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Acta Poloniae Pharmaceutica 2005Formulation of local anesthetics in liposomal topical drug delivery system could provide a sustained and localized anesthesia. The aim of this study was to develop a...
Formulation of local anesthetics in liposomal topical drug delivery system could provide a sustained and localized anesthesia. The aim of this study was to develop a liposomal dibucaine base (DB) local anesthetic delivery system. DB-loaded multilamellar vesicles (MLVs) were prepared through varying lipid composition, induced charge and pH of the hydration medium. Liposomes were characterized for morphology, size, entrapment efficiency (EE), in vitro drug release and stability including leakage stability. The percentage of drug entrapped in liposomes was found to be hydration medium pH dependent and charge dependent and more pronounced for negatively charged liposomes prepared using hydration medium of pH 9. In vitro release studies of liposomes have shown a sustained release of entrapped dibucaine compared to control solution. Results revealed that adjusting the various formulation variables of dibucaine base MLVs could yield stable and effective topical liposomal local anesthetic formulations.
Topics: Anesthetics, Local; Chemistry, Pharmaceutical; Dibucaine; Drug Compounding; Drug Stability; Liposomes; Membranes, Artificial
PubMed: 16459486
DOI: No ID Found -
Journal of Neurochemistry Aug 2005Cleaved or truncated BID (tBID) is known to oligomerize both BAK and BAX. Previously, BAK and BAX lacing the C-terminal fragment (BAXDeltaC) were shown to induce modest...
Cleaved or truncated BID (tBID) is known to oligomerize both BAK and BAX. Previously, BAK and BAX lacing the C-terminal fragment (BAXDeltaC) were shown to induce modest cytochrome c (Cyt c) release from rat brain mitochondria when activated by tBID. We now show that tBID plus monomeric full-length BAX induce extensive release of Cyt c, Smac/DIABLO, and Omi/HtrA2 (but not endonuclease G and the apoptosis inducing factor) comparable to the release induced by alamethicin. This occurs independently of the permeability transition without overt changes in mitochondrial morphology. The mechanism of the release may involve formation of reactive oxygen species (ROS) and activation of calcium-independent phospholipase A(2) (iPLA(2)). Indeed, increased ROS production and activated iPLA(2) were observed prior to massive Cyt c release. Furthermore, the extent of inhibition of Cyt c release correlated with the degree of suppression of iPLA(2) by the inhibitors propranolol, dibucaine, 4-bromophenacyl bromide, and bromenol lactone. Consistent with a requirement for iPLA(2) in Cyt c release from brain mitochondria, synthetic liposomes composed of lipids mimicking the outer mitochondrial membrane (OMM) but lacing iPLA(2) failed to release 10 kDa fluorescent dextran (FD-10) in response to tBID plus BAX. We propose that tBID plus BAX activate ROS generation, which subsequently augments iPLA(2) activity leading to changes in the OMM that allow translocation of certain mitochondrial proteins from the intermembrane space.
Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; BH3 Interacting Domain Death Agonist Protein; Brain; Carrier Proteins; Cytochromes c; Drug Combinations; Enzyme Activation; Group VI Phospholipases A2; High-Temperature Requirement A Serine Peptidase 2; Male; Mitochondria; Mitochondrial Proteins; Peptide Fragments; Phospholipases A; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Recombinant Proteins; Serine Endopeptidases; bcl-2-Associated X Protein
PubMed: 16092941
DOI: 10.1111/j.1471-4159.2005.03248.x -
Anesthesiology Mar 2005Succinylcholine remains the standard neuromuscular blocking drug for tracheal intubation in emergency situations. The short duration of action is due to its rapid...
BACKGROUND
Succinylcholine remains the standard neuromuscular blocking drug for tracheal intubation in emergency situations. The short duration of action is due to its rapid hydrolytic degradation by butyrylcholinesterase (plasmacholinesterase). Multiple variants of this enzyme are known (A, F, S, H, J, K variants) with different effects on enzyme activity. This study was undertaken to evaluate the use of molecular genetic methods in patients with clinically prolonged neuromuscular block.
METHODS
Nine patients with a neuromuscular block of 14 min to 5 h were selected. All four exons of the butyrylcholinesterase were amplified by polymerase chain reaction and analyzed by automated sequencing. Molecular genetic results were compared with clinical relaxation time and with biochemical test results (total butyrylcholinesterase activity, dibucaine and fluoride inhibition).
RESULTS
Seven of nine patients were mutation carriers. Five of these had more than one mutation. The A and K variants were the most frequent variations. Three of four patients who were homozygous for the A variant were also carriers of the K allele. The authors identified one novel mutation (G1294T) introducing a stop codon at amino acid position 432. The duration of neuromuscular block was substantially different between patients with identical butyrylcholinesterase genotypes.
CONCLUSIONS
Variations in the genetic sequence of butyrylcholinesterase are frequent in patients with prolonged duration of action of succinylcholine. Direct sequencing of the whole butyrylcholinesterase gene is an appropriate method for genotyping and, accordingly, should be used in future clinical studies with drugs metabolized by this enzyme (e.g., succinylcholine, mivacurium).
Topics: Adult; Aged; Aged, 80 and over; Butyrylcholinesterase; Child; Female; Genotype; Humans; Male; Mutation; Neuromuscular Blockade; Neuromuscular Depolarizing Agents; Succinylcholine; Time Factors
PubMed: 15731589
DOI: 10.1097/00000542-200503000-00009 -
FEBS Letters Jun 2004The mitochondrial apoptosis-induced channel (MAC) forms in the outer membrane of mitochondria early in apoptosis and this activity is altered by physiological levels of...
The mitochondrial apoptosis-induced channel (MAC) forms in the outer membrane of mitochondria early in apoptosis and this activity is altered by physiological levels of cytochrome c. While cyclosporine A and lidocaine have no effect, dibucaine induces a fast blockade of MAC with an IC(50) of 39 microM. In contrast, the IC(50) for propranolol and trifluoperazine are 52 and 0.9 microM, respectively, and these drugs likely destabilize the open state of MAC. These agents, and others not yet identified, should be valuable tools in the study of apoptosis. Profiling MAC's pharmacology may generate novel therapeutic regimes for disease.
Topics: Apoptosis; Cations; Cell Line; Ion Channels; Mitochondria; Patch-Clamp Techniques
PubMed: 15196916
DOI: 10.1016/j.febslet.2004.05.006 -
Molecular Biology of the Cell Aug 2004Blue light controls the sexual life cycle of Chlamydomonas, mediated by phototropin, a UV-A/blue-light receptor that plays a prominent role in multiple photoresponses....
Blue light controls the sexual life cycle of Chlamydomonas, mediated by phototropin, a UV-A/blue-light receptor that plays a prominent role in multiple photoresponses. By using fractionation experiments and immunolocalization studies, this blue-light receptor, in addition to its known localization to the cell bodies, also was detected in flagella. Within the flagella, it was completely associated with the axonemes, in striking contrast to the situation in higher plants and the Chlamydomonas cell body where phototropin was observed in the plasma membrane. Its localization was not perturbed in mutants lacking several prominent structural components of the axoneme. This led to the conclusion that phototropin may be associated with the outer doublet microtubules. Analysis of a mutant (fla10) in which intraflagellar transport is compromised suggested that phototropin is a cargo for intraflagellar transport. The blue-light receptor thus seems to be an integral constituent of the flagella of this green alga, extending the list of organisms that harbor sensory molecules within this organelle to unicellular algae.
Topics: Animals; Chlamydomonas reinhardtii; Cryptochromes; Dibucaine; Flagella; Flavoproteins; Microtubules; Mutation; Protein Interaction Mapping; Protein Transport
PubMed: 15155806
DOI: 10.1091/mbc.e04-01-0010 -
Anesthesiology Apr 2004Irreversible nerve injury may result from neural membrane lysis due to the detergent properties of local anesthetics. This study aimed to investigate whether local...
BACKGROUND
Irreversible nerve injury may result from neural membrane lysis due to the detergent properties of local anesthetics. This study aimed to investigate whether local anesthetics display the same properties as detergents and whether they disrupt the model membrane at high concentrations.
METHODS
Concentrations at which dodecyltrimethylammonium chloride and four local anesthetic (dibucaine, tetracaine, lidocaine, and procaine) molecules exhibit self-aggregation in aqueous solutions were measured using an anesthetic cation-sensitive electrode. Light-scattering measurements in a model membrane solution were also performed at increasing drug concentrations. The concentration at which drugs caused membrane disruption was determined as the point at which scattering intensity decreased. Osmotic pressures of anesthetic agents at these concentrations were also determined.
RESULTS
Concentrations of dodecyltrimethylammonium chloride, dibucaine, tetracaine, lidocaine, and procaine at which aggregation occurred were 0.15, 0.6, 1.1, 5.3, and 7.6%, respectively. Drug concentrations causing membrane disruption were 0.09% (dodecyltrimethylammonium chloride), 0.5% (dibucaine), 1.0% (tetracaine), 5.0% (lidocaine), 10.2% (procaine), and 20% (glucose), and osmotic pressures at these concentrations were 278, 293, 329, 581, 728, and 1,868 mOsm/kg H2O, respectively.
CONCLUSIONS
These results show that all four local anesthetics form molecular aggregations in the same manner as dodecyltrimethylammonium chloride, a common surfactant. At osmotic pressures insufficient to affect the membrane, local anesthetics caused membrane disruption at the same concentrations at which molecular aggregation occurred. This shows that disruption of the model membrane results from the detergent nature of local anesthetics. Nerve membrane solubilization by highly concentrated local anesthetics may cause irreversible neural injury.
Topics: Anesthetics, Local; Cell Membrane; Detergents; Dose-Response Relationship, Drug; Micelles; Spinal Nerves
PubMed: 15087634
DOI: 10.1097/00000542-200404000-00029 -
Journal of Virology Jan 2004The transmembrane subunits of viral envelope proteins are thought to perform all of the functions required for membrane fusion during entry of enveloped viruses....
The transmembrane subunits of viral envelope proteins are thought to perform all of the functions required for membrane fusion during entry of enveloped viruses. However, changes in a conserved SPHQ motif near the N terminus of the receptor binding subunit of a murine leukemia virus (MLV) envelope protein block infection and induction of cell-cell fusion but not receptor binding. Here we report evidence that a histidine-to-arginine change at position 8 (H8R) in the SPHQ motif of Moloney MLV blocks infection by arresting virus-cell fusion at the hemifusion state. In cell-cell fusion assays, H8R envelope protein induced mixing of membrane outer leaflet lipids but did not lead to content mixing, a finding indicative of fusion pore formation. Kinetic studies of virus-cell fusion showed that lipid mixing of H8R virus membranes begins much later than for wild-type virus. The length of the delay in lipid mixing decreased upon addition of two second-site changes that increase H8R virus infection to 100-fold less than the wild-type virus. Finally, chlorpromazine, dibucaine, and trifluoperazine, agents that induce pores in an arrested hemifusion state, rescued infection by H8R virus to within 2.5-fold of the level of wild-type virus infection and cell-cell fusion to half that mediated by wild-type envelope protein. We interpret these results to indicate that fusion progressed to the hemifusion intermediate but fusion pore formation was inhibited. These results establish that membrane fusion of Moloney MLV occurs via a hemifusion intermediate. We also interpret these findings as evidence that histidine 8 is a key switch-point residue between the receptor-induced conformation changes that expose fusion peptide and those that lead to six-helix bundle formation.
Topics: Animals; Arginine; Cell Fusion; Cell Line; Histidine; Humans; Membrane Fusion; Mice; Moloney murine leukemia virus; Point Mutation; Protein Conformation; Transfection; Viral Envelope Proteins
PubMed: 14671127
DOI: 10.1128/jvi.78.1.473-481.2004 -
The Journal of Neuroscience : the... Apr 2003BH3 (Bcl-2 homology 3)-only proteins of the Bcl-2 family activate Bax or Bak during apoptosis to promote the release of pro-death factors sequestered in the...
BH3 (Bcl-2 homology 3)-only proteins of the Bcl-2 family activate Bax or Bak during apoptosis to promote the release of pro-death factors sequestered in the mitochondrial intermembrane space. Previous results demonstrated that a synthetic BH3 peptide mimics the ability of the BH3-only protein Bid to promote Bax insertion and cytochrome c (cyt c) release from neural cell mitochondria. However, the BH3 peptide was deficient in promoting cyt c release from mitochondria without associated Bax, such as adult rat brain mitochondria. This study tested the hypothesis that the amphiphilic membrane-active cationic drugs dibucaine and propranolol block BH3 peptide-initiated cyt c efflux by preventing the integration of Bax into the mitochondrial outer membrane. BH3 peptide-initiated release of cyt c from GT1-7 neural cell mitochondria was inhibited by dibucaine and propranolol at concentrations of 100-300 microm. Recombinant Bax (100 nm) alone did not release cyt c from adult rat brain mitochondria; however, when BH3 peptide or caspase-8 cleaved Bid (cBid) was added, robust cyt c release was achieved that was inhibited completely by 200 microm dibucaine or propranolol. These drugs at similar concentrations also inhibited release of entrapped 10 kDa dextrans from protein-free liposomes treated with Bax and cBid. Contrary to the hypothesis that dibucaine and propranolol act by inhibiting the insertion of Bax into the mitochondrial outer membrane, membrane insertion of Bax was not inhibited in mitochondria or liposomes, indicating a mechanism of drug action downstream from this event. These results suggest that dibucaine and propranolol inhibit Bax-induced permeability changes through a direct interaction with the lipid membrane and present a novel target for the development of neuroprotective, antiapoptotic therapeutics.
Topics: Amino Acid Sequence; Animals; BH3 Interacting Domain Death Agonist Protein; Biological Transport; Brain; Carrier Proteins; Caspase 8; Caspase 9; Caspases; Cell Line; Cytochrome c Group; Dextrans; Dibucaine; Humans; Intracellular Membranes; Ion Channels; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Molecular Sequence Data; Neurons; PC12 Cells; Peptide Fragments; Propranolol; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; bcl-2-Associated X Protein
PubMed: 12684459
DOI: 10.1523/JNEUROSCI.23-07-02735.2003 -
FEBS Letters Dec 2002The quinoline-based tertiary amine dibucaine has been shown to bind the membrane skeletal protein spectrin with a dissociation constant of 3.5x10(-5) M at 25 degrees C....
The quinoline-based tertiary amine dibucaine has been shown to bind the membrane skeletal protein spectrin with a dissociation constant of 3.5x10(-5) M at 25 degrees C. Such binding is detected by monitoring the quenching of the tryptophan fluorescence intensity with increasing concentrations of dibucaine only and not with the benzene-based local anesthetics procaine, tetracaine and lidocaine. Binding of dibucaine also indicated changes in the tertiary structure of spectrin indicated by a circular dichroism spectrum in the near-UV region due to absorption of the aromatic side chains. The thermodynamic parameters associated with the binding indicated the interaction of dibucaine and spectrin to be enthalpy-driven and insensitive to an increase in the ionic strength of the buffer.
Topics: Amines; Anesthetics, Local; Animals; Circular Dichroism; Dibucaine; Dose-Response Relationship, Drug; Erythrocyte Membrane; Goats; Hot Temperature; Ions; Kinetics; Lidocaine; Procaine; Protein Binding; Protein Structure, Tertiary; Spectrin; Spectrometry, Fluorescence; Temperature; Tetracaine; Thermodynamics; Tryptophan; Ultraviolet Rays
PubMed: 12482599
DOI: 10.1016/s0014-5793(02)03721-3 -
Journal of Neurochemistry Apr 2002We investigated a role of nitric oxide (NO) on ionomycin-evoked [3H]GABA release using mouse cerebral cortical neurons. lonomycin dose-dependently released [3H]GABA up...
We investigated a role of nitric oxide (NO) on ionomycin-evoked [3H]GABA release using mouse cerebral cortical neurons. lonomycin dose-dependently released [3H]GABA up to 1 microM. The extent of the release by 0.1 microM ionomycin was in a range similar to that by 30 mM KCl. The ionomycin (0.1 microM)-evoked [3H]GABA release was dose-dependently inhibited by NO synthase inhibitors and hemoglobin, indicating that the ionomycin-evoked [3H]GABA release is mediated through NO formation. The inhibition of cGMP formation by 1H-[1,2,4] oxodizao [4,3-a] quinoxalin-1-one (ODQ), a selective inhibitor for NO-sensitive guanylate cyclase, showed no affects on the ionomycin-evoked [3H]GABA release. Tetrodotoxin and dibucaine significantly suppressed the ionomycin-evoked [3H]GABA release and ionomycin increased fluorescence intensity of bis-oxonol, suggesting the involvement of membrane depolarization in this release. The ionomycin-evoked [3H]GABA release was maximally reduced by about 50% by GABA uptake inhibitors. The concomitant presence of nifedipine and omega-agatoxin VIA (omega-ATX), inhibitors for L- and P/Q-type voltage-dependent calcium channels, respectively, caused the reduction in the ionomycin-evoked release by about 50%. The simultaneous addition of nifedipine, omega-ATX and nipecotic acid completely abolished the release. Although ionomycin released glutamate, (+)-5-methyl-1-,11-dihydro-5H-dibenzo-[a,d]cycloheptan-5,10-imine (MK-801) and 6,7-dinitroquinoxaline-2,3-dione (DNQX) showed no effects on the ionomycin-induced [3H]GABA release. Based on these results, it is concluded that NO formed by ionomycin plays a critical role in ionomycin-evoked [3H]GABA release from the neurons.
Topics: Animals; Calcium Channel Blockers; Cells, Cultured; Cerebral Cortex; Cyclic GMP; Dibucaine; Dose-Response Relationship, Drug; Enzyme Inhibitors; GABA Agents; Glutamic Acid; Guanylate Cyclase; Hemoglobins; Ionomycin; Ionophores; L-Lactate Dehydrogenase; Magnesium; Mice; Mice, Inbred Strains; Neurons; Nitric Oxide; Nitric Oxide Synthase; Tetrodotoxin; Thiobarbiturates; gamma-Aminobutyric Acid
PubMed: 12067225
DOI: 10.1046/j.1471-4159.2002.00810.x