Identification of dibucaine derivatives as novel potent enterovirus 2C helicase inhibitors: In vitro, in vivo, and combination therapy study.

Enterovirus A71 (EV-A71) is a human pathogen causing hand, foot and mouth disease (HFMD) which seriously threatened the safety and lives of infants and young children. However, there are no licensed direct antiviral agents to cure the HFMD. In this study, a series of quinoline formamide analogues as effective enterovirus inhibitors were developed, subsequent systematic structure-activity relationship (SAR) studies demonstrated that these quinoline formamide analogues exhibited good potency to treat EV-A71 infection. As described, the most efficient EV-A71 inhibitor 6i showed good anti-EV-A71 activity (EC50 = 1.238 µM) in RD cells. Furthermore, compound 6i could effectively prevent death of virus infected mice at dose of 6 mg/kg. When combined with emetine (0.1 mg/kg), this treatment could completely prevent the clinical symptoms and death of virus infected mice. Mechanism study indicated that compound 6i inhibited EV-A71 via targeting 2C helicase, thus impeding RNA remodeling and metabolism. Taken together, these data indicated that 6i is a promising EV-A71 inhibitor and worth extensive preclinical investigation as a lead compound.

Discovery of Quinoline Analogues as Potent Antivirals against Enterovirus D68 (EV-D68).

Enterovirus D68 (EV-D68) is an atypical nonpolio enterovirus that mainly infects the respiratory system of humans, leading to moderate-to-severe respiratory diseases. In rare cases, EV-D68 can spread to the central nervous system and cause paralysis in infected patients, especially young children and immunocompromised individuals. There is currently no approved vaccine or antiviral available for the prevention and treatment of EV-D68. In this study, we aimed to improve the antiviral potency and selectivity of a previously reported EV-D68 inhibitor, dibucaine, through structure-activity relationship studies. In total, 60 compounds were synthesized and tested against EV-D68 using the viral cytopathic effect assay. Three compounds 10a, 12a, and 12c were identified to have significantly improved potency (EC50 < 1 µM) and a high selectivity index (>180) compared with dibucaine against five different strains of EV-D68 viruses. These compounds also showed potent antiviral activity in neuronal cells, such as A172 and SH-SY5Y cells, suggesting they might be further developed for the treatment of both respiratory infection as well as neuronal infection.

Electron Paramagnetic Resonance and Small-Angle X-ray Scattering Characterization of Solid Lipid Nanoparticles and Nanostructured Lipid Carriers for Dibucaine Encapsulation.

Dibucaine (DBC) is one of the most potent long-acting local anesthetics, but it also has significant toxic side effects and low water solubility. Solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) have been proposed as drug-delivery systems to increase the bioavailability of local anesthetics. The purpose of the present study was to characterize SLNs and NLCs composed of cetyl palmitate or myristyl myristate, a mixture of capric and caprylic acids (for NLCs only) plus Pluronic F68 prepared for the encapsulation of DBC. We intended to provide a careful structural characterization of the nanoparticles to identify the relevant architectural parameters that lead to the desirable biological response. Initially, SLNs and NLCs were assessed in terms of their size distribution, morphology, surface charge, and drug loading. Spectroscopic techniques (infrared spectroscopy and electron paramagnetic resonance, EPR) plus small-angle X-ray scattering (SAXS) provided information on the interactions between nanoparticle components and their structural organization. The sizes of nanoparticles were in the 180 nm range with low polydispersity and negative zeta values (-25 to -46 mV). The partition coefficient of DBC between nanoparticles and water at pH 8.2 was very high (>104). EPR (with doxyl-stearate spin labels) data revealed the existence of lamellar arrangements inside the lipid nanoparticles, which was also confirmed by SAXS experiments. Moreover, the addition of DBC increased the molecular packing of both SLN and NLC lipids, indicative of DBC insertion between the lipids, in the milieu assessed by spin labels. Such structural information brings insights into understanding the molecular organization of these versatile drug-delivery systems which have already demonstrated their potential for therapeutic applications in pain control.

Simultaneous Determination of Cinchocaine Hydrochloride and Betamethasone Valerate in Presence of Their Degradation Products.

Cinchocaine hydrochloride (CIN) and betamethasone valerate (BMV) are co-formulated in pharmaceutical formulations that could be used for local treatment of hemorrhoids. Both drugs are susceptible to hydrolytic degradation. Two sensitive and precise stability-indicating chromatographic methods were developed for the simultaneous determination of both active pharmaceutical ingredients. The developed methods were applied for quantitation of CIN and BMV in their pure forms, in presence of their corresponding degradation products and in their pharmaceutical formulation. The first method was a high performance liquid chromatographic (HPLC) one, separation and quantitation was achieved using a Waters Spheriosorb® 5 µm ODS2 C18 analytical column and an isocratic mobile phase formed of acetonitrile-acetate buffer (pH 6.5 ± 0.1) in a ratio of (55:45, v/v). The mobile phase was pumped at a flow rate of 1.2 mL/min. UV-detection was done at 240 nm using photodiode array detector. The second method was based on thin layer chromatography (TLC) fractionation coupled with densitometric determination. Separation was done on high performance thin layer chromatography (HPTLC) silica gel 60F254 plates using a developing system formed of chloroform-toluene-ethanol-acetic acid in a ratio of (4.5:4.5:1:1, by volume). The separated bands were scanned densitometrically at 240 nm. For the HPLC method, linearity was confirmed over concentration ranges of 4-300 and 4-350 µg/mL for CIN and BMV, respectively. For the HPTLC-densitometric method, the obtained ranges were 0.5-12 and 0.5-10 µg/band for CIN and BMV, respectively. The developed methods were optimized and validated according to the ICH guidelines. CIN acid degradation products were separated and identified by mass spectroscopy. The developed HPLC method was used to study the kinetics of acid and alkali degradation of the both drugs. The results obtained were statistically analyzed and compared with those obtained by applying the official methods for both drugs.

Activity and polymorphisms of butyrylcholinesterase in a Polish population.

Butyrylcholinesterase (BChE) activity assay and inhibitor phenotyping can help to identify individuals at risk of prolonged paralysis following the administration of neuromuscular blocking agents, like succinylcholine, pesticides and nerve agents. In this study, the activity of BChE and its sensitivity to inhibition by dibucaine and fluoride was evaluated in 1200 Polish healthy individuals. In addition, molecular analysis of all exons, exon-intron boundaries and the 3'UTR sequence of the BCHE gene was performed in a group of 72 subjects with abnormal BChE activity (<2000 U/L and >5745 U/L) or with DN (Dibucaine Number) or FN (Fluoride-Number) values outside the reference range (DN < 78 and FN < lower than wild type). In a studied group, BChE activity range was similar to those observed in other populations. BChE activity screening allowed to detect UA and UF phenotypes in 26 (2.2%) and 15 (1.2%) individuals, respectively. Observed UA or UF phenotypes were confirmed by direct sequencing and heterozygous c.293A > G or c.1253G > T substitutions were identified in all cases. Nine out of 18 (50%) individuals with BChE activity below 2000 U/L had a mutation in 5'UTR (32G/A), intron 2 (c.1518-121T/C) or exon 4 (c.1699G/A; the K variant mutation). Majority of the individuals with BChE activity ≥6000 U/L were wild type. To summarize, the range of BChE activity in a Polish population is similar to those observed in other countries. We conclude that the BChE phenotyping assay is a reliable method for identification of individuals with the UA and UF genotypes.

History of T-cain: a local anesthetic developed and manufactured in Japan.

In many anesthesia textbooks written in English, lidocaine, tetracaine, bupivacaine, ropivacaine, and chloroprocaine are listed as useful local anesthetics for spinal anesthesia. In contrast, T-cain is not included in these lists, even though it has been reported to be suitable for spinal anesthesia in Japan. T-cain was developed as a local anesthetic in the early 1940s by Teikoku Kagaku Sangyo Inc. in Itami, Japan, by replacing a methyl group on tetracaine (Pantocaine(®)) with an ethyl group. T-cain was clinically approved for topical use in Japan in November 1949, and a mixture of dibucaine and T-cain (Neo-Percamin S(®)) was approved for spinal use in May 1950. Simply because of a lack of foreign marketing strategy, T-cain has never attracted global attention as a local anesthetic. However, in Japan, T-cain has been used topically or intrathecally (as Neo-Percamin S(®)) for more than 60 years. Other than the side effects generally known for all local anesthetics, serious side effects have not been reported for T-cain. In fact, several articles have reported that T-cain decreases the neurotoxicity of dibucaine. In this historical review, the characteristics of T-cain and its rise to become a major spinal anesthetic in Japan are discussed.

Differential interactions of two local anesthetics with phospholipid membrane and nonerythroid spectrin: Localization in presence of cholesterol and ganglioside, GM1.

Interactions of two local anesthetics, dibucaine and tetracaine have been studied with phospholipid vesicles containing cholesterol and/or monosialogangliosides (GM1) using fluorescence spectroscopy. The fluorescence intensity of tetracaine showed a marked increase with the increasing molar ratio of the phospholipid to tetracaine, while that of dibucaine showed opposite effects. Steady state anisotropy and the wavelength of maximum emission (λmax) decreased with the increasing phospholipids to tetracaine ratio. The extent of such changes in anisotropy and λmax in the presence and absence of two important components of neuronal membranes, cholesterol and GM1 indicated differential membrane localization of the two local anesthetics. To understand the intercellular mode of action of local anesthetics, we have also studied the interactions of dibucaine and tetracaine with brain spectrin which indicate differential spectrin interactions with similar binding strength. Thermodynamic parameters associated with such binding reveal that binding is favored by entropy. Tetracaine brings about distinct structural changes in spectrin compared to dibucaine, as reflected in the tryptophan mean lifetime and far-UV CD spectra. Tetracaine also exhibits a detergent-like property inducing concentration dependent decrease in spectrin anisotropy, further indicating structural changes in brain spectrin with probable implications in its anesthetic potential.

Lipid nanoparticles: drug localization is substance-specific and achievable load depends on the size and physical state of the particles.

Lipid nanoemulsions and -suspensions are being intensively investigated as carriers for poorly water soluble drugs. The question on where model compounds or probes are localized within the dispersions has been the subject of several studies. However, only little data exists for pharmaceutically relevant molecules in dispersions composed of pharmaceutically relevant excipients. In this work, the localization of drugs and drug-like substances was studied in lipid nanoemulsions and -suspensions. Conclusions about the drug localization were drawn from the relations between lipid mass, specific particle surface area and drug load in the dispersions. Additionally, the achievable drug loads of the liquid and the solid lipid particles were compared. Nanoemulsions and -suspensions comprised trimyristin as lipid matrix and poloxamer 188 as emulsifier and were prepared with different well-defined particle sizes. These pre-formed dispersions were passively loaded with either amphotericin B, curcumin, dibucaine, fenofibrate, mefenamic acid, propofol, or a porphyrin derivative. The physico-chemical properties of the particles were characterized; drug load and lipid content were quantified by UV spectroscopy and high performance liquid chromatography, respectively. For all drugs the passive loading procedure was successful in both emulsions and suspensions. Solid particles accommodate drug molecules preferably at the particle surface. Liquid particles can accommodate drugs at the particle surface as well as in the core; the distribution between the two sites is drug specific. It is also drug specific whether solid or liquid particles yield higher drug loads. As a general rule, smaller particles led to higher drug loads than larger ones. Propofol and the porphyrin derivative displayed eutectic interaction with the lipid and crystal growth after loading, respectively.

Two-column sequential injection chromatography for fast isocratic separation of two analytes of greatly differing chemical properties.

This paper deals with a novel approach to separate two analytes with different chemical properties and different lipophilicity. The newly described methodology is based on the two column system that was used for isocratic separation of two analytes with very different lipophilicity-dexamethasone and cinchocaine. Simultaneous separation of model compounds cinchocaine and dexamethasone was carried under the following conditions in two-column sequential injection chromatography system (2-C SIC). A 25×4.6 mm C-18 monolithic column was used in the first dimension for retention and separation of dexamethasone with mobile phase acetonitrile:water 30:70 (v/v), flow rate 0.9 mL min(-1) and consumption of 1.7 mL. A 10×4.6 mm C-18 monolithic column with 5×4.6 mm C-18 precolumn was used in the second dimension for retention and separation of cinchocaine using mobile phase acetonitrile:water 60:40 (v/v), flow rate 0.9 mL min(-1) and consumption 1.5 mL. Whole analysis time including both mobile phase's aspirations and both column separations was performed in less than 4 min. The method was fully validated and used for determination of cinchocaine and dexamethasone in pharmaceutical otic drops. The developed 2-C SIC method was compared with HPLC method under the isocratic conditions of separation on monolithic column (25×4.6 mm C-18). Spectrophotometric detection of both compounds was performed at wavelength 240 nm. System repeatability and method precision were found in the range (0.39-3.12%) for both compounds. Linearity of determination was evaluated in the range 50-500 µg mL(-1) and coefficients of determination were found to be r(2)=0.99912 for dexamethasone and r(2)=0.99969 for cinchocaine.

Selective effect of procaine, tetracaine and dibucaine on gold nanoparticles.

An aqueous colloid dispersion of gold nanoparticles (AuNPs) was prepared by reduction of gold(III) chloride and its interaction with three local anesthetics (procaine, dibucaine or tetracaine) was investigated. Optical spectra reveal the modifications in the absorption band of nanoparticles related to their self assembly mediated by anesthetic molecules and depending on the progress in time of the aggregation process. TEM images show the features of the self assemblies formed by the association of gold nanoparticles in presence of anesthetics, and reveal marked differences in the behavior of the AuNPs against the three anesthetics. The main effect of various anesthetics can be described in terms of electrostatic forces between the negatively charged metal nanoparticles and anesthetic molecules, existing in their cationic form at the working pH. Then, the anesthetics functionalized nanoparticles trigger specific interactions to form different self assemblies through a selective combination of hydrophobic and hydrogen bonding interactions between the coated nanoparticles and anesthetics molecular species.

Role of viscosity in influencing the glass-forming ability of organic molecules from the undercooled melt state.

PURPOSE: Understanding the critical factors governing the crystallization tendency of organic compounds is vital when assessing the feasibility of an amorphous formulation to improve oral bioavailability. The objective of this study was to investigate potential links between viscosity and crystallization tendency for organic compounds from the undercooled melt state. METHODS: Steady shear rate viscosities of numerous compounds were measured using standard rheometry as a function of temperature through the undercooled melt regime. Data for each compound were fit to the Vogel-Tamman-Fulcher (VTF) equation; kinetic fragility via strength parameter (D) was determined. RESULTS: Compounds with high crystallization tendencies exhibited lower melt viscosities than compounds with low crystallization tendencies. A correlation was observed between rate of change in viscosity with temperature and crystallization tendency, with slowly crystallizing compounds exhibiting larger increases in viscosity as temperature decreased below T(m). Calculated strength parameters indicated all compounds were kinetically fragile liquids; thus, kinetic fragility may not accurately assess glass-forming ability from undercooled melt state. CONCLUSIONS: A link was observed between the viscosity of a compound through the undercooled melt regime and its resultant crystallization tendency, indicating viscosity is a critical parameter to fully understand crystallization tendency of organic compounds.

Model-free analysis of binding at lipid membranes employing micro-calorimetric measurements.

Based on universal thermodynamic principles (Schwarz in Biophys Chem 86:119-129, 2000) it is shown how measured enthalpy changes can be utilized to determine the relevant binding isotherm as well as the variation of the molar enthalpy change. This is carried out in a novel way involving multiple titration experiments whose evaluation requires no beforehand assumptions or models whatever. An appropriate specific model mechanism may be discussed afterwards and developed in view of the given experimental results. The pertinent procedure is demonstrated using micro-calorimetric data obtained in the case of the local anesthetic dibucaine as it associates with POPC liposomes. Mutual interactions of the bound ligand molecules could be described in terms of repulsive enthalpic and entropic activity coefficients. Apparently these are induced by electrostatic forces and by the finite size of binding sites, respectively.

Amphiphilic effects of dibucaine HCl on rotational mobility of n-(9-anthroyloxy)stearic acid in neuronal and model membranes.

We studied dibucaine's effects on specific locations of n-(9-anthroyloxy)palmitic acid or stearic acid (n-AS) within phospholipids of synaptosomal plasma membrane vesicles isolated from bovine cerebral cortex (SPMV) and model membranes. Giant unilamellar vesicles (GUVs) were prepared with total lipids (SPMVTL) and mixture of several phospholipids (SPMVPL) extracted from SPMV. Dibucaine.HCl increased rotational mobility (increased disordering) of hydrocarbon interior, but it decreased mobility (increased ordering) of membrane interface, in both native and model membranes. The degree of rotational mobility in accordance with the carbon atom numbers of phospholipids comprising neuronal and model membranes was in the order at the 16, 12, 9, 6 and 2 position of aliphatic chain present in phospholipids. The sensitivity of increasing or decreasing effect of rotational mobility of hydrocarbon interior or surface region by dibucaine.HCl differed depending on the neuronal and model membranes in the descending order of SPMV, SPMVPL and SPMVTL.

In vitro release of hydrophilic and hydrophobic drugs from liposomal dispersions and gels.

A method for determining the rate of hydrophilic and hydrophobic drugs release from different types of liposomal dispersions and gels using a dialysis method is described. Dibucaine base and 5-fluorouracil were used as model drugs for a hydrophobic and a hydrophilic drug, respectively. A dialysis technique was employed. Release rates were affected by the rate of rotation of the paddles of the tablet dissolution tester, temperature, and the volume of release medium. The method was used to evaluate the in vitro drug release from hydrophilic and hydrophobic drugs from liposomal dispersions and gels. The in vitro release study of dibucaine base showed no burst effect, while the in vitro release study of 5-fluorouracil showed a clear burst effect with an initial fast release phase followed by a sustained release phase.

Rapid accurate mass desorption electrospray ionisation tandem mass spectrometry of pharmaceutical samples.

Desorption electrospray ionisation (DESI) has been successfully combined with a hybrid quadrupole time-of-flight mass spectrometer to provide mass spectra and product ion mass spectra of active ingredients formulated in pharmaceutical tablets, gels and ointments. Accurate mass data has been obtained from the DESI mass spectra and of the product ion fragments of selected ions, greatly enhancing the selectivity and information content of the experiment. This accurate mass information only takes seconds to acquire since the DESI technique does not require any sample preparation or extraction prior to mass analysis.

Effect of various formulation variables on the encapsulation and stability of dibucaine base in multilamellar vesicles.

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.

Effect of the glycosphingolipid, GM1 on localization of dibucaine in phospholipid vesicles: a fluorescence study.

Interaction of the local anesthetic dibucaine with small unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) and dioleoylphosphatidylcholine (DOPC) containing different mole percents of monosialoganglioside (GM1) has been studied by fluorescence spectroscopy. Fluorescence measurements on dibucaine in the presence of phospholipid vesicles containing various amounts of GM1 yielded a pattern of variation of wavelength at emission maximum and steady-state anisotropy which indicated that the microenvironment of dibucaine is more hydrophobic and rigid in membranes that contain GM1 than in membranes without it. Experiments on quenching of fluorescence from membrane-associated dibucaine by potassium iodide showed reduced quenching efficiency with the increase in GM1 content of the vesicles, demonstrating lesser accessibility of the iodide quenchers to dibucaine in the presence of GM1, when compared to that in its absence. Total emission intensity decay profiles of dibucaine yielded two lifetime components of approximately 1 and approximately 2.8-3.1 ns with mean relative contributions of approximately 25 and approximately 75%, respectively. The mean lifetime in vesicles was 20-30% lower than in the aqueous medium and showed a definite increase in presence of GM1 from that in the absence of it. All the spectral properties point that dibucaine encountered regions of membrane containing significant amount of GM1 and penetrated deeper in hydrophobic core of the bilayer.

Interaction of serotonin1A receptors from bovine hippocampus with tertiary amine local anesthetics.

1. We have examined the interaction of tertiary amine local anesthetics with the bovine hippocampal serotonin1A (5-HT1A) receptor, an important member of the G-protein-coupled receptor superfamily. 2. The local anesthetics inhibit specific agonist and antagonist binding to the 5-HT1A receptor at a clinically relevant concentration range of the anesthetics. This is accompanied by a concomitant reduction in the binding affinity of the 5-HT1A receptor to the agonist. Interestingly, the extent of G-protein coupling of the receptor is reduced in the presence of the local anesthetics. 3. Fluorescence polarization measurements using depth-dependent fluorescent probes show that procaine and lidocaine do not show any significant change in membrane fluidity. On the other hand, tetracaine and dibucaine were found to alter fluidity of the membrane as indicated by a fluorescent probe which monitors the headgroup region of the membrane. 4. The local anesthetics showed inhibition of agonist binding to the 5-HT1A receptor in membranes depleted of cholesterol more or less to the same extent as that of control membranes in all cases. This suggests that the inhibition in ligand binding to the 5-HT1A receptor brought about by local anesthetics is independent of the membrane cholesterol content. 5. Our results on the effects of the local anesthetics on the ligand binding and G-protein coupling of the 5-HT1A receptor support the possibility that G-protein-coupled receptors could be involved in the action of local anesthetics.

Probing the mechanism of drug/lipid membrane interactions using Biacore.

Assay conditions were established to screen a panel of drugs for binding to liposome surfaces using a surface plasmon resonance (SPR) biosensor. Drugs were found to bind negligibly or reversibly or were retained on the liposome surface. Cationic amphiphilic drugs fell into the last class and correlated with drugs that induce phospholipidosis in vivo. To a first approximation, a single-site model yielded apparent binding affinities that adequately described a drug's dose-dependent binding to liposome surfaces. Affinities ranged at least 1000-fold within the drug panel. A liposome's drug-binding capacity and affinity depended on both the lipid headgroup and the drug's structure. Although a drug's charge state generally dominated whether or not it remained bound to the liposome, subtle structural differences between members of certain drug families led to them having widely differing binding affinities. A comparison between the dissociation of drugs from liposome surfaces by Biacore and the lipid retention measurements determined by a parallel artificial membrane permeability assay was drawn. The results from this study demonstrate the potential of using SPR-based assays to characterize drug/liposome-binding interactions.