Use of Pseudomonas protegens to Control Root Rot Disease Caused by Boeremia exigua var. exigua in Industrial Chicory (Cichorium intybus var. sativum Bisch.).

Boeremia exigua var. exigua is a recurrent pathogen causing root rot in industrial chicory. Currently, there is no chemical or varietal control for this disease, and thus, management strategies need to be developed. This study determined the biocontrol effect of strains of Pseudomonas protegens bacteria with antimicrobial compounds on the fungus B. exigua var. exigua under in vitro, in vivo, and field conditions. In addition, root colonization by these bacteria was estimated by the phlD-specific PCR-based dilution end point assay. Eighteen isolates of Pseudomonas spp were evaluated, and the strains that showed the greatest in vitro inhibition of fungal mycelial growth (mm), Ca10A and ChB7, were selected. Inoculation with the strain ChB7 showed less severity (necrotic area) under in vivo conditions (root trials) compared with the control inoculated with the pathogen (p ≤ 0.05). The molecular analysis revealed that the root colonization of plants grown in pots was equal to or greater than 70%. Similar levels were observed in the field trials conducted at the Selva Negra and Canteras experimental stations (2015-2016 season), with values ranging from 85.7 to 70.5% and from 75.0 to 79.5%, respectively. Regarding yield (ton ha-1), values were higher in the treatments inoculated with strains Ca10A and ChB7 (p ≤ 0.05) at both experimental sites, while a lower incidence and severity of root rot were observed at Selva Negra. These results suggest that the Chilean strains of P. protegens are a promising tool for the control of root diseases in industrial chicory.

Effect of physical stimuli on hair follicle deposition of clobetasol-loaded Lipid Nanocarriers.

Clobetasol propionate (CLO) is a potent glucocorticoid used to treat inflammation-based skin, scalp, and hair disorders. In such conditions, hair follicles (HF) are not only the target site but can also act as drug reservoirs when certain formulations are topically applied. Recently, we have demonstrated nanostructured lipid carriers (NLC) containing CLO presenting epidermal-targeting potential. Here, the focus was evaluating the HF uptake provided by such nanoparticles in comparison to a commercial cream and investigating the influence of different physical stimuli [i.e., infrared (IR) irradiation (with and without metallic nanoparticles-MNP), ultrasound (US) (with and without vibration) and mechanical massage] on their follicular targeting potential. Nanosystems presented sizes around 180 nm (PdI < 0.2) and negative zeta potential. The formulation did not alter skin water loss measurements and was stable for at least 30 days at 5 °C. Nanoparticles released the drug in a sustained fashion for more than 3 days and increased passively about 40 times CLO follicular uptake compared to the commercial cream. Confocal images confirmed the enhanced follicular delivery. On the one hand, NLC application followed by IR for heat generation showed no benefit in terms of HF targeting even at higher temperatures generated by metallic nanoparticle heating. On the other hand, upon US treatment, CLO retention was significantly increased in deeper skin layers. The addition of mechanical vibration to the US treatment led to higher follicular accumulation compared to passive exposure to NLC without stimuli. However, from all evaluated stimuli, manual massage presented the highest follicular targeting potential, driving more than double the amount of CLO into the HF than NLC passive application. In conclusion, NLC showed great potential for delivering CLO to HF, and a simple massage was capable of doubling follicular retention.

Versatile chromatographic method for catechin determination in development of topical formulations containing natural extracts.

Catechin is found in several natural sources, as Eugenia dysenterica and Syzygium cumini extracts. Its antioxidant and UV-protective properties suggest a potential use in cosmetic and dermatological formulations. A simple analytical method capable of giving support to experiments performed along the development of topical formulations containing this natural substance (i.e. drug assay, skin permeation and stability studies), however, is still needed. Thus, this work aimed to develop and validate a selective HPLC method for catechin determination during the development of topical formulations. Separation was achieved using an RP-C18 column (300 × 3.9 mm; 10 µm), with a mobile phase of methanol-phosphoric acid 0.01 m (15: 85, v/v), a flow rate of 0.8 mL/min, temperature set at 40°C and UV detection at 230 nm. The method was linear in a range from 0.5 to 10.0 µg/mL (r = 0.9998), precise with an overall variation coefficient of 5.5% and accurate with catechin recovery from the skin layers >85%. Additionally, the method was sensitive (limit of detection, 0.109 µg/mL; limit of quantification, 0.342 µg/mL) and selective against plant extracts, skin matrices and formulation interferents, as well as catechin degradation products. It was also robust regarding both methodology parameters and analytical stability.

Use of mixture design in drug-excipient compatibility determinations: Thymol nanoparticles case study.

The objective of this work was to access thymol-excipient compatibility using an alternative protocol of mixture design subsidizing the development of nanostructures lipid carriers containing this drug. Simultaneous DTA-TG analyses associated with infrared spectroscopy were performed according to simplex centroid mixture designs with three components. Two designs were used: the design A containing stearic acid (SA), soybean lecithin (LC), and sodium taurodeoxycholate (TAU) and the design B, where TAU was replaced by polysorbate 80 (P80). Assays allowed for a quantitative evaluation of thermal events involved with thymol (TML) - melting and evaporation -, as well as events related to excipients decomposition and overall system stability. Although the anionic surfactant TAU did not interact with TML in solid state, chemical and physical stability were compromised after drug melting. Alternatively, nonionic surfactant P80 could be a good excipient option, as TML formulation stability was not influenced by it. Fatty acid SA did not compromise TML stability alone, but, when in combination with other formulation components, negative interaction leading to a possible decomposition of the system was observed. Finally, phospholipid LC solubilizes TML extending its evaporation to higher temperatures; hence, drug stability may be increased. In conclusion, the use of mixture design in the evaluation of multicomponent systems is a valuable tool for identification of synergistic effects of excipients, providing more complete information on formulation development. In addition, the association of techniques employed allowed inferring with certainty if thermal interactions could compromise formulation stability.

Chemical and physical strategies in onychomycosis topical treatment: A review.

Onychomycosis is a fungal infection of the fingernails or toenails caused by dermatophytes, nondermatophytes, moulds, and yeasts. This condition affects around 10-30% people worldwide, negatively influencing patients' quality of life, with severe outcomes in some cases. Since the nail unit acts as a barrier to exogenous substances, its physiological features hampers drug penetration, turning the onychomycosis treatment a challenge. Currently, there are several oral and topical therapies available; nevertheless, cure rates are still low and relapse rates achieves 10-53%. Also, serious side effects may be developed due to long-term treatment. In light of these facts, researchers have focused on improving topical treatments, either by modifying the vehicle or by using some physical technique to improve drug delivery trough the nail plate, hence increasing therapy effectiveness. Therefore, the aim of this paper is to explain these novel alternative approaches. First, the challenges for drug ungual penetration are presented. Then, the chemical and physical strategies developed for overcoming the barriers for drug penetration are discussed. We hope that the information gathered may be useful for the development of safer and more effective treatments for onychomycosis.

Chromatographic method for clobetasol propionate determination in hair follicles and in different skin layers.

Clobetasol propionate (CLO) is a potent steroid used for the treatment of several dermatological diseases. Recent studies suggest its additional use in alopecia topical treatment, generating a demand for novel formulations with specific delivery into hair follicles. Hence, a selective analytical method for drug quantification in follicular structures and skin layers is required. For this, a simple HPLC-UV method was developed. Quantification was performed using a RP-C18 column (4.6 mm × 15 cm, 5 µm), with a mixture of methanol-acetonitrile-water (50:15:35 v/v) as mobile phase, a flow rate of 1.2 mL/min, oven temperature of 30°C, injection volume of 50 µL and detection at 240 nm. The optimized conditions enabled a 12 min running with CLO elution at 10.1 min and resolution of 2.424 from skin matrix interferences. Validation was performed in accordance with International Conference on Harmonization guidelines and fulfilled the criteria of selectivity, linearity (0.5-15.0 µg/mL), robustness, precision, accuracy and limits of detection and quantification (0.02 and 0.07 µg/mL, respectively). The validated method was successfully applied for CLO quantification following in vitro skin permeation experiments and differential tape-stripping for hair follicle deposition determination, demonstrating its suitability.

Infusão subcutânea contínua de insulina: cenário nacional e internacional / Continuous subcutaneous insulin infusion: national and international scenario

Resumo Introdução O sistema de bomba de infusão contínua de insulina (SIC) é uma alternativa à aplicação múltipla diária de insulina por pacientes diabéticos. Embora apresente inúmeras vantagens, a sua disponibilidade no sistema público de saúde ainda é muito baixa. Esse trabalho objetivou identificar práticas e experiências de secretarias e organizações de saúde quanto à utilização do SIC. Método Foram aplicados questionários às secretarias de saúde dos Estados brasileiros (SES) e às organizações internacionais de avaliação de tecnologias em saúde (ATS). Resultados A maioria das SES (83%) tem recebido demandas judiciais, com um aumento de uso do SIC nos últimos cinco anos. As organizações internacionais informaram que, em 47% dos países pesquisados, o SIC é disponibilizado à população pelo governo por meio do sistema de saúde público. Conclusão O uso do SIC resulta em vários benefícios, contudo está atrelado a um custo adicional anual de cerca de R$9.500,00 por paciente. Portanto é evidente a necessidade de se realizar estudos para avaliar a incorporação do SIC e seu impacto orçamentário no sistema de saúde brasileiro, além de definir em quais circunstâncias ele será disponibilizado à população.

Abstract Introduction The Continuous Subcutaneous Insulin Infusion pump system (CSII) is an alternative to multiple daily administration of insulin in diabetic patients. Although it has many advantages, its availability in the public health system is still very low. This study aimed to identify practices and experiences of health departments and organizations on the use of CSII. Method Questionnaires were applied to Brazilian Health Departments of the States (SES) and international organizations for health technologies evaluation. Results Most of the SES (83%) have received lawsuits and the use of CSII has increased in the last 5 years. International organizations reported that in 47% of countries, the CSII is available to patients through the public health system. Conclusion The use of CSII brings several benefits, but also a high annual additional cost of approximately R$ 9,500 per patient. Therefore, there is an evident need to conduct studies assessing the incorporation of CSII, its budgetary impact in the Brazilian health system, and the definition of which circumstances this will be made available to the population.

Development and validation of a selective HPLC-UV method for thymol determination in skin permeation experiments.

Thymol is a natural monoterpene, whose antioxidant and antimicrobial properties suggest a potential use in topical formulations. A simple, precise and selective HPLC method for thymol determination in skin penetration studies was developed and validated in this paper. Separation was achieved with a RP-C18 column, mobile phase comprised of acetonitrile:water (35:65v/v), flow rate of 1.5mL/min, oven temperature at 40°C, injection volume of 30µL and UV detection at 278nm. The validation procedure certified the method was selective for thymol determination even when extracted from skin matrix extracts. It was also linear in a range from 0.5 to 15.0µg/mL, robust, precise and accurate, with recovery rates from the skin layers higher than 90%. Limits of detection and quantification were 0.05 and 0.14µg/mL, respectively. The method showed, therefore, to be adequate for use in further skin permeation studies employing thymol topical formulations.

Topical and Transdermal Delivery of Drug-Loaded Nano/ Microsystems with Application of Physical Enhancement Techniques.

Topical and transdermal delivery has been studied over last decades and it presents advantages for the treatment of several disorders, macromolecules delivery and vaccination. The greatest challenge is to overcome the stratum corneum (SC) barrier. Compared to traditional topical formulation strategies, nano /microsystems offer advantages such as increased stability, increased loading dose, coverage of undesired colors, reduced toxicity and prolonged release of active agents. However, there are no conclusive studies demonstrating the ability of such systems to penetrate the skin in relevant therapeutic amounts. The use of physical methods holds great promise for enhancing skin permeation through the SC and for targeting hair follicles. This review discusses the characteristics and feasibility of using a dual approach employing the application of physical methods of permeation enhancement to enable the topical or transdermal delivery of drug-loaded nano/microsystems.