Novos sistemas farmacêuticos para administração pulmonar

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Design and evaluation of novel microencapsulation technologies to enhance delivery of Ciclosporin

Drug delivery systems influence the various processes of release, absorption, distribution and elimination of drug. Conventional delivery methods administer drug through the mouth, the skin, transmucosal areas, inhalation or injection. However, one of the current challenges is the lack of effective and targeted oral drug administration. Development of sophisticated strategies, such as micro- and nanotechnology that can integrate the design and synthesis of drug delivery systems in a one-step, scalable process is fundamental in advancing the limitations of conventional processing techniques. Thus, the objective of this thesis is to evaluate novel microencapsulation technologies in the production of size-specific and target-specific drug-loaded particles. The first part of this thesis describes the utility of PDMS and silicon microfluidic flow focusing devices (MFFDs) to produce PLGA-based microparticles. The formation of uniform droplets was dependent on the surface of PDMS remaining hydrophilic. However, the durability of PDMS was limited to no more than 1 hour before wetting of the microchannel walls with dichloromethane and subsequent swelling occurred. Critically, silicon MFFDs revealed very good solvent compatibility and was sufficiently robust to withstand elevated fluid flow rates. Silicon MFFDs facilitated experiments to run over days with continuous use and re-use of the device with a narrower microparticle size distribution, relative to conventional production techniques. The second part of this thesis demonstrates an alternative microencapsulation technology, SmPill® minispheres, to target CsA delivery to the colon. Characterisation of CsA release in vitro and in vivo was performed. By modulating the ethylcellulose:pectin coating thickness, release of CsA in-vivo was more effectively controlled compared to current commercial CsA formulations and demonstrated a linear in-vitro in-vivo relationship. Coated minispheres were shown to limit CsA release in the upper small intestine and enhance localised CsA delivery to the colon.

Fabrication of Poly (D,L-Lactic-Co-Glycolic Acid) Microparticles for Improved Human Papillomavirus Vaccine Delivery

Human papillomavirus (HPV) is the leading cause of cervical cancer and the most prevalent sexually transmitted disease worldwide. HPV vaccines require a multi-dose regimen to provide immunity, contributing to low patient compliance. We addressed this problem by formulating biodegradable poly(D,L-lactic-co-glycolic acid) (PLGA) microparticles and assessing their viability for use in controlled-release vaccines. We hypothesized that we could alter fabrication parameters to produce 1-10 μm microparticles in order to encapsulate ovalbumin (OVA) and HPV virus-like particles (VLPs). Microparticles were fabricated using a double emulsion method and used to elicit an immune response in JAWSII cells. Our results contribute to knowledge of vaccine delivery mechanisms and controlled-release technology, and could contribute to the creation of a viable controlled-release HPV vaccine.

Comportamiento de distintos céspedes ante el agregado de fertilizantes nitrogenados de diferente solubilidad

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Intracellular fate of bioresponsive poly(amidoamine)s in vitro and in vivo

Linear poly(amidoamine)s (PAAs) have been designed to exhibit minimal non-specific toxicity, display pH-dependent membrane lysis and deliver genes and toxins in vitro. The aim of this study was to measure PAA cellular uptake using ISA1-OG (and as a reference ISA23-OG) in B16F10 cells in vitro and, by subcellular fractionation, quantitate intracellular trafficking of (125)I-labelled ISA1-tyr in liver cells after intravenous (i.v.) administration to rats. The effect of time after administration (0.5-3h) and ISA1 dose (0.04-100mg/kg) on trafficking, and vesicle permeabilisation (N-acetyl-b-D-glucosaminidase (NAG) release from an isolated vesicular fraction) were also studied. ISA1-OG displayed approximately 60-fold greater B16F10 cell uptake than ISA23-OG. Passage of ISA1 along the liver cell endocytic pathway caused a transient decrease in vesicle buoyant density (also visible by TEM). Increasing ISA1 dose from 10mg/kg to 100mg/kg increased both radioactivity and NAG levels in the cytosolic fraction (5-10 fold) at 1h. Moreover, internalised ISA1 provoked NAG release from an isolated vesicular fraction in a dose-dependent manner. These results provide direct evidence, for the first time, of PAA permeabilisation of endocytic vesicular membranes in vivo, and they have important implications for potential efficacy/toxicity of such polymeric vectors.

The use of fluorescence microscopy to define polymer localisation to the late endocytic compartments in cells that are targets for drug delivery

Macromolecular therapeutics and nano-sized drug delivery systems often require localisation to specific intracellular compartments. In particular, efficient endosomal escape, retrograde trafficking, or late endocytic/lysosomal activation are often prerequisites for pharmacological activity. The aim of this study was to define a fluorescence microscopy technique able to confirm the localisation of water-soluble polymeric carriers to late endocytic intracellular compartments. Three polymeric carriers of different molecular weight and character were studied: dextrin (Mw~50,000 g/mol), a N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer (Mw approximately 35,000 g/mol) and polyethylene glycol (PEG) (Mw 5000 g/mol). They were labelled with Oregon Green (OG) (0.3-3 wt.%; <3% free OG in respect of total). A panel of relevant target cells were used: THP-1, ARPE-19, and MCF-7 cells, and primary bovine chondrocytes (currently being used to evaluate novel polymer therapeutics) as well as NRK and Vero cells as reference controls. Specific intracellular compartments were marked using either endocytosed physiological standards, Marine Blue (MB) or Texas-red (TxR)-Wheat germ agglutinin (WGA), TxR-Bovine Serum Albumin (BSA), TxR-dextran, ricin holotoxin, C6-7-nitro-2,1,3-benzoxadiazol-4-yl (NBD)-labelled ceramide and TxR-shiga toxin B chain, or post-fixation immuno-staining for early endosomal antigen 1 (EEA1), lysosomal-associated membrane proteins (LAMP-1, Lgp-120 or CD63) or the Golgi marker GM130. Co-localisation with polymer-OG conjugates confirmed transfer to discreet, late endocytic (including lysosomal) compartments in all cells types. The technique described here is a particularly powerful tool as it circumvents fixation artefacts ensuring the retention of water-soluble polymers within the vesicles they occupy.

Marine baseline and monitoring strategies for carbon dioxide capture and storage (CCS)

The QICS controlled release experiment demonstrates that leaks of carbon dioxide (CO2) gas can be detected by monitoring acoustic, geochemical and biological parameters within a given marine system. However the natural complexity and variability of marine system responses to (artificial) leakage strongly suggests that there are no absolute indicators of leakage or impact that can unequivocally and universally be used for all potential future storage sites. We suggest a multivariate, hierarchical approach to monitoring, escalating from anomaly detection to attribution, quantification and then impact assessment, as required. Given the spatial heterogeneity of many marine ecosystems it is essential that environmental monitoring programmes are supported by a temporally (tidal, seasonal and annual) and spatially resolved baseline of data from which changes can be accurately identified. In this paper we outline and discuss the options for monitoring methodologies and identify the components of an appropriate baseline survey.

Design of a silicone reservoir intravaginal ring for the delivery of oxybutynin.

Oxybutynin, a drug of choice in the treatment of urinary incontinence, has low oral bioavailability due to extensive first-pass metabolism. A toxic metabolite, N-desethyloxybutynin, has been linked to adverse reactions to oral oxybutynin. This study, therefore, reports on the design of an oxybutynin intravaginal ring (IVR) of reservoir design, comprising an oxybutynin silicone elastomer core encased in a non-medicated silicone sheath, manufactured by reaction injection moulding at 50oC. An unusually high initial burst release of oxybutynin (42.7 mg in 24 h) was observed in vitro with a full length core (100 mg drug loading), with subsequent non-zero order drug release. Use of fractional segment cores substantially reduced the burst effect, yielding linear cumulative drug release versus time plots from days 2 to 14. Thus, a 1/8 fractional segment core gave a 24 h burst of 11.28 mg oxybutynin and, thereafter, zero order release at the target dose of 5 mg/day over 14 days. Two oxybutynin cores, each 1/16 of full length, gave a greater release than a single 1/8 core, due to core segment end effects resulting in an increased surface area for release. The burst release was investigated by determining drug solubilities in the propan-1-ol product of elastomer condensation cure (390 mg/ml) and in the elastomer itself (13.9-20.21 mg/ml, by direct extraction and indirect thermal methods). These high oxybutynin solubilities were considered the major contributors to the burst effect. It was concluded that use of a fractional segment core would allow development of a suitable oxybutynin reservoir IVR.

An investigation into the structure and bioavailability of a-tocopherol dispersions in Gelucire 44/14

In this investigation we describe the preparation, physical characterisation and in vivo behaviour of solid dispersions of a liquid nutraceutical, ±-tocopherol, in Gelucire 44/14 with a view to establishing whether dispersion in this matrix may provide a means of formulating a liquid drug in a solid dosage form while also improving the oral bioavailability. Using Vitamin E Preparation USP as the source of ±-tocopherol, dispersions were prepared using a melt-fusion method with active loadings up to 50% (w/w) and characterised using differential scanning calorimetry and optical microscopy. Capsules containing 300 IU ±-tocopherol were manufactured and the absorption profiles compared to a commercial soft gelatin capsule preparation in healthy human volunteers. Confocal laser scanning microscopy (CLSM) studies were performed in order to elucidate the mechanism by which drug release may be occurring. Differential scanning calorimetry studies indicated that the presence of the active had a negligible effect on the melting profile of the carrier, indicating limited miscibility between the two components, a conclusion supported by the microscopy studies. Similarly, the dispersions were shown to exhibit a glass transition corresponding to the incorporated drug, indicating molecular cooperativity and hence phase separation from the lipid base. Despite the phase separation, it was noted that capsules stored for 18 months under ambient conditions showed no evidence of leakage. Bioavailability studies in six healthy male volunteers indicated that the Gelucire 44/14 formulation showed an approximately two-fold increase in total ±-tocopherol absorption compared to the commercial preparation. Confocal laser scanning microscopy studies indicated that, on contact with water, the dispersions formed two interfacial layers, from which the Gelucire 44/14 disperses in the liquid medium as small particles. Furthermore, evidence was obtained for the dispersed material becoming incorporated into the hydrated lipid. In conclusion, the dispersion of the liquid drug in Gelucire 44/14 appears to allow the dual advantages of the preparation of a solid formulation and improved bioavailability of this material.

Intravaginal ring delivery of the reverse transcriptase inhibitor TMC 120 as an HIV microbicide

TMC 120 (Dapivirine) is a potent non-nucleoside reverse transcriptase inhibitor that is presently being developed as a vaginal HIV microbicide. To date, most vaginal microbicides under clinical investigation have been formulated as single-dose semi-solid gels, designed for application to the vagina before each act of intercourse. However, a clear rationale exists for providing long-term, controlled release of vaginal microbicides in order to afford continuous protection against heterosexually transmitted HIV infection and to improve user compliance. In this study we report on the incorporation of various pharmaceutical excipients into TMC 120 silicone, reservoir-type intravaginal rings (IVRs) in order to modify the controlled release characteristics of the microbicide. The results demonstrate that TMC 120 is released in zero-order fashion from the rings over a 28-day period and that release parameters could be modified by the inclusion of release-modifying excipients in the IVR. The hydrophobic liquid excipient isopropyl myristate had little effect on steady-state daily release rates, but did increase the magnitude and duration of burst release in proportion to excipient loading in the IVR. By comparison, the hydrophobic liquid poly(dimethylsiloxane) had little effect on TMC 120 release parameters. A hydrophilic excipient, lactose, had the surprising effect of decreasing TMC 120 burst release while increasing the apparent steady-state daily release in a concentration-dependent manner. Based on previous cell culture data and vaginal physiology, TMC120 is released from the various ring formulations in amounts potentially capable of maintaining a protective vaginal concentration. It is further predicted that the observed release rates may be maintained for at least a period of 1 year from a single ring device. TMC 120 release profiles and the mechanical properties of rings could be modified by the physicochemical nature of hydrophobic and hydrophilic excipients incorporated into the IVRs.

Characterisation of Silicone Elastomer Vaginal Rings Containing HIV Microbicide TMC120 by Raman Spectroscopy

Silicone elastomer vaginal rings are currently being pursued as a controlled-release strategy for delivering microbicidal substances for the prevention of heterosexual transmission of HIV. Although it is well established that the distribution of drugs in delivery systems influences the release characteristics, in practice the distribution is often difficult to quantify in-situ. Therefore, the aim of this work was to determine whether Raman spectroscopy might provide a rapid, non-contact means of measuring the concentrations of the lead candidate HIV microbicide TMC120 in a silicone elastomer reservoir-type vaginal ring. Vaginal rings loaded with TMC120 were manufactured and sectioned before either Raman mapping an entire ring cross-section (100 µm resolution) or running line scans at appropriate time intervals up to 30 h after manufacture. The results demonstrated that detectable amounts of TMC120, above the silicone elastomer saturation concentration, could be detected up to 1 mm into the sheath, presumably as a consequence of permeation and subsequent reprecipitation. The extent of permeation was found to be similar in rings manufactured at 25 and 80°C.

Design, characterisation and preliminary clinical evaluation of a novel mucoadhesive topical formulation containing tetracycline for the treatment of periodontal disease

This study describes the formulation, characterisation and preliminary clinical evaluation of mucoadhesive, semi-solid formulations containing hydroxyethylcellulose (HEC, 1-5%, w/w), polyvinylpyrrolidine (PVP, 2 or 3%, w/w), poly carbophil (PC, 1 or 3%, w/w) and tetracycline (5%, w/w, as the hydrochloride). Each formulation was characterised in terms of drug release, hardness, compressibility, adhesiveness (using a texture analyser in texture profile analysis mode), syringeability (using a texture analyser in compression mode) and adhesion to a mucin disc (measured as a detachment force using the texture analyser in tensile mode). The release exponent for the formulations ranged from 0.78+/-0.02 to 1.27+/-0.07, indicating that drug release was non-diffusion controlled. Increasing the concentrations of each polymeric component significantly increased the time required for 10 and 30% release of the original mass of tetracycline, due to both increased viscosity and, additionally, the unique swelling properties of the formulations. Increasing concentrations of each polymeric component also increased the hardness, compressibility, adhesiveness, syringeability and mucoadhesion of the formulations. The effects on product hardness, compressibility and syringeability may be due to increased product viscosity and, hence, increased resistance to compression. Similarly, the effects of these polymers on adhesiveness/mucoadhesion highlight their mucoadhesive nature and, importantly, the effects of polymer state (particularly PC) on these properties. Thus, in formulations where the neutralisation of PC was maximally suppressed, adhesiveness and mucoadhesion were also maximal. Interestingly, statistical interactions were primarily observed between the effects of HEC and PC on drug release, mechanical and mucoadhesive properties. These were explained by the effects of HEC on the physical state of PC, namely swollen or unswollen. In the preliminary clinical evaluation, a formulation was selected that offered an appropriate balance of the above physical properties and contained 3% HEC, 3% PVP and 1% PC, in addition to tetracycline 5% (as the hydrochloride). The clinical efficacy of this (test) formulation was compared to an identical tetracycline-devoid (control) formulation in nine periodontal pockets (greater than or equal to 5 mm depth). One week following administration of the test formulation, there was a significant improvement in periodontal health as identified by reduced numbers of sub-gingival microbial pathogens. Therefore, it can be concluded that, when used in combination with mechanical plaque removal, the tetracycline-containing semi-solid systems described in this study would augment such therapy by enhancing the removal of pathogens, thus improving periodontal health. (C) 2000 Elsevier Science B.V. All rights reserved.

Microneedle-mediated intradermal delivery of 5-aminolevulinic acid: potential for enhanced topical photodynamic therapy

Photodynamic therapy of deep or nodular skin tumours is currently limited by the poor tissue penetration of the porphyrin precursor 5-aminolevulinic acid (ALA). In this study, silicon microneedle arrays were used, for the first time, to enhance skin penetration of ALA in vitro and in vivo. Puncturing excised murine skin with 6x7 arrays of microneedles 270 mum in height, with a diameter of 240 mum at the base and an interspacing of 750 mum led to a significant increase in transdermal delivery of ALA released from a bioadhesive patch containing 19 mg ALA cm(-2). Microneedle puncture enhanced ALA delivery to the upper regions of excised porcine skin but, at mean depths of 1.875 mm, ALA concentrations were similar to control values, possibly reflecting binding of ALA by tissue components. However, and importantly, in vivo experiments using nude mice showed that microneedle puncture could reduce application time and ALA dose required to induce high levels of the photosensitiser protoporphyrin IX in skin. This clearly has implications for clinical practice, as shorter application times would mean improved patient and clinician convenience and also that more patients could be treated in the same session. As ALA is expensive and degrades rapidly via a second order reaction, reducing the required dose is also a notable advantage.