PEGylated PLGA nanospheres optimized by design of experiments for ocular administration of dexibuprofen-in vitro, ex vivo and in vivo characterization.

Dexibuprofen-loaded PEGylated PLGA nanospheres have been developed to improve the biopharmaceutical profile of the anti-inflammatory drug for ocular administration. Dexibuprofen is the active enantiomer of ibuprofen and therefore lower doses may be applied to achieve the same therapeutic level. According to this, two batches of nanospheres of different drug concentrations, 0.5 and 1.0mg/ml respectively, have been developed (the latter corresponding to the therapeutic ibuprofen concentration for inflammatory eye diseases). Both batches were composed of negatively charged nanospheres (--14.1 and --15.9mV), with a mean particle size below 200nm, and a high encapsulation efficiency (99%). X-ray, FTIR, and DSC analyses confirmed that the drug was dispersed inside the matrix of the nanospheres. While the in vitro release profile was sustained up to 12h, the ex vivo corneal and scleral permeation profile demonstrated higher drug retention and permeation in the corneal tissue rather than in the sclera. These results were also confirmed by the quantification of dexibuprofen in ocular tissues after the in vivo administration of drug-loaded nanospheres. Cell viability studies confirmed that PEGylated-PLGA nanospheres were less cytotoxic than free dexibuprofen in the majority of the tested concentrations. Ocular in vitro (HET-CAM test) and in vivo (Draize test) tolerance assays demonstrated the non-irritant character of both nanosphere batches. In vivo anti-inflammatory effects were evaluated in albino rabbits before and after inflammation induction. Both batches confirmed to be effective to treat and prevent ocular inflammation.

Bicelles: New Lipid Nanosystems for Dermatological Applications.

Bicelles have emerged as promising membrane models, and due to their attractive combination of lipid composition and physicochemical characteristics, they have become new nanostructures for biomedical research. Depending on the composition, temperature and other experimental factors, these nanosystems exhibit high structural and morphological versatility. Additionally, bicelles are able to modulate the biophysical parameters and barrier function of skin. Given these properties, these nanostructures appear to be smart nanosystems with great potential in biomedicine and dermopharmacy.

Nanoemulsions for dermal controlled release of oleanolic and ursolic acids: In vitro, ex vivo and in vivo characterization.

The aim of the present study was to design and optimize a nanoemulsion for dermal administration of mixtures of natural or synthetic pentacyclic triterpenes with recognized anti-inflammatory activity. The composition of the developed nanoemulsions was obtained from pseudo-ternary phase diagrams, composed of castor oil as the oil phase, labrasol as the surfactant, transcutol-P as co-surfactant and propylene glycol as the aqueous phase. Different ratios of surfactant/co-surfactant mixture (Smix) (4:1, 3:1, 2:1, 1:1, 1:2 and 1:4) were produced, and Smix 4:1 was chosen based on the greater area of optimal nanoemulsion conditions. Two different nanoemulsions of mean droplet size below 600 nm were produced, loading mixtures of natural or synthetic pentacyclic triterpenes, respectively. The viscosity of nanoemulsion containing natural pentacyclic triterpenes was 51.97±4.57 mPas and that loaded with synthetic mixtures was 55.33±0.28 mPas. The studies of release and skin permeation were performed using Franz diffusion cells, adjusting the release kinetics of both formulations to Korsmeyer-Peppas model. No significant differences in permeation parameters between the two nanoemulsions were observed. The amount of drug retained in the skin was higher than the amount of drug that has permeated, favoring a local action. The results of the in vivo tests demonstrated that the developed formulations were not toxic and not irritant to the skin. The formulation loading a mixture of natural triterpenes showed greater ability to inhibit inflammation than that loading the synthetic mixture. The findings clearly corroborate the added value of o/w nanoemulsions for dermal delivery of pentacyclic triterpenes.

Permeation studies through porcine small intestine of furosemide solutions for personalised paediatric administration.

Personalized medicine is a challenging research area in paediatric drug design since no suitable pharmaceutical forms are currently available. Furosemide is an anthranilic acid derivative used in paediatric practice to treat cardiac and pulmonary disorders in premature infants and neonates. However, it is not commercialized in suitable dosage forms for paediatrics. Elaborating new paediatric formulations when no commercial forms are available is a common practice in pharmacy laboratories; amongst these, oral liquid formulations are the most common. We developed two extemporaneous paediatric oral solutions of furosemide (pure powder). The characterization and stability study were also performed. Parameters such as organoleptic characteristics, rheology, pH, content of active substance, and microbial stability were evaluated at three temperatures for two months. Evaluation of all these parameters showed that both solutions were stable for 60 days at 4 and 25 °C. Moreover, ex vivo studies were performed to evaluate the permeation behaviour of developed solutions through porcine small intestine to evaluate the potential paediatric biological parameters influencing the bioavailability and efficacy. A validated spectrofluorometric method was also used for this purpose. Our results guarantee a correct dosification, administration and potential efficacy of furosemide when is formulated in liquid oral forms for the treatment of cardiac and pulmonary disorders in children.

Design and physicochemical stability studies of paediatric oral formulations of sildenafil.

Personalized medicine is a challenging research area in paediatric treatments. Elaborating new paediatric formulations when no commercial forms are available is a common practice in pharmacy laboratories; among these, oral liquid formulations are the most common. But due to the lack of specialized equipment, frequently studies to assure the efficiency and safety of the final medicine cannot be carried out. Thus the purpose of this work was the development, characterization and stability evaluation of two oral formulations of sildenafil for the treatment of neonatal persistent pulmonary hypertension. After the establishment of a standard operating procedure (SOP) and elaboration, the physicochemical stability parameters appearance, pH, particle size, rheological behaviour and drug content of formulations were evaluated at three different temperatures for 90 days. Equally, prediction of long term stability, as well as, microbiological stability was performed. Formulations resulted in a suspension and a solution slightly coloured exhibiting fruity odour. Formulation I (suspension) exhibited the best physicochemical properties including Newtonian behaviour and uniformity of API content above 90% to assure an exact dosification process.

Potential use of nanostructured lipid carriers for topical delivery of flurbiprofen.

The potential use of nanostructured lipid carriers (NLC) composed of a fatty acid [stearic acid (SA)] or a triglyceride (glyceryl behenate) as solid lipids, and a mixture of medium chain triglycerides and castor oil as liquid lipids, for skin administration of flurbiprofen (FB), has been explored. Two different optimized NLC formulations (FB-SANLC based on SA vs. FB-C888NLC based on glyceryl behenate), with respect to the morphometrical properties (particle size and polydispersity index) and the entrapment efficiency, were used in this study. The ex vivo permeation profiles of FB-C888NLC, FB-SANLC and conventional FB solution were evaluated using human skin. An improved FB permeation was observed when the drug was delivered by skin application of FB-C888NLC, attributed to the particle size and matrix crystallinity. The differential scanning calorimetry and X-ray diffraction studies suggested major polymorphic transitions in the lipid matrix of FB-C888NLC. A good correlation between polymorphic transitions and increased drug permeation was observed. However, both NLC dispersions showed a penetration-enhancing ratio (ER) higher than conventional FB solution. The in vitro and in vivo irritancy and local tolerability were assessed by running, respectively, the SKINTEX™ and Draize test. Both FB-C888NLC and FB-SANLC were classified as nonirritant.

Optimizing flurbiprofen-loaded NLC by central composite factorial design for ocular delivery.

The purpose of this study was to design and optimize a new topical delivery system for ocular administration of flurbiprofen (FB), based on lipid nanoparticles. These particles, called nanostructured lipid carriers (NLC), were composed of a fatty acid (stearic acid (SA)) as the solid lipid and a mixture of Miglyol(®) 812 and castor oil (CO) as the liquid lipids, prepared by the hot high pressure homogenization method. After selecting the critical variables influencing the physicochemical characteristics of the NLC (the liquid lipid (i.e. oil) concentration with respect to the total lipid (cOil/L (wt%)), the surfactant and the flurbiprofen concentration, on particle size, polydispersity index and encapsulation efficiency), a three-factor five-level central rotatable composite design was employed to plan and perform the experiments. Morphological examination, crystallinity and stability studies were also performed to accomplish the optimization study. The results showed that increasing cOil/L (wt%) was followed by an enhanced tendency to produce smaller particles, but the liquid to solid lipid proportion should not exceed 30 wt% due to destabilization problems. Therefore, a 70:30 ratio of SA to oil (miglyol + CO) was selected to develop an optimal NLC formulation. The smaller particles obtained when increasing surfactant concentration led to the selection of 3.2 wt% of Tween(®) 80 (non-ionic surfactant). The positive effect of the increase in FB concentration on the encapsulation efficiency (EE) and its total solubilization in the lipid matrix led to the selection of 0.25 wt% of FB in the formulation. The optimal NLC showed an appropriate average size for ophthalmic administration (228.3 nm) with a narrow size distribution (0.156), negatively charged surface (-33.3 mV) and high EE (∼90%). The in vitro experiments proved that sustained release FB was achieved using NLC as drug carriers. Optimal NLC formulation did not show toxicity on ocular tissues.

Rapid human skin permeation and topical anaesthetic activity of a new amethocaine microemulsion.

We developed a fast-acting, topical, 4% (w/w) amethocaine microemulsion and tested its in vitro permeation in isolated human skin. Comparison with a commercial amethocaine gel (Ametop((R)) ) was performed using Franz diffusion cells. Permeability coefficient (k(p)), flux (J) and percentage permeation after 10 h of microemulsion application were, in all cases, 1.5 times higher than those of the gel. The values obtained for the P(1) parameter [1], 1.06.10(-2) cm (microemulsion) and 0.724.10(-2) cm (gel) indicate that the microemulsion excipients favour amethocaine deposition in the skin, increasing the permeability coefficient, amount of drug retained in the skin, and the flux achieved. Analgesic activity was also examined in rats made hyperalgesic or allodynic after carrageenan-induced inflammation. The rats were distributed into four groups (n = 5-9 per group), each group receiving topically either amethocaine microemulsion, amethocaine gel (Ametop), amethocaine subcutaneous infiltration or nothing (controls). In edematous paws, anti-hyperalgesic activity appeared at 4.2 and 13.8 min after application of amethocaine microemulsion and gel, respectively. These effects are lower than after 0.5% w/w amethocaine infiltration. Amethocaine microemulsion was the only topical formulation with an anti-allodynic effect, although this effect was less than with amethocaine infiltration. These results suggest that microemulsion could be a valuable formula for improving amethocaine permeation and thus bringing rapid pain relief.

Rapid skin anesthesia using a new topical amethocaine formulation: a preclinical study.

UNLABELLED: We developed a fast-acting topical amethocaine emulsion and tested its analgesic activity against heat or mechanically induced pain in a rat paw model. The first experiment was performed in rats made hyperalgesic or allodynic after carrageenan-induced inflammation. Rats were distributed in five subgroups, each receiving topically one of the following: amethocaine microemulsion, amethocaine gel (Ametopgel), EMLA (Eutectic Mixture of Local Anesthetics) cream, amethocaine infiltration, or nothing (controls). The second experiment was conducted on healthy, selected heat- or touch-hypersensitive rats, which were distributed as in the first experiment. Paw withdrawal time from a heat and a mechanical stimulus was used as a pain index. In the first experiment, antihyperalgesic activity appeared at 4.2, 13.8, and 14 min after amethocaine microemulsion, gel, or EMLA cream, respectively. Amethocaine microemulsion was the only topical formulation with an antiallodynic effects, although less than with amethocaine infiltration. In healthy rats (second experiment), all topical formulations produced similar analgesic effects in heat-induced pain of the ipsilateral paw. Activity in the contralateral paw appeared earlier with amethocaine microemulsion, which was also the only one that increased touch-induced withdrawal time in the ipsi- and contralateral paws. Therefore, the microemulsion could be valuable for improving amethocaine skin penetration and thus bringing rapid pain relief. IMPLICATIONS: Topical anesthetics are used in several painful clinical procedures, but they tend to have a slow onset time. A new amethocaine microemulsion with a faster onset of analgesia than commercial formulations was developed and its activity tested in pain states induced by heat or mechanical stimulus in inflamed and healthy rat paws.

Comparative study of morphine diffusion from sustained release polymeric suspensions.

In recent years, great efforts have been devoted to the design of drug delivery systems. Many polymeric excipients have been studied in order to make drug release fit the desired profiles. The aim of this work was to design a morphine oral suspension, as sustained release pharmaceutical formulations. To this end, two different ethylcellulose suspensions were prepared: one with the drug incorporated during synthesis (suspension A) so that the drug was inside the polymeric microparticles. In the second group of suspensions the drug was incorporated after synthesis (suspension B), thus resulting in the drug being adsorbed on the surface. The analytical technique used, spectrophotometry, showed that suspensions A were able to spontaneously encapsulate approximately 92% of the drug, whereas suspensions B adsorbed only 15% dose on the particle surface. Moreover, the diffusion results obtained with Franz-cells showed that suspensions A offered the possibility of easy control of the release rate of the active substance. This system transfers morphine hydrocloride during 24 h in accordance with a Weibull kinetic model. This dosage form presents the clinical advantage of less frequent dosing, with increased quality of life for patients. This report documents the suitability of our ethylcellulose polymeric suspension for encapsulated morphine with a controlled release rate.

Influence of the formulation on the in vitro transdermal penetration of sodium diclofenac. Evaluation of the topical and systemic anti-inflammatory activity in the rat.

A study on the transdermal permeation through human skin was performed with a series of 6 semisolid formulations (A-F) containing 1% sodium diclofenac (CAS 15307-79-6) (w/w). A commercially available drug preparation was tested as a reference. Based on permeation characteristics, a study on the topical and systemic anti-inflammatory activities of three formulations (A, F and the reference formulation) was conducted using the model of erythema induced by UV radiation in hairless rats. This is expected, together with the index of topical anti-inflammatory activity to allow the selection of the most suitable formulation for dermal application. The following representative parameters were measured in the permeation study: amount of diclofenac permeated at 24 h, flow, lag time and amount of drug retained in skin at 24 h. Of the formulations tested, diclofenac formulated in the reference formulation showed the highest values of amount of diclofenac permeated at 24 h, amount of drug retained in skin at 24 and flow. As regards the skin inflammation test, no significant differences (p < 0.05) are seen between the topical and systemic anti-inflammatory activities of the three formulations tested. However, in absolute value, formulation F shows a lower systemic activity, which would prevent potential side effects of diclofenac. Since the topical anti-inflammatory index obtained for this formulation was > 1, it is concluded that a good therapeutic performance could be obtained in the treatment of local inflammation with diclofenac by using formulation F.

Influence of formulation on the in vitro transdermal penetration of flutrimazole.

Flutrimazole (1-[(2-fluorophenyl)(4-fluorophenyl)phenylmethyl]-1 H-imidazole, CAS 119006-77-8, UR-4056) is a new wide spectrum local imidazolic antifungal agent that has already been formulated as a dermal cream (FDC). A comparative study was carried out of the release of flutrimazole from two emulsions in which the drug has been incorporated differently: one dissolved in the oily phase (E24) and the other dispersed in the aqueous formulation phase (E25). Based on the E25 formulation, two more dermal creams were prepared, E27 with benzyl alcohol and E28 with diazolidinyl urea as preservative agents. A comparative study of transdermal penetration including E27, E28, FDC (reference 1% flutrimazole dermal cream) and 1% flutrimazole hydroalcoholic solution was also performed. An amount of the sample dosage form containing 10 mg of flutrimazole was applied to a Franz type cell. The penetration membrane used was cellulose acetate in the release studies and human skin provided by a plastic surgery clinic in the transdermal penetration study. The amount released after 7 h was 36.3 +/- 4.9 micrograms when flutrimazole was dissolved (E24) and 35.9 +/- 5.3 micrograms when flutrimazole was dispersed (E25). Although the differences were not significant, the cream with dispersed flutrimazole was selected for further penetration studies due to its better stability observed in previous studies. The amounts of drug penetrated after 44 h were 31.3, 41.5, 38.3 and 186.5 micrograms for E27, E28, FDC dermal creams and topical hydroalcoholic solution, respectively. The solution showed a statistically significant difference (p < 0.05) from the other formulations, however, no differences were observed between the dermal cream formulations. No differences were neither obtained between the different dermal creams when the amount of drug retained in the skin was compared. This allows to assert that the excipients used do not have different influences on transdermal penetration. In all cases, the mean quantity penetrated in relation to the dose applied was at most 0.5%. These results allow to infer that flutrimazole shows scarce transdermal penetration. Further, the amount of flutrimazole retained per gram of skin is more than 100 times the MIC per gram obtained in previous in vitro studies. It may be assumed that the topical application of the new formulations assayed would allow to obtain a good therapeutic response.

Structure-absorption relationships of a series of 6-fluoroquinolones.

The physicochemical constants and some structural parameters (topological, steric, and electronic) of eight third-generation monofluorate quinolones (six uncommercialized and two used clinically [ciprofloxacin and enrofloxacin]) were determined: pKa, intrinsic solubility (S0), chromatographic capacity factor, partition coefficient (P), valency molecular connectivity, molecular volume, molecular surface area, dipolar moment, and charges associated with each atom of the molecule. The apparent intestinal absorption rate constants (K(abs)) in rat (in vivo perfusion) and the MICs at which 90% of the isolates are inhibited (MIC90s) against 100 Escherichia coli strains were also determined. We sought to establish simple nonlinear and multiple linear correlations between K(abs), on the one hand, and lipophilic parameters and other physicochemical and structural parameters estimated. Of the nonlinear functions examined, the hyperbolic had the best correlation between K(abs) and P, which was in accordance with the Wagner-Sedman (J. G. Wagner and A. J. Sedman, J. Pharmacokinet. Biopharm. 1:23-50, 1973) equation, whereas, after application of the stepwise multiple linear regression method, a multiple linear correlation with some predictive value could be established only between K(abs) as a dependent variable and log P and log S0 as independent variables. In conclusion, the K(abs) and MIC90 of the quinolone CNV 8902 suggest that it is a sufficiently interesting compound to warrant the investigation of its potential therapeutic use orally.

A comparative in vitro study of transdermal absorption of antiemetics.

Transdermal absorption of a series of antiemetics (alizapride, bromopride, clebopride, domperidone, metoclopramide, metopimazine, and scopolamine) was studied in vitro with the skin of hairless rats as the membrane. The aim of the study was to determine the permeation parameters [transdermal permeability rate constant (Kp), lag time, and flux] as a measure of the intrinsic permeability of these drugs across the skin, with a view to predicting their potential therapeutic formulation in Transdermal Therapeutic Systems. A linear correlation was established between the log Kp values corresponding to the antiemetics studied and their melting point (r = 0.8120, p < 0.05). The logarithm of Kp for the antiemetics studied can be predicted from the logarithm of the intrinsic partition coefficient (n-octanol-water) by a parabolic function (r = 0.9284, p < 0.01). Bromopride showed the shortest lag time (19.73 h), whereas clebopride was the most suitable drug as a candidate for formulation in transdermal delivery systems.

Influence of the chromatographic capacity factor (log k') as an index of lipophilicity in the antibacterial activity of a series of 6-fluoroquinolones. Relationship between physico-chemical and structural properties and their hydrophobicity.

The aim of this study was to establish the influence of lipophilicity on the antibacterial activity (log 1/MIC50) of 22 fluoroquinolones and to assess the influence of their electronic, steric and topological properties on their hydrophobicity. The lipophilicity of the compounds, expressed as the chromatographic capacity factor (log k'), was determined by ion-pair reversed-phase HPLC. On the basis of the mathematical models developed, an attempt was made to confirm the mechanism of interaction of the quinolones with DNA-gyrase proposed previously.

Gastric, intestinal and colonic absorption of a series of beta-blockers in the rat.

Gastric, intestinal and colonic absorption rates of a series of eleven beta-blockers (alprenolol hydrochloride, atenolol, bunolol hydrochloride, penbutolol sulphate, pronethalol hydrochloride, metoprolol, oxprenolol, bevantolol, bufuralol, propranolol hydrochloride and timolol maleate) were estimated using Doluisio's method. The gastric absorption rate was very low and the absorption rate constant could not be assessed accurately in all cases. In the small intestine, the absorption rate constants, Ka, at pH 6.2 ranged between 0.38 h-1 for atenolol and 4.28 h-1 for penbutolol. In the colon, the rate of drug absorption at pH 7.5 ranged between 0.12 h-1 for atenolol and 2.15 h-1 for penbutolol. In most cases, colonic absorption rate constants were of the same order as those obtained in the small intestine, demonstrating the good penetrability through colonic membrane of the series studied. The relationship between absorption rate constants found in the small intestine and colon and the partition constant ([1/Rf]-1), was studied for this non-homologous series of beta-blocker drugs. In both cases, the functional hyperbolic absorption model proposed by Wagner and Sedman [1973] was the most representative.

Skin density in the hairless rat. Evidence of regional differences.

Skin densities of different regional sites have been determined in the hairless rat. The following regional sites have been considered: abdominal, dorsal, foreleg, rearleg, pectoral, neck (ventral and dorsal) and scrotal. Skin density values were determined by means of an hydrostatic balance. The dorsal regional site and the neck dorsal show the highest density values (1.076 and 1.070 respectively). The scrotal regional site shows the lowest density value 1.041. The abdominal density mean value (1.054) was not different from the foreleg, rearleg, pectoral and neck (ventral) regional sites. The knowledge of these density values will be useful to estimate skin/vehicle partition coefficients.

Intestinal absorption of a series of 6-fluoquinolone derivatives: a comparative study.

The intestinal absorption rate constants of the five 6-fluoquinolones (norfloxacin, ciprofloxacin, pefloxacin, CNV 8802 and CNV 8804), have been estimated in the rat (n = 5) by means of an "in situ" intestinal perfusion method. Remaining 6-fluoquinol one concentrations in the perfusion liquid at fixed time (15, 30, 45, 60, 75, 90 and 120 minutes) were determined using a HPLC procedure with UV detection. Absorptions rate constants were estimated according to first order kinetics. A simple non-linear correlation between Ka and log K' on the one hand and a multiple linear correlation between Ka and the structural theoretical parameters: molar volume, dipolar moment and the charge associated to nitrogen 18 on the other, were performed. Only a correlation between Ka values as dependent variable versus dipolar moment and molecular volume values has been obtained, but this correlation is not statistically significant (p = 0.1808) and not accurate enough to have predictive value.