Cationic Lipid-Based Gene Delivery Systems: Pharmaceutical Perspectives

Gene delivery systems are designed to control the location of administered therapeutic genes within a patient's body. Successful in vivo gene transfer may require (i) the condensation of plasmid and its protection from nuclease degradation, (ii) cellular interaction and internalization of condensed plasmid, (iii) escape of plasmid from endosomes (if endocytosis is involved), and (iv) plasmid entry into cell nuclei. Expression plasmids encoding a therapeutic protein can be, for instance, complexed with cationic liposomes or micelles in order to achieve effective in vivo gene transfer. A thorough knowledge of pharmaceutics and drug delivery, bio-engineering, as well as cell and molecular biology is required to design optimal systems for gene therapy. This mini-review provides a critical discussion on cationic lipid-based gene delivery systems and their possible uses as pharmaceuticals.     

Lactate transport in insulin-secreting beta-cells: contrast between rat islets and HIT-T15 insulinoma cells.

The transport of L- and D-lactate into rat pancreatic islets and HIT-T15 insulinoma cells was studied by measuring uptake of 14C-labelled substrate at room temperature and by following changes in intracellular pH (pHi) in islets and HIT-T15 cells loaded with 2',7'-bis(carboxyethyl)-5'(6')-carboxyfluorescein (BCECF). Uptake of L-lactate into HIT-T15 cells was rapid, reaching equilibrium after 5 min with an apparent Km value of 4.8 mM. Transport was markedly inhibited by alpha-cyano-4-hydroxycinnamate, alpha-fluorocinnamate, quercetin and p-chloromercuribenzenesulphonate (pCMBS), and was enhanced in citrate medium. Uptake of D-lactate was less rapid, apparent equilibrium not being reached within 10 min. In contrast to HIT-T15 cells, rat pancreatic islets showed greatly reduced rates of transport of L- and D-lactate together with a correspondingly lower degree of inhibition by alpha-cyano-4-hydroxycinnamate. The addition of L- or D-lactate to HIT-T15 cells, but not dispersed islet cells, resulted in a marked and rapid intracellular acidification followed by a gradual recovery. In both HIT-T15 cells and isolated islets, the rates of transport of both L- and D-lactate in the presence of alpha-cyano-4-hydroxycinnamate were significantly greater in a depolarising K+ medium compared to the normal Na+ medium. These observations suggest that native rat islet cells have considerably reduced activity of the lactate-/H+ transport system compared to HIT-T15 insulinoma cells. There is evidence in both cell types of an additional electrogenic pathway for lactate which might play a role in coupling lactate efflux to beta-cell depolarisation.     

Cardiovascular collapse during anesthesia in a patient with preoperatively discontinued chronic MAO inhibitor therapy

We describe a patient in whom long-term monoamine oxidase (MAO) inhibitor therapy was discontinued 20 days before surgery with general anesthesia. This patient developed severe perioperative hypotension after administration of 10 mg of bupivacaine through an epidural catheter, which was corrected only after potent vasopressor therapy. We attribute this hemodynamic instability to attenuation of this patient's sympathetic tone based on several mechanisms: (1) residual effect of long-term administration of MAO inhibitor that caused a decrease in the number of β-adrenergic receptors (adrenergic subsensitivity due to receptor down-regulation), (2) recovered MAO activity causing effective degradation of sympathetic amines, and (3) combined attenuating effects of general and epidural anesthesia on sympathetic tone.     

Changes in anal sphincter tone at induction of anaesthesia

Changes in anal pressure have been monitored during the induction of anaesthesia. Falls in pressure accompany loss of consciousness following bolus doses of commonly used intravenous and inhalational anaesthetic agents. Subsequent rises in pressure towards pre-anaesthetic levels are usually associated with the time taken to correct responses and initial recovery. Premedication, including anticholinergic drugs in conventional dosage, does not affect anal pressure.     

Relationship Among MRTA, DXA, and QUS

Dual-energy X-ray absorptiometry (DXA) and quantitative ultrasound (QUS) are the accepted modalities for the evaluation of fracture risk in the clinical setting. However, neither method provides a direct measurement of bone mechanics. In this study, we investigated a prototype device, known as a mechanical response tissue analyzer (MRTA), which provides direct mechanical measurements of mechanical properties of bone. A total of 56 healthy volunteers (20 men and 36 women) between the ages of 18 and 83 were recruited. The MRTA was used to measure the cross-sectional bending stiffness (EI) of the ulna bone. Axial speed of sound (SOS) at the ulna bone was determined by QUS; bone mineral content (BMC) and bone mineral density (BMD) were determined by DXA. Correlations, regression analysis, and analyses of variance (ANOVAs) were used to compare the three modalities. These analyses revealed that although there are strong linear relationships among the data collected by the various technologies, the bone properties reflected by MRTA are not fully explained by DXA and QUS. We conclude that the total information conveyed by MRTA measurements is unique. Further research is needed to delineate the different qualities of bone strength that are captured by MRTA, but not by DXA or QUS.     

RBE and w(R) values of Auger emitters and low-range beta emitters with particular reference to tritium.

An apparent disparity exists between RBE (relative biological effectiveness) values for low-range beta and Auger emitters, and the current value for their radiation weighting factor (w(R)). This paper presents evidence that the current w(R) value of unity for these nuclides is inconsistent with most RBE evidence and should be increased by a factor of two to three. It recommends that the ICRP should clearly state that the most appropriate RBE value for these nuclides, and not the w(R) value, should be used in specific dose calculations, retrospective dose estimations and epidemiological studies. The ICRP should also publish guidance as to the methods and data sources that could be used for these RBE values.     

Stress-Related Changes in β-Endorphin Processing: The Limitations of Slaughterhouse Material

In the sheep, unlike many other species, a significant proportion (>25%) of immunoreactive β-endorphin in the anterior pituitary is post-translationally modified to opioid-inactive, α-N-acetylated forms. In a study to determine the precise molecular nature of α-N-acetylated β-endorphin immunoreactivity, we noted a striking difference in high-performance liquid chromatography profiles of anterior pituitary extracts between sheep killed on the farm, and age-, sex- and strain-matched slaughterhouse animals. These altered patterns of a-N-acetylated β-endorphin processing were reproduced in farm animals by chronic (≤ 4 days) treatment with the synthetic glucocorticoid dexamethasone; in contrast dexamethasone had no effect on a-N-acetylated β-endorphin processing in hypothalamo-pituitary disconnected sheep. These data suggest that (1) the change in processing is a stress response, mediated by prolonged glucocorticoid exposure, (2) this effect is central, rather than a direct effect on the pituitary, and (3) the relative abundance of various peptide sequences in slaughterhouse-derived material may not reflect their abundance under more physiological conditions.     

A mathematical model of volatile release in mouth from the dispersion of gelled emulsion particles.

This paper presents a mathematical model of in-mouth volatile release from gelled emulsion particles dispersed in a continuous aqueous phase. Data based on APCI MS-Breath analysis is presented to demonstrate the effect of particle size, oil content and oil-water partition coefficients. It is shown that in-mouth release of aroma from the dispersion of gelled emulsion particles follows a two-component kinetic equation with fast and slow components. Both the fast and slow rate constants depend on the particle size, oil content and oil water partition coefficient of the aroma. The relative amount of aroma contributing to the fast and slow components also depends on the size of the particles. In order to understand this unexpected behaviour, an analytical model was developed that considers the interplay between the mass transfer of flavour across the interface of the particles and that across the air-liquid interface. Analytical expressions for the two rate constants and the relative ratio of aroma contributing to the fast component have been derived. From this model, three regimes of in-mouth release of aroma from the dispersion of gelled emulsion particles were identified including, the emulsion regime, the transition regime and the gel particle regime. In the emulsion regime, changes in the size of gelled emulsion particles had negligible impact on the overall release. In the transition regime, the release was controlled by the interaction of flavour transfer from the particles with that across the air-water interface. In the gel particle regime, aroma release at long times was governed by the particles and that at short times was governed by the air-water interface, and the two processes were fully decoupled. A simple relationship was derived for the critical size above which the release of aroma from the dispersion of gelled emulsion particles is affected by the size of the particles.