Margarita C. Briñón

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.

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.

SYNTHESIS AND IN VITRO ANTIBACTERIAL ACTIVITY OF NOVEL 5′-O-ANALOG DERIVATIVES OF ZIDOVUDINE AS POTENTIAL PRODRUGS

ABSTRACT An efficient, short synthesis of four potential prodrugs of 3′-azido-3′-deoxythymidine (AZT) and their antibacterial activity are reported. The 5′-OH group of AZT was functionalized with oxalyl chloride obtaining an acyl chloride derivative (AZT-Ox), which by further transformation with leucine, isoleucine and valine amino acids led to the corresponding AZT analogs, namely AZT-Leu, AZT-iLeu and AZT-Val. A carboxyl acid derivative (AZT-Ac) was also obtained by hydrolysis of AZT-Ox. These compounds, which exhibit anti HIV activity, have killed collection and clinical strains of some opportunistic infectious agents in AIDS-related complex. Thus, the clinical strains, K. oxytoca, S. typhi and K. pneumoniae, and collection strain K. pneumoniae ATCC 10031 showed sensitivity to antibiotics. The activity order for the studied compounds against the most sensitive strain (K. pneumoniae ATCC 10031) was AZT-Leu > AZT-iLeu > AZT-Val > AZT-Ac > AZT. On the other hand, the activity order for the second most sensitive strain (K. oxytoca) was AZT-Leu > AZT-Val=AZT-Ac > AZT-iLeu > AZT. The most effective antibacterial drug AZT-Leu, M.I.C.=0.125 µg mL−1) was 16 times more active than AZT (AZT, M.I.C.=2 μg mL−1) against K. pneumoniae ATCC 10031. Thus, this compound may therefore have better clinical potential than AZT for the treatment of AIDS-related complex.

Lipophilicity of 5′‐Carbonates of Lamivudine with Antiretroviral Activity. Correlation Between Different Methods

Abstract This study reports on the lipophilicity of novel 5′‐carbonates of lamivudine with anti‐HIV activity. Reversed‐phase thin layer chromatography (RP‐TLC), reversed‐phase high performance liquid chromatography (RP‐HPLC), conventional shake flask (log Poct), and the theoretical CLOGP methods were used, in order to establish if the linear relationships between the conventional and chromatographic methods permit an extrapolation procedure. The nature of the organic modifiers used in the chromatographic techniques did not substantially affect the measurement of lipophilicity, since a good correlation between experimental log Poct and extrapolated RP‐TLC and RP‐HPLC values (RMw and log k′w, respectively) supported the validity of the extrapolation technique.

3′-AZIDO-3′-DEOXY-5′-O-ISONICOTINOYLTHYMIDINE, A NEW PRODRUG OF ZIDOVUDINE. SYNTHESIS, SOLID STATE CHARACTERIZATION AND ANTI HIV-1 ACTIVITY

ABSTRACT Synthesis, solid state characterization and anti HIV-1 activity of 3′-azido-3′-deoxy-5′-O-isonicotinoylthymidine (2), a new prodrug of zidovudine (AZT, 1), are described. Two solid forms of 2 prepared by crystallization from ethyl acetate-petroleum ether (form α) and from a melt sample of form α (amorphous form) were characterized by X-ray diffractometry, infrared spectroscopy, differential scanning calorimetry (DSC) and thermogravimetry (TGA) techniques. The novel nucleoside exhibited antiviral activity against standard and resistant strain panels of HIV-1 as well as cytotoxicity similar to that of AZT.

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.

Conformational studies of novel antiretroviral analogs of zidovudine.

Abstract Conformational properties of three novel zidovudine analogs, namely 3′-azido-3′-deoxy-5′-O-isonicotinoylthymidine (AZT-Iso, 2), (−)-trans-(5S,6S)-5-bromo-6, 5′-epoxy-5,6-dihydro-3′-azido-3′-deoxythymidine (3) and (+)-trans-(5R,6R)-5-bromo-6,5′-epoxy-5,6-dihydro-3′-azido-3′-deoxythymidine (4), have been investigated by AM1 calculations and NMR studies, and compared with those of the parent nucleoside (AZT, 1). Based on the results obtained the following correlation may be established, a) AZT and AZT-Iso exhibit a conformational behavior analog to other pyrimidinic nucleosides, displaying a dynamic equilibrium in solution where the two conformers (North and South) undergo a constant transformation. b) Compounds 3 and 4 show a different conformational profile. The estimate of the pseudorotation phase angle reveals the rigid structures of the latter compounds, which do not evidence conformational equilibrium in solution; theazide group being the only group free to rotate. c) Diastereoisomers 3 and 4 exhibit an extra conformational parameter compared with other pyrimidinic nucleosides: the chair or boat conformation in the third ring formed between the sugar and the base. In all cases, a reasonable correlation was obtained between theoretical and NMR spectroscopic data.

HYDROPHOBICITY PARAMETERS DETERMINED BY REVERSED-PHASE LIQUID CHROMATOGRAPHY FOR SOME NEW ISOXAZOLYL-NAPHTHOQUINONES

The lipophilic character of a series of new isoxazolyl-naphthoquinones has been studied. Partition chromatography procedures (RP-HPLC and RP-TLC) were developed for determining the log Po-w(n-octanol/water partition coefficient) values. The experimental values from these methods were validated by comparison with those obtained from the conventional shake flask method, except with the more lipophilic because they have poor water solubility. Also, the log Po-w was calculated using a CLOGP computer programme.

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.

Development and validation of a stability indicating method for seven novel derivatives of lamivudine with anti-HIV and anti-HBV activity in simulated gastric and intestinal fluids

A simple micellar liquid chromatography (MLC) method has been developed and validated for use in stability indicating studies of lamivudine and its carbonate derivatives with proved activity against human immunodeficiency and hepatitis B viruses (HIV and HBV, respectively), in simulated gastric (SGF) and intestinal (SIF) fluids samples. The optimized method involves a C18 column thermostated at 30°C, UV detection at 272 nm, a flow rate of 1.0 mL min(-1) and a micellar mobile phase composed by 0.15M sodium dodecyl sulphate (SDS) - 4% (v/v) 1-butanol - 0.01 M KH2PO4-Na2HPO4 (pH 7), using zidovudine (AZT) as internal standard. Validation under Food and Drug Administration (FDA) guideline of the analytical parameters include: linearity (r(2)>0.9996), LODs (1.6 × 10(-7)-6.9 × 10(-6)M) and LOQ (1 × 10(-5)M), intra (0.02-1.48%) and inter-day precision (0.04-1.66%) expressed as relative standard deviation (R.S.D.), and robustness parameters (less than 1.98%). Using this method, recoveries ranging from 92.9 to 119% were obtained for the eight substances. Thus, this method provides a simple, sensitive, accurate and precise assay for the determination of all compounds that can be readily adaptable to routine use by clinical laboratories with standard equipment. In addition, we evaluated the stability of carbonates of lamivudine in buffer pH 1.2 and 6.8; SGF (pH 1.2) and SIF one (pH 6.8), all as indicated in United States Pharmacopeia (USP) 32. Finally, this chromatographic method was applied to stability studies which resulted in all the compounds following a pseudo-first-order kinetics, and in the determination of its kinetic constant and half-life time.