Dibucaine in Ionic-Gradient Liposomes: Biophysical, Toxicological, and Activity Characterization.

Administration of local anesthetics is one of the most effective pain control techniques for postoperative analgesia. However, anesthetic agents easily diffuse into the injection site, limiting the time of anesthesia. One approach to prolong analgesia is to entrap local anesthetic agents in nanostructured carriers (e.g., liposomes). Here, we report that using an ammonium sulphate gradient was the best strategy to improve the encapsulation (62.6%) of dibucaine (DBC) into liposomes. Light scattering and nanotracking analyses were used to characterize vesicle properties, such as, size, polydispersity, zeta potentials, and number. In vitro kinetic experiments revealed the sustained release of DBC (50% in 7 h) from the liposomes. In addition, in vitro (3T3 cells in culture) and in vivo (zebrafish) toxicity assays revealed that ionic-gradient liposomes were able to reduce DBC cyto/cardiotoxicity and morphological changes in zebrafish larvae. Moreover, the anesthesia time attained after infiltrative administration in mice was longer with encapsulated DBC (27 h) than that with free DBC (11 h), at 320 µM (0.012%), confirming it as a promising long-acting liposome formulation for parenteral drug administration of DBC.

Cytotoxicity and type of cell death induced by local anesthetics in human oral normal and tumor cells.

BACKGROUND: Local anesthetics are often administered to tumors and surrounding tissues during the surgery of the head and neck area, however their effects on oral tissues is not well understood. In the present study, the cytotoxicity of a total of seven local anesthetics towards oral tumor and normal cells was compared. MATERIALS AND METHODS: Tumor-specificity index was determined by the ratio of the mean 50% cytotoxic concentration against normal cells to that for tumor cells. Apoptosis induction was monitored by internucleosomal DNA fragmentation and caspase-3, -8, and - 9 activation. Fine cell structure was observed under transmission electron microscopy. RESULTS: All local anesthetics showed slightly higher cytotoxicity towards oral squamous cell carcinoma (OSCC) cell lines than towards normal oral cells. Dibucaine, with a log p-value of approximately 3, was the most cytotoxic, followed by tetracaine, bupivacaine or ethylaminobenzoate, whereas lidocaine, procaine and mepivacain were much less cytotoxic. When the tumor-specificity was evaluated between OSCC and human skin keratinocytes, the index was 6.6. Dibucaine did not induce apoptosis of OSCC cells. On the other hand, dibucaine did induce mitochondrial injury and swelling, formation of secondary lysosomes, and at high concentrations, rupture of the cell membrane. Autophagy inhibitors did not reduce the cytotoxicity of dibucaine. CONCLUSION: Necrosis may be involved in the induction of antitumor activity by dibucaine.

Calpain activator dibucaine induces platelet apoptosis.

Calcium-dependent calpains are a family of cysteine proteases that have been demonstrated to play key roles in both platelet glycoprotein Ibα shedding and platelet activation and altered calpain activity is associated with thrombotic thrombocytopenic purpura. Calpain activators induce apoptosis in several types of nucleated cells. However, it is not clear whether calpain activators induce platelet apoptosis. Here we show that the calpain activator dibucaine induced several platelet apoptotic events including depolarization of the mitochondrial inner transmembrane potential, up-regulation of Bax and Bak, down-regulation of Bcl-2 and Bcl-X(L), caspase-3 activation and phosphatidylserine exposure. Platelet apoptosis elicited by dibucaine was not affected by the broad spectrum metalloproteinase inhibitor GM6001. Furthermore, dibucaine did not induce platelet activation as detected by P-selectin expression and PAC-1 binding. However, platelet aggregation induced by ristocetin or α-thrombin, platelet adhesion and spreading on von Willebrand factor were significantly inhibited in platelets treated with dibucaine. Taken together, these data indicate that dibucaine induces platelet apoptosis and platelet dysfunction.

[Ionic strength influences hemolytic action of dibucaine hydrochloride].

BACKGROUND: Little is known about effect of ionic strength on local anesthetic toxicity. Using human erythrocytes, hemolytic action of dibucaine in solutions of various ionic strength was investigated. METHODS: The critical micellar concentration (CMC) of dibucaine and the dibucaine level that causes destruction of half of the red blood cells in vitro (EC50 value) were determined in solutions of various ionic strength. RESULTS: The mean CMC values of the dibucaine solutions adjusted to ionic strength 0.15, 0.30, 0.45 and 0.90 with NaCl, were 35.3, 22.6, 15.9 and 9.6 mM, respectively. The mean EC50 values of these solutions measured at 5 sec were 22.5, 16.0, 12.6 and 8.2 mM, respectively, and those at 30 min were 4.9, 4.5, 4.5 and 2.6 mM, respectively. There was a significant correlation between mean CMC values and mean EC50 values at 5 sec but not at 30 min in the solution of the same ionic strength. CONCLUSIONS: These findings indicated that the mechanism of dibucaine-induced hemolysis within a few seconds is through membrane lysis, whereas dibucaine-induced hemolysis at 30 min is caused by another mechanism. Because each mechanism is enhanced by high ionic strength, dibucaine dissolved in salt solution should not be administered intrathecally.

Effect of bilirubin on toxicity induced by trifluoperazine, dibucaine and praziquantel to erythrocytes.

Unconjugated bilirubin (UCB), like trifluoperazine (TFP), dibucaine (DBC) and praziquantel (PZQ), induces erythrocyte morphological changes, lysis and lipid exfoliation. In the present study we determined whether TFP, DBC and PZQ toxicity to erythrocytes was potentiated or reverted by UCB. Human erythrocytes were either treated or non-treated with 34.2 micromol/L UCB for 10 min prior to the incubation with toxic concentrations of TFP (0.12 mmol/L), DBC (1.5 mmol/L) or PZQ (3.0 mmol/L), for 1 h (37 degrees C). Studies of toxic effects included morphological analysis of erythrocytes, evaluation of hemoglobin release and loss of membrane lipids. Although UCB has an echinocytogenic effect, its co-incubation with TFP or PZQ did not alter the stomatocytogenic effect of the drug but enhanced DBC-induced stomatocytosis. Cell fusion was a common feature in experiments with DBC. Injurious effect of DBC to erythrocytes was potentiated by UCB as manifested by a marked increase in hemolysis (171%, p<0.05), and in elution of membrane cholesterol (73%, p<0.01) and phospholipids (123%, p<0.01). In opposite, toxic events produced by TFP and PZQ to erythrocytes were not aggravated by UCB. Interestingly, UCB prevented the loss of membrane cholesterol by PZQ (-36%, p<0.01), as well as that of phospholipids by TFP (-28%, p<0.05). These findings indicate that UCB potentiates DBC injury to erythrocytes, while protects membrane lipid elution by PZQ and TFP. Therefore, the relation of the benefits and risks of the administration of DBC to jaundiced patients should be carefully considered.

[Neurotoxicity of dibucaine on the isolated rabbit cervical vagus nerve].

The neurotoxicity of dibucaine was compared with that of commercially available local anesthetics in studies using rabbit desheathed cervical vagus nerve preparation. Dibucaine dose-dependently suppressed the evoked action potential of myelinated A beta nerve component and nonmyelinated C nerve component. The potential of A beta nerve component was more strongly suppressed, compared with that of C nerve component. At low concentrations of 0.0001-0.001%, the suppression was reversible and recovery with C nerve component was faster and more complete. At higher concentrations, the suppression was irreversible. The minimum concentrations of irreversible blockade were 0.003% for A beta nerve component and 0.03% for C nerve component. Electron microscopically, marked damages in the myelin layer and intraaxonal structure were observed in nerves treated with 0.03% dibucaine. When the neurotoxic effect of dibucaine was compared, in terms of safety margins (minimum concentration of irreversible blockade/clinically used concentrations), with those of commercially available local anesthetics, the rank order was dibucaine, tetracaine and bupivacaine; dibucaine showing the lowest safety margin.

Induction of apoptotic cell death in a neuroblastoma cell line by dibucaine.

Dibucaine, a local anesthetic known to interact with cell membranes, induced apoptosis in SK-N-MC human neuroblastoma cells in a dose-dependent manner. Apoptosis was demonstrated by direct visualization of morphological nuclear changes using a DAPI staining technique and confirmed by the production of characteristic ladder patterns of DNA fragmentation on gel electrophoresis. At concentrations which induced apoptosis, dibucaine significantly altered membrane fluidity, indicating that fluidity may be a major target for the cytotoxic action of dibucaine. Also, dibucaine increased intracellular calcium levels more effectively in calcium-containing Krebs-Ringer buffer than in calcium-free Krebs-Ringer buffer. Removal of extracellular calcium or addition of antioxidants or protein synthesis inhibitor effectively blocked dibucaine-induced apoptosis. These results suggest that membrane damage, intracellular calcium levels, and oxygen free radicals may be involved in the apoptosis induced by dibucaine.

The effect of local anaesthetics on the thermal sensitivity of HTC cells.

Local anaesthetics caused death of HTC cells, and the order of toxicity (dibucaine > tetracaine > procaine) correlated with their oil:water partition coefficients. Cytotoxic effects of hyperthermia were enhanced by subtoxic levels of anaesthetics and again, their potency was related to the oil:water partition coefficients. DPH fluidity in plasma membranes at 37 degrees C was increased by dibucaine and tetracaine but not by procaine up to 5mM. At 43 degrees C, plasma membrane fluidity was increased by dibucaine but not by tetracaine or procaine. The results suggest that fluidisation of the hydrophobic core of the membrane may contribute to anaesthetic potentiation of heat cell death.

Reversible suppression of growth and differentiation of cultured chick myogenic cells with very low concentrations of dibucaine.

Dibucaine, a local anaesthetic, reversibly suppressed growth and differentiation of cultured chick myogenic cells. In the presence of 10 microM dibucaine, chick myoblasts multiplied and fused to form myotubes. However, myotubes did not increase their size. Accumulation of creatine kinase (CK) activity per culture dish was also suppressed, showing that myotube growth was suppressed. Concomitantly, myofibril formation and developmental transition of CK isozymes were retarded. The myotubes remained immature. When dibucaine was removed from the culture medium and cells were further incubated, the accumulation of CK activity, especially of muscle type isoform, and formation of myofibrils took place; eventually the myofibrils were organized similarly to those in the control culture. The concentration of dibucaine enough to exert these effects was about one-hundredth of the concentration used in clinical anaesthesia.

Toxicity of local anaesthetics on myogenic cells in culture.

Toxicity of local anaesthetics on chick myogenic cells (mononucleated myoblasts and multinucleated myotubes) in culture was examined. Following treatment with the drugs, myogenic cells showed some morphological changes and finally detached from the culture dishes. In most cases, the toxic effect was estimated by the amount of cells detached quantitatively. The indices used to show the amount of cells were the deoxyribonucleic acid (DNA) and creatine kinase activity content of mono- and multinucleated cells remaining on the dishes, respectively. Dibucaine was more toxic than bupivacaine, mepivacaine, tetracaine and procaine, and was examined in detail. The toxicity was dependent on its concentration, pH and temperature of the reaction medium in both mono- and multinucleated cells, and paralleled the concentration of uncharged form of the drug, suggesting that this form in external medium was actually toxic.