Phylogenetic estimates of diversification rate are affected by molecular rate variation.

Molecular phylogenies are increasingly being used to investigate the patterns and mechanisms of macroevolution. In particular, node heights in a phylogeny can be used to detect changes in rates of diversification over time. Such analyses rest on the assumption that node heights in a phylogeny represent the timing of diversification events, which in turn rests on the assumption that evolutionary time can be accurately predicted from DNA sequence divergence. But there are many influences on the rate of molecular evolution, which might also influence node heights in molecular phylogenies, and thus affect estimates of diversification rate. In particular, a growing number of studies have revealed an association between the net diversification rate estimated from phylogenies and the rate of molecular evolution. Such an association might, by influencing the relative position of node heights, systematically bias estimates of diversification time. We simulated the evolution of DNA sequences under several scenarios where rates of diversification and molecular evolution vary through time, including models where diversification and molecular evolutionary rates are linked. We show that commonly used methods, including metric-based, likelihood and Bayesian approaches, can have a low power to identify changes in diversification rate when molecular substitution rates vary. Furthermore, the association between the rates of speciation and molecular evolution rate can cause the signature of a slowdown or speedup in speciation rates to be lost or misidentified. These results suggest that the multiple sources of variation in molecular evolutionary rates need to be considered when inferring macroevolutionary processes from phylogenies.

[Interactions between cyclodextrins and triglycerides: from emulsion stabilisation to the emergence of a new drug delivery system called "beads"]. / Interactions entre les cyclodextrines et les triglycérides : de la stabilisation des émulsions à l'obtention d'un nouveau système galénique appelé << billes >>.

Natural cyclodextrins are cyclic oligosaccharides which can be modified to obtain more water soluble or insoluble derivatives. The main interest of cyclodextrins results from their ability to form an inclusion complex with hydrophobic molecules. Inclusion constitutes a true molecular encapsulation. This property is employed in pharmaceutical industry to facilitate the formulation of poorly water soluble and/or fragile drugs. A more recent application of cyclodextrins consists in their use in the preparation of dispersed systems such as micro- and nanoparticles or even liposomes. When incorporated in dispersed systems, cyclodextrin can enhance drug solubility, drug stability and drug loading. Interestingly, cyclodextrins themselves can also be employed to form or stabilise dispersed systems (material or emulsifying agent). For example, the interactions between cyclodextrins with components of the vegetable oils (more especially with triglycerides) allow to stabilise simple or multiple emulsions but also to form particles called "beads". Very rich in oil, this novel lipid carrier presents an important potential for the encapsulation of highly lipophilic compounds and their delivery by topical and oral routes. These two applications are more particularly developed in the present paper.

Formulation and characterisation of beads prepared from natural cyclodextrins and vegetable, mineral or synthetic oils.

A continuous external shaking for 2.5 days of a mixture composed of alpha-cyclodextrin (6%), soybean oil (19.6%) and water (74.4%) resulted in a calibrated lipid carrier namely bead with a high fabrication yield. The purpose of this work was to explore the possibility to substitute alpha-cyclodextrin by other natural cyclodextrins, i.e. beta- and gamma-cyclodextrin and then soybean oil by mineral (Primol) 352 and Marcol 82) or synthetic (Silicon 200) fluid 10, 50 or 100cSt) oils. Beads can be successfully prepared using Marcol 82 with alpha-cyclodextrin and Silicon 50 or 100cSt with gamma-cyclodextrin. The area inside oil/cyclodextrin/water ternary diagram corresponding to bead occurrence was superior for the Marcol 82/alpha-cyclodextrin couple compared to that observed with soybean oil/alpha-cyclodextrin couple. Only a few ratios of Silicon 50 and 100cSt/gamma-cyclodextrin/water led to beads. The combinations which did not induce bead occurrence gave either emulsions, two non-miscible liquids or a solid mixture. Whatever the materials used, beads exhibited similarities: presence of a crystalline organisation and viscoelastic properties. Manufacturing process of paraffin- and silicon-based beads need further optimisation to increase fabrication yield and later on, to take advantages from the high stability of both oils for the formulation of drugs with beads.

alpha-Cyclodextrin/oil beads: an innovative self-assembling system.

The aim of this work was to characterise a new type of particulate system, named beads, prepared by a straightforward technique starting from a mixture of alpha-cyclodextrin aqueous solution and soybean oil without the use of any organic solvent or surface-active agent. Mechanisms involved in bead formation were also investigated. Optimal ratio between alpha-cyclodextrin (6%, w/w), soybean oil (19.6%, w/w) and water (74.4%, w/w) led to homogeneous bead size (1.6 mm) with a fabrication yield superior to 80% after a continuous external shaking during 2.5 days. After freeze-drying, oil and alpha-cyclodextrin contents were estimated at 80% (w/w) and 20% (w/w), respectively. X-ray diffraction studies revealed that beads presented a crystalline organisation and microscopic techniques showed that their inner structure was constituted by a matrix containing oily compartments. Beads offer interesting prospects for the microencapsulation of lipophilic and poorly stable molecules. Due to their semi-solid consistency and their ability to be freeze-dried, these beads have great potentialities for pharmaceutical (oral and topical routes) and cosmetic applications.

Effect of membranotropic and mucoadhesive formulations of protein proteinase inhibitors on bovine herpes virus-1 reproduction.

The lipidized derivatives of Bowman-Birk soybean protease inhibitor (BBI) containing one to three oleoyl groups were synthesized and characterized. The (ole)(1)- and (ole)(2)BBI were demonstrated to have 200- and 100-fold higher uptake into Caco-2 cell monolayers compared to native BBI. The acylated BBI had increased affinity to elastase-like proteases. Aprotinin-loaded starch/bovine serum albumin microcapsules were prepared using interfacial cross-linking with terephthaloyl chloride and characterized for their morphology, size and release of the inhibitor. Various formulations of protein proteinase inhibitors were tested for their influence on BHV-1 reproduction in cell cultures. Native aprotinin possessed palpable dose-dependent effect inhibiting the virus reproduction up to 4.0 lg (10,000-fold). The bioadhesive, biodegradable aprotinin-loaded microcapsules were the most effective decreasing virus infectious titer up to 4.0 lg and delaying the cytopathic effect up to 144 h in lesser doses of aprotinin. The lipophilic derivative (ole)(1)BBI was shown to exhibit effective inhibition (>100-fold) of BHV-1 reproduction unlike native BBI.

In vitro inhibition of bovine herpes virus 1 reproduction with native and microencapsulated proteinase inhibitor aprotinin.

This study evaluated the antiviral effect of various dosage forms of proteinase inhibitor-aprotinin as a potential remedy for prophylactics and therapy of infectious bovine rhinotracheitis. Formulations of the inhibitor were tested for their influence on bovine herpes virus reproduction in cell cultures. Starch/bovine serum albumin microcapsules with aprotinin were prepared using interfacial cross-linking with terephthaloyl chloride and characterized for their morphology, size and release of the inhibitor. Two types of these microcapsules-impregnated and loaded with the inhibitor-were used in virus infectious studies. Native aprotinin possessed palpable dose-dependent antiviral effect inhibiting the virus reproduction up to 4.0 lg (10000-fold) and delaying the cytopathic effect up to 96 h in the concentration 800-3300 TIU/ml. The bioadhesive, biodegradable aprotinin-loaded microcapsules were the most effective antiviral drug as this formulation allowed to decrease virus infectious titer up to 4.0 lg and a delay in the cytopathic effect of up to 144 h in lesser doses of inhibitor compared with the native form. In comparison the antiviral effect of microcapsules impregnated with aprotinin was not so appreciable. It was interesting to note that the results of the experiments on diverse cultures were very similar. This was because the drugs influenced the fundamental processes of virus replication cycle.

Gastrointestinal transit and mucoadhesion of colloidal suspensions of Lycopersicon esculentum L. and Lotus tetragonolobus lectin-PLA microsphere conjugates in rats.

PURPOSE: To investigate in vivo the fate and the behavior of lectin-particle conjugates after oral administration. METHODS: Two plant lectins were selected, namely Lycopersicon esculentum L. and Lotus tetragonolobus lectins, which have been reported to be specific for oligomers of N-acetyl-D-glucosamine and L-fucose, respectively, and conjugated to small poly(lactide) microspheres. Their intestinal transit was investigated in detail using radiolabeled particles. The transport and the distribution of the particles along the intestine, as well as their interactions with the intestinal mucosa, were determined after oral administration in rat. RESULTS: The overall transit of the particles was shown to be strongly delayed when the microspheres were conjugated to the lectins, mainly due to the gastric retention of the particles. A significant fraction of the conjugates adhered to the gastric and intestinal mucosae. No significant differences were observed after a preliminary incubation of lectin-microsphere conjugates with specific sugars. CONCLUSION: Although specific interactions could not be excluded, especially in the stomach, it was likely that adhesion was predominantly due to nonspecific interactions. These results could be attributed both to unfavorable physicochemical characteristics of the conjugates and to premature adsorption of soluble mucin glycoproteins, preventing any further specific adhesion.

Combined hydroxypropyl-beta-cyclodextrin and poly(alkylcyanoacrylate) nanoparticles intended for oral administration of saquinavir.

The aim of this study was to prepare and characterize an hydroxypropyl-beta-cyclodextrin-saquinavir inclusion complex with the purpose of incorporating this complex into poly(alkylcyanoacrylate) nanoparticles in order to increase the drug loading. Hydroxypropyl-beta-cyclodextrin-saquinavir complex was characterized by thermal (differential scanning calorimetry), crystallographic (X-ray diffractography) and spectroscopic methods (circular dichroism, H1-NMR). Nanoparticles were prepared by polymerization of alkylcyanoacrylate monomers (isobutyl- and isohexylcyanoacrylate) in a water solution of the complex and further characterized. The apparent solubility of saquinavir was increased 400-fold at pH 7.0 in presence of hydroxypropyl-beta-cyclodextrin owing to the formation of a drug-cyclodextrin complex as demonstrated mainly by 1H NMR and confirmed by other techniques. Saquinavir-loaded nanoparticles could be easily prepared in the presence of a drug-cyclodextrin complex. It was found that large amounts of cyclodextrins remained associated with the particles, resulting in a 20-fold increase in saquinavir loading compared to nanoparticles prepared in the absence of cyclodextrins. This study has shown that the loading in saquinavir of poly(alkylcyanoacrylate) nanospheres could be dramatically improved by simultaneously increasing the apparent solubility of the drug in the preparation medium and the amount of cyclodextrin associated with the particles, making these nanospheres a promising system for oral application.

Formulation of epichlorohydrin cross-linked starch microspheres.

The present work describes a water/oil emulsion technique for the production of microspheres by cross-linking soluble starch with epichlorohyrin, which is a very efficient divalent cross-linking agent for starch. Because they are important features for potential applications, such as pulmonary administration, special attention has been paid to control the mean particle size and size distribution. Microspheres ranging from 0.3-250 microm with narrow size distributions could be obtained. Due to the strongly basic nature of the aqueous phase, no stable emulsions could be obtained in the water/oil emulsion domains. In this context, the stirring rate during the emulsification step was crucial for controlling the particle size. Additionally, a high organic-to-aqueous phase ratio and the presence of a surfactant agent helped to prevent the coalescence of the droplets during the formation of the microspheres. The process was not sensitive to odifications of the chemical conditions, such as the cross-linking ratio, which allows variation of the chemical nature of the polymer forming the core of the microspheres without modifying their morphological characteristics.

Covalent coupling of asparagus pea and tomato lectins to poly(lactide) microspheres.

Lectin-poly(lactide) microsphere conjugates specifically designed for oral administration were prepared and their activity and specificity in presence of mucus were characterized. The presence of hydroxyl or amino groups suitable for covalent coupling of lectins by the glutaraldehyde method at the surface of the microspheres have been ensured by preparing the particles in presence either of poly(vinyl alcohol) (PVA) or bovine serum albumin (BSA). Tomato and asparagus pea lectins could be covalently attached to these particles (1.0-1.3 mg/m(2) of particles). The conjugates demonstrated a 4-10 fold increase in their interactions with mucus compared to control particles. Moreover, the sugar specificity of the lectins was maintained.

[Use of cyclodextrins in the formulation of polyalkylcyanoacrylate nanoparticles charged with different active ingredients]. / Emploi des cyclodextrines dans la formulation de nanoparticules de poly(cyanoacrylate d'alkyle) chargées en divers principes actifs.

Cyclodextrins were used to improve the loading capacity of biodegradable pol(yisobutyl cyanoacrylat)e nanoparticles, which were obtained by anionic polymerization in aqueous medium. We investigated the feasibility of blank nanoparticles in the presence of a series of cyclodextrins (5 mg/ml) and poloxamer 188 (1%). The smaller particles (87 +/- 3 to 103 +/- 6 nm) were obtained in the presence of hydroxypropyl beta- or gamma-cyclodextrin. The nanoparticle loading capacity investigated in the presence of hydroxypropyl beta-cyclodextrin, in the previous conditions, on a series of steroids revealed an increase varying from 5.5 times (megestrol acetate) to 130 times (prednisolone). Differential scanning calorimetry study of the active ingredient (progesterone) in the nanoparticles, revealed an amorphous or molecular state. The in vitro release of the active ingredient occurred very rapidly but reached a plateau depending on the nanoparticle size and the dissolution medium nature. All the active ingredient was released in the presence of esterases. The addition of a preformed hydroxypropyl beta-cyclodextrin/saquinavir inclusion compound to the preparation medium of poly(isobutyl [or] isohexyl cyanoacrylate) nanoparticles, resulted in a 20-fold increase in the encapsulation yield. Presently, poly(isobutyl cyanoacrylate) hydroxypropyl beta-cyclodextrin combined nanoparticles loaded with doxorubicin are in phase II clinical trials.

Bioadhesive potential of gliadin nanoparticulate systems.

The objective of this work was to prepare, characterise and evaluate the adhesive potential of gliadin nanoparticulate carriers. Firstly, lectin-nanoparticle conjugates were obtained by the carbodiimide (CDI) covalent binding of Dolichos biflorus lectin (DBA) to the surface of gliadin nanoparticles (NP) containing carbazole (as a model lipophilic drug). The DBA binding efficiency was favoured in mild acidic conditions. Similarly, a CDI concentration of about 0.63 mg/mg nanoparticles, acting during at least 1 h, provided binding efficiencies of about 50% bulk lectin. Under optimised experimental conditions, the DBA conjugates showed a size of around 500 nm and the amount of loaded carbazole and the DBA content were calculated to be around 15 and 23.5 microg/mg, respectively. The bioadhesive activity of NP and DBA conjugates was determined in samples of small and large rat intestinal mucosa. The amount of adsorbed NP was calculated to be around 8 and 4 g/m(2) in the small and large intestine, respectively. This high capacity to interact with the mucosa may be explained by gliadin composition. In fact, gliadin is rich in neutral and lipophilic residues. Neutral amino acids can promote hydrogen bonding interactions with the mucosa, while the lipophilic components can interact with the biological tissue by hydrophobic interactions. The bioadhesive activity of DBA conjugates was calculated to be about 2 g/m(2) in the small intestine and greater than 4 g/m(2) in the caecum and distal colon. These degrees of interaction were always significantly higher than those obtained with controls. Finally, DBA did not provide the specificity for interaction with Peyer's patches. In summary, gliadin nanoparticles show a high capacity of non-specific interaction with the intestine, whereas DBA binding to the surface of these carriers provided a greater specificity for colonic mucosa.

Phase behavior of fully hydrated DMPC-amphiphilic cyclodextrin systems.

With the aim of exploring relationships between the chemical structure and the physico-chemical properties of amphiphilic beta-cyclodextrin, a reappraisal of the obtaining of pure heptakis (2,3-di-O-hexanoyl)-beta-cyclodextrin (beta-CDC(6)) was undertaken. In this paper the chemical characterization of the newly synthesized beta-CDC(6) and its ability to form mixed structures with dimyristoylphosphatidylcholine (DMPC) are reported. Miscibility of the two amphiphiles is examined: (i) in monolayers formed at the air-water interface by analyzing the surface pressure-area isotherms; and (ii) in fully hydrated mixtures by differential scanning calorimetry (DSC) and X-ray diffraction at small and wide angles. Results demonstrate that the beta-cyclodextrin derivative is partially miscible to the phospholipid: intimate mixing occurs at beta-CDC(6) molar ratios smaller than 7-15 mol%, depending on the dimensional scale considered, while beyond these compositions phase separation is observed. At the air-water interface, the miscibility region of the two compounds shows non-ideal behavior characterized by the non-additivity of the molecular areas in the mixed monolayers. At the three-dimension level, the formation of a beta-CDC(6)/DMPC mixed lamellar phase occurs except at beta-CDC(6) molar ratios close to 5 mol% at which a highly ordered structure is depicted below the solid-to-liquid state transition of the DMPC hydrocarbon chains. At beta-CDC(6) contents higher than 7 mol%, the mixed assemblies coexist with excess amphiphilic cyclodextrin which then forms a separated hexagonal structure.

Poly(vinylbenzyl chloride) microsphere synthesis and their chemical modifications.

Vinylbenzyl chloride (VBC) was dispersion polymerized to give monodisperse microspheres in the presence of poly(vinylpyrrolidone) (PVP) as a steric stabilizer. The effect of PVP concentration on the size and on microsphere stability during the polymerization process was investigated. Microsphere size was examined when co-stabilizer molecules were employed with PVP during the polymerization reaction. The built-in reactive chloromethyl groups of the microspheres were the sites of the nucleophilic reaction of two amino-group model molecules, glucosamine (G), a hexosamine implicated in processes of molecular recognition, and also bovine serum albumin (BSA). Elemental analyses and Fourier transform infrared spectroscopy (FTIR) spectra showed that poly(vinylpyrrolidone) was associated with the microsphere network. Elemental analyses, attenuated total reflection infrared spectroscopy (ATR-FTIR), and zeta potential measurements confirmed G and BSA links at the microsphere surface.

Cyclodextrins and carrier systems.

This paper describes two new possibilities of using cyclodextrins to increase water solubility and bioavailability of poorly water-soluble drugs intended for targeting delivery by the oral or the parenteral route. They use either amphiphilic cyclodextrin nanoparticles or polymeric nanoparticles containing cyclodextrins. Amphiphilic skirt-shaped cyclodextrins, resulting from the esterification of primary hydroxyl groups by hydrocarbon chains varying from C6 to C14, are capable of forming spontaneously nanoparticles which have been loaded with a series of steroid drugs. The drug in the amphiphilic cyclodextrin nanoparticles is molecularly dispersed and can be released very rapidly. Poly(isobutylcyanoacrylate) nanoparticles can be loaded with natural or hydroxypropyl cyclodextrins. This technique results in a significant increase in the loading capacity of nanoparticles with a series of steroids and in a very rapid release of the drug. Both methods are described as well as their potential interest for water-insoluble drugs.

Biodegradable cross-linked starch/protein microcapsules containing proteinase inhibitor for oral protein administration.

The objective of this study is to demonstrate the feasibility of microcapsules containing a protein and a proteinase inhibitor in order to allow the oral administration of proteic or peptidic drug. Starch/bovine serum albumin mixed-walled microcapsules were prepared using interfacial cross-linking with terephthaloyl chloride. The microcapsules were loaded with native or amino-protected aprotinin by incorporating protease inhibitors in the aqueous phase during the cross-linking process. Microcapsules can be degraded in the presence of alpha-amylase. The influence of the formulation parameters on the in vitro release of the inhibitor activity and the protein was studied. The protective effect of microcapsules with aprotinin for bovine serum albumin was revealed in vitro. The presence of the native bovine serum albumin was demonstrated after incubation of the microcapsules with aprotinin in a mixture of alpha-amylase (5.4 U/ml) and trypsin (900 spectrophotometric BAEE units/ml) for 3 h at 37 degrees C, whereas the protein was completely degraded in the release medium of the microcapsules without aprotinin.

Preparation and characterization of microencapsulated proteinase inhibitor aprotinin.

Preparation of microcapsules through interfacial cross-linking of soluble starch/hydroxyethyl starch and bovine serum albumin (BSA) with terephthaloyl chloride is described. The proteinase inhibitor aprotinin, either native or active site protected, was microencapsulated, being incorporated in the aqueous phase. The influence of aqueous phase pH, BSA, and terephthaloyl chloride concentrations as well as stirring rate on microcapsule morphology and size was studied. The polycondensation pH was shown to be the determining factor for tough microcapsule production with a high encapsulation yield. The size of the microcapsules ranged between 10-30 and 50-100 microm at stirring speed 1500 and 500 rpm, respectively. Fourier transform infrared spectroscopic studies were performed on microcapsules prepared under various conditions. A correlation was established between spectral changes and microcapsule morphology and size. The optimal conditions for microcapsule degradation by alpha-amylase were found. Active site-protected aprotinin was shown to fully retain its activity after microencapsulation.

Cyclodextrins in targeting. Application to nanoparticles.

For some years cyclodextrins and their hydrophilic derivatives have been described in the literature as solubilizers capable of enhancing the loading capacity of liposomes and microparticles. We present here two new possibilities of using cyclodextrins in the design of colloidal carriers. The first possibility consists in increasing the loading capacity of poly(isobutyl cyanoacrylate) nanospheres prepared by anionic polymerization, by employing hydroxypropyl cyclodextrins. The second possibility consists in the spontaneous formation of either nanocapsules or nanospheres by the nanoprecipitation of amphiphilic cyclodextrin diesters. These two new techniques are very promising because of the great interest presented by nanoparticles for drug administration by the oral or parenteral routes.

An original method for studying in vitro the enzymatic degradation of cross-linked starch microspheres.

A reproducible technique based on microvolume measurements has been described which can be used for the assessment of the enzymatic degradation of small samples of microspheres (typically 104 to 105 micrometer3). As a model, the degradation pattern of epichlorohydrin cross-linked starch microspheres by alpha-amylase has been studied in the range of 5 to 100 IU/l by this technique. On the one hand, analysis of the decrease in volume of the microspheres by a cubic root law suggested that the degradation profiles were dependent on the initial size distribution of the microspheres. On the other hand, no internal rupture of the microspheres was detected from size distribution data, suggesting that enzymatic degradation of starch microspheres is surface-controlled.