Joana Sousa

Intranasal delivery of systemic-acting drugs: small-molecules and biomacromolecules.

As a non-invasive route, intranasal administration offers patient comfort and compliance which are hurdled in parenteral drug therapy. In addition, the current recognition that the high permeability and vascularization of nasal mucosa coupled to the avoidance of the first-pass elimination and/or gastrointestinal decomposition ensure higher systemic drug absorption than oral route has contributed to the growing interest for intranasal delivery of drugs that require considerable systemic exposure to exert their therapeutic actions (systemic-acting drugs). Nevertheless, several features may hamper drug absorption through the nasal mucosa, particularly the drug molecular weight and intrinsic permeability, and, therefore, several strategies have been employed to improve it, propelling a constant challenge during nasal drug (formulation) development. This review will firstly provide an anatomical, histological and mechanistic overview of drug systemic absorption after nasal administration and the relevant aspects of the therapeutic interest and limitations of the intranasal systemic delivery. The current studies regarding the nasal application of systemic-acting small drugs (analgesic drugs, cardiovascular drugs and antiviral drugs) and biomacromolecular drugs (peptide/protein drugs and vaccines) will also be outlined, addressing drug pharmacokinetics and pharmacodynamic improvements.

Individual Spatial Responses towards Roads: Implications for Mortality Risk

Background Understanding the ecological consequences of roads and developing ways to mitigate their negative effects has become an important goal for many conservation biologists. Most mitigation measures are based on road mortality and barrier effects data. However, studying fine-scale individual spatial responses in roaded landscapes may help develop more cohesive road planning strategies for wildlife conservation. Methodology/Principal Findings We investigated how individuals respond in their spatial behavior toward a highway and its traffic intensity by radio-tracking two common species particularly vulnerable to road mortality (barn owl Tyto alba and stone marten Martes foina). We addressed the following questions: 1) how highways affected home-range location and size in the immediate vicinity of these structures, 2) which road-related features influenced habitat selection, 3) what was the role of different road-related features on movement properties, and 4) which characteristics were associated with crossing events and road-kills. The main findings were: 1) if there was available habitat, barn owls and stone martens may not avoid highways and may even include highways within their home-ranges; 2) both species avoided using areas near the highway when traffic was high, but tended to move toward the highway when streams were in close proximity and where verges offered suitable habitat; and 3) barn owls tended to cross above-grade highway sections while stone martens tended to avoid crossing at leveled highway sections. Conclusions Mortality may be the main road-mediated mechanism that affects barn owl and stone marten populations. Fine-scale movements strongly indicated that a decrease in road mortality risk can be realized by reducing sources of attraction, and by increasing road permeability through measures that promote safe crossings.

Analytical methods for determination of new fluoroquinolones in biological matrices and pharmaceutical formulations by liquid chromatography: a review

AbstractFluoroquinolones are one of the most promising and intensively studied drugs of contemporary anti-infective chemotherapy. New fluoroquinolone antibacterials with improved pharmacokinetic properties and a broad spectrum of activity have been developed, opening new windows of opportunity for clinical use. To our knowledge, no comprehensive and critical review of the analytical methods for the determination of these agents, which correspond to the third- and fourth-generation quinolones, has yet been published. This work summarizes for the first time most of the liquid chromatographic methods reported in the literature for the separation and quantification of the new fluoroquinolones in biological matrices and pharmaceutical formulations. A systematic and detailed survey of physicochemical properties, sample preparation procedures, and chromatographic and detection conditions is presented herein. In the course of this review several liquid chromatographic methods are discussed: reversed-phase high-performance liquid chromatography (RP-HPLC), ion-exchange high-performance liquid chromatography (IEX-HPLC), hydrophilic interaction liquid chromatography (HILIC), high-performance thin-layer chromatography (HPTLC) and other chiral chromatographic methods. Their advantages, applicability and limitations are also examined. FigureLiquid chromatographic methods for determination of new fluoroquinolones in biological matrices and pharmaceutical formulations.

Starch-based coatings for colon-specific delivery. Part II: physicochemical properties and in vitro drug release from high amylose maize starch films.

This work reports an investigation into free-film properties of a high amylose maize starch-based film coating that has been used in the preparation of formulations for drug delivery to the colon (WO 2008/012573 A1) and relates these properties to in vitro drug release from pellets. Maize starch/ethylcellulose free films were prepared and characterised by scanning electron microscopy (SEM), light microscopy, modulated differential scanning calorimetry (mDSC), Fourier-transform infrared (FT-IR), X-ray and % swelling in aqueous fluids with pH conditions similar to the stomach and small intestine. 5-ASA release from film-coated pellets was tested in enzyme free simulated gastric fluid and phosphate buffer pH 7.2. Selected formulations were further assessed in simulated gastric and intestinal fluids containing pepsin and pancreatin, respectively. The free films prepared were smooth and homogeneous in their appearance. The two polymers are immiscible, and neither mDSC nor FT-IR could detect interactions between them. Films made from high amylose starches were found to have a considerably lower swelling ability than high amylopectin-based films, and they suppressed drug release in the enzyme free media successfully. 5-ASA release from pellets coated with mixtures of high amylose starches (Hylon VII, Hylon V or LAPS) and Surelease in a ratio of 1 to 2 w/w was found to be minimal in simulated gastric and intestinal fluids. This suggests that these mixed films provide starch domains that are resistant to the enzymes present in the upper GI tract and thus can potentially be used in the preparation of colon-specific delivery devices. Starches with a minimum amylose content of 56% such as the starches used in this study (Hylon VII and Hylon V) are preferred, and although pure amylose can also be used this is not essential.

Antioxidant Versus Cytotoxic Properties of Hydroxycinnamic Acid Derivatives – A New Paradigm in Phenolic Research

Trihydroxycinnamic derivatives were synthesized and evaluated for their antioxidant and cytotoxic activities. The ester derivatives exhibited a higher radical‐scavenging activity, when liposomes were used as target systems, a fact which may be related to their lipophilicity and conformational preferences. These compounds were found to display significant growth inhibition and cytotoxic effects towards a human cervix adenocarcinoma cell line (HeLa). The partition coefficients presently obtained for the trihydroxycinnamic derivatives correlate well both with their structural characteristics and with their antioxidant/cytotoxic activities. A positive structure‐activity‐property relationship between cytotoxic and antioxidant activities, which is intrinsically related with physico‐chemical and conformational properties, is anticipated, as a noteworthy study that must be done for phenolic systems. As damage events are frequently correlated with oxidative stress, the prevalence of both properties in a single compound could be beneficial in terms of rationale preventive or therapeutic purposes.

Third and Fourth Generation Fluoroquinolone Antibacterials: A Systematic Review of Safety and Toxicity Profiles

In the last decade, several third and fourth generation fluoroquinolones (FQs) have been approved for clinical use. These new agents exhibit a more potent and broader-spectrum antibacterial activity and improved pharmacokinetic properties in comparison to the earlier FQs. Although new FQs are generally safe and well tolerated, moderate-to-severe toxicity events have been reported for some of them, leading to their restriction, suspension or even withdrawal from the market. The most common FQ-related adverse effects (AEs) are usually mild and involve the gastrointestinal tract (e.g. nausea and diarrhea) and the central nervous system (e.g. headache and dizziness). Uncommon, but severe AEs (e.g. arthropathy, QTc interval prolongation, dysglycaemia and phototoxicity) and idiosyncratic reactions (e.g. hepatitis and hemolytic anemia) have also been reported and will be discussed throughout this paper. The evidence currently available suggests that AEs can be inherent to the FQ class or can be associated with a particular chemical moiety of the molecular structure of each FQ, thus varying in frequency, severity and nature. The main goal of this review is to provide a systematic evaluation of safety and tolerability data of the newer FQs with emphasis on those currently marketed.

Development and validation of a fast isocratic liquid chromatography method for the simultaneous determination of norfloxacin, lomefloxacin and ciprofloxacin in human plasma.

A simple and fast liquid chromatographic method coupled with fluorescence detection (LC-FD) is reported, for the first time, for the simultaneous quantification of norfloxacin (NOR), ciprofloxacin (CIP) and lomefloxacin (LOM) in human plasma, using levofloxacin as internal standard (IS). Sample preparation consists of a single-step precipitation of plasma proteins followed by vortex-mixing and centrifugation. Chromatographic separation was achieved within 7  min on a reversed-phase C(18) column with a mobile phase consisting of 0.1% aqueous formic acid (pH = 3.0, triethylamine)-methanol (82:18, v/v) pumped isocratically at 1.2  mL/min. The detector was set at excitation/emission wavelengths of 278/450  nm. Calibration curves were linear (r(2) ≥ 0.994) in the range of 0.02-5.0 µg/mL, and the limit of quantification was established at 0.02 µg/mL for all analytes (NOR, CIP and LOM). The overall precision did not exceed 8.19% and accuracy was within ±10.91%. NOR, CIP and LOM were extracted from human plasma with an overall mean recovery ranged from 90.1 to 111.5%. No interferences were observed at the retention times of the analytes and IS. This novel LC-FD method enables the reliable determination of NOR, CIP and LOM in a single chromatographic run, which may be suitable to support human pharmacokinetic-based studies with those antimicrobial agents.

First liquid chromatography method for the simultaneous determination of levofloxacin, pazufloxacin, gatifloxacin, moxifloxacin and trovafloxacin in human plasma

For the first time a simple, selective and sensitive liquid chromatography method was developed and validated for the simultaneous determination of levofloxacin (LEV), pazufloxacin (PAZ), gatifloxacin (GAT), moxifloxacin (MOX) and trovafloxacin (TRO) in human plasma. Samples were pre-treated with acetonitrile for precipitation of plasma proteins followed by evaporation and reconstitution steps. Chromatographic separation of the analytes and norfloxacin, used as internal standard (IS), was performed under gradient elution on a LiChroCART(®) Purospher Star C18 column (55mm×4mm, 3μm). The mobile phase comprised a mixture of 0.1% aqueous formic acid adjusted to pH 3.0 with triethylamine, acetonitrile and methanol pumped at a flow rate of 1.0mL/min. The detector was set at excitation/emission wavelengths of 260/455nm. Calibration curves were linear (r(2)≥0.9923) in the ranges of 0.005-5μg/mL for GAT, 0.02-5μg/mL for LEV, PAZ and MOX and 0.04-5μg/mL for TRO. The intra and interday precision did not exceed 7.32% and the intra and interday accuracy ranged from -11.73 to 8.92%. The limits of quantification were established at 0.005μg/mL for GAT, 0.02μg/mL for LEV, PAZ and MOX and 0.04μg/mL for TRO. No endogenous or tested exogenous compounds were found to interfere at the retention times of the analytes and IS. Since the proposed method proved to be reliable for the quantitative determination of LEV, PAZ, GAT, MOX and TRO it may be a useful tool for routine analysis and to support clinical pharmacokinetic and toxicological studies involving these antibiotics.

Lack of Endogenous Adenosine Tonus on Sympathetic Neurotransmission in Spontaneously Hypertensive Rat Mesenteric Artery

Background Increased sympathetic activity has been implicated in hypertension. Adenosine has been shown to play a role in blood flow regulation. In the present study, the endogenous adenosine neuromodulatory role, in mesenteric arteries from normotensive and spontaneously hypertensive rats, was investigated. Methods and Results The role of endogenous adenosine in sympathetic neurotransmission was studied using electrically-evoked [3H]-noradrenaline release experiments. Purine content was determined by HPLC with fluorescence detection. Localization of adenosine A1 or A2A receptors in adventitia of mesenteric arteries was investigated by Laser Scanning Confocal Microscopy. Results indicate a higher electrically-evoked noradrenaline release from hypertensive mesenteric arteries. The tonic inhibitory modulation of noradrenaline release is mediated by adenosine A1 receptors and is lacking in arteries from hypertensive animals, despite their purine levels being higher comparatively to those determined in normotensive ones. Tonic facilitatory adenosine A2A receptor-mediated effects were absent in arteries from both strains. Immunohistochemistry revealed an adenosine A1 receptors redistribution from sympathetic fibers to Schwann cells, in adventitia of hypertensive mesenteric arteries which can explain, at least in part, the absence of effects observed for these receptors. Conclusion Data highlight the role of purines in hypertension revealing that an increase in sympathetic activity in hypertensive arteries is occurring due to a higher noradrenaline/ATP release from sympathetic nerves and the loss of endogenous adenosine inhibitory tonus. The observed nerve-to-glial redistribution of inhibitory adenosine A1 receptors in hypertensive arteries may explain the latter effect.

The Adenosinergic System as a Therapeutic Target in the Vasculature: New Ligands and Challenges

Adenosine is an adenine base purine with actions as a modulator of neurotransmission, smooth muscle contraction, and immune response in several systems of the human body, including the cardiovascular system. In the vasculature, four P1-receptors or adenosine receptors—A1, A2A, A2B and A3—have been identified. Adenosine receptors are membrane G-protein receptors that trigger their actions through several signaling pathways and present differential affinity requirements. Adenosine is an endogenous ligand whose extracellular levels can reach concentrations high enough to activate the adenosine receptors. This nucleoside is a product of enzymatic breakdown of extra and intracellular adenine nucleotides and also of S-adenosylhomocysteine. Adenosine availability is also dependent on the activity of nucleoside transporters (NTs). The interplay between NTs and adenosine receptors’ activities are debated and a particular attention is given to the paramount importance of the disruption of this interplay in vascular pathophysiology, namely in hypertension., The integration of important functional aspects of individual adenosine receptor pharmacology (such as in vasoconstriction/vasodilation) and morphological features (within the three vascular layers) in vessels will be discussed, hopefully clarifying the importance of adenosine receptors/NTs for modulating peripheral mesenteric vascular resistance. In recent years, an increase interest in purine physiology/pharmacology has led to the development of new ligands for adenosine receptors. Some of them have been patented as having promising therapeutic activities and some have been chosen to undergo on clinical trials. Increased levels of endogenous adenosine near a specific subtype can lead to its activation, constituting an indirect receptor targeting approach either by inhibition of NT or, alternatively, by increasing the activity of enzymes responsible for ATP breakdown. These findings highlight the putative role of adenosinergic players as attractive therapeutic targets for cardiovascular pathologies, namely hypertension, heart failure or stroke. Nevertheless, several aspects are still to be explored, creating new challenges to be addressed in future studies, particularly the development of strategies able to circumvent the predicted side effects of these therapies.