Josefina Prat

Stability of carboplatin in 5% glucose solution in glass, polyethylene and polypropylene containers.

The degradation of carboplatin (3.2 mg ml-1) in 5% glucose infusion solution at 25 degrees C and protected from light was investigated. The effects of the material of the container and temperature were also studied. Solutions were prepared in 5% glucose solution and stored in glass bottles, polyethylene (PE) and polypropylene (PP) containers at 40, 50 and 60 degrees C and at 25 degrees C +/- 1 degrees C. Samples were assayed by an HPLC method to determine the residual carboplatin concentration at each time of sampling. Carboplatin degradation followed pseudo-first-order kinetics and no dependence on the nature of the container was found. After 1 month at 25 degrees (+/- 1 degrees)C the change in carboplatin concentration was < 2% of the initial concentration in 5% glucose. These results are in agreement with those predicted by the application of the Arrhenius equation.

Study of the inhibition capacity of an 18-mer peptide domain of GBV-C virus on gp41-FP HIV-1 activity

The peptide sequence (175-192) RFPFHRCGAGPKLTKDLE (P59) of the E2 envelope protein of GB virus C (GBV-C) has been proved to decrease cellular membrane fusion and interfere with the HIV-1 infectivity in a dose-dependent manner. Based on these previous results, the main objective of this study was to deepen in the physicochemical aspects involved in this interaction. First, we analyzed the surface activity of P59 at the air-water interface as well as its interaction with zwitterionic or negatively charged lipid monolayers. Then we performed the same experiments with mixtures of P59/gp41-FP. Studies on lipid monolayers helped us to understand the lipid-peptide interaction and the influence of phospholipids on peptide penetration into lipid media. On another hand, studies with lipid bilayers showed that P59 decreased gp41-FP binding to anionic Large Unilamellar Vesicles. Results can be attributed to the differences in morphology of the peptides, as observed by Atomic Force Microscopy. When P59 and gp41-FP were incubated together, annular structures of about 200 nm in diameter appeared on the mica surface, thus indicating a peptide-peptide interaction. All these results confirm the gp41-FP-P59 interaction and thus support the hypothesis that gp41-FP is inhibited by P59.

Degradation pathway of carboplatin in aqueous solution

A degradation pathway for carboplatin in aqueous solution is described. Degraded solutions of carboplatin in water and in 5% glucose solution were analysed by high performance liquid chromatography; carboplatin and its degradation products were well separated. Three degradation products of carboplatin have been determined either in pure water and 5% glucose solution and they have been identified as 1,1-cyclobutanedicarboxilate anion, its protonated forms and cis-diamminediaquoplatinum (II) complex.

Degradation kinetics of ifosfamide in aqueous solution

The degradation process of ifosfamide in aqueous solution at 50°C was investigated at different concentrations, different pH ranges (3.6–9.8), different molarity and constant ionic strength. The observed rate obtained by measuring the remaining intact ifosfamide was shown to observe first-order kinetics and was shown to be a specific acid and base catalysis. The pH rate profile in buffer solutions showed a plateau between pH 4 and 7.9. In order to determine the effect of temperature, the kinetic process at four temperatures (40, 50, 60 and 70°C) was studied. The apparent heat of activation for ifosfamide degradation in solution was found to be 118 kJ/mol and by application of the Arrhenius equation the stability at 25°C (k25) and the shelf-life (t90) has been predicted. The agreement between the thin-layer chromatography (TLC), nuclear magnetic resonance (NMR) and high performance liquid chromatography (HPLC) supports the hypothesis presented concerning the reactions involved in the degradation of ifosfamide solutions.

Surface behavior of peptides from E1 GBV-C protein: Interaction with anionic model membranes and importance in HIV-1 FP inhibition

The interaction between a peptide sequence from GB virus C E1 protein (E1P8) and its structural analogs (E1P8-12), (E1P8-13), and (E1P8-21) with anionic lipid membranes (POPG vesicles and POPG, DPPG or DPPC/DPPG (2:1) monolayers) and their association with HIV-1 fusion peptide (HIV-1 FP) inhibition at the membrane level were studied using biophysical methods. All peptides showed surface activity but leakage experiments in vesicles as well as insertion kinetics in monolayers and lipid/peptide miscibility indicated a low level of interaction: neither E1P8 nor its analogs induced the release of vesicular content and the exclusion pressure values (πe) were clearly lower than the biological membrane pressure (24-30 mN m(-1)) and the HIV-1 FP (35 mN m(-1)). Miscibility was elucidated in terms of the additivity rule and excess free energy of mixing (GE). E1P8, E1P8-12 and E1P8-21 (but not E1P8-13) induced expansion of the POPG monolayer. The mixing process is not thermodynamically favored as the positive GE values indicate. To determine how E1 peptides interfere in the action of HIV-1 FP at the membrane level, mixed monolayers of HIV-1 FP/E1 peptides (2:1) and POPG were obtained. E1P8 and its derivative E1P8-21 showed the greatest HIV-1 FP inhibition. The LC-LE phase lipid behavior was morphologically examined via fluorescence microscopy (FM) and atomic force microscopy (AFM). Images revealed that the E1 peptides modify HIV-1 FP-lipid interaction. This fact may be attributed to a peptide/peptide interaction as indicated by AFM results. Finally, hemolysis assay demonstrated that E1 peptides inhibit HIV-1 FP activity.

Spectroscopic studies of the interfacial binding of Humicola lanuginosa lipase

The interaction of Humicola lanuginosa lipase (HLL) with small unilamellar vesicles of 1-palmitoyl-2-oleoylglycero-sn-3-phosphoglycerol (POPG) and in the presence of tributyrin (TB) as a substrate was studied by the use of steady-state fluorescence techniques. An inactive mutant with the serine from the catalytic triad changed by alanine (S146A) was used in experiments with TB to avoid interferences from product formation. HLL binds to POPG vesicles in an active or open form for the catalytic turnover, and therefore POPG provides a suitable system for studying the conformational changes involving the movement of the loop of amino acids that covers the active site of the enzyme in solution. Tryptophan (Trp) fluorescence shows that HLL binding to POPG occurs with a change in the environment of Trp residue(s) and that there is only one type of bound form, even in the presence of TB. Accessibility to aqueous quenchers indicates shielding of Trp in the membrane. Fluorescence anisotropy of the enzyme increases on binding to the vesicles, indicating restricted rotational freedom for the Trp due to penetration in the bilayer. Resonance energy transfer experiments using an interfacial membrane probe, 1-[4-(trimethylammonio)phenyl]-6-phenylhexa-1,3,5-triene p-toluenesulfonate (TMA-DPH), and an internal membrane probe, 1,6-diphenylhexa-1,3,5-triene (DPH), indicate that HLL does not penetrate very deeply in the hydrophobic core of the membrane, but preferentially stays close to the lipid interface. Addition of substrate (TB) does not result in any additional changes in the spectroscopic properties of HLL. It is suggested that the observed changes are due to the ‘opening of the lid’ on binding to POPG vesicles, leaving the active site accessible for the substrate to bind.

Stability of carboplatin in 5% glucose solution exposed to light

Abstract The degradation of carboplatin (3.2 mg ml −1 and 0.8 mg ml −1 ) in 5% glucose infusion solution at different experimental conditions was studied. It was observed that the degradation rate of carboplatin under illumination increases notably with respect to the rate obtained in the dark. The time course of carboplatin in solution follows a first-order kinetics with rate constants that depend on the incident light intensity and little with temperature. The time in which 10% of carboplatin has been degraded ( t 90 ) lies between 1 and 10 h depending on the experimental conditions. From the results obtained in the present work it is concluded that under clinical conditions, protection from light must be necessary.

Time-lapse atomic force microscopy observations of the morphology, growth rate, and spontaneous alignment of nanofibers containing a peptide-amphiphile from the hepatitis G virus (NS3 protein).

Time-lapse atomic force microscopy is used in this contribution to directly watch the growth of nanofibers of a lipidated peptide on a mica surface. Specifically, the studied lipopeptide is the palmitoyl derivative of the fragment 505-514 of NS3 protein from the hepatitis G virus, abbreviated as Palmitoyl-NS3 (505-514). Data on the morphology, growth rate, and orientation of these peptide-amphiphile nanofibers have been obtained. From these data, it can be concluded that this synthetic lipopeptide forms two types of fiber-like aggregates: (i) half-spherical fibrous aggregates with lengths of hundreds of nanometers and (ii) spherical fibrous aggregates with lengths of several micrometers. In addition, when a fresh lipopeptide aqueous solution is deposited onto a mica surface, the aggregates spontaneously orient parallel to each other, yielding well-aligned nanofibers on large areas of the mica surface. A significant growth in both the length and the number of the fibers was observed during the first minutes after the solution deposition. Elongation of the fibrous aggregates from one end is more frequent, though elongation from both ends also occurs, with growth rates in the 4-5 nm/s range. The effects of dilution, mechanical perturbation, and pH on the aggregation behavior of Palmitoyl-NS3 (505-514) are also detailed in this paper.

Stability study of epirubicin in NaCl 0.9% injection.

Objective To determine the stability of epirubicin in NaCl 0.9% injection under hospital storage conditions. Methods NaCl 0.9% solution was added to epirubicin iyophilized powder to make a final concentration of 1 mg/mL to study the degradation kinetics and 2 mg/mL to study the stability in polypropylene syringes under hospital conditions. Setting Physical chemistry laboratory, Unitat de Fisicoquímica, Universitat de Barcelona. Main outcome Measures Solutions of epirubicin at 2 mg/mL in NaCl 0.9% solutions stored in plastic syringes were studied under hospital conditions at room temperature (25 ± 1 °C) and under refrigeration (4 ± 1 °C) both protected from light and exposed to room light (~50 lumens/m2). All samples were studied in triplicate and epirubicin concentrations were obtained periodically throughout each storage/time condition via a specific stability-indicating HPLC method. To determine the degradation kinetics, solutions of epirubicin in NaCl 0.9% at 1 mg/mL were stored at different temperatures (40, 50, and 60 °C) to obtain the rate degradation constant and the shelf life at room temperature and under refrigeration. Results The degradation of epirubicin in NaCl 0.9% solutions follows first-order kinetics. The shelf life was defined as the time by which the epirubicin concentration had decreased by 10% from the initial concentration. In this study, epirubicin was stable in NaCl 0.9% injection stored in polypropylene containers for all time periods and all conditions. That results in a shelf life of at least 14 and 180 days at 25 and 4 °C, respectively. The maximum decrease in epirubicin concentration observed at 25 °C and 14 days was 4%, and at 4 °C and 180 days was 8%. The predicted shelf life obtained from the Arrhenius equation was 72.9 ± 0.2 and 3070 ± 15 days at 25 and 4 °C, respectively, in both dark and illuminated conditions. Conclusions Solutions of epirubicin in NaCl 0.9% at 2 mg/mL are chemically stable when they are stored in polypropylene syringes under hospital storage conditions. No special precaution is neccessary to protect epirubicin solutions (2 mg/mL) from light.

Modification of FP-HIV activity by peptide sequences of GB virus C: a biophysical approach.

Three synthetic peptide sequences of 18 amino acid each, corresponding to different fragments of the E2 capsid protein of GB virus C (GBV-C): SDRDTVVELSEWGVPCAT (P45), GSVRFPFHRCGAGPKLTK (P58) and RFPFHRCGAGPKLTKDLE (P59) have been characterized in order to find a relationship between their physicochemical properties and the results obtained in cellular models. Experiments were performed in presence and absence of the HIV fusion peptide (FP-HIV) due to the evidences that GBV-C inhibits AIDS progression. P45 peptide showed lower surface activity and less extent of penetration into 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS) (3:2, mol/mol) lipid monolayers than P58 and P59. However, P45 peptide presented higher capacity to inhibit FP-HIV induced cell-cell fusion than the other two sequences. These results were supported by fluorescence anisotropy measurements which indicated that P45 had a significant effect on the inhibition of FP-HIV perturbation of liposomes of the same lipid composition. Finally, atomic force microscopy (AFM) studies have evidenced the modification of the changes induced by the FP-HIV in the morphology of lipid bilayers when P45 was present in the medium.