FORMULATION AND IN VITRO EVALUATION OF METOPROLOL SUCCINATE FLOATING TABLETS

Gastroretentive dosage forms extend significantly the period of time over which the drug may be released. This prolonged gastric retention improves bioavailability, decrease drug waste and improve solubility of drugs that are less soluble in a high pH environment due to their availability in gastric pH for longer duration of time.Floating drug delivery systems have a bulk density less than gastric fluids and hence remain buoyant in the stomach. The main objective of the present study was to develop Gastroretentive (GR) controlled release ormulations of Metoprolol to prolong the gastric retention time so that its bioavailability can be improved. The formulations were prepared by using swellable polymers like HPMC K4M, HPMC K15M, HPMC K100M, Guar Gum, Xanthan Gum, Sodium carboxymethyl cellulose and various effervescent compounds, e.g. sodium bicarbonate, and citric acid by the direct compression method. All the formulations were evaluated for different parameters like floating lag time, total floating time, hardness, weight variation, density measurements, drug content and water uptake/swelling index. Dissolution studies were done for all formulations in 0.1N HCl (pH 1.2). Formulations F3, F4 and F10 were found to provide maximum sustained release of metoprolol s uccinate up to 24 h with optimum floating properties.Key words : Controlled release; Gastro retentive; HPMC; Guargum; Xantham gu

IN VITRO ABSORPTION STUDY OF CARBAMAZEPINE SOLID DISPERSION USING EVERTED GUT SAC METHOD

The oral Bioavailability of BCS (Bio Pharmaceutical Classification System) class II drug with poor solubility and reasonable permeability is limited by drug dissolution. In order to improve the aqueous solubility of the drug and dissolution of thedrug, the solid dispersion was prepared and evaluated for its absorption in intestine using modified everted gut sac method. The solid dispersion of carbamezepine (CBZ) was prepared using polaxomer and guargum by kneading method. The CBZ and CBZSD (Solid Disposisi) shows 2.329% and 3.948% drug absorption, respectively. The data show that solid dispersion increase the absorption of the CBZ in CBZ-SD is more than 70% in comparison to pure CBZ. The increase in CBZ solubility of the SD could be attributed to several factors such as improved wettability, local solubilization, drug particle size reduction and crystalline or, interstitial solid solution reduction. Key words: Everted gut sac method, solid dispersion, absorption, solubilit

Design and evaluation of guargum - based timolol maleate ocular insert For the treatment of glaucoma

Purpose: To prepare ocular inserts of timolol maleate using guar gum as a polymer for treatment of glaucoma. Methods: Timolol maleate ocular inserts were prepared by solvent casting method using guar gum in different proportions (0.25% w/v, 0.50% w/v , 75 % w/v and 1.0% w/v). The prepared formulations were evaluated for thickness, weight variation, percentage drug content, surface pH, folding endurance, percentage moisture absorption and loss, percentage swelling, mechanical strength and in vitro transcorneal permeation. In-vitro transcorneal permeation study was performed on goat cornea using modified Franz diffusion cell. Results: The inserts were found to be of uniform thickness (ranging from 41.12±0.04µm to 79.90±0.03µm) and weight (0.84±0.07 mg to 2.11±0.09 mg). The % drug content in the inserts was found to be varied between 98.69±0.58to 96.37±0.58. The cumulative % drug releases from the formulation ranged from 50.22±1.41 to 97.72±0.67over a period of 24 h. In-vitro transcorneal study revealed that an increase in concentration of polymer slows down the release of timolol maleate from the formulation. Conclusion: Ocular inserts using guar gum as a polymer were successfully prepared and can be effectively used for sustain ocular delivery over a period of 24 h

IN VITRO ABSORPTION STUDY OF CARBAMAZEPINE SOLID DISPERSION USING EVERTED GUT SAC METHOD

The oral Bioavailability of BCS (Bio Pharmaceutical Classification System) class II drug with poor solubility and reasonable permeability is limited by drug dissolution. In order to improve the aqueous solubility of the drug and dissolution of thedrug, the solid dispersion was prepared and evaluated for its absorption in intestine using modified everted gut sac method. The solid dispersion of carbamezepine (CBZ) was prepared using polaxomer and guargum by kneading method. The CBZ and CBZSD (Solid Disposisi) shows 2.329% and 3.948% drug absorption, respectively. The data show that solid dispersion increase the absorption of the CBZ in CBZ-SD is more than 70% in comparison to pure CBZ. The increase in CBZ solubility of the SD could be attributed to several factors such as improved wettability, local solubilization, drug particle size reduction and crystalline or, interstitial solid solution reduction

The Human CD8β M-4 Isoform Dominant in Effector Memory T Cells Has Distinct Cytoplasmic Motifs That Confer Unique Properties

<div><p>The CD8 co-receptor influences T cell recognition and responses in both anti-tumor and anti-viral immunity. During evolution in the ancestor of humans and chimpanzees, the CD8B gene acquired two additional exons. As a result, in humans, there are four CD8β splice variants (M1 to M4) that differ in their cytoplasmic tails. The M-1 isoform which is the equivalent of murine CD8β, is predominantly expressed in naïve T cells, whereas, the M-4 isoform is predominantly expressed in effector memory T cells. The characteristics of the M-4 isoform conferred by its unique 36 amino acid cytoplasmic tail are not known. In this study, we identified a dihydrophobic leucine-based receptor internalization motif in the cytoplasmic tail of M-4 that regulated its cell surface expression and downregulation after activation. Further the M-4 cytoplasmic tail was able to associate with ubiquitinated targets in 293T cells and mutations in the amino acids NPW, a potential EH domain binding site, either enhanced or inhibited the interaction. In addition, the M-4 tail was itself mono-ubiquitinated on a lysine residue in both 293T cells and a human T cell line. When peripheral blood human T cells expressed CD8αβ M-4, the frequency of MIP-1β secreting cells responding to antigen presenting cells was two-fold higher as compared to CD8αβ M-1 expressing T cells. Thus, the cytoplasmic tail of the CD8β M-4 isoform has unique characteristics, which likely contributed to its selective expression and function in human effector memory T cells.</p> </div

Identification of motifs signals in the cytoplasmic tail of M-4 isoform that modulate downregulation from the cell surface.

<p>(<b>A</b>) Receptor downregulation was measured by determining surface expression for CD8β M-4 wild-type and M-4 mutant levels by flow cytometry before and after stimulation of cells with PMA (100 ng/ml) for 60 minutes. One representative of 3 independent experiments is shown. (<b>B</b>) Receptor downregulation relative to CD8β M4 wild type was as in (A) quantitatively represented. Levels of CD3 protein after stimulation are indicated as well. Analyzing three experiments values that are statistically different from the wild type M-4 protein are indicated as * for p<0.05 and ** p<0.01.</p

Motifs in the cytoplasmic tail of the M-4 isoform that regulate cell surface expression.

<p>(<b>A</b>) Potential amino acid motifs in the cytoplasmic tail of M-4 isoform (<a href="http://www.expasy.org" target="_blank">www.expasy.org</a>). (<b>B</b>) Quantitative analysis of the surface expression of the M-4 wild-type and mutant proteins expressed in H9 cell line using the anti-CD8β antibody (5F2) by flow cytometry. The amount of CD8β binding was normalized to GFP expression. Each value corresponds to an average of three experiments. The standard deviation and two-population Student’s paired t-test was used to determine statistical differences of the mutants relative to the wild type M-4 isoform, indicated as one star * for p<0.05 and ** p<0.01. (<b>C</b>) Surface expression levels of M-4 wild-type and mutant proteins normalized to GFP expression in primary CD4⁺ T cells. Peripheral blood CD4⁺ T cells were stimulated with antibodies against CD3 and CD28 and transduced with lentiviruses expressing GFP and wild type or mutant M-4 proteins. On day 8 cell surface staining with CD8β antibody was analyzed by flow cytometry. The data are the mean +/− S.D. from three independent experiments. Values that are statistically different from the wild type M-4 protein are indicated as * p<0.05 and ** for p<0.01. (<b>D</b>) CD8αβ expression on CD4⁺ T cells prepared as in (C) expressing M-4 wild-type or mutants S²³²A or LL^235–6AG/IL^240–1AA. One representative experiment of five experiments is shown.</p

CD4⁺ T cells transduced with the M-4 CD8β isoform showed increased frequency of cells producing MIP-1β after stimulation.

<p>Peripheral blood CD4⁺ T cells were stimulated with anti-CD3 and anti-CD28 antibodies for 24 hours, and then co-transduced with a lentivirus expressing a NY-ESO-1 TCR and another lentivirus expressing CD8α and one of the CD8β isoforms. Cells were stimulated and analyzed for cytokine/chemokine production after day 10–12. (<b>A</b>) Schematic representation of lentiviral vectors used for co-transduction of primary CD4⁺ T cells is followed by histograms for surface expression of CD8αβ, CD8α and TCR and dot plots showing cell population co-expressing NY-ESO-1 TCR and CD8α. Data were collected by flow cytometry using antibodies against CD8 and an MHC tetramer specific to NY-ESO-1 (NY-ESO- tetramer). Live CD3⁺ lymphocytes were gated using side vs. forward scatter, anti-CD3 antibody and live/dead cell dye. (<b>B</b>) Frequency of transduced CD4⁺ T cells producing MIP-1β (top panel) after stimulation with K562 target cells expressing the NY-ESO-1 antigen. T cells without targets served as negative control and cells stimulated with PMA and Ionomycin (bottom panel) were used as positive control. One representative of three independent experiments is shown. Values that are statistically different from the wild type M-1 protein as determined by two-population Student’s paired t-test are indicated as one star (*) for p<0.05.</p

The NPW motif in the M-4 cytoplasmic tail mediates binding to ubiquitinated proteins.

<p>(<b>A</b>) HEK-293T cells co-transfected with plasmids expressing HA-tagged ubiquitin, and CD8β wild type M1, M4 or chimeric protein M1 with 15 amino acids of the C terminus of the M4 cytoplasmic tail. After 48 hrs cells were lysed with 1% BRIJ 97, precipitated with anti-CD8β mAb and run on a polyacrylamide gel. Western blotting was performed with the indicated antibody. The membrane was then stripped and re-probed with the anti-CD8β antibody. The experiment was repeated three times. (<b>B</b>) HEK-293T cells co-transfected with plasmids expressing HA-tagged ubiquitin, and CD8β wild type or mutant proteins. The immunoprecipitation and Western blotting experiments were performed as in (A). A representative of four experiments is depicted. (<b>C</b>) The intensity of the 30 kDa band was analyzed and the amount of the 30 kDa band of the wild type relative to each mutant protein is represented. The levels of CD8β protein detected after reprobing with anti-CD8β antibody were used to correct for differences in protein expression between experiments. Student’s paired t-test was used to determine statistical differences of the mutants relative to the wild type M-4 isoform, indicated as one star * for p<0.05.</p