and tube formation in human umbilical vein cells (HUVECs) and induced vascularlization in the in ovo chick chorioallantoic membrane (CAM) assay.

Mechanism of H2S Formation from the Metabolism of Anetholedithiolethione and Anetholedithiolone by Rat Liver Microsomes.

The drug anetholedithiolethione (ADT) and its analogs have been extensively used as H2S donors. However, the mechanism of H2S formation from ADT under biologic conditions remains almost completely unknown. This article shows that only small amounts of H2S are formed during incubation of ADT and of its metabolite anetholedithiolone (ADO) with rat liver cytosol or with rat liver microsomes (RLM) in the absence of NADPH, indicating that H2S formation under these conditions is of hydrolytic origin only to a minor extent. By contrast, much greater amounts of H2S are formed upon incubation of ADT and ADO with RLM in the presence of NADPH and dioxygen, with a concomitant formation of H2S and para-methoxy-acetophenone (pMA). Moreover, H2S and pMA formation under those conditions are greatly inhibited in the presence of N-benzyl-imidazole indicating the involvement of cytochrome P450-dependent monooxygenases. Mechanistic studies show the intermediate formation of the ADT-derived 1,2-dithiolium cation and of the ADO sulfoxide during microsomal metabolism of ADT and ADO, respectively. This article proposes the first detailed mechanisms for the formation of H2S from microsomal metabolism of ADT and ADO in agreement with those data and with previously published data on the metabolism of compounds involving a C=S bond. Finally, this article shows for the first time that ADO is a better H2S donor than ADT under those conditions. SIGNIFICANCE STATEMENT: Incubation of anetholedithiolethione (ADT) or its metabolite anetholedithiolone (ADO) in the presence of rat liver microsomes, NADPH, and O2 leads to H2S. This article shows for the first time that this H2S formation involves several steps catalyzed by microsomal monooxygenases and that ADO is a better H2S donor than ADT. We propose the first detailed mechanisms for the formation of H2S from the microsomal metabolism of ADT and ADO.

Metabolism of Anethole Dithiolethione by Rat and Human Liver Microsomes: Formation of Various Products Deriving from ItsO-Demethylation andS-Oxidation. Involvement of Cytochromes P450 and Flavin Monooxygenases in These Pathways

A study of the metabolism of anethole dithiolethione (ADT, 5-(p-methoxyphenyl)-3H-1,2-dithiole-3-thione) by rat and human liver microsomes showed the formation of the corresponding S-oxide and the S-oxide of desmethyl-ADT (dmADT, 5-(p-hydroxyphenyl)-3H-1,2-dithiole-3-thione), and of p-methoxy-acetophenone (pMA) and p-hydroxy-acetophenone (pHA), in addition to the previously described metabolites, dmADT, anethole dithiolone (ADO, 5-(p-methoxyphenyl)-3H-1,2-dithiole-3-one) and its demethylated derivative dmADO [5-(p-hydroxyphenyl)-3H-1,2-dithiole-3-one]. The microsomal metabolism of ADO under identical conditions led to dmADO and to pMA and pHA. The metabolites of ADT derive from two competing oxidative pathways: an O-demethylation catalyzed by cytochromes P450 and an S-oxidation mainly catalyzed by flavin-dependent monooxygenases (FMO) and, to a minor extent, by CYP enzymes. The most active human CYP enzymes for ADT demethylation appeared to be CYP1A1, 1A2, 1B1, 2C9, 2C19, and 2E1. ADT S-oxidation is catalyzed by FMO 1 and 3, and to a minor extent by CYP enzymes such as CYP3A4.

176 - Mitochondria-targeted Hydrogen Sulfide (H2S), but not Untargeted H2S, Reverses Ventricular Arrhythmia at Reperfusion

The ‘gasomediator’ hydrogen sulfide has rapidly grown in importance as a potential therapeutic approach for cardiovascular and ischemia-reperfusion conditions including myocardial infarction. Indeed, infarct limitation by postconditioning the heart against myocardial infarction has been demonstrated with either slow-releasing or mitochondrial-targeted H2S donor in vivo such as GYY4137 and AP39 respectively. In this current study we characterised the effects of H2S donors, and their respective control compounds, on reperfusion-induced arrhythmia, an additional form of myocardial reperfusion injury. Inactin®-anaesthetised Sprague Dawley rats underwent 30 minutes regional myocardial ischaemia and 120 minutes reperfusion with all interventions were given 10 minutes before reperfusion. We utilised different H2S donors; two which target mitochondria (AP39 and AP123; 1 µmol/kg) and one non-targeted H2S donor (GYY4137; 266 µmol/kg). We also evaluated the effects of donor controls at the same doses to ensure our observations were to due H2S and not the parent compound or decomposition products formed during/after H2S release. For example, for mitochondrial targeting without H2S-releasing moieties we used AP219 (TPP+-decanoate), for mitochondrial H2S donors alone, anethole dithiolethione (for AP39) and thiohydroxybenzamide (for AP123) and for GYY4137 we used its decomposition product, (4-methoxyphenylphosphon-amidothioate). We also used ischemic preconditioning (IPC) as a positive control. Hemodynamics and electrocardiographic records, obtained using lead II electrodes, were examined to identify ventricular premature beats (VPBs), ventricular tachycardia (VT), sustained VT (>30 seconds) and ventricular fibrillation (VF) during the first 10 minutes of reperfusion in the presence and absence of H2S donors or their control compounds. AP39 significantly (p<0.01; ANOVA with post hoc Dunnett’s test) attenuated the occurrence of VPBs in the early reperfusion by 56% compared to the vehicle group. Moreover, postconditioning with GYY4137 and AP123 reduced the incidence of VPBs by 41% and 31%, respectively, however, this did not achieve statistical significance. AP39 also significantly limited the incidence of reperfusion-triggered sustained VT and VF by 69% and 67%, respectively, compared to the control (each p<0.01; ANOVA). There were no detectable effects for any of the controls on any form of ventricular arrhythmia. In sharp contrast to previously published findings with ‘non-targeted H2S’ by sulfide or GYY4137, the reversal of ventricular arrthymia by AP39 was independent of the salvage kinase pathways since L-NAME (to inhibit nitric oxide synthase), LY294002 (to inhibit phosphoinositide-3 kinases) or ODQ (to inhibit guanylyl cyclase) failed to reverse the effects of AP39 on VT, VF or VPBs. Additionally AP39 did not induce the phosphorylation of eNOS, Akt, GSK-3β or ERK1/2. Presumably this was because H2S delivery was mitochondrial rather than ‘global’. Indeed, AP39 (but not GYY4137) also reversed ‘oxidant’ production and increased calcium retention, potentiating the effect of cyclosporine A, in subsarcolemmal and interfibrillar mitochondria isolated from the left ventricle. Our results show for the first time the potential inhibitory effect of selective mitochondrial delivery of H2S on reperfusion-induced ventricular arrhythmia in vivo and add to the increasing body of evidence for the therapeutic potential of H2S, and mitochondria-targeted H2S, for the treatment of cardiovascular disorders.

228 - Evaluation of a Novel Mitochondria-targeted Peptide-based H2S Donor Compound (RTP-10) in Hyperglycaemia-induced Microvascular Endothelial Cell Dysfunction

An overproduction of mitochondrial reactive oxygen species (ROS) in endothelial cells, is a major contributor to vascular endothelial dysfunction (VED) and angiopathy in diabetes. Hyperglycaemia (HG) induces metabolic changes in mitochondria, notably superoxide production, membrane hyperpolarisation and loss of ATP synthesis. Supplementation of cells and diabetic animals with sources of hydrogen sulfide (H2S) such as crude sulfide salts (e.g. NaSH) or the slow-release compound GYY4137 have been shown to protect the vasculature from the detrimental effects of oxidative stress (e.g. in hypoxia/ischaemia-reperfusion injury, diabetic angiopathy, stroke etc). However, since the major intracellular effect of H2S is on mitochondria (e.g. stimulation of cellular bioenergetics) massive concentrations (e.g. >200 µM) or doses in vivo (e.g. up to 1 g/kg for GYY4137) are required for this vascular protection as these approaches do not target H2S to where it is needed, i.e. the mitochondria. Two previously mitochondria-targeted H2S (mtH2SD) donor organic compounds based around a triphenylphosphonium (TPP+) targeting scaffold, AP39 and AP123 have been described in the literature which contain anethole dithiolethione and thiohydroxybenzamide as the H2S generating moieties. These compounds have shown significant efficacy when used therapeutically in several animal models of mitochondrial dysfunction at very low doses (7-300 µg / kg). Since the TPP+-based scaffolds require an active mitochondrial ΔΨm to accumulate within mitochondria, (a possible limitation) we now describe an alternative approach to target H2S to mitochondria using a novel H2S donor derivative of D-Arg-L-Tyr-L-Lys-L-Phe-NH2 (RTP10). We therefore exposed murine b.End3 brain microvascular endothelial cells to hyperglycaemia (HG) for 7 days to induce mitochondrial ‘stress’ and then added RTP10 (0.1 nM to 1 µM) for 3 days. After this time, the reversal of HG-induced metabolic changes, specifically mitochondrial ΔΨm (JC-1), mitochondrial superoxide, (mitosox) and ATP synthesis (by luminescence) were measured. RTP10 caused a concentration-dependent increase in mitochondrial H2S levels as well as reversed HG-induced mitochondrial hyperpolarisation and oxidant production, and restored ATP synthesis. Our study further suggests that targeting H2S to mitochondria may be a useful therapeutic strategy for reversing oxidative stress-induced mitochondrial damage in diabetes and other vascular and metabolic pathologies.

Magnetic Nanoliposomes as in Situ Microbubble Bombers for Multimodality Image-Guided Cancer Theranostics

Nanosized drug delivery systems have offered promising approaches for cancer theranostics. However, few are effective to simultaneously maximize tumor-specific uptake, imaging, and therapy in a single nanoplatform. Here, we report a simple yet stimuli-responsive anethole dithiolethione (ADT)-loaded magnetic nanoliposome (AML) delivery system, which consists of ADT, hydrogen sulfide (H2S) pro-drug, doped in the lipid bilayer, and superparamagnetic nanoparticles encapsulated inside. HepG2 cells could be effectively bombed after 6 h co-incubation with AMLs. For in vivo applications, after preferentially targeting the tumor tissue when spatiotemporally navigated by an external magnetic field, the nanoscaled AMLs can intratumorally convert to microsized H2S bubbles. This dynamic process can be monitored by magnetic resonance and ultrasound dual modal imaging. Importantly, the intratumoral generated H2S bubbles imaged by real-time ultrasound imaging first can bomb to ablate the tumor tissue when exposed to higher acoustic intensity; then as gasotransmitters, intratumoral generated high-concentration H2S molecules can diffuse into the inner tumor regions to further have a synergetic antitumor effect. After 7-day follow-up observation, AMLs with magnetic field treatments have indicated extremely significantly higher inhibitions of tumor growth. Therefore, such elaborately designed intratumoral conversion of nanostructures to microstructures has exhibited an improved anticancer efficacy, which may be promising for multimodal image-guided accurate cancer therapy.

432 - Mitochondria-Targeted Hydrogen Sulfide Donors Protect Microvascular Endothelial Cells from Hyperglycaemia-Inducted Metabolic Changes and Oxidative Damage

There is decreased bioavailability of the gas hydrogen sulfide (H 2 S) in humans with diabetes and several animal models of this disease. The loss of H 2 S is thought to be due, at least in part, to its consumption by detrimental oxidants induced in the diabetic vasculature by periods of hyperglycaemia (HG) and results in mitochondrial and endothelial dysfunction, contributing to the development of diabetic vascular complications. Therefore, strategies which either protect or reverse HG-induced mitochondrial damage may represent a novel approach to treat diabetic angiopathies. We have previously shown that mitochondria-targeted H 2 S donor compounds such as AP39, prevented and/or reversed mitochondrial and cellular damage in in vivo models of cardiac arrest, myocardial infarction, hypertension, kidney IRI and organ transplantation leading us to propose that mitochondrial- H 2 S would also be beneficial in HG-induced endothelial toxicity. We have therefore synthesised a series of novel mitochondria-targeted hydrogen sulfide donors containing mitochondria-targeting motifs (such as triphenylphosphonium decanoate) coupled to molecules we have identified as H 2 S releasing agents (a) anethole dithiolethione (e.g. AP39), (b) thiohydroxybenzamide (e.g. AP123) as well as a novel H 2 S donor moiety (c) 5-(4-hydroxyphenyl)-3H-1,2-dithiol-3-one (e.g. RT-01). We investigated the protective effects of the compounds against HG-induced oxidative stress and metabolic changes in isolated mouse brain microvascular endothelial cells. HG induced significant increase TCA cycle activity, elevated mitochondrial membrane potential and mitochondrial oxidant production and lower mitochondrial electron transport. AP39, AP123 and RT-01 (30-300nM) decreased these detrimental metabolic changes, increased electron transport at respiratory complex III and improved the cellular metabolism. Mitochondrial targeting of H 2 S also induced >1000-fold increase in the potency of H 2 S against HG-induced injury c.f. non-targeted H 2 S. The high potency and long-lasting effect elicited by these H 2 S donors strongly suggests mitochondria-targeted H 2 S could be useful against diabetic angiopathies and other vascular disorders.

The novel mitochondria-targeted hydrogen sulfide (H2S) donors AP123 and AP39 protect against hyperglycemic injury in microvascular endothelial cells in vitro

The development of diabetic vascular complications is initiated, at least in part, by mitochondrial reactive oxygen species (ROS) production in endothelial cells. Hyperglycemia induces superoxide production in the mitochondria and initiates changes in the mitochondrial membrane potential that leads to mitochondrial dysfunction. Hydrogen sulfide (H2S) supplementation has been shown to reduce the mitochondrial oxidant production and shows efficacy against diabetic vascular damage in vivo. However, the half-life of H2S is very short and it is not specific for the mitochondria. We have therefore evaluated two novel mitochondria-targeted anethole dithiolethione and hydroxythiobenzamide H2S donors (AP39 and AP123 respectively) at preventing hyperglycemia-induced oxidative stress and metabolic changes in microvascular endothelial cells in vitro. Hyperglycemia (HG) induced significant increase in the activity of the citric acid cycle and led to elevated mitochondrial membrane potential. Mitochondrial oxidant production was increased and the mitochondrial electron transport decreased in hyperglycemic cells. AP39 and AP123 (30–300nM) decreased HG-induced hyperpolarisation of the mitochondrial membrane and inhibited the mitochondrial oxidant production. Both H2S donors (30–300nM) increased the electron transport at respiratory complex III and improved the cellular metabolism. Targeting H2S to mitochondria retained the cytoprotective effect of H2S against glucose-induced damage in endothelial cells suggesting that the molecular target of H2S action is within the mitochondria. Mitochondrial targeting of H2S also induced >1000-fold increase in the potency of H2S against hyperglycemia-induced injury. The high potency and long-lasting effect elicited by these H2S donors strongly suggests that these compounds could be useful against diabetic vascular complications.

Synthesis of Amino-ADT Provides Access to Hydrolytically Stable Amide-Coupled Hydrogen Sulfide Releasing Drug Targets.

As additional physiological functions of hydrogen sulfide (H2S) are discovered, developing practical methods for exogenous H2S delivery is important. In particular, nonsteroidal anti-inflammatory drugs (NSAIDs) functionalized with H2S-releasing anethole dithiolethione (ADT-OH) through ester bonds are being investigated for their combined anti-inflammatory and antioxidant potential. The chemical robustness of the connection between drug and H2S-delivery components, however, is a key and controllable linkage in these compounds. Because esters are susceptible to hydrolysis, particularly under acidic conditions such as stomach acid in oral drug delivery applications, we report here a simple synthesis of amino-ADT (ADT-NH2 ) and provide conditions for successful ADT-NH2 derivatization with the drugs naproxen and valproic acid. Using UV-vis spectroscopy and HPLC analysis, we demonstrate that amide-functionalized ADT derivatives are significantly more resistant to hydrolysis than ester-functionalized ADT derivatives.

Back Cover: Macromol. Biosci. 11/2015.

Back Cover: The micellar prodrugs of desmethyl anethole dithiolethione (ADT-OH) with different hydrolysis rates prepared from block copolymers having ADT-OH linked via an ester bond using glycine and isoleucine linkers are presented. Micelles having a glycine linker inhibit proliferation of cancer cells. Further details can be found in the article by U. Hasegawa, N. Tateishi, H. Uyama, A. J. van der Vlies on page 1512.

Effects of AP39, a novel triphenylphosphonium derivatised anethole dithiolethione hydrogen sulfide donor, on rat haemodynamic parameters and chloride and calcium Cav3 and RyR2 channels

H2S donor molecules have the potential to be viable therapeutic agents. The aim of this current study was (i) to investigate the effects of a novel triphenylphosphonium derivatised dithiolethione (AP39), in the presence and absence of reduced nitric oxide bioavailability and (ii) to determine the effects of AP39 on myocardial membrane channels; CaV3, RyR2 and Cl–. Normotensive, L-NAME- or phenylephrine-treated rats were administered Na2S, AP39 or control compounds (AP219 and ADT-OH) (0.25–1 µmol kg–1i.v.) and haemodynamic parameters measured. The involvement of membrane channels T-type Ca2+ channels CaV3.1, CaV3.2 and CaV3.3 as well as Ca2+ ryanodine (RyR2) and Cl– single channels derived from rat heart sarcoplasmic reticulum were also investigated. In anaesthetised Wistar rats, AP39 (0.25–1 µmol kg–1 i.v) transiently decreased blood pressure, heart rate and pulse wave velocity, whereas AP219 and ADT-OH and Na2S had no significant effect. In L-NAME treated rats, AP39 significantly lowered systolic blood pressure for a prolonged period, decreased heart rate and arterial stiffness. In electrophysiological studies, AP39 significantly inhibited Ca2+ current through all three CaV3 channels. AP39 decreased RyR2 channels activity and increased conductance and mean open time of Cl– channels. This study suggests that AP39 may offer a novel therapeutic opportunity in conditions whereby •NO and H2S bioavailability are deficient such as hypertension, and that CaV3, RyR2 and Cl– cardiac membrane channels might be involved in its biological actions.

114 - Effects of a Novel Triphenylphosphonium Derivatised Anethole Dithiolethione Hydrogen Sulfide (H2S) Donor, AP39, on Cardiac Ion Channels and on Haemodynamic Parameters in Normotensive and Hypertensive Rats in Vivo

114 - Effects of a Novel Triphenylphosphonium Derivatised Anethole Dithiolethione Hydrogen Sulfide (H2S) Donor, AP39, on Cardiac Ion Channels and on Haemodynamic Parameters in Normotensive and Hypertensive Rats in Vivo

P22: Mitochondira-targeted hydrogen sulfide donors AP39 and AP123 attenuate hyperglycaemia-induced oxidative stress and loss of bioenergetics in microvascular endothelial cells

Mitochondria are key mediators in determining cell fate and some mitochondrial functions are regulated by endogenous H2S. 3-MST is constitutively expressed in mitochondria and CSE/CBS translocate to mitochondria during oxidative/hypoxic insult and mitochondrial H2S synthesis is cytoprotective. With this in mind, we have designed a series of novel compounds to generate H2S within the mitochondria containing triphenylphosphonium (mitochondria-targeting) and either anethole dithiolethione (AP39) or hydroxythiobenzamide (AP123). We have evaluated the effectiveness of AP39, AP123 (and respective their control compounds) at preventing/reversing hyperglycaemia (HG)-induced oxidative stress, and metabolic changes in brain microvascular endothelial cells in vitro. Incubation of endothelial cells with AP39 and AP123 (30–300 nM) increased intracellular H2S levels, with a preferential response seen in the mitochondrial regions confirming mitochondrial H2S delivery. HG (10 days, 25 mM), induced significant mitochondrial ‘oxidant’ production (MitoSox and CM-DCF), hyperpolarisation of the mitochondrial membrane (JC-1) and inhibition of mitochondrial electron transport, ATP production and cellular bioenergetics (Seahorse, XF24 Extracellular Flux Analyzer). These metabolic changes were significantly attenuated by treatment of cells with 200 microM of the non-targeted H2S donor GYY4137. This study suggests that very low concentrations of mitochondrial-targeted H2S (sub nM) induce positive bioenergetic effects, preserve mitochondrial function and cell integrity. Moreover, by targeting mitochondria, the mitochondria- and cytoprotective potency of H2S (e.g with AP39 and AP123) is increased by >2000 fold. Mitochondria-targeted H2S donor molecules may be useful in the treatment/prevention in pathologies associated with mitochondrial dysfunction (e.g. diabetes, sepsis, hypertension, stroke etc).

Anethole dithiolethione lowers the homocysteine and raises the glutathone levels in solid tissues and plasma of rats: A novel non-vitamin homocysteine-lowering agent

High homocysteine (Hcys) levels are suspected to contribute to the pathogenesis of cardiovascular disease and of other chronic conditions. Failure of B vitamins to reduce the incidence of cardiovascular events while lowering the Hcys levels, has prompted the search for alternative treatments. We tested the ability of anethole dithiolethione (ADT) to lower the Hcys levels in rats and we explored possible underlying mechanisms. Parenteral administration of 10mg/kg ADT to normal rats for 3 days lowered the Hcys levels between 51.4% and 31.5% in kidneys, liver, testis and plasma. Concomitantly, glutathione (GSH) increased between 112% and 28% in kidneys, brain, liver and plasma whereas protein thiolation index decreased 30%. In hyperhomocysteinemic rats, the plasma Hcys levels dropped 70% following a single ip injection of 10mg/kg ADT, while they decreased 55% following oral administration of 2mg/kg/day ADT for one week. Significant additive effects occurred when sub-therapeutic doses of ADT and folic acid were used in combination. To test the possible mechanism(s) of these actions, we perfused isolated rat livers and kidneys with albumin-bound Hcys, the prevalent form of plasma Hcys, and physiological thiols and disulfides at different ratios. In both organ preparations, the elimination rate of albumin-bound Hcys was progressively faster as the amount of reduced thiols was increased in the perfusate. These findings indicate that ADT shifts the redox ratio of GSH and other thiols with their oxidized forms toward the reduced forms, thus favoring the dissociation of albumin-bound Hcys and its transfer to renal and hepatic cells for further processing.

P51 Polymeric micelles for controlled delivery of hydrogen sulfide

With the discovery of the important biological roles of hydrogen sulfide (H2S), therapeutic applications of this gas have attracted growing attention. For medical applications of H2S, an increasing number of H2S donors, compounds which release H2S under physiological conditions, have been developed to evaluate the therapeutic potential of H2S. However, these low molecular weight molecules have intrinsic pharmacokinetic problems including poorly-controlled tissue distribution and rapid elimination from the blood circulation which may result in adverse effects and low therapeutic efficiency for practical applications. To overcome these limitations, polymeric micelles, spherical supramolecular assemblies formed from amphiphilic block copolymers with a diameter of 10–100 nm, have been used to improve pharmacokinetics, bioavailability and stability of drugs. In view of these feature, we sought to develop a micellar H2S delivery system. Here we report on novel polymeric micelles carrying H2S-releasing anethole dithiolethione (ADT) moieties. We prepared diblock copolymers having a hydrophilic poly (ethylene glycol) block and a hydrophobic ADT-bearing H2S-releasing block using reversible addition-fragmentation transfer (RAFT) polymerization. The resulting polymers self-assembled into monodisperse micelles with a diameter of 40 nm according to dynamic light scattering (DLS). Further characterization by transmission electron microscopy (TEM) confirmed that the polymers formed spherical micelles with narrow size distributions. The in vitro experiments using murine macrophages showed that the micelles enhanced lipopolysaccharide-induced nuclear factor-κB (NF-κB) activation and proinflammatory cytokine production. MTT assay revealed that the micelles have much lower toxicity compared to the small H2S donor ADT. This micellar system may offer a safe and efficient delivery of H2S in immunotherapy.

Determining the Effect of Dithiolethione Compounds on the Activity of Human Glyceraldehyde-3-Phosphate Dehydrogenase

Dithiolethione compounds, like anethole dithiolethione (ADT) have chemopreventive, cytoprotective, and antimitogenic effects. The chemopreventive effects are thought to be mediated by the activation of antioxidant response elements and of the tumor repressor PP2a. We have shown that ADT interacts with the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Cancer cells usually exhibit increased glycolysis (Warburg effect) that generates ATP to meet their energy needs. Mounting evidence suggests that inhibition of glycolysis in cancer cells severely depletes ATP and may be an effective strategy in chemoprevention. We hypothesize that the chemopreventive effects of ADT may be in part due to inhibition of glycolysis via inhibition of GAPDH. Dithiolethiones can directly or indirectly modify cysteine residues in protein targets. GAPDH contains three cysteine residues that are susceptible to thiol-modifying agents. Therefore, we set out to examine how ADT affects GAPDH activity. Human GAPDH was overexpressed in bacterial cells and purified by ion-exchange and affinity chromatography. We measured the catalytic activity of GAPDH in the absence and the presence of ADT and showed a concentration-dependent inhibition of GAPDH catalytic activity by ADT. In order to determine the exact mechanism of ADT modification of GAPDH we are coupling site-directed mutagenesis with enzymatic assay, and mass-spectrometry.This investigation was sponsored in part by NIH/NIGMS MARC U∗STAR T34 08663 National Research Service Award to UMBC and also by the Howard Hughes Medical Institute's Precollege and Undergraduate Science Education Program (JC) and UMBC DRIF SRAIS (EG).

ChemInform Abstract: Synthesis and Biological Evaluation of Lipid‐Soluble Prodrugs of Anethole Dithiolthione.

AbstractCompound (Ia) is a potential hepatic protectant which exhibits better bioavailability and potency than anethol trithione.