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.

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.

Preformulation studies of Zidovudine derivatives: Acid dissociation constants, differential scanning calorimetry, thermogravimetry, x-ray powder diffractometry and aqueous stability studies.

As part as of the preformulation studies of new 5'-OH derivatives of zidovudine, compounds 2-6, their acid dissociation constants, Differential Scanning Calorimetry (DSC) and Thermogravimetry (TG) curves, X-Ray Powder diffractograms and aqueous stability are reported. A sensitive technique such as differential scanning potentiometry was used to determine the pKa constants of the above mentioned compounds. In addition, pKa values were calculated from theoretical methods, and no significant differences with those of experimental ones were observed. X-Ray Powder Diffractometry data demonstrated that compounds 2-4 were crystalline while 5 and 6 were amorphous. DSC analysis indicated that all of them presented an exothermic decomposition peak above 150 °C which is accompanied by a weight loss in the respective TG curves. The stability of these compounds in aqueous medium at different pH values was investigated, using a validated High Performance Liquid Chromatography (HPLC) method, which demonstrated to be rapid, selective, sensitive, accurate and stability-indicating. Good recovery, linearity and precision were also achieved. For all compounds the aqueous hydrolysis followed a pseudo-first-order kinetics, depending on pH and the union existing between AZT and the associate moiety. The hydrolysis was catalyzed by hydroxide ion in the 7.4-13.2 pH range, while all compounds exhibited pH-independent stability from acidic to neutral media (pHs 1.0-7.4).

Stability-indicating micellar liquid chromatography method for three novel derivatives of zidovudine in aqueous and simulated gastric and intestinal fluids matrices.

This work studies the stability of three new anti-HIV agents which were obtained by the association of zidovudine (AZT) with different amino acids, such as leucine (AZT-Leu) and valine (AZT-Val), and one with an acid group (AZT-Ac). Before commercialisation, their stability in different matrices - simulated gastric fluid (SGF, pH 1.2) and simulated intestinal fluid (SIF, pH 6.8), both as the USP 32 Guideline indicates, and buffers (pH 1.2 and 6.8) - must be studied. To this end, a new stability-indicating micellar liquid chromatography (MLC) method has been optimised and validated. Measurements were based on the disappearance of reagents and the appearance of the only degradation product (AZT). This optimised and validated method used a C18 column and a mobile phase containing 0.05 M sodium dodecyl sulphate-1% (v/v)1-butanol-0.01 M NaH(2)PO(4) (pH 3.0) at 30°C, and a flow rate of 1 mL min(-1). Under these conditions, retention times were 1.4, 3.6, 6.3 and 9.5 min for AZT-Ac, AZT, AZT-Val and AZT-Leu, respectively. Calibrations better than 0.9995, intra- and inter-day precisions below 1.08% and good recoveries (94.47-116.52%) and robustness (RSD less that 1.08%) were obtained and were adequate to analyse the four compounds. Finally, this MLC method was applied to achieve stability studies which resulted in the evidence that all the compounds followed a pseudo-first-order kinetics, and in the determination of their kinetic constants and half-life time. A reference method, applied in the same studies, validated the MLC method reported herein.

Determination of liposome permeability of ionizable carbamates of zidovudine by steady state fluorescence spectroscopy.

In the present paper the relative permeabilities of AZT-Pyp and AZT-Ethy across a phospholipid bilayer were estimated by the means of fluorescence spectroscopy. The center of spectral mass of both non-encapsulated AZT-derivatives (AZT-der) emission spectra increased as a function of the illumination time inside the spectrofluorimeter cell. This phenomenon was even more evident when drugs were incubated under an UV mercury lamp, suggesting its photolytic origin. AZT-der were protected from photolysis inside liposomes and decomposed upon irradiation when they were free in the aqueous phase. The time-dependent decrease in the fluorescence intensity at a constant wavelength was fitted to a two-exponential equation and the values of rate constants for permeability and photolysis were calculated. It was concluded that AZT-Pyp but not AZT-Ethy diffused across the bilayer. This behavior correlated with the molecular volumes of AZT-Pyp (379.6A(3)) and AZT-Ethy (450.5A(3)), determined from the minimum energy conformations but not with previously reported logP values. These results reinforce the concept that not only lipophilicity but also membrane structure and AZT-der molecular size had a critical influence in passive diffusion across bilayers and may help in future refinements of other AZT-der molecular design.

3'-Azido-3'-deoxy-5'-O-isonicotinoylthymidine: a novel antiretroviral analog of zidovudine. II. Stability in aqueous media and experimental and theoretical ionization constants.

Degradation of 3'-azido-3'-deoxy-5'-O-isonicotinoylthymidine (AZT-Iso), an antiretroviral derivative of zidovudine, was investigated in buffer pH 7.4, mu = 300 mOsm at 37, 50 and 60 degrees C, and in water (pH 6.6, 37 degrees C), giving zidovudine (AZT) and isonicotinic acid (INA) as products. The rate constants were determined by reversed-phase HPLC showing pseudo-first-order kinetics related to the residual amount of AZT-Iso. In this way, the studied compound was demonstrated to be 153 times more stable in water than in buffer solution at 37 degrees C. The analytical method was conveniently validated demonstrating to be a rapid and accurate stability-indicating technique. In addition, experimental and theoretical values of pKa were determined.