Characterization of 5-fluorouracil loaded liposomes prepared by reverse-phase evaporation or freezing-thawing extrusion methods: study of drug release.

Entrapment of the anti-tumoral drug 5-fluorouracil (5-FU) in unilamellar liposomes prepared by freeze-thawing extrusion technique (FATVET) and the reverse-phase evaporation method (REV) from natural (bovine brain) sphingomyelin (SM) and synthetic distearoylphosphatidylcholine (DSPC) phospholipids was studied. Reverse-phase evaporation vesicles obtained from DSPC sized through polycarbonate membranes of 0.2 micron pore size were found to entrap roughly double amounts of drug than did extruded liposomes (0.1 micron pore size); however, s-REV in these preparations were more heterogenous in vesicle size than FATVET. The rate of in vitro drug release from the liposomes was found to be dependent of the bilayer composition and the method used to prepare the vesicles. The permeability coefficient P obtained was approx. 10(-11) m/s. The results suggest that 5-FU release is kinetically controlled by an interfacial process seemingly dependent on the surface activity of the drug. Also, the physical state of the bilayer determines the retention capacity of the vesicles. Thus, liposomes consisting of distearoylphosphatidylcholine whose acyl chains were in a gel state at the working temperature (37 degrees C) retained 70% of encapsulated 5-FU after 1 h, whereas liposomes composed of natural bovine brain sphingomyelin retained only 15% over the same period.

Analysis of the particle size distribution and internal volume of liposomal preparations.

In this work we studied the particle size distribution of three liposomal preparations by quasi-elastic light scattering spectroscopy. Sized unilamellar vesicles of small diameter (s-SUV) were prepared by ultrasonication and subsequent centrifugation followed by extrusion through polycarbonate membranes of 0.2-micron pore size. Large unilamellar vesicles were obtained by reversed-phase evaporation (REV) and extrusion through polycarbonate filters with or without preliminary freezing-thawing cycles (VETI and VETII, respectively). After preparation, REV were sized to small diameter REV (s-REV) by extrusion through 0.4- and 0.2-micron polycarbonate membranes. According to the results, the s-SUV preparations were made up of two subpopulations, the major of which consisted of vesicles that were 26 nm in mean diameter and accounted for 95% of the overall s-SUV population. The s-REV dispersions always resolved into two populations centered at 120 and 380 nm, the relative proportions of which depended on the pore size of the filters used. VET structures were composed of a single population of vesicles that were approximately 100 nm in mean diameter. Cholesterol inclusion into the bilayer composition extended the distribution without altering its mean value. On the other hand, the internal volumes calculated from mean diameters or assuming a Gaussian distribution were inconsistent with experimental data obtained by usual techniques.

Evaluation of the "Auto-Stat 6010" automatic osmometer and its comparison with the "digimatic-advanced 3DII" manual osmometer.

The results are presented of a comparative study between two osmometers, a manual one, Digimatic-Advanced 3DII (Advanced Instruments, Medical Europa, Barcelona), and an automatic one, Auto-Stat 6010 (Kyoto, Daiichi, Kagaku, Menarini, Firenze). Both osmometers have the same operation principle, but they employ different systems for determination of the freezing point depression. Both instruments showed good precision (CV < 1%), accuracy, and a wide range of linearity. In the assay of 133 sera and 101 24-h urines, the methods showed a good correlation (r = 0.945 and r = 0.999) and the Anova test showed no statistical difference between the means (p > 0.05). The carry over effect in the Auto-Stat was statistically significant (p < 0.001) but within the range of imprecision of the osmometers. Variation due to evaporation is lower than 0.8%. In conclusion, we are sure that both osmometers are useful in the laboratory, although the Auto-Stat 6010 osmometer has the advantage over the Digimatic-Advanced 3DII of automatic sample processing.

Platelet integrin alpha IIb beta 3 (GPIIb-IIIa) is not implicated in the binding of LDL to intact resting platelets.

It has been suggested that the fibrinogen receptor (glycoprotein [GP] IIb-IIIa or platelet integrin alpha IIb beta 3) could be the binding site for low-density lipoprotein (LDL); however, recent data do not support this. Furthermore, GPIIb and not the GPIIb-IIIa complex is the main binding protein for lipoprotein(a) [Lp(a)]. In the present study, we have investigated the interaction between Lp(a) particles and platelet LDL binding sites and whether platelet integrin alpha IIb beta 3 is implicated. Displacement experiments showed that 125I-LDL binding to intact resting platelets was inhibited with the same apparent affinity by both unlabeled LDL and apolipoprotein(a)-free lipoprotein particles [Lp(a)-, an LDL-like particle prepared from Lp(a)]. Hill coefficients for displacement curves suggested that a single set of binding sites was involved. In contrast, both native and oxidized Lp(a) particles were unable to inhibit platelet LDL binding. Furthermore, platelets bound 125I-Lp(a)- particles to a class of saturable binding sites numbering approximately 1958 +/- 235 binding sites per platelet with a dissociation constant (Kd) of 48.3 +/- 12 x 10(-9) mol/L. These values were similar to those obtained for LDL. In contrast to Lp(a), evidence indicates that platelet integrin alpha IIb beta 3 was not involved in the interaction of LDL and intact resting platelets. First, specific ligands for platelet integrin alpha IIb beta 3, such as fibrinogen, vitronectin, and fibronectin, were unable to inhibit the binding of LDL to intact resting platelets. Second, similar LDL binding characteristics (Kd and Bmax values) were found in platelets from control subjects and patients with type I and type II Glanzmann's thrombasthenia, characterized by total and partial lack of GPIIb-IIIa and fibrinogen, respectively. Third, polyclonal antibodies against the GPIIb-IIIa complex (edu-3 and 5B12), human antiserums against platelet alloantigens (anti-Baka/B and anti-PLA1/2), anti-integrin subunits (anti-alpha v and anti-beta 3), and a wide panel of monoclonal antibodies (mAbs) against well-known epitopes of GPIIb (M3, M4, M5, M6, and M95-2b) and GPIIIa (P23-7, P33, P37, P40, and P97) did not affect platelet LDL binding. Finally, in contrast to the proaggregatory effect of native and oxidized LDL, both native and oxidized Lp(a) particles caused a significant dose-dependent decrease of collagen-induced platelet aggregation. In conclusion, we demonstrate that neither the GPIIb-IIIa complex nor GPIIb and GPIIIa individually are membrane binding proteins for LDL on intact resting platelets. Lp(a) particles do not interact with platelet LDL binding sites, and their biological response is clearly different from that of LDL.

Comparison of particle size and encapsulation parameters of three liposomal preparations.

Liposomes can be produced by using various techniques such as mechanical agitation, ultrasonication, ether injection, detergent dialysis, reversed-phase evaporation, extrusion through polycarbonate filters of appropriate pore size, etc. Each procedure provides liposomes with their own perculiar features so the choice of a given one to obtain suitable vesicles for use as drug delivery systems should be based on careful experimentation. In this work we tested three liposome preparation methods that provide unilamellar structures with high yields. In fact, unilamellar vesicles of small diameters (SUV) were prepared by ultrasonication and large unilamellar vesicles (LUV) were obtained by (a) reversed-phase evaporation (REV) and (b) extrusion through polycarbonate filters--with or without preliminary freezing-thawing (VETI and VETII, respectively). According to the results obtained, subsequent filtration of the SUV and REV liposomal preparations results in dramatically decreased average diameters and polydispersity indices, as well as in marked changes in their encapsulation parameters and in lipid losses. Among extruded liposomes, those obtained with preliminary freezing-thawing (VETII) resulted in a higher lipid recovery, even though their average hydrodynamic diameter and polydispersity index as measured by quasi-elastic light-scattering spectroscopy (QELSS) were quite similar to those of the VETI counterparts.

[AM3 (a biological response modifier) in the treatment of recurrent aphthous stomatitis. Clinical evaluation and preliminary studies on NK (CD16) cells and their pathogenic role in the syndrome]. / AM3 (modificador de la respuesta biológica) en el tratamiento de la estomatitis aftosa recidivante. Valoración clínica y estudios preliminares sobre células NK (CD16) y su relación con la patogenia del síndrome.

In an open-controlled trial--oral washes (20 patients) versus test (19 patients)--, we have studied the effects of AM3 (a new oral BRM) on clinical evolution of the recurrent stomatitis (RAS) syndrome. The results obtained at 6th month showed significant decreases on ulcer numbers (p less than 0.001) as well as in their mean duration time (p less than 0.001) due to the AM3 treatment. From a pathophysiologic point of view, the study of the NK peripheral blood cells (Leu 11/CD16) suggests the existence of two kinds of RAS-patients: those showing normal NK cell numbers (approximately 33%) and those ones showing a partial lack in the NK numbers (approximately 67%). These results suggest different rational new approaches to treatment, based on new pathophysiological concepts.

Angiotensin converting enzyme in patients with sleep apnoea syndrome: plasma activity and gene polymorphisms.

The prevalence of several cardiovascular diseases is increased with obstructive sleep apnoea syndrome (OSAS), due to, as yet, unclear reasons. Angiotensin converting enzyme (ACE) abnormalities have been implicated in the pathogenesis of various cardiovascular diseases. In this study, plasma ACE activity and the distribution of an insertion (I)/deletion (D) polymorphism of the ACE gene were determined in OSAS patients and in healthy controls. A total of 63 patients with OSAS (mean+/-SEM 54.5+/-2.5 apnoea/hypopnoeas.h(-1)) and 32 healthy subjects were studied. To avoid potential confounding factors, patients treated with ACE inhibitors or continuous positive airway pressure were excluded, as well as controls in whom a blood sample was not obtained early in the morning. ACE activity was determined spectrophotometrically in 46 OSAS patients and 25 controls. The I/D ACE polymorphism was determined by polymerase chain reaction in 44 patients and 32 controls. ACE activity was higher in OSAS patients (53.9+/-2.5 IU.L(-1)) than in healthy controls (42.4+/-3.1 IU.L(-1), p<0.01). This was independent of the presence of arterial hypertension. The frequency distribution of the DD, II and ID genotypes in OSAS patients (30%, 16%, 54%, respectively) was not significantly different from that seen in healthy subjects (31%, 28%, 41%, respectively, p=0.356). These results indicate that ACE plasma activity is increased in untreated OSAS patients. This increased activity may contribute to the pathogenesis of the cardiovascular disease in these patients.