Systematic study of liposomes composition towards efficient delivery of plasmid DNA as potential application of dermal fibroblasts targeting.

The use of non-viral DNA vectors to topically treat skin diseases has demonstrated a high potential. However, vectors applied on the skin face extracellular barriers including the stratum corneum and intracellular barriers such as the endosomal escape and the nuclear targeting of the plasmid DNA. The aim of this study was to develop a formulation suitable for dermal application and effective for delivering plasmid DNA into cells. Different formulations were prepared using different cationic lipids (DOTAP, DC-Chol, DOTMA) and co-lipids (DOPE, DSPE). Lipoplexes were produced by complexing liposomes with plasmid DNA at different pDNA/CL (w/w) ratios. Our results showed that appropriate pDNA/CL ratios allowing total complexation of plasmid DNA differed depending on the structure of the lipid used. The transfection rates showed that (i) higher rates were obtained with DOTMA lipoplexes, (ii) DC-Chol lipoplexes provided a transfection twice as important as DOTAP lipoplexes and (iii) when DSPE was added, the cytotoxicity decreased while transfection rates were similar. We found that formulations composed of DC-Chol:DOPE:DSPE or DOTMA:DOPE were appropriate to complex plasmid DNA and to transfect human primary dermal fibroblasts with efficacy and limited cytotoxicity. Therefore, these formulations are highly promising in the context of gene therapy to treat skin diseases.

ESR technique for noninvasive way to quantify cyclodextrins effect on cell membranes.

A new way to study the action of cyclodextrin was developed to quantify the damage caused on cell membrane and lipid bilayer. The Electron Spin Resonance (ESR) spectroscopy was used to study the action of Randomly methylated-beta-cyclodextrin (Rameb) on living cells (HCT-116). The relative anisotropy observed in ESR spectrum of nitroxide spin probe (5-DSA and cholestane) is directly related to the rotational mobility of the probe, which can be further correlated with the microviscosity. The use of ESR probes clearly shows a close correlation between cholesterol contained in cells and cellular membrane microviscosity. This study also demonstrates the Rameb ability to extract cholesterol and phospholipids in time- and dose-dependent ways. In addition, ESR spectra enabled to establish that cholesterol is extracted from lipid rafts to form stable aggregates. The present work supports that ESR is an easy, reproducible and noninvasive technique to study the effect of cyclodextrins on cell membranes.

Quantification of Randomly-methylated-beta-cyclodextrin effect on liposome: an ESR study.

In the present work, the effect of Randomly-methylated-beta-cyclodextrin (Rameb) on the microviscosity of dimyristoyl-l-alpha phosphatidylcholine (DMPC) bilayer was investigated using the electron spin resonance (ESR) technique. The ability of Rameb to extract membrane cholesterol was demonstrated. For the first time, the percentage of cholesterol extracted by Rameb from cholesterol doped DMPC bilayer was monitored and quantified throughout a wide Rameb concentration range. The effect of cholesterol on the inner part of the membrane was also investigated using 16-doxyl stearic acid spin label (16-DSA). 16-DSA seems to explore two different membrane domains and report their respective microviscosities. ESR experiments also establish that the presence of 30% of cholesterol in DMPC liposomes suppresses the jump in membrane fluidity at lipids phase-transition temperature (23.9 degrees C).

Natural philosophy in the constitution.

The natural philosophers who wrote the U.S. social contract held the advancement of science to be the supreme exercise of citizen sovereignty. The rising nation, in the late 19th century, established the seat of that sovereignty in its universities. Today those institutions have come to be regarded as contract research centers at the service of the federal government. Research contracts in support of the proposed Strategic Defense Initiative are pressed on them against the consensus of the scientific community that holds this "Star Wars" enterprise to be technically infeasible. The time has come to reconstruct the relation between the federal government and university science in the spirit of our social contract.

In vitro skin penetration enhancement techniques: A combined approach of ethosomes and microneedles.

Dermal administration of different macromolecules, such as nucleic acids, remains a real challenge because of the difficulty of crossing the main skin barrier, the stratum corneum (SC). To overcome this barrier, the use of deformable lipid-based nanovectors were developed to increase topical penetration through the SC and to promote the intercellular delivery of drugs. The purpose of this study is to compare the skin penetration of different liposome formulations according to their composition. In vitro and ex vivo experiments using Franz diffusion cells were performed to highlight the effect of (i) lipid charge, (ii) edge activators (EA) and (iii) ethanol on the diffusion properties of nanovectors. We showed that all formulations were not able to cross the SC. However, on a tape stripped skin, we showed that cationic formulations containing an EA and ethanol improved the skin penetration. The use of microneedles was considered to bypass the SC. We have shown that sodium cholate and ethanol were necessary to ensure an appropriate diffusion of liposomes into the dermis when applied by means of microneedles. This could be a promising approach to further deliver efficiently macromolecules such as genes into the skin.

Study of the relationship between lipid binding properties of cyclodextrins and their effect on the integrity of liposomes.

It is well known that cyclodextrins are able to extract lipids constituting membranes, increasing their fluidity and permeability. This behaviour towards biological membranes is directly linked to the toxicological effects of methylated cyclodextrins. However, confusion is currently made in the literature between the different methylated cyclodextrin derivatives. Moreover, a new methylated cyclodextrin derivative recently occurred in the market, the Crysmeb. We wanted to compare and understand the effect of the most currently used cyclodextrins on a model membrane. We studied the influence of natural cyclodextrins (betaCD and gammaCD), methylated derivatives (2,6-dimethyl-betaCD (Dimeb), 2,3,6-trimethyl-betaCD (Trimeb) and randomly methylated-betaCD (Rameb), as well as the new derivative Crysmeb), hydroxypropylated derivatives (HPbetaCD of different substitution degrees and HPgammaCD) and the sulfobutylated derivative (SBEbetaCD) on the release of a fluorescent marker encapsulated in the inner cavity of liposomes. It was shown that the observed effect on calcein release can be directly related to the affinity of cyclodextrins for both lipid components of liposomes, cholesterol and phosphatidylcholine. From this relationship, we were able to determine, for each cyclodextrin, a theoretical concentration giving rise to 50% or 100% calcein release. This theoretical concentration was confirmed experimentally. We have also showed that cyclodextrins which provoke calcein release also induce large structure modifications of liposomes.

Automated method for the determination of a new matrix metalloproteinase inhibitor in ovine plasma and serum by coupling of restricted access material for on-line sample clean-up to liquid chromatography.

A fully automated liquid chromatographic method was developed for the determination of Ro 28-2653, a new synthetic inhibitor of matrix metalloproteinases (MMPs), in ovine serum and plasma. The method was based on the coupling of a pre-column packed with restricted access material, namely LiChrospher RP-8 ADS (alkyl diol silica), for sample clean-up to an analytical column containing octyl silica stationary phase. One hundred microl of biological sample, to which 2-propanol was automatically added, were injected onto the ADS pre-column, which was then washed with a washing liquid consisting of a mixture of 25 mM phosphate buffer (pH 7.0) and acetonitrile (90:10; v/v) for 10 min. By rotation of the switching valve, the analyte was then eluted in the back-flush mode with the LC mobile phase composed of a mixture of acetonitrile and 25 mM phosphate buffer (pH 7.0) (57:43; v/v). The UV detection was performed at 395 nm. The main parameters likely to influence the sample preparation technique were investigated. The method was then validated over a concentration range from 17.5 to 1950 ng/ml, the first concentration level corresponding to the lower limit of quantitation. At this concentration level, the mean bias and the R.S.D. value for intermediate precision were -2.4% and 4.2%, respectively.

Cyclodextrins as a potential carrier in drug nebulization.

The inhalation route is widely studied for many drug applications focusing on either local or systemic distributions. One matter of concern is the solubilization of hydrophobic drugs. We have studied the feasibility of using different cyclodextrins (CDs) to elaborate pharmaceutical formulations for the inhalation route and tested the short-term toxicity of such formulations administered by inhalation to C57BL/6 mice. We have shown that HP-beta-CD, gamma-CD, as well as RAMEB aqueous solutions can undergo aerosolization and that the resulting droplet-size ranges are compatible with pulmonary deposition. In vivo, we have demonstrated that short-term exposure to inhaled HP-beta-CD, gamma-CD and RAMEB solutions are non-toxic after assessing bronchoalveolar lavage (BAL), lung and kidney histology, bronchial responsiveness to methacholine and blood urea. The only change noted is a slight increase in lymphocyte count in the BAL after HP-beta-CD and gamma-CD inhalation. We conclude that CDs are useful in significantly enhancing the solubility of apolar drugs with a view to inhalation therapy although an increase in lymphocyte counts in the BAL after CDs inhalations needs further investigations.

Oral bioavailability in sheep of albendazole from a suspension and from a solution containing hydroxypropyl-beta-cyclodextrin.

Albendazole (ABZ) is a benzimidazole derivative with a broad spectrum of activity against human and animal helminthe parasites. ABZ has a very poor aqueous solubility. This study shows that hydroxypropyl-beta-cyclodextrin (HP-beta-CD) is able to form inclusion complexes with ABZ and that is able to increase its aqueous solubility. A synergistic effect exists between HP-beta-CD and citric acid. The combination of HP-beta-CD (200 mM) and citric acid (50 mM) allows dissolution of more than 1.5 mg of ABZ per ml. The aim of this study is the in vivo evaluation in sheep of a solution of the inclusion complex of ABZ with HP-beta-CD in comparison with a suspension of the same drug. A significant (P<0.05) increase in the relative bioavailability is obtained with the solution containing the ABZ-HP-beta-CD complex as measured by ABZSO plasma levels. The area under the curve (AUC(0--> proportional, variant )) of the solution is 37% higher than that obtained with the suspension. Likewise the peak plasma concentration (C(max)) is twice that of the solution while the time to reach C(max) (T(max)) is reduced.

Skin compatibility of cyclodextrins and their derivatives: a comparative assessment using a corneoxenometry bioassay.

Few studies have been performed to assess the risk of skin damage by cyclodextrins (CD) and they have yielded contradictory results. The present study was conducted using the corneoxenometry bioassay on human stratum corneum to compare the skin compatibility of CD currently used in pharmaceutical preparations (betaCD, gammaCD, Rameb, Dimeb, Trimeb, HP-betaCD and HP-gammaCD) and that of new amphiphilic CD derivatives, namely, the phospholipidyl-CD (DMPE-Dimeb and DMPE-Trimeb). All the tested CD were well tolerated by the stratum corneum at a concentration of 5%. However, inter-individual reactivity was larger for DMPE-Dimeb, suggesting a more aggressive trend for this compound. Cutaneous Index of Mildness values obtained confirm that Dimeb is able to extract some skin components and shows that DMPE-Dimeb performs similarly.

Study of the influence of both cyclodextrins and L-lysine on the aqueous solubility of nimesulide; isolation and characterization of nimesulide-L-lysine-cyclodextrin complexes.

Nimesulide is a nonsteroidal antiinflammatory drug that exhibits a very poor water solubility (0.01 mg.mL-1). A nimesulide-beta-cyclodextrin complex prepared according to patent application WO 94/ 02177 has an aqueous solubility of approximately 16 mg.mL-1 of nimesulide. A nimesulide-L-lysine salt has also been prepared and increases the aqueous solubility of nimesulide to approximately 5.0-7.5 mg.mL-1. The purpose of the present study was to investigate the interaction of both cyclodextrins and L-lysine on the aqueous solubility of nimesulide. Nimesulide-L-lysine-beta- or gamma-cyclodextrin complexes were prepared by spray-drying. The inclusion of the nimesulide-L-lysine salt into the cyclodextrin cavity was confirmed by differential scanning calorimetry and proton nuclear magnetic resonance spectroscopy. These complexes offered remarkable aqueous solubility. The incorporation of nimesulide in a nimesulide-L-lysine-beta-cyclodextrin complex increased its water solubility by a factor of 10 at pH 1.5 (0.050 mg.mL-1 for the complex versus 0.005 mg.mL-1 for nimesulide), 160 at pH 6.8 (2.373 mg.mL-1 for the complex versus 0.015 mg.mL-1 for nimesulide), and 3600 in purified water (36.400 mg.mL-1 for the complex versus 0.01 mg.mL-1 for nimesulide).

Molecular modeling study of beta- and gamma-cyclodextrin complexes with miconazole.

Different authors have demonstrated the inclusion of miconazole in cyclodextrins (CD). Miconazole can be included in the CD cavity both in the neutral and in the ionized form. The present study tries to understand which fragment of the miconazole molecule is involved in the inclusion. Austin Model 1 approximate molecular orbital calculations have been performed on several complexes between beta-cyclodextrin (betaCD) or gamma-cyclodextrin (gammaCD) and miconazole in the ionized and the non-ionized forms of the two R and S enantiomers in three different orientations. We observed that betaCD is a good vehicle to transport miconazole which can be very easily released. The complexation energy between miconazole and betaCD is not very high but the entropic factor has a great incidence on the stability of the formed complex. The inclusion of the dichlorobenzene-CH(2)-O- and of the imidazole part of the S isomer gives rise to the most probable complex in acidic conditions (ionized miconazole). Nevertheless, the inclusion should be considered as a dynamic process in which different parts of the molecule could be alternatively included in betaCD. The present work demonstrates the high capability of deformation of betaCD which could easily accommodate several types of ligand. By opposite, the cycle extension in gammaCD leads to a more rigid vehicle with regards to miconazole.

Systematic characterization of oil-in-water emulsions for formulation design.

Oil-in-water emulsions varying in surfactant concentration and manufacturing process were prepared. About 10 experiments were performed to characterize them. The goal of this research was to find out which tests should systematically be carried out to assess efficiently the stability and the properties of an emulsified preparation. Thus, formulation design requires at least the measurement of the droplet size, the determination of the zeta potential, a TurbiScan analysis, the investigation of the stability under centrifugation and freeze/thaw cycles. If the emulsion contains an active substance, stability under storage at 4 degrees C and microscopic analysis are relevant. Quality control should be improved by measurements of viscosity and pH.

Comparison of the IV pharmacokinetics in sheep of miconazole-cyclodextrin solutions and a micellar solution.

The pharmacokinetics of miconazole were studied after intravenous administration to six sheep (4 mg/kg) of three aqueous solutions: a marketed micellar solution containing polyoxyl-35 castor oil (Daktarin IV(R)) was compared with two solutions both containing 50 mM lactic acid and a cyclodextrin derivative (100 mM HP-betaCD or 50 mM SBE7-betaCD). The aim of this work was to demonstrate that these cyclodextrin derivatives (CDs) have no effect on the pharmacokinetics of miconazole by comparison with the micellar solution. The plasma concentration time curves have shown that there is no significant difference between the three solutions.