Minimal hepatic encephalopathy: consensus statement of a working party of the Indian National Association for study of the liver

Hepatic encephalopathy (HE) is a major complication that develops in some form and at some stage in a majority of patients with liver cirrhosis. Overt HE occurs in approximately 30-45% of cirrhotic patients. Minimal HE (MHE), the mildest form of HE, is characterized by subtle motor and cognitive deficits and impairs health-related quality of life. The Indian National Association for Study of the Liver (INASL) set up a Working Party on MHE in 2008 with a mandate to develop consensus guidelines on various aspects of MHE relevant to clinical practice. Questions related to the definition of MHE, its prevalence, diagnosis, clinical characteristics, pathogenesis, natural history and treatment were addressed by the members of the Working Party

Targeted Delivery of Doxorubicin-Loaded Poly (ε-caprolactone)-b-Poly (N-vinylpyrrolidone) Micelles Enhances Antitumor Effect in Lymphoma

<div><p>Background</p><p>The present study was motivated by the need to design a safe nano-carrier for the delivery of doxorubicin which could be tolerant to normal cells. PCL₆₃-b-PNVP₉₀ was loaded with doxorubicin (6 mg/ml), and with 49.8% drug loading efficiency; it offers a unique platform providing selective immune responses against lymphoma.</p><p>Methods</p><p>In this study, we have used micelles of amphiphilic PCL₆₃-b-PNVP₉₀ block copolymer as nano-carrier for controlled release of doxorubicin (DOX). DOX is physically entrapped and stabilized in the hydrophobic cores of the micelles and biological roles of these micelles were evaluated in lymphoma.</p><p>Results</p><p>DOX loaded PCL₆₃-b-PNVP₉₀ block copolymer micelles (DOX-PCL₆₃-b-PNVP₉₀) shows enhanced growth inhibition and cytotoxicity against human (K-562, JE6.1 and Raji) and mice lymphoma cells (Dalton's lymphoma, DL). DOX-PCL₆₃-b-PNVP₉₀ demonstrates higher levels of tumoricidal effect against DOX-resistant tumor cells compared to free DOX. DOX-PCL₆₃-b-PNVP₉₀ demonstrated effective drug loading and a pH-responsive drug release character besides exhibiting sustained drug release performance in in-vitro and intracellular drug release experiments.</p><p>Conclusion</p><p>Unlike free DOX, DOX-PCL₆₃-b-PNVP₉₀ does not show cytotoxicity against normal cells. DOX-PCL₆₃-b-PNVP₉₀ prolonged the survival of tumor (DL) bearing mice by enhancing the apoptosis of the tumor cells in targeted organs like liver and spleen.</p></div

Synthesis and loading of Doxorubicin in PCL₆₃-<i>b</i>-PNVP₉₀.

<p>(A) Schematic illustration of the synthesis of well-defined amphiphilic poly (<i>ε</i>-caprolactone)-<i>b</i>-poly (<i>N</i>-vinylpyrrolidone) block copolymer via the combination of ROP and xanthate-mediated RAFT polymerization. (B) Schematic illustration of the formation of micelle or, drug-loaded micelle. (C) Details illustration of the synthesis of DOX- PCL₆₃-<i>b</i>-PNVP₉₀.</p

DOX-PCL₆₃-b-PNVP₉₀ hinders colony formation in DL.

<p>Clonogenic assay was performed in six-well plates following seeding of 100 cells with clones produced by DL tumor cells. Untreated controls and PCL₆₃-b-PNVP₉₀ treated cells formed colonies (A and C). Smaller size colonies with less number of cells were observed following doxorubicin (0.5 μM) treatment (B). Cells treated with DOX-PCL₆₃-b-PNVP₉₀ treatment (0.5 μM) unable to form colony (D). Survival analysis of DL cells treated with free DOX (Red Line) or, DOX-PCL₆₃-b-PNVP₉₀ (Blue line), (p = 0.01) (E). DOX-PCL₆₃-b-PNVP₉₀ induced growth inhibition in DL cells is caspase dependent. Growth inhibition study in presence and absence of pan caspase inhibitor ZVAD-FMK for 48 h (n = 3, mean ±SD). MTT assay was performed as described above (G).Data presented as mean ± SD, n = 3.</p

Effect of free DOX and DOX- PCL63-b-PNVP90 on normal Cell viability.

<p>Normal human lymphocytes (A), DC (B), and monocytes (C) were treated with serial concentrations of doxorubicin (0.0001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, and 5 μM). Plates were incubated at 37°C, 5% CO₂, for 48 h. The cell viability was measured by XTT assay kit (Cell Signaling, USA). Data shown as mean ± SD, n = 3. Effects on leukocytes number following in vivo administration of free DOX and DOX-PCL₆₃-b-PNVP₉₀ was studied in normal mice (D) tumor bearing mice (E) mice treated with free DOX (F), or DOX- PCL₆₃-b-PNVP₉₀ (G). RBC (H) and differential leukocyte count (I) of individual treatment are shown, mean ± SD, n = 3. (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 in control versus experimental group).</p

Induction of apoptosis by DOX-PCL₆₃-b-PNVP₉₀ micelles.

<p>The effect of free DOX or, DOX-PCL₆₃-b-PNVP₉₀ on the induction of apoptosis in parental (A) and doxorubicin resistant (DOX-R) (B) tumor cells was quantified by flow cytometric analysis of Annexin V FITC staining in tumor cells following 18 h treatment with indicated drugs. The lower left (LL) quadrant represents live/healthy cells, the lower right (LR) quadrant represents early apoptosis, and the upper right (UR) represents cells in late apoptosis, while the upper left (UL) represents the percent DOX uptake. The percentage of cells undergoing early or, late apoptosis is indicated in the respective quadrate. Data shown are mean ± SD, n = 3.</p

In vitro anti-tumor efficacy of DOX-PCL₆₃-b-PNVP₉₀.

<p>Graphs show doxorubicin concentration response on cell survival after treatment with free doxorubicin or, DOX-PCL₆₃-b-PNVP₉₀ in parental and doxorubicin resistant human erytholeukemic cell line K-562 (A and B), T cell leukemia line JE6.1 (C and D), Burkitt lymphoma cell line Raji (E and F) and mouse lymphoma cell line, Dalton lymphoma (DL) (G and H). Data presented as mean ± SD, n = 5. Differences in IC₅₀ values between parental and doxorubicin resistant cell lines are mentioned. (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 in control versus experimental group).</p

Doxorubicin distribution in organs following therapy with DOX-PCL₆₃-b-PNVP₉₀.

<p>Uptake of free DOX and DOX-PCL₆₃-b-PNVP₉₀ micelles by tumor cells and organs were studied using fluorescence plate reader (A). Data presented as mean ± SD of triplicate determination, n = 3. Distribution profile of DOX in Tumor cell (B), Spleen (C), Liver (D), Lung (E), Kidney (F), and Heart (G) by FACS analysis, n = 3.</p

Cytotoxicity of DOX-PCL₆₃-b-PNVP₉₀.

<p>Cytotoxic effect of DOX-PCL₆₃-b-PNVP₉₀ against parental and DOX-resistant lymphoma cells was determined by 18 h LDH release assay. Cells were incubated with free polymer (carrier), DOX-PCL₆₃-b-PNVP₉₀ micelles or free DOX solution at different concentrations (0.0001, 0.0002, 0.0005, 0.001, 0.01, 0.05, 5 μM) for 18 h followed by measurement of the released LDH according to the manufacture's protocol. (A and B) K-562 and K-562/DOX-R, (C and D) JE6.1 and JE6.1/DOX-R (E and F) Raji and Raji/DOX-R (G and H) DL and DL/DOX-R. (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 in control versus experimental group) data represents mean ± SD, n = 3.</p

Time dependent uptake of free doxorubicin and DOX-PCL₆₃-b-PNVP₉₀ micelles.

<p>Uptake of free DOX or DOX-PCL₆₃-b-PNVP₉₀ micelles by parental and doxorubicin-resistant K-562 (A). JE6.1 (B) Raji (C) or, DL (D) cells were studied using fluorescence plate reader. Intracellular DOX concentration (μM) was measured in triplicate at different time points up to 30 h. Data presented as Mean ± SD, n = 4. Flow cytometric measurements of cellular DOX levels in parental K-562 (E) JE6.1 (F), Raji (G) and DL (H) (n = 3). Temporal uptake of free DOX or DOX-PCL₆₃-b-PNVP₉₀ micelles into K-562 (I) and DL (J) cells were studied by incubating in 24-well plates at a concentration of 50,000 cells per well. Representative images shown were obtained using a fluorescence microscope Eclipse 80i (Nikon, Japan) (Plan Fluor, 40X, NA 0.75 objective) equipped with blue and red filters for, Hoechst and Doxorubicin respectively (n = 3).</p