Cartilage oligomeric matrix protein (COMP) is modified by intra-articular liposomal clodronate in an experimental model of arthritis.

OBJECTIVE: High-dose liposomal bisphosphonates exert apoptotic effects. This work studies the chondroprotective and anti-inflammatory properties of intra-articularly administered low-dose, non-cytotoxic liposomal clodronate. METHODS: Antigen induced arthritis in rabbits was treated with intra-articular injections of liposomal clodronate. Drug effects on cartilage oligomeric matrix protein COMP was assessed using immunohistochemistry and morphometry of synovial membrane and hyaline articular cartilage. RESULTS: COMP remained close to normal in liposomal clodronate treated superficial articular cartilage compared to a significant loss of COMP in arthritis controls treated with empty liposomes. The middle and deep layers of the hyaline articular cartilage were characterized by highly increased COMP expression in liposomal clodronate treated AIA joints compared to controls. In contrast to cartilage, synovial COMP expression was slightly decreased as a result of liposomal clodronate treatment. CONCLUSION: Low-dose, non-cytotoxic liposomal clodronate exerts a dichotomous effect on synovial membrane and articular cartilage COMP in the AIA model. COMP is a useful inflammation marker in the synovial tissue, but it also contributes to the structural integrity of the hyaline articular cartilage forming bridges between type II and IX collagens. Enhancement of COMP in clodronate treated AIA cartilage suggests a chondroprotective and anti-inflammatory effect in the inflammatorily damaged and mechanically strained cartilage.

Oligonucleotide-cationic liposome interactions. A physicochemical study.

Cationic liposomes are effective in delivering antisense oligonucleotides into cells in culture, but their interactions with the oligonucleotides are poorly understood. We studied the aggregation and fusion reactions during the formation of cationic lipid/oligonucleotide complexes in solution and their interactions with lipid bilayers. Phosphorothioate oligonucleotides (15-mer) were complexed with cationic liposomes composed of dimethyldioctadecylammonium bromide (DDAB) and dioleoylphosphatidylethanolamine (DOPE) at 8:15 molar ratio or of a commercial formulation DOTAP (N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammoniummethylsul fate), at different ratios with apparent -/+ charge ratios of 0.03-5.6. Mean size of the complexes increased with -/+ ratio so that at charge ratios 0.4-2.0 the size increased by at least an order magnitude due to the oligonucleotide induced aggregation. Resonance energy transfer experiments showed that in addition to aggregation oligonucleotides induced fusion of cationic liposomes, but the fusion was rate-controlled by the initial aggregation step. Rate constants for oligonucleotide induced aggregation were dependent on lipid concentration and were in the range of (0.2-1).10(7) M-1 s-1 and (1-10).10(7) M-1 s-1 for DDAB/DOPE and DOTAP, respectively. Increase in oligonucleotide concentration induced the aggregation and fusion until at high -/+ ratios electrostatic repulsion of negative surfaces inhibited further aggregation and fusion. DOTAP/oligonucleotide complexes did not induce leakage of calcein from neutral EPC liposomes, but did cause leakage at -/+ charge ratios of < 0.7 and > 2.0 from EPC/DOPE liposomes. Also at -/+ charge ratios below 0.8 DOTAP/oligonucleotide complexes induced leaking from negatively charged DPPC/DPPG liposomes. These results indicate that either phosphatidylethanolamine or negative charge are required in the cell membrane for fusion of cationic liposome-oligonucleotide complexes. The ratio of oligonucleotide to cationic lipid is critical in determining the physicochemical properties of the mixture.

Interaction of liposomes with human skin in vitro--the influence of lipid composition and structure.

Liposomes have been suggested as a vehicle for dermal and transdermal drug delivery, but the knowledge about the interaction between lipid vesicles and human skin is poor. Therefore, we visualized liposome penetration into the human skin by confocal laser scanning microscopy (CLSM) in vitro. Liposomes were prepared from phospholipids in different compositions and labeled with a fluorescent lipid bilayer marker, N-Rh-PE (L-alpha-phosphatidylethanolamine-N-lissamine rhodamine B sulfonyl). Fluorescently labelled liposomes were not able to penetrate into the granular layers of epidermis. However, the fluorescence from liposome compositions containing DOPE (dioleylphosphatidyl ethanolamine) was able to penetrate deeper into the stratum corneum than that from liposomes without DOPE. Pretreatment of skin with unlabeled liposomes containing DOPE or lyso-phosphatidyl choline (lyso-PC) enhanced the subsequent penetration of the fluorescent markers, N-Rh-PE and sulforhodamine B into the skin, suggesting possible enhancer activity, while most liposomes did not show such enhancement. Resonance energy transfer (RET) and calcein release assay between stratum corneum lipid liposomes (SCLLs) and the phospholipid vesicles suggested that the liposomes containing DOPE may fuse or mix with skin lipids in vitro and loosen the SCLL bilayers, respectively. Among the factors not affecting stratum corneum penetration were: negative charge, cholesterol inclusion and acyl chain length of the phospholipids. In conclusion, fusogenicity of the liposome composition appears to be a prerequisite for the skin penetration.

Upregulation of the mevalonate pathway by cholesterol depletion abolishes tolerance to N-bisphosphonate induced Vγ9Vδ2 T cell cytotoxicity in PC-3 prostate cancer cells.

Zoledronate (ZOL) inhibits farnesyl pyrophosphate synthase leading to intracellular accumulation of isopentenyl pyrophosphate/triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester (IPP/ApppI). Cytotoxic Vγ9Vδ2 T cells have been shown to recognize IPP/ApppI in breast cancer cells. Further, human breast cancer cells have been shown to differ remarkably in their ZOL treatment induced IPP/ApppI production and responses to that. In this communication we analysed the responsiveness of prostate cancer cells PC-3 and DU-145, Caki-2 renal carcinoma cells and U87MG glioblastoma cells to ZOL treatment, and the subsequent activation of Vγ9Vδ2 T-cell cytotoxicity. Of the cell lines tested, PC-3 cells were not susceptible to Vγ9Vδ2 T-cell cytotoxicity due to low activity of the mevalonate pathway and low amount of IPP formed. However, the resistance of PC-3 cells to Vγ9Vδ2 T-cell cytotoxicity could be abrogated by upregulation of the mevalonate pathway through cholesterol depletion.

Clodronate and liposome-encapsulated clodronate are metabolized to a toxic ATP analog, adenosine 5'-(beta, gamma-dichloromethylene) triphosphate, by mammalian cells in vitro.

Clodronate, alendronate, and other bisphosphonates are widely used in the treatment of bone diseases characterized by excessive osteoclastic bone resorption. The exact mechanisms of action of bisphosphonates have not been identified but may involve a toxic effect on mature osteoclasts due to the induction of apoptosis. Clodronate encapsulated in liposomes is also toxic to macrophages in vivo and may therefore be of use in the treatment of inflammatory diseases. It is generally believed that bisphosphonates are not metabolized. However, we have found that mammalian cells in vitro (murine J774 macrophage-like cells and human MG63 osteosarcoma cells) can metabolize clodronate (dichloromethylenebisphosphonate) to a nonhydrolyzable adenosine triphosphate (ATP) analog, adenosine 5'-(beta, gamma-dichloromethylene) triphosphate, which could be detected in cell extracts by using fast protein liquid chromatography. J774 cells could also metabolize liposome-encapsulated clodronate to the same ATP analog. Liposome-encapsulated adenosine 5'-(beta, gamma-dichloromethylene) triphosphate was more potent than liposome-encapsulated clodronate at reducing the viability of cultures of J774 cells and caused both necrotic and apoptotic cell death. Neither alendronate nor liposome-encapsulated alendronate were metabolized. These results demonstrate that the toxic effect of clodronate on J774 macrophages, and probably on osteoclasts, is due to the metabolism of clodronate to a nonhydrolyzable ATP analog. Alendronate appears to act by a different mechanism.

Conditioned medium of estrogen-treated osteoblasts inhibits osteoclast maturation and function in vitro.

The increase of bone resorption and reduction of bone mass in postmenopausal women can be prevented by treatment with estrogen. Although it is well established that estrogen treatment normalizes the increased bone turnover, the mechanism by which estrogen exerts its protective influence at the cellular and molecular level in bone remains elusive. It has been shown that osteoblasts are involved in osteoclast development and osteoclastic bone resorption. In this work we examine the effect of estrogen (E2) on osteoclast-mediated bone resorption via the medium conditioned by osteoblast cultures. The conditioned medium collected from osteoblast cultures without (CM) or with 0.1 nmol/L 17beta-estradiol (E-CM) was mixed in a 1:1 ratio with fresh osteoclast culture medium. Osteoclasts were isolated from the bone marrow of 3-day-old NMRI mice and cultured on bovine bone slices. The total number of multinucleated tartrate-resistant alkaline phosphatase (TRAP)-positive cells in cultures with CM and E-CM was similar to that of cells incubated in control medium. However, the number of osteoclasts containing more than three nuclei was significantly smaller in the cultures containing E-CM. The total area of resorption was only slightly decreased in cultures containing CM, but was markedly inhibited in cultures with E-CM. In osteoblast cultures, the production of interleukin (IL)-1 and IL-6, but not of TNF-alpha, was reduced by 0.1 nmol/L E2. Our data suggest that E2 treatment of osteoblasts decreases the production of factor(s) that induces osteoclast differentiation to multinucleated cells with a higher capacity for bone resorption.

Nitrogen-containing bisphosphonates induce apoptosis of Caco-2 cells in vitro by inhibiting the mevalonate pathway: a model of bisphosphonate-induced gastrointestinal toxicity.

Bisphosphonates have become an important addition to the pharmacological armamentarium against postmenopausal osteoporosis. One of the major side effects of oral therapy with some nitrogen-containing bisphosphonates appears to be gastrointestinal (GI) intolerability, particularly esophageal irritation and ulceration. Because nitrogen-containing bisphosphonates can cause apoptosis in a variety of cell types in vitro, by inhibiting the mevalonate pathway, we hypothesized that the effect of these agents on the GI tract may be due to apoptosis or inhibition of growth of gut epithelial cells. A comparison between clodronate, etidronate, pamidronate, alendronate, and risedronate demonstrated that only the nitrogen-containing bisphosphonates were effective at inducing apoptosis or inhibiting proliferation of Caco-2 human epithelial cells in vitro, at concentrations of between 10 and 1000 micromol/L. The ability of nitrogen-containing bisphosphonates to cause apoptosis and inhibit Caco-2 cell proliferation was due to inhibition of the mevalonate pathway, because the addition of farnesol, oxidized low-density lipoprotein (LDL) cholesterol, or especially geranylgeraniol suppressed the effects. Furthermore, pamidronate, alendronate, and risedronate inhibited protein prenylation in Caco-2 cells, as determined by analysis of the processing of Rap1A, a prenylated small GTPase. These studies suggest that the effects of nitrogen-containing bisphosphonates observed in the GI tract may be due to inhibition of proliferation or apoptosis of gut epithelial cells, following loss of prenylated proteins and sterols.

A peptide prodrug approach for improving bisphosphonate oral absorption.

This work was aimed at improving the absorption of bisphosphonates by targeting carrier systems in the intestine and the intestinal peptide carrier system (hPEPT1), in particular. (14)C-Labeled pamidronate and alendronate as well as radiolabeled and "cold" peptidyl-bisphosphonates, Pro-[(3)H]Phe-[(14)C]pamidronate, and Pro-[(3)H]Phe-[(14)C]alendronate were synthesized. In situ single-pass perfusion studies revealed competitive inhibition of transport by Pro-Phe, suggesting peptide carrier-mediated transport. Prodrug transport in the Caco-2 cell line was significantly better than that of the parent drugs, and the prodrugs exhibited high affinity to the intestinal tissue. Oral administration of the dipeptidyl prodrugs resulted in a 3-fold increase in drug absorption following oral administration in rats, and the bioavailability of Pro-Phe-alendronate was 3.3 (F(TIBIA)) and 1.9 (F(URINE)) times higher than that of the parent drug. The results indicate that the oral absorption of bisphosphonates can be improved by peptidyl prodrugs via the hPEPT1; however, other transporters may also be involved.

Calcitonin promotes osteoclast survival in vitro.

Inactivation of resorbing osteoclasts by calcitonin is associated with typical morphological changes and alteration of the specific organization of osteoclast cytoskeleton. Here we show that calcitonin also promotes the survival of rat osteoclasts in vitro, cultured either on glass or bone, by delaying the onset of apoptosis. Parathyroid hormone had no effect on osteoclasts cultured on glass but it slightly increased apoptosis index of osteoclasts cultured on bone. Calcitonin was also able to rescue osteoclasts in calvarial explant cultures. The survival effect of calcitonin was mimicked by dibutyryl cAMP and could not be blocked by various metabolic inhibitors known to affect the apoptotic pathway. However, clodronate-induced apoptosis of osteoclasts could not be reversed by calcitonin and neither could calcitonin rescue osteoclasts already committed to apoptosis. It did not alter the distribution of Bcl-2 in osteoclasts. Our results show that at least in vitro calcitonin protects osteoclasts from apoptosis and suggest that it regulates the onset of apoptosis.

Phospholipids affect stratum corneum lipid bilayer fluidity and drug partitioning into the bilayers.

Phospholipids, e.g. fluid-state EPC (l-alpha-phosphatidylcholine from egg yolk), may diffuse into the stratum corneum and enhance dermal and transdermal drug penetration, while many other phospholipids, e.g. gel-state DSPC (distearoylphosphatidyl choline), are not able to do this. These effects are suggested to be due to the interactions between the phospholipids and the skin lipid bilayers, and so an in vitro method was developed to evaluate the influence of phospholipids on the distribution of drugs to stratum corneum lipids. The distribution coefficients of estradiol, progesterone and propranolol between stratum corneum lipid liposomes (SCLLs) without phospholipids or with EPC, DSPC, SPC (l-alpha-phosphatidylcholine from soybean) or DOPE (dioleylphosphatidyl ethanolamine), and pH 7.4 buffer were determined. Fluid-state phospholipids in SCLLs increased the partitioning of drugs into SCLLs, while gel-state lipid, DSPC, did not. The increased distribution of drugs into the SCLLs was at least partially due to the increased fluidity of SCLL bilayers by phospholipids, which was shown using steady-state fluorescence anisotropy. This in vitro method enables screening of the effects of phospholipids and other permeation enhancers on stratum corneum bilayer fluidity and drug partitioning.

Clodronate (dichloromethylene bisphosphonate) inhibits LPS-stimulated IL-6 and TNF production by RAW 264 cells.

Effect of liposome-encapsulated and free clodronate on the IL-6 and TNF production by macrophages was studied using RAW 264 cell line as a macrophage model, and dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) for analysis of secreted cytokines. LPS-stimulated RAW 264 cells proved to produce notable amounts of these two cytokines, and DELFIA was sensitive and reliable method for analysis. Liposome-encapsulated clodronate inhibited the production of both cytokines, IL-6 being affected more than TNF, and the effect was mostly due to the drug itself, not to liposomal lipid. More than ten times higher concentration of free clodronate than liposomal clodronate was needed to inhibit cytokine production. This is the first report on the cytokine inhibitory property of clodronate, and the results support the idea of the use of liposomal clodronate as a macrophage suppressive agent in autoimmune diseases.

Effects of liposome-encapsulated bisphosphonates on acetylated LDL metabolism, lipid accumulation and viability of phagocyting cells.

Bisphosphonates, the drugs used for the treatment of e.g. osteoporosis, inhibit the development of experimental atherosclerosis. When encapsulated in liposomes, they also inactivate macrophages, which have a key role in atherogenesis. We studied the effects of three clinically used bisphosphonates, i.e. clodronate, etidronate and pamidronate, on 1) the viability of mouse peritoneal macrophages and macrophage-like RAW 264 cells, 2) the degradation of 125I-labeled acetylated LDL by RAW 264 cells, and 3) the formation of LDL-derived foam cells in vitro. Liposome-encapsulated clodronate and pamidronate, but not etidronate, decreased the fraction of viable peritoneal macrophages in a concentration-dependent manner, whereas RAW 264 cells were much more resistant to the cytotoxic effects of bisphosphonates. Preincubation with liposomal clodronate and etidronate inhibited in a concentration-dependent manner the degradation of acetylated LDL in RAW 264 cells, but non-cytotoxic concentrations of liposomal pamidronate had only a weak inhibitory effect. The inhibition was more pronounced by liposomal clodronate than by liposomal etidronate. At high concentrations (500 microg protein/ml) of acetylated and aggregated LDL, RAW 264 cells transformed to foam cells. Preincubation with liposomal clodronate and etidronate reduced the cellular accumulation of acetylated LDL-derived lipids, but the drugs had no effect on the lipid accumulation caused by aggregated LDL. The results suggest that liposomal clodronate and etidronate inhibit the activity of phagocyting cells in internalizing and degrading atherogenic modified LDL.

Effects of tiludronate and ibandronate on the secretion of proinflammatory cytokines and nitric oxide from macrophages in vitro.

Bisphosphonates inhibit osteoclastic bone resorption and are used for the treatment of bone diseases. Some bisphosphonates, such as clodronate and tiludronate, can be incorporated into non-hydrolysable ATP analogues in cells, whereas the more potent anti-resorptive aminoalkylbisphosphonates are not metabolised. Furthermore, clodronate inhibits proinflammatory cytokine and nitric oxide (NO) secretion from activated macrophages in vitro and has anti-inflammatory properties in vivo, especially when delivered into cells by liposomes. By contrast, aminobisphosphonates can induce an acute phase response and fever in vivo, which appears to involve the induction of cytokine secretion. In this study we examined the effect of liposome-mediated intracellular delivery of one aminobisphosphonate, ibandronate, and one metabolizable bisphosphonate, tiludronate, on the secretion of inflammatory mediators. The intracellular uptake of bisphosphonates by macrophages was enhanced by a factor of 20-200 by using liposomes. Tiludronate dose-dependently inhibited both cytokine and NO secretion from activated macrophages, and liposomal tiludronate was more potent than the free drug. By contrast, ibandronate enhanced LPS-induced secretion of IL-1beta and IL-6 but did not affect TNFalpha or NO secretion at non-cytotoxic concentrations. The present results, together with our previous studies, strongly suggest that bisphosphonates can be grouped into those that are metabolised by cells and that are capable of inhibiting cytokine and NO secretion from macrophages, thus having potential anti-inflammatory properties, and those that are not metabolised but can actually enhance the production of cytokines following macrophage activation.

Regulation of MMP-9 (gelatinase B) in activated human monocyte/macrophages by two different types of bisphosphonates.

Metalloproteinases (MMP), particularly MMP-9 produced by the intratumor monocyte/macrophages, play an important role in tumor invasion and metastases. Recent clinical trials in patients with primary breast cancer suggest that bisphosphonates (BP), above all clodronate, may reduce bone metastases. The aim of the present study was to evaluate whether the effects of BPs on cancer dissemination include inhibition of MMP-9 production in human monocyte/macrophages. The effects of clodronate and pamidronate on the MMP-9 expression in and secretion from stimulated human monocyte/macrophages were measured using quantitative reverse transcriptase - polymerase chain reaction (RT-PCR) and enzyme-linked immunoadsorbent assay (ELISA), respectively. The MMP-9 mRNA levels remained relatively stable in the presence of clodronate. In contrast, pamidronate at 30 microM-300 microM increased the mRNA levels 5- to 10-fold. MMP-9 secretion was dose-dependently down-regulated by clodronate whereas pamidronate at 30 microM induced a 50% increase on MMP-9 secretion (p < 0.05), followed by a down-regulation at higher concentrations. The results suggest that MMP-9 is differentially regulated at mRNA and enzyme protein level by BPs, which affect ATP-dependent intracellular enzymes (clodronate) or post-translational modification of GTPases (pamidronate). These findings may have implications for the therapeutic use of these compounds.

Growth inhibition of macrophage-like and other cell types by liposome-encapsulated, calcium-bound, and free bisphosphonates in vitro.

Bisphosphonates effectively inhibit osteoclastic bone resorption in diseases characterized by excessive bone loss. Liposome-encapsulated clodronate (dichloromethylene bisphosphonate) also is known to inactivate phagocytic cells in vivo, and inhibit the growth of macrophage-like RAW 264 cells in vitro. The macrophage suppressive effect of liposomal clodronate is of interest in autoimmune diseases, like rheumatoid arthritis, in which phagocytic cells are involved in inflammatory processes. Earlier in vivo studies suggested that liposomal clodronate is a far more potent inactivator of macrophages than liposomal forms of two other bisphosphonate compounds, pamidronate (3-amino-1-hydroxypropylidene bisphosphonate), and etidronate (1-hydroxyethylidene-1,1-bisphosphonate). We examined the growth inhibitory properties of these three bisphosphonates with macrophage-like RAW 264 cells and with other types of cells in vitro. All three bisphosphonates encapsulated in liposomes effectively inhibited the growth of RAW 264 and CV1-P cells, while free drugs were 20-1000 times less potent growth inhibitors. Also, high extracellular calcium concentrations enhanced the potency of bisphosphonates for RAW 264 cells, indicating that, in addition to liposomes, the uptake of bisphosphonates by macrophages is mediated also by calcium. In all formulations, pamidronate was the most potent compound for the cells, with the exception of CV1-P cells, for which liposomal clodronate was the most potent. The effects of liposomal drugs were selective for highly endocytotic cells. The results suggest that liposome-encapsulated bisphosphonates could provide a specific tool to affect the function of macrophages and all three of these bisphosphonates are potentially effective as macrophage suppressors in autoimmune diseases.

In vitro and in vivo effects of tetrakisphosphonates on bone resorption, tumor osteolysis, ectopic calcification, and macrophages.

The biological effects of bisphosphonates in calcium-related disorders are attributed to the incorporation of the bisphosphonates in bone, enabling direct interaction with osteoclasts and/or osteoblasts. The high accumulation of bisphosphonates in bone, due to their high affinity to hydroxyapatite (HAP), is essential for mediating in vitro and in vivo activity. In this study we examined the activity of tetrakisphosphonates, molecules containing two P-C-P type bisphosphonate moieties connected by a carbon chain. The novel compounds were examined in a battery of in vitro and in vivo models including HAP formation and dissolution, ectopic calcification, bone resorption, tumor osteolysis, and of macrophage-like cells (anti- or pro-inflammatory properties). The inhibition of ectopic calcification was ranked as follows: geminal bisphosphonates > bisacylphosphonates > tetrakisphosphonates. Pamidronate, but not the tetrakisphosphonates, was an effective antiosteolytic agent. Neither DNTP (tetrasodium 1,9-dihydroxynonane 1,1,9,9-tetrakisphosphonate) nor the bisacylphosphonate, PiBP (pimeloylbisphosphonate) seem to possess strong macrophage suppressive or inductive effects and can be considered to be relatively inactive in terms of anti- or pro-inflammatory action. A significant anticalcification effect was caused by various phosphonates, such as the tetrakisphosphonates, but DNTP, a tetrakisphosphonate, was found toxic as it impeded somatic growth and bone development.

Influence of lipids on the mannitol flux during transdermal iontophoresis in vitro.

The aim of the present study was to evaluate the influence of the lipids EPC (L-alpha-phosphatidylcholine, egg lecithin), DSPC (distearoylphosphatidylcholine), and SA (stearylamine) on the iontophoretic mannitol transport through human skin in vitro. The skin was pretreated with 1 mM lipid suspension with ethanol (32%) for 24 h prior to the iontophoretic experiment with mannitol. In addition, the penetration of fluorescent lipids into the epidermis during the pretreatment was studied by confocal laser scanning microscopy (CLSM). The results of the present study show that pretreatment of the skin with zwitterionic EPC increases the iontophoretic transdermal mannitol flux about three-fold compared to iontophoretic control without pretreatment. However, skin pretreatment with another zwitterionic phospholipid, DSPC, did not influence the iontophoretic flux of mannitol. In contrast, pretreatment of the skin with cationic SA decreased the iontophoretic mannitol flow from the anode. It is concluded that EPC works as a penetration enhancer further increasing the transdermal mannitol flux during iontophoresis. In contrast, the cationic stearylamine changes the charge of the skin, thus leading to decreased electroosmosis and decreased mannitol flux. Hence, the effects of stearylamine are assumed to be mediated by the alterations in the charge of the stratum corneum structures, while EPC is suggested to decrease the permeability barrier of the skin.

A lipid carrier with a membrane active component and a small complex size are required for efficient cellular delivery of anti-sense phosphorothioate oligonucleotides.

Anti-sense oligonucleotides are potential therapeutic agents that are used to block protein expression from mRNA. To assess the essential properties for an efficient cellular delivery system of phosphorothioate oligonucleotides (PS-ODNs), different cationic carriers were compared. The carriers were complexed with oligonucleotides at various +/- charge ratios in MES-Hepes buffer. Cationic polymers, polylysines (PLL, mean MWs 4000, 20000, 200000 kDa), polyethyleneimines (PEI, mean MWs 25 and 800 kDa) and fractured sixth-generation polyamidoamine dendrimer (PAMAM) were tested for ODN delivery into a D 407 cell line (human retinal pigment epithelial cells) with stably transfected luciferase gene. Anti-sense ODN was directed against the luciferase gene, and the anti-sense effect was determined using a luminometric method. Lipid-based vehicles included DOTAP, DOTAP/DOPE (1/1 by mol), DOTAP/Chol (1/1 by mol), DOTAP/DOPE/Chol (2/1/1 by mol), DOGS and Cytofectin GS/DOPE (2/1 by mol). Additionally a membrane-active peptide JTS-1 (NH(2) -GLFEALLELLESLWELLLEA-COOH) was added to the complexes containing DOTAP, PEI or PLL. In D 407 and CV-1 cells, the anti-sense effect was seen only with lipid-based carriers with a membrane-active component (DOPE or JTS-1). The polymeric systems were ineffective. The effect of the complexation medium was further studied on CV-1 cells. Complexes were prepared in either water, MES-Hepes buffer or cell growth medium (DMEM). Complexes prepared in water were generally most effective and the greater activity is probably due to the smaller complex size. Complex sizes differed greatly in buffer and DMEM, especially in the case of DOPE containing complexes. In conclusion, lipid carrier with a membrane active component and small complex size are required for an efficient cellular delivery of phosphorothioate oligonucleotides.

Liposome-skin interactions and their effects on the skin permeation of drugs.

The aim of the study was to evaluate the interaction of phospholipid liposomes with skin and stratum corneum lipid liposomes (SCLLs). The influence of phospholipid liposomes on the skin permeability of model drugs was also studied. The transdermal flux of the drugs applied in various phospholipid containing formulations through human epidermis was studied in diffusion chambers. Liposomes in water solutions did not enhance the skin permeability of the drugs, but when ethanol (32% w/v) was present in the donor with EPC (egg yolk lecithin), permeabilities of some model drugs were substantially increased. Confocal microscopy studies revealed that EPC do not penetrate into the skin from water solutions, while from ethanol solutions, EPC penetrates deeply into the stratum corneum. Also, resonance energy transfer between different liposome compositions and the release of calcein from SCLLs showed that interactions between phospholipid liposomes and SCLLs increased with increasing ethanol concentration in the liposome solutions.

Contrasting effects of alendronate and clodronate on RAW 264 macrophages: the role of a bisphosphonate metabolite.

Clodronate (dichloromethylidene-bisphosphonate), a halogen-containing bisphosphonate, can inhibit the release of cytokines from RAW 264 macrophages and has anti-inflammatory properties in rheumatoid arthritis, whilst amino-containing bisphosphonates such as alendronate (4-amino-1-hydroxybutylidene-bisphosphonate), have pro-inflammatory properties and can cause an acute phase response. The basis for these pharmacological properties is unclear. Recently, it was demonstrated that clodronate is metabolised by certain cell lines in vitro to an analogue of ATP, whereas amino-bisphosphonates are not. We therefore investigated whether clodronate can also be metabolised by RAW 264 macrophages and whether intracellular accumulation of the metabolite (AppCCl2p) could account for the anti-inflammatory properties of clodronate. The effect of alendronate and AppCCl2p on the release of cytokines (IL-1beta, IL-6, and TNFalpha) from RAW 264 cells was compared, and the effect of the bisphosphonates and AppCCl2p on the DNA binding activities of transcription factors, NF-kappaB and AP-1, was investigated. Pretreatment of RAW 264 macrophages with alendronate augmented the LPS-stimulated release of IL-1beta and increased the binding of NF-kappaB to DNA in an electrophoretic mobility shift assay. Without LPS-induction, alendronate did not affect cytokine release or NF-kappaB binding. Clodronate was metabolised by RAW 264 cells to AppCCl2p. Like clodronate, AppCCl2p inhibited the LPS-induced release of cytokines and NO from RAW 264 macrophages. Both clodronate and its metabolite also inhibited the LPS-stimulated binding of NF-kappaB to DNA. In conclusion, these results suggest that the metabolite of clodronate may be responsible for the anti-inflammatory properties of clodronate, and that the contrasting effects of different bisphosphonates on the release of cytokines could be mediated partly through changes in the DNA binding activity of NF-kappaB.