Göran Ocklind

Chitosans as absorption enhancers of poorly absorbable drugs 3: Influence of mucus on absorption enhancement

Chitosans are potent nontoxic absorption enhancers after nasal administration but their effects on the intestinal epithelium in vivo has not been studied in detail. In this study, the effects of chitosans with varying molecular weights and degrees of acetylation on the absorption of a poorly absorbed model drug (atenolol) were studied in intestinal epithelial cell layers with or without a mucus layer and in an in situ perfusion model of rat ileum. The effects of the chitosans on epithelial morphology and release of lactate dehydrogenase (LDH) into the perfusate were investigated in the in situ model. The chitosans had pronounced effects on the permeability of mucus-free Caco-2 layers and enhanced the permeation of atenolol 10- to 15-fold, with different absorption kinetics for different chitosans, in accordance with previous results. In contrast, enhancement of atenolol absorption through rat ileum was modest. LDH release from the tissues perfused with chitosans did not increase, indicating that the chitosans were used at nontoxic concentrations. Morphological examination of the perfused ileal tissues revealed more mucus discharge from the tissues exposed to chitosans than from controls, which suggested that the discharged mucus may inhibit the binding of chitosan to the epithelial surface and hence decrease the absorption-enhancing effect. This hypothesis was supported by studies with intestinal epithelial HT29-H goblet cells covered with a mucus layer. The binding of chitosan to the epithelial cell surface and subsequent absorption-enhancing effects were significantly reduced in mucus-covered HT29-H cultures. When the mucus layer was removed prior to the addition of chitosan, the cell surface binding and absorption-enhancing effects of the chitosans were increased. We conclude that the modest absorption-enhancing effects of unformulated chitosan solutions in the perfused rat ileum are a result of the mucus barrier in this tissue. This effect may be overcome by increasing the local concentrations of both chitosan and drug, i.e,. through formulation of the chitosan into a particulate dosage form.

Mechanism of Absorption Enhancement in Humans After Rectal Administration of Ampicillin in Suppositories Containing Sodium Caprate

AbstractPurpose. The medium chain fatty acid sodium caprate (C10) is approved as an absorption enhancer but its mechanism of action has not been studied in humans. The aim of this study was to investigate the mechanism of action of C10 in human subjects after rectal administration. Methods. Twelve healthy human subjects were randomised to receive ampicillin suppositories with (AM-C10) or without (AM) C10. Serum and urine samples were collected and analysed for ampicillin by HPLC. Rectal biopsies were taken before and 25 min (approximate time of maximum serum concentration, Cmax, for ampicillin) and 185 min (during the final part of the elimination phase) after rectal administration of the suppositories. The osmolality of the rectal fluid was also measured. Results. AM-C10 administration increased Cmax, area under the serum concentration-time curve (AUC) and urinary recovery of ampicillin 2.6-, 2.3- and 1.8-fold, respectively, compared to AM. Histological examination of the biopsies showed that AM-C10 exposure resulted in reversible mucosal damage that occurred at the same time as the Cmax for ampicillin while AM prolonged mucosal damage. A reversible increase in rectal fluid osmolality was observed with both treatments. Conclusions. AM-ClO-enhanced absorption of ampicillin coincides with non-specific damage to the rectal mucosa. C10 itself as well as the suppository base and the hyperosmolality of the rectal fluid contributed to this effect. However, the histological damage was reversible with AM-C10, suggesting that C10 also has a protective effect on the rectal mucosa.

Freeze-thaw immobilization of liposomes in chromatographic gel beads: evaluation by confocal microscopy and effects of freezing rate.

Biological membranes immobilized in chromatographic gel beads constitute a multifunctional affinity matrix. Membrane protein–solute interactions and drug partitioning into the lipid bilayers can conveniently be studied. By the use of confocal laser‐scanning microscopy (CLSM) the distribution of immobilized model membranes in the beads has been visualized for the first time. Freeze–thaw‐immobilized liposomes in Superdex 200 gel beads were situated in a thick shell surrounding a liposome‐free core. The amount of phospholipids immobilized by freeze–thawing was dependent on the temperature in the cooling bath and the type of test tube used. A bath temperature of −25 °C gave higher immobilization yield than freezing at −75 or −8 °C did. Freeze–thawing in the presence of liposomes did not affect the gel bead shape or the refractive index homogeneity of the agarose network of the beads, as shown by confocal microscopy. Copyright

An improved method for preparing a pollen concentrate suitable for 14C-dating

Abstract An improved procedure for preparing a pollen concentrate from clayey sediments is presented. Two new steps have been added compared with earlier procedures. Firstly an initial dispersion which enhances the pollen yield from a sample so that a smaller amount of material is required for the separation, and secondly, the use of CsCl for sequential density separation which results in high purity pollen concentrates. Our separation technique was tested through radiocarbon dating a sediment sequence from Old Uppsala, central Sweden.

Expression of CD54, CD58, CD14, and HLA-DR on macrophages and macrophage-derived accessory cells and their accessory capacity

Human peripheral monocytes can differentiate in vitro into macrophages (Mph) possessing a low accessory activity in T cell stimulation. Mph can be converted into a state of high accessory activity by treatment with dibutyryl cyclic AMP. This finding was used in this study to achieve Mph-derived AC (MphAC). Among the surface antigens on AC which have been shown to participate in accessory events leading to T cell proliferation, MHC class II antigens, CD58 (LFA-3) and CD54 (ICAM-1) seem to be especially important. We show here that the high accessory capacity of MphAC was not correlated with a high level of the surface antigens HLA-DR, CD58, and CD54. The amount of CD54 molecules was, in fact, lower on the MphAC than on the Mph.

Stimulation of human lymphocytes by oxidized red blood cells (RBC) in the presence of polyethylene glycol (PEG).

In the present work, it is shown that oxidized red blood cells (RBC) in the presence of polyethylene glycol (PEG) is a powerful cellular class-II MHC-free mitogen for human lymphocytes. Human lymphocytes were stimulated with oxidized autologous human RBC or sheep RBC in the absence or presence of PEG, M.W. 10,000. The RBC were oxidized with the enzyme combination neuraminidase-galactose oxidase or with sodium periodate. The presence of PEG strongly potentiated the response. Optimal stimulatory conditions were obtained with a 10:1 ratio of oxidized RBC to lymphocytes and a PEG concentration of 25 +/- 5%.

Stimulation of human lymphocytes by phytohemagglutinin (PHA) in a new ultra-microtest plate

The requirements for monocytes in lymphocyte proliferation were studied in ultra-microcultures. For this purpose, an ultra-microtest plate was developed which comprises 121 wells, each having 0.23 microliter volume and 0.28 mm2 culture area. Human peripheral lymphocytes were seeded in the wells in numbers ranging from 1-57 cells/well and subsequently stimulated with phytohemagglutinin (PHA). Proliferation, assessed by microscopy in situ, was established in 46% of the wells where adherent non-specific, esterase-positive cells were present and in 6% where such cells were absent. The results indicate that PHA-stimulated human lymphocytes can proliferate in the absence of monocytes. The new microplate should be a valuable tool for dissecting the early events in T cell activation, especially if combined with various analytical methods such as time-lapse video, autoradiography and surface-marker techniques.

Activation of Human T Cells by Neuraminidase-Galactose Oxidase-Treated Erythrocytes Involving CD2 (T11) and its Complementary Structure

Human T lymphocyte proliferation induced by neuraminidase‐galactose oxidase (NAGO)‐treated autologous erythrocytes (HENAGO) plus polyethylene glycol (PEG) has previously been shown to be independent of accessory cells. Here, we show that the response to HENAGO+PEG was accompanied by interleukin 2 (IL‐2) release and was inhibited by anti‐IL‐2 and anti‐IL‐2 receptor antibodies. HENAGO alone initiated DNA synthesis together with phorbol ester (12‐O‐tetradecanoyl‐phorbol‐13‐acetate; TPA). To elucidate the nature of the stimulatory signals NAGO‐treated sheep erythrocytes (SENAGO) were used in additional experiments. In parallel to the superior rosetting capacity of SE compared to HE, SENAGO were by themselves stimulatory, and the response was further enhanced by PEG or TPA. Antibody L180/1, specific for the T11 (CD2) target structure (T11TS) on SE, homologous to the human CD2 ligand LFA‐3, abolished the response to SENAGO alone or when combined with PEG or TPA. The results suggest that ENAGO induce T‐cell response through CD2‐LFA‐3‐T11TS interaction, and via other surface antigens bound by the oxidatively induced aldehyde groups on ENAGO.

Accessory cell-independent activation of T cells by oxidized red blood cells plus polyethylene glycol

We have previously shown that the combination of neuraminidase-galactose oxidase (NAGO)-treated autologous erythrocytes (EOX) and polyethylene glycol (PEG) is highly mitogenic for human peripheral blood lymphocytes (PBL). In this report, we show that EOX plus PEG-induced T lymphocyte proliferation is independent of HLA-DR and Leu M3-positive accessory cells (AC). Purified T (pT) cells and PBL were equally stimulated by EOX + PEG, while pT cells were unresponsive to the mitogens phytohemagglutinin (PHA), NAGO, and the anti-CD3 antibody UCHT1, even in the presence of PEG. These findings indicate that specific signals from AC may be replaced by unspecific stimuli in T cell activation.

Conditionally Immortalized Intestinal Epithelial Cells: A New Model for Studying Intestinal Epithelial Cell Turnover

Conditionally immortalized intestinal epithelial cells - A new model for studying intestinal epithelial cell turnover