[An investigation and analysis on a poisoning tetramine accident].

From August 21 to December 13, 2018, a tetramine poisoning incident in Wenzhou, Zhejiang Province was investigated, and the clinical diagnosis and treatment of tetramine poisoning was analyzed. There were 6 cases of poisoning caused by artificial tetramine poisoning. The diagnosis was delayed, coma and convulsions were severe manifestations continuous renal replacement therapy (CRRT) was effective in the treatment of severe cases, and all 6 cases were cured. The possibility of poisoning should be considered for unexplained coma and/or convulsions. Although tetramine is banned, it still needs to be highly vigilant and avoids the recurrence of delayed diagnosis and treatment.

[Role and clinical significance of Th17/Treg balance in patients with severe exacerbation of hepatitis B].

OBJECTIVE: To investigate the role of Th17/Treg balance in immune mechanism in severe exacerbation of hepatitis B. METHODS: The clinical data of 41 patients with chronic hepatitis B were collected, and according to the conditions during hospitalization, these patients were divided into exacerbation group (19 patients) and improvement group (22 patients). On admission, at weeks 1 and 2 of treatment, and at the end of treatment, flow cytometry was used to measure the frequencies of Th17 and Treg cells in peripheral blood, and enzyme-linked immunosorbent assay was used to determine the serum levels of interleukin-17 (IL-17), interleukin-10 (IL-10), and transforming growth factor-ß (TGF-ß). The dynamic changes in the frequencies of Th17 and Treg cells were compared between the two groups, and the correlation between clinical indices for hepatitis and cytokines was analyzed. The t-test was used for comparison between groups, a one-way analysis of variance was used for comparison within one group across different time points, and Pearson correlation analysis was performed. RESULTS: With disease progression, the exacerbation group showed an increase in the frequency of Th17 cells and a relatively low frequency of Treg cells; compared with the improvement group, the exacerbation group had a higher frequency of Th17 cells and a lower frequency of Treg cells. Th17/Treg ratio gradually increased with exacerbation and decreased with improvement in conditions; in the exacerbation group and the improvement group, Th17/Treg ratio was positively correlated with total bilirubin and negatively correlated with prothrombin activity. In the exacerbation group and the improvement group, Th17 cells were positively correlated with IL-17, and Treg cells were positively correlated with IL-10 and TGF-ß. CONCLUSION: Th17 and Treg cells play important roles in severe exacerbation of hepatitis B, and Th17/Treg ratio may be used as an immunobiological marker for the judgment of severity during severe exacerbation of hepatitis B.

A quantitative model for the dependence of solute permeability on peptide and cholesterol content in biomembranes.

The influence of varying concentrations of a transmembrane peptide, gramicidin A (gA), and cholesterol (Chol) on the passive permeation of p-methylhippuric acid (MHA) and alpha-carbamoyl-p-methylhippuric acid (CMHA) across egg-lecithin membranes (EPC) has been investigated in vesicle efflux experiments. Incorporation of 0.25 volume fraction of gA in its nonchannel conformation increased the permeability coefficient (Pm) for CMHA by a factor of 6.0 +/- 1.8 but did not alter Pm for MHA, a more lipophilic permeant. In contrast, incorporation of 0.26 volume fraction Chol with no added protein decreased the Pm values for both CMHA and MHA by similar factors of 4.2 +/- 1.1 and 3.5 +/- 1.2, respectively. A quantitative structure-transport model has been developed to account for the dependence of Pm on the membrane concentrations of gA and Chol in terms of induced changes in both membrane chain ordering and hydrophobicity. Chain ordering is assumed to affect Pm for both permeants similarly since they are comparable in molecular size, while changes in Pm ratios in the presence of gA or Chol are attributed to alterations in membrane hydrophobicity. Changes in lipid chain ordering were detected by monitoring membrane fluidity using fluorescence anisotropy of 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene incorporated into the membranes. The influence of additives on membrane hydrophobicity, which governs Pm ratios through effects on solute partitioning into the barrier domain, were rationalized within the framework of regular solution theory using solubility parameters as a measure of membrane hydrophobicity. Fits of the Pm ratios using the theoretical model yielded solubility parameters for gA and Chol in EPC membranes of 13.2 and 7.7 (cal/ml)(1/2), respectively, suggesting that gA decreases the barrier domain hydrophobicity while Chol has a minimal effect on barrier hydrophobicity. After correcting for barrier domain hydrophobicity, permeability decrements due to membrane ordering induced by gA or Chol were found to exhibit a strong correlation with membrane order as predicted by free-surface-area theory, regardless of whether gA or Chol is used as the ordering agent.

Independence of substituent contributions to the transport of small molecule permeants in lipid bilayers.

To explore the independence of functional group contributions to permeability of nonelectrolytes across egg lecithin bilayers, the transport rates were measured of a series of alpha-substituted p-methylhippuric acids (-H, -Cl, -OCH3, -CN, -OH, -COOH, and -CONH2) across egg lecithin lipid bilayers, in the form of large unilamellar vesicles (LUVs) at 25 degrees C. Intrinsic permeability coefficients (P(HA)) were calculated from apparent permeability coefficients (P(app)) measured as a function of pH. Group contributions to the free energy of transfer from water into the barrier domain Delta (Delta G degrees)P,X, were calculated for the substituents and compared to the contributions of these groups when attached top-toluic acid measured earlier. The Delta (Delta G degrees)P,X values from permeability data were also correlated with Delta (Delta G degrees)PC,X values of partitioning from water into organic solvents to determine the physicochemical selectivity of the barrier domain. P(app) values in LUVs were found to vary approximately linearly with the fraction of neutral permeant over a pH range of 5.5 to 10.5, suggesting that the transport of the ionized species is negligible over this pH range. The Delta (Delta G degrees)P,X values from the 2 series of compounds appear to be the same, indicating that the functional group contributions are independent. 1,9-decadiene was found to be the most similar to the chemical environment of the barrier domain. Functional group contributions to transport across egg lecithin bilayers appear to be independent of the compound to which they are attached, even though the thickness of the barrier domain in lipid bilayers is approximately the same as the extended length of the permeant.

Influence of chain ordering on the selectivity of dipalmitoylphosphatidylcholine bilayer membranes for permeant size and shape.

The effects of lipid chain packing and permeant size and shape on permeability across lipid bilayers have been investigated in gel and liquid crystalline dipalmitoylphosphatidylcholine (DPPC) bilayers by a combined NMR line-broadening/dynamic light scattering method using seven short-chain monocarboxylic acids (formic acid, acetic acid, propionic acid, butyric acid, valeric acid, isovaleric acid, and trimethylacetic acid) as permeants. The experimental permeability coefficients are compared with the predictions of a bulk solubility diffusion model in which the bilayer membrane is represented as a slab of bulk hexadecane. Deviations of the observed permeability coefficients (Pm) from the values predicted from solubility diffusion theory (Po) lead to the determination of a correction factor, the permeability decrement f (= Pm/Po), to account for the effects of chain ordering. The natural logarithm of f has been found to correlate linearly with the inverse of the bilayer free surface area with slopes of 25 +/- 2, 36 +/- 3, 45 +/- 8, 32 +/- 12, 33 +/- 4, 49 +/- 12, and 75 +/- 6 A2 for formic acid, acetic acid, propionic acid, butyric acid, valeric acid, isovaleric acid, and trimethylacetic acid, respectively. The slope, which measures the sensitivity of the permeability coefficient of a given permeant to bilayer chain packing, exhibits an excellent linear correlation (r = 0.94) with the minimum cross-sectional area of the permeant and a poor correlation (r = 0.59) with molecular volume, suggesting that in the bilayer interior the permeants prefer to move with their long principal axis along the bilayer normal. Based on these studies, a permeability model combining the effects of bilayer chain packing and permeant size and shape on permeability across lipid membranes is developed.

Phase structures of binary lipid bilayers as revealed by permeability of small molecules.

The effects of changes in bilayer phase structure on the permeability of acetic acid and trimethylacetic acid were studied in large unilamellar vesicles (LUVs) composed of dipalmitoylphosphatidylcholine (DPPC)/cholesterol (CHOL), dihexadecylphosphatidylcholine (DHPC)/CHOL, or DPPC/dimyristoylphosphatidylcholine (DMPC) using an NMR line-broadening method. Phase transitions were induced by changes in temperature and lipid composition (i.e., XCHOL was varied from 0.0 to 0.5 and XDMPC from 0.0 to 1.0). In DPPC/CHOL and DHPC/CHOL bilayers, the addition of CHOL induces only a modest change in the permeability coefficient (Pm) of acetic acid in the gel-phase (Pbeta') but significantly reduces Pm in ordered and disordered liquid-crystalline phases (Lo and Lalpha). Abrupt changes in slopes in semi-logarithmic plots of Pm vs. XCHOL occur at specific values of XCHOL and temperature corresponding to the boundaries between Pbeta' and Lo or between Lalpha and Lo phases. In most respects, phase diagrams generated from the break points in plots of Pm vs. XCHOL obtained at various temperatures in DHPC/CHOL and DPPC/CHOL bilayers closely resemble those constructed previously for DPPC/CHOL bilayers using NMR and DSC methods. Above Tm, the phase diagrams generated from permeability data reveal the presence of both the disordered (Lalpha) and the ordered (Lo) liquid-crystalline phases, as well as the two-phase coexistence region. In DPPC/DMPC bilayers, the addition of DMPC increases Pm dramatically in the gel phase but only slightly in the liquid-crystalline phase. Abrupt changes in slopes in semi-logarithmic plots of Pm vs. XDMPC also occur at specific values of XDMPC and temperature, from which a phase diagram can be constructed which closely resembles diagrams obtained previously by other methods. These correlations indicate that trans-bilayer permeability measurements can be used to construct lipid bilayer phase diagrams. Positive deviations of Pm from predicted values based on the phase lever rule are observed in the two-phase coexistence regions with the degree of the deviation depending on bilayer chemical composition and temperature. These results may reflect a specific contribution of the interfacial region between two phases to higher solute permeability or may be due to the higher lateral compressibility of lipid bilayers in the two-phase coexistence region.

Permeability of acetic acid across gel and liquid-crystalline lipid bilayers conforms to free-surface-area theory.

Solubility-diffusion theory, which treats the lipid bilayer membrane as a bulk lipid solvent into which permeants must partition and diffuse across, fails to account for the effects of lipid bilayer chain order on the permeability coefficient of any given permeant. This study addresses the scaling factor that must be applied to predictions from solubility-diffusion theory to correct for chain ordering. The effects of bilayer chemical composition, temperature, and phase structure on the permeability coefficient (Pm) of acetic acid were investigated in large unilamellar vesicles by a combined method of NMR line broadening and dynamic light scattering. Permeability values were obtained in distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, and dilauroylphosphatidylcholine bilayers, and their mixtures with cholesterol, at various temperatures both above and below the gel-->liquid-crystalline phase transition temperatures (Tm). A new scaling factor, the permeability decrement f, is introduced to account for the decrease in permeability coefficient from that predicted by solubility-diffusion theory owing to chain ordering in lipid bilayers. Values of f were obtained by division of the observed Pm by the permeability coefficient predicted from a bulk solubility-diffusion model. In liquid-crystalline phases, a strong correlation (r = 0.94) between f and the normalized surface density sigma was obtained: in f = 5.3 - 10.6 sigma. Activation energies (Ea) for the permeability of acetic acid decreased with decreasing phospholipid chain length and correlated with the sensitivity of chain ordering to temperature, [symbol: see text] sigma/[symbol: see text](1/T), as chain length was varied. Pm values decreased abruptly at temperatures below the main phase transition temperatures in pure dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine bilayers (30-60-fold) and below the pretransition in dipalmitoylphosphatidylcholine bilayers (8-fold), and the linear relationship between in f and sigma established for liquid-crystalline bilayers was no longer followed. However, in both gel and liquid-crystalline phases in f was found to exhibit an inverse correlation with free surface area (in f = -0.31 - 29.1/af, where af is the average free area (in square angstroms) per lipid molecule). Thus, the lipid bilayer permeability of acetic acid can be predicted from the relevant chain-packing properties in the bilayer (free surface area), regardless of whether chain ordering is varied by changes in temperature, lipid chain length, cholesterol concentration, or bilayer phase structure, provided that temperature effects on permeant dehydration and diffusion and the chain-length effects on bilayer barrier thickness are properly taken into account.

Phospholipid surface density determines the partitioning and permeability of acetic acid in DMPC:cholesterol bilayers.

Relationships between the permeability coefficient (PHA) and partition coefficient (K m/w) of acetic acid and the surface density of DMPC:cholesterol bilayers have been investigated. Permeability coefficients were measured in large unilamellar vesicles by NMR line broadening. Bilayer surface density, sigma, was varied over a range of 0.5-0.9 by changing cholesterol concentration and temperature. The temperature dependence of PHA for acetic acid exhibits Arrhenius behavior with an average apparent activation energy (Ea) of 22 +/- 3 kcal/mole over a cholesterol mole fraction range of 0.00-0.40. This value is much greater than the enthalpy change for acetic acid partitioning between bulk decane and water (delta H degree = 4.8 +/- 0.8 kcal/mole) and the calculated Ea (= 8.0 kcal/mole) assuming a "bulk phase" permeability model which includes the enthalpy of transfer from water to decane and the temperature dependence of acetic acid's diffusion coefficient in decane. These results suggest that dehydration, previously considered to be a dominant component, is a minor factor in determining Ea. Values of 1n PHA decrease linearly with the normalized phospholipid surface density with a slope of kappa = -12.4 +/- 1.1 (r = 0.90). Correction of PHA for those temperature effects considered to be independent of lipid chain order (i.e., enthalpy of transfer from water to decane and activation energy for diffusion in bulk hydrocarbon) yielded an improved correlation (kappa = -11.7 +/- 0.5 (r = 0.96)). The temperature dependence of Km/w is substantially smaller than that for PHA and dependent on cholesterol composition. Values of 1n K m/w decrease linearly with the surface density with a slope of kappa = -4.6 +/- 0.3 (r = 0.95), which is 2.7-fold smaller than the slope of the plot of 1n PHA vs. sigma. Thus, chain ordering is a major determinant for molecular partitioning into and transport across lipid bilayers, regardless of whether it is varied by lipid composition or temperature.

Development of a combined NMR paramagnetic ion-induced line-broadening/dynamic light scattering method for permeability measurements across lipid bilayer membranes.

A combined method using NMR line-broadening for permeant lifetime determination and dynamic light scattering for vesicle size determination has been developed for the measurement of permeability coefficients of ionizable permeants across phospholipid:cholesterol large unilamellar vesicles. The method has been validated by examining its reproducibility and the influence of various factors that might affect the permeability measurements. The vesicle hydrodynamic diameter was varied between 0.1 and 0.2 micron by extruding multilamellar vesicles through polycarbonate membranes with different pore sizes (0.03-0.2 microns). For these large unilamellar vesicles, the normalized size distributions analyzed by the CONTIN method had standard deviations < 0.36, which led to errors in permeability coefficients < 10% as predicted from a theoretical model developed here. The permeability coefficient for acetic acid is independent of its concentration, vesicle hydrodynamic diameter, the concentration of Pr3+, and ionic strength over the ranges 0.01-0.2 M, 0.1-0.2 microns, 0.004-0.04 M, and 0.03-0.3, respectively. Membrane/water and decane/water partition coefficient measurements of acetic acid indicate that the effects of permeant binding onto the bilayer membrane and self-association are negligible within the permeant concentration range 0.01-0.2 M. The addition of Pr3+ ions induces vesicle fusion with rates increasing with temperature and decreasing with cholesterol concentration in the membranes. While the intravesicular resonance intensity for acetic acid decreases continuously with time due to vesicle fusion under certain conditions, the corresponding line width and chemical shift remain constant over the same period, highlighting an important advantage of this NMR method over those based on detecting net flux in response to a concentration gradient as there is no means in the latter experiments of discerning vesicle leakiness from passive diffusion rates. The effective chemical nature of a dimristoylphosphatidylcholine:cholesterol bilayer barrier microenvironment was explored by comparing the transport of two permeants, D-(-)-mandelic acid and phenylacetic acid, to their relative bulk solvent/water partition coefficients using three reference solvents (n-decane, 1,9-decadiene, and isoamyl alcohol). Using the NMR line-broadening method, the permeability coefficients for these two permeants were determined to be (2.9 +/- 0.4) x 10(-4) cm/s and (3.9 +/- 0.7) x 10(-2) cm/s, respectively, at 294 K and Xchol = 0.3. The incremental free energy of transport for the additional OH group in D-(-)-mandelic acid, delta delta G0 = +2.9 kcal/mol, resembles most closely that for the transfer of this group from water to 1,9-decadiene, suggesting that the barrier domain resides in the acyl chain region and is slightly more polar/polarizable than a saturated hydrocarbon, possibly due to the presence of a double bond in cholesterol and/or the proximity of the barrier domain to the hydrophilic interface.

Substituent contributions to the transport of substituted p-toluic acids across lipid bilayer membranes.

The fluxes of p-toluic acid and seven alpha-methylene-substituted analogs have been determined as a function of pH across planar egg lecithin/decane bilayers to construct a set of well-isolated polar functional group contributions to the free energy of transfer from water to the bilayer transport barrier domain. Nonlinear regression analyses of flux-pH profiles using a model which accounts for unstirred layer effects yielded membrane permeability coefficients (PRX) that varied from 1.1 cm/s for p-toluic acid to 4.1 x 10(-5) cm/s for the alpha-carbamoyl-p-toluic acid. Bulk organic solvent/water partition coefficients (KRX) were obtained for the same set of permeants using four solvent systems to identify a bulk solvent which closely resembles the chemical nature of the bilayer barrier microenvironment for these permeants. The slopes of plots of log PRX vs log KRX were 0.85, 0.91, 0.99, and 2.4, respectively, for hexadecane/water, hexadecene/water, 1,9-decadiene/water, and octanol/water with the best model solvent being that which yielded a slope closest to unity. A significant deviation in the slope from 1, as observed in the correlation with octanol/water partition coefficients, reveals that this relatively polar, hydrogen-bonding solvent is a poor model solvent for describing the barrier microenvironment for these permeants. Thus, the polar interfacial regions occupied by phospholipid head groups are not the barrier domain for the transport of the series examined in this study. The incremental group contributions to the free energy of transfer to the barrier domain (cal/mol) for the functional groups, CI, OCH3, CN, OH, COOH, and CONH2, were found to be 325, 687, 2170, 3860, 5170, and 6060, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

The relationship between permeant size and permeability in lipid bilayer membranes.

Permeability coefficients (Pm) across planar egg lecithin/decane bilayers and bulk hydrocarbon/water partition coefficients (Kw-->hc) have been measured for 24 solutes with molecular volumes, V, varying by a factor of 22 and Pm values varying by a factor of 10(7) to explore the chemical nature of the bilayer barrier and the effects of permeant size on permeability. A proper bulk solvent which correctly mimics the microenvironment of the barrier domain was sought. Changes in Pm/Kw-->hc were then ascribed to size-dependent partitioning and/or size-dependent diffusivity. The diffusion coefficient-size dependency was described by Dbarrier = Do/Vn. When n-decane was used as a reference solvent, the correlation between log Pm/Kw-->hc and log V was poor (r = 0.56) with most of the lipophilic (hydrophilic) permeants lying below (above) the regression line. Correlations improved significantly (r = 0.87 and 0.90, respectively) with more polarizable solvents, 1-hexadecene and 1,9-decadiene. Values of the size selectivity parameter n were sensitive to the reference solvent (n = 0.8 +/- 0.3, 1.2 +/- 0.1 and 1.4 +/- 0.2, respectively, for decane, hexadecene, and decadiene). Decadiene was selected as the most suitable reference solvent. The value for n in bilayer transport is higher than that for bulk diffusion in decane (n = 0.74 +/- 0.10), confirming the steep dependence of bilayer permeability on molecular size. Statistical mechanical theory recently developed by the authors suggests that a component of this steep size dependence may reside in size-dependent solute partitioning into the ordered chain region of bilayers. This theory, combined with the above diffusion model, yielded the relationship, Pm/Kw-->hc = D(o)exp(-alpha V)Vn. A fit of the experimental data to this model gave the best fit (r = 0.93) with alpha = 0.0053 +/- 0.0021 and n = 0.8 +/- 0.3, suggesting that both diffusion and partitioning mechanisms may play a role in determining the size dependence of lipid bilayer permeabilities.

Molecular distributions in interphases: statistical mechanical theory combined with molecular dynamics simulation of a model lipid bilayer.

A mean-field statistical mechanical theory has been developed to describe molecular distributions in interphases. The excluded volume interaction has been modeled in terms of a reversible work that is required to create a cavity of the solute size against a pressure tensor exerted by the surrounding interphase molecules. The free energy change associated with this compression process includes the configuration entropy as well as the change in conformational energy of the surrounding chain molecules. The lateral pressure profile in a model lipid bilayer (30.5 A2/chain molecule) has been calculated as a function of depth in the bilayer interior by molecular dynamics simulation. The lateral pressure has a plateau value of 309 +/- 48 bar in the highly ordered region and decreases abruptly in the center of the bilayer. Model calculations have shown that for solute molecules with ellipsoidal symmetry, the orientational order increases with the ratio of the long to short molecular axes at a given solute volume and increases with solute volume at a given axial ratio, in accordance with recent experimental data. Increased lateral pressure (p perpendicular) results in higher local order and exclusion of solute from the interphase, in parallel with the effect of surface density on the partitioning and local order. The logarithm of the interphase/water partition coefficient for spherical solutes decreases linearly with solute volume. This is also an excellent approximation for elongated solutes because of the relatively weak dependence of solute partitioning on molecular shape. The slope is equal to (2p perpendicular - p parallel)/3KBT, where p parallel is the normal pressure component, and different from that predicted by the mean-field lattice theory. Finally, the lattice theory has been extended herein to incorporate an additional constraint on chain packing in the interphase and to account for the effect of solute size on partitioning.

Diffusion of ionizable solutes across planar lipid bilayer membranes: boundary-layer pH gradients and the effect of buffers.

The diffusion of weak acids or bases across planar lipid bilayer membranes results in aqueous boundary layer pH gradients. If not properly taken into account, such pH gradients will lead to errors in estimated membrane permeability coefficients, Pm. The role of the permeant concentration, the buffer capacity, and the physicochemical properties of both permeant and buffer on the magnitude and impact of such pH gradients have been explored. A theoretical model has been developed to describe the diffusion of both permeant and buffer species. Significant pH gradients develop depending on solution pH and the pKa's, concentrations, and Pm values of both permeant and buffer. The relative error in experimentally determined Pm values was calculated as the ratio, r, between apparent Pm values (obtained from flux measurements using an equation which neglected boundary layer pH gradients) and its true value. Simulated r values ranged from 1 (0% error) to < 0.01 (> 100% error) for weak acids, decreasing with decreasing buffer capacity and increasing solute flux. The buffer capacity required for an r > 0.95 was calculated versus pH for permeants varying in pKa and Pm. Membrane-permeable buffers significantly reduce boundary layer pH gradients through a feedback effect due to buffer cotransport. Apparent Pm values of p-hydroxymethyl benzoic acid across lecithin bilayer membranes at 25 degrees C were obtained as a function of permeant concentration in various buffers [glycolic, 2-(N-morpholino)ethane-sulfonic, and formic acids]. Predictions agreed closely with experimental fluxes.

A computer simulation of free-volume distributions and related structural properties in a model lipid bilayer.

A novel combined approach of molecular dynamics (MD) and Monte Carlo simulations is developed to calculate various free-volume distributions as a function of position in a lipid bilayer membrane at 323 K. The model bilayer consists of 2 x 100 chain molecules with each chain molecule having 15 carbon segments and one head group and subject to forces restricting bond stretching, bending, and torsional motions. At a surface density of 30 A2/chain molecule, the probability density of finding effective free volume available to spherical permeants displays a distribution with two exponential components. Both pre-exponential factors, p1 and p2, remain roughly constant in the highly ordered chain region with average values of 0.012 and 0.00039 A-3, respectively, and increase to 0.049 and 0.0067 A-3 at the mid-plane. The first characteristic cavity size V1 is only weakly dependent on position in the bilayer interior with an average value of 3.4 A3, while the second characteristic cavity size V2 varies more dramatically from a plateau value of 12.9 A3 in the highly ordered chain region to 9.0 A3 in the center of the bilayer. The mean cavity shape is described in terms of a probability distribution for the angle at which the test permeant is in contact with one of and does not overlap with anyone of the chain segments in the bilayer. The results show that (a) free volume is elongated in the highly ordered chain region with its long axis normal to the bilayer interface approaching spherical symmetry in the center of the bilayer and (b) small free volume is more elongated than large free volume. The order and conformational structures relevant to the free-volume distributions are also examined. It is found that both overall and internal motions have comparable contributions to local disorder and couple strongly with each other, and the occurrence of kink defects has higher probability than predicted from an independent-transition model.

Solubilization of thiazolobenzimidazole using a combination of pH adjustment and complexation with 2-hydroxypropyl-beta-cyclodextrin.

The thiazolobenzimidazole 1-(2,6-difluorophenyl)-1H,3H-thiazolo[3,4-a] benzimidazole, TBI, is an experimental drug for the treatment of AIDS which exhibits a low water solubility (11 micrograms/mL) and is therefore difficult to administer in an injectable solution dosage form at a target solution concentration of 10 mg/mL. The compound has a single ionizable functional group and exhibits an increase in solubility with decreasing pH consistent with a pKa of 3.55, but the maximum solubility attainable by pH adjustment has been shown to be only 0.4 mg/mL (at pH 2). TBI has been found to form inclusion complexes in either its neutral or its protonated form with 2-hydroxypropyl-beta-cyclodextrin (HPCD). The equilibrium constants for 1:1 complex formation were found to be 81 and 1033 M-1 for the protonated and neutral species, respectively. Although the formation of protonated complex is less favored in comparison to the neutral complex, the contribution of this species to the overall solubility of TBI predominates at low pH. Thus, using a combined approach of pH adjustment and complexation with HPCD, a solubility enhancement of 3 orders of magnitude is possible. NMR proton spectroscopy and molecular modeling studies, conducted to understand the orientation of TBI in the complex and the effect of protonation, are described.

Transport methods for probing the barrier domain of lipid bilayer membranes.

Two experimental techniques have been utilized to explore the barrier properties of lecithin/decane bilayer membranes with the aim of determining the contributions of various domains within the bilayer to the overall barrier. The thickness of lecithin/decane bilayers was systematically varied by modulating the chemical potential of decane in the annulus surrounding the bilayer using different mole fractions of squalene in decane. The dependence of permeability of a model permeant (acetamide) on the thickness of the solvent-filled region of the bilayer was assessed in these bilayers to determine the contribution of this region to the overall barrier. The flux of acetamide was found to vary linearly with bilayer area with Pm = (2.9 +/- 0.3) x 10(-4) cm s-1, after correcting for diffusion through unstirred water layers. The ratio between the overall membrane permeability coefficient and that calculated for diffusion through the hydrocarbon core in membranes having maximum thickness was 0.24, suggesting that the solvent domain contributes only slightly to the overall barrier properties. Consistent with these results, the permeability of acetamide was found to be independent of bilayer thickness. The relative contributions of the bilayer interface and ordered hydrocarbon regions to the transport barrier may be evaluated qualitatively by exploring the effective chemical nature of the barrier microenvironment. This may be probed by comparing functional group contributions to transport with those obtained for partitioning between water and various model bulk solvents ranging in polarity or hydrogen-bonding potential. A novel approach is described for obtaining group contributions to transport using ionizable permeants and pH adjustment. Using this approach, bilayer permeability coefficients of p-toluic acid and p-hydroxymethyl benzoic acid were determined to be 1.1 +/- 0.2 cm s-1 and (1.6 +/- 0.4) x 10(-3) cm s-1, respectively. From these values, the -OH group contribution to bilayer transport [delta(delta G0-OH)] was found to be 3.9 kcal/mol. This result suggests that the barrier region of the bilayer does not resemble the hydrogen-bonding environment found in octanol, but is somewhat less selective (more polar) than a hydrocarbon solvent.