Hironori Tsuchiya

Local anesthetics structure-dependently interact with anionic phospholipid membranes to modify the fluidity.

While bupivacaine is more cardiotoxic than other local anesthetics, the mechanistic background for different toxic effects remains unclear. Several cardiotoxic compounds act on lipid bilayers to change the physicochemical properties of membranes. We comparatively studied the interaction of local anesthetics with lipid membranous systems which might be related to their structure-selective cardiotoxicity. Amide local anesthetics (10-300 microM) were reacted with unilamellar vesicles which were prepared with different phospholipids and cholesterol of varying lipid compositions. They were compared on the potencies to modify membrane fluidity by measuring fluorescence polarization. Local anesthetics interacted with liposomal membranes to increase the fluidity. Increasing anionic phospholipids in membranes enhanced the membrane-fluidizing effects of local anesthetics with the potency being cardiolipin>>phosphatidic acid>phosphatidylglycerol>phosphatidylserine. Cardiolipin was most effective on bupivacaine, followed by ropivacaine. Local anesthetics interacted differently with biomimetic membranes consisting of 10mol% cardiolipin, 50mol% other phospholipids and 40mol% cholesterol with the potency being bupivacaine>>ropivacaine>lidocaine>prilocaine, which agreed with the rank order of cardiotoxicity. Bupivacaine significantly fluidized 2.5-12.5mol% cardiolipin-containing membranes at cardiotoxicologically relevant concentrations. Bupivacaine is considered to affect lipid bilayers by interacting electrostatically with negatively charged cardiolipin head groups and hydrophobically with phospholipid acyl chains. The structure-dependent interaction with lipid membranes containing cardiolipin, which is preferentially localized in cardiomyocyte mitochondrial membranes, may be a mechanistic clue to explain the structure-selective cardiotoxicity of local anesthetics.

Local anesthetic failure associated with inflammation: verification of the acidosis mechanism and the hypothetic participation of inflammatory peroxynitrite

The presence of inflammation decreases local anesthetic efficacy, especially in dental anesthesia. Although inflammatory acidosis is most frequently cited as the cause of such clinical phenomena, this has not been experimentally proved. We verified the acidosis mechanism by studying the drug and membrane lipid interaction under acidic conditions together with proposing an alternative hypothesis. Liposomes and nerve cell model membranes consisting of phospholipids and cholesterol were treated at different pH with lidocaine, prilocaine and bupivacaine (0.05%–0.2%, w/v). Their membrane-interactive potencies were compared by the induced-changes in membrane fluidity. Local anesthetics fluidized phosphatidylcholine membranes with the potency being significantly lower at pH 6.4 than at pH 7.4 (p < 0.01), supporting the acidosis theory. However, they greatly fluidized nerve cell model membranes even at pH 6.4 corresponding to inflamed tissues, challenging the conventional mechanism. Local anesthetics acted on phosphatidylserine liposomes, as well as nerve cell model membranes, at pH 6.4 with almost the same potency as that at pH 7.4, but not on phosphatidylcholine, phosphatidylethanolamine and sphingomyelin liposomes. Since the positively charged anesthetic molecules are able to interact with nerve cell membranes by ion-paring with anionic components like phosphatidylserine, tissue acidosis is not essentially responsible for the local anesthetic failure associated with inflammation. The effects of local anesthetics on nerve cell model membranes were inhibited by treating with peroxynitrite (50 μM), suggesting that inflammatory cells producing peroxynitrite may affect local anesthesia.

Membrane interactivity of charged local anesthetic derivative and stereoselectivity in membrane interaction of local anesthetic enantiomers.

With respect to the membrane lipid theory as a molecular mechanism for local anesthetics, two critical subjects, the negligible effects of charged drugs when applied extracellularly and the stereoselective effects of enantiomers, were verified by paying particular attention to membrane components, phospholipids with the anionic property, and cholesterol with several chiral carbons. The membrane interactivities of structurally-different anesthetics were determined by their induced fluidity changes of liposomal membranes. Lidocaine (3.0 μmol/mL) fluidized phosphatidylcholine membranes, but not its quaternary derivative QX-314 (3.0 μmol/mL). Similarly to the mother molecule lidocaine, however, QX-314 fluidized phosphatidylserine-containing nerve cell model membranes and acidic phospholipids-constituting membranes depending on the acidity of membrane lipids. Positively charged local anesthetics are able to act on lipid bilayers by ion-pairing with anionic (acidic) phospholipids. Bupivacaine (0.75 mol/mL) and ropivacaine (0.75 and 1.0 μmol/mL) fluidized nerve cell model membranes with the potency being S(−)-enantiomer < racemate < R(+)-enantiomer (P < 0.01, vs antipode and racemate) and cardiac cell model membranes with the potency being S(−)-ropivacaine < S(−)-bupivacaine < R(+)-bupivacaine (P < 0.01). However, their membrane effects were not different when removing cholesterol from the model membranes. Stereoselectivity is producible by cholesterol which increases the chirality of lipid bilayers and enables to discriminate anesthetic enantiomers. The membrane lipid interaction should be reevaluated as the mode of action of local anesthetics.

Stereostructure-based differences in the interactions of cardiotoxic local anesthetics with cholesterol-containing biomimetic membranes

Amide-type pipecoloxylidide local anesthetics, bupivacaine, and ropivacaine, show cardiotoxic effects with the potency depending on stereostructures. Cardiotoxic drugs not only bind to cardiomyocyte membrane channels to block them but also modify the physicochemical property of membrane lipid bilayers in which channels are embedded. The opposite configurations allow enantiomers to be discriminated by their enantiospecific interactions with another chiral molecule in membranes. We compared the interactions of local anesthetic stereoisomers with biomimetic membranes consisting of chiral lipid components, the differences of which might be indicative of the drug design for reducing cardiotoxicity. Fluorescent probe-labeled biomimetic membranes were prepared with cardiolipin and cholesterol of varying compositions and different phospholipids. Local anesthetics were reacted with the membrane preparations at a cardiotoxically relevant concentration of 200 μM. The potencies to interact with biomimetic membranes and change their fluidity were compared by measuring fluorescence polarization. All local anesthetics acted on lipid bilayers to increase membrane fluidity. Chiral cardiolipin was ineffective in discriminating S(-)-enantiomers from their antipodes. On the other hand, cholesterol produced the enantiospecific membrane interactions of bupivacaine and ropivacaine with increasing its composition in membranes. In 40 mol% and more cholesterol-containing membranes, the membrane-interacting potency was S(-)-bupivacaine<racemic bupivacaine<R(+)-bupivacaine, and S(-)-ropivacaine<R(+)-ropivacaine. Ropivacaine (S(-)-enantiomer), levobupivacaine (S(-)-enantiomeric), and bupivacaine (racemic) interacted with biomimetic membranes in increasing order of intensity. The rank order of membrane interactivity agreed with that of known cardiotoxicity. The stereoselective membrane interactions determined by cholesterol with higher chirality appears to be associated with the stereoselective cardiotoxic effects of local anesthetics. The stereostructure and membrane interactivity relationship supports the clinical use and development of S(-)-enantiomers to decrease the adverse effects of pipecoloxylidide local anesthetics on the cardiovascular system.

Comparative study on determination of antioxidant and membrane activities of propofol and its related compounds

Certain anesthetics have been suggested to protect against the pathological states associated with oxidative stress. We compared the antioxidant and membrane activities of propofol (2,6-diisopropylphenol) and its related compounds to address the structure-activity relationship especially in a lipid membrane phase. They were studied for the effects on 1,1-diphenyl-2-picrylhydrazyl radicals, nitro blue tetrazolium reduction by superoxide anions and membrane lipid peroxidation by peroxynitrite, and also for the induced changes in membrane fluidity of liposomes. 2-Isopropylphenols scavenged free radicals with the potency being propofol>2,5-diisopropylphenol>2-isopropylphenol>2,4-diisopropylphenol, but not 3- and 4-isopropylphenols and 1,3- and 1,4-diisopropylbenzenes. The tested compounds showed no significant superoxide dismutase-like effects. Propofol inhibited membrane lipid peroxidation more intensively than 2,5-diisopropylphenol, 2,4-diisopropylphenol and 2-isopropylphenol. Despite structurally resembling antioxidant alpha-tocopherol, 2,6-dimethylphenol was less potent than propofol. Propofol produced 50% inhibition of the lipid peroxidation in unsaturated phosphatidylcholine liposomal membranes and cell-mimetic membranes at 4.0 and 10.1 microM, respectively. Propofol and 2-alkylphenolic compounds interacted with membranes to increase their fluidity with the potency correlating with lipid peroxidation inhibiting activity. The 2-isopropylphenol structure is a requisite for both lipid peroxidation inhibition and membrane fluidity modification. The structure-specific membrane interactivity appears to be one of possible antioxidant mechanisms for propofol.

Stereospecific interaction of bupivacaine enantiomers with lipid membranes.

Background and Objectives: S(−)‐Bupivacaine has the pharmacotoxicological advantage over its antipode and racemate. The interaction with lipid membranes was compared between S(−)‐, R(+)‐ and racemic bupivacaine. Methods: The bupivacaine‐induced changes in membrane property were determined by turbidity and fluorescence polarization measurements of membrane preparations to which bupivacaine stereoisomers of 1.0‐5.0 mmol/L were applied. Liposomal membranes were made of 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine without or with cholesterol (5 to 15 mol%), and nerve cell model membranes of 55 mol% different phospholipids and 45 mol% cholesterol. The purity and hydrophobic interaction of bupivacaine were analyzed by reversed‐phase high‐performance liquid chromatography. Results: Both S(−)‐ and R(+)‐bupivacaine were not different in lowering the phase transition temperature of membrane 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine. S(−)‐, R(+)‐ and racemic bupivacaine disordered 100 mol% 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine liposomal membranes, although the potency was indistinguishable between stereoisomers. By adding cholesterol to membranes, however, the membrane‐disordering effects showed stereostructure‐specificity that was enhanced with increasing the cholesterol content (0 to 15 mol%). The enantio‐differentiating effects resulted from neither impurities in enantiomers nor hydrophobic interaction with phosphatidylcholine acyl chains. Bupivacaine disordered nerve cell model membranes with the potency being S(−)‐enantiomer < racemate < R(+)‐enantiomer, which resembled their relative stereopotency in nerve and cardiac channel inhibition. Membrane‐disordering stereospecificity disappeared in the membranes without containing cholesterol. Conclusions: Bupivacaine stereostructure‐specifically interacts with membranes containing cholesterol, which is consistent with the clinical features of S(−)‐bupivacaine. Membrane cholesterol appears to increase the chirality of lipid bilayers and enable them to interact with S(−)‐, racemic and R(+)‐bupivacaine differently.

R(+)-, Rac-, and S(-)-bupivacaine stereostructure-specifically interact with membrane lipids at cardiotoxically relevant concentrations.

It remains questionable whether local anesthetics can interact with membrane lipids at clinically relevant concentrations to show the difference between enantiomers. We compared the effects of bupivacaine stereoisomers on biomimetic membranes containing cardiolipin and cholesterol. Bupivacaine interacted with the membranes at cardiotoxic 5 &mgr;M with the potency being S(–)-enantiomer < racemate < R(+)-enantiomer, which agreed with the rank order of their cardiotoxicity. Such differences became greater with decreasing drug concentrations, possibly explaining the inconsistent cardiotoxic potencies of bupivacaine stereoisomers reported previously. The interactivity with biomembranes may in part contribute to the mode of toxic action of local anesthetics.

The membrane interaction of drugs as one of mechanisms for their enantioselective effects

The discrimination between different enantiomers of chiral compounds by the biological system is medically important as the pharmacological and toxicological effects of enantiomeric drugs significantly differ depending on their stereostructures. One enantiomer is preferred over its enantiomeric counterpart and a racemic mixture for higher activity or lower toxicity. Such enantioselectivity has been exclusively explained by the stereostructure-specific interactions with receptors, channels and enzymes of drugs including general and local anesthetics, sedatives, hypnotics, anti-inflammatory drugs, analgesics and β-adrenergic antagonists. These drugs can act on not only protein targets but also lipid biomembranes. Almost all of the relevant proteins are embedded in or associated with membrane lipid bilayers. Therefore, we propose one of possible mechanisms that drugs might enantioselectively interact with membrane lipids and induce changes in membrane property like fluidity which are discriminable between enantiomers. If the induced changes are different between enantiomers, enantiomeric drugs would differently influence the membrane lipid environments for receptors, channels and enzymes, resulting in the enantioselectivity of drug effects. The enantioselective membrane interactions of drugs could be mediated by membrane component cholesterol and phospholipids, both of which have chiral centers in structure as well as drug enantiomers. Chiral membrane lipids possibly exhibit the preference for the interactions with drug molecules of either the same chirality or the different chirality, producing the selectivity to one drug enantiomer. The proposed hypothesis may be available to investigate more useful medicines based on the novel concept of drug enantioselectivity.

Characteristic interactivity of landiolol, an ultra-short-acting highly selective β1-blocker, with biomimetic membranes: Comparisons with β1-selective esmolol and non-selective propranolol and alprenolol.

Although β1-blockers have been perioperatively used to reduce the cardiac disorders associated with general anesthesia, little is known about the mechanistic characteristics of ultra-short-acting highly selective β1-blocker landiolol. We studied its membrane-interacting property in comparison with other selective and non-selective β1-blockers. Biomimetic membranes prepared with phospholipids and cholesterol of varying compositions were treated with β1-selective landiolol and esmolol and non-selective propranolol and alprenolol at 0.5–200 μM. The membrane interactivity and the antioxidant activity were determined by measuring fluorescence polarization and by peroxidizing membrane lipids with peroxynitrite, respectively. Non-selective β1-blockers, but not selective ones, intensively acted on 1,2-dipalmitoylphosphatidylcholine (DPPC) liposomal membranes and cardiomyocyte-mimetic membranes to increase the membrane fluidity. Landiolol and its inactive metabolite distinctively decreased the fluidity of DPPC liposomal membranes, suggesting that a membrane-rigidifying effect is attributed to the morpholine moiety in landiolol structure but unlikely to clinically contribute to the β1-blocking effect of landiolol. Propranolol and alprenolol interacted with lipid raft model membranes, whereas neither landiolol nor esmolol. All drugs fluidized mitochondria-mimetic membranes and inhibited the membrane lipid peroxidation with the potency correlating to their membrane interactivity. Landiolol is characterized as a drug devoid of the interactivity with membrane lipid rafts relating to β2-adrenergic receptor blockade. The differentiation between β1-blocking selectivity and non-selectivity is compatible with that between membrane non-interactivity and interactivity. The mitochondrial membrane fluidization by landiolol independent of blocking β1-adrenergic receptors is responsible for the antioxidant cardioprotection common to non-selective and selective β1-blockers.

Comparative Effects of α-, β-, and γ-Carbolines on Platelet Aggregation and Lipid Membranes

Cigarette smoking and alcohol consumption possibly affect platelet functions. To verify the hypothesis that some α-, β-, and γ-carboline components in cigarette smoke and alcoholic beverages may change platelet aggregability, their effects on human platelets were determined by aggregometry together with investigating their membrane effects by turbidimetry. Carbolines inhibited platelet aggregation induced by five agents with the potency being 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole > 3-amino-1-methyl-5H-pyrido[4,3-b]indole > 1-methyl-9H-pyrido[3,4-b]indole. The most potent 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole showed 50% aggregation-inhibitory concentrations of 6–172 μM. Both γ-carbolines interacted with phosphatidylcholine membranes to lower the lipid phase transition temperature with the potency correlating to the antiplatelet activity, suggesting that the interaction with platelet membranes to increase their fluidity underlies antiplatelet effects. Given their possible concentration and accumulation in platelets, γ- and β-carbolines would provide cigarette smokers and alcohol drinkers with reduced platelet aggregability, and they may be responsible for the occurrence of hemorrhagic diseases associated with heavy smoking and alcoholics.