Mónica A. Raviolo

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.

Comparative study of hydrophobicity parameters of novel 5'-carbamates of zidovudine

Abstract The lipophilic character of a series of 5′‐carbamates of zidovudina has been studied. The lipophilicity was measured by means of reversed‐phase thin layer chromatography (RP‐TLC) and reversed‐phase high performance liquid chromatography (RP‐HPLC) techniques giving the corresponding RMw and log k′w parameters, respectively. These values were compared with those obtained by the classical shake flask methodology. RP‐TLC assays were performed on the basis of thermodynamically true RM values, and buffer pHs 7.4 and 12.03 were chosen to determine the influence of the pH on the lipophilicity of the studied compounds. In addition, the influence of the organic modifier was studied in RP‐TLC, showing the superiority of methanol as compared with acetone as the organic modifier, and physicochemical reasons have been discussed. Based on RP‐TLC results, RP‐HPLC studies were carried out using methanol and buffer pH 7.4 as mobile phase. Chromatographic data (RMw and log k′w) proved to be reliable parameters for describing the lipophilic properties of the test compounds.

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

Abstract 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.

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.

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

Abstract Degradation of 3′-azido-3′-deoxy-5′-O-isonicotinoylthymidine (AZT-Iso), an antiretroviral derivative of zidovudine, was investigated in buffer pH 7.4, µ = 300 mOsm at 37, 50 and 60°C, and in water (pH 6.6, 37°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°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.

Synthesis and pharmaceutical properties of N-acyloxymethyl prodrugs of Allop with potential anti-trypanosomal activity

Abstract We report herein the synthesis, and the physicochemical and pharmacokinetic properties of N-acyloxymethyl prodrugs of allopurinol (Allop) (2a–f). Allop is a compound with activity against Trypanosoma cruzi, a causative agent of Chagas disease. Its pathology leads to a huge number of infections and deaths per year, because in addition to many sufferers only having limited access to health services only an inefficient chemotherapy is available. Relevant pharmaceutical properties (pKa, stability, solubility, lipophilicity, in vitro permeability, binding protein, xanthine oxidase binding) were also determined. The results obtained showed that derivatives behave as prodrugs of Allop, since they exhibit improved physicochemical and pharmacokinetic properties relative to their precursor. This behavior turns these compounds into active reservoirs of Allop, and reduces its unfavorable characteristics, so 2a–f compounds are excellent candidates for the treatment of Chagas disease. This work is therefore an important contribution leading to the suppression of Chagas disease.