Molecular properties of ciprofloxacin-indion 234 complexes

The purpose of this research was to formulate tasteless complexes of ciprofloxacin with Indion 234 and to evaluate molecular properties of drug complexes. The effect of batch and column process, complexation time, temperature, and pH on ciprofloxacin loading on Indion 234 is reported. Drug resin complexes (DRC) were characterized by infrared spectroscopy, thermal analysis, and x-ray diffraction pattern. Ciprofloxacin release from DRC is obtained at salivary and gastric pH and in the presence of electrolytes. The efficient drug loading was evident in batch process using activated Indion 234 with a drug-resin ratio of 1∶1.3. Drug complexation enhanced with pH from 1.2 to 6, while temperature did not affect the complexation process. Infrared spectroscopy revealed complexation of—NH (drug) with Indion 234. DRC are amorphous in nature. Drug release from DRC in salivary pH was insufficient to impart bitter taste. Volunteers rated the complex as tasteless and agreeable. Complete drug release was observed at gastric pH in 2 hours. The drug release was accelerated in the presence of electrolytes. Indion 234 is inexpensive, and the simple technique is effective for bitterness masking of ciprofloxacin.     

Bacteriocin release proteins: mode of action, structure, and biotechnological application

Abstract: The mechanisms by which Gram-negative bacteria like Escherichia coli secrete bacteriocins into the culture medium is unique and quite different from the mechanism by which other proteins are translocated across the two bacterial membranes, namely through the known branches of the general secretory pathway. The release of bacteriocins requires the expression and activity of a so-called bacteriocin release protein and the presence of the detergent-resistant phospholipase A in the outer membrane. The bacteriocin release proteins are highly expressed small lipoproteins which are synthesized with a signal peptide that remains stable and which accumulates in the cytoplasmic membrane after cleavage. The combined action of these stable, accumulated signal peptides, the lipid-modified mature bacteriocin release proteins (BRPs) and phospholipase A cause the release of bacteriocins. The structure and mode of action of these BRPs as well as their application in the release of heterologous proteins by E. coli is described in this review.     

A Ciprofloxacin Extended Release Tablet Based on Swellable Drug Polyelectrolyte Matrices

The aim of this work was the development of extended release tablets of 500 mg of ciprofloxacin based on swellable drug polyelectrolyte matrices (SDPM). A set of complexes of carbomer, ciprofloxacin and sodium, (CB–Cip)₅₀Na _x , having a molar ratio Cip/CB acid groups of 0.5 and variable proportions of Na⁺ was used to prepare SDPM. Characterization of complexes by FT-IR, powder X-ray diffraction and thermal analysis revealed that Cip, in its protonated form, is ionically bonded to the functional groups of CB. Rates of fluid uptake of (CB–Cip)₅₀Na _x matrices as well as Cip release in simulated gastric fluid were modulated by changes in the proportion of Na⁺ incorporated in the complexes. A direct correlation between fluid uptake and delivery rate was observed along the series of matrices. Release rates were modulated from 1.4 mg/min to 25 mg/min in going from (CB–Cip)₅₀Na₁₀ to (CB–Cip)₅₀Na₁₄. The analysis of kinetic data suggest that rates of swelling, ionic pair dissociation and drug diffusion play a role in the kinetic control of delivery. Complexes were satisfactorily prepared and processed together with small amounts of antiadherent and lubricant excipients to obtain a series of extended release SDPM tablets through the current tableting technology processes. Cip release from matrices was widely modulated by the composition of the complexes yielding a flexible system that allows selecting a composition that releases in 120 min 90% of the dose in simulated gastric fluid.     

Changes of synaptotagmin interaction with t-SNARE proteins in vitro after calcium/calmodulin-dependent phosphorylation

The regulation of multiple phases of the life cycle of synaptic vesicles is carried out by a complex series of protein-protein interactions. According to the SNARE hypothesis the core of these interactions is a heterotrimeric complex formed by syntaxin, SNAP-25, and VAMP-synaptobrevin. Other proteins interacting with the core of the SNARE complex, such as voltage-activated calcium channels and synaptotagmin (a putative calcium sensor), are considered crucial for the calcium dependence of release and also molecular mediators of synaptic plasticity. Here the interaction of synaptotagmin with SNARE proteins was studied in immunoprecipitated native complexes, and the effects of previous phosphorylation-dephosphorylation on this interaction were analyzed. It is surprising that the interaction of synaptotagmin with syntaxin and SNAP-25 in native complexes was not found to be calcium-dependent. However, previous incubation under dephosphorylating conditions decreased the synaptotagmin-syntaxin interaction. Stimulation of Ca2+/calmodulin-dependent protein kinase II, which endogenously phosphorylates synaptotagmin in synaptic vesicles, increased the interaction of syntaxin and SNAP-25 with synaptotagmin (particularly when measured in the presence of calcium), as well as increasing the binding of the kinase itself. These results suggest that calcium decreases synaptotagmin-t-SNARE interactions after dephosphorylation and increases them after phosphorylation. Overall, these results imply a phosphorylation-dephosphorylation balance in regulation of the synaptotagmin-t-SNARE interaction and suggest a role for protein phosphorylation in the modulation of calcium sensitivity in transmitter release.     

Flavonoid Glycosides Extracted from Hop (Humulus lupulus L.) as Inhibitors of Chemical Mediator Release from Human Basophilic KU812 Cells

The antiallergic properties of hop water extract (HWE) were studied by evaluating histamine release from human basophilic KU812 cells induced by calcium ionophore A23187. HWE significantly inhibited histamine release, but boiling water extract and chloroform–methanol extract did not show any inhibitory effect on it. A 50% methanol-eluted fraction separated from HWE by XAD-4 column chromatography (MFH) had a strong inhibitory effect as compared with HWE. Quercetin glycosides and kaempherol glycosides were identified in MFH, of which quercetin glycosides contributed to the inhibition of histamine release. Most quercetin in HWE existed in glycoside form and its quercetin content, obtained by acid hydrolysis, was about 200 μg/g. HWE and MFH significantly inhibited protein kinase C, which plays a pivotal role in the degranulation of chemical mediators. These results indicate that HWE can inhibit type-I allergic reactions.     

Use of cyclodextrins as a cosmetic delivery system for fragrance materials: Linalool and benzyl acetate

The aim of this study was to increase the stability and water solubility of fragrance materials, to provide controlled release of these compounds, and to convert these substances from liquid to powder form by preparing their inclusion complexes with cyclodextrins (CDs). For this purpose, linalool and benzyl acetate were chosen as the fragrance materials. The use of beta-cyclodextrin (beta CD) and 2-hydroxypropyl-beta-cyclodextrin (2-HP beta CD) for increasing the solubility of these 2 fragrance materials was studied. Linalool and benzyl acetate gave a B-type diagram with beta CD, whereas they gave an A(L)-type diagram with 2-HP beta CD. Therefore, complexes of fragrance materials with 2-HP beta CD at 1:1 and 1:2 molar ratios (guest:host) were prepared. The formation of inclusion complexes was confirmed using proton nuclear magnetic resonance ((1)H-NMR) spectroscopy and circular dichroism spectroscopy. The results of the solubility studies showed that preparing the inclusion complex with 2-HP beta CD at a 1:1 molar ratio increased the solubility of linalool 5.9-fold and that of benzyl acetate 4.2-fold, whereas the complexes at a 1:2 molar ratio increased the solubility 6.4- and 4.5-fold for linalool and benzyl acetate, respectively. The stability and in vitro release studies were performed on the gel formulations prepared using uncomplexed fragrance materials or inclusion complexes of fragrance materials at a 1:1 molar ratio. It was observed that the volatility of both fragrance materials was decreased by preparing the inclusion complexes with 2-HP beta CD. Also, in vitro release data indicated that controlled release of fragrances could be possible if inclusion complexes were prepared.     

Juvenile Hormone biosynthesis and release during oocyte development in Phormia regina Meigen

A radiochemical assay for Juvenile Hormone (JH) biosynthesis and release by the corpus allatum (CA) was used to assess the effects of diet on CA activity of adult female Phormia regina (Meigen) fed either sugar-water or sugar-water-liver. CC-CA complexes were incubated in L-methionine-free medium 199 supplemented with ³H-L-methionine. The rate of JH release by the CC-CA complexes is linear for 3 h and declines slightly thereafter. JH III appears to be one of the major components of the isooctane-extractable product from incubated CC-CA. High pressure liquid chromatographic analysis indicates that 10% of the released radiolabelled product is JH III. Rates of JH release show a strict dependence on L-methionine concentration in the incubation medium, with optimal rates occurring between 100 and 150 μM L-methionine. JH release is at a low level (<0.02pmolh^-1 per pair of CC-CA) in flies fed only sugar-water, but increases dramatically in flies fed sugar-water-liver (average release rate of 0.2pmolh^-1 per pair of CA, 24h after a liver meal). The rate of JH release increases steadily to more than 1.2pmolh^-1 per pair at 128h of age (i.e. 56h after a liver meal) at which time oocytes are mature. Elevated rates of JH release in vitro appear to be correlated in vivo with the appearance of vitellogenin in the haemolymph and its uptake by the developing oocytes.     

A late phase of exocytosis from synaptosomes induced by elevated [Ca2+]i is not blocked by Clostridial neurotoxins

Treatment of rat cerebrocortical synaptosomes with botulinum toxin types E and C1 or tetanus toxin removed the majority of intact SNAP-25, syntaxin 1A/1B, and synaptobrevin and diminished Ca(2+)-dependent K+ depolarization-induced noradrenaline secretion. With botulinum toxin type E, <10% of intact SNAP-25 remained and K(+)-evoked release of glutamate and GABA was inhibited. The large component of noradrenaline release evoked within 120 s by inclusion of the Ca2+ ionophore A23187 with the K+ stimulus was also attenuated by these toxins; additionally, botulinium neurotoxin type E blocked the first 60 s of ionophore-induced GABA and glutamate exocytosis. However, exposure to A23187 for longer periods induced a phase of secretion nonsusceptible to any of these toxins (>120 s for noradrenaline; >60 s for glutamate or GABA). Most of this late phase of release represented exocytosis because of its Ca2+ dependency, ATP requirement, and sensitivity to a phosphatidylinositol 4-kinase inhibitor. Based on these collective findings, we suggest that the ionophore-induced elevation of [Ca2+]i culminates in the disassembly of complexes containing nonproteolyzed SNAP receptors protected from the toxins that can then contribute to neuroexocytosis.     

蓝舌病毒释放机制的研究进展

蓝舌病毒(Bluetongue virus,BTV)作为由媒介昆虫库蠓传播的引起反刍动物蓝舌病(Bluetongue,BT)的病原微生物,同时也是研究无囊膜病毒(Non-enveloped virus)释放机制的经典模型。文中以BTV侵染细胞及组装为始,对BTV诱导细胞自噬并通过多囊泡体以细胞外囊泡形式释放、BTV诱导细胞凋亡而裂解释放、BTV从质膜出芽释放的不同途径以及BTV关键非结构蛋白NS3在调控BTV释放过程中作用机制的研究进展进行综述,为进一步了解BTV感染、增殖、释放的分子机制提供参考。     

Release of a neurosecretory protein from the corpora cardiaca of Locusta migratoria induced by high potassium saline and compound action potentials

Electrical stimulation of nervus corpus cardiacum I (NCCI) resulted in the propagation of a compound action potential into the storage and glandular lobes of the corpus cardiacum (CC) of Locusta migratoria. The compound action potential was abolished in the presence of both sodium-free saline and tetrodotoxin (TTX). Calcium-free saline had variable effects.The release of a neurosecretory protein (estimated following precipitation with an antiserum directed against neurosectetory protein) was examined after treatment with high potassium saline and electrical stimulation of NCC I. Release was induced by elevated potassium salines and by the propagation of a compound action potential along NCC I. The release was calcium-dependent. TTX and sodium-free saline abolished the electrically-induced release of protein. Concomitant with the release of protein was the release of a factor with diuretic activity, illustrating that hormones are also being released along with the neurosecretory protein. The release of this protein was dependent upon the frequency of electrical stimulation (up to approx. 5 Hz) and the patterning of electrical stimulation. This neurosecretory protein which has previously been shown to be very similar in both size and amino acid composition to the pituitary neurophysins, now also shares the characteristic of being released along with hormone.     

Calcium-dependent dissociation of synaptotagmin from synaptic SNARE complexes

The formation of the synaptic core (SNARE) complex constitutes a crucial step in synaptic vesicle fusion at the nerve terminal. The interaction of synaptotagmin I with this complex potentially provides a means of conferring Ca2+-dependent regulation of exocytosis. However, the subcellular compartments in which interactions occur and their modulation by Ca2+ influx remain obscure. Sodium dodecyl sulfate (SDS)-resistant core complexes, associated with synaptotagmin I, were enriched in rat brain fractions containing plasma membranes and docked synaptic vesicles. Depolarization of synaptosomes triggered [3H]GABA release and Ca2+-dependent dissociation of synaptotagmin from the core complex. In perforated synaptosomes, synaptotagmin dissociation was induced by Ca2+ (30-300 microM) but not Sr2+ (1 mM); it apparently required intact membrane bilayers but did not result in disassembly of trimeric SNARE complexes. Synaptotagmin was not associated with unstable v-SNARE/t-SNARE complexes, present in fractions containing synaptic vesicles and cytoplasm. These complexes acquired SDS resistance when N-ethylmaleimide-sensitive fusion protein (NSF) was inhibited with N-ethylmaleimide or adenosine 5'-O-(3-thiotriphosphate), suggesting that constitutive SNARE complex disassembly occurs in undocked synaptic vesicles. Our findings are consistent with models in which the Ca2+ triggered release of synaptotagmin precedes vesicle fusion. NSF may then dissociate ternary core complexes captured by endocytosis and recycle/prime individual SNARE proteins.     

Lateral release of proteins from the TOM complex into the outer membrane of mitochondria

The TOM complex of the outer membrane of mitochondria is the entry gate for the vast majority of precursor proteins that are imported into the mitochondria. It is made up by receptors and a protein conducting channel. Although precursor proteins of all subcompartments of mitochondria use the TOM complex, it is not known whether its channel can only mediate passage across the outer membrane or also lateral release into the outer membrane. To study this, we have generated fusion proteins of GFP and Tim23 which are inserted into the inner membrane and, at the same time, are spanning either the TOM complex or are integrated into the outer membrane. Our results demonstrate that the TOM complex, depending on sequence determinants in the precursors, can act both as a protein conducting pore and as an insertase mediating lateral release into the outer membrane.     

Acid-controlled release complexes of podophyllotoxin and etoposide with acyclic cucurbit[n]urils for low cytotoxicity

The targeted or responsive systems are appealing therapeutic platforms for the development of next-generation precision medications. So, we design and prepare acid-controlled release complexes of podophyllotoxin (POD) and etoposide (VP-16) with pH-labile acyclic cucurbit[n]urils, and their characteristics and inclusion complexation behaviors were investigated via fluorescence spectroscopy, nuclear magnetic resonance and X-ray power diffraction. Cells incubated with complexes have been analyzed by high-content analysis (HCA), and cytotoxicity tests have been completed by MTT assay. The results showed that complexes with different binding constants can release the drug substance in the physiological pH environment of cancer cells, maintain good anticancer activity, and have low cytotoxicity. This provides a strategy about targeted and responsive systems of POD and VP-16 for clinical application.     

The Effects of pH and Intraliposomal Buffer Strength on the Rate of Liposome Content Release and Intracellular Drug Delivery

Targeted liposomal drug formulations may enter cells by receptor-mediated endocytosis and then traffick by membrane flow into acidic intracellular compartments. In order to understand the impact of these intracellular pH changes on liposomal drug unloading, the effect of pH on the release from folate-targeted liposomes of three model compounds with distinct pH dependencies was examined. 5(6)-carboxyfluorescein, which titrates from its anionic to uncharged form following internalization by KB cells, displays strong endocytosis-dependent release, since only its uncharged (endosomal) form is membrane permeable. Endocytosis-triggered unloading of drugs of this sort is enhanced by encapsulating the drug in a weak buffer at neutral pH, so that acidification of the intraliposomal compartment following cellular uptake can occur rapidly. Sulforhodamine B, in contrast, retains both anionic and cationic charges at endosomal pH (~pH 5), and consequently, escapes the endosomes only very slowly. Doxorubicin, which is commonly loaded into liposomes in its membrane-impermeable (cationic) form using an acidic buffer, still displays endocytosis-triggered unloading, since sufficient uncharged doxorubicin remains at endosomal pHs to allow rapid re-equilibration of the drug according to the new proton gradient across the membrane. In this case, when the extraliposomal [H⁺] increases 250-fold from 4 × 10^–8 M (pH 7.4, outside the cell) to 10^–5 M (pH 5, inside the endosome), the ratio of doxorubicin inside to outside the liposome must decrease by a factor of 250. Therefore, the collapse of the transliposomal pH gradient indirectly drives an efflux of the drug molecule from the liposome. Since a change in intraliposomal pH is not required to unload drugs of this type, the intraliposomal compartment can be buffered strongly at acidic pH to prevent premature release of the drug outside the cell. In summary, pH triggered release of liposome-encapsulated drugs can be achieved both with drugs that increase as well as decrease their membrane permeabilities upon acidification, as long as the intraliposomal buffer strength and pH is rationally selected.     

Co-activation of PKA and PKC in cerebrocortical nerve terminals synergistically facilitates glutamate release

Protein kinase A and protein kinase C are involved in processes that enhance glutamate release at glutamatergic nerve terminals. However, it is not known whether these two kinases co-exist within the same nerve terminal, nor is it clear what impact their simultaneous activation may have on neurotransmitter release. In cerebrocortical nerve terminals, co-application of forskolin, which increases cAMP levels and activates protein kinase A, and 4beta-phorbol dibutyrate, a direct activator of protein kinase C, synergistically enhanced the spontaneous release of glutamate. This enhancement exhibited both tetrodotoxin-sensitive and tetrodotoxin-resistant components. Interestingly, the tetrodotoxin-resistant component of release was not observed when cyclic AMP-dependent protein kinase (PKA) and calcium- and phospholipid-dependent protein kinase (PKC) were activated separately, but developed slowly after the co-activation of the two kinases, accounting for 50% of the facilitated release. This release component was dependent on voltage-dependent Ca2+ channels that opened spontaneously after PKA and PKC activation and occurred in the absence of Na+ channel firing. These data provide functional evidence for the co-existence of PKA- and PKC-signalling pathways in a subpopulation of glutamatergic nerve terminals.     

Evidence for the involvement of cAMP-GEF (Epac) pathway in amylase release from the rat parotid gland

Amylase release from the rat parotid gland is mainly mediated in a cAMP-dependent protein kinase (PKA)-dependent manner. In the present study, amylase release mediated in cAMP-dependent and PKA-independent manners was investigated with a cAMP-regulated guanine nucleotide exchange factor (cAMP-GEF: Epac)-selective cAMP analogue, 8CPT-2Me-cAMP. The Epac was localized in the intracellular and the plasma membrane fractions. PKA activation by 8CPT-2Me-cAMP was 100-fold lower than that by cAMP. The amylase release (% of the total) from the intact parotid acinar cells was 16 and 3.6% by isoproterenol (1microM) and 8CPT-2Me-cAMP (200microM), respectively, and that from the saponin-permeabilized cells was 15 and 3% by cAMP (100microM) and 8CTP-2Me-cAMP (10microM), respectively. H-89 inhibited cAMP-induced amylase release, but did not inhibit 8CPT-2Me-cAMP-induced amylase release. These results indicated that amylase release by beta-adrenergic stimulation is mediated through both the cAMP/PKA and cAMP/Epac signal pathways.     

Polymer-Based Sustained-Release Dosage Forms for Protein Drugs, Challenges, and Recent Advances

While the concept of using polymer-based sustained-release delivery systems to maintain therapeutic concentration of protein drugs for extended periods of time has been well accepted for decades, there has not been a single product in this category successfully commercialized to date despite clinical and market demands. To achieve successful systems, technical difficulties ranging from protein denaturing during formulation process and the course of prolonged in vivo release, burst release, and incomplete release, to low encapsulation efficiency and formulation complexity have to be simultaneously resolved. Based on this updated understanding, formulation strategies attempting to address these aspects comprehensively were reported in recent years. This review article (with 134 citations) aims to summarize recent studies addressing the issues above, especially those targeting practical industrial solutions. Formulation strategies representative of three areas, microsphere technology using degradable hydrophobic polymers, microspheres made of water soluble polymers, and hydrophilic in vivo gelling systems will be selected and introduced. To better understand the observations and conclusions from different studies for different systems and proteins, physicochemical basis of the technical challenges and the pros and cons of the corresponding formulation methods will be discussed.     

Characterization of the Release of Cholecystokinin-8 from Isolated Nerve Terminals and Comparison with Exocytosis of Classical Transmitters

In the present study, the release of the neuropeptide cholecystokinin-8 (CCK) from purified nerve terminals (synaptosomes) of the rat hippocampus was characterized with respect to the subcellular distribution, the release upon addition of various agents, the release kinetics, the Ca2+ and ATP dependence of release, and the relationship between CCK release and elevations of intraterminal free Ca2+ concentration ([Ca]i). These characteristics were compared with those for the release of classical transmitters in similar preparations. CCK-like immunoreactivity (CCK-LI) is enriched in the purified synaptosomal fraction of hippocampus homogenates and released in a strictly Ca2(+)-dependent manner upon chemical depolarization, addition of 4-aminopyridine, or stimulation with the Ca2+ ionophore ionomycin. The presence of Ca2+ in the medium significantly stimulates the basal efflux of CCK-LI from synaptosomes. The release upon stimulation develops gradually in time with no significant release in the first 10 s and levels off after 3 min of depolarization. At this time, a large amount of CCK-LI is still present inside the synaptosomes. A correlation exists between the release of CCK-LI and the elevations of [Ca]i. The release of CCK-LI is decreased, but not blocked, upon ATP depletion. These characteristics markedly differ from those for classical transmitters, which show a fast component of Ca2(+)-dependent (exocytotic) release, an absolute dependence on cellular ATP, and no marked stimulation of basal efflux in the presence of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

A Remote Controlled Valve in Liposomes for Triggered Liposomal Release

In order to reduce the toxicity and increase the efficacy of drugs, there is a need for smart drug delivery systems. Liposomes are one of the promising tools for this purpose. An ideal liposomal delivery system should be stable, long-circulating, accumulate at the target site and release its drug in a controlled manner. Even though there have been many developments to this end, the dilemma of having a stable liposome during circulation but converting it into a leaky structure at the target site is still a major challenge. So far, most attempts have focused on destabilizing the liposome in response to a specific stimulus at a target site, but with limited success. Our approach is to keep the stable liposome but build in a remote-controlled valve as a new release mechanism, instead. The valve is a pore-forming bacterial membrane protein. It has been engineered such that, after being reconstituted into the liposomes, its opening and closing can be controlled on command by the ambient pH, light or a combination of both. In addition, a much higher degree of flexibility for fine-tuning of the liposome's response to its environment is achieved.