Biomedical subjects

Alex Avdeef

Publications and source records attributed to Alex Avdeef.

15 recordsLinked to original sources

PubMed2006

PAMPA - excipient classification gradient map.

The effect of excipients on the artificial membrane permeability (Double-Sink PAMPA) properties of eight sparingly soluble drugs was studied. Quantities of excipient were selected to match the concentrations expected in the gastrointestinal fluid under clinically relevant conditions. Over 1,200 measurements were performed. To correct for the effects of the aqueous boundary layer and determine the intrinsic permeability, precisely measured ionization constants were used. The intrinsic permeability of weak acids was enhanced (up to 100 fold) but that of weak bases depressed (up to 270 fold) by the excipients: mefenamic acid > glybenclamide > progesterone > griseofulvin > clotrimazole > astemizole > dipyridamole > butacaine. Excipient enhancement ranked: 3 mM NaTC > 0.24% PEG400 > 0.2 M KCl > 0.24% NMP > 5% PEG400 > 0.24% PG > 1% PEG400 > 0.1M KCl > 1% PG > 1% NMP > 5% PG > 0.24% HP-beta-CD > 1% HP-beta-CD > 15 mM NaTC. The study clearly indicates that the method is suitable for use in preclinical development to assess the effect of excipients on the permeability of sparingly soluble drug candidates. The method is quick, cost-effective, and reasonably accurate. The self-rank-ordered PAMPA-Mapping may be a helpful visualization tool for delivery screening.

Cell Membrane Permeability↗

PubMed2006

PAMPA--a drug absorption in vitro model 13. Chemical selectivity due to membrane hydrogen bonding: in combo comparisons of HDM-, DOPC-, and DS-PAMPA models.

This study compares the intrinsic permeability coefficients of 40 drug molecules, obtained by three popular variants of the PAMPA assay, based on: (a) n-hexadecane, (b) 2% w/v dioleyoylphosphatidylcholine in n-dodecane, and (c) 20% w/v lecithin in n-dodecane, the HDM-, DOPC-, DS-PAMPA models, respectively. It was shown that PAMPA permeability values consistently rank in magnitude according to: DS>DOPC>HDM, with molecules like metoprolol showing 1000-fold greater permeability in DS than in HDM. Abraham descriptors were used to rationalize these observations. Water-solubilized polar molecules form very strong H-bonds with the solvent. Such molecules need to break these bonds in order to enter the pure alkane phase, which, in turn, offers no compensating H-bond solvation. Thus, more energy appears to be needed for a polar molecule to penetrate a pure alkane barrier, compared to a barrier possessing some H-bond interactions. The 20% phospholipid content of the DS-PAMPA lipid may be thought to ease the permeation process, by offering a compensating source of H-bonding within the membrane phase.

Algorithms↗

PubMed2005

Caco-2 permeability of weakly basic drugs predicted with the double-sink PAMPA pKa(flux) method.

The aim of this study was to analyze pH-dependent permeability of cationic drugs in Caco-2 cell monolayers using the pK(a)(flux) method and to correlate the results with those obtained in PAMPA (parallel artificial membrane permeability assay). The pH-dependent permeability of verapamil and propranolol was studied in Caco-2 cell monolayers. The data were subsequently processed using software developed for the PAMPA pK(a)(flux) method. Literature values for an additional nine cationic drugs were also analyzed. Double-Sink PAMPA data were also obtained for the same cationic drugs, to compare with the Caco-2 data. The Algorithm Builder program was then used to develop a predictive model of Caco-2 permeability based on PAMPA permeability and calculated Abraham molecular descriptors. From the relationship between permeability and pH it was shown that in PAMPA only the uncharged form of the drugs permeated across the membrane barrier, while charged and ionized forms of the drugs were significantly permeable in Caco-2. The charged-form permeability, P(i), was therefore determined and subsequently subtracted from all permeability coefficients in Caco-2 prior to the comparison with PAMPA. The resulting intrinsic permeability coefficients (P(o)) obtained in Caco-2 were successfully related to those derived from the PAMPA model. In this study we have shown that permeability coefficients obtained in PAMPA can predict the passive transcellular permeability in Caco-2.

Caco-2 Cells↗

PubMed2005

The rise of PAMPA.

The parallel artificial membrane permeability assay (PAMPA), as a passive-permeability screen, is a possible low-cost alternative to cellular models for the earliest ADME primary screening of research compounds. Its popularity in the industry has risen rapidly. This review examines state-of-the-art PAMPA methods. The various covered topics include: different lipid formulations, the quantitative relationships between hexadecane, dioyleyoylphosphatidycholine and Double-Sink PAMPA measurements, the use of individual-well stirring, issues of ultraviolet sensitivity, timing strategies, reproducibility of measurements, the correct pH to perform the measurement to avoid aqueous boundary layer problems, the pKa(flux) method for determining intrinsic permeability coefficients and the cosolvent method for very insoluble molecules. Examples of the determination of permeability of very difficult molecules, but molecules that are well absorbed, are given. Carefully gathered evidence in support of the use of the Double-Sink PAMPA model is presented. The review concludes with a binning strategy to predict human intestinal absorption, based on the use of the sum of permeability coefficients, measured at gradient pH 5.0, 6.2 and 7.4. Opinions regarding the future of PAMPA are offered.

Cell Membrane Permeability↗

PubMed2004

Ionization, lipophilicity, and molecular modeling to investigate permeability and other biological properties of amlodipine.

This paper uses a recent approach toward drug discovery, in which in silico tools and experimental data are combined together to study the structural features of amlodipine and their relevance in the peculiar pharmacodynamic and pharmacokinetic profiles of this long acting calcium antagonist. Results reveal for amlodipine two families of conformers (folded and extended) but also demonstrate that protonation is the predominant factor governing amlodipine intermolecular interactions among which ionic forces play a major role.

Amlodipine↗

PubMed2004

PAMPA--a drug absorption in vitro model 7. Comparing rat in situ, Caco-2, and PAMPA permeability of fluoroquinolones.

Parallel artificial membrane permeability assay (PAMPA) was used to measure the effective permeability, P(e), as a function of pH from 4 to 10, of 17 fluoroquinolones, including three congeneric series with systematically varied alkyl chain length at the 4'N-position of the piperazine residue. The permeability values spanned over three orders of magnitude. The intrinsic permeability, P(o), and the membrane permeability, P(m), were determined from the pH dependence of the effective permeability. The pK(a) values were determined potentiometrically. The PAMPA method employed stirring, adjusted such that the unstirred water layer (UWL) thickness matched the 30-100 microm range estimated to be in the human small intestine. The intrinsic permeability coefficients (10(-6)cm/s), representing the permeability of the uncharged form of the drug, are for 4'N-R-norfloxacin: 0.7 (R=H), 49 (Me), 132 (n-Pr), 365 (n-Bu); 4'N-R-ciprofloxacin: 2.7 (H), 37 (Me), 137 (n-Pr), 302 (n-Bu); 4'N-R-3'-methylciprofloxacin: 3.8 (H), 20 (Me), 51 (Et), 160 (n-Pr), 418 (n-Bu). Increasing the alkyl chain length in the congeneric series resulted in increased permeability, averaging about 0.34 log units per methylene group, except that of the first (H-to-Me), which was about 1.2 log units. These results were compared to Caco-2 and rat in situ permeability measurements. The in situ closed loop technique used for obtaining permeability values in rat showed a water layer thickness effect quite consistent with in vivo expectations. The rat-PAMPA correlation (r2=0.87) was better than that of rat-Caco-2 (r2=0.63). Caco-2-PAMPA correlation indicated r2=0.66. The latter correlation improved significantly (r2=0.82) when the Caco-2 data were corrected for the UWL effect.

Animals↗

PubMed2004

PAMPA--a drug absorption in vitro model 8. Apparent filter porosity and the unstirred water layer.

In the parallel artificial membrane permeability assay (PAMPA), if more lipid is used than needed to fill all the pores of a microfilter, the excess lipid layer on both sides of the lipophilic filter increases the "apparent" porosity, epsilon(a), of the filter. The specific resistance of the artificial membrane barrier is lowered with increasing lipid excess. If this effect is not recognized, and the uncorrected value of filter porosity, epsilon, is used, then the calculated intrinsic permeability and the unstirred water layer (UWL) permeability coefficient of the permeating molecule can be significantly overestimated, resulting in underestimates of the thickness of the unstirred water layer. Unstirred water layer corrections are important in pharmaceutical research for in vitro-in vivo correlations aimed at predicting oral absorption and blood-brain barrier penetration characteristics of lead candidate compounds. The novel concept of the apparent porosity is introduced, described, and its utility demonstrated with the drugs diclofenac, desipramine, caffeine, and piroxicam. The PAMPA data of Wohnsland and Faller [J. Med. Chem. 44 (2001) 923] is taken as an example, where the reported extraordinarily efficient stirring is thought to be better explained in terms of normal stirring when apparent porosity is taken into account in the calculation of the effective permeability coefficient.

Adsorption↗

PubMed2004

PAMPA--a drug absorption in vitro model 11. Matching the in vivo unstirred water layer thickness by individual-well stirring in microtitre plates.

Many plate-based in vitro assays of membrane permeability (e.g., Caco-2, MDCK, PAMPA) of sparingly soluble candidate molecules report permeability of water, and not of the intended membrane barrier. This is so because the unstirred water layer on both sides of the membrane barrier is rate limiting for these highly permeable molecules. The thickness of this water layer can be 1500-4000 microm in unstirred assays. Under in vivo conditions, however, the unstirred water layer is believed to be 30-100 microm thick. Lightly stirred in vitro assays, using plate shakers, cannot lower the thickness of the water layer to match that found in vivo. In this study, 55 lipophilic drugs were employed to characterize the effect of stirring in parallel artificial membrane permeability assay (PAMPA). Highly efficient individual-well magnetic stirring at speeds greater than 110 rpm has been demonstrated to lower the unstirred water layer thickness to the in vivo range. Stirring at 622 rpm has lowered the layer thickness to 13 microm in some cases, which had not been previously achieved for plate-based permeability assays. With diminished water layer contribution at 622 rpm, for example, the effective permeability of progesterone is 2754 x 10(-6) cm/s. The new stirring apparatus used in this study is not only suitable for PAMPA, but can also be used in Caco-2 assays. Because of the diminished resistance of the thinner water layer, the stirred PAMPA permeation time has decreased from the usual 15 h to about 15 min for lipophilic compounds.

Absorption↗

PubMed2004

Acid-base cosolvent method for determining aqueous permeability of amiodarone, itraconazole, tamoxifen, terfenadine and other very insoluble molecules.

A high-throughput, UV-detection PAMPA (parallel artificial membrane permeability assay) cosolvent procedure is described, based on the use of 20% v/v acetonitrile in aqueous buffer. A training set of 32 drugs (17 bases, 13 acids, 2 ampholytes) was studied both in aqueous buffer and in cosolvent-buffer solutions. A procedure was devised, where intrinsic permeability values, log P(o)(COS), measured in cosolvent solution, are converted to values expected under cosolvent-free conditions, using an in silico model based on Abraham H-bond acidity (alpha) and basicity (beta) descriptors, developed with the Algorithm Builder computer program, to obtain aqueous intrinsic permeability values: log P(o)=0.738+0.885 log P(o)(COS)-1.262alpha+0.436beta, r(2)=0.97, q(2)=0.96, s=0.38, n=32, F=279. Five sparingly-soluble weak bases (solubility <1 microg/ml), which could not be characterized without cosolvent, had their aqueous intrinsic permeability, P(o), estimated: miconazole 0.32 cm/s; itraconazole 3.2 cm/s; amiodarone 13 cm/s; tamoxifen 28 cm/s; terfenadine 162 cm/s.

Acids↗

PubMed2003

In vitro trans-monolayer permeability calculations: often forgotten assumptions.

In designing effective therapeutic strategies, novel drugs must exhibit favorable pharmacokinetic properties. The physicochemical characteristics of a drug, such as pK(a), molecular weight, solubility and lipophilicity, will influence the way the drug partitions from the aqueous phase into membranes, and thus, will influence its ability to cross cellular barriers, such as the lining of the gastrointestinal tract and the blood-brain barrier. Physicochemical characteristics also influence the degree to which a drug is able to cross a barrier layer, and the route by which it does this; whether transcellular (across the cells)-by diffusion, carrier-mediated transport or transcytosis-or paracellular-by diffusing through the tight junctions between the cells. The in vitro model systems that are currently employed to screen the permeation characteristics of a drug often represent a compromise between high throughput with low predictive potential and low throughput with high predictive potential. Here, we will examine the way in which in vitro cellular permeability assays are often performed and the assumptions that are implied but sometimes forgotten, and we will make simple suggestions for improving the methodological techniques and mathematical equations used to determine drug permeability.

Cell Membrane Permeability↗

PubMed2003

Absorption classification of oral drugs based on molecular surface properties.

The aim of this study was to investigate whether easily calculated and comprehended molecular surface properties can predict drug solubility and permeability with sufficient accuracy to allow theoretical absorption classification of drug molecules. For this purpose, structurally diverse, orally administered model drugs were selected from the World Health Organization (WHO)'s list of essential drugs. The solubility and permeability of the drugs were determined using well-established in vitro methods in highly accurate experimental settings. Descriptors for molecular surface area were generated from low-energy conformations obtained by conformational analysis using molecular mechanics calculations. Correlations between the calculated molecular surface area descriptors, on one hand, and solubility and permeability, on the other, were established with multivariate data analysis (partial least squares projection to latent structures (PLS)) using training and test sets. The obtained models were challenged with external test sets. Both solubility and permeability of the druglike molecules could be predicted with high accuracy from the calculated molecular surface properties alone. The established correlations were used to perform a theoretical biopharmaceutical classification of the WHO-listed drugs into six classes, resulting in a correct prediction for 87% of the essential drugs. An external test set consisting of Food and Drug Administration (FDA) standard compounds for biopharmaceutical classification was predicted with 77% accuracy. We conclude that PLS models of easily comprehended molecular surface properties can be used to rapidly provide absorption profiles of druglike molecules early on in drug discovery.

Administration, Oral↗

PubMed2003

Classification structure-activity relations (C-SAR) in prediction of human intestinal absorption.

AB/HIA is a "soft" filter for identifying compounds with poor intestinal membrane permeability. The analyzed data set included over 1000 drug-like compounds with experimental human intestinal absorption (HIA) values. A sequence of recursive partitioning analyses based on multiple physicochemical and structural descriptors led to the derivation of the rule-based algorithm (filter). The obtained rules reveal a simple physicochemical model of intestinal permeability; they also account for the specific effects caused by quarternary nitrogens and biphosphonate groups. Comparison of the observed and predicted values revealed very low percent of disagreement (15% false-positives and 3% false-negatives). The unusual absorption of compounds that deviated from the predicted values was explained in terms of active transport, efflux, chemical stability, chelating ability, and solubility. Most of these effects can be accounted for by new, substructure-specific rules that can be added into the existing filter. This can lead to the development of a reliable theoretical model for predicting human intestinal absorption. If combined with other models for predicting first pass metabolism, the updated AB/HIA filter can be very useful in predicting oral bioavailability.

Humans↗

PubMed2003

PAMPA--a drug absorption in vitro model. 5. Unstirred water layer in iso-pH mapping assays and pKa(flux)--optimized design (pOD-PAMPA).

Iso-pH mapping unstirred parallel artificial membrane permeability assay (PAMPA) was used to measure the effective permeability, P(e), as a function of pH from 3 to 10, of five weak monoprotic acids (ibuprofen, naproxen, ketoprofen, salicylic acid, benzoic acid), an ampholyte (piroxicam), five monoprotic weak bases (imipramine, verapamil, propranolol, phenazopyridine, metoprolol), and a diprotic weak base (quinine). The intrinsic permeability, P(o), the unstirred water layer (UWL) permeability, P(u), and the apparent pK(a) (pK(a)(flux)) were determined from the pH dependence of logP(e). The underlying permeability-pH equations were derived for multiprotic weak acids, weak bases and ampholytes. The average thickness of the unstirred water layer on each side of the membrane was estimated to be nearly 2000 microm, somewhat larger than that found in Caco-2 permeability assays (unstirred). Since the UWL thickness in the human intestine is believed to be about forty times smaller, it is critical to correct the in vitro permeability data for the effect of the UWL. Without such correction, the in vitro permeability coefficient of lipophilic molecules would be indicative only of the property of water. In single-pH PAMPA (e.g. pH 7.4), the uncertainty of the UWL contribution can be minimized if a specially-selected pH (possibly different from 7.4) were used in the assay. From the analysis of the shapes of the log P(e)-pH plots, a method to improve the selection of the assay pH, called pK(a)(flux)-optimized design (pOD-PAMPA), was described and tested. From an optimally-selected assay pH, it is possible to estimate P(o), as well as the entire membrane permeability-pH profile.

Algorithms↗

PubMed2003

In vitro permeability of poorly aqueous soluble compounds using different solubilizers in the PAMPA assay with liquid chromatography/mass spectrometry detection.

PURPOSE: This study compares the use of UV-VIS detection with liquid chromatography/mass spectrometry (LC/MS) detection for the PAMPA (Parallel Artificial Membrane Permeability Assay) permeability determination of compounds in the drug discovery stage. LC/MS detection offers a selective and sensitive method for the determination of the PAMPA permeability for compounds that do not contain a UV chromophore or possess a low UV extinction coefficient. To enhance the reliability of our permeability measurements for compounds with low aqueous solubility, we demonstrated the use of LC/MS detection as a means for facilitating the study of solubilizing agents to enhance aqueous solubility that normally would interfere with UV-VIS detection. In doing so, the PAMPA assay can be expanded to study the in vitro permeability of poorly water soluble compounds and evaluate the effects of solubilizers' on the membrane permeability of different compounds. This might be useful in selecting solubilizers for poorly water soluble compounds to be used for further in vivo studies. METHODS: A diverse set of 20 drugs using UV-VIS detection were compared with data using LC/MS detection. A PAMPA screening method was designed which used solubilizers (Brij 35, Cremophor EL, ethanol, and Tween 80) for compounds with low aqueous solubility. The stability of the artificial membrane was determined using various solubilizer concentrations (0.1-5% w/v) to ensure the phospholipid membrane was not disrupted. Two compounds, amiodarone and miconazole, with low aqueous solubility yielding an undetected response in the PAMPA assay using UV-VIS detection were subjected to the different solubilizing agents and their PAMPA permeability was measured using LC/MS detection. RESULTS: Most of the compounds showed similar PAMPA permeability using the two detection systems. However, for compounds lacking a UV chromophore or with a low UV extinction coefficient, LC/MS was the detection method of choice for determination of PAMPA permeability values. LC/MS also gave reliable quantification data for compounds containing impurities, as well as compounds that were not stable during the assay. Although many solubilizers were found to interfere with UV-VIS detection, the LC/MS approach was applicable to determine the permeability values of compounds with normally low aqueous solubility. CONCLUSIONS: LC/MS detection offered greater sensitivity and selectivity as compared with UV-VIS detection for the PAMPA assay. With this added versatility in detection, PAMPA can be used in both discovery and pre-formulation applications, which has not been described before.

Gas Chromatography-Mass Spectrometry↗

PubMed2003

Classification analysis of P-glycoprotein substrate specificity.

Prediction of P-glycoprotein substrate specificity (S(PGP)) can be viewed as a constituent part of a compound's "pharmaceutical profiling" in drug design. This task is difficult to achieve due to several factors that raised many contradictory opinions: (i) the disparity between the S(PGP) values obtained in different assays, (ii) the confusion between Pgp substrates and inhibitors, (iii) the confusion between lipophilicity and amphiphilicity of Pgp substrates, and (iv) the dilemma of describing class-specific relationships when Pgp has no binding sites of high ligand specificity. In this work, we compiled S(PGP) data for 1000 compounds. All data were represented in a binary format, assigning S(PGP) = 1 for substrates and S(PGP) = 0 for non-substrates. Each value was ranked according to the reliability of experimental assay. Two data sets were considered. Set 1 included 220 compounds with S(PGP) from polarized transport across MDR1 transfected cell monolayers. Set 2 included the entire list of 1000 compounds, with S(PGP) values of generally lower reliability. Both sets were analysed using a stepwise classification structure-activity relationship (C-SAR) method, leading to derivation of simple rules for crude estimation of S(PGP) values. The obtained rules are based on the following factors: (i) compound's size expressed through molar weight or volume, (ii) H-accepting given by the Abraham's beta (that can be crudely approximated by the sum of O and N atoms), and (iii) ionization given by the acid and base pKa values. Very roughly, S(PGP) can be estimated by the "rule of fours". Compounds with (N + O) > or = 8, MW > 400 and acid pKa > 4 are likely to be Pgp substrates, whereas compounds with (N + O) < or = 4, MW < 400 and base pKa < 8 are likely to be non-substrates. The obtained results support the view that Pgp functioning can be compared to a complex "mini-pharmacokinetic" system with fuzzy specificity. This system can be described by a probabilistic version of Abraham's solvation equation, suggesting a certain similarity between Pgp transport and chromatographic retention. The chromatographic model does not work in the case of "marginal" compounds with properties close to the "global" physicochemical cut-offs. In the latter case various class-specific rules must be considered. These can be associated with the "amphiphilicity" and "biological similarity" of compounds. The definition of class-specific effects entails construction of the knowledge base that can be very useful in ADME profiling of new drugs.

ATP Binding Cassette Transporter, Subfamily B, Mem↗