Increased efficacy of acyclovir-loaded microparticles against herpes simplex virus type 1 in cell culture.

Acyclovir is one of the most effective and selective agents against viruses of the herpes group. In order to increase its antiviral activity, acyclovir loaded microparticles, prepared by an O/W solvent evaporation method were developed. Their antiviral activity against herpes simplex virus type 1 (HSV-1) and toxicity were evaluated on Vero cells and then compared with those presented by a drug solution. The 50% inhibitory concentration (IC(50)) values for acyclovir loaded microspheres determined by plaque reduction assays at 48 and 96 h, were found to be 1.06 +/- 0.01 microM and 0.15 +/- 0.03 microM, respectively, while the equivalent values obtained for an acyclovir solution were 1.28 +/- 0.04 microM at 48 h and 0.27 +/- 0.02 microM at 96 h. These results indicate that acyclovir shows a higher antiviral activity, against herpes simplex virus type 1, when this drug was loaded in microparticles rather than as a drug solution, especially after 96 h of incubation. The toxicity of these microparticles was determined by the MTT test at 48 and 96 h. At 48 h only a small toxicity was found (cell viability ranged from 72 to 82%, with the higher concentration tested) and it could not be attributed to the microparticles, since the acyclovir control solution showed similar toxicity values. However, after 96 h a higher toxicity was observed with acyclovir microparticles as well as with the unloaded ones (cell viability located between 60 and 70%). In summary, acyclovir-loaded microparticles have shown to be promising carriers for the effective delivery of acyclovir in the treatment of HSV-1 infections in cells so they can have a potential use in vivo.

Optimization of topical cidofovir penetration using microparticles.

Cidofovir is a new class of antiviral agent with potent in vitro and in vivo activity against a broad spectrum of herpes viruses. The aim of this work was to obtain a prolonged therapeutic effect of cidofovir in the basal epidermis after its topical application. For this purpose, poly(lactide-co-glycolide) (PLGA) microparticles were prepared by solvent evaporation and spray-drying methods. Microparticles prepared by spray-drying showed a encapsulation efficiency of 80%. Conversely, for all the microspheres prepared by the W/O/W solvent evaporation method the encapsulation efficiency was low. Also, microparticles prepared by spray-drying showed a higher burst release. Skin penetration and distribution experiments were carried out with cidofovir-loaded microparticles prepared by spray-drying, since these carriers presented the best characteristics in terms of size and encapsulation efficiency. A cidofovir solution in 0.2% PVA served for comparison. Penetration experiments were carried out in Franz type diffusion cells with an available diffusion area of 1.76 cm(2), using porcine skin. The results obtained showed that the amount of cidofovir penetrated, over a 24 h time period, was higher with the drug solution than with microparticles. Cidofovir distribution in porcine skin, after topical application of microparticles and drug solution for 24 h, was determined by horizontal slicing of the skin. The profiles obtained for the two formulations showed that the quantity of cidofovir retained in the skin decreased with the depth. Besides the amount of cidofovir found in the basal epidermis (120-150 microm) was much higher with microparticles than with the control solution. These data showed that cidofovir-loaded microparticles could improve cidofovir topical therapy since these vehicles increased drug retention in the basal epidermis and decreased its penetration through the skin.

Determination of cidofovir in both skin layers and percutaneous penetration samples by HPLC.

The aim of this study was to develop a direct, simple and rapid high performance liquid chromatographic method for the determination of cidofovir in both skin layers and percutaneous penetration experiments. Samples were chromatographed on a reversed phase encapped column 250 x 4 mm C(8) LiChrospher Select B. The phase mobile consisted on 3% of acetonitrile and 97% of 1.5 mM of tetrabutylammonium dihydrogen phosphate (TADP) and 3.5 mM of disodium hydrogenphosphate adjusted to pH 6. Detection was at 274 nm and the run time was 14 min. The limit of detection was 0.06 microg/ml. The detector response was found to be linear in the concentration range 0.1-10 microg/ml. This assay is a selective, sensitive and reproducible method for the quantification of cidofovir in skin layers and in the receptor compartment of Franz-type diffusion cells after percutaneous studies.

Transdermal lontophoresis and skin retention of piroxicam from gels containing piroxicam: hydroxypropyl-beta-cyclodextrin complexes.

Iontophoretic transport of piroxicam (Px) across porcine ear skin in vitro was investigated. Cathodal iontophoresis of negatively charged Px was carried out from gel formulations containing Px as an inclusion complex with hydroxypropl-beta-cyclodextrin (HP-beta-CD). From the gels, following a 7h application period at 0.4 mA/cm2, iontophoresis delivered 3.4 times more drug than passive diffusion. The formation of Px:-HP-beta-CD complexes did not increase the iontophoretic Px flux through the skin. However, Px complexation with HP-beta-CD allowed us to increase the drug concentration in the gel; because of that, the amount of Px transported across the skin increased considerably. After iontophoretic experiments, the amount of Px retained in skin seemed to be related to the flux values obtained in each case. Skin pretreatment with 20% HP-beta-CD, tested passively and iontophoretically for 3h, followed by the application of gel containing Px: HP-beta-CD complexes, showed no enhancing capacity in any case. The amount of Px retained in the skin after pretreatment experiments was found to be very similar to that obtained without skin pretreatment and was observed to be related to the Px flux through the skin.

Effect of passive and iontophoretic skin pretreatments with terpenes on the in vitro skin transport of piroxicam.

The enhancing effect of several terpenes (thymol, menthone and 1,8-cineole) in the percutaneous permeation of piroxicam (Px), either passive or iontophoretically, was investigated. These terpenes were applied, on the skin membrane, as a passive and iontophoretic skin pretreatment. Px was delivered from carbopol gels containing hydroxypropyl-beta-cyclodextrin (2% w/w Px). An increase in Px flux values, both passive and iontophoretic after skin pretreatment with 5% terpenes/50% EtOH, was found to be in the following order: thymol>menthone>1,8-cineole. Iontophoretic skin pretreatment with terpenes produced a slight increase in the passive flux of Px, in comparison with the passive skin pretreatment. This result indicated that iontophoresis could modify the skin morphology and consequently, increase the passive transport of Px. However, when Px was transported iontophoretically, passive skin pretreatment with terpenes, produced higher flux values than iontophoretic skin pretreatment. These results could be explained by the fact that with the iontophoretic pretreatment, terpenes could penetrate into the skin and limitate the movement of the ionized species, across the skin, during the iontophoretic experiments. The amount of Px retained in the skin after all experiments was related to flux values across skin.

PLGA microparticles: possible vehicles for topical drug delivery.

Distribution of PLGA-microparticles in porcine skin, after its topical application, was studied in vitro using microparticles containing rhodamine as a fluorescent probe. PLGA-microparticles loaded with rhodamine were prepared using a solvent evaporation technique. Skin distribution of fluorescent microparticles was performed, by horizontal and vertical slicing of frozen skin. Fluorescence photomicrographs revealed that PLGA-microparticles could penetrate through the stratum corneum and reach the epidermis. However, permeation experiments showed that these microparticles were not able to reach the receptor compartment of the diffusion cells, even in a period of 24 h. The carriers described in this work could be used as vehicles for topical drug delivery, in order to obtain a sustained drug release into the skin, improving therapy by reduction of time intervals between doses.

Sensitive LC determination of piroxicam after in vitro transdermal permeation studies.

A direct, very sensitive, simple and rapid high-performance liquid chromatographic (HPLC) method for the determination of piroxicam, with tenoxicam as internal standard, has been developed and validated. Samples were chromatographed on a 5 microm Scharlau C(18) column. The mobile phase was a mixture of acetonitrile-acetic acid 4% (pH 2.8) (45:55, v/v). Detection was at 354 nm and the run time was 7 min. The limit of detection was 0.025 microg/ml. The detector response was found to be linear in the concentration range 0.05-9 microg/ml. This HPLC assay has been applied to measure the 'in vitro' percutaneous permeation of piroxicam through abdominal hairless rat skin, using Franz-type diffusion cells, in order to obtain the concentration-time profiles of piroxicam.

Combined effect of oleic acid and propylene glycol on the percutaneous penetration of tenoxicam and its retention in the skin.

The influence of oleic acid (OA) on the in vitro percutaneous absorption of tenoxicam (TEN) and its combined effect with propylene glycol (PG) was studied using Franz-type diffusion cells. Furthermore, at defined concentrations of OA, complexes of the drug with cyclodextrins (MbetaCD and gammaCD) were added because their combined use may be an interesting approach to raise TEN flux. In addition, the amount of TEN retained in the skin after topical administration of several formulations was determined. It was found that OA content markedly increased TEN absorption when compared to the control gel; the highest drug flux was obtained by 15% of OA. The absorption rate of TEN increased in parallel with increasing OA concentration, due to the alteration of the stratum corneum caused by this enhancer. Moreover, the action of OA is likely to be strongly dependent on the vehicle used since drug penetration tended to increase with increasing PG content in the vehicle, especially at the high OA concentrations. Contrary to our expectations, addition of CD complexes did not produce a significant further enhancement. Skin pretreatment with OA, independently of the vehicle used to dissolve the fatty acid, dramatically improved TEN percutaneous penetration. The amount of TEN retained in the skin was related to the flux values obtained with each formulation.

Topical application of acyclovir-loaded microparticles: quantification of the drug in porcine skin layers.

The goal of this work was to increase the amount of acyclovir (ACV) in the basal epidermis, site of Herpes virus simplex infections, using microparticles as carriers. Poly(D,L-lactic-co-glycolic acid) microparticles loaded with ACV were prepared using a solvent evaporation technique. ACV distribution into porcine skin after topical application of microparticles for 6, 24 and 88 h, was determined by horizontal slicing of the skin. An ACV suspension served for comparison. The results showed that, at 6 and 24 h, the quantity of the drug in the basal epidermis with the microparticles, is similar to that obtained with the ACV suspension. However, after 88 h, the ACV reservoir in the basal epidermis was higher with the microparticles compared with the control suspension. This could be explained by the controlled drug release produced by the vector in the basal epidermis. Besides, at 88 h the amount of ACV detected in the receptor chamber of the diffusion cells was much lower with the microparticles than with the suspension. This type of carrier can improve acyclovir topical therapy since it increases drug retention in the basal epidermis and consequently increases the time intervals between doses.

Influence of piroxicam: hydroxypropyl-beta-cyclodextrin complexation on the in vitro permeation and skin retention of piroxicam.

The interactions between piroxicam (Px) and hydroxypropyl-beta-cyclodextrin (HPbetaCD) were thoroughly investigated both in solution and the solid state. The solubility studies have demonstrated the formation of a Px:HPbetaCD inclusion complex with 1:1 stoichiometry. The addition of propylene glycol to the medium produced less stable complexes, revealing the fact of this co-solvent probably acting as a competing agent. Equimolecular Px:HPbetaCD solid systems were prepared using the co-precipitation method and then fully characterized by X-ray diffractometry, infrared spectra and differential scanning calorimetry. A topical gel formulation containing Px, as inclusion complex with HPbetaCD, was developed in order to study the influence of Px complexation on its release rate and skin percutaneous permeation. The formation of Px:HPbetaCD complexes did not increase either Px release from the vehicle or its skin permeation. However, Px complexation with HPbetaCD allowed the incorporation of a higher quantity of Px into the gel, which resulted in a considerable increase in the Px released and permeated. Skin pretreatment with different HPbetaCD solutions, followed by the application of control gel, showed no enhancing capacity. The amount of Px retained in the skin, after pretreatment experiments, was found to be very similar to that obtained without skin pretreatment and was observed to be related to flux values through the skin.

Interaction of tenoxicam with cyclodextrins and its influence on the in vitro percutaneous penetration of the drug.

Solid complexes of tenoxicam (TEN) with cyclodextrins (CDs), in a 1:1 molar ratio, were obtained by the coprecipitation method and characterized by x-ray diffractometry, infrared spectroscopy, and differential scanning calorimetry. The binding capacity of the CDs with TEN was also demonstrated in aqueous solution and in water-propylene glycol mixtures. The purpose of this study was to determine the effect of CDs on the in vitro percutaneous penetration of TEN from carbopol gels, taking into account the role of the CD cavity size and the nature of the substituents. The effect of pretreatment was studied too. In vitro permeation experiments were carried out on Franz diffusion cells using cellulose nitrate membranes and abdominal rat skin. In these results, the release rates of the drug scarcely decreased when the CDs were added, probably because of a lower concentration of the free drug and an increased gel viscosity. However, it was also found that CDs, particularly gamma-CD and M-beta-CD, can improve slightly TEN absorption through the skin. Pretreatment studies with CDs, however, provided no effects on TEN permeation, but lag time was markedly reduced, suggesting a faster partitioning of TEN into the skin. Therefore, the use of pretreatment with CDs would be interesting when a quick action of the drug is desired.

Effect of skin pretreatment with fatty acids on percutaneous absorption and skin retention of piroxicam after its topical application.

The enhancing effect of several fatty acids from different subclasses: saturated (lauric acid), mono-unsaturated (oleic acid) and poly-unsaturated (linoleic and linolenic acids) in the percutaneous absorption of piroxicam was investigated. These fatty acids were applied on the skin membrane in three different ways: included in the vehicle, as a pretreatment or both. An increase in piroxicam flux value was found for lauric and oleic acids in the following order: skin pretreatment with 5% fatty acids followed by application of gels containing 5% fatty acids>skin pretreatment with 5% fatty acids followed application of control gel>gel containing 5% fatty acids without skin pretreatment. For linoleic and linolenic acids, the piroxicam flux in the two pretreatment experiments was almost the same, although higher than when fatty acids were included in the formulation. Skin pretreatment with 5% linolenic acid in propylene glycol followed by application of control gel or a gel containing 5% linolenic acid, showed the highest enhancing capacity. After skin pretreatment with fatty acids, the lag time values decreased nearly three times compared to those obtained when the same fatty acids were included in the formulation. The amount of piroxicam retained in the skin after pretreatment with fatty acids was found to be very similar for all fatty acids and 3-fold higher than in the experiments without skin pretreatment.

Determination by high-performance liquid chromatography of ketoprofen in vitro in rat skin permeation samples.

A direct, simple and rapid high-performance liquid chromatographic method has been developed for the determination of ketoprofen with ibuprofen as internal standard. Samples were chromatographed on a 5 microm Kromasil 100 C18 column. The mobile phase was a mixture of acetonitrile-0.01 M KH2PO4 adjusted to pH 1.5 with orthophosphoric acid 85% (60:40, v/v). Detection was at 260 nm and the run time was 10 min. The detector response was found to be linear in the concentration range 0.02 to 40 microg/ml. This HPLC assay has been applied to measure the "in vitro" percutaneous penetration of ketoprofen through rat skin.

Influence of propylene glycol and isopropyl myristate on the in vitro percutaneous penetration of diclofenac sodium from carbopol gels.

The influence of propylene glycol (PG) on the in vitro penetration of diclofenac sodium (DFS) through a synthetic membrane and abdominal rat skin from carbopol gels was investigated using Franz-type diffusion cells. The combined effect of isopropyl myristate (IPM) and PG was also evaluated. It was found that the penetration through the synthetic membrane was well described by the Higuchi model. The gel containing 40% PG showed the highest release rate, indicating that a releasing maximum exists for PG content which provides the fully solubilized drug in the vehicle. When using rat skin as the barrier, the penetration rate was controlled by the membrane. DFS flux decreased with increasing PG content of the gels due to an increase of the drug affinity to the vehicle. A cosolvent action of PG was evident. However, the combination of PG and IPM resulted in a synergistic enhancement of DFS flux. Maximum enhancing activity was obtained from gels containing 40% PG, which yielded an enhancement ratio of about 8. Increasing IPM content from 3 to 5% increased the flux and decreased the lag time taken to reach a steady-state level.

Surfactant effects on the in vitro percutaneous absorption of diclofenac sodium.

Nonionic surfactants, which are a safe class of enhancers, may offer means of enhancing drug permeation through the skin. In order to determine this effect, the influence of four nonionic surfactants on the percutaneous absorption of diclofenac sodium from carbopol gels containing 40% propylene glycol was investigated. In vitro diffusion experiments were carried out using excised full-thickness abdominal rat skin as well as cellulose nitrate membranes. The data of this study clearly revealed that Tween 80 decreased diclofenac penetration rate. This was due to a decrease in thermodynamic activity as a result of micellar complexation. In contrast, the more hydrophobic sorbitans enhanced diclofenac skin penetration, probably due to changes in the barrier properties of the skin and in the vehicle-stratum corneum partition coefficient. The most enhancing effect was induced by Span 20, a surfactant with a C12 saturated hydrophobic group. However, diffusional lag times for all the tested surfactants were longer than for the control gel.

In vitro percutaneous absorption of piroxicam through synthetic membranes and abdominal rat skin.

The in vitro release of piroxicam from carbomer gels and its penetration through isopropyl myristate impregnated membranes and abdominal rat skin were investigated. Attempts were made to relate the differences in the release rate with physicochemical properties of the drug and the vehicle. The results showed that piroxicam is released from the topical gel formulations and diffuses through skin. It is suggested that although piroxicam flux across abdominal rat skin was lower than through isopropyl myristate membranes, this kind of membranes can be used in preliminary screening among the different piroxicam formulations.

Role of surfactants in the bioavailability of intranasal insulin.

The nasal mucosa has good physiological characteristics to allow the administration through other routes presents some problems, such is the case of insulin. The presence of surfactants is necessary to get high bioavailability.