Guilherme Martins Gelfuso

Liposomal voriconazole (VOR) formulation for improved ocular delivery.

Treating infectious eye diseases topically requires a drug delivery system capable of overcoming the eyes defense mechanisms, which efficiently reduce the drug residence time right after its administration, therefore reducing absorption. In order to try to surpass such administration issues and improve life quality for patients with fungal keratitis, liposomal voriconazol (VOR) formulations were prepared. Formulations were composed of soy phosphatidylcholine (PC) containing or not 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and cholesterol. Liposomes were characterized by their drug entrapment efficiency (EE), drug recovery (DR), average diameter (size) and polydispersivity index (PdI). In vitro mucosal interaction and irritancy levels, ex vivo permeation, as well as the short-term stability were also assessed. Liposomal VOR formulation produced with 7.2:40mM VOR:PC showed to be the most promising formulation: mean size of 116.6±5.9nm, narrow PdI (0.17±0.06), negative zeta potential (∼-7mV) and over 80% of EE and yield, remaining stable for at least 30 days in solution and 90 days after lyophilization. This formulation was classified as non-irritant after HET-CAMs test and was able to deliver about 47.85±5.72μg/cm(2) of VOR into porcine cornea after 30min of permeation test. Such drug levels are higher than the minimal inhibitory concentrations (MIC) of several fungi species isolated from clinical cases of corneal keratitis. Overall results suggest VOR can be effectively incorporated in liposomes for potential topical treatment of fungal keratitis.

Chitosan nanoparticles for targeting and sustaining minoxidil sulphate delivery to hair follicles

This work developed minoxidil sulphate-loaded chitosan nanoparticles (MXS-NP) for targeted delivery to hair follicles, which could sustain drug release and improve the topical treatment of alopecia. Chitosan nanoparticles were obtained using low-molecular weight chitosan and tripolyphosphate as crosslink agent. MXS-NP presented a monomodal distribution with hydrodynamic diameter of 235.5 ± 99.9 nm (PDI of 0.31 ± 0.01) and positive zeta potential (+38.6 ± 6.0 mV). SEM analysis confirmed nanoparticles average size and spherical shape. A drug loading efficiency of 73.0 ± 0.3% was obtained with polymer:drug ratio of 1:1 (w/w). Drug release through cellulose acetate membranes from MXS-NP was sustained in about 5 times in comparison to the diffusion rate of MXS from the solution (188.9 ± 6.0 μg/cm(2)/h and 35.4 ± 1.8 μg/cm(2)/h). Drug permeation studies through the skin in vitro, followed by selective recovery of MXS from the hair follicles, showed that MXS-NP application resulted in a two-fold MXS increase into hair follicles after 6h in comparison to the control solution (5.9 ± 0.6 μg/cm(2) and 2.9 ± 0.8 μg/cm(2)). MXS-loading in nanoparticles appears as a promising and easy strategy to target and sustain drug delivery to hair follicles, which may improve the topical treatment of alopecia.

Iontophoretic transport of zinc phthalocyanine tetrasulfonic acid as a tool to improve drug topical delivery.

Phthalocyanines have been used as systemic photosensitizers because of their high affinity towards tumour tissue, and the high rates of reactive oxygen species produced when they are irradiated during photodynamic therapy. However, the topical administration of these compounds is limited by their large size, poor hydrosolubility and ionic character. This study aimed to investigate the iontophoretic delivery of charged zinc phthalocyanine tetrasulfonic acid (ZnPcS4) from a hydrophilic gel to different skin layers by means of in-vitro and in-vivo studies. Six hours of passive administration was insufficient for ZnPcS4 to cross the stratum corneum (SC) and to reach the epidermis and dermis. No positive effect was reached when anodal iontophoresis was performed, showing that the drug–electrode attraction effect was higher than the electro-osmosis contribution at a pH of 5.5. Cathodal iontophoresis, however, was able to transport significant amounts of the drug to the viable epidermis. In addition, the absence of NaCl in the formulation significantly increased (by five-fold) the amount of ZnPcS4 that crossed the SC and accumulated in the epidermis and dermis. It was possible to visualize the drug accumulation in the follicle openings and in the epidermis, even after SC removal. In-vivo experiments in rat skin showed that these results were maintained in an in-vivo model, even with only 15 min of iontophoresis. In addition, confocal analysis of the treated skin showed a homogeneous distribution of ZnPcS4 in the viable epidermis after this short period of cathodal iontophoresis.

Iontophoresis-targeted, follicular delivery of minoxidil sulfate for the treatment of alopecia

Although minoxidil (MX) is a drug known to stimulate hair growth, the treatment of androgenic alopecia could be improved by delivery strategies that would favor drug accumulation into the hair follicles. This work investigated in vitro the potential of iontophoresis to achieve this objective using MX sulfate (MXS), a more water-soluble derivative of MX. Passive delivery of MXS was first determined from an ethanol-water solution and from a thermosensitive gel. The latter formulation resulted in greater accumulation of MXS in the stratum corneum (skins outermost layer) and hair follicles and an overall decrease in absorption through the skin. Anodal iontophoresis of MXS from the same gel formulation was then investigated at pH 3.5 and pH 5.5. Compared with passive delivery, iontophoresis increased the amount of drug reaching the follicular infundibula from 120 to 600 ng per follicle. In addition, drug recovery from follicular casts was threefold higher following iontophoresis at pH 5.5 compared with that at pH 3.5. Preliminary in vivo experiments in rats confirmed that iontophoretic delivery of MXS facilitated drug accumulation in hair follicles. Overall, therefore, iontophoresis successfully and significantly enhanced follicular delivery of MX suggesting a useful opportunity for the improved treatment of alopecia.

Iontophoretic transport kinetics of ketorolac in vitro and in vivo: demonstrating local enhanced topical drug delivery to muscle.

The objective of the study was to investigate the iontophoretic delivery kinetics of ketorolac (KT), a highly potent NSAID and peripherally-acting analgesic that is currently indicated to treat moderate to severe acute pain. It was envisaged that, depending on the amounts delivered, transdermal iontophoretic administration might have two distinct therapeutic applications: (i) more effective and faster local therapy with shorter onset times (e.g. to treat trauma-related pain/inflammation in muscle) or (ii) a non-parenteral, gastrointestinal tract sparing approach for systemic pain relief. The first part of the study investigated the effect of experimental conditions on KT iontophoresis using porcine and human skin in vitro. These results demonstrated that KT electrotransport was linearly dependent on current density - from 0.1875 to 0.5mA/cm(2) - (r(2)>0.99) and drug concentration - from 5 to 20mg/ml (r(2)>0.99). Iontophoretic permeation of KT from a 2% hydroxymethyl cellulose gel was comparable to that from an aqueous solution with equivalent drug loading (584.59±114.67 and 462.05±66.56μg/cm(2), respectively). Cumulative permeation (462.05±66.56 and 416.28±95.71μg/cm(2)) and steady state flux (106.72±11.70 and 94.28±15.47μg/cm(2)h), across porcine and human skin, were statistically equivalent confirming the validity of the model. Based on the results in vitro, it was decided to focus on topical rather than systemic applications of KT iontophoresis in vivo. Subsequent experiments, in male Wistar rats, investigated the local enhancement of KT delivery to muscle by iontophoresis. Drug biodistribution was assessed in skin, in the biceps femoris muscle beneath the site of iontophoresis (treated muscle; TM), in the contralateral muscle (non-treated muscle; NTM) and in plasma (P). Passive topical delivery and oral administration served as negative and positive controls, respectively. Iontophoretic administration for 30min was superior to passive topical delivery for 1h and resulted in statistically significant increases in KT levels in the skin (91.04±15.48 vs. 20.16±8.58μg/cm(2)), in the biceps femoris at the treatment site (TM; 6.74±3.80 vs. <LOQ), in the contralateral site (NTM; 1.26±0.54 vs. <LOQ) and in plasma (P; 8.58±2.37μg/ml vs. <LOD). In addition to increasing bioavailability, iontophoretic administration of KT showed clear selectivity for local delivery to the biceps femoris at the treatment site - the TM:NTM ratio was 5.26±1.45, and the TM:P and NTM:P ratios were 0.75±0.32 and 0.14±0.04, respectively. Furthermore, the post-iontophoretic concentration of KT in the treated biceps femoris muscle and the muscle:plasma ratio were also superior to those following oral administration of a 4mg/kg dose (6.74±3.80 vs. 0.62±0.14μg/g and 0.75±0.32 vs. 0.14±0.03, respectively). In conclusion, the results demonstrate that iontophoresis of ketorolac enables local enhanced topical delivery to subjacent muscle; this may have clinical application in the treatment of localised inflammation and pain.

Iontophoresis of minoxidil sulphate loaded microparticles, a strategy for follicular drug targeting?

The feasibility of targeting drugs to hair follicles by a combination of microencapsulation and iontophoresis has been evaluated. Minoxidil sulphate (MXS), which is used in the treatment of alopecia, was selected as a relevant drug with respect to follicular penetration. The skin permeation and disposition of MXS encapsulated in chitosan microparticles (MXS-MP) was evaluated in vitro after passive and iontophoretic delivery. Uptake of MXS was quantified at different exposure times in the stratum corneum (SC) and hair follicles. Microencapsulation resulted in increased (6-fold) drug accumulation in the hair follicles relative to delivery from a simple MXS solution. Application of iontophoresis enhanced follicular delivery for both the solution and the microparticle formulations. It appears, therefore, that microencapsulation and iontophoresis can act synergistically to enhance topical drug targeting to hair follicles.

Microparticles prepared with 50–190 kDa chitosan as promising non-toxic carriers for pulmonary delivery of isoniazid

Chitosan biocompatibility and mucoadhesiveness make it an ideal polymer for antituberculotic drugs microcapsulation for pulmonary delivery. Yet, previous study indicated toxicity problems to J-774.1-cells treated with some medium molecular weight (190-310kDa) chitosan microparticles. As polymer molecular weight is a crucial factor to be considered, this paper describes the preparation and characterization of chitosan (50-190kDa) microparticles containing isoniazid (INH). Cytotoxicity assays were also performed on murine peritoneal (J-774.1) and alveolar (AMJ2-C11) macrophages cell lines, followed by cytokines detection from AMJ2-C11 cells. Spray-drying process produced mucoadhesive microparticles from 3.2μm to 3.9μm, entrapping more than 89% of the drug and preserving their chemical stability. Drug release behavior could be controlled by the use of cross-linked or uncross-linked chitosan, the latter leading to a rapid drug release. Mucoadhesive potential of the microparticles was characterized following in vitro and ex vivo assays. Finally, a significant reduction on toxicity against peritoneal macrophages and no toxic effect on alveolar macrophages with use of such microparticles were observed. In conclusion, 50-190kDa chitosan microparticles may act as promising non-cytotoxic carriers for pulmonary delivery of INH showing marked alveoli macrophage activation.

Microspheres prepared with different co-polymers of poly(lactic-glycolic acid) (PLGA) or with chitosan cause distinct effects on macrophages.

Microencapsulation of bioactive molecules for modulating the immune response during infectious or inflammatory events is a promising approach, since microspheres (MS) protect these labile biomolecules against fast degradation, prolong the delivery over longer periods of time and, in many situations, target their delivery to site of action, avoiding toxic side effects. Little is known, however, about the influence of different polymers used to prepare MS on macrophages. This paper aims to address this issue by evaluating in vitro cytotoxicity, phagocytosis profile and cytokines release from alveolar macrophages (J-774.1) treated with MS prepared with chitosan, and four different co-polymers of PLGA [poly (lactic-co-glycolic acid)]. The five MS prepared presented similar diameter and zeta potential each other. Chitosan-MS showed to be cytotoxic to J-774.1 cells, in contrast to PLGA-MS, which were all innocuous to this cell linage. PLGA 5000-MS was more efficiently phagocytized by macrophages compared to the other MS tested. PLGA 5000-MS and 5002-MS induced significant production of TNF-α, while 5000-MS, 5004-MS and 7502-MS decreased spontaneous IL-6 release. Nevertheless, only PLGA 5002-MS induced significant NFkB/SEAP activation. These findings together show that MS prepared with distinct PLGA co-polymers are differently recognized by macrophages, depending on proportion of lactic and glycolic acid in polymeric chain, and on molecular weight of the co-polymer used. Selection of the most adequate polymer to prepare a microparticulate drug delivery system to modulate immunologic system may take into account, therefore, which kind of immunomodulatory response is more adequate for the required treatment.

Current efforts and the potential of nanomedicine in treating fungal keratitis

Fungal infection of the cornea (mycotic or fungal keratitis, keratomycosis) is a serious disease that can lead to loss of vision if not diagnosed and treated promptly and effectively. The pharmacological approach of management of fungal keratitis involves administration of antifungal agents. However, owing to the physiologic constraints of the eye, only a few drugs define sufficient bioavailability. The need for more potent antifungals with increased activity, shorter treatment durations and fewer adverse effects simultaneously stimulates the drive for the development of new antifungal agents with a broader spectrum and improved pharmacokinetic profile, and the development of advanced novel formulations for drug delivery that could increase drug bioavailability while reducing the adverse effects. In this article, the efforts and scientific potential of these two avenues are discussed. First, the classical and novel antifungal drugs are presented. Second, the classical formulations are compared with the advanced novel nanomedicines, and their potential clinical applications are discussed.

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

&NA; 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. Graphical abstract Figure. No caption available. HighlightsDrug‐excipient compatibility protocol using mixture design was proposed.Mixture design showed excipients synergistic effects in thymol formulations.Thermal and spectroscopy assays allowed inferring which interactions compromise stability.Taurodeoxycolate jeopardized thymol stability in multicomponent systems.Lecithin physically and chemically stabilized thymol.