Single Oral Dose Pharmacokinetics of Decursin and Decursinol Angelate in Healthy Adult Men and Women

The ethanol extract of Angelica gigas Nakai (AGN) root has promising anti-cancer and other bioactivities in rodent models. It is currently believed that the pyranocoumarin isomers decursin (D) and decursinol angelate (DA) contribute to these activities. We and others have documented that D and DA were rapidly converted to decursinol (DOH) in rodents. However, our in vitro metabolism studies suggested that D and DA might be metabolized differently in humans. To test this hypothesis and address a key question for human translatability of animal model studies of D and DA or AGN extract, we conducted a single oral dose human pharmacokinetic study of D and DA delivered through an AGN-based dietary supplement Cogni.Q (purchased from Quality of Life Labs, Purchase, NY) in twenty healthy subjects, i.e., 10 men and 10 women, each consuming 119 mg D and 77 mg DA from 4 vegicaps. Analyses of plasma samples using UHPLC-MS/MS showed mean time to peak concentration (T_max) of 2.1, 2.4 and 3.3 h and mean peak concentration (C_max) of 5.3, 48.1 and 2,480 nmol/L for D, DA and DOH, respectively. The terminal elimination half-life (t_1/2) for D and DA was similar (17.4 and 19.3 h) and each was much longer than that of DOH (7.4 h). The mean area under the curve (AUC_0-48h) for D, DA and DOH was estimated as 37, 335 and 27,579 h∙nmol/L, respectively. Gender-wise, men absorbed the parent compounds faster and took shorter time to reach DOH peak concentration. The human data supported an extensive conversion of D and DA to DOH, even though they metabolized DA slightly slower than rodents. Therefore, the data generated in rodent models concerning anti-cancer efficacy, safety, tissue distribution and pharmacodynamic biomarkers will likely be relevant for human translation.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT02114957","term_id":"NCT02114957"}}NCT02114957     

Annexin-V promotes anti-tumor immunity and inhibits neuroblastoma growth in vivo

The goal of the current study is to determine the effects of blocking phosphatidylserine (PS) on the growth of neuroblastoma in mice. PS, an anionic phospholipid restricted to the cytoplasmic surface of plasma membranes in most cells, is externalized to the surface of apoptotic cells. PS has been shown to induce immune tolerance to self-antigens. PS can also be found on the surface of live cells and in particular tumor cells. Annexin-V (AnV) is a protein that specifically binds and blocks PS. To determine the effects of blocking PS with AnV on tumor growth and immunogenicity, mice were inoculated with AGN2a, a poorly immunogenic murine neuroblastoma that expresses high level of PS on the cell surface. Survival and anti-tumor T cell response were determined. AGN2a were engineered to secrete AnV. Secreted protein effectively blocked tumor PS. 40?% of mice inoculated with AnV-expressing AGN2a cells survived free of tumor, whereas none of the mice inoculated with control cells survived (p?=?0.0062). The benefits of AnV were lost when mice were depleted of T cells. The findings suggest that AnV could protect mice from tumor challenge through an immune mediated mechanism. Mice were then immunized with irradiated AnV-secreting or control cells, and challenged with wild-type AGN2a cells. AnV-secreting cell vaccine protected 80?% of mice from AGN2a challenge, while control cell vaccine prevented tumor growth in only 30?% of animals (p?=?0.012). ELISPOT analysis demonstrated that AnV-secreting cell vaccine induced a greater frequency of interferon-gamma producing splenic T cells. T cells isolated from mice immunized with AnV-secreting but not control vaccine lysed AGN2a. In summary, AnV blocked PS, enhanced T cell mediated tumor immunity, and inhibited tumor growth.     

Hot Melt Extrusion: Development of an Amorphous Solid Dispersion for an Insoluble Drug from Mini-scale to Clinical Scale

The objective of the study was to develop an amorphous solid dispersion (ASD) for an insoluble compound X by hot melt extrusion (HME) process. The focus was to identify material-sparing approaches to develop bioavailable and stable ASD including scale up of HME process using minimal drug. Mixtures of compound X and polymers with and without surfactants or pH modifiers were evaluated by hot stage microscopy (HSM), polarized light microscopy (PLM), and modulated differential scanning calorimetry (mDSC), which enabled systematic selection of ASD components. Formulation blends of compound X with PVP K12 and PVP VA64 polymers were extruded through a 9-mm twin screw mini-extruder. Physical characterization of extrudates by PLM, XRPD, and mDSC indicated formation of single-phase ASD’s. Accelerated stability testing was performed that allowed rapid selection of stable ASD’s and suitable packaging configurations. Dissolution testing by a discriminating two-step non-sink dissolution method showed 70–80% drug release from prototype ASD’s, which was around twofold higher compared to crystalline tablet formulations. The in vivo pharmacokinetic study in dogs showed that bioavailability from ASD of compound X with PVP VA64 was four times higher compared to crystalline tablet formulations. The HME process was scaled up from lab scale to clinical scale using volumetric scale up approach and scale-independent-specific energy parameter. The present study demonstrated systematic development of ASD dosage form and scale up of HME process to clinical scale using minimal drug (～500 g), which allowed successful clinical batch manufacture of enabled formulation within 7 months.     

Controlled Release of 5-Fluorouracil from Alginate Beads Encapsulated in 3D Printed pH-Responsive Solid Dosage Forms

Three-dimensional printing is being steadily deployed as manufacturing technology for the development of personalized pharmaceutical dosage forms. In the present study, we developed a hollow pH-responsive 3D printed tablet encapsulating drug loaded non-coated and chitosan-coated alginate beads for the targeted colonic delivery of 5-fluorouracil (5-FU). A mixture of Eudragit® L100-55 and Eudragit® S100 was fabricated by means of hot-melt extrusion (HME) and the produced filaments were printed utilizing a fused deposition modeling (FDM) 3D printer to form the pH-responsive layer of the tablet with the rest comprising of a water-insoluble poly-lactic acid (PLA) layer. The filaments and alginate particles were characterized for their physicochemical properties (thermogravimetric analysis, differential scanning calorimetry, X-ray diffraction), their surface topography was visualized by scanning electron microscopy and the filaments’ mechanical properties were assessed by instrumented indentation testing and tensile testing. The optimized filament formulation was 3D printed and the structural integrity of the hollow tablet in increasing pH media (pH 1.2 to pH 7.4) was assessed by means of time-lapsed microfocus computed tomography (μCT). In vitro release studies demonstrated controlled release of 5-FU from the alginate beads encapsulated within the hollow pH-sensitive tablet matrix at pH values corresponding to the colonic environment (pH 7.4). The present study highlights the potential of additive manufacturing in fabricating controlled-release dosage forms rendering them pertinent formulations for further in vivo evaluation.     

Antinociceptive mechanisms of orally administered decursinol in the mouse

Antinociceptive profiles of decursinol were examined in ICR mice. Decursinol administered orally (from 5 to 200 mg/kg) showed an antinociceptive effect in a dose-dependent manner as measured by the tail-flick and hot-plate tests. In addition, decursinol attenuated dose-dependently the writhing numbers in the acetic acid-induced writhing test. Moreover, the cumulative response time of nociceptive behaviors induced by an intraplantar formalin injection was reduced by decursinol treatment during the both 1st and 2nd phases in a dose-dependent manner. Furthermore, the cumulative nociceptive response time for intrathecal (i.t.) injection of TNF-alpha (100 pg), IL-1 beta (100 pg), IFN-gamma (100 pg), substance P (0.7 microg) or glutamate (20 microg) was dose-dependently diminished by decursinol. Intraperitoneal (i.p.) pretreatment with yohimbine, methysergide, cyproheptadine, ranitidine, or 3,7-dimethyl-1-propargylxanthine (DMPX) attenuated inhibition of the tail-flick response induced by decursinol. However, naloxone, thioperamide, or 1,3-dipropyl-8-(2-amino-4-chloro-phenyl)-xanthine (PACPX) did not affect inhibition of the tail-flick response induced by decursinol. Our results suggests that decursinol shows an antinociceptive property in various pain models. Furthermore, antinociception of decursinol may be mediated by noradrenergic, serotonergic, adenosine A(2), histamine H(1) and H(2) receptors.     

Region-specific enhancement of basal extracellular and cocaine-evoked dopamine levels following constitutive deletion of the Serotonin(1B) receptor

The behavioral effects of cocaine are enhanced following constitutive deletion of the serotonin(1B) receptor. The neural substrates mediating the enhanced response to cocaine are unknown. The present studies determined whether basal dopamine dynamics or cocaine-evoked dopamine levels are altered in projection areas of mesostriatal or mesoaccumbens dopamine neurons following serotonin(1B) receptor deletion. Male wild-type and serotonin(1B) knockout mice were implanted with microdialysis guide cannulas aimed at the dorsal striatum or nucleus accumbens. The zero net flux method of quantitative microdialysis was used to quantify basal extracellular dopamine concentrations (DA(ext)) and the extraction fraction of dopamine (E(d)), which provides an index of dopamine uptake. Conventional microdialysis techniques were used to quantify cocaine (0, 5.0, and 20.0 mg/kg)-evoked dopamine overflow. Basal DA(ext) and E(d) did not differ in striatum of wild-type and knockout mice. Similarly, cocaine-stimulated dopamine overflow did not differ between genotype. The basal E(d) did not differ in the nucleus accumbens of wild-type and knockout mice. However, DA(ext) was significantly elevated in the nucleus accumbens of knockout mice. Cocaine-evoked dopamine overflow (nM) was also enhanced in the nucleus accumbens of knockout mice. However, the cocaine-induced increase in dopamine levels, relative to basal values, did not differ between genotype. These data demonstrate that deletion of the serotonin(1B) receptor is associated with increases in basal DA(ext) in the nucleus accumbens. This increase is not associated with an alteration in E(d), suggesting increased basal dopamine release in these animals. It is hypothesized that these alterations in presynaptic neuronal activity are a compensatory response to constitutive deletion of the serotonin(1B) receptor and may contribute to the enhanced behavioral effects of psychostimulants observed in knockout mice.     

Development of Tablet Formulation of Amorphous Solid Dispersions Prepared by Hot Melt Extrusion Using Quality by Design Approach

The objective of the study was to identify the extragranular component requirements (level and type of excipients) to develop an immediate release tablet of solid dispersions prepared by hot melt extrusion (HME) process using commonly used HME polymers. Solid dispersions of compound X were prepared using polyvinyl pyrrolidone co-vinyl acetate 64 (PVP VA64), Soluplus, and hypromellose acetate succinate (HPMCAS-LF) polymers in 1:2 ratio by HME through 18 mm extruder. A mixture design was employed to study effect of type of polymer, filler (microcrystalline cellulose (MCC), lactose, and dicalcium phosphate anhydrous (DCPA)), and disintegrant (Crospovidone, croscarmellose sodium, and sodium starch glycolate (SSG)) as well as level of extrudates, filler, and disintegrant on tablet properties such as disintegration time (DT), tensile strength (TS), compactibility, and dissolution. Higher extrudate level resulted in longer DT and lower TS so 60–70% was the maximum amount of acceptable extrudate level in tablets. Fast disintegration was achieved with HPMCAS-containing tablets, whereas Soluplus- and PVP VA64-containing tablets had higher TS. Crospovidone and croscarmellose sodium were more suitable disintegrant than SSG to achieve short DT, and MCC was a suitable filler to prepare tablets with acceptable TS for each studied HME polymer. The influence of extragranular components on dissolution from tablets should be carefully evaluated while finalizing tablet composition, as it varies for each HME polymer. The developed statistical models identified suitable level of fillers and disintegrants for each studied HME polymer to achieve tablets with rapid DT (<15 min) and acceptable TS (≥1 MPa at 10–15% tablet porosity), and their predictivity was confirmed by conducting internal and external validation studies.     

Alcohol Enhances Acinetobacter baumannii-Associated Pneumonia and Systemic Dissemination by Impairing Neutrophil Antimicrobial Activity in a Murine Model of Infection

Acinetobacter baumannii (Ab) is a common cause of community-acquired pneumonia (CAP) in chronic alcoholics in tropical and sub-tropical climates and associated with a >50% mortality rate. Using a murine model of alcohol (EtOH) administration, we demonstrated that EtOH enhances Ab-mediated pneumonia leading to systemic infection. Although EtOH did not affect neutrophil recruitment to the lungs of treated mice, it decreased phagocytosis and killing of bacteria by these leukocytes leading to increased microbial burden and severity of disease. Moreover, we determined that mice that received EtOH prior to Ab infection were immunologically impaired, which was reflected in increased pulmonary inflammation, sequential dissemination to the liver and kidneys, and decreased survival. Furthermore, immunosuppression by EtOH was associated with deregulation of cytokine production in the organs of infected mice. This study establishes that EtOH impairs immunity in vivo exacerbating Ab infection and disease progression. The ability of Ab to cause disease in alcoholics warrants the study of its virulence mechanisms and host interactions.     

Chronic binge-like moderate ethanol drinking in rats results in widespread decreases in brain serotonin, dopamine, and norepinephrine turnover rates reversed by ethanol intake

This research was initiated to assess the turnover rates (TORs) of dopamine (DA), norepinephrine (NA), serotonin (5-HT), aspartate, glutamate, and GABA in brain regions during rodent ethanol/sucrose (EtOH) and sucrose (SUC) drinking and in animals with a history of EtOH or SUC drinking to further characterize the neuronal systems that underlie compulsive consumption. Groups of five male rats were used, with two trained to drink EtOH solutions, two to drink SUC and one to serve as a non-drinking control. When stable drinking patterns were obtained, rats were pulse labeled intravenously and killed 60 or 90 min later and the TORs of DA, norepinephrine, 5-HT, aspartate, glutamate, and GABA determined in brain regions. Changes in the TOR of 5-HT, DA, and NA were detected specific to EtOH drinking, SUC drinking or a history of EtOH or SUC drinking. An acute EtOH deprivation effect was detected that was mostly reversed with EtOH drinking. These results suggest that binge-like drinking of moderate amounts of EtOH produces a deficit in neuronal function that could set the stage for the alleviation of anhedonic stimuli with further EtOH intake that strengthen EtOH seeking behaviors which may contribute to increased EtOH use in at risk individuals.     

Topical Formulations Containing Pimenta pseudocaryophyllus Extract: In Vitro Antioxidant Activity and In Vivo Efficacy Against UV-B-Induced Oxidative Stress

Pimenta pseudocaryophyllus is a Brazilian native plant that presents high concentrations of flavonoids and other polyphenolic compounds. Herein, we evaluated: (1) the chemical properties of P. pseudocaryophyllus ethanolic extract (PPE), (2) the in vitro antioxidant activity (AA) of PPE and of two different topical formulations (F1 and F2) containing PPE, (3) physico-chemical and functional stability, (4) in vitro release of PPE, and (5) in vivo capacity of formulations to prevent UV-B irradiation-induced skin damage. Results show that the polyphenol and flavonoid contents in PPE were 199.33 and 28.32 mg/g, respectively, and HPLC results show the presence of eugenol, tannic acid, and rutin. Evaluation of the in vitro AA of PPE demonstrated a dose-dependent effect and an IC₅₀ of 4.75 μg/mL in 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 3.0 μg/mL in 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays. The ferric-reducing antioxidant power (FRAP assay) was 0.046 μmol/L trolox equivalent/μg/mL of extract. Among the AA, only the capacity to scavenge DPPH radical of PPE was maintained in F1 and F2. In addition, both formulations satisfactorily released the extract. The evaluation of the functional stability of F1 and F2 did not demonstrate loss of activity by storage at room temperature and at 4°C/6 months. In irradiated mice, treatment with F1 and F2 added with PPE significantly increased the capacity to scavenge ABTS radical and the FRAP of skin compared to vehicle-treated mice. In conclusion, the present results suggest that formulations containing PPE may be a topical source of antioxidant compounds to decrease oxidative damages of the skin.     

Influence of Processing Parameters and Formulation Factors on the Bioadhesive,Temperature Stability and Drug Release Properties of Hot-Melt Extruded Films Containing Miconazole

This study investigated the processing parameters and formulation factors on the bioadhesive properties, temperature stability properties, and drug release properties of miconazole in PolyOx® and Klucel® matrix systems produced by Hot-melt Extrusion (HME) technology. Miconazole incorporated into these matrix systems were found to be stable for 8 months by X-ray diffraction (XRD). The addition of miconazole increased area under the curve (AUC) at contact time intervals of 30 and 60 sec, while the bioadhesion decreased with an increase in processing temperatures. The release profiles suggest that a sustained release of miconazole was observed from all of the tested HME film formulations for approximately 10 h. The release from the optimal HME film extruded at 205°C was found to be significantly different than that extruded at 190°C. Therefore, this matrix system may address the present shortcomings of currently available therapy for oral and pharyngeal candidiasis.     

Molecular Interaction Studies of Amorphous Solid Dispersions of the Antimelanoma Agent Betulinic Acid

Betulinic acid (BA), a novel natural product with antimelanoma activity, has poor aqueous solubility (<0.1 μg/mL) and therefore exhibits poor bioavailability. The purpose of this study was to explore the feasibility of preparing BA solid dispersions (BA-SDs) with hydrophilic polymers to enhance the aqueous solubility of BA. Melt-quenched solid dispersions (MQ-SDs) of BA were prepared at various ratios with the hydrophilic polymers including Soluplus, HPMCAS-HF, Kollidon VA64, Kollidon K90, and Eudragit RLPO. BA was found to be miscible in all polymers at a 1:4 (w/w) ratio by modulated differential scanning calorimetry (MDSC). BA/Soluplus MQ-SD exhibited the highest solubility in simulated body fluids followed by BA/Kollidon VA64 MQ-SD. The MQ-SDs of BA/Soluplus, BA/HPMCAS-HF, and BA/Kollidon VA64 were found to be amorphous as indicated by X-ray powder diffraction (XRPD) studies. Fourier transform infra-red (FT-IR) studies indicated molecular interactions between BA and Soluplus. Our preliminary screening of polymers indicates that Soluplus and Kollidon VA64 exhibit the greatest potential to form BA-SDs.

Electronic supplementary material

The online version of this article (doi:10.1208/s12249-014-0220-x) contains supplementary material, which is available to authorized users.KEY WORDS: betulinic acid, DSC, FT-IR, HPLC, SEM, solid dispersions, solubility, XRPD     

Whole-Genome Sequences of DA and F344 Rats with Different Susceptibilities to Arthritis,Autoimmunity, Inflammation and Cancer

DA (D-blood group of Palm and Agouti, also known as Dark Agouti) and F344 (Fischer) are two inbred rat strains with differences in several phenotypes, including susceptibility to autoimmune disease models and inflammatory responses. While these strains have been extensively studied, little information is available about the DA and F344 genomes, as only the Brown Norway (BN) and spontaneously hypertensive rat strains have been sequenced to date. Here we report the sequencing of the DA and F344 genomes using next-generation Illumina paired-end read technology and the first de novo assembly of a rat genome. DA and F344 were sequenced with an average depth of 32-fold, covered 98.9% of the BN reference genome, and included 97.97% of known rat ESTs. New sequences could be assigned to 59 million positions with previously unknown data in the BN reference genome. Differences between DA, F344, and BN included 19 million positions in novel scaffolds, 4.09 million single nucleotide polymorphisms (SNPs) (including 1.37 million new SNPs), 458,224 short insertions and deletions, and 58,174 structural variants. Genetic differences between DA, F344, and BN, including high-impact SNPs and short insertions and deletions affecting >2500 genes, are likely to account for most of the phenotypic variation between these strains. The new DA and F344 genome sequencing data should facilitate gene discovery efforts in rat models of human disease.     

Effects of maternal strain on ethanol responses in reciprocal F1 C57BL/6J and DBA/2J hybrid mice

Variations in maternal behavior, either occurring naturally or in response to experimental manipulations, have been shown to exert long-lasting consequences on offspring behavior and physiology. Despite previous research examining the effects of developmental manipulations on drug-related phenotypes, few studies have specifically investigated the influence of strain-based differences in maternal behavior on drug responses in mice. The current experiments used reciprocal F1 hybrids of two inbred mouse strains (i.e. DBA/2J and C57BL/6J) that differ in both ethanol (EtOH) responses and maternal behavior to assess the effects of maternal environment on EtOH-related phenotypes. Male and female DBA/2J and C57BL/6J mice and their reciprocal F1 hybrids reared by either DBA/2J or C57BL/6J dams were tested in adulthood for EtOH intake (choice, forced), EtOH-induced hypothermia, EtOH-induced activity and EtOH-induced conditioned place preference (CPP). C57BL/6J and DBA/2J mice showed differences on all EtOH responses. Consistent with previous reports that maternal strain can influence EtOH intake, F1 hybrids reared by C57BL/6J dams consumed more EtOH during forced exposure than did F1 hybrids reared by DBA/2J dams. Maternal strain also influenced EtOH-induced hypothermic responses in F1 hybrids, producing differences in hybrid mice that paralleled those of the inbred strains. In contrast, maternal strain did not influence EtOH-induced activity or CPP in hybrid mice. The current findings indicate that maternal environment may contribute to variance in EtOH-induced hypothermia and EtOH intake, although effects on EtOH intake appear to be dependent upon the type of EtOH exposure.     

Evaluation of the hypomagnetic environment effects on capillary blood circulation,blood pressure,and heart rate

The impact of attenuated magnetic field (МF) on human health is a burning issue of present-day cosmonautics. A series of experiments with animals exposed to attenuated MF revealed violent disorders in the development of the cardiovascular system. The purpose of this study was to estimate the effects of hypomagnetic environment (HME) on capillary blood circulation, blood pressure (BP), and heart rate (HR) in healthy humans. Participants (n = 34) were 24 men and 10 women without cardiovascular symptoms. The mean age was 43.3 ± 15.4 years. Thirteen participants, eight men and five women, were randomly selected for a repeated investigation under natural conditions (sham exposure). The mean age in this group was 47.9 ± 18 years. Cardiac rhythm and heart rate were recorded using an Astrocard cardiac monitor (Russia). BP was measured by means of a Tonocard automatic blood pressure monitor (Russia). Capillary circulation was determined using a digital capillaroscope (Russia) with a high-speed CMOS camera (100 frames/s). The duration of HME exposure was 60 min. It has been demonstrated that HME increases capillary circulation rate by 22.4% in healthy humans without cardiovascular symptoms as compared to the records made under natural conditions. There was a significant HR reduction by the end of HME exposure as compared to the measurements taken at the beginning. At the end of the exposure, diastolic BP dropped considerably relative to mid-exposure values; on the contrary, systolic BP significantly increased by the end.     

Optimization of <Emphasis Type="Italic">Curcuma</Emphasis> Oil/Quinine-Loaded Nanocapsules for Malaria Treatment

Quinine, a treatment used in chloroquine-resistant falciparum malaria, was loaded into poly(?-caprolactone) or Eudragit^® RS100 nanocapsules using Curcuma oil as the oil-based core. Until now, the effect of cationic nanocapsules on malaria has not been reported. A 2⁴ factorial design was adopted using, as independent variables, the concentration of Curcuma oil, presence of quinine, type of polymer, and aqueous surfactant. Diameter, zeta potential, and pH were the responses studied. The formulations were also evaluated for drug content, encapsulation efficiency, photostability, and antimalarial activity against Plasmodium berghei-infected mice. The type of polymer influenced all of the responses studied. Quinine-loaded Eudragit^® RS100 (F13) and PCL nanocapsules (F9), both with polysorbate 80 coating, showed nanometric particle size, positive zeta potential, neutral pH, high drug content, and quinine photoprotection ability; thus, these nanocapsules were selected for in vivo tests. Both formulations showed lower levels of parasitemia from the beginning of the experiment (5.78 ± 3.60 and 4.76 ± 3.46% for F9 and F13, respectively) and highest survival mean time (15.3 ± 2.0 and 14.9 ± 5.6 days for F9 and F13, respectively). F9 and F13 showed significant survival curve compared to saline, thus demonstrating that nanoencapsulation improved bioefficacy of QN and co-encapsulated curcuminoids, regardless of the surface charge.     

Alcohol enhances characteristic releases of dopamine and serotonin in the central nucleus of the amygdala

The amygdaloid complex (AMY) is implicated in emotional and motivational aspects of behavior, including the formation of positive reinforcement association. AMY may also associated with brain rewarding circuitry. In the present study, the effect of ethanol (EtOH) on the release of dopamine (DA) and serotonin (5-HT) was studied in the central amygdaloid nucleus (CeAMY), and projecting excitatory afferents to the ventral tegmental area (VTA), of freely moving Wistar rats by brain microdialysis. Within 20 min of i.p. injection of EtOH (2 g/kg), the levels of DA and 5-HT in the CeAMY dialysate increased over the baseline value by 270 and 160% (N = 6-7), respectively. Addition of EtOH (25, 50 and 100 mM) to the microdialysis perfusion medium for 1 h caused a 115-150% dose-related increase in the extracellular level of DA in the CeAMY. 100 mM EtOH-induced CeAMY DA release continued to increase for 1 h after the perfusion medium was returned to normal perfusion medium. In contrast, the CeAMY 5-HT level was increased only by the addition of 100 mM EtOH for 1 h to 130% for 80 min. The stimulation of the CeAMY by EtOH through the microdialysis membrane showed delayed responses of DA and 5-HT compared with the i.p. injection of EtOH. Overall, the present findings are not sufficient to conclude whether EtOH acts directly or indirectly on the major monoamine nerve cells in the CeAMY, but the degree of acute EtOH action affected the differences in time at the peak response on EtOH-induced DA and 5-HT releases in the CeAMY via VTA.     

Lack of Cyclin E1 in hepatocytes aggravates ethanol-induced liver injury and hepatic steatosis in experimental murine model of acute and chronic alcohol-associated liver disease

BackgroundCyclin E1 is the regulatory subunit of cyclin-dependent kinase 2 (Cdk2) and one of the central players in cell cycle progression. We recently showed its crucial role for initiation of liver fibrosis and hepatocarcinogenesis. In the present study, we investigated the role of Cyclin E1 in the development of alcohol-associated liver disease (ALD).MethodsMice with constitutive (E1^?/?), hepatocyte-specific (Cyclin E1^Δhepa), or intestinal-epithelial-cell-specific (Cyclin E1^ΔIEC) inactivation of Cyclin E1 and corresponding wild type littermate controls (WT) were administered either a Lieber-DeCarli ethanol diet (LDE) for 3 weeks or acute ethanol binges (6 g/kg) through oral gavage. Serum parameters of liver functionality were measured; hepatic tissues were collected for biochemical and histological analyses.ResultsThe administration of acute EtOH binge and chronic LDE diet to E1^?/? mice enhanced hepatic steatosis, worsened liver damage and triggered body weight loss. Similarly, in the acute EtOH binge model, Cyclin E1^Δhepa mice revealed a significantly worsened liver phenotype. In contrast, inactivation of Cyclin E1 only in intestinal epithelial cell (IECs)did not lead to any significant changes in comparison to WT mice after acute EtOH challenge. Remarkably, both acute and chronic EtOH administration in E1^?/? animals resulted in increased levels of ADH and decreased expression of ALDH1/2. The additional application of a pan-Cdk inhibitor (S-CR8) further promoted liver damage in EtOH-treated WT mice.ConclusionOur data point to a novel unexpected role of Cyclin E1 in hepatocytes for alcohol metabolism, which seems to be independent of the canonical Cyclin E1/Cdk2 function as a cell cycle regulator.     

Effect of alcohol on the interleukin 6-mediated inflammatory response in a new mouse model of acute-on-chronic liver injury

Background and Aims

ACLF is usually associated with a precipitant in the setting of a chronically damaged liver. We aim to combine a mouse model with a pre-injured liver (Abcb4/Mdr2^?/?) with a recently standardized ethanol feeding model to dissect alcohol-related inflammatory responses in this model.