Marjoram extract prevents ischemia reperfusion-induced myocardial damage and exerts anti-contractile effects in aorta segments of male wistar rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Marjoram (Origanum majorana L.) is an herb traditionally used as a medicine in different countries, as Morocco and Iran, because of its beneficial cardiovascular effects. Some studies suggest that these effects are due, at least in part, to the presence of phenolic compounds such as rosmarinic acid (RA) and luteolin. AIM OF THE STUDY: To analyze the possible cardiprotective effects of a marjoram extract (ME) reducing myocardial damage after coronary ischemia-reperfusion (IR) and its possible antihypertensive effects reducing the response of aorta segments to the vasoconstrictors noradrenaline (NA) and endothelin-1 (ET-1). MATERIALS AND METHODS: Male Wistar rats (300g) were used. After sacrifice, the heart was immediately removed and mounted in a perfusion system (Langendorff). The aorta was carefully dissected and cut in 2 mm segments to perform vascular reactivity experiments. RESULTS: In the heart, ME perfusion after IR reduced heart rate and prevented IR-induced decrease of cardiac contractility, possibly through vasodilation of coronary arteries and through the upregulation of antioxidant markers in the myocardium that led to reduced apoptosis of cardiomyocytes. In the aorta, ME decreased the vasoconstrictor response to NA and ET-1 and exerted a potent anti-inflammatory and antioxidant effect. Neither RA nor 6-hydroxi-luteolin-O-glucoside, major compounds of this ME, were effective in improving cardiac contractility after IR or attenuating vasoconstriction to NA and ET-1 in aorta segments. CONCLUSION: In conclusion, ME reduces the myocardial damage induced by IR and the contractile response to vasoconstrictors in the aorta. Thus, it may be useful for the treatment of cardiovascular diseases such as myocardial infarction and hypertension.

Nanoemulsions and acidified milk gels as a strategy for improving stability and antioxidant activity of yarrow phenolic compounds after gastrointestinal digestion.

The aim of this study was to improve the stability and antioxidant activity of yarrow phenolic compounds upon an in vitro simulated gastrointestinal digestion. Therefore, two types of caseins-based delivery systems, sodium caseinate stabilized nanoemulsions (NEs) and glucono delta-lactone acidified milk gels (MGs), were formulated containing an ultrasound-assisted yarrow extract (YE) at two concentrations (1 and 2.5 mg/mL). Formulations with 1 mg/mL of YE were chosen based on their higher encapsulation efficiency to perform the in vitro digestion experiments. After digestion, YE-loaded NEs only partially protected phenolic compounds from degradation; meanwhile the phenolic composition of YE including in MGs after digestion was quite similar to undigested YE. Moreover, the antioxidant activity of MGs after digestion was higher than NEs digested samples, which confirms the higher protection of YE phenolic compound by the milk gels systems. This research demonstrated the potential use of acidified MGs as carriers to improve the stability and antioxidant activity of yarrow phenolic compounds. Therefore, these matrices could be employed to develop new dairy products enriched with phenolic compounds.

Supercritical anti-solvent fractionation for improving antioxidant and anti-inflammatory activities of an Achillea millefolium L. extract.

Achillea millefolium L. is a plant widely used in traditional medicine. Nowadays, there is a growing concern about the study of its bioactive properties in order to develop food and nutraceutical formulations. Supercritical anti-solvent fractionation (SAF) of an A. millefollium extract was carried out to improve its antioxidant and anti-inflammatory activities. A selective precipitation of phenolic compounds was achieved in the precipitation vessel fractions, which presented an antioxidant activity twice than original extract, especially when fractionation was carried out at 10 MPa. The main phenolic components identified in this fraction were luteolin-7-O-glucoside, 3,5-dicaffeoylquinic acid, 6-hidroxyluteolin-7-O-glucoside and apigenin-7-O-glucoside. However, separator fractions presented higher anti-inflammatory activity than precipitation vessel ones, particularly at 15 MPa. This fact could be related to separator fractions enrichment in anti-inflammatory compounds, mainly camphor, artemisia ketone and borneol. Therefore, SAF produced a concentration of antioxidant and anti-inflammatory compounds that could be used as high-added valued ingredients.

Insuficiencia hepática crónica y hemostasia / Chronic Liver Failure and Hemostasis

Resumen La insuficiencia hepática es un estado patológico que condiciona la síntesis y metabolismo de diversas biomoléculas, siendo las alteraciones a la hemostasia una de las primeras consecuencias a nivel sistémico que se hacen presentes; debido a esto y por la dimensión de los riesgos de esta situación, no es raro que las pruebas de coagulación sean indispensables para formular las escalas pronósticas en pacientes hepatópatas. Los conocimientos sobre hemostasia han avanzado en la última década; la clásica cascada de activación de los factores de la coagulación ha sido perfeccionada hasta conformarse el ahora vigente modelo celular que considera la valiosa e indispensable participación del endotelio y las plaquetas. Gracias a esto es posible comprender que, en pacientes con insuficiencia hepática, el riesgo de sangrado no obedece únicamente a la deficiencia en la producción de los factores de la coagulación y, por tanto, es cuestionable la administración de vitamina K. Más relevante aún es que, gracias a estos conocimientos, se puede comprender el a veces contradictorio riesgo de trombosis en estos pacientes, complicación potencialmente mortal.

Abstract Hepatic insufficiency is a pathology that conditions synthesis and metabolism of various biomolecules. Alterations of hemostasis is one of its first systemic consequences. Because of this and the size of the risks, it is not uncommon for clotting tests to be indispensable for formulating prognostic scales in patients with liver disease. Knowledge about hemostasis has advanced in the last decade, and the classic cascade of activation of coagulation factors has been perfected until it has become the now-current cellular model that considers the valuable and indispensable participation of the endothelium and platelets. Thanks to this, it is possible to understand that the risk of bleeding in patients who have hepatic insufficiency is not only due to deficiencies in production of coagulation factors, and that for this reason administration of vitamin K is questionable. Even more relevant, is the fact that, thanks to this this knowledge, we can understand the sometimes contradictory and potentially life-threatening complication of thrombosis in these patients.

Phenylalanine hydroxylase deficiency in Mexico: genotype-phenotype correlations, BH4 responsiveness and evidence of a founder effect.

The mutational spectrum of the phenylalanine hydroxylase gene (PAH) in Mexico is unknown, although it has been suggested that PKU variants could have a differential geographical distribution. Genotype-phenotype correlations and genotype-based predictions of responsiveness to tetrahydrobiopterin (BH4 ) have never been performed. We sequenced the PAH gene and determined the geographic origin of each allele, mini-haplotype associated, genotype-phenotype correlations and genotype-based prediction of BH4 responsiveness in 48 Mexican patients. The mutational spectrum included 34 variants with c.60+5G>T being the most frequent (20.8%) and linked to haplotype 4.3 possibly because of a founder effect and/or genetic drift. Two new variants were found c.1A>T and c.969+6T>C. The genotype-phenotype correlation was concordant in 70.8%. The genotype-based prediction to BH4 -responsiveness was 41.7%, this information could be useful for the rational selection of candidates for BH4 testing and therapy.

Caracterización de errores innatos del metabolismo intermediario en pacientes mexicanos / Characterization of inborn errors of intermediary metabolism in mexican patients

Introducción: Los errores innatos del metabolismo intermediario (EIMi) son enfermedades genéticas heterogéneas que causan importante morbimortalidad y representan un reto diagnóstico. El objetivo de este trabajo es describir el número, el tipo y las características clínicas de los pacientes con EIMi en un hospital pediátrico de alta especialidad. Material y métodos: Estudio retrospectivo de 204 expedientes de pacientes diagnosticados con EIMi por sospecha clínica, de enero del 2000 a diciembre del 2012, analizados antes y después de la implementación de la espectrometría de masas en tándem (MS/MS) como herramienta de tamiz selectivo. Resultados: En los 204 casos analizados, se encontraron 25 diferentes tipos de EIMi: 102 con acidurias orgánicas y 100 con aminoacidopatías y 2 con defectos de la beta oxidación. La introducción de la MS/MS incrementó el número de casos detectados en 50%. Los pacientes fueron enviados por 13 diferentes servicios médicos, siendo los pediatras los que remitieron más casos. El intervalo promedio entre el inicio de los síntomas y el diagnóstico fue de 18 meses. Conclusión: En los niños enfermos mexicanos estudiados se encontró una gran variedad de EIMi, destacando los defectos del propionato y la enfermedad de orina de jarabe de arce. En esta población analizada, el diagnóstico de la enfermedad metabólica se realizó en forma muy tardía. Estos resultados pueden servir como evidencia para incorporar los EIMi al tamiz neonatal ampliado, o en su defecto para que se realice el diagnóstico selectivo en todos los niños hospitalizados con datos clínicos indicativos (AU)

Introduction: Inborn errors of intermediary metabolism (IEiM) are a group of heterogeneous genetic diseases that are diagnostically challenging and cause significant morbidity and mortality. The aim of this study is to perform a descriptive analysis of the number, type, and clinical features, in a series of cases with IEiM identified through selective diagnosis in a highly specialized pediatric hospital. Materials and methods: A retrospective study was performed from January of 2000 to December of 2012 by analyzing the files of 204 patients with an IEiM, by selective screening, before and after the implementation of tandem mass spectrometry (MS/MS).Results: A total of 25 different types of IEiM were found in the 204 files; 102 organic acidurias, 100 aminoacidopathies, and 2 fatty acid oxidation disorders. The introduction of MS/MS increased the number of cases detected by 50%. Patients were referred from 13 different specialists, with pediatricians being the most active. The average interval between onset of symptoms and diagnosis was 18 months. Conclusion: Among the sick Mexican children studied, a wide variety of IEiM was found, propionate defects and maple syrup urine disease being noteworthy. The diagnosis of metabolic disease was delayed in the population studied. These results present evidence to perhaps incorporate IEiM into an expanded newborn screening, or else to perform selective diagnosis in all hospitalized children with suggestive clinical data (AU)

[Characterization of inborn errors of intermediary metabolism in mexican patients]. / Caracterización de errores innatos del metabolismo intermediario en pacientes mexicanos.

INTRODUCTION: Inborn errors of intermediary metabolism (IEiM) are a group of heterogeneous genetic diseases that are diagnostically challenging and cause significant morbidity and mortality. The aim of this study is to perform a descriptive analysis of the number, type, and clinical features, in a series of cases with IEiM identified through selective diagnosis in a highly specialized pediatric hospital. MATERIALS AND METHODS: A retrospective study was performed from January of 2000 to December of 2012 by analyzing the files of 204 patients with an IEiM, by selective screening, before and after the implementation of tandem mass spectrometry (MS/MS). RESULTS: A total of 25 different types of IEiM were found in the 204 files; 102 organic acidurias, 100 aminoacidopathies, and 2 fatty acid oxidation disorders. The introduction of MS/MS increased the number of cases detected by 50%. Patients were referred from 13 different specialists, with pediatricians being the most active. The average interval between onset of symptoms and diagnosis was 18 months. CONCLUSION: Among the sick Mexican children studied, a wide variety of IEiM was found, propionate defects and maple syrup urine disease being noteworthy. The diagnosis of metabolic disease was delayed in the population studied. These results present evidence to perhaps incorporate IEiM into an expanded newborn screening, or else to perform selective diagnosis in all hospitalized children with suggestive clinical data.

Causes of delay in referral of patients with phenylketonuria to a specialized reference centre in Mexico.

OBJECTIVE: To expose causes leading to the delayed arrival of phenylketonuria (PKU) patients at a governmental reference centre (RC), and to describe their clinical characteristics. Material and methods PKU files registered during the past 18 years at the National Institute of Pediatrics in Mexico City were evaluated. Patients were classified into two groups according to their age at arrival: Group I (early reference), patients arriving during the first month of life; and Group II (late reference), those who arrived after thirty days of age. Time and causes of delay were documented. RESULTS: Of 57 recorded files, 10 were classified in Group I and 47 in Group II. Causes leading to the late arrival of Group II patients were absence of routine newborn screening (NBS), PKU not included in the routine NBS, sampling after the recommended age, false negative result, results without interpretation and/or instructions to follow, delayed notification of results, poor medical criteria of attending physician, difficulties in obtaining confirmatory tests, and administrative failures. CONCLUSION: The main cause of late referral of PKU patients was the absence of PKU testing. As a developing country, Mexico still faces challenges in the proper functioning and expansion of the NBS programme. Most PKU patients arrived at the RC late, presenting with varying degrees of the clinical spectrum. Incorporating PKU testing into the already established Mexican NBS system and adding quality indicators to guarantee proper operation in all NBS phases is necessary to achieve the goal of identifying, referring, diagnosing, and treating patients promptly.

[External validity of a relevant model for Irritable Bowel Syndrome (IBS) using chronic stress by water avoidance in Wistar rats]. / Validez externa de un modelo experimental para síndrome de intestino irritable (SII) mediante estrés crónico por evitación al agua en ratas Wistar.

BACKGROUND: Development of animal models has been one of the most remarkable achievements for Irritable Bowel Syndrome (IBS) research. These models need to exhibit face validity, construct and predictive validity, as well as internal (replicability) and external validity (generalizability). Among these models, chronic stress induced by water avoidance exposure (WA) has been validated in rats through increasing visceral hypersensitivity, motility impairment, anxiety and, colonic immune activity, all related to IBS. OBJECTIVE: To assess the external validity of WA indirectly on colonic motility in Wistar rats. METHOD: Ten male-Wistar rats were exposed to WA and compared with ten Wistar rats not exposed (controls). At the end of each exposure, the quantity of fecal pellets were determined and considered as a sign of autonomic regulation of colonic motility. Data was analyzed with a general linear model for repeated measures. RESULTS: Rats exposed to WA had higher number or pellets than controls: 7.46 ± 0.45 (95%CI: 6.51, 8.41) vs. 2.88 ± 0.45 (1.93, 3.83), p < 0.001. The higher number of pellets was related to WA exposure as there were no other significant interactions. In both groups, the number of pellets was higher during the first day and then decreased progressively. CONCLUSIONS: Chronic stress induced through WA in Wistar rats, exhibits external validity as an experimental model for IBS research and our findings of increased number of fecal pellets coincide with the appearance hypermotility related to IBS. The model is optimum for research studies on this disorder.

Screening for bioactive compounds from algae.

In the present work, a comprehensive methodology to carry out the screening for novel natural functional compounds is presented. To do that, a new strategy has been developed including the use of unexplored natural sources (i.e., algae and microalgae) together with environmentally clean extraction techniques and advanced analytical tools. The developed procedure allows also estimating the functional activities of the different extracts obtained and even more important, to correlate these activities with their particular chemical composition. By applying this methodology it has been possible to carry out the screening for bioactive compounds in the algae Himanthalia elongata and the microalgae Synechocystis sp. Both algae produced active extracts in terms of both antioxidant and antimicrobial activity. The obtained pressurized liquid extracts were chemically characterized by GC-MS and HPLC-DAD. Different fatty acids and volatile compounds with antimicrobial activity were identified, such as phytol, fucosterol, neophytadiene or palmitic, palmitoleic and oleic acids. Based on the results obtained, ethanol was selected as the most appropriate solvent to extract this kind of compounds from the natural sources studied.

Subcritical water extraction and characterization of bioactive compounds from Haematococcus pluvialis microalga.

In this work, extraction and characterization of compounds with antioxidant and antimicrobial activity from Haematococcus pluvialis microalga in red phase have been carried out. To do this, subcritical water extraction (SWE) has been combined with analytical techniques such as HPLC-DAD, HPLC-QqQ-MS and GC-MS and in vitro assays (i.e., for antioxidant and antimicrobial activity). The effect of the extraction temperature (50, 100, 150 and 200 degrees C) and solvent polarity has been studied in terms of yield and activity of the extracts. Results demonstrate that the extraction temperature has a positive influence in the extraction yield and antioxidant activity. Thus, the extraction yield achieved with this process was higher than 30% of dry weight at 200 degrees C as extraction temperature. Moreover, the extract obtained at 200 degrees C presented the highest antioxidant activity by far, while temperature does not seem to significatively affect the antimicrobial activity. Chemical composition was determined by HPLC-DAD, HPLC-QqQ-MS and GC-MS. Short chain fatty acids turned out to be responsible of the antimicrobial activity, whereas the antioxidant activity was correlated to vitamin E (present exclusively in the 200 degrees C extract), together with simple phenols, caramelization products and possible Maillard reaction products obtained during the extraction at high temperatures.

Pressurized fluid extraction of bioactive compounds from Phormidium species.

In the search for new functional ingredients with potential use in the food industry, extracts of unknown species of microalgae, such as Phormidium species have been studied. Three solvents of different polarities (i.e., hexane, ethanol, and water) have been used to obtain pressurized liquid extracts with different compositions. Moreover, extractions were performed at four different extraction temperatures (50, 100, 150, and 200 degrees C) with 20 min as extraction time. Antioxidant activity of the extracts has been measured by the TEAC assay. In general, hexane and ethanol extracts showed a higher antioxidant capacity that was mainly attributed to carotenoid compounds, as the TEAC value trend seems to be similar to the carotenoid content of the extracts. On the other hand, the high antioxidant activity of the 200 degrees C water extracts is likely related to the presence of Maillard reaction compounds produced by thermal degradation of the sample. beta-Carotene, lutein, violaxanthin, and neoxanthin were identified in 150 degrees C ethanol extracts. Four different microbial species ( Escherichia coli, Staphylococcus aureus, Candida albicans, and Aspergillus niger) were used to screen the potential antimicrobial activity of the Phormidium sp. extracts. The most sensitive microorganism was the yeast, C. albicans, whereas the fungus, A. niger, was the most resistant. In general, no drastic differences were found for solvents and temperatures tested, showing a very diverse nature of the compounds responsible for the antimicrobial activity of these microalgae. In ethanol extracts, antimicrobial activity could be mainly attributed to the presence of terpenes (i.e., beta-ionone, neophytadiene) and fatty acids (i.e., palmitoleic and linoleic acids) in the samples. Toxicity studies carried out with the extracts evaluated in the present work showed a cellular toxicity lower than those of other cyanobacteria such as Spirulina plantensis.

Supercritical carbon dioxide extraction of compounds with antimicrobial activity from Origanum vulgare L.: determination of optimal extraction parameters.

Oregano leaves were extracted using a pilot-scale supercritical fluid extraction plant under a wide range of extraction conditions, with the goal of determining the extraction and fractionation conditions to obtain extracts with optimal antimicrobial activity. In this investigation, the essential oil-rich fractions were selectively precipitated in the second separator, and their chemical composition and antimicrobial activity were investigated. Gas chromatography-mass spectrometry analysis of the various fractions resulted in the identification of 27 compounds of the essential oil. The main components of these fractions were carvacrol, trans-sabinene hydrate, cis-piperitol, borneol, terpinen-4-ol, and linalool. Antimicrobial activity was investigated by the disk diffusion and broth dilution methods against six different microbial species, including two gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis), two gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa), a yeast (Candida albicans), and a fungus (Aspergillus niger). All of the supercritical fluid extraction fractions obtained showed antimicrobial activity against all of the microorganisms tested, although the most active fraction was the one obtained in experiment 5 (fraction was obtained with 7% ethanol at 150 bar and 40 degrees C). C. albicans was the most sensitive microorganism to the oregano extracts, whereas the least susceptible was A. niger. Carvacrol, sabinene hydrate, borneol, and linalool standards also showed antimicrobial activity against all of the microorganisms tested, with carvacrol being the most effective. Consequently, it was confirmed that essential oil from experiment 5, with the best antimicrobial activity, also presented the highest quantity of carvacrol.

Chemical composition and antimicrobial activity of Rosmarinus officinalis L. essential oil obtained via supercritical fluid extraction.

The chemical composition and antimicrobial activity of essential oil-rich fractions obtained by supercritical CO2 extraction from Rosmarinus officinalis L. were investigated. Gas chromatography-mass spectroscopy analysis of these fractions resulted in the identification of 33 compounds of the essential oil. The main components of these fractions were alpha-pinene, 1,8-cineole, camphor, verbenone, and borneol, constituting ca. 80% of the total oil. The antimicrobial activity was investigated by the disc diffusion and broth dilution methods against six microbial species, including gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis), gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa), a yeast (Candida albicans), and a fungus (Aspergillus niger). All of the essential oil-rich fractions obtained showed antimicrobial activity against all of the microorganisms tested, with inhibition zones and minimal bactericidal and fungicidal concentration values in the range of 17 to 33 mm and 2.25 to 0.25 mg/ml, respectively. The most active fraction was the one obtained in experiment 4 (4% ethanol as modifier; extraction pressure, 25 MPa; extraction temperature, 60 degrees C). S. aureus was found to be the most sensitive bacteria to the rosemary extracts, whereas the least susceptible was A. niger. alpha-Pinene, 1,8-cineole, camphor, verbenone, and borneol standards also showed antimicrobial activity against all the microorganisms tested, borneol being the most effective followed by camphor and verbenone. In that way, it was confirmed that essential oil from experiment 4, with the best antimicrobial activity, presented the highest quantity of camphor, borneol, and verbenone.

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.