M. B. Calvo

Enhanced immune response after subcutaneous and oral immunization with biodegradable PLGA microspheres.

PLGA microspheres containing bovine serum albumin (BSA) as a model antigen, were prepared by a double emulsion/solvent extraction method and their in vitro characterization was performed. The same microspheres were used in a series of in vivo studies to evaluate the immune response induced after subcutaneous or oral inoculation following different immunization protocols. The in vivo data confirm that the immunogenicity of the albumin is not affected by the encapsulation procedure. The subcutaneous administration of microspheres showed an immune response (serum IgG levels by ELISA) statistically above BSA solution, even when the dose administered was 10 times lower. The adjuvanticity of the microspheres was found to be comparable to that of Freunds complete adjuvant (FCA), but in contrast to FCA they are biocompatible and did not induce any adverse reaction at the site of injection. A single oral administration of the microspheres was not a successful strategy for the induction of a reproducible response. Therefore, microspheres of 1 and 5 micrometer were orally administered on 3 consecutive days and the response obtained showed that the use of a boosting dose was not necessary for the 1 micrometer particles. These results suggest the possibility of simplifying the immunization schedule to a primary immunization if 1 micrometer particles are administered.

Influence of formulation variables on the in-vitro release of albumin from biodegradable microparticulate systems

Poly(D,L-lactide-co-glycolide) microspheres containing BSA were prepared by a modified solvent evaporation method using a double emulsion. These microspheres were characterized for size, morphology, surface absorbed protein, encapsulation efficiency and release kinetics. The influence of two formulation variables (the procedure to obtain the first emulsions and the lyophilization of the microspheres once obtained) on the physical characteristics and release behaviour of the microspheres was also investigated. Sonicated microspheres were smooth and spherical, with a mean particle size of 20 microns and an encapsulation efficiency of 81%. When the first emulsion was prepared by vortex mixing the particles were irregular and porous, with a mean size of 31 microns and a lower encapsulation efficiency (56%). The sonication allows a more homogeneous emulsion as well as a lower percentage of albumin adsorbed on the surface. The in vitro release profile was described as a biexponential process with an initial burst effect due to the release of the protein adsorbed on the microsphere surface and a second sustained release phase due to protein diffusion through the channels or pores formed in the polymer coat. The release of BSA was dependent on the preparation method. The greatest burst release was found for microspheres formulated using the vortex mixer, 58% of the encapsulated protein was released during the first 24 h, whereas sonicated microspheres released 32.2%. This burst effect could be reduced by lyophilizing the microspheres following their preparation. The amount of protein released decreased to 28.3% and 51.6% in sonicated and non-sonicated microspheres respectively, when they were lyophilized.

Stability of BSA encapsulated into PLGA microspheres using PAGE and capillary electrophoresis

Bovine serum albumin (BSA, Mw 66 200 Da) has been encapsulated as a model protein drug within poly(d,l-lactide-co-glycolide) (PLGA 50:50) microspheres using a w/o/w double emulsion method. The microspheres prepared were smooth and spherical with a mean particle size of 1.32 μm. The total protein loading and surface-associated protein were 8.61 and 16.60%, respectively. The microspheres showed a triphasic in vitro release profile with an initial burst effect due to the release of the protein adsorbed on the microsphere surface, a second sustained release phase due to protein diffusion through the pores or channels formed in the polymer matrix, and a third phase due to polymer bioerosion. The purpose of this paper was to evaluate the effect of the microencapsulation process on the integrity of the entrapped protein using polyacrylamide gel electrophoresis and capillary electrophoresis. The stability of the protein released during in vitro assays was also assessed. The results obtained showed that there was no apparent effect of the drastic encapsulation conditions (contact with dichloromethane (DCM), probe sonication, and vigorous shaking) on the structural integrity of the protein. On the other hand, it was found that after 1 week of incubation the protein released from the microspheres starts to hydrolyze to smaller fragments, probably due to a significant decrease in the medium pH as a result of the accumulation of the polymer degradation products.

Effect of aging on the release of salbutamol sulfate from lipid matrices.

The aim of the present work was to evaluate the influence of aging that might condition the release of salbutamol sulfate from oral formulations (lipid matrices) using Gelucire as lipid excipients. Gelucires are essentially characterized by their melting point and their hydrophilic-lipophilic balance. The release profiles of salbutamol sulfate from the capsules elaborated were dependent on the type of Gelucires, fast release, in the case of Gelucire 35/10, a slower release for Gelucire 48/09 and a slow release for Gelucire 46/07. Differential scanning calorimetry was used to study the physical state of drugs in the matrices. Gelucires may exhibit aging effects, whereby a range of physical properties may change upon storage. In the case of Gelucire 35/10, which presents a fast release of salbutamol sulfate, storage produces a decrease in the values of dissolution constant for all capsule sizes. Gelucire 48/09 showed a slower release rate than Gelucire 35/10, and after 1 year of storage, a decrease in the salbutamol dissolution rate for capsule number 3 and 4 was observed. Gelucire 46/07 presented the slowest dissolution rate, but there were not statistically significant differences. These results show that the faster the dissolution rate, and the larger the capsule size, the higher is the influence of storage.

Influence of shaking and surfactants on the release of bsa from plga microspheres

SummaryThe aim of the present work was to study the release of a model protein, bovine serum albumin (BSA) encapsulated within biodegradable poly (D,L-lactide-co-glycolide) (PLGA) microspheres prepared by a modified solvent evaporation method using a double emulsion. These microspheres were characterized for size, morphology, surface adsorbed protein, encapsulation efficiency and release kinetics. Two types of in vitro assays were developed to evaluate the influence of shaking and the addition of surfactants on the release profile of encapsulated protein. Scanning electron microscopy (SEM) observation showed spherical and smooth surface particles, with a mean particle size of 20 μm and an encapsulation efficiency of 81%. Surface associated protein was about 25%. The in vitro release profile showed a biphasic pattern described by means of a biexponential equation. There was an initial burst effect due to the release of the protein adsorbed on the microsphere surface and a sustained release phase due to protein diffusion through the channels or pores formed in the polymer coat. The release obtained profiles in static and dynamic assays showed statistically significant differences in the amount of the released protein, whereas the release rate was not affected. The burst effect was 28.30±1.63% and 35.20±1.50% of the total encapsulated protein for the static and dynamic assays respectively. The addition of surfactants (SDS) to the release medium increased the rate and the amount of drug released. In both assays the value of the slow release rate constant, β, was 0.029±0.002 days−1 when the surfactant was added, and 0.017±0.0014 days−1 in the samples without surfactant. It is believed that the surfactant leads to an increase in the microsphere surface polarity which allows channel and pore formation inside the polymer through which the protein diffuses easily.

Correlation of 'in vitro' release and 'in vivo' absorption characteristics of four salbutamol sulphate formulations

The purpose of this study was to investigate the possibility to develop different levels of correlation between ‘in vitro’ dissolution parameters and ‘in vivo’ pharmacokinetic parameters for four salbutamol sulphate formulations: two commercially available formulations (Ventolin® Oral and Volmax®) and two sustained-release formulations (SG7 and SG14) developed in our laboratory. A level A correlation of ‘in vitro’ release and ‘in vivo’ absorption could be set up for individual plasma level data by means of the deconvolution method. Linear correlations could be obtained when dose fraction released ‘in vitro’ was plotted vs. dose fraction absorbed ‘in vivo’, with correlation coefficients between 0.97 and 0.99 for the formulations studied. A second level of correlation between mean ‘in vitro’ dissolution time (MDT) and mean ‘in vivo’ residence time (MRT) was performed with a correlation coefficient of 0.81. Finally, it was also possible to establish a good ‘in vitro’-‘in vivo’ correlation when the mean dissolution time ‘in vitro’ and Tmax or Cmax ‘in vivo’ were compared, but it appeared impossible to establish any correlation between mean dissolution time (MDT) and AUC.

Influence of route of administration and dosage form in the pharmacokinetics and bioavailability of salbutamol

SummaryThe present study was carried out to define the pharmacokinetics of salbutamol sulfate administered to mongrel dogs in five pharmaceutical forms via two routes of administration. One pharmaceutical form was administered intravenously (Ventolin® i.v.) while the other four were administered orally (Ventolin®: immediate-release formulation, Volmax®: commercial osmotic pump, SG7 and SG14: sustained-release hydrophilic matrices developed in our laboratory). We obtained a first-order release kinetic of the salbutamol from Ventolin® and SG7, whereas a zero-order release kinetic was observed for SG14 and Volmax® formulations. Oral bioavailability was 80% and there were neither significant differences (P>0.05) in terms of the calculation method used (relation of the areas under the plasma level curve Loo-Riegelman, deconvolution) nor in terms of the dosage form (Ventolin®, Volmax®, SG7 and SG14). The elimination half-life value of salbutamol was 1.2 h when administered intravenously; this parameter had a value of 3.0 h for the immediate-release formulation and ranged between 5.4 and 7.2 h in the sustained-release formulations when administered orally. These changes in the half-life value of the sustained-release formulations will allow us to modify the frequency of administration in relation to immediate-release formulations.

Influence of hepatic regeneration after partial hepatectomy on theophylline pharmacokinetics in rats

SummaryThe influence of hepatic regeneration after partial hepatectomy on theophylline pharmacokinetics has been studied on the rat. At different times after partial hepatectomy, theophylline was administered intravenously as a single dose of 6 mg/Kg. Drug plasma levels were determined by HPLC and pharmacokinetic parameters were obtained. Physiological parameters were also measured. Following hepatectomy, an increase in mass liver was observed and 15 days after surgery, liver mass was 78% of nonhepatectomized rats. Initial theophylline concentrations varied during the regeneration period, as well as the distribution volume at steady-estate (Vss). Elimination half-life (t1/2), notably increased after hepatectomy (7.27±1.38 h), decreased with time (6.70±1.18 h, 6.47±0.69 and 5.17±0.87 h after 24 h, 3 days and 15 days post-hepatectomy, respectively) to reach a value closeto that of the control group (4.30±1.37h). The increase in elimination half-life led to a decrease in the mean residence time during the period of liver regeneration. However, the intrinsic clearance hardly varied during regeneration period. We could establish the following relationship between liver weight (LW) and the elimination half-life: t1/2(h)=0.358*LW (g)+8.6168 (R2=0.9906). For the mean residence time (MRT) this relationship was: MRT (h)=−0.5173*LW (g)+12.433 (R2=0.991).

Influence of partial hepatectomy in rats on the activity of hepatic microsomal enzymatic systems

SummaryThe influence of partial hepatectomy on the activity of the hepatic microsomal enzymatic systems was determined in rats. Cytochrome P-450, cytochrome b5, four mixed functional oxidase (MFO) activities (microsomal aniline hydroxylase,p-nitroanisole O-demethylase, aminopyrine N-demethylase and NADPH cytochrome c reductase) and glutathione levels were measured in unhepatectomized rats (control group) and in hepatectomized rats 12 h, 24 h, 3 days and 6 days after 70% hepatectomy. Following surgery the remaining lobes of the liver grow rapidly in order to restore the original liver mass. Partial hepatectomy significantly reduces cytochrome P-450 and b5 content in the remaining liver as well as the four MFO activities studied. But when the enzymatic systems are expressed as nmoles/mg microsomal protein, only cytochrome P-450 shows statistical differences. The hepatic biotransformation capacity of drugs and xenobiotics decreases during the regeneration period due to the reduction of hepatic mass rather than because of a reduction of their metabolic capacity. Glutathione levels are increased after partial hepatectomy but increased glutathione-dependent protector mechanisms are not expected.