Lecithin vesicles for topical delivery of diclofenac.

Skin penetration of topically applied diclofenac is important for the treatment of rheumatic diseases and actinic keratoses. We have studied the permeation of diclofenac across human cadaver epidermis in-vitro from four lecithin vesicle formulations and a few marketed semi-solid preparations. The lecithin vesicle formulations were prepared by dissolving the lipid contents (lecithin and sodium cholate) in a 1:1 mixture of methanol-chloroform, evaporating the solvents under vacuum, and hydrating the lipid layer with the drug solution in water or 10% ethanol. The vesicles were sonicated for 5 min to reduce the vesicle size and their size and Zeta potential were characterized. The cumulative amount and maximum flux of diclofenac was 69.7+/-40.3 micrograms and 4.77+/-3.16 micrograms/hcm(2) from lecithin vesicles containing sodium cholate and 10% ethanol, and is the highest of all formulations studied. The cumulative amount and mean maximum flux obtained from other formulations were in the range of 2.46+/-1.98-29.9+/-10.1 micrograms and 0.53+/-0.46-3.61+/-0.86 micrograms/hcm(2). Based on the results, lecithin vesicles of diclofenac appear to be advantageous for the topical delivery of diclofenac.     

Inhibition of in vivo tumorigenicity and invasiveness of a human glioblastoma cell line transfected with antisense uPAR vectors

Our previous studies showed that glioblastomas express increased urokinase-type plasminogen activator receptors (uPARs) in comparison to low-grade gliomas (Yamamoto et al., Cancer Res., 54, 5016-5020, 1994). To explore whether downregulation of uPAR inhibits tumor formation and invasiveness, a human glioblastoma cell line was transfected with a cDNA construct corresponding to 300 bp of the human uPAR's 5¢ end in an antisense orientation, resulting in a reduced number of uPA receptors. Co-culture studies with tumor spheroids and fetal rat brain aggregates showed that antisense SNB19-AS1 cells expressing reduced uPAR failed to invade fetal rat brain aggregates. Intracerebral injection of SNB19-AS1 stable transfectants failed to form tumors and were negative for uPAR expression in nude mice. Thus uPAR appears in this model to be essential for tumorigenicity and invasion of glioblastomas in vivo.     

Expression of cysteine protease inhibitors in human gliomas and meningiomas

Increased levels of human cysteine proteases have been implicated in the progression of tumors from the premalignant to the malignant state. The physiological activities of these proteases are regulated by their interactions with specific inhibitors. To our knowledge there have been no previous reports about the cysteine protease inhibitors (CPIs) in human brain tumors. In the study reported here, we determined CPI activity during glioma progression and compared that with normal human brain tissue. We also determined CPI activities in meningioma and glioblastoma cell lines in vitro. This activity was significantly higher in normal brain tissue and low-grade glioma than in anaplastic astrocytoma and glioblastoma. CPI activity was significantly higher in benign and atypical meningioma cell extracts in comparison with those from malignant meningiomas and with those from glioblastoma cell lines. After several passages, one benign meningioma cell line showed reduced levels of CPI and increased levels of cathepsin. Our results suggest that decreases in the activities of CPI may contribute to the malignant properties of brain tumors.     

Proteolysis and invasiveness of brain tumors: Role of urokinase-type plasminogen activator receptor

Summary The cellular receptor for urokinase-type plasminogen activator (uPAR) in glioblastoma cell lines has been identified and found to be similar to the uPAR expressed by other tumor cell lines. Increased levels of uPAR have been found in primary malignant brain tumor tissues, especially highly malignant glioblastoma, and, to a lesser degree, in malignant astrocytomas, suggesting that this receptor might be involved in efficient activation of pro-uPA and confinement of uPA activity on the cell surface of invading brain tumors. The cell surface uPARs in gliomas could constitute an optimum environment for the generation and activity of plasmin, which is known to play a crucial role in the dissolution of the extracellular matrix during tumor cell invasion.In situ hybridization studies have shown that uPAR mRNA is expressed abundantly in tumor cells and is consistently present at the invasive edges of malignant gliomas. These results imply that uPAR is involved in plasmincatalyzed proteolysis during glioma invasion and that interference with the uPAuPAR interactions could constitute a novel approach for developing therapeutic strategies to counteract invasion of brain tumors.     

Glial cell-induced endothelial morphogenesis is inhibited by interfering with extracellular signal-regulated kinase signaling.

PURPOSE: Tumor vasculature provides the infrastructure by which malignant tissue can be nourished; therefore, targeting angiogenesis may be an effective means of treating cancer. We showed previously that SNB19 glioblastoma cells modulate bovine retinal endothelial cells in cocultures to form capillary-like network structures, that matrix metalloproteinase-9 (MMP-9) expression is critical for endothelial morphogenesis, and that MMP-9 expression in glioblastoma cells is regulated by extracellular signal-regulated kinase-1 (ERK-1). In the present study, we investigated whether interfering with the activation of this mitogen-activated protein (MAP) kinase would repress MMP-9 synthesis and inhibit capillary formation. EXPERIMENTAL DESIGN: Cocultures of bovine retinal endothelial and SNB19 cells were analyzed for MMP-9 secretion, and phospho- and total ERK levels. These cocultures were treated with PD98059, a specific inhibitor of MAP/ERK kinase 1, or transfected with dominant-negative ERK-1 mutant containing expression vector. Alterations in capillary-like structure formation, and actin cytoskeleton and secretion of vascular endothelial growth factor (VEGF), MMP-9, and tissue inhibitor of metalloproteinase-1 were determined by immunofluorescence, gelatin zymography, and Western blotting. RESULTS: We found that inhibition of the ERK-1/2 pathway with PD98059 abrogated glial cell-mediated capillary formation by the endothelial cells and reduced the levels of MMP-9 in the coculture. Strikingly, the abrogation of MAP kinase signaling by a dominant-negative ERK-1 mutant inhibited glial-induced capillary network formation by reducing VEGF levels and MMP-9 activity and increasing the levels of tissue inhibitor of metalloproteinase-1. Inhibition of ERK activity also disrupted the formation of the actin cytoskeleton, a prerequisite for endothelial cell migration. CONCLUSION: The mechanism underlying activation of ERK is involved in reorganization of the actin cytoskeleton, and induction of VEGF and MMP-9, thereby stimulating endothelial cell morphogenesis. These studies clearly provide experimental evidence that ERK inhibition diminishes glial-induced endothelial-cell morphogenesis; therefore, interfering with ERK signaling may be a viable approach to target angiogenesis.     

Biphasic intra-thoracic pressure regulation augments cardiac index during porcine peritonitis: a feasibility study

Preservation of cardiac output (CO) and pulmonary artery pressure (PAP) is vital to maintaining tissue oxygenation in sepsis. This feasibility study tested the hypothesis that therapeutic intra-thoracic pressure regulation (tIPR), delivered with a novel device, was designed to non-invasively enhance venous return by creating sub-atmospheric intra-thoracic pressure during the expiratory phase of mechanical ventilation, improves CO without fluid resuscitation in a porcine E. coli peritonitis model of sepsis. Seven pigs were intubated, anaesthetized and instrumented with a Swan-Ganz and femoral artery catheter. After a 30?min basal period, a fibrin clot containing 4–5?×?10⁹ cfu kg^?1 E. coli O111.B4 was implanted in the peritoneum. One hour after clot implantation, tIPR was utilized for 30?min and then removed from the ventilator circuit for 30?min. This tIPR cycle was repeated 4-times. Changes in haemodynamic parameters were calculated by comparing pre-tIPR values to peak values during tIPR administration. Following peritonitis, tIPR significantly increased the peak cardiac index (mean?±?SEM) (14.8?±?2.6 vs 7.9?±?2.3?ml kg^?1) and mean arterial pressure (10.2?±?1.5 vs 4.9?±?1.1?mmHg) and simultaneously decreased PAP (?7.7?±?1.5 vs ?2.7?±?0.8?mmHg). These results support the feasibility of the concept that therapeutic application of negative expiratory pressure may provide a non-invasive and complementary approach to increase cardiac output and organ perfusion in the setting of septic shock.     

Replication-competent Lentivirus Analysis of Clinical Grade Vector Products

Lentiviral vectors are now in clinical trials for a variety of inherited and acquired disorders. A challenge for moving any viral vector into the clinic is the ability to screen the vector product for the presence of replication-competent virus. Assay development for replication-competent lentivirus (RCL) is particularly challenging because recombination of vector packaging plasmids and cellular DNA leading to RCL has not been reported with the current viral vector systems. Therefore, the genomic structure of a RCL remains theoretical. In this report, we describe a highly sensitive RCL assay suitable for screening vector product and have screened large-scale vector supernatant, cells used in vector production, and cells transduced with clinical grade vector. We discuss the limitations and challenges of the current assay, and suggest modifications that may improve the suitability of this assay for screening US Food and Drug Administration (US FDA)-licensed products.