Evaluation of the lipophilic properties of opioids, amphetamine-like drugs, and metabolites through electrochemical studies at the interface between two immiscible solutions

For the first time, the partition coefficients of the ionized forms of several opioids, amphetamine-like drugs, and their metabolites were determined by studying their ionic transfer process across the bare interface water/organic solvent. The ionic partition coefficients of the monocationic forms of 12 compounds--heroin, 6-monoacetylmorphine (6-MAM), morphine, acetylcodeine, codeine, dihydrocodeine, methamphetamine, amphetamine, 3,4-methylenedioxymethamphetamine (MDMA or "ecstasy"), 3,4-methylenedioxyamphetamine (MDA), 3-methoxy-alpha-methyldopamine (3-OMe-alpha-MeDA), and alpha-methyldopamine (alpha-MeDA)-were attained using electrochemical measurements, by cyclic voltammetry, at the interface between two immiscible electrolyte solutions (ITIES). Then the acquired lipophilicity values were correlated to the chemical structure of the compounds and with the metabolic pathways central to each class of drugs. Although the mechanisms of biotoxicity of this type of drugs are still unclear, the data obtained evidence that the lipophilicity of metabolites may be a contributing factor for the qualitative differences found in their activity. In addition, the partition coefficients of the ionic drugs were calculated using three available software packages: ModesLab, Dragon, and HyperChem. As shown by cross-comparison of the experimental and calculated values, HyperChem was the most reliable software for achieving the main goal. The data obtained so far seem to be correlated to the proposed metabolic pathways of the drugs and could be of great value in understanding their pharmacological and/or toxicological profiles at the molecular level. This study may also contribute to gaining an insight into the mechanisms of biotransportation of this type of compounds given that the ionic partition coefficients reflect their ability to cross the membrane barriers.     

Purification of Extracellular Cholesterol Oxidase with High Activity in the Presence of Organic Solvents from Pseudomonas sp. Strain ST-200

Extracellular cholesterol oxidase of Pseudomonas sp. strain ST-200 was purified from the culture supernatant. This oxidase contained bound flavin and was categorized as a 3β-hydroxysteroid oxidase, converting 3β-hydroxyl groups to keto groups. The molecular mass was 60 kDa. The enzyme was stable at pH 4 to 11 and active at pH 5.0 to 8.5, showing optimal activity at pH 7 at 60°C. The Michaelis constant of the ST-200 cholesterol oxidase was lower than those of commercially available oxidases. The cholesterol oxidation rate was enhanced 3- to 3.5-fold in the presence of organic solvents, with log P_ow values (partition coefficients of the organic solvent between n-octanol and water), in the range of 2.1 to 4.2, compared with that in the absence of organic solvents.     

Effects of barbiturates on ultrastructure and polymerization of microtubules in vitro

Summary Barbiturates were examined for in vitro effects on ultrastructure of the frog sciatic system and polymerization of microtubules (MT) in a brain supernatant. Exposure for 5–17 h to 2.0 mM barbiturates caused a considerable loss of MT in ganglionic cell bodies and sciatic axons. This was mostly followed by a proliferation of 10 nm filaments. Under similar conditions treatment with 1 mM NaCN or 0.1 mM 2,4-DNP did not change the number or ultrastructure of MT and filaments.Eight barbiturates, varying in binding ratios to serum albumin and partition coefficients, were tested for effects on polymerization of MT using viscometry. Inhibitory effects were found which correlated with their reported ability to bind to albumin and brain fractions. Dimethylsulphoxide and ethanol were used as solvents for some of the barbiturates. These solvents at 1% had stabilizing effects on MT.The present results are discussed in relation to previous findings of inhibition of rapid axonal transport in vitro in the frog sciatic system by barbiturates.The present work was supported by grants from Statens Naturvetenskapliga Forskningsråd (No. 2535-0011), Statens Medicinska Forskningsråd (B 75-12x-2543-07), Wilhelm och Martina Lundgrens Vetenskapsfond, Magnus Bergwalls Stifteise och Göteborgs Kungl. Vetenskapsoch Vitterhetssamhälle. Thanks are due to Miss Monica Lindhé for her expert technical assistance.     

Electronic speckle pattern interferometry: a tool for determining diffusion and partition coefficients for proteins in gels

The aim of this study was to demonstrate electronic speckle pattern interferometry (ESPI) as a powerful tool in determining diffusion coefficients and partition coefficients for proteins in gels. ESPI employs a CCD camera instead of a holographic plate as in conventional holographic interferometry. This gives the advantage of being able to choose the reference state freely. If a hologram at the reference state is taken and compared to a hologram during the diffusion process, an interferometric picture can be generated that describes the refraction index gradients and thus the concentration gradients in the gel as well as in the liquid. MATLAB is then used to fit Fick's law to the experimental data to obtain the diffusion coefficients in gel and liquid. The partition coefficient is obtained from the same experiment from the flux condition at the interface between gel and liquid. This makes the comparison between the different diffusants more reliable than when the measurements are performed in separate experiments. The diffusion and partitioning coefficients of lysozyme, BSA, and IgG in 4% agarose gel at pH 5.6 and in 0.1 M NaCl have been determined. In the gel the diffusion coefficients were 11.2 +/- 1.6, 4.8 +/- 0.6, and 3.0 +/- 0.3 m(2)/s for lysozyme, BSA, and IgG, respectively. The partition coefficients were determined to be 0.65 +/- 0.04, 0.44 +/- 0.06, and 0.51 +/- 0.04 for lysozyme, BSA, and IgG, respectively. The current study shows that ESPI is easy to use and gives diffusion coefficients and partition coefficients for proteins with sufficient accuracy from the same experiment.     

Monitoring proton dissociation and solution conformation of chiral ytterbium complexes with near-IR CD

The ytterbium complex [Yb((S)-THP)](3+) ((S)-THP = (1S,4S,7S,10S-tetrakis(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane) is investigated in solution through NMR, near-IR absorption, and CD spectroscopy. Quantitative analysis of the paramagnetic pseudocontact NMR shift shows Lambda helicity of the ligand cage around the metal. The NIR CD spectrum recorded at acidic pH is found to be very similar to that of [Yb((R)-DOTMA)](-) ((R)-DOTMA = (1R,4R,7R,10R)-alpha,alpha',alpha',alpha'-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), which in solution assumes a twisted square antiprism (TSA) conformation. The similarity of the NIR CD spectra is discussed, and it is the first proof of the Lambda(lambda,lambda,lambda,lambda) conformation of [Yb((S)-THP)](3+). NIR CD spectra recorded in the pH range of 2-9 allow one to easily follow proton dissociation and to calculate the pK of this equilibrium in water (pK(A) = 6.4 +/- 0.1). This value agrees well with that determined for [Lu((S)-THP)](3+) using potentiometric methods. This demonstrates once again that NIR CD spectroscopy is a powerful technique for investigating the solution structure and dynamics of these complexes.     

Interfacial ionization and partitioning of membrane-bound local anesthetics.

Consideration of the interfacial protonation equilibria of membrane-associated amphiphiles indicates that the partition coefficients of the protonated and unprotonated species will differ considerably. The partition coefficients of the charged and uncharged forms of spin-labelled myristic acid in dimyristoylphosphatidylcholine bilayer dispersions have been measured by EPR spectroscopy and found to be approximately 140-fold higher for the protonated acid than for the dissociated salt form. This ratio of partition coefficients is found to be in good agreement with that predicted from the interfacial shift in pKa of the fatty acid on its partitioning into the membrane. The latter was determined from the changes in the EPR spectra of the membrane-associated fatty acid with pH and was found to be +2.1 pH units. The interfacial shifts in pKa for a series of spin-labelled analogues of tertiary amine local anaesthetics have been determined from the pH dependence of the partition coefficients in dimyristoylphosphatidylcholine bilayer dispersions and are found mostly to be in the range of approx. -1.0 to -1.5 pH units, corresponding to a 10- to 30-fold higher partition coefficient of the uncharged base compared with that of the charged ammonium form.     

A caveat for the use of log P values for the assessment of the biocompatibility of organic solvents

The degree of enzyme deactivation for lipases from Candida rugosa and Pseudomonas sp., hydroxynitrile lyase and mandelate racemase upon exposure to organic solvents can be correlated to their respective partition coefficients (log P values). However, three unexpected results were obtained: (1) the deactivation exerted by protic solvents, e.g., methanol, is severely underestimated; (2) little deactivation by an organic solvent cannot neccessarily be correlated to catalytic activity in this medium, and (3) in contrast to other enzymes, hydroxynitrile lyase is exceptionally stable towards deactivation by DMF.     

Phospholipid lateral phase separation and the partition of cis-parinaric acid and trans-parinaric acid among aqueous, solid lipid, and fluid lipid phases.

The partition of cis-parinaric acid (9,11,13,15-cis, trans, trans,cis-octadecatetraenoic acid, cis-PnA) and trans-parinaric acid (9,11,13,15-all-trans-octadecatetraenoic acid, trans-PnA) among aqueous, solid lipid, and fluid lipid phases has been measured by three spectroscopic parameters: absorption spectral shifts, fluorescence quantum yield, and fluorescence polarization. The solid lipid was dipalmitoylphosphatidylcholine (DPPC); the fluid lipid was palmitoyldocosahexaenoylphosphatidylcholine (PDPC). Mole fraction partition coefficients between lipid and water were determined by absorption spectroscopy to be for ci--PnA, 5.3 X 10(5) with a solid lipid and 9 X 10(5) with fluid lipid and, for trans-PnA, 5 X 10(6) with solid lipid and 1.7 X 10(6) with fluid lipid. Ratios of the solid to the fluid partition coefficients (Kps/f) are 0.6 +/- 0.2 for cis-PnA and 3 +/- 1 for trans-PnA. A phase diagram for codispersions of DPPC and PDPC has been constructed from the measurements of the temperature dependence of the fluorescence quantum yield and polarization of cis-PnA and trans-PnA and their methyl ester derivatives. A simple analysis based on the phase diagram and fluorescence data allows additional calculations of Kps/f's which are determined to be 0.7 +/- 0.2 for the cis probes and 4 +/- 1 for the trans probes. The relative preference of trans-PnA for solid phase lipids and its enhanced quantum yield in solid phase lipids make it sensitive to a few percent solid. The trans probes provide evidence that structural order may persist in dispersions of these phospholipids 10 degrees C or more above their transition temperature. It is concluded that measurements of PnA fluorescence polarization vs. temperature are better suited than measurements of quantum yield vs. temperature for determining phospholipid phase separation.     

Selection of organic solvents for in situ extraction of fermentation products fromClostridium thermohydrosulfuricum cultures

Summary Fifteen organic solvents were examined to determine their biocompatibility for in situ extraction of fermentation products from cultures of the thermophilic anaerobeClostridium thermohydrosul furicum. Five solvents (hexadecane, isooctane, kerosene, oleyl alcohol, Shellsol TD) were found to be non-toxic toClostridium thermohydrosul furicum. Interfacial tensions, phase separation and partition coefficients for ethanol of the biocompatible solvents were compared. With the exception of kerosene, these solvents showed good separation from the aqueous phase. Oleyl alcohol had the highest partition coefficient for ethanol (K_D=0.34 at 65°C) and appears to be suitable for extractive ethanol fermentation.     

Apparent monomer activity of saturated fatty acids im micellar bile salt solutions measured by a polyethylene partitioning system

Partitioning of saturated fatty acids between discs of polyethylene film and aqueous buffer has been characterized and subsequently used to measure monomer activities of fatty acids in micellar solutions of bile salt. Partitioning of fatty acids between polyethylene and buffer achieved equilibrium in about 24-48 hr. Partition coefficients for fatty acids 10:0 and 16:0 were essentially independent of concentration, as expected for true partitioning. Experiments with various pH buffers showed that only the protonated form of fatty acids 12:0 and 16:0 participated in partitioning, and the midpoints of the partition coefficients vs. pH curves were 4.5-5.0 and 6.5-7.0, respectively. Experimentally determined partition coefficients at pH 7.4 ranged from 2.03 +/- 0.09 for 9:0 to 56,100 +/- 13,850 for 17:0. The addition of each methylene group increased the partition coefficient by a factor of about 3.75, corresponding to an incremental free energy change for each methylene group of -3433 J.mole(-1) (-820 cal.mole(-1)). Monomer activities of solutions of 14:0 and 16:0 dissolved in 20 mM taurodeoxycholate were linearly dependent on the total fatty acid concentration. 1 mM 14:0 and 16:0 in 20 mM taurodeoxycholate had monomer activities of 1.3 x 10(-5) M and 5.6 x 10(-7) M, respectively. Solutions prepared with a constant concentration ratio of fatty acid to taurodeoxycholate had essentially constant monomer activities between 8 and 20 mM taurodeoxycholate. These studies support the hypothesis that fatty acid interaction with bile acid micelles is similar to a phase distribution system, so that some measurable monomer activity of fatty acid exists in equilibrium with the mass of fatty acid contained in the micelle.     

Lipase catalyzed esterification of glycidol in organic solvents

We studied the resolution of racemic glycidol through esterification with butyric acid catalyzed by porcine pancreatic lipase in organic media. A screening of seven solvents (log P values between 0.49 and 3.0, P being the n-octanol-water partition coefficient of the solvent) showed that neither log P nor the logarithm of the molar solubility of water in the solvent provides good correlations between enantioselectivity and the properties of the organic media. Chloroform was one of the best solvents as regards the enantiomeric purity (e. p.) of the ester produced. In this solvent, the optimum temperature for the reaction was determined to be 35 degrees C. The enzyme exhibited maximum activity at a water content of 13 +/- 2% (w/w). The enantiomeric purity obtained was 83 +/- 2% of (S)-glycidyl butyrate and did not depend on the alcohol concentration or the enzyme water content for values of these parameters up to 200 mM and 25% (w/w), respectively. The reaction was found to follow a BiBi mechanism. (c) 1993 John Wiley & Sons, Inc.     

A solvent-partition method for measuring the binding of drugs to DNA. Application to the quinoxaline antibiotics echinomycin and triostin A.

The development of a novel solvent-partition method for measuring the interaction between nucleic acids and drugs of limited water solubility is described. Factors relevant to the choice of a suitable water-immiscible solvent are summarised. i-Amyl acetate was selected for studying the binding of echinomycin and triostin A to DNA. Details of the experimental determination of extinction and partition coefficients are given; in the i-amyl acetate/buffer system employed for most experiments, the partition coefficients for echinomycin and triostin A were 111 +/- 4 and 943 +/- 23, respectively. Equilibration of echinomycin between the organic and aqueous phases was 90% complete within a few minutes, and a period of 2 h shaking was found satisfactory to ensure full attainment of equilibrium. Representative results are presented showing specific binding of the quinoxaline antibiotics to DNA, strong preference for double-helical as opposed to heat-denatured or single-stranded DNA, and restricted uptake by closed circular duplex PM2 DNA. The method is potentially applicable, with appropriate modifications, to the study of interactions between other ligands and DNA.     

Kinetic theory of enzymatic reactions in reversed micellar systems. Application of the pseudophase approach for partitioning substrates

A kinetic theory is proposed for enzymatic reactions proceeding in reversed micellar systems in organic solvents, and involving substrates capable of partitioning among all pseudophases of the micellar system i.e. aqueous cores of reversed micelles, micellar membranes and organic solvent. The theory permits determination of true (i.e. with reference to the aqueous phase, where solubilized enzyme is localized) catalytic parameters of the enzyme, provided partition coefficients of the substrate between different phases are known. The validity of the kinetic theory was verified by the example of oxidation of aliphatic alcohols catalyzed by horse liver alcohol dehydrogenase in the system of reversed sodium bis(2-ethylhexyl)sulfosuccinate (AOT, aerosol OT) micelles in octane. In order to determine partition coefficients of alcohols between phases of the micellar system, flow microcalorimetry technique was used. It was shown that in the first approximation, the partition coefficient of the substrate in a simple biphasic system consisting of water and corresponding organic solvent can be used as an estimate for the partition coefficient of the substrate between aqueous and organic solvent phases of the micellar system. True values of the Michaelis constant of alcohols in the micellar system, determined using suggested approach, are equal to those obtained in aqueous solution and differ from apparent values referred to the total volume of the system. The results clearly show that the previously reported shift in the substrate specificity of HLADH, observed on changing from aqueous solution to the system of reversed aerosol OT micelles in octane, is apparent and can be explained on the basis of partitioning effects of alcoholic substrates between phases of the micellar system.     

Solubility of carbon dioxide in lipid bilayer membranes and organic solvents

Partition coefficients of carbon dioxide into lipid bilayers (liposomes) and organic solvents were measured as a function of temperature. The molar partition coefficient of CO2 into liposomes of egg lecithin at 25 degrees C was 0.95 (ml CO2/ml lipid)/(ml CO2/ml saline). The addition of an equimolar amount of cholesterol to the egg lecithin decreased the partition coefficient by about 25%. The partition coefficients for CO2 into liposomes at 25 degrees C were lower than the partition coefficients into octanol (1.3), hexadecane (1.5) and olive oil (1.7). The results are discussed in terms of the solubility-diffusion model of non-electrolyte transport through lipid bilayer membranes.     

Can the stereoselective effects of the anesthetic isoflurane be accounted for by lipid solubility?

Isoflurane is an inhalational general anesthetic widely used in surgical operations as a racemic mixture of its two optical isomers. The recent availability of pure enantiomers of isoflurane has encouraged their use in experimental studies, and stereoselective effects have now been observed on anesthetic-sensitive neuronal ion channels. Although it has been assumed that such chiral effects demonstrate direct interactions with proteins, it is possible that they could be due to stereoselective interactions with chiral membrane lipids. We have determined the partition coefficients of the two optical isomers of isoflurane between lipid bilayers and water, using racemic isoflurane and gas chromatography with a chiral column. For lipid bilayers of phosphatidylcholine (PC) and 4 mol% phosphatidic acid (PA), both with and without cholesterol (CHOL), we found equal partitioning of the isoflurane optical isomers. The ratios of the S(+) to R(-) isoflurane partition coefficients were (mean +/- SEM): 1.018 +/- 0.010 for bilayers of PC/CHOL/PA (mole ratios 56:40:4) and 1.011 +/- 0.002 for bilayers of PC/PA (mole ratio 96:4). Molar partition coefficients for racemic isoflurane were 49 +/- 4 and 165 +/- 10, respectively. These findings support the view that the stereoselective effects on ion channels observed with isoflurane are due to direct actions on proteins rather than lipids.     

Benzyl alcohol penetration into micelles, dielectric constant of the binding site, partition coefficient and high-pressure squeeze-out

The absorbance maximum, lambda max, of a local anesthetic, benzyl alcohol, is shifted to longer wavelengths when solvent polarity is decreased. The shift was approximately a linear function of the dielectric constant of the solvent. This transition in electronic spectra according to the microenvironmental polarity is used to analyze benzyl alcohol binding to surfactant micelles. A facile method is devised to estimate the micelle/water partition coefficient from the dependence of lambda max of benzyl alcohol on surfactant concentrations. The effective dielectric constants of the sodium decyl sulfate, dodecyl sulfate and tetradecyl sulfate micelles were 29, 31 and 33, respectively. The partition coefficient of benzyl alcohol between the micelles and the aqueous phase was 417, 610 and 1089, respectively, in the mole fraction unit. The pressure dependence of the partition coefficient was estimated from the depression of the critical micelle concentration of sodium dodecyl sulfate by benzyl alcohol under high pressure up to 200 MPa. High pressure squeezed out benzyl alcohol molecules from the micelle until about 120 MPa, then started to squeeze in when the pressure was further increased. The volume change of benzyl alcohol by transfer from the aqueous to the micellar phase was calculated from the pressure dependence of the partition coefficient. The volume change, estimated from the thermodynamic argument, was 3.5 +/- 1.1 cm3.mol-1 at 298.15 K, which was in reasonable agreement with the partial molal volume change determined directly from the solution density measurements, 3.1 +/- 0.2 cm3.mol-1. Benzyl alcohol apparently solvates into the micelles close to surface without losing contact with the aqueous phase.     

Solvent effect on the synergic extraction of thulium(III) with acetylacetone and 1,10-phenanthroline

The synergic extraction equilibrium of Tm(III) with acetylacetone (Hacac) and 1,10-phenanthroline (phen) in various organic solvents has been studied. The adduct formation constants, β_s, for Tm(acac)₃^? phen, were determined in heptane, cyclohexane, carbon tetrachloride, benzene and chloroform. The solvent effect on β_s is explained in connection with the activity coefficients of the neutral ligand, the chelate, and the adduct in the organic solvent. The activity coefficients can be calculated from the corresponding solubility parameters on the basis of the regular solution theory, and the solubility parameters of the solutes were estimated from their two-phase partition coefficients. It is demonstrated that β_s in different organic solvents except those having a specific interaction with the solute, such as chloroform, can be calculated by the present approach.     

Ovine myometrial cytosol inhibits phospholipase A activity, in vitro

Myometrium obtained from pregnant ewes (30-80 days gestation) contains a factor which inhibits phospholipase A2 (PLA2) activity. The activity of this moiety was assessed using an in vitro porcine pancreatic PLA2 assay system. Inhibitory activity was associated with a 35-45000 dalton molecular weight fraction, heat-labile, sensitive to protease degradation and did not partition into organic solvents. These data are indicative that PLA2-inhibitory activity resides in a protein moiety. Dixon-plot analysis of myometrial-inhibitory activity was indicative that the inhibition of PLA2 activity was of a non-competitive nature (Ki = 4.1 +/- 0.7 micrograms/ml, ca 118 nmol/l). Myometrial phospholipase-inhibitory protein(s) may be involved in the suppression of eicosanoid biosynthesis by the uterine tissues throughout gestation thus inhibiting uterine contractile activity.     

Tetracaine-membrane interactions: effects of lipid composition and phase on drug partitioning, location, and ionization

Interactions of the local anesthetic tetracaine with unilamellar vesicles made of dimyristoyl or dipalmitoyl phosphatidylcholine (DMPC or DPPC), the latter without or with cholesterol, were examined by following changes in the drug's fluorescent properties. Tetracaine's location within the membrane (as indicated by the equivalent dielectric constant around the aromatic fluorophore), its membrane:buffer partition coefficients for protonated and base forms, and its apparent pK(a) when adsorbed to the membrane were determined by measuring, respectively, the saturating blue shifts of fluorescence emission at high lipid:tetracaine, the corresponding increases in fluorescence intensity at this lower wavelength with increasing lipid, and the dependence of fluorescence intensity of membrane-bound tetracaine (TTC) on solution pH. Results show that partition coefficients were greater for liquid-crystalline than solid-gel phase membranes, whether the phase was set by temperature or lipid composition, and were decreased by cholesterol; neutral TTC partitioned into membranes more strongly than the protonated species (TTCH(+)). Tetracaine's location in the membrane placed the drug's tertiary amine near the phosphate of the headgroup, its ester bond in the region of the lipids' ester bonds, and associated dipole field and the aromatic moiety near fatty acyl carbons 2-5; importantly, this location was unaffected by cholesterol and was the same for neutral and protonated tetracaine, showing that the dipole-dipole and hydrophobic interactions are the critical determinants of tetracaine's location. Tetracaine's effective pK(a) was reduced by 0.3-0.4 pH units from the solution pK(a) upon adsorption to these neutral bilayers, regardless of physical state or composition. We propose that the partitioning of tetracaine into solid-gel membranes is determined primarily by its steric accommodation between lipids, whereas in the liquid-crystalline membrane, in which the distance between lipid molecules is larger and steric hindrance is less important, hydrophobic and ionic interactions between tetracaine and lipid molecules predominate.