Synthetic biodegradable ionomers that engulf, store, and deliver intact proteins

Telechelic anionic and cationic biodegradable ionomers capable of loading, storing, and releasing proteins are presented. Two different ionomers have been synthesized with either anionic or cationic end groups. The reaction was done quantitatively as shown by (1)H NMR. The swelling properties of the hydrophobic poly(trimethylene carbonate) polymer are contributed to the ionic end groups that display hydrophilic properties. Depending on the molecular weight of the ionomer, and also on the ionic charge, the materials swell differently in water, from approximately 50% for M(w) = 12 000 g/mol to approximately 500% when dealing with 2000 g/mol. The high swelling led us to believe that it would be possible to load and release proteins preferably in a still active form. As models, two different proteins were chosen: hemoglobin and cytochrome c. The swelling and release study shows that both ionomers possess the capability to adsorb and later release the proteins with retained structure. Release measurements from both the swollen and dried states have been evaluated with similar results, showing that the dried state seems to release a little bit less than the swollen one. These kinds of materials should be interesting for a wide variety of applications where drug and protein release is wanted, as well as in applications such as protein separation media.     

Evidence for a protein kinase C-directed mechanism in the phorbol diester-induced phospholipase D pathway of diacylglycerol generation from phosphatidylcholine

In this study we provide evidence for the involvement of protein kinase C (PKC) in phorbol diester-induced phosphatidylcholine (PC) hydrolysis by the phospholipase D pathway. Rat embryo fibroblasts (REF52) were prelabeled with either tritiated choline or myristic acid; these compounds are preferentially incorporated into cellular PC. Phorbol diester-induced PC degradation was determined by measuring the release of [3H]choline, and the formation of [3H]myristoyl-containing phosphatidate (PA), diacylglycerol (DG), and phosphatidylethanol (PE). Staurosporine, a PKC inhibitor, blocked from 73 to 90% of the phorbol diester-induced PC hydrolysis. The inhibition of phorbol diester-induced choline release by staurosporine was dose dependent with an approximate ED50 of 150 nM. Pretreatment of cells with phorbol diester inhibited subsequent phorbol diester-induced PC degradation by 78-92%. A close correlation between the ED50 for phorbol diester-stimulated choline release and the Kd for phorbol diester binding was demonstrated. Neither forskolin nor dibutyryl cAMP elicited cellular PC degradation. In vitro experiments using phospholipase D from Streptomyces chromofuscus showed that staurosporine did not inhibit and TPA did not stimulate enzyme activity.     

Hexadecylphosphocholine inhibits phosphatidylcholine biosynthesis and the proliferation of HepG2 cells.

Hexadecylphosphocholine (HePC) is a synthetic lipid representative of a new group of antiproliferative agents, alkylphosphocholines (APC), which are promising candidates in anticancer therapy. Thus we have studied the action of HePC on the human hepatoblastoma cell line HepG2, which is frequently used as a model for studies into hepatic lipid metabolism. Non-toxic, micromolar concentrations of HePC exerted an antiproliferative effect on this hepatoma cell line. The incorporation into phosphatidylcholine (PC) of the exogenous precursor [methyl-14C]choline was substantially reduced by HePC. This effect was not due to any alteration in choline uptake by the cells, the degradation rate of PC or the release of PC into the culture medium. As anaccumulation of soluble choline derivatives points to CTP:phosphocholine cytidylyltransferase (CT) as the target of HePC activity we examined its effects on the different enzymes involved in the biosynthesis of PC via CDP-choline. Treatment with HePC altered neither the activity of choline kinase (CK) nor that of diacylglycerol cholinephosphotransferase (CPT), but it did inhibit CT activity in HepG2 cells. In vitro HePC also inhibited the activity of cytosolic but not membrane-bound CT. Taken together our results suggest that HePC interferes specifically with the biosynthesis of PC in HepG2 cells by depressing CT translocation to the membrane, which may well impair their proliferation.     

Controlled release of tamoxifen citrate encapsulated in cross-linked guar gum nanoparticles

Natural polysaccharides, due to their outstanding merits, have received more and more attention in the field of drug delivery. In the present study tamoxifen citrate, TMX (a non-steroidal antiestrogenic drug) loaded guar gum nanoparticles, GG NPs, crosslinked with glutaraldehyde were prepared for treatment of breast cancer. An oil in water (o/w) emulsion polymer cross-linking method was employed for preparation of blank and drug loaded sustained release nature biodegradable nanoparticles. Prepared nanoparticles were characterized by morphology in scanning electron microscope (SEM), size distribution in transmission electron microscope (TEM), TMX loading by high performance liquid chromatography (HPLC) and in vitro drug release characteristics. An overall sustained release of the drug from the biodegradable nanoparticles was observed in in vitro release studies. The release of TMX from GG NPs was found to be effected by guar gum and glutaraldehyde concentration. Regression coefficient (R²) analysis suggested that the predominant mechanism behind the drug release from the nanoparticles was time dependent release and diffusion. In vivo studies on female albino mice demonstrated maximum uptake of the drug by mammary tissue after 24 h of administration with drug loaded guar gum nanoparticles in comparison with that with the tablet form of the drug. These findings demonstrate that controlled release of TMX from GG NPs could be a potential alternative pharmaceutical formulation in passive targeting of TMX in breast cancer treatments.     

In silico Structure Modeling and Comparative Analysis of Characterization Properties of Protein Polymers Useful for Protein-Based Nano Particulate Drug Delivery Systems (NPDDS): A Bioinformatics Approach

With an increasing interest in nanoparticulate delivery systems, there is a greater need to identify biomaterials that are biocompatible and safe for human applications. Protein polymers from animal and plant sources are promising materials for designing nanocarriers. Composition of the protein plays an important role for specific drug delivery applications such as drug release, targeting, and stimuli responsive drug release. An important issue in protein polymers is characteristics such as size, charge, and hydrophobicity may play a significant role in phagocytic uptake and initiating a subsequent immune response. This remains to be investigated systematically by analyzing factors that influence nanoparticle characteristics of protein and reduce phagocytic uptake and does not initiate immune response too. Although protein polymers are biodegradable, it is essential to ensure that there must not be premature enzymatic breakdown of the protein nanoparticles in the systemic circulation. Surface modification of the protein nanoparticles can be used to address this issue to propose the necessary modification in the surface of the protein would be great contribution in the nano particulate drug delivery systems (NPPDS). Of the various proteins, gelatin and albumin have been widely studied for drug delivery applications. Plant proteins are yet to be investigated widely for drug delivery applications so there is need to find out the plant proteins capable to act as nanoparticles. The commercial success of albumin-based nanoparticles has created an interest in other proteins. An increased understanding of the physicochemical properties coupled with the developments in rDNA technology will open up new opportunities for protein-based nanoparticulate systems. In the present studies several proteins currently useful for drug delivery system were structurally modeled and has been analyzed to propose the essential characteristics of protein for protein-based NPDDS.     

Lidocaine inhibits choline uptake and phosphatidylcholine biosynthesis in human leukemic monocyte-like U937 cells

The effect of lidocaine on [³H]choline uptake and the incorporation of label into phosphatidylcholine (PC) in human monocyte-like U937 cells was investigated. Lidocaine inhibited the rate of choline uptake in a dose-dependent manner; at 3·2 mM it resulted in a drastic reduction, by as much as 65 per cent (n = 10; p < 0·0005) or 55 per cent (n = 10; p < 0·0006) in a 3- or 6-h incubation, respectively. Lidocaine also decreased the rate of choline incorporation into PC in a dose-dependent manner. At the highest dose, nearly 70 per cent or 45 per cent reduction was seen in a 3- or 6-h incubation, respectively. Analysis of choline-containing metabolites showed that the major label association with phosphocholine and PC was reduced to a similar extent which was also parallel to the inhibition of choline uptake. At 3·2 mM lidocaine, the reduction of choline uptake was shown to follow a competitive inhibition. In the case of [³H] choline incorporation into PC, the inhibitory pattern was shown to be of a mixed type. The pulse-chase study dissecting the effect on choline metabolism from that on total choline uptake indicated that lidocaine exerted an additionally inhibitory effect on intracellular choline metabolism into PC. In a separate protocol in which the labelled cells were first allowed to be chased until ³H-incorporation into PC reached a steady state, lidocaine no longer showed any effect. These results seem to exclude the possibility of enhanced PC breakdown and further suggest that the main inhibitory effect is on the CDP-choline pathway for PC biosynthesis. After a 3-h treatment, CTP: cholinephosphate cytidylyltransferase (CYT) in both the cytosolic and microsomal fractions was inhibited by approximately 20 per cent, while choline kinase (CK) and choline phosphotransferase (CPT) remain relatively unchanged. There was no evidence for translocation of CYT between cytosol and microsomes. Taken together, we have demonstrated a dual inhibitory function of lidocaine which inhibits PC biosynthesis in addition to its ability to block choline uptake profoundly in U937 cells.     

The metabolism of [Me-14C]choline in the brain of the rat in vivo

[Me-(14)C]Choline was injected intracerebrally into the adult rat, and its uptake into the lipids and their water-soluble precursors in brain was studied. The radioactivity could be detected only in the choline-containing lipids and was confined to the base choline. The results indicated that initial phosphorylation of the free choline followed by the formation of CDP-choline and the subsequent transfer of the phosphorylcholine to a diglyceride is one of the principal routes by which choline lipids in brain are formed. Further evidence for this was obtained in experiments in which either phosphoryl[Me-(14)C]choline or [(32)P]orthophosphate was injected and the radioactivity in the choline-containing water-soluble and lipidbound components studied.     

Effect of feeding protein deficient diet on phospholipid turnover and protein kinase C mediated protein phosphorylation in rat brain

Feeding of protein deficient diet is known to alter the transmembrane signalling in brain of rat by reducing total protein kinase C (PKC) activity. Phospholipid metabolism regulates the activation of PKC through generation of second messengers and the extent of PKC activation accordingly influences the magnitude of phosphorylation of its endogenous substrate proteins. Thus it was speculated that ingestion of protein deficient diet may modify the turnover rate of membrane phospholipids and magnitude of phosphorylation of endogenous substrate proteins of PKC. The experiments were conducted on rats fed on three different types of laboratory prepared diets viz. casein (20% casein), deficient (4% protein, rice flour as source of protein) and supplemented (deficient diet supplemented with L-lysine and DL-threonine) for 28 days. The metabolism of phosphoinositides (PIs) and phosphatidyl choline (PC) was studied by equilibrium labeling with [3H] myo inositol and [14C methyl] choline chloride respectively. The phosphorylation of endogenous substrate proteins of PKC was studied by using 32P-gamma-ATP followed by SDS-PAGE and autoradiography. The results suggest that in deficient group, there is an increased incorporation of [3H] myo inositol in PIs and inositol phosphate pool in comparison to the casein group. The phosphatidyl inositol (PI) turnover reduced, although there was a marginal increase in the phosphatidyl inositol monophosphate (PIP) and phosphatidyl inositol bis phosphate (PIP2). Supplementation of diet showed a reversal of the pattern towards control to a considerable extent. In the deficient group, PC metabolism showed an increased incorporation of [14C methyl] choline in choline phospholipids but decreased incorporation in phosphoryl choline in comparison with the casein group. The increase in total PC contents was significant but marginal in residue contents. The turnover rate of PC increased only marginally and that of residue declined. Supplementation of diet reduced the total contents of PC and residue, but the turnover rate of PC and residue remained still higher. Phosphorylation of endogenous proteins showed four different proteins of 78, 46, 33 and 16 kDa to be the substrates of PKC in casein group. In deficient group, phosphorylation of these proteins increased markedly while supplementation of diet had a reversing effect rendering the values to be intermediate between casein and the supplemented group. The changes in phospholipid metabolism and in phosphorylation of endogenous substrate proteins of PKC suggest that dietary protein deficiency causes alterations in transmembrane signalling mechanism in rat brain. These effects are partially reversed by improving the quality of proteins in the diet.     

Identification and Characterization of a High-Affinity Choline Uptake System of Brucella abortus

Phosphatidylcholine (PC), a common phospholipid of the eukaryotic cell membrane, is present in the cell envelope of the intracellular pathogen Brucella abortus, the etiological agent of bovine brucellosis. In this pathogen, the biosynthesis of PC proceeds mainly through the phosphatidylcholine synthase pathway; hence, it relies on the presence of choline in the milieu. These observations imply that B. abortus encodes an as-yet-unknown choline uptake system. Taking advantage of the requirement of choline uptake for PC synthesis, we devised a method that allowed us to identify a homologue of ChoX, the high-affinity periplasmic binding protein of the ABC transporter ChoXWV. Disruption of the choX gene completely abrogated PC synthesis at low choline concentrations in the medium, thus indicating that it is a high-affinity transporter needed for PC synthesis via the PC synthase (PCS) pathway. However, the synthesis of PC was restored when the mutant was incubated in media with higher choline concentrations, suggesting the presence of an alternative low-affinity choline uptake activity. By means of a fluorescence-based equilibrium-binding assay and using the kinetics of radiolabeled choline uptake, we show that ChoX binds choline with an extremely high affinity, and we also demonstrate that its activity is inhibited by increasing choline concentrations. Cell infection assays indicate that ChoX activity is required during the first phase of B. abortus intracellular traffic, suggesting that choline concentrations in the early and intermediate Brucella-containing vacuoles are limited. Altogether, these results suggest that choline transport and PC synthesis are strictly regulated in B. abortus.     

Membrane formation and drug loading effects in high amylose starch tablets studied by NMR imaging

Cross-linked high amylose starch is used as an excipient in the preparation of pharmaceutical tablets for the sustained release of drugs. NMR imaging with contrast enhanced by proton density and by self-diffusion coefficient was used to follow the water uptake and swelling, two critical parameters controlling the drug release of the cross-linked starch tablets containing 10 wt % of ciprofloxacin and of acetaminophen, respectively. The drug-loaded tablets were studied in a H2O/D2O mixture at 37 degrees C in comparison to the tablets without any drug loading. The diffusion of water in the tablets all showed a Fickian behavior, but the kinetics of water uptake was faster in the case of the drug-loaded tablets. The formation of a membrane at the water/tablet interface was observed.     

The protein phosphatase inhibitor, okadaic acid, inhibits phosphatidylcholine biosynthesis in isolated rat hepatocytes.

There is evidence that phosphatidylcholine (PC) biosynthesis in hepatocytes is regulated by a phosphorylation-dephosphorylation mechanism. The phosphatases involved have not been identified. We, therefore, investigated the effect of okadaic acid, a potent protein phosphatase inhibitor, on PC biosynthesis via the CDP-choline pathway in suspension cultures of isolated rat hepatocytes. Okadaic acid caused a 15% decrease (P less than 0.05) in [Me-3H]choline uptake in continuous-pulse labeling experiments. After 120 min of treatment, the labeling of PC was decreased 46% (P less than 0.05) with a corresponding 20% increase (P less than 0.05) in labeling of phosphocholine. Cells were pulsed with [Me-3H]choline for 30 min and subsequently chased for up to 120 min with choline in the absence or presence of okadaic acid. The labeling of phosphocholine was increased 86% (P less than 0.05) and labeling of PC decreased 29% (P less than 0.05) by 120 min of chase in okadaic acid-treated hepatocytes. The decrease of label in PC was quantitatively accounted for in the phosphocholine fraction. Incubation of hepatocytes with both okadaic acid and CPT-cAMP did not produce an additive inhibition in labeling of PC. Choline kinase and cholinephosphotransferase activities were unaltered by treatment with okadaic acid. Hepatocytes were incubated with digitonin to cause release of cytosolic components. Cell ghost membrane cytidylyltransferase (CT) activity was decreased 37% (P less than 0.005) with a concomitant 33% increase (P less than 0.05) in released cytosolic cytidylyltransferase activity in okadaic acid-treated hepatocytes. We postulate that CT activity and PC biosynthesis are regulated by protein phosphatase activity in isolated rat hepatocytes.     

Actions of ginsenoside Rb1 on choline uptake in central cholinergic nerve endings.

The ginsenoside Rb1 has previously been reported to improve memory deficits induced by anticholinergic drug treatment, and to facilitate acetylcholine (Ach) release from rat brain hippocampal slices. The increase in ACh release was not associated with an increase in calcium uptake into nerve terminals, but was associated with an increase in uptake of the precursor choline. In the present studies, analysis of choline uptake kinetics indicated that Rb1 increased the maximum velocity of choline uptake, while the affinity of the choline uptake carrier for choline (Km) was not significantly altered. Acute treatment with Rb1 did not alter the number of [3H]hemicholinium-3 (HC-3) binding sites in any of three cholinergic brain regions examined, suggesting that the increase in the maximum velocity of choline uptake was not associated with an increase in the number of choline carriers. However, chronic (3 day) administration of Rb1 did increase the number of choline uptake sites in the hippocampus, and to a lesser extent in the cortex.     

Stimulation of phosphatidylcholine biosynthesis by hemicholinium-3, a potent inhibitor of choline uptake in human leukemic monocyte-like U937 cells

The effect of hemicholinium-3 (HC-3) on choline uptake and phosphatidylcholine (PC) biosynthesis was examined in human leukemic monocyte-like U937 cells. HC-3 inhibited [³H]choline uptake in a dose- and time-dependent manner. After a 3 h treatment, HC-3 (100 μM) decreased choline uptake by as much as 80 per cent (p < 0·0001; n = 4). Reduction of incorporation of label into PC was also detected in a dose-dependent manner; the extent of inhibition, however, was always 10–20 per cent less than that observed in the total uptake. At 3 h HC-3 decreased the incorporation into PC by 65 per cent (p < 0·0001; n = 5). Kinetic studies in vivo showed that HC-3 inhibited total uptake and incorporation into PC differently, suggesting that the labelling of PC is not simply dictated by [³H]choline uptake. In separate experiments, cells were pretreated with 100 μM HC-3 for 3 h. After washing, the inhibitory effect on total uptake was no longer observed, while a 20 per cent stimulation of the incorporation into PC was obtained in these pretreated cells. In pulse-chase studies, the cells were prelabelled with [³H]choline for 30 min and chased with HC-3 for up to 3 h; the results showed a significant stimulation of incorporation into PC in a longer chase with 100 μM HC-3. After a 3 h treatment, the cytosolic CTP:cholinephosphate cytidylytransferase (CT) was activated by 56 per cent, while choline kinase (CK) was inhibited slightly. The stimulation of CT was not simply due to the intact HC-3 molecule, and there was no redistribution of CT between cytosol and microsomes. Taken together, the results suggest that HC-3 activates PC biosynthesis apart from the inhibitory effect on choline uptake.     

Effects of (dihydro)cytochalasin B, colchicine, monensin and trifluoperazine on uptake and processing of liposomes by Kupffer cells in culture

We investigated the effects of (dihydro)cytochalasin B, colchicine, monensin and trifluoperazine on uptake and processing of large unilamellar liposomes by rat Kupffer cells in maintenance culture. The phospholipid vesicles were labeled in the lipid moiety with phosphatidyl[14C]choline and contained [3H]inulin or [125I]iodoalbumin as nondegradable and degradable markers of the aqueous vesicle content, respectively. Cytochalasin B and dihydrocytochalasin B, inhibitors of microfilament function, reduced inert inulin label uptake by 75% maximally, but residual uptake was not followed by release of lipid degradation products from the cells. By contrast, colchicine, an inhibitor of microtubule assembly, reduced uptake of liposomal inulin by maximally 55% but could not inhibit release of lipid degradation products from the cells. It is concluded that the cytochalasins partly inhibit uptake but fully prevent the arrival of internalized liposomes in the lysosomal compartment, while the action of colchicine is to slow down the overall process of uptake and subsequent transportation to the lysosomes. Monensin reduced inulin uptake to an extent similar to that found with colchicine, but reversibly blocked degradation of liposomal lipid and encapsulated protein. The kinetics of degradation of liposomal constituents suggests that residual uptake in the presence of monensin represents accumulation in an intracellular compartment. Trifluoperazine did not affect binding, internalization or degradation of encapsulated protein at low concentration (6 microM), but completely inhibited release of liposomal lipid degradation products under these conditions. At intermediate concentration (14 microM), the drug also reduced the internalization, while a high concentration (22 microM) was required to inhibit protein degradation as well. We conclude that trifluoperazine has multiple sites of action in the uptake and processing of liposomal constituents by Kupffer cells.     

Phospholipid uptake by Plasmodium knowlesi infected erythrocytes

The uptake of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) in Plasmodium knowlesi infected erythrocytes has been studied. Whereas uptake of phospholipids, in the absence of phospholipid transfer proteins, is negligible in control cells, the infected cells can incorporate considerable amounts of added phospholipids. The uptake is enhanced by the presence of lipid transfer proteins. Doubly labeled [3H]oleate, [14C]choline) PC does not undergo any appreciable remodelling following uptake, which strongly suggests that plasma PC is used as such for the biogenesis of the parasite membranes. Transport of extracellularly offered PS and PE towards the intraerythrocytic parasite and utilization of these lipids by the parasite are confirmed by the observation that these lipids are converted into respectively PE and PC. The extent and rate of these conversions depend on the way the phospholipids are introduced into the infected cells.     

Effect of Formulation and Process Parameters on Chitosan Microparticles Prepared by an Emulsion Crosslinking Technique

There are many studies about the synthesis of chitosan microparticles; however, most of them have very low production rate, have wide size distribution, are difficult to reproduce, and use harsh crosslinking agents. Uniform microparticles are necessary to obtain repeatable drug release behavior. The main focus of this investigation was to study the effect of the process and formulation parameters during the preparation of chitosan microparticles in order to produce particles with narrow size distribution. The technique evaluated during this study was emulsion crosslinking technique. Chitosan is a biocompatible and biodegradable material but lacks good mechanical properties; for that reason, chitosan was ionically crosslinked with sodium tripolyphosphate (TPP) at three different ratios (32, 64, and 100%). The model drug used was acetylsalicylic acid (ASA). During the preparation of the microparticles, chitosan was first mixed with ASA and then dispersed in oil containing an emulsifier. The evaporation of the solvents hardened the hydrophilic droplets forming microparticles with spherical shape. The process and formulation parameters were varied, and the microparticles were characterized by their morphology, particle size, drug loading efficiency, and drug release behavior. The higher drug loading efficiency was achieved by using 32% mass ratio of TPP to chitosan. The average microparticle size was 18.7 μm. The optimum formulation conditions to prepare uniform spherical microparticles were determined and represented by a region in a triangular phase diagram. The drug release analyses were evaluated in phosphate buffer solution at pH 7.4 and were mainly completed at 24 h.     

Regulatory networks,genes and glycerophospholipid biosynthesis pathway in schistosomiasis: A systems biology view for pharmacological intervention

Understanding network topology through embracing the global dynamical regulation of genes in an active state space rather than traditional one-gene–one trait approach facilitates the rational drug development process. Schistosomiasis, a neglected tropical disease, has glycerophospholipids as abundant molecules present on its surface. Lack of effective clinical solutions to treat pathogens encourages us to carry out systems-level studies that could contribute to the development of an effective therapy. Development of a strategy for identifying drug targets by combined genome-scale metabolic network and essentiality analyses through in silico approaches provides tantalizing opportunity to investigate the role of protein/substrate metabolism. A genome-scale metabolic network model reconstruction represents choline–phosphate cytidyltransferase as the rate limiting enzyme and regulates the rate of phosphatidylcholine (PC) biosynthesis. The uptake of choline was regulated by choline concentration, promoting the regulation of phosphocholine synthesis. In Schistosoma, the change in developmental stage could result from the availability of choline, hampering its developmental cycle. There are no structural reports for this protein. In order to inhibit the activity of choline–phosphate cytidyltransferase (CCT), it was modeled by homology modeling using 1COZ as the template from Bacillus subtilis. The transition-state stabilization and catalytic residues were mapped as ‘HXGH’ and ‘RTEGISTT’ motif. CCT catalyzes the formation of CDP-choline from phosphocholine in which nucleotidyltransferase adds CTP to phosphocholine. The presence of phosphocholine permits the parasite to survive in an immunologically hostile environment. This feature endeavors development of an inhibitor specific for cytidyltransferase in Schistosoma. Flavonolignans were used to inhibit this activity in which hydnowightin showed the highest affinity as compared to miltefosine.     

Cellulose Acetate Butyrate: Ammonio Methacrylate Copolymer Blends as a Novel Coating in Osmotic Tablets

The objective of this work was the preparation of osmotic tablets using polymer blends of cellulose acetate butyrate (CAB) or ethylcellulose with ammonio methacrylate copolymer (Eudragit® RL). The advantage of these coatings in comparison to the traditionally used cellulose acetate is their solubility in safer organic solvents like ethanol. Polymer films were characterized with respect to their water uptake, dry mass loss, and mechanical properties. The effect of the polymer blend ratio on drug release and on the rupture force of the coating was investigated. In addition, the effect of drug solubility and content, pH and agitation rate of the release medium, and coating level and plasticizer content on the release were studied. With increased Eudragit® RL content in the coating blends, higher medium uptake of the film was observed, resulting in shorter lag times and faster drug release from the osmotic tablets. Replacing ethylcellulose with cellulose acetate butyrate as a coating material led to shorter lag times and faster drug release due to increased film permeability. In addition, CAB-based films had a higher strength and flexibility. The drug release was osmotically controlled and decreased with increasing coating level. It increased with increased drug solubility, plasticizer content, change of buffer species (acetate > phosphate), and decreased coating level. Agitation rate and drug content had no effect on the drug release. A 20% w/w coating level was sufficient for the tablet to tolerate forces of more than five times of the gastric destructive force reported in literature.     

Acetylcholine Mobilization in a Sympathetic Ganglion in the Presence and Absence of 2-(4-Phenylpiperidino)Cyclohexanol (AH5183)

The present experiments measured the release of acetylcholine (ACh) by the cat superior cervical ganglia in the presence of, and after exposure to, 2-(4-phenylpiperidino)cyclohexanol (AH5183), a compound known to block the uptake of ACh by cholinergic synaptic vesicles. We confirmed that AH5183 blocks evoked ACh release during preganglionic nerve stimulation when approximately 13-14% of the initial ganglial ACh stores had been released; periods of rest in the presence of the drug did not promote recovery from the block, but ACh release recovered following the washout of AH5183. ACh was synthesized in AH5183-treated ganglia, as determined by the synthesis of [3H]ACh from [3H]choline, and this [3H]ACh could be released by stimulation following drug washout. The specific activity of the released ACh matched that of the tissue's ACh, and thus we conclude that ACh synthesized in the presence of AH5183 is a releasable as pre-existing ACh stores once the drug is removed. We tested the relative releasability of ACh synthesized during AH5183 exposure (perfusion with [3H]choline) and that synthesized during recovery from the drug's effects (perfusion with [14C]choline: the ratio of [3H]ACh to [14C]ACh released by stimulation was similar to the ratio in the tissue. These results suggest that the mobilization of ACh for release by ganglia during recovery from an AH5183-induced block is independent of the conditions under which the ACh was synthesized. Unlike nerve impulses, black widow spider venom (BWSV) induced the release of ACh from AH5183-blocked ganglia, even in the drug's continued presence. Venom-induced release of ACh from AH5183-treated ganglia was not less than the venom-induced release from tissues not exposed to AH5183. This effect of BWSV was attributed to the action of the protein, alpha-latrotoxin, because an anti-alpha-latrotoxin antiserum blocked the venom's action. ACh synthesized during AH5183 exposure was labelled from [3H]choline, and subsequent treatment with BWSV released [3H]ACh with the same temporal pattern as the release of total ACh. To exclude a nonexocytotic origin for the [3H]ACh released by BWSV, ganglia were preloaded with [3H]diethylhomocholine to form [3H]acetyldiethylhomocholine, an ACh analogue excluded from vesicles; the venom did not increase the rate of [3H]acetyldiethylhomocholine efflux. It is concluded that a vesicular ACh pool insensitive to the inhibitory action of AH5183 might exist and that this vesicular pool is not mobilized by electrical stimulation to exocytose in the presence of AH5183, but it is by BWSV.     

Acetylcholine release and choline uptake by cuttlefish (Sepia officinalis) optic lobe synaptosomes

Acetylcholine (ACh), which is synthesized from choline (Ch), is believed to hold a central place in signaling mechanisms within the central nervous system (CNS) of cuttlefish (Sepia officinalis) and other coleoid cephalopods. Although the main elements required for cholinergic function have been identified in cephalopods, the transmembrane translocation events promoting the release of ACh and the uptake of Ch remain largely unsolved. The ACh release and Ch uptake were quantitatively studied through the use of in vitro chemiluminescence and isotopic methods on a subcellular fraction enriched in synaptic nerve endings (synaptosomes) isolated from cuttlefish optic lobe. The ACh release evoked by K+ depolarization was found to be very high (0.04 pmol ACh.s(-1).mg(-1) protein). In response to stimulation by veratridine, a secretagogue (a substance that induces secretion) that targets voltage-gated Na+ channels, the release rate and the total amount of ACh released were significantly lower, by 10-fold, than the response induced by KCl. The high-affinity uptake of choline was also very high (31 pmol Ch.min(-1).mg(-1) protein). The observed ACh release and Ch uptake patterns are in good agreement with published data on preparations characterized by high levels of ACh metabolism, adding further evidence that ACh acts as a neurotransmitter in cuttlefish optic lobe.