Teresa M. Garrigues

Skin-PAMPA: a new method for fast prediction of skin penetration.

The goal of this study was to develop a quick, reliable, and cost-effective permeability model for predicting transdermal penetration of compounds. The Parallel Artificial Membrane Permeability Assay (PAMPA) was chosen for this purpose, as it already has been successfully used for estimating passive gastrointestinal absorption and blood-brain barrier permeability. To match the permeability of the rate-limiting barrier in human skin, synthetic certramides, which are analogs of the ceramides present in the stratum corneum, were selected for the skin-PAMPA model. The final skin-PAMPA membrane lipid mixture (certramide, free fatty acid, and cholesterol) was selected and optimized based on data from three different human skin databases and the final model was found to correlate well to all of the databases. The reproducibility of the skin-PAMPA model was investigated and compared to that of other PAMPA models. The homogeneity of the filter-impregnated lipid mixture membrane was confirmed with Raman microscopy. It was shown that skin-PAMPA is a quick and cost-effective research tool that can serve as a useful model of skin penetration in pharmaceutical and cosmetic research.

Provisional Classification and in Silico Study of Biopharmaceutical System Based on Caco-2 Cell Permeability and Dose Number

Today, early characterization of drug properties by the Biopharmaceutics Classification System (BCS) has attracted significant attention in pharmaceutical discovery and development. In this direction, the present report provides a systematic study of the development of a BCS-based provisional classification (PBC) for a set of 322 oral drugs. This classification, based on the revised aqueous solubility and the apparent permeability across Caco-2 cell monolayers, displays a high correlation (overall 76%) with the provisional BCS classification published by World Health Organization (WHO). Current database contains 91 (28.3%) PBC class I drugs, 76 (23.6%) class II drugs, 97 (31.1%) class III drugs, and 58 (18.0%) class IV drugs. Other approaches for provisional classification of drugs have been surveyed. The use of a calculated polar surface area with a labetalol value as a high permeable cutoff limit and aqueous solubility higher than 0.1 mg/mL could be used as alternative criteria for provisionally classifying BCS permeability and solubility in early drug discovery. To develop QSPR models that allow screening PBC and BCS classes of new molecular entities (NMEs), 18 statistical linear and nonlinear models have been constructed based on 803 0-2D Dragon and 126 Volsurf+ molecular descriptors to classify the PBC solubility and permeability. The voting consensus model of solubility (VoteS) showed a high accuracy of 88.7% in training and 92.3% in the test set. Likewise, for the permeability model (VoteP), accuracy was 85.3% in training and 96.9% in the test set. A combination of VoteS and VoteP appropriately predicts the PBC class of drugs (overall 73% with class I precision of 77.2%). This consensus system predicts an external set of 57 WHO BCS classified drugs with 87.5% of accuracy. Interestingly, computational assignments of the PBC class reasonably correspond to the Biopharmaceutics Drug Disposition Classification System (BDDCS) allocations of drugs (accuracy of 63.3-69.8%). A screening assay has been simulated using a large data set of compounds in different drug development phases (1, 2, 3, and launched) and NMEs. Distributions of PBC forecasts illustrate the current status in drug discovery and development. It is anticipated that a combination of the QSPR approach and well-validated in vitro experimentations could offer the best estimation of BCS for NMEs in the early stages of drug discovery.

In Silico Prediction of Caco-2 Cell Permeability by a Classification QSAR Approach.

In the present study, 21 validated QSAR models that discriminate compounds with high Caco‐2 permeability (Papp≥8×10−6 cm/s) from those with moderate‐poor permeability (Papp<8×10−6 cm/s) were developed on a novel large dataset of 674 compounds. 20 DRAGON descriptor families were used. The global accuracies of obtained models were ranking between 78–82 %. A general model combining all types of molecular descriptors was developed and it classified correctly 81.56 % and 83.94 % for training and test sets, respectively. An external set of 10 compounds was predicted and 80 % was correctly assessed by in vitro Caco‐2 assays. The potential use of the final model was evaluated by a virtual screening of a human intestinal absorption database of 269 compounds. The model predicted 121 compounds with high Caco‐2 permeability and the 90 % of them had high values of human intestinal absorption (HIA≥80). This study provides the most comprehensive database of Caco‐2 permeability and evidenced the utility of the combined methodology (in silico+in vitro) in the prediction of Caco‐2 permeability. It suggests that the present methodology can be used in the design of large libraries of compounds with appropriate values of permeability and to perform virtual screening in the early stages of drug development.

Transintestinal secretion of ciprofloxacin, grepafloxacin and sparfloxacin: in vitro and in situ inhibition studies.

The influence of the secretion process on the absorption of ciprofloxacin, grepafloxacin and sparfloxacin has been evaluated by means of inhibition studies. Two well known P-glycoprotein inhibitors (cyclosporine, verapamil), a mixed inhibitor of P-glycoprotein and the organic cation transporter OCT1 (quinidine) and a well established MRP substrate (p-aminohipuric acid) have been selected in order to distinguish the possible carriers implicated. An in situ rat gut perfusion model and CACO-2 permeability studies are used. Both methods suggest the involvement of several types of efflux transporters for every fluoroquinolone. The relevance of the secretory pathway depends on the intrinsic permeability of the quinolone. The in vitro model seems to be more suitable for discriminating mechanisms underlying the absorption process, while in situ studies are less sensitive to inhibition studies.

Kinetic modelling of passive transport and active efflux of a fluoroquinolone across Caco-2 cells using a compartmental approach in NONMEM

The purpose was to develop a general mathematical model for estimating passive permeability and efflux transport parameters from in vitro cell culture experiments. The procedure is applicable for linear and non-linear transport of drug with time, <10 or >10% of drug transport, negligible or relevant back flow, and would allow the adequate correction in the case of relevant mass balance problems. A compartmental kinetic approach was used and the transport barriers were described quantitatively in terms of apical and basolateral clearances. The method can be applied when sink conditions are not achieved and it allows the evaluation of the location of the transporter and its binding site. In this work it was possible to demonstrate, from a functional point of view, the higher efflux capacity of the TC7 clone and to identify the apical membrane as the main resistance for the xenobiotic transport. This methodology can be extremely useful as a complementary tool for molecular biology approaches in order to establish meaningful hypotheses about transport mechanisms.

The Use of Rule-Based and QSPR Approaches in ADME Profiling: A Case Study on Caco-2 Permeability

During the early ADME profiling the development of simple, interpretable and reliable in silico tools is very important. In this study, rule‐based and QSPR approaches were investigated using a large Caco‐2 permeability database. Three permeability classes were determined: high (H), moderate (M) and low (L). The main physicochemical properties related with permeability were ranked as follows: Polar Surface Area (PSA)>Lipophilicity (logP/logD)>Molecular Weight (MW)>number of Hydrogen Bond donors and acceptors>Ionization State>number of Rotatable Bonds>number of Rings. The best rule, based on the combination of PSA‐MW‐logD (3PRule), was able to identify the H, M and L classes with accuracy of 72.2, 72.9 and 70.6 %, respectively. Subsequently, a consensus system based on three voting binary classification trees was constructed. It accurately predicted 78.4/76.1/79.1 % of H/M/L compounds on training and 78.6/71.1/77.6 % on test set. Finally, the 3PRule and multiclassifier were validated with 23 drugs in a Caco‐2 assay. The rule is very useful to improve assay design and prioritize the high absorption candidates. Meanwhile the QSPR model exhibits appropriate classification performance. Due to the simplicity, easy interpretation and accuracy, the 3PRule and consensus model developed here can be used in early ADME profiling.

Quantification of nortriptyline in plasma by HPLC and fluorescence detection.

A simple, sensitive and specific high-performance liquid chromatography method has been developed for the determination of nortriptyline (NT) in plasma samples. The assay involved derivatization with 9H-fluoren-9-ylmethyl chloroformate (Fmoc-Cl) and isocratic reversed-phase (C(18)) chromatography with fluorescence detection. The developed method required only 100 microl of plasma sample, deproteinized and derivatized in one step. Calibration curves were lineal over the concentration range of 5-5000 ng/ml. The derivatization reaction was performed at room temperature in 20 min and the obtained NT derivative was stable for at least 48 h at room temperature. The within-day and between-day relative standard deviation was below 8%. The limit of detection (LOD) was 2 ng/ml, and the lower limit of quantification (LLOQ) was established at 10 ng/ml. The method was applied on plasma collected from rats, at different time intervals, after intravenous administration of 0.5 mg of NT.

Pharmacokinetics, bioavailability and absorption of flumequine in the rat

The study demonstrates that the oral extent of bioavailability of flumequine in the rat, relative to the intravenous injection, is complete (0.94 +/- 0.04) and not significantly different from that found by the intraduodenal route (0.95 +/- 0.04). The rate of oral bioavailability, however, is slow (ka = 1.20 +/- 0.07 h-1; Tmax = 2.0 h), but enough to maintain plasma levels above the minimal inhibitory concentration of the most common pathogens for an extended period of time (about 10 h). The reason for the oral absorption slowness could be a slow gastric emptying, an adsorption to the gastric mucosae, a precipitation in the gastric medium or any other feature concerning the stomach as the intraduodenal administration is very quick (kid = 38.1 +/- 4.7 h-1; Tmax = 0.05 h). A possible precipitation of flumequine cannot be discarded as the solubility of flumequine is very low in the pH range of 3 to 6 (mean pH values for rat stomach and rat intestine, respectively; T.T. Kararli, Biopharm. Drug Dispos. 16 (1995) 351-380). Flumequine was shown to be not substantially excreted in bile (2-3% of the dose). Surprisingly, plasma levels and AUC values found for animals with interrupted bile flow always surpass those found for animals with enterohepatic circulation. This could be due to experimental model features, which might bias plasmatic flumequine concentrations if the homeostatic equilibrium of the animal is not completely restored due to the volume reduction induced by biliary extraction.

Polymeric microcontainers improve oral bioavailability of furosemide.

Microcontainers with an inner diameter of 223 μm are fabricated using the polymer SU-8, and evaluated in vitro, in situ and in vivo for their application as an advanced oral drug delivery system for the poorly water soluble drug furosemide. An amorphous sodium salt of furosemide (ASSF) is filled into the microcontainers followed by applying a lid using Eudragit L100. It is possible to control the drug release in vitro, and in vitro absorption studies show that the microcontainers are not a hindrance for absorption of ASSF. In situ perfusion studies in rats are performed with ASSF-filled microcontainers coated with Eudragit and compared to a furosemide solution. The absorption rate constant of ASSF confined in microcontainers is found to be significantly different from the solution, and by light microscopy, it is observed that the microcontainers are engulfed by the intestinal mucus. An oral bioavailability study in rats is performed with ASSF confined in microcontainers coated with Eudragit and a control group with ASSF in Eudragit-coated capsules. A relative bioavailability of 220% for the ASSF in microcontainers compared to ASSF in capsules is found. These studies indicate that the microcontainers could serve as a promising oral drug delivery system.

Harmonization of QSAR Best Practices and Molecular Docking Provides an Efficient Virtual Screening Tool for Discovering New G-Quadruplex Ligands.

Telomeres and telomerase are key players in tumorogenesis. Among the various strategies proposed for telomerase inhibition or telomere uncapping, the stabilization of telomeric G-quadruplex (G4) structures is a very promising one. Additionally, G4 stabilizing ligands also act over tumors mediated by the alternative elongation of telomeres. Accordingly, the discovery of novel compounds able to act on telomeres and/or inhibit the telomerase enzyme by stabilizing DNA telomeric G4 structures as well as the development of approaches efficiently prioritizing such compounds constitute active areas of research in computational medicinal chemistry and anticancer drug discovery. In this direction, we applied a virtual screening strategy based on the rigorous application of QSAR best practices and its harmonized integration with structure-based methods. More than 600,000 compounds from commercial databases were screened, the first 99 compounds were prioritized, and 21 commercially available and structurally diverse candidates were purchased and submitted to experimental assays. Such strategy proved to be highly efficient in the prioritization of G4 stabilizer hits, with a hit rate of 23.5%. The best G4 stabilizer hit found exhibited a shift in melting temperature from FRET assay of +7.3 °C at 5 μM, while three other candidates also exhibited a promising stabilizing profile. The two most promising candidates also exhibited a good telomerase inhibitory ability and a mild inhibition of HeLa cells growth. None of these candidates showed antiproliferative effects in normal fibroblasts. Finally, the proposed virtual screening strategy proved to be a practical and reliable tool for the discovery of novel G4 ligands which can be used as starting points of further optimization campaigns.