Evaluation of physicochemical properties and bacterial photoinactivation of phenothiazine photosensitizers.

We report herein the physicochemical properties and antimicrobial activity of a new monobrominated derivative of Azure B and its parent compound. These dyes are used as photosensitizers for photodynamic therapy and photodynamic antimicrobial chemotherapy. Relevant pharmaceutical properties (pKa, chemical and photochemical stability, and in vitro antimicrobial activity) were determined. A UV-visible spectrophotometry method was developed and validated according to the International Conference on Harmonization (ICH) guidelines for use in stability indicating studies and determination of the acid dissociation constant of Azure B and its monobrominated derivative. The results showed that both dyes were chemically stable. In addition, bromination of the phenothiazine dye decreased its photochemical stability and pKa value without affecting the ionization rate at physiological pH. The analytical parameters for validation of the method were linearity (r2 > 0.9981), limit of detection (LOD) (0.2-0.9 µM), limit of quantification (LOQ) (0.6-2.7 µM), and intra-day precision (0.76-1.40%) expressed as relative standard deviation (RSD). Recoveries ranging from 99.5 to 100.9% were obtained for the two dyes. Thus, this method provides a simple, sensitive, accurate, and precise assay for the determination of all compounds. The effect of photosensitizer concentration and visible irradiation time on lethal photosensitization against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli was investigated. Both photosensitizers were active against the evaluated bacteria. However, the new monobrominated derivative was more effective than its predecessor and managed to eradicate these microorganisms by using different doses of the dye and light. In other words, a lower concentration of AzBBr and irradiation time were required to cause bacterial death equal to or greater than its precursor. The photodynamic efficacy of the two photosensitizers presented the following order: S. aureus > E. coli > P. aeruginosa. These studies indicated that the tested dyes satisfy the conditions of potential photosensitizers in terms of physicochemical and antimicrobial properties.

Preformulation studies of novel 5'-O-carbonates of lamivudine with biological activity: solubility and stability assays.

As a part of preformulation studies, the aim of this work was to examine the solubility and stability of a series of 5'-O-carbonates of lamivudine with proven antihuman immunodeficiency virus activity. Solubility studies were carried out using pure solvents (water, ethanol and polyethylene glycol 400 [PEG 400]), as well as cosolvents in binary mixture systems (water-ethanol and water-PEG 400). These ionizable compounds showed that their aqueous solubility is decreasing as the carbon length of the substituent moiety increases, but being enhanced as the pH was reduced from 7.4 to 1.2. Thus, 3TC-Metha an active compound of the series, with an intrinsic solubility at 25 °C of 17 mg/mL, was about 70 times more soluble than 3TC-Octa (0.24 mg/mL), and at pHs of 1.2, 5.8 and 7.4 had intrinsic solubilities of 36.48, 19.20 and 15.40 mg/mL, respectively. In addition, the solubility was enhanced significantly by using ethanol and PEG 400 as cosolvents. A stability study was conducted in buffer solutions at pH 1.2, 5.8, 7.4 and 13.0 and in human plasma at 37 °C. Stability-indicating high-performance liquid chromatography procedure was found to be selective, sensitive and accurate for these compounds and good recovery, linearity and precision were also observed.

Synthesis and anti-HIV activity of novel 2',3'-dideoxy-3'-thiacytidine prodrugs.

We report here the synthesis of a novel series of 5'-O-carbonates of 3TC, using different aliphatic alcohols and N,N-carbonyldiimidazol. Its antiviral activity was determined in peripheral blood mononuclear cells (PBMCs) showing some carbonate derivatives with an activity similar to or better than 3TC, except 3TC-Metha and 3TC-2Pro with less activity. In vitro assays in PBMCs have demonstrated that cytotoxicity increases as the carbon chain length of the alcohol moiety increases, showing compounds with a normal chain length of n=2-5 good selective index, compared to the parent drug. Thus, this work is an important contribution leading to the suppression of HIV replication.

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.

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.