Skin drug delivery using lipid vesicles: A starting guideline for their development.

Lipid vesicles can provide a cost-effective enhancement of skin drug absorption when vesicle production process is optimised. It is an important challenge to design the ideal vesicle, since their properties and features are related, as changes in one affect the others. Here, we review the main components, preparation and characterization methods commonly used, and the key properties that lead to highly efficient vesicles for transdermal drug delivery purposes. We stand by size, deformability degree and drug loading, as the most important vesicle features that determine the further transdermal drug absorption. The interest in this technology is increasing, as demonstrated by the exponential growth of publications on the topic. Although long-term preservation and scalability issues have limited the commercialization of lipid vesicle products, freeze-drying and modern escalation methods overcome these difficulties, thus predicting a higher use of these technologies in the market and clinical practice.

Cyanocobalamin Ultraflexible Lipid Vesicles: Characterization and In Vitro Evaluation of Drug-Skin Depth Profiles.

Atopic dermatitis (AD) and psoriasis are the most common chronic inflammatory skin disorders, which importantly affect the quality of life of patients who suffer them. Among other causes, nitric oxide has been reported as part of the triggering factors in the pathogenesis of both conditions. Cyanocobalamin (vitamin B12) has shown efficacy as a nitric oxide scavenger and some clinical trials have given positive outcomes in its use for treating skin pathologies. Passive skin diffusion is possible only for drugs with low molecular weights and intermediate lipophilicity. Unfortunately, the molecular weight and hydrophilicity of vitamin B12 do not predict its effective diffusion through the skin. The aim of this work was to design new lipid vesicles to encapsulate the vitamin B12 to enhance its skin penetration. Nine prototypes of vesicles were generated and characterized in terms of size, polydispersity, surface charge, drug encapsulation, flexibility, and stability with positive results. Additionally, their ability to release the drug content in a controlled manner was demonstrated. Finally, we found that these lipid vesicle formulations facilitated the penetration of cyanocobalamin to the deeper layers of the skin. The present work shows a promising system to effectively administer vitamin B12 topically, which could be of interest in the treatment of skin diseases such as AD and psoriasis.

Ciprofloxacin self-dissolvable Soluplus based polymeric films: a novel proposal to improve the management of eye infections.

Infections of the eye are among the leading causes of vision impairment and vision loss worldwide. The ability of a drug to access the anterior parts of the eye is negligible after systemic administration. Effective drug delivery to the eye is a major challenge due to the presence of protective mechanisms and physiological barriers that result in low ocular availability after topical application. The main purpose of this work was the improvement of the corneal and conjunctival permeation of the antibiotic Ciprofloxacin, a wide spectrum antibiotic used for the most common eye infection, using a self-dissolving polymeric film. Films were prepared by the solvent casting technique, using polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft co-polymer (Soluplus), polyvynyl alcohol, and propylene glycol. Films were homogeneous in drug content and thickness, as demonstrated by adapting the Swiss Roll technique followed by microscopy observation. These films proved in vitro to control the release of the Ciprofloxacin. Ex vivo permeability studies using Franz diffusion cells and porcine cornea and sclera showed an effective permeability of the drug without inducing irritation of the tissues. Films swelled in contact with artificial tears forming an in situ gel over 20 min, which will improve drug contact and reduce the need of multiple dosing. The antibiotic activity was also tested in vitro in five types of bacterial cultures, assuring the pharmacological efficacy of the films. The developed films are a promising drug delivery system to topically treat or prevent ocular infections.

Microneedle-Based Delivery: An Overview of Current Applications and Trends.

Microneedle arrays (MNA) are considered as one of the most promising resources to achieve systemic effects by transdermal delivery of drugs. They are designed as a minimally invasive, painless system which can bypass the stratum corneum, overcoming the potential drawbacks of subcutaneous injections and other transdermal delivery systems such as chemical enhancers, nano and microparticles, or physical treatments. As a trendy field in pharmaceutical and biomedical research, its applications are constantly evolving, even though they are based on very well-established techniques. The number of molecules administered by MNA are also increasing, with insulin and vaccines administration being the most investigated. Furthermore, MNA are being used to deliver cells and applied in other organs and tissues like the eyes and buccal mucosae. This review intends to offer a general overview of the current state of MNA research, focusing on the strategies, applications, and types of molecules delivered recently by these systems. In addition, some information about the materials and manufacturing processes is presented and safety data is discussed.

Cubic Microcontainers Improve In Situ Colonic Mucoadhesion and Absorption of Amoxicillin in Rats.

An increased interest in colonic drug delivery has led to a higher focus on the design of delivery devices targeting this part of the gastrointestinal tract. Microcontainers have previously facilitated an increase in oral bioavailability of drugs. The surface texture and shape of microcontainers have proven to influence the mucoadhesion ex vivo. In the present work, these findings were further investigated using an in situ closed-loop perfusion technique in the rat colon, which allowed for simultaneous evaluation of mucoadhesion of the microcontainers as well as drug absorption. Cylindrical, triangular and cubic microcontainers, with the same exterior surface area, were evaluated based on in vitro release, in situ mucoadhesion and in situ absorption of amoxicillin. Additionally, the mucoadhesion of empty cylindrical microcontainers with and without pillars on the top surface was investigated. From the microscopy analysis of the colon sections after the in situ study, it was evident that a significantly higher percentage of cubic microcontainers than cylindrical microcontainers adhered to the intestinal mucus. Furthermore, the absorption rate constants and blood samples indicated that amoxicillin in cubic microcontainers was absorbed more readily than when cylindrical or triangular microcontainers were dosed. This could be due to a higher degree of mucoadhesion for these particular microcontainers.

Ultraflexible lipid vesicles allow topical absorption of cyclosporin A.

Psoriasis and atopic dermatitis are widespread pathologies with a need to improve their treatment. Topical administration of cyclosporine A (CyA) could be used if targeted to the skin, thus avoiding systemic levels. Unfortunately, CyA molecular weight and lipophilicity prevent its diffusion through human skin. Four novel lipid vesicles have been prepared by different methodologies to overcome this problem. The vesicles were characterized in terms of particle size, size polydispersity, Z-potential, morphology, drug encapsulation, phospholipid content, and vesicle flexibility. Freeze-drying in presence and absence of cryoprotective agents was also performed, to guarantee long-term stability. The ability to deliver CyA into the skin was assessed using human epidermis in Franz diffusion cells and compared with the delivery of drug solutions with enhancers. The technical characteristics of four types of vesicle make them suitable to carry drugs. Moreover, these liposomal formulations were able to effectively deliver CyA in vitro into the skin. The present work introduces a promising approach for the topical treatment of skin pathologies with an immune component.

Effect of freezing and storage temperature on stability and antimicrobial activity of an antibiotic mixture used for decontamination of tissue allografts.

One of the most important risks to be controlled in tissue banking is the infection associated with the clinical use of auto- and allografts. Thus, tissue disinfection protocols are used, in addition to processing in controlled environments. For this purpose, combinations of antibiotics are designed to ensure a broad spectrum of antimicrobial activity. This type of protocol is usually validated by testing its antimicrobial efficacy. In this work, we have studied the effect of several factors on the potential of an antibiotic mixture: container, freezing, storage at 4 °C, storage at - 30 °C and storage at - 80 °C. The molecular stability of the compounds has also been tested, additionally to their efficacy. Our findings show that storage conditions affect the molecular stability of Fungizone and Tobramycin (only in case of frozen storage for the last one). Nevertheless, the solution retains its antimicrobial activity for several weeks. The availability of stored aliquots of disinfectant solution and defining expiry dates for different storage conditions can help to schedule tissue bank tasks.

Closed-Loop Doluisio (Colon, Small Intestine) and Single-Pass Intestinal Perfusion (Colon, Jejunum) in Rat-Biophysical Model and Predictions Based on Caco-2.

PURPOSE: The effective rat intestinal permeability (P eff ) was deconvolved using a biophysical model based on parameterized paracellular, aqueous boundary layer, transcellular permeabilities, and the villus-fold surface area expansion factor. METHODS: Four types of rat intestinal perfusion data were considered: single-pass intestinal perfusion (SPIP) in the jejunum (n = 40), and colon (n = 15), closed-loop (Doluisio type) in the small intestine (n = 78), and colon (n = 74). Moreover, in vitro Caco-2 permeability values were used to predict rat in vivo values in the rat data studied. RESULTS: Comparable number of molecules permeate via paracellular water channels as by the lipoidal transcellular route in the SPIP method, although in the closed-loop method, the paracellular route appears dominant in the colon. The aqueous boundary layer thickness in the small intestine is comparable to that found in unstirred in vitro monolayer assays; it is thinner in the colon. The mucosal surface area in anaesthetized rats is 0.96-1.4 times the smooth cylinder calculated value in the colon, and it is 3.1-3.6 times in the small intestine. The paracellular permeability of the intestine appeared to be greater in rat than human, with the colon showing more leakiness (higher P para ) than the small intestine. CONCLUSION: Based on log intrinsic permeability values, the correlations between the in vitro and in vivo models ranged from r2 0.82 to 0.92. The SPIP-Doluisio method comparison indicated identical log permeability selectivity trend with negligible bias.

Skin-PAMPA: a new method for fast prediction of skin penetration.

The goal of this study was to develop a quick, reliable, and cost-effective permeability model for predicting transdermal penetration of compounds. The Parallel Artificial Membrane Permeability Assay (PAMPA) was chosen for this purpose, as it already has been successfully used for estimating passive gastrointestinal absorption and blood-brain barrier permeability. To match the permeability of the rate-limiting barrier in human skin, synthetic certramides, which are analogs of the ceramides present in the stratum corneum, were selected for the skin-PAMPA model. The final skin-PAMPA membrane lipid mixture (certramide, free fatty acid, and cholesterol) was selected and optimized based on data from three different human skin databases and the final model was found to correlate well to all of the databases. The reproducibility of the skin-PAMPA model was investigated and compared to that of other PAMPA models. The homogeneity of the filter-impregnated lipid mixture membrane was confirmed with Raman microscopy. It was shown that skin-PAMPA is a quick and cost-effective research tool that can serve as a useful model of skin penetration in pharmaceutical and cosmetic research.

In Silico Prediction of Caco-2 Cell Permeability by a Classification QSAR Approach.

In the present study, 21 validated QSAR models that discriminate compounds with high Caco-2 permeability (Papp ≥8×10(-6)  cm/s) from those with moderate-poor permeability (Papp <8×10(-6)  cm/s) were developed on a novel large dataset of 674 compounds. 20 DRAGON descriptor families were used. The global accuracies of obtained models were ranking between 78-82 %. A general model combining all types of molecular descriptors was developed and it classified correctly 81.56 % and 83.94 % for training and test sets, respectively. An external set of 10 compounds was predicted and 80 % was correctly assessed by in vitro Caco-2 assays. The potential use of the final model was evaluated by a virtual screening of a human intestinal absorption database of 269 compounds. The model predicted 121 compounds with high Caco-2 permeability and the 90 % of them had high values of human intestinal absorption (HIA≥80). This study provides the most comprehensive database of Caco-2 permeability and evidenced the utility of the combined methodology (in silico+in vitro) in the prediction of Caco-2 permeability. It suggests that the present methodology can be used in the design of large libraries of compounds with appropriate values of permeability and to perform virtual screening in the early stages of drug development.

Wistar rat skin as surrogate for human skin in nortriptyline hydrochloride patch studies.

Six different matrices were prepared containing nortriptyline hydrochloride (NTH) with hydroxypropyl-methyl-cellulose as polymer. A mixture of transdermal enhancers was included as part of the vehicle. Diffusion studies were carried out through Wistar rat full thickness skin using Franz cells. They were compared with previously determined human heat separated epidermis in order to test if this animal can be used as model for in vivo assays. A linear correlation was obtained between NTH diffusion coefficients through both skin types (r2=0.996).

In situ kinetic modelling of intestinal efflux in rats: functional characterization of segmental differences and correlation with in vitro results.

The objective was to devise and apply a novel modelling approach to combine segmental in situ rat perfusion data and in vitro cell culture data, in order to elucidate the contribution of efflux in drug absorption kinetics. The fluoroquinolone CNV97100 was used as a model P-gp substrate. In situ intestinal perfusion was performed in rat duodenum, jejunum, ileum and colon to measure the influence of P-gp expression on efflux. Inhibition studies of CNV97100 were performed in the presence of verapamil, quinidine, cyclosporin A and p-aminohippuric acid. Absorption/efflux parameters were modelled simultaneously, using data from both in situ studies as well as in vitro studies. The maximal efflux velocity was modelled as a baseline value, corrected for each segment based on the expression level. CNV97100 passive diffusional permeability (P(diff)) and its affinity for the efflux carrier (K(m)) were assumed to be the same in all segments. The results indicate the new approach to combine in situ data and in vitro data succeed in yielding a unified, quantitative model for absorption/efflux. The model incorporated a quantitative relationship between P-gp expression level and the efflux functionality, both across in situ and in vitro systems, as well across different intestinal segments in the in situ studies. Permeability values decreased from duodenum to ileum in accordance with the increasing P-gp expression levels in rat intestine. The developed model reflects a strong correlation between in vitro and in situ results, including intrinsic differences in surface area. The successful application of a model approach to combine absorption data from two different experimental systems holds promise for future efforts to predict absorption results from one system to a second system.

PAMPA--a drug absorption in vitro model 7. Comparing rat in situ, Caco-2, and PAMPA permeability of fluoroquinolones.

Parallel artificial membrane permeability assay (PAMPA) was used to measure the effective permeability, P(e), as a function of pH from 4 to 10, of 17 fluoroquinolones, including three congeneric series with systematically varied alkyl chain length at the 4'N-position of the piperazine residue. The permeability values spanned over three orders of magnitude. The intrinsic permeability, P(o), and the membrane permeability, P(m), were determined from the pH dependence of the effective permeability. The pK(a) values were determined potentiometrically. The PAMPA method employed stirring, adjusted such that the unstirred water layer (UWL) thickness matched the 30-100 microm range estimated to be in the human small intestine. The intrinsic permeability coefficients (10(-6)cm/s), representing the permeability of the uncharged form of the drug, are for 4'N-R-norfloxacin: 0.7 (R=H), 49 (Me), 132 (n-Pr), 365 (n-Bu); 4'N-R-ciprofloxacin: 2.7 (H), 37 (Me), 137 (n-Pr), 302 (n-Bu); 4'N-R-3'-methylciprofloxacin: 3.8 (H), 20 (Me), 51 (Et), 160 (n-Pr), 418 (n-Bu). Increasing the alkyl chain length in the congeneric series resulted in increased permeability, averaging about 0.34 log units per methylene group, except that of the first (H-to-Me), which was about 1.2 log units. These results were compared to Caco-2 and rat in situ permeability measurements. The in situ closed loop technique used for obtaining permeability values in rat showed a water layer thickness effect quite consistent with in vivo expectations. The rat-PAMPA correlation (r2=0.87) was better than that of rat-Caco-2 (r2=0.63). Caco-2-PAMPA correlation indicated r2=0.66. The latter correlation improved significantly (r2=0.82) when the Caco-2 data were corrected for the UWL effect.

Activity-bioavailability balance in oral drug development for a selected group of 6-fluoroquinolones.

A nomogram is proposed to select the best candidate in drug development studies with quinolones and is intended to substitute other possible models. The nomogram is referred to as an activity-bioavailability balance (ABB) because it includes the following two criteria: ABB = [(1/gm MIC drug candidate)/ (1/gm MIC ciprofloxacin)].[(F(calc) drug candidate)/( F(calc) ciproflaxacin)]. The in vitro activity of a group of 4'N-alkyl-ciprofloxacin derivatives was determined together with that of ciprofloxacin, initially against some reference strains and subsequently against 159 clinical isolates of eight selected species. The inverse of the geometric mean of the lowest concentration of drug at which the original inoculum was reduced to no more than two colonies (1/gm MIC), as an antimicrobial activity parameter, and the absolute oral bioavailability index (F(calc)), as predicted from in situ intestinal absorption rate constants, were used for calculation of the ABB values, which ranged from 0.1 to 17 for the species and compounds tested. Ciprofloxacin was the best candidate only against Escherichia coli, whereas 4'N-methyl- and/or 4'N-ethyl-ciprofloxacin showed better or much better ABB values than the model drug, and can be selected as potential drug candidates against the remaining clinical strains. The procedure described could be a useful technique for further drug development studies.