Role of P-glycoprotein and permeability upon the brain distribution and pharmacodynamics of etamicastat: a comparison with nepicastat.

1. This study explores the impact of permeability and P-glycoprotein (P-gp) efflux, upon brain exposure to etamicastat, a new dopamine-ß-hydroxylase (DBH) inhibitor and consequently brain levels of catecholamines. 2. Brain exposure to etamicastat (10 mg/kg), following intravenous administration to mice, was residual and upon oral administration of the same dose no compound was detected, concurring with the absence of effects upon brain catecholamines. The intravenous co-administration of elacridar (1.0 mg/kg), a known P-gp/BCRP dual modulator, significantly increased brain etamicastat exposure, but the levels attained were very low when compared to those of nepicastat, a centrally active DBH inhibitor. 3. In vitro permeability studies from apical-to-basal direction conducted in Caco-2 cells and MDCK-II cells showed that etamicastat apparent permeability was 1.2 × 10(-5) and 1.1 × 10(-6 )cm/s, respectively, 5- and 50-fold lower as compared to nepicastat. The secretory efflux ratio in MDCK-II cells overexpressing human P-gp showed an efflux ratio greater than 2, for both compounds, which was significantly decreased by elacridar. Despite its lower bioavailability and higher clearance, as compared to nepicastat, etamicastat showed preferential distribution to peripheral tissues and high plasma free fraction (15.5%), which may explain its effects upon peripheral DBH and catecholamine levels. 4. Though P-gp-mediated efflux may contribute to the limited brain penetration of etamicastat, the low permeability along with the pharmacokinetic properties of etamicastat may be perceived as the main contributors for its peripheral selectivity, which is advantageous for a cardiovascular drug candidate.

Hydrogen sulfide protects against myocardial ischemia and reperfusion injury by activating AMP-activated protein kinase to restore autophagic flux.

How hydrogen sulfide (H2S) protects against myocardial ischemia-reperfusion (I/R) injury is poorly understood. By using a slow-releasing H2S donor, we investigated if H2S protected against myocardial I/R injury by activating AMPK and restoring I/R-impaired autophagic flux. Male rats received anterior descending coronary artery occlusion followed by reperfusion. The H2S donor ADT and/or the AMPK inhibitor, compound C (CC), were administered after occlusion. Infarction was analyzed histologically. AMPK activation was assessed in the ischemic heart by analyzing phosphorylation of AMPK and S6 ribosomal protein. Autophagy was assessed by analyzing the following markers: microtubule-associated protein 1 light chain 3 (LC3) I and II, lysosome associated membrane protein-2 (LAMP-2), P62 and beclin-1. We further investigated if blocking autophagic flux with chloroquine abolished ADT cardioprotection in vivo. Myocardial I/R reduced serum H2S levels, which was elevated by ADT. ADT enhanced AMPK activation and reduced infarction following I/R, and both effects were abolished by AMPK inhibition. Myocardial I/R induced autophagosome accumulation, as evidenced by the increased ratios of LC3-II/LC3-I, upregulation of beclin-1 and P62 and reduction in LAMP-2. ADT blunted these autophagic changes induced by I/R, indicating that ADT restored I/R-impaired autophagic flux. The AMPK inhibitor CC blocked ADT effects on restoring I/R-impaired autophagy flux. Moreover, chloroquine pretreatment abolished cardioprotection of ADT and increased autophagosome accumulation in the ADT-treated heart following I/R. In conclusion, AMPK activation and subsequent restoration of I/R-impaired autophagic flux are unrecognized mechanisms underlying cardioprotective effects conferred by H2S donors.

Evaluation of the dopamine ß-hydroxylase (DßH) inhibitor nepicastat in participants who meet criteria for cocaine use disorder.

In the present study, we tested the hypothesis that the potent and selective dopamine-ß-hydroxylase (DßH) inhibitor nepicastat would have minimal effects on cardiovascular and pharmacokinetic parameters associated with cocaine administration and would reduce the positive subjective effects produced by cocaine. We conducted a double-blind, placebo-controlled, inpatient study of oral nepicastat (0, 80 and 160mg) concurrent with intravenous (IV) cocaine (0, 10, 20 and 40mg) in non-treatment seeking participants who metcriteria for cocaine use disorder. Safety analyses revealed that nepicastat was well-tolerated and there were no differences in adverse events observed after nepicastat plus cocaine vs. cocaine alone. In addition, the pharmacokinetic properties of cocaine administration were not altered by nepicastat treatment. Cocaine-induced cardiovascular and subjective effects were evaluated for completers in the cohort randomized to nepicastat (n=13) using a within-subjects statistical analysis strategy. Specifically, the cardiovascular and subjective effects of cocaine were assessed in the presence of placebo (0mg), 80mg of nepicastat or 160mg of nepicastat on study Days 4, 8 and 12, respectively. Analyses revealed a main effect of nepicastat to reduce several cocaine-induced positive subjective effects. Taken together, these data indicate that nepicastat is safe when co-administered with cocaine and may suppress its positive subjective effects, and may be viable as a pharmacotherapy for treatment of cocaine use disorder.

HPLC determination of novel dithiolethione containing drugs and its application for in vivo studies in rats.

A panel of new drugs obtained by grafting a sulfurated moiety, i.e. 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADTOH) onto existing drugs have been synthesized and their in vivo action is under preclinical evaluation. In the present paper we describe rapid HPLC methods to detect ADTOH derivatives of valproic acid (ACS2), sildenafil (ACS6), aspirin (ACS14) and diclofenac (ACS15) in plasma. These methods allow the simultaneous detection of the potential drugs and of ADTOH moiety. In the case of ACS14 the de-acetylated metabolite (ACS21) can also be concomitantly measured. The chromatographic separation was performed on a C18 column, applying a mobile phase consisting of a mixture of trifluoroacetic acid and acetonitrile. ADTOH, ACS6, ACS14, ACS21 were separated isocratically whereas ACS2 and ACS15 were separated applying gradient elution. The methods are precise and accurate, with a low quantification limit of 200 nM for ACS2, ACS15 and ACS21 or 100 nM for ADTOH, ACS6 and ACS14. The mean absolute recovery for all tested molecules was always found to be close to 100%. The methods are shown to be selective and linear in the range 0.2-50 microM and thus appear suitable for pharmacokinetic studies with ADTOH containing compounds, as indicated by exemplificative experiments performed with intravenous administration of the drugs to rats.

Globin monoadducts and cross-links provide evidence for the presence of S-(1,2-dichlorovinyl)-L-cysteine sulfoxide, chlorothioketene, and 2-chlorothionoacetyl chloride in the circulation in rats administered S-(1,2-dichlorovinyl)-L-cysteine.

S-(1,2-Dichlorovinyl)-L-cysteine (DCVC), a mutagenic and nephrotoxic metabolite of trichloroethylene, is bioactivated to S-(1,2-dichlorovinyl)-L-cysteine sulfoxide (DCVCS) and chlorothioketene and/or 2-chlorothionoacetyl chloride by cysteine conjugate S-oxidase (S-oxidase) and cysteine conjugate beta-lyase (beta-lyase), respectively. Previously, we identified DCVCS-globin monoadducts and cross-links upon treating rats with DCVCS or incubating erythrocytes with DCVCS. In this study, the formation of DCVC-derived reactive intermediates was investigated after rats were given a single (230 or 460 micromol/kg, i.p.) or multiple (3 or 30 micromol/kg daily for 5 days) DCVC doses. LC/ESI/MS of trypsin-digested globin peptides revealed both S-oxidase and beta-lyase-derived globin monoadducts and cross-links consistent with in vivo DCVC bioactivation by both pathways. MS/MS analyses of trypsin-digested fractions of globin from one of the rats treated with multiple 30 micromol/kg DCVC doses led to identification of beta-lyase-derived monoadducts on both Cys93 and Cys125 of the beta-chains. While rats dosed with the 230 micromol/kg DCVC dose exhibited beta-lyase-dependent monoadducts and cross-links only (four out of four rats), rats given the 460 micromol/kg DCVC dose (two out of four) and rats administered the multiple DCVC doses (two out of four) exhibited both beta-lyase- and S-oxidase-derived monoadducts and cross-links. Because previous incubations of erythrocytes with DCVC did not result in detection of DCVCS-derived monoadducts or cross-links and had only resulted in detection of beta-lyase-derived monoadducts and cross-links, the DCVCS-globin monoadducts and cross-links detected in this study are likely the result of DCVC bioactivation outside the circulation and subsequent translocation of DCVCS and N-acetylated DCVCS into the erythrocytes.

Metabolism of a sulfur-containing heteroarotionoid antitumor agent, SHetA2, using liquid chromatography/tandem mass spectrometry.

SHetA2 {[(4-nitrophenyl)amino][2,2,4,4-tetramethylthiochroman-6-yl)amino]methanethione], NSC 726189}, a sulfur-containing heteroarotinoid, selectively inhibits cancer cell growth and induces apoptosis without activation of nuclear retinoic acid receptors (RARs). The objective of this study was to investigate its in vitro metabolism in rat and human liver microsomes and in vivo metabolism in the mouse and rat using liquid chromatography-ultraviolet/multi-stage mass spectrometry (LC-UV/MS(n)) on an ion-trap mass spectrometer coupled with a photo-diode array (PDA) detector. In vitro, in the absence of glutathione (GSH), oxidation of the four aliphatic methyl groups of SHetA2 yielded one mono-, two di-, and one tri-hydroxylated SHetA2 metabolites, which were identified based on their UV and multi-stage mass spectra. In the presence of GSH, in addition to these primary oxidative metabolites, four GSH adducts of SHetA2 and a novel rare form thioether GSH adduct was detected and characterized. In vivo, the monohydroxylated SHetA2 metabolites were also detected in mouse and rat plasma and two GSH adducts were detected in rat liver following intravenous (i.v.) bolus administration of SHetA2 at 40 mg/kg.

Toluene-3,4-dithiol analysis of blood for assessing carbon disulfide exposure.

Carbon disulfide is a neurotoxic compound used in the production of viscose rayon, and is a major decomposition product of dithiocarbamates used in industry, agriculture, and medicine. Methods used currently for assessing exposure to CS2 are limited in their ability to evaluate cumulative exposures and provide useful information for relatively short periods of time after exposure has ended. The present investigation evaluates a method for monitoring CS2 exposure that consists of cleaving the thiocarbonyl function of free CS2 or certain CS2-generated modifications on proteins using toluene-3,4-dithiol. The resulting toluene trithiocarbonate product is then quantified using reverse-phase high-performance liquid chromatography. The sensitivity, dose response, kinetics and specificity of this biomarker in blood were examined in rats administered CS2 by inhalation, intraperitoneal injection, or gavage for acute through subchronic periods. Dithiol reactive functions in plasma and hemolysate demonstrated a linear dose response over a wide range of exposure levels, were dependent upon the duration of exposure, and appeared to have an appropriate sensitivity for evaluating occupational levels of exposure. Elimination rates of dithiol reactive functions may also be dependent upon exposure duration and exhibit different kinetics for plasma and hemolysate suggesting that elimination rates may be useful for estimating cumulative exposure and intervals between exposure and sample procurement. Dithiol analysis, used in conjunction with previously established erythrocyte protein cross-linking biomarkers, may provide a means to characterize the internal dose of CS2 resulting from acute through chronic periods, and may provide insight into the level of CS2-mediated covalent protein modifications occurring within the nervous system.