Iontophoretic skin delivery systems: Success and failures.

Iontophoretic transdermal delivery uses a small electric current to push charged molecules into the skin under an electrode of same polarity and offers an attractive option to facilitate the delivery of macromolecules or hydrophilic molecules and to improve patient compliance. This technique has been used in physical therapy clinics for several decades, though the science was not always there to support claims of clinical effectiveness. Recently, this modality of treatment has undergone more systematic and rigorous investigations to withstand the scrutiny of regulatory authorities. In recent years various drugs have gained FDA approval for iontophoretic patches. This technique is gaining recognition due to better compliance rates, non-invasive drug delivery leading to fewer side effects, and sustained release of the drug. Furthermore, programmed delivery and bolus delivery systems have helped with customizing the drug dosage and frequency of dosage based on the patient's need.

Stability indicating UPLC method for simultaneous determination of atorvastatin, fenofibrate and their degradation products in tablets.

A stability indicating UPLC method was developed and validated for the simultaneous determination of atorvastatin, fenofibrate and their impurities in tablets. The chromatographic separation was performed on acquity UPLC BEH C18 column (1.7 microm, 2.1 mmx100 mm) using gradient elution of acetonitrile and ammonium acetate buffer (pH 4.7; 0.01 M) at flow rate of 0.5 ml/min. UV detection was performed at 247 nm. Total run time was 3 min within which main compounds and six other known and major unknown impurities were separated. Stability indicating capability was established by forced degradation experiments and separation of known degradation products. The method was validated for accuracy, repeatability, reproducibility and robustness. Linearity, LOD and LOQ was established for atorvastatin, fenofibrate and their known impurities.

Minimally modified low-density lipoprotein induces monocyte adhesion to endothelial connecting segment-1 by activating beta1 integrin.

We have shown previously that treatment of human aortic endothelial cells (HAECs) with minimally modified low-density lipoprotein (MM-LDL) induces monocyte but not neutrophil binding. This monocyte binding was not mediated by endothelial E-selectin, P-selectin, vascular cell adhesion molecule-I, or intercellular adhesion molecule-I, suggesting an alternative monocyte-specific adhesion molecule. We now show that moncytic alpha4beta1 integrins mediate binding to MM-LDL-treated endothelial cells. We present data suggesting that the expression of the connecting segment-1 (CS-1) domain of fibronectin (FN) is induced on the apical surface of HAEC by MM-LDL and is the endothelial alpha4beta1 ligand in MM-LDL-treated cells. Although the levels of CS-1 mRNA and protein were not increased, we show that MM-LDL treatment causes deposition of FN on the apical surface by activation of beta1integrins, particularly those associated with alpha5 integrins. Activation of beta1 by antibody 8A2 also induced CS-1-mediated monocyte binding. Confocal microscopy demonstrated the activated beta1 and CS-1colocalize in concentrated filamentous patches on the apical surface of HAEC. Both anti-CS-1 and an antibody to activated beta1 showed increased staining on the luminal endothelium of human coronary lesions with active monocyte entry. These results suggest the importance of these integrin ligand interactions in human atherosclerosis.

Increased transcription of IL-8 in endothelial cells is differentially regulated by TNF-alpha and oxidized phospholipids.

Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (Ox-PAPC) upregulates a spectrum of inflammatory cytokines and adhesion molecules different from those induced by classic inflammatory mediators such as tumor necrosis factor-alpha (TNF-alpha) or lipopolysaccharide. Interestingly, Ox-PAPC also induces the expression of a set of proteins similar to those induced by TNF-alpha or lipopolysaccharide, which include the chemokines monocyte chemotactic protein-1 (MCP-1) and interleukin (IL)-8. To elucidate the molecular mechanisms of Ox-PAPC-induced gene expression and to determine whether Ox-PAPC and other inflammatory mediators such as TNF-alpha utilize common signaling pathways, we examined the transcriptional regulation of IL-8 by Ox-PAPC and TNF-alpha in human aortic endothelial cells. Both Ox-PAPC and TNF-alpha induced the expression of IL-8 mRNA in a dose-dependent fashion; however, the kinetics of IL-8 mRNA accumulation between the 2 ligands differed. Ox-PAPC-induced IL-8 mRNA was seen as early as 30 minutes, peaked between 4 and 8 hours, and decreased substantially by 24 hours. In contrast, TNF-alpha-induced IL-8 mRNA synthesis was elevated at 30 minutes, peaked at 2 hours, and reached basal/undetectable levels by 6 hours. Actinomycin D experiments suggested that both Ox-PAPC and TNF-alpha regulate the expression of IL-8 at the transcriptional level. Furthermore, the half-life of IL-8 mRNA for both ligands was similar (<30 minutes), suggesting that mRNA stability was not responsible for the differences in the kinetics of IL-8 accumulation between the 2 ligands. Transient transfection studies with reporter constructs containing 1.48 kb of the IL-8 promoter identified an Ox-PAPC-specific response region between -133 and -1481 bp of the IL-8 promoter. In contrast, TNF-alpha activation of the IL-8 promoter was mediated almost entirely through the nuclear factor-kappaB and activation protein-1 response elements present between -70 and -133 bp of the IL-8 promoter. Thus, although Ox-PAPC and TNF-alpha both induced IL-8 synthesis, our data suggest that the 2 ligands utilize different mechanisms in the regulation of IL-8 transcription.

HDL and the inflammatory response induced by LDL-derived oxidized phospholipids.

Oxidation of low density lipoprotein (LDL) phospholipids containing arachidonic acid at the sn-2 position occurs when a critical concentration of "seeding molecules" derived from the lipoxygenase pathway is reached in LDL. When this critical concentration is reached, the nonenzymatic oxidation of LDL phospholipids produces a series of biologically active, oxidized phospholipids that mediate the cellular events seen in the developing fatty streak. Normal high density lipoprotein (HDL) contains at least 4 enzymes as well as apolipoproteins that can prevent the formation of the LDL-derived oxidized phospholipids or inactivate them after they are formed. In the sense that normal HDL can prevent the formation of or inactivate these inflammatory LDL-derived oxidized phospholipids, normal HDL is anti-inflammatory. HDL from mice that are genetically predisposed to diet-induced atherosclerosis became proinflammatory when the mice are fed an atherogenic diet, injected with LDL-derived oxidized phospholipids, or infected with influenza A virus. Mice that were genetically engineered to be hyperlipidemic on a chow diet and patients with coronary atherosclerosis, despite normal lipid levels, also had proinflammatory HDL. It is proposed that LDL-derived oxidized phospholipids and HDL may be part of a system of nonspecific innate immunity and that the detection of proinflammatory HDL may be a useful marker of susceptibility to atherosclerosis.

Evidence for a role of phospholipid oxidation products in atherogenesis.

There is growing evidence for the accumulation of phospholipid oxidation products (some of which can also be formed enzymatically) in several chronic disease processes including atherosclerosis. There also is considerable evidence that enzymes involved in hydrolysis of these phospholipids (present in both lipoproteins and cells) may be important in regulation of atherogenesis. In vitro studies suggest that these lipids can activate vascular wall cells to states that contribute to the atherosclerotic process. This review focuses on two types of bioactive phospholipids: phosphatidyl cholines in which the sn-2 fatty acid has been modified by oxidation and lysophosphatidic acid in which both the sn-2 and sn-3 positions have been modified. The mechanism by which these phospholipid oxidation products activate cells has revealed the presence of several different receptors and signal transduction pathways.

Development and validation of a simultaneous HPLC method for estimation of bisoprolol fumarate and amlodipine besylate from tablets.

A fast, robust and stability indicating RP-HPLC method was developed for simultaneous determination of bisoprolol fumarate and amlodipine besylate in tablets. The mobile phase was mixture of 25 mM ammonium acetate adjusted to pH 5.0 and methanol (65: 35) at 0.8 ml/min. The stationary phase was Luna C18-2 column (3 mu, 50x4.6 mm ID). UV detection was performed at 230 nm. Retention time was 1.45 min and 3.91 min for bisoprolol and amlodipine, respectively. Linearity was established in the range of 8-33 mug/ml. Mean recovery was 99.1% and 98.6% for bisoprolol fumarate and amlodipine besylate, respectively.

A novel complication of peritoneovenous shunt: cardiac tamponade.

For the last two decades, peritoneovenous shunts (PVS) have commonly been used in the management of refractory ascites. However, these devices are associated with a wide variety of complications. We report a case of cardiac tamponade due to ventricular perforation by a PVS. Given the substantial morbidity associated with PVS, other therapeutic options should be considered before proceeding with PVS placement.

Hemoperitoneum and disseminated intravascular coagulation in two neonates with congenital bilateral neuroblastoma.

We report two neonates with anemia, hemoperitoneum, and bilateral palpable abdominal masses. Both developed bleeding: a hemoperitoneum in one and visceral hemorrhages secondary to disseminated intravascular coagulation in the other. Each child was thought to have an unusual complication of adrenal hemorrhage, as the masses were of mixed echogenicity. However, histological evaluation revealed neuroblastoma. These cases point out the diagnostic dilemma of a mixed echogenicity suprarenal mass in the neonatal period and emphasize that local and disseminated bleeding can occur as a major manifestation of neuroblastoma.

Polymorphisms and linkage analysis for ICAM-1 and the selectin gene cluster.

Genetic polymorphisms in leukocyte and endothelial cell adhesion molecules may be important variables with regard to susceptibility to multifactorial disease processes that include an inflammatory component. For this reason, polymorphisms were sought for intercellular adhesion molecule-1 (ICAM-1; gene symbol ICAM1) and for the three genes in the selectin cluster, P-selectin, L-selectin, and E-selectin (gene symbols SELP, SELL, and SELE, respectively). Two amino acid polymorphisms were identified for ICAM-1; Gly or Arg at codon 241 and Lys or Glu at codon 469. Dinucleotide repeat polymorphisms were identified in the 3'-untranslated region for ICAM-1 and in intron 9 for P-selectin. Restriction fragment length polymorphisms were found using cDNAs for each of the three selectin genes as probes; E-selectin with BglII, P-selectin with ScaI, and L-selectin with HincII. Linkage analysis was performed for the selectin gene cluster and for ICAM-1 using the CEPH families; ICAM-1 is very tightly linked to the LDL receptor on chromosome 19, and the selectin cluster is linked to markers at chromosome 1q23.

Intercellular adhesion molecule 1 gene associations with immunologic subsets of inflammatory bowel disease.

BACKGROUND & AIMS: Inflammatory bowel disease is characterized by a failure to down-regulate the usual self-limited gut inflammatory response, suggesting that one or more of the predisposing genes could be those that determine the level of the immune response along the inflammatory pathway. The aim of this study was to examine potential associations of intercellular adhesion molecule 1 (ICAM-1) gene polymorphisms with inflammatory bowel disease or subsets of inflammatory bowel disease. METHODS: One hundred eighteen patients with ulcerative colitis (UC) and 130 patients with Crohn's disease (CD) as well as 77 ethnically matched controls were tested for antineutrophil cytoplasmic antibodies (ANCAs) and genotyped by polymerase chain reaction and allele-specific oligonucleotide techniques for ICAM-1 polymorphisms at codon 241 (exon 4) and codon 469 (exon 6). RESULTS: There was no significant difference between all patients with UC, patients with CD, and controls in either polymorphism. However, when stratified by ANCA status, ANCA-negative UC showed a significantly increased frequency of allele R241 compared with ANCA-positive UC (16.0% vs. 6.6%; P = 0.047, Fisher's Exact Test). In contrast, it was ANCA-positive CD that had an increased allele frequency compared with ANCA-negative CD (19.6% vs. 8.4%; P = 0.027, Fisher's Exact Test). CONCLUSIONS: Because the codon 241 polymorphism is in a functionally important domain III of ICAM-1, we may have identified an actual responsible genetic variation for genetically heterogeneous subsets of both UC and CD.

Blocking very late antigen-4 integrin decreases leukocyte entry and fatty streak formation in mice fed an atherogenic diet.

Atherosclerotic lesion development is characterized by the recruitment of leukocytes, principally monocytes, to the vessel wall. Considerable interest has been focused on the adhesion molecule(s) involved in leukocyte/endothelial interactions. The goal of the present study was to determine the role of the very late antigen-4 (VLA-4) integrin/ligand interaction in fatty streak development using murine models. Because alpha4 null mice are not viable, a peptidomimetic was used to block VLA-4-mediated leukocyte binding. The ability of a synthetic peptidomimetic of connecting segment-1 (CS-1 peptide) to block the recruitment of leukocytes and the accumulation of lipid in the aortic sinus of either wild-type mice (strain C57BL/6J) or mice with a low-density lipoprotein null mutation (LDLR-/-) maintained on an atherogenic diet was assessed. The active (Ac) CS-1 peptide or scrambled (Sc) CS-1 peptide was delivered subcutaneously into mice using a mini osmotic pump. Mice were exposed to the peptide for 24 to 36 hours before the onset of the atherogenic diet. In C57BL/6J mice, leukocyte entry into the aortic sinus, as assessed by en face preparations, was inhibited by the active peptide (Ac=28+/-4, Sc=54+/-6 monocytes/valve; P=0.004). Additionally, frozen sections stained with Oil Red O were analyzed to assess lipid accumulation in the aortic sinus. C57BL/6J mice that received the (Ac) compound demonstrated significantly reduced lesion areas as compared with mice that received the (Sc) peptide (Ac=4887+/-4438 microm2, Sc=15 009 +/-5619 microm2; P<0.0001). In a separate study, LDLR-/- mice were implanted with pumps containing either the (Ac) or (Sc) peptide before initiation of the atherogenic diet. Because LDLR-/- mice fed a chow diet displayed small lesions at 14 weeks, the effects of the peptide seen in these animals represented a change in early lipid accumulation rather than initiation. By using whole-mount preparations, the (Ac) but not the (Sc) peptide significantly reduced the area of lipid accumulation in the aortic sinus, resulting in an approximate 66% decrease. Plasma analysis from all studies revealed concentrations of peptide to be present at levels previously determined by in vitro analysis to block adhesion. (Ac) CS-1 peptide, which blocks VLA-4 on the leukocyte surface, is effective in reducing leukocyte recruitment and lipid accumulation in the aortic sinus. The present study provides in vivo evidence that the VLA-4 integrin plays an important role in the initiation of the atherosclerotic lesion and lipid accumulation, and it suggests a potential therapeutic strategy for this disease.

Structurally similar oxidized phospholipids differentially regulate endothelial binding of monocytes and neutrophils.

We previously have demonstrated that oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC), a component of minimally modified low density lipoprotein (MM-LDL), activates endothelial cells to bind monocytes. 1-Palmitoyl-2- (5-oxovaleroyl)-sn-glycero-3-phosphorylcholine (POVPC) and 1- palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (PGPC), which are present in OxPAPC, MM-LDL, and atherosclerotic lesions, were shown to have a major role in the activation of endothelial cells. We now demonstrate that these two highly similar molecules have dramatically different effects on leukocyte endothelial interactions. POVPC is a potent regulator of monocyte-specific endothelial interactions. Treatment of endothelial cells with POVPC increased monocyte binding by inducing the surface expression of the connecting segment 1 domain of fibronectin; no increase in neutrophil binding was observed. In addition, POVPC strongly inhibited lipopolysaccharide-mediated induction of neutrophil binding and expression of E-selectin protein and mRNA. This inhibition was mediated by a protein kinase A-dependent pathway, resulting in down-regulation of NF-kappaB-dependent transcription. In contrast, PGPC induced both monocyte and neutrophil binding and expression of E-selectin and vascular cell adhesion molecule 1. We present evidence to suggest that the two phospholipids act by different novel receptors present in Xenopus laevis oocytes and that POVPC, but not PGPC, stimulates a cAMP-mediated pathway. At concentrations equal to that present in MM-LDL, the effect of POVPC dominates and inhibits PGPC-induced neutrophil binding and E-selectin expression in endothelial cells. In summary, our data provide evidence that both POVPC and PGPC are important regulators of leukocyte-endothelial interactions and that POVPC may play a dominant role in a number of chronic inflammatory processes where oxidized phospholipids are known to be present.

Induction of P-selectin by oxidized lipoproteins. Separate effects on synthesis and surface expression.

Leukocyte binding to the endothelium is one of the earliest events in the occurrence of atherosclerosis. Leukocyte adhesion molecules involved in this process have not been definitely identified. We have found that treatment of human aortic endothelial cells (HAECs) with minimally modified low-density lipoprotein (MM-LDL) for 24 hours caused a 2- to 3-fold increase of P-selectin protein, with little change in P-selectin surface expression. A 15-minute histamine treatment of cells exposed to MM-LDL caused a 50% to 100% increase in P-selectin surface expression compared with cells not treated with the lipoprotein. This increase resulted in a 2-fold increase in binding of leukocytes to the endothelium. Immunostaining of permeabilized HAECs after MM-LDL treatment also revealed a highly reproducible increase in intracellular P-selectin associated with rod-shaped structures, typical of Weibel-Palade bodies. Oxidized phospholipids were shown to be mainly responsible for the action of MM-LDL. This increased P-selectin expression was associated with MM-LDL-induced cAMP elevation. Like histamine, highly oxidized low-density lipoprotein, especially the oxidized fatty acids, caused immediate redistribution of P-selectin to the cell surface followed by reinternalization. Immunohistochemical staining showed that endothelial cells on human fatty streak lesions expressed increased levels of P-selectin compared with nonlesion areas. These studies suggest that P-selectin may play an important role in early recruitment of mononuclear cells to the subendothelium in human atherosclerosis and that oxidized lipoproteins may contribute to the increased expression of this molecule by increasing intracellular stores and causing redistribution to the cell surface.