Caveolae as potential macromolecule trafficking compartments within alveolar epithelium.

With inhalational delivery the alveolar epithelium appears to be the appropriate lung surface to target for the systemic delivery of macromolecules, such as therapeutic proteins. The existence of a high numerical density of smooth-coated or non-coated plasma membrane vesicles or invaginations within the alveolar epithelial type I cell has long been recognised. The putative function of these vesicles in macromolecule transport remains the focus of research in both pulmonary physiology and pharmaceutical science disciplines. These vesicles, or subpopulations thereof, have been shown to biochemically possess caveolin, a marker protein for caveolae. This review considers the morphometric and biochemical studies that have progressed the characterisation of the vesicle populations within alveolar type I epithelium. Parallel research findings from the endothelial literature have been considered to contrast the state of progress of caveolae research in alveolar epithelium. Speculation is made on a model of caveolae vesicle-mediated transport that may satisfy some of the pulmonary pharmacokinetic data that has been generated for macromolecule absorption. The putative transport function of caveolae within alveolar epithelium is reviewed with respect to in-situ tracer studies conducted within the alveolar airspace. Finally, the functional characterisation of in-vitro alveolar epithelial cell cultures is considered with respect to the role of caveolae in macromolecule transport. A potentially significant role for alveolar caveolae in mediating the alveolar airspace to blood transport of macromolecules cannot be dismissed. Considerable research is required, however, to address this issue in a quantitative manner. A better understanding of the membrane dynamics of caveolae in alveolar epithelium will help resolve the function of these vesicular compartments and may lead to the development of more specific drug targeting approaches for promoting pulmonary drug delivery.

Caveolae and the caveolins in human disease.

There has been an exponential growth in caveolae research since the early 1990s. The caveolae membrane system comprises unique lipid and protein domains, and fulfills a role in a wide range of processes. At the plasma membrane caveolae serve to compartmentalise and integrate a wide range of signal transduction processes. A key structural and functional protein for caveolae is caveolin. Caveolin proteins possess a 'scaffolding' domain which for caveolins-1 and -3 appear central to many of the reported signal regulation functions for caveolae. Caveolae or caveolin protein are increasingly implicated in the molecular pathology of a number of diseases. Opportunities exist for basic and applied investigators working within the pharmaceutical sciences to exploit the caveolae membrane system to identify novel pharmacological targets and therapeutic strategies, including the delivery of pharmacologically active caveolin based peptides.

Comparison of vasodilatory responses to nitroglycerin and its dinitrate metabolites in human veins.

Independent of the route of nitroglycerin administration, substantial amounts of 1,2-glyceryl dinitrate (1,2-GDN) and 1,3-glyceryl dinitrate (1,3-GDN) metabolites accumulate in humans. Thus far their pharmacologic activity in comparison to nitroglycerin in humans is unknown. To compare the venodilatory potency of nitroglycerin and of 1,2-GDN and 1,3-GDN in vivo, cumulative dose-response curves were established in nine healthy volunteers by use of the dorsal hand vein compliance technique. Separated by a washout period, two of the three venodilators were infused in randomized order after preconstriction with phenylephrine. Values for maximum vasodilation were similar for all compounds: nitroglycerin, 109%; 1,2-GDN, 100%; and 1,3-GDN, 106%. The respective values for the dose rate exerting 50% of maximum vasodilation were 5.1, 43, and 60 ng/min, indicating that the dinitrates were about 10 times less potent than nitroglycerin (p < 0.001) but not significantly different from each other. The findings support the hypothesis that activity of nitroglycerin metabolites is related to the number of nitrate groups in the molecule and are in agreement with lower dinitrate potencies found in animal experiments.

Synthesis, anti-human immunodeficiency virus activity and esterase lability of some novel carboxylic ester-modified phosphoramidate derivatives of stavudine (d4T).

We report the design, synthesis and antiviral evaluation of a series of lipophilic, masked phosphoramidate derivatives of the anti-human immunodeficiency virus (HIV) nucleoside analogue d4T, designed to act as membrane-soluble prodrug forms for the free nucleotide. In particular, we report a series of 12 novel compounds with systematic variation in the structure of the carboxylate ester function. In order to rationalize the changes in antiviral action with variation of this moiety we applied our recently developed 31P NMR-based assay for carboxyesterase lability to this series. However, no clear positive correlation emerged, indicating that, at least within this series, factors other than simple esterase lability may be the major determinants of antiviral potency.

Polylysine and polyornithine gene transfer complexes: a study of complex stability and cellular uptake as a basis for their differential in-vitro transfection efficiency.

Gene transfer vectors formed between the cationic polyamino acid, poly-(L)-omithine (PLO) and plasmid DNA (pDNA) have demonstrated superior transfection efficiency (up to x 10-fold) compared to equivalent polylysine-based systems in-vitro. The mechanism(s) underlying this observation remains to be elucidated. We previously reported no significant difference in colloidal particle size or zeta potential of polycation/pDNA complexes formed with poly-(L)-lysine (PLL), poly-(D)-lysine (PDL) or PLO. Here we report spectrofluorometric analysis indicating that PLO condenses pDNA at lower charge (+/-) ratios than PLL or PDL (cf. 0.8:1, 1.2:1 and 1.5:1). Moreover, PLO/pDNA complexes proved more stable to disruption by the polyanions, poly-(L)-aspartic acid (PAA) and heparin. There were no qualitative differences in the ability of the polycations to protect complexed pDNA from enzymatic degradation both in the presence and in the absence of polyanions. The superior transfection efficiency of PLO/pDNA complexes did not appear to be mediated by an increased cellular delivery of pDNA. The data suggests a greater affinity of PLO for pDNA as an important parameter for the observed superior in-vitro transfection efficiency.

The effect of fatty acids and analogues upon intracellular levels of doxorubicin in cells displaying P-glycoprotein mediated multidrug resistance.

Multidrug resistance mediated by overexpression of P-glycoprotein (P-gp) is a major obstacle in the chemotherapeutic management of cancer. The objectives of the current work were to examine if fatty acids affect the intracellular transport and dynamics of doxorubicin in drug-resistant cancer cell lines, and to assess if such effects were mediated through modulation of P-gp efflux pump activity. Among the range of fatty acids tested in this study, eicosapentaenoic acid diester (EPADI) increased doxorubicin accumulation [A] to 137% and retention [R] to 212% in doxorubicin-resistant MCF-7/ADR breast carcinoma cells, and [A] to 147% and [R] to 163% in vinblastine-resistant KBVI nasopharyngeal carcinoma cells. Consistent with EPADI-induced increases in intracellular doxorubicin concentrations, EPADI (10 microg/ml) sensitized MCF-7/ADR cells to the cytotoxic effects of doxorubicin (1 microg/ml) as assessed by MTT assay (viability < 50% of control), while EPADI itself displayed no cytotoxicity. The combination of EPADI (10 microg/ml) with verapamil (1 microM) resulted in a considerable increase in the [A] and [R] of the model P-gp substrate rhodamine-123 within drug-resistant cells compared to when either agent were used alone. KBV1 cells treated with combination of EPADI (10 microg/ml) and verapamil (1 microM) achieved 160% and 1120% greater [A] and [R] of rhodamine-123, respectively, compared to untreated cells. The P-gp modulatory effects of EPADI either alone, or as part of a combination with more potent inhibitors, should be further investigated.

Statistical modelling of the formulation variables in non-viral gene delivery systems.

Traditionally, optimisation of a gene delivery formulation utilises a study design that involves altering only one formulation variable at any one time whilst keeping the other variables constant. As gene delivery formulations become more complex, e.g. to include multiple cellular and sub-cellular targeting elements, there will be an increasing requirement to generate and analyse data more efficiently and allow examination of the interaction between variables. This study aims to demonstrate the utility of multifactorial design, specifically a Central Composite Design, in modelling the responses size, zeta potential and in vitro transfection efficiency of some prototypic non-viral gene delivery vectors. i.e. cationic liposome-pDNA complexes, and extending the application of the design strategy to more complex vectors, i.e. tri-component lipid:polycation:DNA (LPD). The modelled predictions of how the above responses change as a function of formulation show consistency with an extensive literature base of data obtained using more traditional approaches, and highlight the robustness and utility of the Central Composite Design in examining key formulation variables in non-viral gene delivery systems. The approach should be further developed to maximise the predictive impact of data across the full range of pharmaceutical sciences.

Progress and limitations in the use of in vitro cell cultures to serve as a permeability screen for the blood-brain barrier.

A relatively simple, widely applicable, and robust in vitro method of predicting blood-brain barrier (BBB) permeability to central nervous system-acting drugs is an increasing need. A cell-based model offers the potential to account for transcellular and paracellular drug diffusional processes, metabolism, and active transport processes, as well as nondefined interactions between a drug and cellular material that may impact upon a membrane's overall permeability profile. Any in vitro BBB cell model to be utilized for the transendothelial BBB permeability screening of potential central nervous system drugs must display reproducible solute permeability, and a number of other general criteria including: a restrictive paracellular barrier; a physiologically realistic cell architecture; the functional expression of key transporter mechanisms; and allow ease of culture to meet the technical and time constraints of a screening program. This article reviews the range of in vitro cell-based BBB models available, including the primary/low passage bovine and porcine brain endothelial cultures as well as the spectrum of immortalized brain endothelial cell lines that have been established. The article further discusses the benefits and limitations of exploiting such systems as in vitro BBB permeability screens.

Examination of the biophysical interaction between plasmid DNA and the polycations, polylysine and polyornithine, as a basis for their differential gene transfection in-vitro.

The impetus to develop non-viral gene delivery vectors has led to examination of synthetic polycationic polymers as plasmid DNA (pDNA) condensing agents. Previous reports have highlighted superiority (up to x 10-fold) in the in-vitro transfection of pDNA complexes formed by poly-(L)-ornithine (PLO) compared to those formed with poly-(L)-lysine (PLL). The apparent basis for this consistent superiority of PLO complexes remains to be established. This comparative study investigates whether physico chemical differences in the supramolecular properties of polycation:pDNA complexes provide a basis for their observed differential gene transfection. Specifically, particle size distribution and zeta potential of the above complexes formulated over a wide range of polycation:pDNA ratios were found to be consistent with a condensed (150-200 nm) cationic ( + 30-40 mV) system but not influenced by the type of cationic polymer used. A spectrofluorimetric EtBr exclusion assay showed that polycation:pDNA complexes display different pDNA condensation behaviour, with PLO able to condense pDNA at a lower polycation mass compared to both polylysine isomers, and form complexes that were more resistant to disruption following challenge with anionic counter species, i.e. poly-(L)-aspartic acid and the glycosaminoglycan molecule. heparin. We conclude that particle size and surface potential as gross supramolecular properties of these complexes do not represent, at least in a non-biological system, the basis for the differential transfection behaviour observed between these condensing polymers. However, differences in the ability of the polylysine and polyornithine polymers to interact with pDNA and to stabilise the polymer-pDNA assembly could have profound effects upon the cellular and sub-cellular biological processing of pDNA molecules and contribute to the disparity in cell transfection efficiency observed between these complexes.

Temporal dependence of ectopeptidase expression in alveolar epithelial cell culture: implications for study of peptide absorption.

There is little data available regarding the extent of peptide metabolism encountered following inhalation to the lung. We have studied the activity of five ectopeptidases in primary rat alveolar epithelial cells, A549 cells and pulmonary macrophages. Peptidase activity was assayed in the plasma membrane fractions (PMF) of primary type II alveolar epithelial cells (ATII cells) after 2 days in culture and after 7 days in culture when they had formed monolayers of type I-like cells (ATI-like cells). Dipeptidyl peptidase IV (DPP) activity fell from 36.65 mU/mg protein to 16.29 mU/mg protein between day 2 and day 7 in culture, aminopeptidase N (AMN) activity increased from 16.16 mU/mg protein to 23.99 mU/mg protein, angiotensin-converting enzyme (ACE) activity was lost (4.29 mU/mg protein at day 2, not detected at day 7), and carboxypeptidase M (CPM) activity was acquired (not detected at day 2, 5.20 mU/mg protein at day 7). The profile of exopeptidase expression in A549 cells was similar to that of primary rat alveolar cells at day 7 in culture (DPP 24.24 mU/mg protein, AMN 47.74 mU/mg protein, CPM 4.28 mU/mg protein, ACE not detected). Macrophages expressed high levels of aminopeptidases (DPP 46.85 mU/mg protein, AMN 28.28 mU/mg protein) but carboxypeptidase activity was not detected. Low neutral endopeptidase 24.11 (NEP) activity was found in all cell types studied (0.96-2.41 mU/mg protein). The qualitative and quantitative changes in the peptidase activity of primary cultured rat alveolar cells between day 2 and day 7 in culture has implications for the use of alveolar cell monolayers as drug absorption models to investigate peptide absorption from the lung. Ectopeptidase activity in cultured alveolar cells can be used to infer the peptide-metabolising capacity of the surface of the alveolar epithelium. The aminopeptidase activity in particular, if representative of enzyme activity in vivo, would offer a significant metabolic barrier to systemic delivery of peptides via the lung.

Physical stability and in-vitro gene expression efficiency of nebulised lipid-peptide-DNA complexes.

The lower respiratory tract provides a number of disease targets for gene therapy. Nebulisation is the most practical system for the aerosolisation of non-viral gene delivery systems. The aerosolisation process represents a significant challenge to the maintenance of the physical stability and biological activity of the gene vector. In this study we investigate the role of a condensing polycationic peptide on the stability and efficiency of nebulised lipid-DNA complexes. Complexes prepared from the cationic lipid 1, 2-dioleoyl-3-trimethylammonium propane (DOTAP) and plasmid DNA (pDNA) at mass (w/w) ratios of 12:1, 6:1 and 3:1, and complexes prepared from DOTAP, the polycationic peptide, protamine, and pDNA (LPD) at 3:2:1 w/w ratio were nebulised using a Pari LC Plus jet nebuliser. Samples from the nebuliser reservoir (pre- and post-nebulisation) and from the aerosol mist were collected and investigated for changes, including: particle diameter, retention of in-vitro transfection activity and the relative concentration and nature of the complexed pDNA remaining after the nebulisation procedure. The process of jet nebulisation adversely affected the physical stability of lipid:pDNA complexes with only those formulated at 12:1 w/w DOTAP:pDNA able to maintain their pre-nebulisation particle size distribution (145+/-3 nm pre-nebulisation vs. 142+/-2 nm aerosol mist) and preserve significant pDNA integrity in the reservoir (35% of pre-nebulisation pDNA band intensity). The LPD complexes were smaller (102+/-1 nm pre-nebulisation vs. 113+/-2 nm aerosol mist) with considerably greater retention of pDNA integrity in the reservoir (90% of pre-nebulisation pDNA band intensity). In contrast the concentration of pDNA in the aerosol mist for both the 12:1 w/w DOTAP:pDNA and LPD complexes were significantly reduced (10 and 12% of pre-nebulised values, respectively). Despite reduced pDNA concentration the transfection (% cells transfected) mediated by aerosol mist for the nebulised complexes was comparatively efficient (LPD aerosol mist 26 vs. 40% for pre-nebulised complex; the respective values for 12: 1 w/w DOTAP:pDNA were 12 vs. 28%). The physical stability and biological activity of nebulised lipid:pDNA complexes can be improved by inclusion of a condensing polycationic peptide such as protamine. The incorporation of the peptide precludes the use of potentially toxic excesses of lipid and charge and may act as a platform for the covalent attachment of peptide signals mediating sub-cellular targetting.

Microcalorimetry does not predict the cellular phagocytosis of latex microspheres.

Current literature highlights the potential suitability of microcalorimetry for the investigation of cell-drug interactions. Previous work using bacteria or antigens derived from infectious organisms yielded conclusions that heat production is a quantitative means of measuring phagocytosis. In this study we evaluated the potential of flow-through microcalorimetry as a method of quantifying the phagocytosis of microsphere particulates. The technique avoids the need to incorporate radioactive or fluorescent markers into the particulate formulation, and would be widely applicable in biopharmaceutical research. Using the monocyte cell line Mono Mac 6 a power output of 9.00 microW per million cells was increased significantly on addition of zymosan, lipopolysaccaride (LPS) and phorbol myristate acetate but not following exposure to FITC labelled latex microspheres (LM). TNFalpha production increased on exposure to zymosan, LPS and LPS-phorbol myristate acetate, though not on exposure to LB. An assay was developed which allowed the quantification of internalised particulates in phagocytic cells using fluorescent activated cell sorting (FACS). In contrast to the microcalorimetric and TNFalpha data FACS revealed that 20% of the MM6 population phagocytosed a mean of 1.35 LM. Microcalorimetry and measurements of TNFalpha production are assays of cellular activation a phenomenon not necessarily associated with phagocytosis. FACS, however, serves as a specific and quantitative measure of phagocytosis. Microcalorimetry may not be a suitable technique for the quantitative assessment of the phagocytosis of drug delivery particulates.

The inhibition of phagocytosis of respirable microspheres by alveolar and peritoneal macrophages.

Respirable poly(lactic co-glycolic acid) (PLGA) microspheres (2-3 microm diameter), were fabricated as a model drug delivery system whose uptake by macrophages could be quantified by fluorescent activated cell sorting. The microspheres exhibited minimal release of the entrapped flourophore (rhodamine B) and thus avoided possible fluid phase uptake of the flourophore. Externally bound microspheres were removed from the cell membrane by acid washing. The fluorescent intensity associated with the cells arose, therefore, from the internalised microspheres. NR8383 continuous culture alveolar macrophages were verified against primary cultures as a good model of alveolar phagocytosis. Peritoneal macrophages were also isolated and systemic and alveolar phagocytosis compared. Poloxamer 338 adsorbed at the microsphere surface did not reduce phagocytosis by NR8383 macrophages. It did, however, reduce the number of microspheres contained in primary alveolar macrophages but did not reduce the percentage of phagocytic cells. Poloxamer coatings did not reduce phagocytosis by peritoneal macrophages once the ratio of five microspheres per cell was exceeded. Dipalmitoylphosphatidylcholine (DPPC), the major component of lung surfactant, was added to cultures to model the alveolar environment where it was observed to reduce phagocytosis. In light of this finding, microspheres were coated in DPPC, which reduced their uptake by all cell types at all microsphere to cell ratios.

Gene expression in an intact ex-vivo skin tissue model following percutaneous delivery of cationic liposome-plasmid DNA complexes.

The skin represents an attractive site for the localised gene therapy of dermatological pathologies and as a potential antigen bioreactor following transdermal delivery. Potential also exists for the gene therapy of skin as a cosmetic intervention. The most exploited non-viral gene delivery system involves the complexation of cationic liposomes with plasmid DNA (pDNA) to form lipid:pDNA vectors that protect the DNA from nuclease-mediated degradation and improve transgene-cell interactions. Despite numerous studies examining the potential for these vectors in delivering genes to a variety of keratinocyte models, investigations into the topical application of such complexes to intact skin tissue is limited. This ex-vivo study, conducted with intact skin tissue derived from hairless mice, provides quantitative confirmation that topical administration of cationic lipid:pDNA complexes can mediate uptake and expression of reporter pDNA (33-fold higher compared with control) in viable epidermal tissue. The ex-vivo study design provides for intact skin tissue that has not been subjected to depilatory procedures of potential detriment to stratum corneum barrier function, and can be utilised for the quantitative and efficient examination of a potentially wide range of non-viral gene vectors designed for epidermal expression.

Dose-response and time-response biochemical and histological study of potassium dichromate-induced nephrotoxicity in the rat.

This study provides quantitative toxicological data on potassium dichromate-induced renal damage and considers the possible difficulties arising from the non-invasive in vivo assessment of renal damage, with particular attention to enzymuria. Renal damage induced in male Wistar rats by single sc injections of potassium dichromate was assessed 52 to 72 hr after doses ranging from 3 to 20 mg potassium dichromate/kg body weight and throughout a 9-day period following a dose of 20 mg potassium dichromate/kg. The earliest and most sensitive non-invasive functional change in the dose-response and time-response studies was an elevation in the rate of urinary excretion of protein. Evidence of tissue damage was observed with elevations in the urinary excretion rates of the brush border enzymes, gamma-glutamyltransferase, alkaline phosphatase and leucine aminopeptidase, the cytosolic enzymes, aspartate aminotransferase and lactate dehydrogenase and the lysosomal enzyme, N-acetyl-beta-D-glucosaminidase. Such changes occurred as early as the abnormal urinary protein excretion, but returned to control or sub-control values sooner. Urinary brush border enzyme excretion returned to control values within 48 hr following potassium dichromate injection, despite histological and histochemical evidence of extensive renal damage and renal dysfunction. Elevations in plasma aspartate aminotransferase and lactate dehydrogenase levels were observed, but histochemical and isoenzyme studies would be needed to determine the source of these increases. The simplest and most persistent indicators of renal damage were the urinary excretion of protein and N-acetyl-beta-D-glucosaminidase.

Differential influence of laboratory anaesthetic regimens upon renal and hepatosplanchnic haemodynamics in the rat.

Renal blood flow in rats anaesthetized with the combination alphaxolone/alphadolone (3.90 mL min-1 (g tissue)-1) was significantly (P less than 0.05) greater than in rats anaesthetized with ketamine midazolam (3.24 mL min-1 (g tissue)-1, pentobarbitone (3.19 mL min-1 (g tissue)-1), fentanyl/fluanisone midazolam (2.84 mL min-1 (g tissue)-1) or urethane (1.99 mL min-1 (g tissue)-1). Renal blood flow in the urethane anaesthetized rats was significantly (P less than 0.05) lower than in animals anaesthetized with the other anaesthetic regimens, and is consistent with literature reports of a depressive effect of urethane anaesthesia upon xenobiotic renal clearance in the rat. Hepatosplanchnic blood flow was highest in the alphaxolone/alphadolone anaesthetized animals (71.7 mL min-1 kg-1), with the urethane anaesthetized animals demonstrating a significantly (P less than 0.05) lower (33.4 mL min-1 kg-1) blood flow. The fentanyl fluanisone/midazolam (65.4 mL min-1 kg-1), pentobarbitone (61.1 mL min-1 kg-1), and ketamine/midazolam (51.4 mL min-1 kg-1) regimens resulted in hepatosplanchnic blood flows of intermediate magnitude. The observed marked differential effects of the anaesthetic regimens upon renal and hepatosplanchnic blood flows may dramatically influence drug disposition in the experimental animal, and be of significance to laboratory pharmacokinetic studies in which anaesthesia is used.

Lung surfactant phospholipids inhibit the uptake of respirable microspheres by the alveolar macrophage NR8383.

Fluorescent poly(lactic-co-glycolic acid) microspheres of a respirable size were fabricated for use in a fluorescent activated cell sorting assay utilizing the continuous alveolar macrophage NR8383. This is a suitable model of alveolar phagocytosis, which permitted an investigation of the influence of phospholipid structure on the inhibition of phagocytosis of microspheres. Phospholipid inhibition was found to be independent of phosphatidylcholine alkyl chain length. Head group effects were investigated by studies employing phosphatidyl-choline, -serine, and -ethanolamine, and inhibition was shown to be independent of head group. Closer modelling of the lung environment by co-culturing NR8383 on A549 alveolar epithelium showed type II secretions to also down-regulate phagocytosis. In addition, pre-incubation with microspheres coated with dipalmitoylphosphatidylcholine reduced the uptake of a second microsphere (fluorescein isothiocyanate-labelled latex).