Do Liposome‐binding Constants of Porphyrins Correlate with Their Measured and Predicted Partitioning Between Octanol and Water?¶

We tested correlations between lipophilicity parameters and the partitioning of sensitizers into membranes. For this purpose we investigated 17 porphyrins and two chlorins having various chemical structures. Some of these compounds possess an amphiphilic structure (including hematoporphyrin, deuteroporphyrin, mesoporphyrin, chlorin e6 and more). The others are very symmetrical sensitizers [meso-tetra(N-methyl-4-pyridyl)porphyrin, tetra-benzoporphyrin, coproporphyrin I dihydrochloride (CP), meso-tetra(4-carboxyphenyl)porphyrin (TCP) and meso-tetra(m-hydroxyphenyl)chlorin]. Our investigation also included two series of hematoporphyrins and protoporphyrins with varying lengths of alkylcarboxylate side groups. The partitioning of these compounds between the bulk aqueous phase and liposomes was studied by fluorescence methods, and a liposome-binding constant, Kb, was obtained. It was found that CP and TCP do not incorporate into the lipid phase at pH 7.3. An n-octanol-water partition coefficient (log P) and a distribution coefficient (log D) were predicted with a modeling software. The values of log D were also obtained experimentally. We found that for the studied molecules, the predicted log D correlated well with the measured values. The values of log D as well as log P, in turn, did not correlate nicely, for the whole group of studied compounds, with the binding constants to liposomes. However, in the case of porphyrins that share a similar structure, the Kb showed good linear correlation with both log P and log D. For the series of hematoporphyrins and protoporphyrins with different lengths of alkylcarboxyl groups, it was shown that prolongation of this group caused an increase in the lipophilicity and the liposome-binding constant. This effect is more pronounced for the proto- than for the hematoporphyrin series. The results highlight the possible use, as well as limitations, of lipophilicity parameters for the prediction of membrane binding.     

Binding of amino acids to "smart" sorbents: where does hydrophobicity come into play?

Poly(N-isopropylacrylamide) (PNIPA)-based sorbents have been successfully used as sorbents in temperature-sensitive chromatography. Yet, the mechanisms controlling the binding of biochemicals to these sorbents and, therefore, the separation process are not fully understood. In the current work, the role of hydrophobic interactions in the binding of amino acids of different hydrophobicities to PNIPA microgels was studied. Binding experiments were conducted both below (25 degrees C) and above (37 degrees C) the volume-phase transition temperature of the gel. At 25 degrees C, no straightforward correlation between the partition coefficient and the hydrophobicity could be suggested for low hydrophobicity values. Contrary, at higher hydrophobicities the partition coefficient increases with increasing hydrophobicity. This correlation holds for the whole hydrophobicity range at 37 degrees C; however, the binding data suggests two different binding mechanisms of the hydrophilic amino acids and the hydrophobic ones. Isothermal titration calorimetry measurements confirmed this suggestion: The binding of hydrophobic amino acids seems to be driven by hydrophobic interactions, as evident from the positive binding enthalpy and the clear correlation between the amino acid's hydrophobicity and the binding entropy. Contrary, the binding of the hydrophilic amino acids was exothermic, implying a binding mechanism based on specific interactions, most probably hydrogen bonding.     

Binding studies of porphyrins to human serum albumin using affinity capillary electrophoresis

The present work demonstrates that affinity capillary electrophoresis (ACE) can be employed as a valuable and powerful tool for studying the interactions between porphyrins and proteins in biological and biomedical research, such as the development of porphyrins and related compounds as efficient and selective photosensitizers in the photodynamic therapy of cancers. Binding constants of human serum albumin (HSA) to four biological porphyrins (uroporphyrin I, heptacarboxylporphyrin, coproporphyrin I, protoporphyrin IX), which possess a wide range of hydrophobicity, were estimated by ACE. Based on 1:1 molecular association between these individual porphyrins and HSA, the change of the electrophoretic mobility of HSA as a function of porphyrin concentration in the run buffer was measured and the binding constants were calculated from the slope of the Scatchard plots. The binding constant values were found to be 8.80 +/- 0.51 x 10(4) M(-1), 2.39 +/- 0.16 x 10(5) M(-1), 1.61 +/- 0.11 x 10(6) M(-1), and 9.34 +/- 0.30 x 10(6) M(-1) for uroporphyrin I, heptacarboxylporphyrin, coproporphyrin I, and protoporphyrin IX, respectively, and most of these results are in good agreement with those reported in the literature using conventional methods for binding measurements. Additionally, experimental binding constant data obtained using ACE was found to exhibit very good correlation with theoretical hydrophobicity values calculated using the Rekker's hydrophobic fragmental constant method, thus further supporting the hypothesis that the hydrophobicity of the porphyrin side chains play an important role in governing the hydrophobic interaction of porphyrins with serum proteins such as HSA.     

EVALUATION OF PORPHYRIN CHARACTERISTICS REQUIRED FOR PHOTODYNAMIC THERAPY

The cytotoxicity (in the dark), phototoxicity (red light) and subcellular localization (using confocal laser scanning microscopy) were determined for 15 porphyrins (1-15) in C6 glioma cells. The partition coefficient in 2-octanol was also determined for each porphyrin at pH 7.4. The cytotoxicity increased with pi (log of partition coefficient) up to pi values of +2. The 7 porphyrins with cationic side chains exhibited a classical parabolic correlation between phototoxicity and pi, with maximal activity at a pi value of approximately 1.0. There was also a significant correlation between subcellular localization and degree of phototoxicity, with the three most photosensitive porphyrins all possessing cationic side chains, and all three localizing in mitochondria.     

Evaluation of the hydrophobicity of perfluoroalkyl chains in amphiphilic compounds that are incorporated into cell membrane

Incubation of animal cells in the presence of amphiphilic glycosides having a lactoside residue (hydrophilic moiety) and various kinds of perfluoroalkyl chain (hydrophobic moiety) resulted to glycosylation of the saccharide chain by cellular enzymes and afforded glycolipid-like compounds. The amounts of amphiphilic glycoside and glycolipid-like compounds found in the cell and culture medium fractions, respectively, varied depending on the fluorine content of the perfluoroalkyl chain. To investigate further, the hydrophobicity of the perfluoroalkyl chain was estimated from the critical micelle concentration values and was found to be 1.5 times larger in hydrophobicity than the hydrocarbon chain [CF₂ = 1.5CH₂]. Liposomes resembling cells in size were also prepared and the amphiphilic glycosides were introduced. Results showed a positive correlation between hydrophobicity and localization of amphiphilic glycoside into liposomes. The amount of amphiphilic glycosides localized in liposomes increased with increasing hydrophobicity that is attributed mainly to the fluorine content of the aglycon. In spite of the low affinity of fluorous chain for hydrocarbon chain, the amphiphilic fluorous compounds showed high affinity for cell membrane that is composed of amphiphilic phospholipids.     

Factors influencing the distribution pattern of porphyrins in cell membranes

The mechanism of the sensitizer-membrane interactions has been studied by following the distribution properties of selected porphyrins, including haematoporphyrin (HP) and protoporphyrin (PP), into unilamellar liposomes of dipalmitoyl phosphatidylcholine (DPPC). The endomembrane distribution of HP and PP has been checked as a function of the membrane fluidity and composition by fluorescence polarization and quenching techniques. At porphyrin concentrations below 0.5 microM, HP and PP exclusively localize in the inner phospholipid monolayer; at higher concentrations, the outer monolayer also becomes populated. The porphyrin binding sites in liposomes, however, are different for HP and PP: HP preferentially distributes into water-accessible lipid regions, while PP localizes in the most hydrophobic loci of the lipid matrix. A porphyrin redistribution occurs when the fluidity properties of the liposomes are changed by addition of cholesterol or cardiolipin. In DPPC-cholesterol vesicles, all HP molecules dissolve in DPPC-rich regions while all PP molecules partition in cholesterol-rich environments. In DPPC-cardiolipin vesicles both porphyrins preferentially localize in regions accessible to the external medium. The effect of the nature of the carrier on porphyrin distribution in membranes has been studied by following the uptake and photosensitization properties of free and DPPC-incorporated PP and HP with rat liver mitochondria. The porphyrin photosensitizing efficiency has been checked by following the impairment of the respiratory function of mitochondria upon irradiation. Liposome-bound HP is less active than aqueous HP in determining membrane photodamage in mitochondria. On the contrary, aqueous PP is a very poor sensitizer as compared to a DPPC liposome-entrapped drug.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular docking using the molecular lipophilicity potential as hydrophobic descriptor: impact on GOLD docking performance

GOLD is a molecular docking software widely used in drug design. In the initial steps of docking, it creates a list of hydrophobic fitting points inside protein cavities that steer the positioning of ligand hydrophobic moieties. These points are generated based on the Lennard-Jones potential between a carbon probe and each atom of the residues delimitating the binding site. To thoroughly describe hydrophobic regions in protein pockets and properly guide ligand hydrophobic moieties toward favorable areas, an in-house tool, the MLP filter, was developed and herein applied. This strategy only retains GOLD hydrophobic fitting points that match the rigorous definition of hydrophobicity given by the molecular lipophilicity potential (MLP), a molecular interaction field that relies on an atomic fragmental system based on 1-octanol/water experimental partition coefficients (log P(oct)). MLP computations in the binding sites of crystallographic protein structures revealed that a significant number of points considered hydrophobic by GOLD were actually polar according to the MLP definition of hydrophobicity. To examine the impact of this new tool, ligand-protein complexes from the Astex Diverse Set and the PDB bind core database were redocked with and without the use of the MLP filter. Reliable docking results were obtained by using the MLP filter that increased the quality of docking in nonpolar cavities and outperformed the standard GOLD docking approach.     

Interaction of liposomes with cultured cells: possible involvement of lipid peroxidation in cell growth inhibition

Systematic analyses of the interaction between liposomes and cells were examined. Liposomes were found to affect the growth of mouse NIH 3T3 cells depending upon their size, net charge, and cholesterol content. Among the charged compounds, stearylamine was the most inhibitory and showed complete inhibition of cell growth at 100 microM. The cholesterol-rich and small unilamellar vesicles were more suppressive compared to the cholesterol-poor and multilamellar ones, respectively. The binding assay of liposomes to the cells showed a positive correlation between liposome binding and the extent of growth inhibition. Suppression of liposome uptake by inhibitors of the cytoskeletal system and energy metabolism were suggestive of an endocytotic mechanism for the cellular uptake of liposomes. The growth inhibitory effect seemed secondary to the intracellular uptake of liposomes, and peroxidation of incorporated lipids would lead to cellular damage. Therefore, it is highly recommended that potential growth inhibitory effects associated with the particular composition and other properties of liposomes should be carefully assessed in any human studies, especially for long-term use.     

Quantitative structure-retention and retention-activity relationships of beta-blocking agents by micellar liquid chromatography

Sixteen beta-blocking agents (acebutolol, alprenolol, atenolol, bisoprolol, carteolol, celiprolol, esmolol, labetalol, metoprolol, nadolol, oxprenolol, pindolol, practolol, propranolol, sotalol and timolol) showing a large range of hydrophobicity (octanol-water partition coefficients, log P between -0.026 and 2.81) were subjected to micellar liquid chromatography with sodium dodecyl sulfate as micelle forming agent, and n-propanol as organic modifier. The correlation between log P and the retention factor extrapolated to a mobile phase free of micelles and organic modifier was investigated. The use of an interpolated retention factor or the retention factor for specific individual experimental mobile phases was however advantageous since the retention factors of all beta-blocking agents were measurable in the selected mobile phases. Good correlations with log P and with in vitro biological parameters (cellular permeability coefficients in Caco-2 monolayers and apparent permeability coefficients in rat intestinal segments) were found.     

Effect of ionization and the nature of the mobile phase in quantitative structure-retention relationship studies

The octanol-water distribution constant, commonly called partition coefficient, Po/w, is a parameter often retained as a measure of the hydrophobicity of a molecule. log Po/w, for a given molecule, can be conveniently evaluated constructing correlation lines between standard retention factor logarithms (log k) in reversed-phase liquid chromatography (RPLC) and standard log Po/w values. Many compounds of pharmaceutical interest can be quite hydrophobic and have, simultaneously, basic nitrogen atoms or acidic sulfur containing groups in their structure. This renders them ionizable. The hydrophobicity of the molecular drug form (Po/w value) is completely different from its ionic form (log Po/w(+ or -) value). The actual hydrophobicity of such ionizable molecule depends on the pH. It can be represented by an apparent Papp value that takes into account the amount of compound in its molecular and ionic state combining the Po/w and Po/w(+ or -) values. In this work, log k in RPLC for ionizable as well as non-ionizable pharmaceutical compounds with different therapeutic properties (10 beta-blockers, seven tricyclic antidepressants (TA), eight steroids and 12 sulfonamides) were correlated with log Po/w. Similar correlations were done between log k and the corrected log Papp values at pH 3. Aqueous-organic mobile phases containing acetonitrile (conventional RPLC) and micellar-organic mobile phases (micellar liquid chromatography, MLC), prepared with the anionic surfactant sodium dodecyl sulfate and the organic solvents acetonitrile, propanol or pentanol, were also used to elute the compounds. All mobile phases were buffered at pH 3. Using conventional retention RPLC data, the correlation of log k with log Po/w, was satisfactory for steroids because they cannot ionize. For ionizable beta-blockers and TAs, the use of log Papp values improved the quality of the correlations, but yielded similar results for sulfonamides. In MLC, since an electrostatic interaction is added to hydrophobic forces, poorer correlations were obtained in all cases. The retention data obtained in RPLC also seems to correlate better with the biological activity of the drugs.     

ACCUMULATION OF PORPHYRINS IN CELLS: INFLUENCE OF HYDROPHOBICITY AGGREGATION AND PROTEIN BINDING

Abstract— For a variety of chemically defined, synthetic and natural porphyrins, the tendency for self aggregation, binding to serum albumin, distribution coefficient betweenoctanol–1 and water and uptake in V79 Chinese hamster cells have been determined. A strong correlation was found between cell uptake and distribution into octanol. None of the other factors could be correlated with cell uptake. These observations might have an impact on the use of porphyrins in photodynamic and boron neutron capture therapy of tumors.     

Can Cellular Phototoxicity be Accurately Predicted on the Basis of Sensitizer Photophysics?

The phototoxicity of three structurally related photosensitizers (PS), deuteroporphyrin IX (DP) and monobromo (Br-DP) and dibromo (Br2-DP) derivatives, was studied in murine L1210 leukemia cells. These compounds were chosen on the basis of heavy-atom-induced differences in triplet yield, phi T, and lifetime, tau T, and used as tools to test a model for phototoxicity based on photophysical parameters. All three porphyrins were found to localize preferentially in the plasma membrane of L1210 cells by confocal fluorescence microscopy. A poor correlation was observed between the measured photodynamic efficacies of these PS and a model using photophysical parameters determined by laser flash photolysis in homogeneous solution. However, an excellent correlation was obtained when the same parameters measured directly in the cells were used. The biological microenvironment of the porphyrins in cells induces significant changes in the photophysics of the PS. Reduction in fluorescence yield, phi F, and phi T observed for Br2-DP in cell suspensions arises from self association of the molecule due to increased hydrophobicity and high local concentrations. The photophysical model was also tested for its ability to handle variations in the oxygen dependence of cellular phototoxicity of these PS. The good correlation achieved between laser flash photolysis data determined in cells and the measured phototoxicity under air, 1.5% and 0.5% O2-saturated conditions, proves the intermediacy of singlet oxygen. This study gives further credence to the direct use of photophysical techniques to elucidate photochemical mechanisms in biological media while highlighting the potential pitfalls of using solution data to predict photosensitizing potential.     

Investigation of the chiral surfactant N-dodecoxycarbonylvaline in electrokinetic chromatography: improvements in elution range and pH stability via mixed micelles and vesicles, and the hydrophobicity determination of basic pharmaceutical drugs

The chiral surfactant dodecoxycarbonylvaline (DDCV) has proven to be an effective pseudostationary phase for the separation of many enantiomeric pharmaceutical compounds. In this study the elution range and the prediction of octanol-water partitioning for the DDCV micellar system was examined. Through incorporation of DDCV in mixed micelles and unilamellar vesicles, enhancement of the elution range was observed. The mixed micelles contained a second anionic surfactant, sodium dodecyl sulfate (SDS), while the vesicles were composed of DDCV and the cationic surfactant cetyltrimethylammonium bromide (CTAB). Enantioselectivity, as well as other chromatographic and electrophoretic parameters, were compared between the mixed micelles, vesicles, and DDCV micelles. The hydrophobicity of the DDCV system was also evaluated as a predictor of n-octanol-water partition coefficients for 15 beta amino alcohols. The correlation between the logarithm of the retention factor (log k) and log P(ow) for seven hydrophobic beta-blockers and eight beta-agonists were r2 = 0.964 and r2 = 0.814, respectively.     

Interaction of dicarboxylic metalloporphyrins with liposomes. The effect of pH on membrane binding revisited

The acid-base properties of Zn-hematoporphyrin IX (ZnHP) and Zn-mesoporphyrin IX (ZnMP) and the effect of pH on their binding to liposomes have been studied. The ionization constants for the two carboxylate groups of ZnHP were calculated by principal component analysis and are 5.7 +/- 0.1 and 6.9 +/- 0.05. The neutral species and the mono- and dianionic forms all bind to liposomes, but a strong pH effect on the binding constant was observed for both the investigated compounds. We also observed a decrease in the binding of the two anionic species when the membranes carried a negative charge. These results indicate that the porphyrins partition into the membrane with their carboxylic moieties near the lipid-water interface so that their deprotonation, leading to a charged molecule, does not prevent the insertion of the tetrapyrrole ring into the lipid environment of neutral liposomes.     

Liquid chromatographic measurement of hydrophobicity constants for N-arylsulfonylglycine aldose reductase inhibitors

The hydrophobicity constants for a series of aldose reductase inhibitors (ARIs) are determined by reversed-phase liquid chromatography. A series of reference compounds consisting of 23 barbituric acid derivatives are separated on two phenylsilica stationary phases over a range of methanol concentrations (30-80%) in 0.05 M phosphate buffer. Linear regression analysis of the measured log k' data is used to estimate the capacity factor in 100% water (log k'w) for each compound. The log k'w values are regressed against the shake-flask-measured 1-octanol-water partition coefficients, producing a correlation of 0.953. The same procedure is then used to estimate the log k'w values for a large group of ARIs and their log P values, calculated from the established relationship between log k'w and log P from the reference compounds. An initial analysis of the aldose reductase inhibitory activity of these compounds as a function of hydrophobicity alone fails to reveal a clear relationship, demonstrating the need for a multivariant approach for quantitative structure-activity analysis in this series of compounds.     

In vitro UPTAKE OF DICARBOXYLIC PORPHYRINS BY HUMAN ATHEROMA. KINETIC and ANALYTICAL STUDIES

Human atheromatous aorta segments as well as presumably disease-free control aorta were obtained at autopsy. They were incubated with solutions of various purified dicarboxylic porphyrins including hematoporphyrin (HP) and hydroxyethylvinyldeuteroporphyrin (HVD), and with solutions of Photofrin. Selective labelling of the atheroma was shown by macroscopic and microscopic observations of the characteristic porphyrin fluorescence associated with the atheromatous plaques. The time dependence of the uptake, monitored by absorption spectrophotometry or by high performance liquid chromatography, was inferred from the disappearance of the porphyrins in the incubation medium. Significant binding was observed in the absence of albumin or serum proteins. The uptake of HP was less than that of the more hydrophobic compounds HVD or Photofrin when these porphyrins were used alone. The presence of albumin or serum drastically reduces atheroma labelling. Some competition between HP and HVD for binding sites is also seen. The present results do indicate that hydrophobic porphyrins have an intrinsic affinity for atheroma and that they can be taken up through passive processes. Taking into account previous data on animal models (Photochem. Photobiol. (1989), 731-737), it appears however that, in vivo, interactions with proteins and pharmacokinetics will primarily determine plaque labelling.     

疏水分配常数用于反相液相色谱保留值的预测

在反相液相色谱保留值基本方程log k_′=a+_cC_B的基础上,描述了采用疏水分配常数及氢键作用能来预测a、c参数的方法,并系统讨论了疏水分配常数对参数a、c的影响,借此对反相液相色谱宽浓度范围内的保留值进行了预测。