Ana Santoveña

Effect of high shear rate on stability of proteins: kinetic study.

Size-exclusion chromatography (SEC) was used to monitor the time-course of protein degradation induced by high shear rates during the formulation and manufacture of controlled-release pharmaceutical dosage forms. SEC with multi-angle laser light-scattering (MALLS) detection was used to characterize the aggregation products, determining their absolute molecular weight. A stability-indicating method was developed and validated to obtain reliable drug degradation data. The results obtained according to the ICH guidelines confirm that the system and methods proposed are suitable for their intended use. The degradation kinetics are influenced by the type of protein and the effect of the shear rate on their stability. Reversible pseudo-first order degradation kinetics were observed for bovine beta-lactoglobulin, whereas for human (HSA) and bovine serum albumin (BSA), a monomer-dimer transition was observed, independently of the rate of shear. However, trimer formation was also observed for HSA, especially at high shear rates. The kinetic model may thus be described as a two-step process: a monomer-dimer, and dimer-trimer transition.

Potential applications of PLGA film-implants in modulating in vitro drugs release

In this work we evaluate poly(lactic/glycolic) acid (PLGA) film-implants as potential biodegradable devices for controlled release of two different drugs: 5-Fluorouridine (5-FUR), a conventional low molecular weight water-soluble compound and SPf66 malaria vaccine, a therapeutic synthetic polypeptide. Three types of devices were prepared by solvent-casting techniques alone or combined with compression method: simple monolithic discs (SMD), multilayer discs with a central monolithic layer (MLDM), and multilayer discs with a central drug-reservoir (MLDR). For the highly water-soluble drug, 5-FUR, in vitro release from SMD showed an initial burst (24% in 2 h) followed by prolonged release over 20 days. In contrast, from a MLDM (two drug-free PLGA discs were added to the SMD) showed an initial lag-time of 12 days followed by a very fast second release phase. Finally, when the load of this system was increased from 3 to 9%, an extended release over 20 days with a low burst effect was obtained. For SPf66, the central reservoir containing the synthetic polypeptide MLDR reduces the possibility of degradation due to peptide contact with polymer solution. When four layers were added, 10 days sustained-release was obtained without any burst effect. With six layers a moderate pulse was obtained, 18-22 days from the beginning of the release. The results show the suitability of the proposed devices to control release and avoid the burst effect with highly water-soluble drugs; as well as modulate in vitro peptide release.

Stability Study of Human Serum Albumin Pharmaceutical Preparations

The influence of temperature on the stability of human serum albumin (HSA) pharmaceutical preparations has been studied by size‐exclusion high‐performance liquid chromatography with multi‐angle laser‐light‐scattering detection and by particle‐size analysis.

Design of a pediatric oral formulation with a low proportion of hydrochlorothiazide

It is a normal pediatric practice in community and hospital pharmacies to prepare a new drug formulation when no commercial forms of it are available. Any dose or stability control is usually done for these types of compounding formulations due to the effort which means to develop these types of tests in pharmacies. We have studied five different hydrochlorothiazide oral formulations prepared with traditional compounding techniques in pharmacies to treat heart failure and edemas in babies. A Standard Operating Procedure (SOP) was done for every suspension. After the strictly monitoring of the SOP, every suspension was subjected to quality control tests (pH, particle size, viscosity, dose content and stability). There is only one studied formulation that guarantees the correct dose administering and stability after 3 weeks stored at 5 °C and light protected. Both, the percentage of wetting agent and the viscosity of the suspensor vehicle in this formulation make the correct dose administering possible after the formulation is shaken.

Structural properties of biodegradable polyesters and rheological behaviour of their dispersions and films

This paper focuses on the dependence of the rheological properties of PLA-PEG and PLGA dispersions and films on the polymer structural properties, in order to obtain useful information to predict and explain the performance of polyester films as drug-delivery systems. In this study, one PLA-PEG and three PLGA polymers of different molecular mass were synthesized and characterized by NMR, GPC, DSC and TGA–FT-IR. To characterize the viscoelastic behaviour of concentrated solutions in dichloromethane and of the films obtained by a solvent-casting technique, oscillatory shear rheometry was used. The polymer dispersions showed a characteristic Newtonian viscous behaviour, but with different consistency index depending on the nature of the polymer. Freshly prepared, PLGA and PLA-PEG films had elastic modulus (G′) greater than viscous modulus (G″). The decrease in both moduli caused by an increase in temperature from 25 to 37°C was especially marked for the polymers with T g below or around 25°C (PLGA 27 kDa and PLA-PEG 27 kDa). After being immersed in pH 7.4 aqueous solution for one week, PLGA films showed a significant increase in both G′ and G″, due to the promotion of polymer–polymer interactions in a non-solvent medium. In contrast, the PLA-PEG film became softer and more hydrated, due to the amphiphilic character of the polymer. The water taken up by the film acted as a plasticizer and induced the softening of the system. These results suggest that the presence of PEG chains exerts a strong influence on the mechanical properties of polyesters films and, possibly, the performance as coating or matrices of drug-delivery systems.

Chromatographic characterization of synthetic peptides: SPf66 malaria vaccine.

The development and validation of a quantitative size-exclusion chromatography (SEC) method for SPf66 malaria vaccine was achieved. The results show the reliability of the analytical method for the intended use. SPf66 malaria vaccine characterization was perforrmed using both relative techniques such as the conventional SEC and absolute techniques: mass spectrometry and multi-angle laser-light scattering detection. The relative and absolute molecular masses were in the 4600-18,000 Da range. The results clearly indicate the presence of the monomer and dimer species, whereas the third species could be the trimer or tetramer.

Pharmacokinetics analysis of sustained release hGH biodegradable implantable tablets using a mouse model of human ovarian cancer

This paper presents the pharmacokinetic of human growth hormone (hGH) implantable tablets tested on a human ovarian cancer mouse model. In order to obtain a sustained release device which permits to administer a high dose of the hormone that keeps its integrity and stability, three different formulations of hGH-poly (d,l-lactic-co-glycolic acid) (PLGA) were elaborated by direct compression method varying hormone load, PLGA content and compactation time. In vitro studies showed that drug release was mainly controlled by hormone load. Pharmacokinetic studies were conducted by using immunodeficient female mice. Four days before the insertion of hGH implantable tablets in the peritoneal cavity, every mouse received 5x10(6) human ovarian cancer cells (SKOV3.ip1). Hormone serum levels were monitored through bleeding from eye orbital vessels. The population pharmacokinetic model used was based on the in series tank model and model parameters were estimated using the maximum likelihood method. The null hypothesis test about differences between formulations leads us to the conclusion that the three formulations showed the same kinetic behavior except for the hGH load. The hormone release was extended all over 2 weeks but no increase or decrease in survival time was observed. These results suggest that hGH serum levels do not facilitate tumoral cells proliferation, an expected effect of hGH and this could explain why survival times of mice treated with implantable tablets are not shorter than those treated with the control ones.

Characterization of antimalarial SPf66 peptide using MALDI-TOF MS, CD and SEC

SPf66 is the first chemically synthesized peptide to elicit a partial protective immune response against malaria. Size-exclusion chromatography (SEC) with multi-angle laser light-scattering (MALLS) detection and hydrogen/deuterium (H/D) exchange monitored by (matrix-assisted laser desorption/ionization) MALDI-TOF (time-of-flight) mass spectrometry (MS) were used to assess the conformation and stability in aqueous solution after storage at different temperatures. Moreover, the feasible conformational changes of this peptide were also measured by circular dichroism (CD)-spectroscopy. The absolute molecular weight of SPf66 monomer and dimer species were 4765 and 8960Da using SEC with MALLS detection, and 4643 and 9490Da by MALDI-TOF MS, the discrepancy being between both methods lower than 5.7%, a value quite close to those found in other proteins. The results from H/D exchange monitored by MALDI-TOF MS and CD-spectroscopy show that the SPf66 monomer lacks ordered structure, whereas the SPf66 dimer species presents segments of secondary structure as a determined by CD measurements.

Development of an ultra high performance liquid chromatography method for determining triamcinolone acetonide in hydrogels using the design of experiments/design space strategy in combination with process capability index

An ultra high performance liquid chromatography method was developed and validated for the quantitation of triamcinolone acetonide in an injectable ophthalmic hydrogel to determine the contribution of analytical method error in the content uniformity measurement. During the development phase, the design of experiments/design space strategy was used. For this, the free R-program was used as a commercial software alternative, a fast efficient tool for data analysis. The process capability index was used to find the permitted level of variation for each factor and to define the design space. All these aspects were analyzed and discussed under different experimental conditions by the Monte Carlo simulation method. Second, a pre-study validation procedure was performed in accordance with the International Conference on Harmonization guidelines. The validated method was applied for the determination of uniformity of dosage units and the reasons for variability (inhomogeneity and the analytical method error) were analyzed based on the overall uncertainty.

Study of quality and stability of ursodeoxycholic acid formulations for oral pediatric administration

This paper describes a rational method of characterizing the biopharmaceutical stability of two oral suspensions of ursodeoxycholic acid (UDCA) used in pediatrics. Because there is no commercial presentation of UDCA that can administer appropriate doses for infants and children, an active pharmaceutical ingredient (API) formulation is required. Due to its very low solubility and low dose in the formula (1.5%), two different suspensions with minimal use of excipients were studied, avoiding the use of complex additives and those not recommended by the European Medicines Agency (EMA). Adherence to Standard Operating Procedure (SOP) allows the preparation of formulations with appropriately sized and stable particles, and suitable rheological behavior in withdrawing the dose after stirring. Dose uniformity, expressed as mass and content variability, was determined using the criteria of the European and the United States Pharmacopoeia. Additionally, dose content variation of every mass determined was studied. A rational method was developed for determining the dose uniformity of UDCA in suspensions, whether freshly prepared or after storage under different conditions for 30 and 60 days. This method permits detection of differences between doses taken at different heights in the vessel at various times and storage conditions. UDCA was stable under all conditions studied, requiring the presence of glycerol in the formulation to obtain the declared API value after stirring. Storage of UDCA suspensions in a refrigerator increased variability between doses.