A cell-based approach for the early assessment of the phospholipidogenic potential in pharmaceutical research and drug development.

Phospholipidosis is a term commonly used to indicate a phospholipid storage disorder; in affected cells, phospholipids accumulate in lysosomes that acquire a multilamellar morphological appearance. Cationic amphiphilic drugs (CADs) are suggested to induce phospholipidosis by direct interaction of xenobiotics with intracellular phospholipids or by the action of xenobiotics on the synthesis and metabolism of phospholipids. To date, electron microscopy (EM) represents the most reliable and the preferred method for the demonstration of phospholipidotic cell damage. Nevertheless, EM has a low throughput, it is expensive, and it is not suitable for screening purposes. We discuss here the assessment of the the phospholipidogenic potential of drugs using a cell culture-based model. In this test, intracellular phospholipids of treated U-937 cells (a human monocyte-derived cell line) were measured using the fluorescent probe Nile red. Eleven CADs reported to induce phospholipidosis in vivo and eight nonphospholipidogenic drugs were tested. Results obtained with the U-937 model confirmed the phospholipidogenic potential of drugs tested as described in the literature. Results have also been correlated with data obtained with a physical-chemical model (chromatographic hydrophobicity index measurement). Good correlation was obtained, confirming that the physical-chemical properties of CADs play a crucial role in the development of phospholipidosis. This work demonstrates that the U-937 model is a rapid and sensitive method for the determination of phospholipidosis-mediated cell damage. The specificity and the predictive potency observed make this method suitable for screening purposes in pharmaceutical development.

Retention of ionizable compounds on HPLC. 8. Influence of mobile-phase pH change on the chromatographic retention of acids and bases during gradient elution.

The relationships between retention and mobile-phase pH in gradient elution are studied for acids and bases. The apparent pH shift caused by the increasing amount of acetonitrile and methanol has been determined starting from a wide range of pH values. It is shown that good relationships between the retention of ionizable compounds and the pH of the aqueous buffer can be established if the same type of buffer (ammonium acetate in this work) is used for all pH points. Equations are proposed to fit the gradient retention data to the pH of the aqueous buffer. The proposed equation gives an account of the relative variation of the pKa of the compound in the reference to the variation of the pH of the buffer as both parameters change during gradient elution.

Rapid method for the estimation of octanol/water partition coefficient (log P(oct)) from gradient RP-HPLC retention and a hydrogen bond acidity term (zetaalpha(2)(H)).

We propose a rapid method for the measurement of octanol/water partition coefficients (log P(oct)) via fast gradient reversed phase retention and the calculation of the hydrogen bond acidity of the compounds. The cycle time of the generic gradient HPLC method is 5 minutes. The general solvation equation obtained for the log Poct values and the fast gradient Chromatographic Hydrophobicity Indices with acetonitrile (CHI(ACN)) and methanol

Unique selectivity of perfluorinated stationary phases with 2,2,2-trifluoroethanol as organic mobile phase modifier.

The selectivity of Luna C18 Xterra C18 and Fluophase (perfluorinated C6) stationary phases has been investigated with aqueous acetonitrile, methanol and 2,2,2-trifluoroethanol mobile phases using linear solvation equations. The gradient retention times of a set of 60 compounds with known molecular descriptors have been determined. Linear solvation equations have been set up to describe the relationship between the gradient retention times and the molecular properties. The selectivity of the stationary phase/mobile phase systems was characterised by the regression coefficients of the molecular descriptors. The perfluorinated stationary phase showed very different selectivity using 2,2,2-trifluoroethanol (TFE) as co-solvent. Compounds with H-bond donor functionality were retained much less than in the other investigated high-performance liquid chromatography (HPLC) systems. This unique selectivity can be explained by the stronger adsorption of trifluoroethanol on the perfluorinated stationary phase surface, than on the hydrocarbon surface. It suggests the importance of the adsorbed organic modifiers in the separation mechanism during reversed-phase HPLC.

Rapid-gradient HPLC method for measuring drug interactions with immobilized artificial membrane: comparison with other lipophilicity measures.

A fast-gradient high-performance liquid chromatographic (HPLC) method has been suggested to characterize the interactions of drugs with an immobilized artificial membrane (IAM). With a set of standards, the gradient retention times can be converted to Chromatographic Hydrophobicity Index values referring to IAM chromatography (CHI(IAM)) that approximates an acetonitrile concentration with which the equal distribution of compound can be achieved between the mobile phase and IAM. The CHI(IAM) values are more suitable for interlaboratory comparison and for high throughput screening of new molecular entities than the log k(IAM) values (isocratic retention factor on IAM). The fast-gradient method has been validated against the isocratic log k(IAM) values using the linear free energy relationship solvation equations based on the data from 48 compounds. The compound set was selected to provide a wide range and the least cross-correlation between the molecular descriptors in the solvation equation: (2) where SP is a solute property (e.g., logarithm of partition coefficients, reversed-phase (RP)-HPLC retention parameters, such as log k, log k(w), etc.) and the explanatory variables are solute descriptors as follows: R(2) is an excess molar refraction that can be obtained from the measured refractive index of a compound, pi(2)(H) is the solute dipolarity/polarizability, summation operatoralpha(2)(H) and summation operatorbeta(2)(0) are the solute overall or effective hydrogen-bond acidity and basicity, respectively, and V(x) is the McGowan characteristic volume (in cm(3)/100 mol) that can be calculated for any solute simply from molecular structure using a table of atomic constants. It was found that the relative constants of the solvation equation were very similar for the CHI(IAM) and for the log k(IAM). The IAM lipophilicity scale was quite similar to the octanol/water lipophilicity scale for neutral compounds. The effect of charge on the interaction with IAM was studied by varying the mobile phase pH.

Characterizing the selectivity of stationary phases and organic modifiers in reversed-phase high-performance liquid chromatographic systems by a general solvation equation using gradient elution.

Retention data for a set of 69 compounds using rapid gradient elution are obtained on a wide range of reversed-phase stationary phases and organic modifiers. The chromatographic stationary phases studied are Inertsil (IN)-ODS, pentafluorophenyl, fluoro-octyl, n-propylcyano, Polymer (PLRP-S 100), and hexylphenyl. The organic solvent modifiers are 2,2,2-trifluoroethanol (TFE); 1,1,1,3,3,3-hexafluoropropan-2-ol (HFIP); isopropanol; methanol (MeOH); acetonitrile (AcN); tetrahydrofuran; 1,4-dioxane; N,N-dimethylformamide; and mixed solvents of dimethylsulfoxide (DMSO) with AcN and DMSO with MeOH (1:1). A total of 25 chromatographic systems are analyzed using a solvation equation. In general, most of the systems give reasonable statistics. The selectivity of the reversed phase-high-performance liquid chromatographic (HPLC) systems with respect to the solute's dipolarity-polarity, hydrogen-bond acidity, and basicity are reflected in correspondingly large coefficients in the solvation equation. We wanted to find the most orthogonal HPLC systems, showing the highest possible selectivity difference in order to derive molecular descriptors using the gradient retention times of a compound. We selected eight chromatographic systems that have a large range of coefficients of interest (s, a, and b) similar to those found in water-solvent partitions used previously to derive molecular descriptors. The systems selected are IN-ODS phases with AcN, MeOH, TFE, and HFIP as mobile phase, PLRP-S 100 phase with AcN, propylcyano phase with AcN and MeOH, and fluorooctyl phase with TFE. Using the retention data obtained for a compound in the selected chromatographic systems, we can estimate the molecular descriptors with the faster and simpler gradient elution method.

Detection of the principal synthetic route indicative impurity in lamotrigine.

An analytical method has been developed for the detection of trace amounts of the principal synthetic route indicative impurity in lamotrigine (3,5-diamino-6-(2,3-dichlorophenyl)-1,2,4-triazine). A sample extract was preconcentrated by normal-phase high-performance liquid chromatography (HPLC) and analysed by subsequent on-line reversed-phase HPLC-thermospray mass spectrometry (TSP-MS). During the sample extraction and concentration step, carried out by semipreparative normal-phase chromatography, the preliminary separation of the impurity from the lamotrigine takes place. The organic solvent (dichloroethane-methanol, 90:10, v/v) is evaporated from the collected fraction and the material is redissolved in a smaller volume of the reversed-phase mobile phase. The collected fraction is then subjected to reversed-phase HPLC-TSP-MS. The influence of an ultrasonic extraction step has been examined. When the method was applied to lamotrigine tablets, a shake flask partitioning step using 1 mg/ml EDTA in water-dichloroethane was used instead of the ultrasonic extraction. Detection limit and recovery measurements showed that the route indicative impurity formed during the synthesis could be detected in the 50-100 ppb (w/w) range.

Chromatographic Hydrophobicity Index by Fast-Gradient RP-HPLC: A High-Throughput Alternative to log P/log D.

A new chromatographic hydrophobicity index (CHI) is described which can be used as part of a protocol for high-throughput (50-100 compounds/day) physicochemical property profiling for rational drug design. The index is derived from retention times (t(R)) observed in a fast gradient reversed-phase HPLC method. The isocratic retention factors (log k') were measured for a series of 76 structurally unrelated compounds by using various concentrations of acetonitrile in the mobile phase. By plotting the log k' as a function of the acetonitrile concentration, the slope (S) and the intercept (log k'(w)) values were calculated. The previously validated index of hydrophobicity φ(0) was calculated as -log k'(w)/S. A good linear correlation was obtained between the gradient retention time values, t(R) and the isocratically determined φ(0) values for the 76 compounds. The constants of this linear correlation can be used to calculate CHI. For most compounds, CHI is between 0 and 100 and in this range it approximates to the percentage (by volume) of acetonitrile required to achieve an equal distribution of compound between the mobile and the stationary phases. CHI values can be measured using acidic, neutral, or slightly basic eluents. Values corresponding to the neutral form of molecules could be measured for 52 of the compounds and showed good correlation (r = 0.851) to the calculated octanol/water partition coefficient (c log P) values.

Binding measurements of indolocarbazole derivatives to immobilised human serum albumin by high-performance liquid chromatography.

The binding properties of six indolocarbazole derivative have been measured using immobilised human serum albumin (HSA) in an HPLC column. The compounds showed very strong binding to HSA which necessitated the application of a 30 to 40% concentration of 2-propanol in the mobile phase. This represents a much higher concentration than is recommended by the column manufacturers. This HSA column had not changed its binding property when it was used again with 4% 2-propanol and 96% phosphate buffer. The binding parameters were estimated by extrapolation to 0% 2-propanol and were above 99% for each indolocarbazole derivative. The correlation analysis, including the calculated octanol/water partition coefficient (log P), pKa values as well as measured reversed-phase retention data of the compounds revealed that the extremely strong binding can be explained by the hydrophobic and acidic properties of the compounds.

Analytical and semi-preparative separation of diastereomeric lipid amino acid conjugates.

A series of biologically active peptides and their conjugates with lipidic amino acids were investigated by systematic change of the mobile phase composition using traditional octadecylsilica stationary phase and the newly developed Supelcosil LC-ABZ column. The mobile phases contained various concentrations of methanol and acetonitrile combined with 0.1% trifluoroacetic acid (TFA). Better peak shapes and higher resolution of the isomers could be observed when the mobile phase contained 0.1% TFA. More symmetrical peaks and much higher S values (slope of the log k' vs organic phase concentration plots) were obtained on the special reversed-phase column developed for anionic, basic or zwitterionic compounds. The optimum separation conditions were scaled up to a semi-preparative reversed-phase column (15 mm i.d.) to collect mg quantities of isomers for further studies.

Two-dimensional high-performance liquid chromatographic method for assaying S-adenosyl-L-methionine and its related metabolites in tissues.

S-Adenosyl-L-methionine (SAM) is a methyl-donor compound which is actively involved in a variety of biochemical reactions. An assay has been developed permitting the quantitative measurement of SAM and its related metabolites (S-adenosylhomocysteine, decarboxylated SAM, methylthioadenosine, adenosine and adenine) in liver and cell cultures. As gradient reversed-phase chromatographic or cation-exchange chromatographic methods often resulted in overlapping peaks, a two-dimensional high-performance liquid chromatographic (HPLC) procedure was developed involving gradient reversed-phase chromatographic separation followed by ion-exchange chromatography. After precipitating large molecules in the sample by perchloric acid, gel permeation was carried out on a Sephadex G 25 column to separate small water-soluble metabolites from proteins and membrane fragments. The freeze-dried sample was injected onto an ODS column and a 0-10% acetonitrile gradient in 10 mM ammonium formate buffer (pH 2.9) (20 min, linear) was applied. The relevant fractions were collected and injected onto a cation-exchange column (Partisil SCX, 10 microns, 250 mm x 4.6 mm I.D.). Elution and quantification were carried out using ammonium formate buffers of various concentration (15-400 mM), pH 2.9. The detector response (254 nm) as a function of concentration was linear over the concentration range 30-500 pmol. The detection limits of the compounds after the two-dimensional chromatographic procedure ranged from 10 to 60 pmol and the recovery was higher than 70%. The reproducibility of the results obtained from given samples was within 9-22% for rat liver and 6-24% for mast cells.

[Development of methods for the chromatographic analysis of Hevizos]. / A hevizos kromatográfiás analitikai módszereinek kifejlesztése.

Normal phase HPLC and OPTLC methods are described for the analysis of Hevizos (5-isopropyl-2'-beta-deoxyuridine) and it's impurities. The eluent is 50% water saturated ethyl-acetate to which 0.5-1.5% methanol is added depending on the silica used. The water content is important as it practically eliminates tailing. The methods can also be used for the determination of 5-isopropyl-2'-beta-deoxyuridine and it's impurities in Hevizos ointment after a simple extraction procedure. The OPTLC method uses a CHROMPRES 10 apparatus, aluminum backed HPTLC plates and the same eluent-system as the HPLC method. The reproducibility of the HPLC method (rel. st. dev.) is 0.8%, the correlation coefficient is > 0.999 for the main component, while the impurities can be determined at the 0.1% level with a reproducibility of 20%, and a correlation coefficient of > 0.997. The reproducibility of the OPTLC method (rel. st. dev.) is 4% for the main component, while for the impurities at the 0.5% level the reproducibility is 20%.

A comparative study of the reversed-phase HPLC retention behaviour of S-adenosyl-L-methionine and its related metabolites on Hypersil ODS and Supelcosil LC-ABZ stationary phases.

S-Adenosyl-L-methionine (SAM) and its metabolites S-adenosyl-L-homocysteine (SAH) and methyl-thioadenosine (MTA) are endogenous compounds that are heavily involved in a variety of biochemical processes, and have therefore been the target for several assays in body fluids and tissues. Reversed-phase chromatographic behaviour of SAM and its metabolites has been studied by using Supelcosil LC-ABZ column, specially designed for analysis of acidic, basic, zwitterionic and neutral compounds, and on a Hypersil ODS column as a function of mobile phase pH. The retentions of the compounds, expressed by the capacity ratio (k'), are measured on both column with mobile phases comprised of 10% acetonitrile and 10 mM ammonium formate buffer with pH values ranging from 2 to 9. Higher selectivity is observed on Supelcosil LC-ABZ within pH range 4-6. Different retention properties are observed at very low pH and seemed as if the Supelcosil LC-ABZ column reduced the effect of the mobile phase pH by about 1 pH unit. Whilst the Supelcosil column can be recommended for the routine analysis of SAM and its related metabolites in biological fluids by using mobile phase pH 5, the Hypersil ODS column may be suggested for use with mobile phase pH values of 3-4.

Relationships between the chromatographic retention data and the effects of nucleoside derivatives in highly metastatic 3LL cells.

The effect of 21 nucleoside derivatives on the [3H]-thymidine cellular uptake and on the incorporation into DNA of highly metastatic 3LL (Lewis lung carcinoma) cells has been measured. Hydrophobic and hydrophilic molecular parameters (the adsorption capacity, specific adsorption surface, lipophilicity and specific hydrophobic surface area) have been determined by using thin-layer chromatography. Stepwise linear regression analysis and principal component analysis have been applied in order to reveal the relationships between the molecular parameters and the effect of the nucleoside derivatives on highly metastatic 3LL cells. The first principal component obtained from the measured activity data could be attributed to the change of [3H]-thymidine cellular uptake caused by the nucleoside, while the second principal component could be regarded as the measure of the effect on the DNA incorporation of [3H]-thymidine. The effect of the nucleosides on the [3H]-thymidine uptake could be explained by the specific hydrophobic and adsorption surface area of the nucleoside, on the other hand the effect on the DNA incorporation could be described by the adsorption characteristics (specific hydrophilic surface area and adsorption capacity) of the derivatives.

Chromatographic separation and molecular modelling of triazines with respect to their inhibition of the growth of L1210/R71 cells.

The potential anti-cancer activity of triazines was characterized by the inhibition of the growth of L1210/R71 cells. The retention times for fifteen triazine derivatives were measured by high-performance liquid chromatography on octyl silica and silica gel columns. The slope and intercept values of the plot of the logarithmic capacity factor versus acetonitrile concentration were calculated from the reversed-phase retention measurements. The adsorption properties of the compounds were characterized by the retention data obtained on silica gel columns using high and low concentrations of ammonium salts in the hydro-organic mobile phase. The non-polar, non-polar unsaturated and polar surface areas, the surface energies, the dipole moments and the Van der Waals radii of the molecules were calculated from their chemical structures after energy minimization on the basis of molecular mechanics. Correlation analysis of these parameters showed that the inhibitory effect is dependent on the polar and non-polar surface areas of the molecules. The reversed-phase slope showed a significant correlation with the difference between the accessible and the total non-polar surface areas of the compounds, whereas the intercept values correlated with the non-polar accessible surface area. The adsorption properties of the triazines on silica gel cannot be described by the molecular parameters investigated here.

Relationships between nucleotide incorporation rates and molecular parameters obtained by molecular modelling and chromatography.

Deoxyuridine derivatives play an important role in pharmaceutical chemistry as they are potential antiviral and antitumour agents. Their pharmacological activity depends on their ability to incorporate into DNA in their triphosphate forms. High-performance liquid chromatographic (HPLC) retention behaviour of a series of 5-alkyl, alkenyl and alkynyl substituted deoxyuridine derivatives were investigated on reversed-phase stationary phase using various mixtures of methanol and water as mobile phases. The slope and the intercept values of the linear relationships between the logarithmic capacity ratio (log k') values and methanol concentration have been calculated. Non-polar, non-polar unsaturated and polar surface areas, surface energies, dipole moments, van der Waals radii of the derivatives have been calculated on the bases of molecular mechanics by PC Model approach. The correlation study of the above-mentioned parameters revealed that hydrophobic and hydrophilic surface areas and the electronic effects of the substituents determine not only the retention behaviour of the derivatives but also their incorporation rate into DNA in their triphosphate forms.

Relationship between the hydrophobic and hydrophilic molecular parameters of some synthetic nucleosides, determined by means of adsorptive and reversed-phase thin-layer chromatography.

The adsorption capacity, the specific adsorptive surface, the lipophilicity and the specific hydrophobic surface of 59 natural and synthetic nucleoside derivatives were determined by means of adsorptive chromatography and reversed-phase thin-layer chromatography for the future application of these molecular parameters in quantitative structure-activity relationship studies. Stepwise regression analysis and principal component analysis proved that each of the physico-chemical parameters has a different information content, and their application in the design of new bioactive derivatives is therefore justified.

Structure-activity relationships of new muscarinergic dibenzodioxazocines.

1H-NMR and X-ray conformation studies of new muscarinergic dibenzodioxazocines have been carried out. It is suggested that EGYT-2347 (2-chloro-12-/2-piperidino-ethyl/-dibenzo [d,g] [1, 3, 6] dioxazocine hydrochloride) may exist in solution in at least two distinct conformations, unlike other tricyclic or non-tricyclic compounds having antimuscarinergic activity. One of these conformations possessing an asymmetric, twisted central hetero-ring confined between two phenyl rings is probably the energetically more stable form, while the other having a butterfly-like structure, with mirror symmetry-related phenyl rings as in phenothiazines seems to be more suitable for receptor binding. The importance of the hydrophobic pocket at the receptor site was revealed by the good correlation of the calculated and measured hydrophobic parameters to the muscarinic activity of these newly synthesized and other known muscarinergic compounds.