Development and Characterization of Pharmaceutical Systems Containing Rifampicin.

Rifampicin is a potent antimicrobial drug with some suboptimal properties, such as poor stability, low solubility, and variable bioavailability. Therefore, in the current study, a multicomponent complex between rifampicin, γ-cyclodextrin, and arginine was prepared with the aim of improving drug properties. Solubility was evaluated by phase-solubility studies. The mechanism of interaction was established through proton nuclear magnetic resonance spectroscopy and molecular modeling. Physicochemical characterization was investigated using Fourier transform-infrared spectroscopy, powder X-ray diffraction, and scanning electron microscopy. The dissolution properties, antimicrobial activity (antibacterial, antibiofilm, and antileishmanial), and stability of the different samples were studied. The results obtained in this investigation demonstrate that multicomponent complexes can improve the water solubility and dissolution rate of rifampicin, as well as its antibacterial and antileishmanial action, and present suitable stability. In conclusion, rifampicin complexed with γ-cyclodextrin and arginine is an attractive approach for developing pharmaceutical dosage forms of rifampicin with increased antimicrobial activities.

Structure-Activity Relationship Study of an Alkynylphosphonate and Vynilphosphonate Analogues of Calcitriol.

BACKGROUND: 1α,25-dihydroxy vitamin D3 (calcitriol) shows potent growth-inhibitory properties on different cancer cell lines, but its hypercalcemic effects have severely hampered its therapeutic application. Therefore, it is important to develop synthetic calcitriol analogues that retain or even increase its antitumoral effects and lack hypercalcemic activity. Based on previous evidence of the potent antitumor effects of the synthetic alkynylphosphonate EM1 analogue, we have now synthesized a derivative called SG. OBJECTIVE: The aim of the present work is to evaluate the calcemic activity and the antitumor effect of SG, comparing these effects with those exerted by calcitriol and with those previously published for EM1. In addition, we propose to analyze by in silico studies, the chemical structure-biological function relationship of these molecules. METHODS: We performed the synthesis of vinylphosphonate SG analogue; in vitro assays on different cancer cell lines; in vivo assays on mice; and in silico assays applying computational molecular modeling. RESULTS: The SG compound lacks hypercalcemic activity, similar to the parent compound EM1. However, the antitumor activity was blunted, as no antiproliferative or anti-migratory effects were observed. By in silico assays, we demonstrated that SG analogue has a lower affinity for the VDRligand- binding domain than the EM1 compound due to lack of interaction with the important residues His305 and His397. CONCLUSION: These results demonstrate that the chemical modification in the lateral side chain of the SG analogue affects the antitumoral activity observed previously for EM1 but does not affect the calcemic activity. These results contribute to the rational design and synthesis of novel calcitriol analogues.

Improved Activity of Rifampicin Against Biofilms of Staphylococcus aureus by Multicomponent Complexation.

The aim of this study was to evaluate a multicomponent complex (MC) between rifampicin (RIF), ß-cyclodextrin (ß-CD), and selected amino acids to enhance the solubility and antibiofilm activity of RIF. After performing phase-solubility studies that demonstrated a considerable increase in the solubility of RIF for the MC, the corresponding solid system was prepared by a freeze-drying method. Characterization of the MC was performed by Fourier transform-infrared spectroscopy, thermal analysis, powder X-ray diffraction, and scanning electron microscopy. Structural analyses evidenced molecular interactions between the components, resulting in a MC with amorphous solid features. Structural studies involving both experimental (i.e., 1H NMR) and theoretical (i.e., molecular modeling) methodologies demonstrated the inclusion of the RIF piperazine ring in the ß-CD cavity. The bioactivity of the MC measured against biofilms of Staphylococcus aureus showed a significant reduction in the metabolic activity of the bacterium. Overall, the studied MC exhibited promising properties for the development of pharmaceutical formulations to treat bacterial infections.

Vitamin D receptor exhibits different pharmacodynamic features in tumoral and normal microenvironments: A molecular modeling study.

The vitamin D receptor (VDR) constitutes a promising therapeutic target for the treatment of cancer. Unfortunately, its natural agonist calcitriol does not have clinical utility due to its potential to induce hypercalcemic effects at the concentrations required to display antitumoral activity. For this reason, the search for new calcitriol analogues with adequate therapeutic profiles has been actively pursued by the scientific community. We have previously reported the obtaining and the biological activity evaluation of new calcitriol analogues by modification of its sidechain, which exhibited relevant antiproliferative and selectivity profiles against tumoral and normal cells. In this work we conducted molecular modeling studies (i.e. molecular docking, molecular dynamics, constant pH molecular dynamics (CpHMD) and free energy of binding analysis) to elucidate at an atomistic level the molecular basis related to the potential of the new calcitriol analogues to achieve selectivity between tumoral and normal cells. Two histidine residues (His305 and His397) were found to exhibit a particular tautomeric configuration that produces the observed bioactivity. Also, different acid-based properties were observed for His305 and His307 with His305 showing an increased acidity (pKa 5.2) compared to His397 (pKa 6.8) and to the typical histidine residue. This behavior favored the pharmacodynamic interaction of the calcitriol analogues exhibiting selectivity for tumoral cells when VDR was modeled at the more acidic tumoral environment (pH ≅ 6) compared to the case when VDR was modeled at pH 7.4 (normal cell environment). On the other hand, non-selective compounds, including calcitriol, exhibited a similar interaction pattern with VDR when the receptor was modeled at both pH conditions. The results presented constitute the first evidence on the properties of the VDR receptor in different physicochemical environments and thus represent a significant contribution to the in silico screening and design of new calcitriol analogues.

Identification of the potential biological target of N-benzenesulfonyl-1,2,3,4-tetrahydroquinoline compounds active against gram-positive and gram-negative bacteria.

The development of new antibiotics with activity towards a broad spectrum of bacteria, including multiresistant strains, is a very important topic for global public health. As part of previous works, N-benzenesulfonyl-1,2,3,4-tetrahydroquinoline (BSTHQ) derivatives were described as antimicrobial agents active against gram-positive and gram-negative pathogens. In this work, experimental and molecular modelling studies were performed in order to identify their potential biological target in the light of structure-based design efforts towards further BSTHQ derivatives. First, a carboxyfluorescein leakage assay was performed using liposomes to mimic bacterial membranes, which found no significative membrane disruption effects with respect to control samples. These results support a non-surfactant antimicrobial activity of the tested compounds. In a second stage, the inhibition of potential antimicrobial targets was screened using molecular modelling methods, taking into account previously reported druggable targets deposited in the ChEMBL database for Escherichia coli and Staphylococcus aureus. Two enzymes, namely D-glutamic acid-adding enzyme (MurD) and N-acetylglucosamine-1-phophate-uridyltransferase (GlmU), both involved in bacterial membrane synthesis, were identified as potential targets. Pharmacodynamic interaction models were developed using known MurD and GlmU inhibitors by applying state-of-the-art chemoinformatic methods (molecular docking, molecular dynamics and free energy of interaction analyses). These models were further extended to the analysis of the studied BSTHQ derivatives. Overall, our results demonstrated that the studied BSTHQ derivatives elicit their antibacterial activity by interacting with a specific molecular target, GlmU being the highly feasible one. Based on the presented results, further structure-aided design efforts towards the obtaining of novel BSTHQ derivatives are envisioned.Communicated by Ramaswamy H. Sarma.

Novel calcitriol analogue with an oxolane group: In vitro, in vivo, and in silico studies.

The active form of vitamin D3 , calcitriol, is a potent antiproliferative compound. However, when effective antitumor doses of calcitriol are used, hypercalcemic effects are observed, thus blocking its therapeutic application. To overcome this problem, structural analogues have been designed with the aim of retaining or even increasing the antitumor effects while decreasing its calcemic activity. This report aims at gaining insights into the structure-activity relationships of the novel oxolane-containing analogue, AM-27, recently synthesized. We herein demonstrate that this compound has antiproliferative and antimigratory effects in squamous cell carcinoma, glioblastoma, and breast cancer cell lines. Analyses of the mechanisms underlying the AM-27 effects on cell viability revealed induction of apoptosis by the analogue. Importantly, nonmalignant cell lines were little or not affected by the compound. In addition, the analogue did not produce hypercalcemia in mice. Also, in silico studies involving docking and molecular dynamics techniques showed that AM-27 is able to bind to the human vitamin D receptor with a higher affinity than the natural ligand calcitriol, a feature that is mostly derived from an electrostatic interaction pattern. Altogether, the proapoptotic effect observed in cancer cells, the lack of calcemic activity in mice, and the differential effects in normal cells suggest the potential of AM-27 as a therapeutic compound for cancer treatment.

Design, synthesis and molecular docking studies of novel N-arylsulfonyl-benzimidazoles with anti Trypanosoma cruzi activity.

Currently, only two drugs (i.e. benznidazole (BZN) and nifurtimox (NFX)) have been approved for the treatment of Trypanosoma cruzi (Tc) infection, the etiological agent causing Chagas disease. Since both drugs exhibit severe side effects, patients frequently abandon therapy, resulting in an inefficient pharmacotherapeutic treatment. In this context, there is an urgent need to develop new, safer and optimised anti-Tc agents. In this report, we present the synthesis and biological activity of 11 novel and 3 already reported N-arylsulfonyl-benzimidazole derivatives (NBSBZD,1-14) currently in development as potential anti-Tc compounds. These compounds were designed as part of a library of synthetic arylsulfonyl heterocycle derivatives constructed from privileged structures exhibiting drug-like properties. Based on bioactivity assays against Tc, (in both the extracellular and intracellular forms), we observed that 10 compounds exhibited bioactivity against the epimastigote form, while six of them exhibited activity against the amastigote counterpart. Also, the compounds showed less cytotoxicity compared to the reference drug BZN as measured in Vero cell culture. In order to elucidate the potential mechanism of action, metabolite excretion profiles studies were performed, and complemented with molecular modeling studies performed over known Tc druggable targets. Consistency was observed between experimental and theoretical findings, with metabolic profiles showing that compounds 1, 2, 9, 12 and 14 interfered with the normal glycolysis cycle of Tc, while molecular modeling studies were able to establish a solid structure-activity relationship towards the inhibition of 6-phospho-1-fructokinase, a key enzyme involved in the parasite glycolytic cascade. Overall, the present study constitutes a multidisciplinary contribution to the development of new anti-Chagas compounds.

Synthesis, biological evaluation and molecular modeling of a novel series of fused 1,2,3-triazoles as potential anti-coronavirus agents.

Synthesis and biological evaluation of a novel library of fused 1,2,3-triazole derivatives are described. The in-house developed multicomponent reaction based on commercially available starting materials was applied and broad biological screening against various viruses was performed, showing promising antiviral properties for compounds 14d, 14n, 14q, 18f and 18i against human coronavirus 229E. Further in silico studies identified the key molecular interactions between those compounds and the 3-chymotrypsin-like protease, which is essential to the intracellular replication of the virus, supporting the hypothesis that the protease is the target molecule of the potential antiviral derivatives.

Stability and plasmatic protein binding of novel zidovudine prodrugs: Targeting site ii of human serum albumin.

Despite its vastly demonstrated clinical efficacy, zidovudine (AZT) exhibits several suboptimal pharmacokinetic properties. In particular, its short plasmatic half-life (t1/2 ~ 1 h) is related to its low bound fraction to whole plasmatic proteins and in particular to human serum albumin (HSA). The design of prodrugs constitutes a promising strategy to enhance AZT pharmacokinetic properties, including its affinity for HSA. Recently, we reported the synthesis and chemical stability evaluation of three novel prodrugs of AZT obtained by derivatization with dicarboxylic acids (1-3). In this work, we present the design, synthesis and evaluation of chemical and enzymatic stabilities of a novel series of double prodrugs of AZT obtained by derivatization of 1-3 with a methylated l-phenylalanine moiety (4-6). In addition, the plasmatic protein binding properties were studied both by experimental and theoretical techniques. Prodrugs 4-6 were found to be relatively stable at pH 7.4 (t1/2 between 4.1 and 57.8 h), while also demonstrated adequate stabilities in human plasma at 37 °C (t1/2 between 1.0 and 2.1 h). Also, prodrugs 4-6 were able to regenerate AZT at a rate that depended on the length of the alkyl chain in 1-3. Additionally, 4-6 exhibited a significantly increased binding to plasmatic proteins (between 52.1 and 72.5%) with respect to AZT (12%) and 1-3 (between 26 and 34%). It is noteworthy that the displacement experiments with HSA site I and II markers, demonstrated that 4-6 bound to a different site than that of AZT and 1-3. Molecular modeling studies (i.e. molecular docking and free energy of binding analysis) were applied to shed light at an atomistic level on the pharmacodynamic properties driving the interaction of 4-6 with HSA. Overall, the present work provides a state of the art contribution to the design and development of novel prodrugs of AZT with optimized pharmacokinetic properties.

Structural basis for the potent inhibition of the HIV integrase-LEDGF/p75 protein-protein interaction.

Integrase (IN) constitutes one of the key enzymes involved in the lifecycle of the Human Immunodeficiency Virus (HIV), the etiological agent of AIDS. The biological role of IN strongly depends on the recognition and binding of cellular cofactors belonging to the infected host cell. Thus, the inhibition of the protein-protein interaction (PPI) between IN and cellular cofactors has been envisioned as a promising therapeutic target. In the present work we explore a structure-activity relationship for a set of 14 compounds reported as inhibitors of the PPI between IN and the lens epithelium-derived growth factor (LEDGF/p75). Our results demonstrate that the possibility to adopt the bioactive conformation capable of interacting with the hotspots IN-LEDGF/p75 hotspots residues constitutes a critical feature to obtain a potent inhibition. A ligand efficiency (|Lig-Eff|) quantitative descriptor combining both interaction energetics and conformational requirements was developed and correlated with the reported biological activity. Our results contribute to the rational development of IN-LEDGF/p75 interaction inhibitors providing a solid quantitative structure-activity relationship aimed for the screening of new IN-LEDGF/p75 interaction inhibitors.

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

The interaction of methotrexate (MTX) with beta-cyclodextrin (ß-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 ß-CD cavity, in which TEA contributes by intensifying MTX interaction with ß-CD and stabilizes MTX:ß-CD:TEA ternary complex by electrostatic interaction. The maintenance of these interactions in solid phase was also studied in ternary MTX:ß-CD:TEA and comparisons were made with freeze dried binary MTX:ß-CD and physical mixtures. FTIR studies evidenced that MTX-ß-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.

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.

Characterization of the hydrochlorothiazide: ß-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 ß-cyclodextrin (ß-CD) with HCT, we performed multiple-temperature-pH isothermal titration calorimetric measurements of the HCT:ß-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 ß-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 ß-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:ß-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 ß-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.

Intestinal permeability of lamivudine (3TC) and two novel 3TC prodrugs. Experimental and theoretical analyses.

Lamivudine (3TC) is an antiviral drug with a widely demonstrated clinical efficacy used to treat Acquired Immunodeficiency Syndrome (AIDS) in humans. However, since the rapid emergence of resistant viral strains has limited its effect, several new strategies such as the design of prodrugs have been applied to try to optimize its pharmacotherapeutic properties. The present study deals with the intestinal permeation of 3TC and two novel prodrugs of 3TC, namely 3TC-Etha and 3TC-Buta, by using rat intestinal segments and applying the gut sac in vitro technique. An adequate bioanalytical method (sample preparation and quantitative analysis) was fully developed and validated for the quantitation of these three compounds. A low permeability coefficient (P(app) 0.408 ± 0.049 × 10(-4) cm/min) was found for 3TC if compared to that previously reported for zidovudine (2.38 ± 0.12 × 10(-4) cm/min), while no statistically significant differences were observed in its apical-to-basal or basal-to-apical permeabilities. Our data suggests that 3TC permeates through the intestinal tissue by passive diffusion, with no intervention of efflux mechanism during this process as determined applying the gut sac technique. Regarding the prodrugs, both 3TC-Etha and 3TC-Buta were able to increase 3TC permeability (2 and 10 times, respectively), but none of these compounds were capable of crossing the intestinal tissue in their intact forms. In the case of 3TC-Etha, the permeability of the intact compound (P(app) 0.093 ± 0.010 × 10(-4) cm/min) was impaired by a P-glycoprotein (P-gp) mediated efflux, evidenced by the higher permeability coefficient (6.933 ± 0.586 × 10(-4) cm/min) determined in the presence of verapamil on the apical side of the enterocyte. In contrast, 3TC-Buta was not subjected to efflux mechanisms on the apical side of the enterocyte, but was efficiently converted to 3TC by enzymatic hydrolysis during the permeation process. In the light of these results, molecular modeling (docking and molecular dynamics) techniques were applied to study further the structural features that may confer the different behaviors of these two compounds with respect to P-gp mediated efflux. The results also highlight the potential of combining experimental and theoretical studies in the design of 3TC prodrugs with enhanced intestinal permeation properties.

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.

Rational approaches for the design of effective human immunodeficiency virus type 1 nonnucleoside reverse transcriptase inhibitors.

The binding of several classes of nonnucleoside reverse transcriptase inhibitors (NNRTIs) to wild-type (wtRT) and K103N mutant (mRT) human immunodeficiency virus type 1 (HIV-1) reverse transcriptase is studied by molecular dynamics and energy decomposition techniques. The imidoylthiourea (ITU), diaryltriazine (DATA), and diarylpyrimidine (DAPY) NNRTIs studied maintain the hydrogen bond with Lys101 during the 3 ns molecular dynamics trajectories. When bound to mRT, all the DAPYs studied establish hydrogen bonds with Glu138; among these, those of the potent inhibitors TMC120 and TMC125 are water-mediated. The molecular interactions of the NNRTIs in the binding pocket are correlated to the drugs' potency. Quantitative free energy analyses show a linear relationship between the van der Waals energetic component and the potency against wtRT. The molecular basis of the interaction between NNRTIs and RT presented here provide quantitative approaches for the design of novel effective anti-HIV drugs.

Complexation of sulfonamides with beta-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.

Specific binding capacity of beta-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.

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.

Quantitative plasma determination of a novel antiretroviral derivative of zidovudine by solid-phase extraction and high-performance liquid chromatography.

A solid-phase extraction methodology, followed by high-performance liquid chromatography (HPLC) quantification with UV absorbance detection (lambda = 267 nm), was developed in order to study the stability of 3'-azido-3'-deoxy-5'-O-isonicotinoylthymidine (AZT-Iso), a novel derivative of the antiretroviral AZT, in different matrixes. The half-lives (t1/2) for AZT-Iso were 1.19, 1.13 and 0.30 h for human, rat and rabbit plasma, respectively, and 14.91 and 25.49 h for potassium phosphate buffer (pH 7.4) and human serum albumin solution, respectively. The HPLC method proved to be selective, sensitive and accurate. Good recovery, linearity and precision were achieved using p-fluorophenol as an internal standard. The validity of this method was tested using synthetic mixtures of the intact drug with its decomposition products. In conclusion, the method presented is applicable to in vivo pharmacokinetics studies of AZT-Iso in rats.