Development and validation of a rapid RP-HPLC method for simultaneous quantification of paclitaxel and cetuximab in immunoliposomes.

The development of rational therapies against complex diseases, such as cancer, has increased in the past few years due to the advances of 'omics' technologies. Concomitantly, several efforts have been made to design sophisticated drug delivery systems in order to increase specificity and drug accumulation in tumor sites. The complexity of these drug delivery systems highlights the need for suitable analytical methods to determine encapsulation/conjugation efficiency of drugs and molecules responsible for the targeted delivery. Therefore, this study focuses on the development and validation of a RP-HPLC-DAD methodology for concurrent quantification of paclitaxel (PTX) and cetuximab (CTX) in immunoliposomes. Chromatographic separation was achieved using a wide pore C8 column, and a gradient mobile phase consisting of 0.1% trifluoroacetic acid (TFA) in Milli-Q water/acetonitrile/isopropanol with a flow rate of 1 mL min-1. Drug peaks were fully separated and detected at 280 nm using UV detector. The method was validated according to ICH and FDA guidelines in terms of specificity and forced degradation studies, system suitability, linearity, limit of detection, limit of quantification, repeatability, intermediate precision, accuracy, robustness, and short-term stability. The developed method was linear over the concentration range of 37.5-150 µg mL-1 of PTX and 75-300 µg mL-1 of CTX. All parameters evaluated satisfied the acceptance criteria, according to both FDA and ICH guidelines. The applicability of the analytical method was assessed following the development of PTX-loaded immunoliposomes conjugated with CTX. Thus, the present study shows a novel, simple, stability-indicating and suitable method to quantify simultaneously PTX and CTX in immunoliposomes.

Wound healing treatment using insulin within polymeric nanoparticles in the diabetes animal model.

The aim of this work was to prepare chitosan nanoparticles containing insulin and to evaluate its therapeutic activity during wound healing in diabetic rats. The hypothesis that guided this study was that the combination of insulin within chitosan nanoparticles could stimulate the signaling pathway for wound healing. The chitosan nanoparticles were prepared by the ionotropic gelation method presenting average size of 183.3 ± 8.32 nm, polydispersity index (PDI) 0.397 ± 0.07 and zeta potential of 33.7 ± 2.45 mV for empty chitosan nanoparticles (EC) and 245.9 ± 25.46 nm and PDI 0.463 ± 0.01, and zeta potential of 39.3 ± 4.88 mV for chitosan nanoparticles containing insulin (IC). The insulin association efficiency was 97.19% ± 2.18. These nanoparticles and free insulin (FI) were incorporated within a hydrogel (Sepigel®) for topical application in the wound of 72 diabetic rats distributed in four groups: Sepigel® (S, control), free insulin (FI), empty chitosan nanoparticles (EC), and chitosan nanoparticles containing insulin (IC). The animals in each group were reorganized into three subgroups (n = 6) to assess their clinical signs after days 3, 7, and 14 from the beginning of treatments. Intense fibroplasias were observed in the free or insulin-chitosan nanoparticles groups. In the latter, a large number of blood vessels were observed at day 7th. Our data indicated that both empty and insulin-containing chitosan nanoparticles were able to stimulate inflammatory cell proliferation, and angiogenesis, followed by wound maturation.

Microemulsion based on Pterodon emarginatus oil and its anti-inflammatory potential

This article reports the development of a pharmaceutical product containing vegetable actives from a Brazilian medicinal plant. The possibility of forming a microemulsion using Pterodon emarginatus ("sucupira") oil was evaluated and the anti-inflammatory potential of this microemulsion was also examined. A formulation was developed using P. emarginatus oil, a mixture of ethoxylated Castor Oil (Ultramone(r) R-540/propylene glycol 2:1) (surfactant/cosurfactant) and distilled water at a ratio of 10:15:75, respectively. The microemulsion which was selected was then subjected to the preliminary stability test and analyzed in terms of average diameter of droplets, pH, zeta potential, and polydispersity index, on the 1st, 7th, 15th, and 30th days after preparation and stored at different temperatures (5 ± 2 °C, 25 ± 2 °C, and 40 ± 2 °C). The anti-inflammatory in vivo activity of both oil and formulation were evaluated, using the experimental model of croton oil-induced ear edema. The preliminary stability test showed that the microemulsion stored at 5 and 25 °C retained its original features throughout the 30-day period. The anti-inflammatory potential of both oil and formulation was shown to be statistically significant (p < 0.001), when compared to the control group, however, the microemulsion proved to be more effective (p < 0.05) than the oil when applied directly to the ear.

Descrevemos o desenvolvimento de um produto farmacêutico contendo ativo vegetal baseado em uma planta medicinal brasileira. Foi avaliada a habilidade de formação de uma microemulsão à base do óleo de Pterodon emarginatus (sucupira) e seu potential anti-inflamatório. Uma formulação foi desenvolvida utilizando o óleo de P. emarginatus, mistura de óleo de mamona etoxilado (Ultramona(r) R-540)/propilenoglicol (2:1) (tensoativo/cotensoativo) e água destilada, na proporção de 10:15:75, respectivamente. A microemulsão selecionada foi submetida ao teste preliminar de estabilidade e foi analisada quanto ao diâmetro médio das gotículas, pH, potential zeta e índice de polidispersão, no 1º, 7º, 15º e 30º dias após o preparo e foram estocadas em diferentes temperaturas (5±2 °C, 25±2 °C e 40±2 °C). Avaliaram-se a atividade anti-inflamatória in vivo do óleo de sucupira e da formulação, usando o modelo experimental do edema de orelha induzido pelo óleo de cróton. No teste preliminar de estabilidade observou-se que a formulação estocada a 5 °C e a 25 °C mantiveram suas características originais durante 30 dias. O potencial anti-inflamatório de ambos, óleo de sucupira e formulação foi estatisticamente significativo (p <0.001), quando comparado ao controle, porém a microemulsão foi mais eficiente (p <0.05) que o óleo aplicado diretamente nas orelhas dos animais.

Biodegradable polymeric nanocapsules based on poly(DL-lactide) for genistein topical delivery: obtention, characterization and skin permeation studies.

The isoflavone genistein (GEN) is a natural product with potential applications for skin cancer treatment and chemoprevention; however its high lipophilicity and chemical instability limits its clinical use. Therefore, attempts towards protecting GEN against degradation and increasing its penetration in the skin might be a valid approach. In this work, GEN loaded-PLA nanocapsules (GEN-NC) were prepared by interfacial deposition of preformed polymer (nanoprecipitation); physicochemical characterization and stability studies for 90 days were conducted. GEN-NC were incorporated into semi-solid formulations and permeation experiments were carried out using porcine ear skin. GEN-NC optimized formulation presented a mean diameter of 139 +/- 7.31 nm, polydispersity index of 0.128 +/- 0.08, encapsulation efficiency of 89.63 +/- 2.27% and drug loading from 0.6 to 1.4 w/w%. Stability studies demonstrated that nanocapsules did not exhibit aggregation during the 90 days of the assay, however, a drop in encapsulation efficiency was observed in the first 10 days. Permeation experiments demonstrated that a higher amount of GEN reaches deeper layers of the skin and increased penetration was achieved when GEN-NC were incorporated in a semi-solid gel formulation, indicating that GEN-NC might be a promising nanocarrier system for skin delivery of GEN.

Phytochemical Analysis and Antimicrobial, Antinociceptive, and Anti-Inflammatory Activities of Two Chemotypes of Pimenta pseudocaryophyllus (Myrtaceae).

Preparations from Pimenta pseudocaryophyllus (Gomes) L.R. Landrum (Myrtaceae) have been widely used in Brazilian folk medicine. This study aims to evaluate the antimicrobial activity of the crude ethanol extracts, fractions, semipurified substances, and essential oils obtained from leaves of two chemotypes of P. pseudocaryophyllus and to perform the antinociceptive and anti-inflammatory screening. The ethanol extracts were purified by column chromatography and main compounds were spectrally characterised (1D and 2D (1)H and (13)C NMR). The essential oils constituents were identified by GC/MS. The broth microdilution method was used for testing the antimicrobial activity. The abdominal contortions induced by acetic acid and the ear oedema induced by croton oil were used for screening of antinociceptive and anti-inflammatory activities, respectively. The phytochemical analysis resulted in the isolation of pentacyclic triterpenes, flavonoids, and phenol acids. The oleanolic acid showed the best profile of antibacterial activity for Gram-positive bacteria (31.2-125 µg mL(-1)), followed by the essential oil of the citral chemotype (62.5-250 µg mL(-1)). Among the semipurified substances, Ppm5, which contained gallic acid, was the most active for Candida spp. (31.2 µg mL(-1)) and Cryptococcus spp. (3.9-15.6 µg mL(-1)). The crude ethanol extract and fractions from citral chemotype showed antinociceptive and anti-inflammatory effects.

Dissolution test of herbal medicines containing Paullinia cupana: validation of methods for quantification and assessment of dissolution / Ensaio de dissolução de medicamentos fitoterápicos contendo Paullinia cupana: validação de métodos de quantificação e avaliação de dissolução

"Guaraná" (Paullinia cupana) is used as a physical activity enhancer and stimulator due to its methylxanthines and condensed tannins. The aim of this work was to evaluate the dissolution behavior of five herbal medicines in the form of capsules and tablets containing guaraná. Assay and dissolution methods were validated and results obtained allowed simultaneous marker quantification with precision, accuracy, selectivity and robustness. Findings showed that 100 percent of the herbal medicinal products analyzed did not provide satisfactory results concerning the presence of four markers, 60 percent had three markers (caffeine, catechin and epicatechin), while 40 percent had only caffeine at tested dosage forms. In addition, after 30 minutes, only capsule A showed at least 80 percent of the dissolved markers. In other capsules, marker dissolution did not exceed 60 percent whereas 60 percent of the samples had some characteristic pharmacotechnical problems. These results evidence the need for rigorous quality control to help ensure the therapeutic action of these drugs. To this end, dissolution studies are an essential tool for quality assurance of herbal medicines.

Guaraná (Paullinia cupana) é utilizado como revigorante e estimulante devido à presença de metilxantinas e taninos condensados. Este trabalho visou avaliar o comportamento de dissolução de cinco fitoterápicos, na forma de cápsulas e comprimidos, contendo guaraná. O método de quantificação e de dissolução foram validados e os resultados obtidos permitiram a quantificação dos marcadores simultaneamente, com precisão, exatidão, seletividade e robustez. Foi verificado que 100 por cento dos fitoterápicos analisados encontravam em desacordo quanto à presença dos quatro marcadores, sendo que 60 por cento apresentaram três marcadores (cafeína, catequina e epicatequina) e 40 por cento apresentaram somente a cafeína. Além disso, após o tempo de 30 minutos de ensaio foi possível observar que somente a cápsula A apresentou pelo menos 80 por cento dos marcadores dissolvidos. Nas demais cápsulas o comportamento de dissolução apresentado pelos marcadores não ultrapassou 60 por cento e, além disso, 60 por cento das amostras apresentaram alguns problemas farmacotécnicos característicos. Frente aos resultados obtidos torna-se evidente a necessidade de um rigoroso controle de qualidade que contribua para assegurar a ação terapêutica desses medicamentos e, nesse sentido, o estudo de dissolução constitui uma ferramenta essencial para a garantia de qualidade dos fitoterápicos.