Mario A. Quevedo

P-glycoprotein limits the absorption of the anti-HIV drug zidovudine through rat intestinal segments.

Zidovudine (AZT) was the first drug approved for the treatment of Acquired Immunodeficiency Syndrome (AIDS) in humans, and although its clinical efficacy has been demonstrated, suboptimal pharmacokinetic aspects still remain a concern. To assess the basis of its highly variable oral bioavailability, this work deals with the study of AZT intestinal absorption by applying the gut sac technique. Permeation through the rat jejunum and ileum segments was analyzed at different drug concentrations and gut regions, with higher apparent permeability coefficients (P(app)) being found for the proximal regions of the small intestine compared to distal ones. Bi-directional permeation assays demonstrated that AZT is subjected to efflux mechanisms in distal regions of small intestine, which are blocked by verapamil (VER), thus demonstrating a P-glycoprotein (P-gp) mediated mechanism. The efficiency of AZT efflux increased in the distal ileum as consequence of exposure to AZT, with the amount of drug permeating from the mucosal to the serosal side diminishing after 35 min. Molecular modeling techniques were applied to analyze the binding mode of AZT to P-gp, which was compared to that of VER and AZT-Ac, a novel prodrug of AZT. The energy required for their solvation was found to constitute a critical feature in their binding to this efflux protein. The present work updates the impact of P-gp in AZT oral bioavailability, highlighting the need for further study of the dynamic nature of its expression at intestinal level.

Triethanolamine stabilization of methotrexate-β-cyclodextrin interactions in ternary complexes.

The interaction of methotrexate (MTX) with beta-cyclodextrin (β-CD) in the presence of triethanolamine (TEA) was investigated with the aim to elucidate the mechanism whereby self-assembly cyclodextrin systems work in association with this third component. Solubility diagram studies showed synergic increment of the MTX solubility to be about thirty-fold. Experiments using 2D ROESY and molecular modeling studies revealed the inclusion of aromatic ring III of the drug into β-CD cavity, in which TEA contributes by intensifying MTX interaction with β-CD and stabilizes MTX:β-CD:TEA ternary complex by electrostatic interaction. The maintenance of these interactions in solid phase was also studied in ternary MTX:β-CD:TEA and comparisons were made with freeze dried binary MTX:β-CD and physical mixtures. FTIR studies evidenced that MTX–β-CD interaction remained in solid ternary complexes, which was also supported by thermal (differential scanning calorimetry (DSC), thermogravimetric analysis (TG)/first derivative of TG analysis (DTG) and C,N,H elementary analysis) and structural (X-ray diffraction analysis, (XRD)) studies, mainly regarding the increment of drug stability. The efficient in vitro drug dissolution studies successfully demonstrated the contribution of ternary complexes, which highlights the importance of this possible new raw material for further applications in drug delivery systems.

Complexation of Sulfonamides With β-Cyclodextrin Studied by Experimental and Theoretical Methods

The complex formation between three structurally related sulfonamides (sulfadiazine (SDZ), sulfamerazine (SMR), and sulfamethazine (SMT)) and beta-cyclodextrin (beta-CD) was studied, by exploring its structure affinity relationship. In all the cases, 1:1 stoichiometries were determined with different relative affinities found by phase solubility (SDZ:beta-CD > SMR:beta-CD > SMT:beta-CD) and nuclear magnetic resonance (NMR) (SMT:beta-CD > SMR:beta-CD > SDZ:beta-CD) studies. The spatial configurations determined by NMR were in agreement with those obtained by molecular modeling, showing that SDZ included its aniline ring into beta-CD, while SMR and SMT included the substituted pyrimidine ring. Energetic analyses demonstrated that hydrophobicity is the main driving force to complex formation.

Binding to human serum albumin of zidovudine (AZT) and novel AZT derivatives. Experimental and theoretical analyses

This work presents the binding of AZT and nine novel AZT derivatives to human serum albumin (HSA), both defatted (HSA(D)) and complexed with fatty acids (HSA(FA)). The bound fractions and binding site were determined by applying an ultrafiltration procedure, with an increased affinity for the majority of these derivatives to HSA(D) being found with respect to that of AZT, while only one derivative exhibited an increased affinity for HSA(FA). By means of computational methods, we observed that specific electrostatic interactions are responsible for the increased affinity for HSA(D), while the presence of fatty acids complexed to HSA caused an intense electrostatic repulsion with negatively charged ligands located in Sudlow site I, thus diminishing their bound fractions. A strong relationship between the calculated energetic components and the observed experimental affinity was identified.

Specific binding capacity of β-cyclodextrin with cis and trans enalapril: Physicochemical characterization and structural studies by molecular modeling

The main objective of this work was to study an inclusion complex between enalapril (ENA), and beta-cyclodextrin (beta-CD). From nuclear magnetic resonance (NMR) we determined that the complex showed a 1:1 stoichiometry, with an apparent formation constant (K(C)) of 439 and 290 M(-1) for the cis and trans isomers, respectively. The molecular modeling and NMR techniques demonstrated that the aromatic moiety of ENA was inserted into the hydrophobic cavity of beta-CD. When studying the chemical stability of ENA complexed to beta-CD, a clear stabilizing effect was observed in both the aqueous solution and solid state.

Increasing Doxycycline Hyclate Photostability by Complexation with β-Cyclodextrin

Doxycycline hyclate (DOX) is a highly photosensitive drug, a feature that limits the stability of the corresponding dosage forms. The main objectives of this work were the preparation and characterization of an inclusion complex of DOX with β-cyclodextrin (βCD) and to investigate if this approach could improve the photostability of the drug. Guest-host interactions were investigated using nuclear magnetic resonance, which were afterwards combined with molecular modeling methods to study the complex formation and its three-dimensional structure was proposed. A freeze-drying method was applied to obtain the complex in the solid state, which was further confirmed by thermal and spectroscopic techniques. To evaluate the complexation effect on DOX integrity, the photostability of the inclusion complex was studied, with a significant decrease in the photodegradation of DOX being found in aqueous solution upon complexation. Finally, the photoprotection produced by the complexation was evaluated by means of an antimicrobial assay. Overall, the presented results suggest that the formulation of DOX complexed with βCD constitutes an interesting approach for the preparation of pharmaceutical dosage forms of DOX with enhanced stability properties.

Characterization of the hydrochlorothiazide: β-cyclodextrin inclusion complex. Experimental and theoretical methods.

Hydrochlorothiazide (HCT) is one of the most commonly prescribed antihypertensive drugs. In an attempt to gain an insight into the physicochemical and molecular aspects controlling the complex architecture of native β-cyclodextrin (β-CD) with HCT, we performed multiple-temperature-pH isothermal titration calorimetric measurements of the HCT:β-CD system, together with proton nuclear magnetic resonance spectroscopy ((1)H NMR), phase solubility analysis, and molecular modeling methods. The A(L)-type diagrams, obtained at different pH values and temperatures, suggested the formation of soluble 1:1 inclusion complexes of β-CD with HCT. The corresponding stability constants (K(1:1)) were determined by phase solubility studies and compared with those obtained by ITC, with good agreement between these two techniques being found. The three-dimensional array of the complex was studied by (1)H NMR and molecular modeling methods. Both techniques confirmed the formation of the inclusion complex, with good agreement between the experimental and theoretical techniques regarding the HCT binding mode to β-CD. Also, the forces involved in the association process were determined, both from the thermodynamic parameters obtained by ITC (association enthalpy, binding constant, Gibbs free energy, and entropy) and from energetic decomposition analyses derived from computational methods. We concluded that the formation of the HCT:β-CD complex was enthalpy driven, with the inclusion mode of HCT being highly dependent on its ionization state. In all cases, sustained hydrogen bond interactions with hydroxyl groups of β-CD were identified, with the solvation energy limiting the affinity. Regarding the pH and temperature dependence, lower affinity constants were found at higher HCT ionization states and temperatures.

LIPOPHILIC CHARACTER OF NOVEL AMINO ACID DERIVATIVES OF ZIDOVUDINE WITH ANTI HIV ACTIVITY

ABSTRACT The lipophilic properties of a novel series of zidovudine amino acid derivatives were measured using chromatographic techniques, reversed-phase thin layer chromatography (RP-TLC) and reversed-phase high performance liquid chromatography (RP-HPLC), as well as the classic shake flask (log Po/w) and theoretical CLOGP methods. These novel derivatives, obtained by association of zidovudine (AZT) with the essential amino acids leucine (AZT-Leu), isoleucine (AZT-iLeu), phenylalanine (AZT-Phe), valine (AZT-Val), proline (AZT-Prol) and tryptophane (AZT-Tryp), exhibited an increased log Po/w as compared with the parent compound as follows: AZT-iLeu>AZT-Leu>AZT-Tryp>AZT-Val>AZT-Phe>AZT-Prol>AZT>Thym. All assays were performed using a buffer, pH 2, as mobile phase, at which the mentioned compounds were completely as their non-ionized forms. In addition, good linear relationships were observed between log P values determined by the shake flask method (log Po/w), and those obtained by chromatographic techniques (log PRP-TLC and log PRP-HPLC) and from theoretical calculations using the CLOGP program (log PCLOGP). These results demonstrate the applicability of the chromatographic methods to describe the lipophilic properties of this family of compounds.

Development of a receptor model for efficient in silico screening of HIV-1 integrase inhibitors.

Integrase (IN) is a key viral enzyme for the replication of the type-1 human immunodeficiency virus (HIV-1), and as such constitutes a relevant therapeutic target for the development of anti-HIV agents. However, the lack of crystallographic data of HIV IN complexed with the corresponding viral DNA has historically hindered the application of modern structure-based drug design techniques to the discovery of new potent IN inhibitors (INIs). Consequently, the development and validation of reliable HIV IN structural models that may be useful for the screening of large databases of chemical compounds is of particular interest. In this study, four HIV-1 IN homology models were evaluated respect to their capability to predict the inhibition potency of a training set comprising 36 previously reported INIs with IC50 values in the low nanomolar to the high micromolar range. Also, 9 inactive structurally related compounds were included in this training set. In addition, a crystallographic structure of the IN-DNA complex corresponding to the prototype foamy virus (PFV) was also evaluated as structural model for the screening of inhibitors. The applicability of high throughput screening techniques, such as blind and ligand-guided exhaustive rigid docking was assessed. The receptor models were also refined by molecular dynamics and clustering techniques to assess protein sidechain flexibility and solvent effect on inhibitor binding. Among the studied models, we conclude that the one derived from the X-ray structure of the PFV integrase exhibited the best performance to rank the potencies of the compounds in the training set, with the predictive power being further improved by explicitly modeling five water molecules within the catalytic side of IN. Also, accounting for protein sidechain flexibility enhanced the prediction of inhibition potencies among the studied compounds. Finally, an interaction fingerprint pattern was established for the fast identification of potent IN inhibitors. In conclusion, we report an exhaustively validated receptor model if IN that is useful for the efficient screening of large chemical compounds databases in the search of potent HIV-1 IN inhibitors.

Homodiselenacalix[4]arenes: Molecules with Unique Channelled Crystal Structures.

A synthetic route towards homodiselenacalix[4]arene macrocycles is presented, based on the dynamic covalent chemistry of diselenides. The calixarene inner rim is decorated with either alkoxy or tert-butyl ester groups. Single-crystal X-ray analysis of two THF solvates with methoxy and ethoxy substituents reveals the high similarity of their molecular structures and alterations on the supramolecular level. In both crystal structures, solvent channels are present and differ in both shape and capacity. Furthermore, the methoxy-substituted macrocycle undergoes a single-crystal-to-single-crystal transformation during which the molecular structure changes its conformation from 1,3-alternate (loaded with THF/water) to 1,2-alternate (apohost form). Molecular modelling techniques were applied to explore the conformational and energetic behaviour of the macrocycles.