Reduction of protein-bound uraemic toxins in plasma of chronic renal failure patients: A systematic review.

BACKGROUND: Protein-bound uraemic toxins (PBUTs) accumulate in patients with chronic kidney disease and impose detrimental effects on the vascular system. However, a unanimous consensus on the most optimum approach for the reduction of plasma PBUTs is still lacking. METHODS: In this systematic review, we aimed to identify the most efficient clinically available plasma PBUT reduction method reported in the literature between 1980 and 2020. The literature was screened for clinical studies describing approaches to reduce the plasma concentration of known uraemic toxins. There were no limits on the number of patients studied or on the duration or design of the studies. RESULTS: Out of 1274 identified publications, 101 studies describing therapeutic options aiming at the reduction of PBUTs in CKD patients were included in this review. We stratified the studies by the PBUTs and the duration of the analysis into acute (data from a single procedure) and longitudinal (several treatment interventions) trials. Reduction ratio (RR) was used as the measure of plasma PBUTs lowering efficiency. For indoxyl sulphate and p-cresyl sulphate, the highest RR in the acute studies was demonstrated for fractionated plasma separation, adsorption and dialysis system. In the longitudinal trials, supplementation of haemodialysis patients with AST-120 (Kremezin®) adsorbent showed the highest RR. However, no superior method for the reduction of all types of PBUTs was identified based on the published studies. CONCLUSIONS: Our study shows that there is presently no technique universally suitable for optimum reduction of all PBUTs. There is a clear need for further research in this field.

CC chemokine ligands in patients presenting with stable chest pain: association with atherosclerosis and future cardiovascular events.

BACKGROUND: CC chemokine ligands (CCLs) are elevated during acute coronary syndrome (ACS) and correlate with secondary events. Their involvement in plaque inflammation led us to investigate whether CCL3-5-18 are linked to the extent of coronary artery disease (CAD) and prognostic for primary events during follow-up. METHODS: We measured CCL3-5-18 serum concentrations in 712 patients with chest discomfort referred for cardiac CT angiography. Obstructive CAD was defined as ≥50 % stenosis. The extent of CAD was measured by calcium score and segment involvement score (number of coronary segments with any CAD, range 0-16). Patients were followed up for all-cause mortality, ACS and revascularisation, for a mean 26 ± 7 months. RESULTS: Patients with obstructive CAD had significantly higher CCL5 (p = 0.02), and borderline significantly elevated CCL18 plasma levels as compared with patients with <50 % stenosis (p = 0.06). CCL18 levels were associated with coronary calcification (p = 0.002) and segment involvement score (p = 0.007). Corrected for traditional risk factors, only CCL5 provided independent predictive value for obstructive CAD: odds ratio (OR) 1.27 (1.02-1.59), p = 0.04. CCL5 provided independent predictive value for primary events during follow-up: OR 1.62 (1.03-2.57), p = 0.04. CONCLUSIONS: While CCL18 serum levels correlated with extent of CAD, CCL5 demonstrated an independent association with the presence of obstructive CAD, and occurrence of primary cardiac events.

Local atherosclerotic plaques are a source of prognostic biomarkers for adverse cardiovascular events.

OBJECTIVE: Atherosclerotic cardiovascular disease is a major burden to health care. Because atherosclerosis is considered a systemic disease, we hypothesized that one single atherosclerotic plaque contains ample molecular information that predicts future cardiovascular events in all vascular territories. METHODS AND RESULTS: AtheroExpress is a biobank collecting atherosclerotic lesions during surgery, with a 3-year follow-up. The composite primary outcome encompasses all cardiovascular events and interventions, eg, cardiovascular death, myocardial infarction, stroke, and endovascular interventions. A proteomics search identified osteopontin as a potential plaque biomarker. Patients undergoing carotid surgery (n=574) served as the cohort in which plaque osteopontin levels were examined in relation to their outcome during follow-up and was validated in a cohort of patients undergoing femoral endarterectomy (n=151). Comparing the highest quartile of carotid plaque osteopontin levels with quartile 1 showed a hazard ratio for the primary outcome of 3.8 (95% confidence interval, 2.6-5.9). The outcome did not change after adjustment for plaque characteristics and traditional risk factors (hazard ratio, 3.5; 95% confidence interval, 2.0-5.9). The femoral validation cohort showed a hazard ratio of 3.8 (95% confidence interval 2.0 to 7.4) comparing osteopontin levels in quartile 4 with quartile 1. CONCLUSIONS: Plaque osteopontin levels in single lesions are predictive for cardiovascular events in other vascular territories. Local atherosclerotic plaques are a source of prognostic biomarkers with a high predictive value for secondary manifestations of atherosclerotic disease.

The hypoxia-sensor carbonic anhydrase IX affects macrophage metabolism, but is not a suitable biomarker for human cardiovascular disease.

Hypoxia is prevalent in atherosclerotic plaques, promoting plaque aggravation and subsequent cardiovascular disease (CVD). Transmembrane protein carbonic anhydrase IX (CAIX) is hypoxia-induced and can be shed into the circulation as soluble CAIX (sCAIX). As plaque macrophages are hypoxic, we hypothesized a role for CAIX in macrophage function, and as biomarker of hypoxic plaque burden and CVD. As tumor patients with probable CVD are treated with CAIX inhibitors, this study will shed light on their safety profile. CAIX co-localized with macrophages (CD68) and hypoxia (pimonidazole), and correlated with lipid core size and pro-inflammatory iNOS+ macrophages in unstable human carotid artery plaques. Although elevated pH and reduced lactate levels in culture medium of CAIX knock-out (CAIXko) macrophages confirmed its role as pH-regulator, only spare respiratory capacity of CAIXko macrophages was reduced. Proliferation, apoptosis, lipid uptake and expression of pro- and anti-inflammatory genes were not altered. Plasma sCAIX levels and plaque-resident CAIX were below the detection threshold in 50 and 90% of asymptomatic and symptomatic cases, respectively, while detectable levels did not associate with primary or secondary events, or intraplaque hemorrhage. Initial findings show that CAIX deficiency interferes with macrophage metabolism. Despite a correlation with inflammatory macrophages, plaque-resident and sCAIX expression levels are too low to serve as biomarkers of future CVD.

Leukocyte cathepsin S is a potent regulator of both cell and matrix turnover in advanced atherosclerosis.

OBJECTIVE: A dysbalance of proteases and their inhibitors is instrumental in remodeling of atherosclerotic plaques. One of the proteases implicated in matrix degradation is cathepsin-S (CatS). To address its role in advanced lesion composition, we generated chimeric LDLr(-/-) mice deficient in leukocyte CatS by transplantation with CatS(-/-)xLDLr(-/-) or with LDLr(-/-) bone marrow and administered a high-fat diet. METHODS AND RESULTS: No difference in aortic root lesion size could be detected between CatS(+/+) and CatS(-/-) chimeras. However, leukocyte CatS deficiency markedly changed plaque morphology and led to a dramatic reduction in necrotic core area by 77% and an abundance of large foam cells. Plaques of CatS(-/-) chimeras contained 17% more macrophages, 62% less SMCs, and 33% less intimal collagen. The latter two could be explained by a reduced number of elastic lamina fractures. Moreover, macrophage apoptosis was reduced by 60% with CatS deficiency. In vitro, CatS was found to be involved in cholesterol metabolism and in macrophage apoptosis in a collagen and fibronectin matrix. CONCLUSIONS: Leukocyte CatS deficiency results in considerably altered plaque morphology, with smaller necrotic cores, reduced apoptosis, and decreased SMC content and collagen deposition and may thus be critical in plaque stability.

In vitro and in vivo analysis of expression cassettes designed for vascular gene transfer.

Increasing the level and duration of transgene expression and restricting expression to vascular cells are important goals for clinically useful gene therapy vectors. We evaluated several promoters, enhancers and introns in endothelial, smooth muscle and liver cells in tissue culture and in vivo, comparing local delivery to the carotid artery with intravenous delivery to the liver. A 1800-bp fragment of the oxidized LDL receptor (LOX-1) promoter showed highest in vivo activity in the carotid artery, achieving 39% the activity of the reference cytomegalovirus promoter, with 188-fold greater specificity for carotid artery over liver. An enhancer from the Tie2 gene in combination with the intracellular adhesion molecule-2 promoter improved endothelial specificity of plasmid vectors, increased the expression from adenoviral vectors in cultured endothelial cells and doubled the specificity for carotid artery over liver in vivo. Adding a short intron to expression cassettes increased expression in both endothelial and smooth muscle cells in vitro; however, the eNOS enhancer failed to consistently increase the expression or endothelial specificity of the vector. In conclusion, elements from the LOX-1 promoter and Tie2 enhancer together with an intron can be used to improve vectors for vascular gene transfer.

CC chemokine ligand-5 (CCL5/RANTES) and CC chemokine ligand-18 (CCL18/PARC) are specific markers of refractory unstable angina pectoris and are transiently raised during severe ischemic symptoms.

BACKGROUND: Chemokines play an important role in atherogenesis and in ischemic injury and repair; however, prospective data on individual chemokines in unstable angina pectoris (UAP) are scarce. Therefore, we assessed chemokine patterns in a prospective cohort of patients with UAP. METHODS AND RESULTS: Plasma samples of 54 patients with Braunwald class IIIB UAP were examined at baseline for 11 chemokines and 5 inflammatory mediators via multiplex analysis. Levels of CC chemokine ligand (CCL)-5 (also known as RANTES [regulated on activation, normally T-cell expressed, and secreted]; 32.7 versus 23.1 ng/mL, P=0.018) and CCL18 (also known as PARC [pulmonary and activation-regulated chemokine]; 104.4 versus 53.7 ng/mL, P=0.011) were significantly elevated in patients with refractory ischemic symptoms versus stabilized patients. Temporal monitoring by ELISA of CCL5, CCL18, and soluble CD40 ligand (sCD40) levels revealed a drop in CCL5 and sCD40L levels in all UAP patients from day 2 onward (CCL5 12.1 ng/mL, P<0.001; sCD40L 1.35 ng/mL, P<0.05), whereas elevated CCL18 levels were sustained for at least 2 days, then were decreased at 180 days after inclusion (34.5 ng/mL, P<0.001). Peripheral blood mononuclear cells showed increased protein expression of chemokine receptors CCR3 and CCR5 in CD3+ and CD14+ cells at baseline compared with 180 days after inclusion, whereas mRNA levels were downregulated, which was attributable in part to a postischemic release of human neutrophil peptide-3-positive neutrophils and in part to negative feedback. Finally, elevated CCL5 and CCL18 levels predicted future cardiovascular adverse events, whereas C-reactive protein and sCD40L levels did not. CONCLUSIONS: We are the first to report that CCL18 and CCL5 are transiently raised during episodes of UAP, and peak levels of both chemokines are indicative of refractory symptoms. Because levels of both chemokines, as well as of cognate receptor expression by circulating peripheral blood mononuclear cells, are increased during cardiac ischemia, this may point to an involvement of CCL5/CCL18 in the pathophysiology of UAP and/or post-UAP responses.

Reduced leucocyte cholesteryl ester transfer protein expression in acute coronary syndromes.

OBJECTIVE: Cholesterol ester transfer protein (CETP) plays an important role in HDL cholesterol metabolism. Leucocytes, including monocyte-derived macrophages in the arterial wall synthesize and secrete CETP, but its role in atherosclerosis is unclear. The aim of the current study was to investigate the effect of acute coronary syndromes (ACS) on leucocyte CETP expression. RESEARCH DESIGN: Peripheral blood mononuclear cells (PBMCs) were freshly isolated from hospitalized ACS patients displaying Braunwald class IIIB unstable angina pectoris (UAP) on admission (t = 0) and at 180 days post inclusion (t = 180) for analysis of CETP expression. In addition, to prove the potential correlation between leucocyte CETP and ACS the effect of acute myocardial infarction on leucocyte CETP expression was studied in CETP transgenic mice. RESULTS: Upon admission, UAP patients displayed approximately 3-6 fold (P < 0.01) lower CETP mRNA and nearly absent CETP protein expression in PBMCs, as compared to healthy age-/sex-matched controls. Interestingly, CETP mRNA and protein levels were significantly elevated in PBMCs isolated from UAP patients (both stabilized and refractory) at t = 180 as compared to t = 0 (P < 0.01), which was correlated with a reduced inflammatory status after medical treatment. In agreement with the data obtained in UAP patients, markedly down-regulated leucocyte CETP mRNA expression was observed after coronary artery ligation in CETP transgenic mice, which also correlated with increased serum amyloid A levels. CONCLUSIONS: We are the first to report that episodes of UAP in humans and myocardial infarction in CETP transgenic mice are associated with reduced leucocyte CETP expression. We propose that the impairment in leucocyte CETP production is associated with an enhanced inflammatory status, which could be clinically relevant for the pathogenesis of ACS.

Mdm2, but not Mdm4, protects terminally differentiated smooth muscle cells from p53-mediated caspase-3-independent cell death.

p53 is a potent inhibitor of cell growth and an inducer of apoptosis. During embryonic development, Mdm2 and Mdm4 inhibit the growth suppressive activities of p53. However, whether tight surveillance of p53 activity is required in quiescent cells is unknown. To test this, conditional inactivation of mdm2 and mdm4 was carried out in smooth muscle cells (SMCs). Upon SMC-specific inactivation of mdm2, and not of mdm4, mice rapidly became ill and died. Necropsy showed small intestinal dilation, and histological analyses indicated a severe reduction in the number of intestinal SMCs. Increased p53 levels and activity were detected in the remaining SMCs, and the phenotype was completely rescued on a p53-null background. Interestingly, intestinal SMCs are caspase-3-negative and therefore did not undergo caspase-3-dependent apoptotic cell death. Together, Mdm2, but not Mdm4, prevents accumulation of active p53 in quiescent SMCs and thereby the induction of p53-mediated caspase-3-independent cell death.

Fibrin and activated platelets cooperatively guide stem cells to a vascular injury and promote differentiation towards an endothelial cell phenotype.

OBJECTIVE: Bone marrow-derived progenitor cells play a role in vascular regeneration. However, their homing to areas of vascular injury is poorly understood. One of the earliest responses to an injury is the activation of coagulation and platelets. In this study we assessed the role of hemostatic components in the recruitment of CD34+ cells to sites of injury. METHODS AND RESULTS: Using an ex vivo injury model, representing endothelial cell (EC) injury or vessel denudation, we studied homing of CD34+ under flow. Platelet aggregates facilitated initial tethering and rolling of CD34+ cells through interaction of P-selectin expressed by platelets and P-selectin glycoprotein ligand-1 (PSGL-1), expressed by CD34+ cells. Ligation of PSGL-1 activated adhesion molecules on CD34+ cells, ultimately leading to firm adhesion of CD34+ cells to tissue factor-expressing ECs or to fibrin-containing thrombi formed on subendothelium. We also demonstrate that fibrin-containing thrombi can support migration of CD34+ cells to the site of injury and subsequent differentiation toward a mature EC phenotype. Additionally, intravenously injected CD34+ cells homed in vivo to denuded arteries in the presence of endogenous leukocytes. CONCLUSIONS: We provide evidence that hemostatic factors, associated with vascular injury, provide a regulatory microenvironment for re-endothelialization mediated by circulating progenitor cells.

Lesional overexpression of matrix metalloproteinase-9 promotes intraplaque hemorrhage in advanced lesions but not at earlier stages of atherogenesis.

BACKGROUND: Matrix metalloproteinase-9 (MMP-9) is involved in atherosclerosis and elevated MMP-9 activity has been found in unstable plaques, suggesting a crucial role in plaque rupture. This study aims to assess the effect of MMP-9 on plaque stability in apolipoprotein E-deficient mice at different stages of plaque progression. METHODS AND RESULTS: Atherosclerotic lesions were elicited in carotid arteries by perivascular collar placement. MMP-9 overexpression in intermediate or advanced plaques was effected by intraluminal incubation with an adenovirus (Ad.MMP-9). A subset was coincubated with Ad.TIMP-1. Mock virus served as a control. Plaques were analyzed histologically. In intermediate lesions, MMP-9 overexpression induced outward remodeling, as shown by a 30% increase in media size (p=0.03). In both intermediate and advanced lesions, prevalence of vulnerable plaque morphology tended to be increased. Half of MMP-9-treated lesions displayed intraplaque hemorrhage, whereas in controls and the Ad.MMP-9/Ad.TIMP-1 group this was 8% and 16%, respectively (p=0.007). Colocalization with neovessels may point to neo-angiogenesis as a source for intraplaque hemorrhage. CONCLUSIONS: These data show a differential effect of MMP-9 at various stages of plaque progression and suggest that lesion-targeted MMP-9 inhibition might be a valuable therapeutic modality in stabilizing advanced plaques, but not at earlier stages of lesion progression.

Modulation of plasma protein binding and in vivo liver cell uptake of phosphorothioate oligodeoxynucleotides by cholesterol conjugation.

Several studies have shown improved efficacy of cholesteryl-conjugated phosphorothioate antisense oligodeoxynucleotides. To gain insight into the mechanisms of the improved efficacy in vivo, we investigated the disposition of ISIS-9388, the 3'-cholesterol analog of the ICAM-1-specific phosphorothioate oligodeoxynucleotide ISIS-3082, in rats. Intravenously injected [(3)H]ISIS-9388 was cleared from the circulation with a half-life of 49.9 +/- 2.2 min (ISIS-3082, 23.3 +/- 3.8 min). At 3 h after injection, the liver contained 63.7 +/- 3. 3% of the dose. Compared to ISIS-3082, the hepatic uptake of ISIS-9388 is approximately 2-fold higher. Endothelial, Kupffer and parenchymal cells accounted for 45.7 +/- 5.7, 33.0 +/- 5.9 and 21.3 +/- 2.6% of the liver uptake of [(3)H]ISIS-9388, respectively, and intracellular concentrations of approximately 2, 75 and 50 microM, respectively, could be reached in these cells (1 mg/kg dose). Preinjection with polyinosinic acid or poly-adenylic acid reduced the hepatic uptake of [(3)H]ISIS-9388, which suggests the involvement of (multiple) scavenger receptors. Size exclusion chromatography of mixtures of the oligonucleotides and rat plasma indicated that ISIS-9388 binds to a larger extent to high molecular weight proteins than ISIS-3082. Analysis by agarose gel electrophoresis indicated that ISIS-9388 binds more tightly to plasma proteins than ISIS-3082. The different interaction of the oligonucleotides with plasma proteins possibly explains their different dispositions. We conclude that cholesterol conjugation results in high accumulation of phosphorothioate oligodeoxynucleotides in various liver cell types, which is likely to be beneficial for antisense therapy of liver-associated diseases.

Induction of rapid atherogenesis by perivascular carotid collar placement in apolipoprotein E-deficient and low-density lipoprotein receptor-deficient mice.

BACKGROUND: Perivascular collar placement has been used as a means for localized atherosclerosis induction in a variety of experimental animal species. In mice, however, atherosclerosis-like lesions have thus far not been obtained by this method. The aim of this study was the development of a mouse model of rapid, site-controlled atherogenesis. METHODS AND RESULTS: Silastic collars were placed around the carotid arteries of apolipoprotein E-deficient (apoE-/-) and LDL receptor-deficient (LDLr-/-) mice. The development of collar-induced lesions was found to occur predominantly in the area proximal to the collar and to be dependent on a high-cholesterol diet. Lesions were evident in apoE-/- mice after 3 weeks and in LDLr-/- mice after 6 weeks and were overtly atherosclerotic in appearance. Lumen stenosis reached 85% in apoE-/- mice and 61% in LDLr-/- mice 6 weeks after collar insertion. Expression levels of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 were increased both proximal and distal to the collar, whereas endothelial nitric oxide synthase expression was downregulated at the proximal site. CONCLUSIONS: We propose that this model of collar-induced acceleration of carotid atherogenesis is of hemodynamic cause. It may serve as a substrate for sequential mechanistic studies concerned with the underlying cause and pathogenesis of atherosclerosis. The rapidity of lesion development will also aid the efficient screening of new potentially antiatherogenic chemical entities and the evaluation of therapies with limited duration of effectiveness, such as adenoviral gene therapy.

Attenuation of atherogenesis by systemic and local adenovirus-mediated gene transfer of interleukin-10 in LDLr-/- mice.

In view of its multifaceted anti-inflammatory properties, interleukin-10 (IL-10) has been deemed to be potentially anti-atherogenic. We have evaluated the capacity of adenoviral gene transfer of IL-10 for the modulation of de novo atherosclerotic lesion formation by systemic and by local overexpression. Atherogenesis was initiated in the carotid arteries of low-density lipoprotein receptor deficient mice by perivascular placement of silastic collars. One week after collar placement, mice were injected intravenously with 1 x 109 plaque-forming units (pfu's) of IL-10 (AdV.IL-10) or control adenovirus (AdV.empty). Administration of AdV.IL-10 resulted in extended systemic expression of IL-10 (peak serum level 3.0 +/- 1.1 ng/ml) and a reduction in atherosclerotic lumen stenosis by 62.2% (P<0.02). This finding was accompanied by monocyte deactivation and lowering of serum cholesterol levels (maximum decrease 44%). In a second experiment, collared arteries were transfected locally by transluminal instillation of adenovirus (titer 1.5x1010 pfu/ml). Systemic parameters remained unchanged following local transfection, but the degree of stenosis was, nonetheless, decreased by 44.9% (P<0.05). We conclude that a marked inhibition of atherogenesis can be achieved by systemic overexpression of AdV.IL-10, owing to its metabolic and immunomodulatory effects. Local IL-10 transfer is virtually equipotent, however, and it may represent a valuable addition to the armory of anti-atherosclerotic therapies.

Overexpression of IL-18 decreases intimal collagen content and promotes a vulnerable plaque phenotype in apolipoprotein-E-deficient mice.

OBJECTIVE: Although IL-18 has been implicated in atherosclerotic lesion development, little is known about its role in advanced atherosclerotic plaques. This study aims to assess the effect of IL-18 overexpression on the stability of preexisting plaques. METHODS AND RESULTS: Atherosclerotic lesions were elicited in carotid arteries of apolipoprotein E (apoE)-deficient mice (n=32) by placement of a perivascular collar. Overexpression of IL-18 was effected by intravenous injection of an adenoviral vector 5 weeks after surgery. Two weeks after transduction, lesions were analyzed histologically with regard to plaque morphology and composition or by real-time polymerase chain reaction. No difference in plaque size was detected between groups. In the Ad.IL-18-treated group, 62% of lesions displayed a vulnerable morphology or even intraplaque hemorrhage as compared with only 24% in the controls (P=0.037). In agreement, IL-18 overexpression reduced intimal collagen by 44% (P<0.003) and cap-to-core ratio by 41% (P<0.002). Although IL-18 did not affect the expression of collagen synthesis-related genes, it was found to enhance the collagenolytic activity of vascular smooth muscle cells in vitro, suggesting that the low collagen content is attributable to matrix degradation rather than to decreased synthesis. CONCLUSIONS: Systemic IL-18 overexpression markedly decreases intimal collagen content and cap thickness, leading to a vulnerable plaque morphology.

Recombinant lipoproteins: lipoprotein-like lipid particles for drug targeting.

Lipoproteins are endogenous particles that transport lipids through the blood to various cell types, where they are recognised and taken up via specific receptors. These particles are, therefore, excellent candidates for the targeted delivery of drugs to various tissues. For example, the remnant receptor and the asialoglycoprotein receptor (ASGPr), which are uniquely localised on hepatocytes, recognise chylomicrons and lactosylated high density lipopoteins (HDL), respectively. In addition, tumour cells of various origins overexpress the low density lipoprotein (LDL) receptor that recognises apolipoprotein E (apoE) on small triglyceride-rich particles and apoB-100 on LDL. Being endogenous, lipoproteins are biodegradable, do not trigger immune reactions, and are not recognised by the reticuloendothelial system (RES). However, their endogenous nature also hampers large-scale pharmaceutical application. In the past two decades, various research groups have successfully synthesised recombinant lipoproteins from commercially available natural and synthetic lipids and serum-derived or recombinant apolipoproteins, which closely mimic the metabolic behaviour of their native counterparts in animal models as well as humans. In this paper, we will summarise the studies that led to the development of these recombinant lipoproteins, and we will address the possibility of using these lipidic particles to selectively deliver a wide range of lipophilic, amphiphilic, and polyanionic compounds to hepatocytes and tumour cells. In addition, the intrinsic therapeutic activities of recombinant chylomicrons and HDL in sepsis and atherosclerosis will be discussed.

The cholesterol derivative of a triantennary galactoside with high affinity for hepatic asialoglycoprotein receptor: a potent cholesterol lowering agent.

Cholesterol-derivatized galactosides have been devised in order to induce liver uptake of lipoproteins via the galactose-recognizing asialoglycoprotein receptor in the liver. In this study we describe the derivatization of a newly developed triantennary cluster galactoside having high affinity for the asialoglycoprotein receptor, N-[[tris-O-(3,6,9-trioxaundecanyl-beta-D-galactopyranosyl)metho xym ethyl] -N alpha-[1-(6-methyladipyl)]glycinamide (TG(20A)) with cholesterol. Hereto, TG(20A) was coupled to glycine-(5-cholesten-3 beta-yl ester) in the presence of (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate, affording N-[[tris-O-(3,6,9-trioxaundecanyl-beta-D- galactopyranosyl)methoxymethyl]methyl]-N alpha-[1-(6-(5-cholesten-3 beta-yloxy)glycyl)adipyl]glycinamide (TG(20A)C) in 46% yield. This compound is an amphiphilic, water-soluble compound. In aqueous solution it readily formed small micelles (4.9 +/- 1.2 nm) consisting of approximately 20 molecules. Upon incubation with human serum, TG(20A)C spontaneously incorporated into the most prominent serum lipoproteins, i.e., low-density lipoprotein (LDL) and high-density lipoprotein (HDL), thereby inducing an increase in buoyant density of these lipoproteins. The integrity of HDL and LDL, as judged from particle size analysis of both lipoproteins, was not altered by incubation with up to 0.33% of TG(20A)C (w/v). Following intravenous bolus injection into rats, TG(20A)C induced a dose-dependent decrease in the serum cholesterol content of maximally 44%, at a dose of 1.9 mg kg-1. This makes TG(20A)C at least 30-fold more effective than the previously developed N-[[tris-O-(beta-D-galactopyranosyl)methyl]methyl]-N alpha-[4-(5- cholesten-3 beta-yloxy)succinyl]glycinamide (TG(4A)C), provided with a cluster galactoside that displayed a 2000-fold lower affinity for the asialoglycoprotein receptor than TG(20A). In conclusion, the hypocholesterolemic activity of a cholesterylated galactoside can be strongly enhanced by using a cluster galactoside with higher affinity for the asialoglycoprotein receptor.

Synthesis of cluster galactosides with high affinity for the hepatic asialoglycoprotein receptor.

High-affinity ligands for the asialoglycoprotein receptor, which is uniquely localized on the parenchymal liver cell and recognizes oligoantennary galactosides, might be utilized as homing device to specifically target drugs or genes to parenchymal liver cells. In the present study, the synthesis of galactose-terminated triantennary glycosides, provided with various spacers between the beta-galactopyranosyl moieties and the branching point of the dendrite, is described. N-[Tris[[(methylthio)methoxy]methyl]methyl]-N alpha-[1-(6- methyladipy)]glycinamide (3b) was glycosylated with monogalactosyl derivatives, containing propanediol or ethylene glycol units as hydrophilic spacer moieties, to yield the corresponding cluster galactosides. To determine the affinity of the cluster galactosides for the asialoglycoprotein receptor, we have performed competition studies of [125I]ASOR binding, a specific ligand for the asialoglycoprotein receptor, to isolated parenchymal cells. The affinity for the asialoglycoprotein receptor significantly increased with increasing spacer length. N-[[[Tris-O-(beta-D-galactopyranosyl)-3,6,9-trioxaunde- canoxy]methoxy]methyl]-N-alpha-[1-(6-methyladipyl)]glycinami de (4e), a cluster galactoside provided with a 20 A spacer, possessed an at least 2000-fold higher affinity for the receptor than N-[[tris-O-(beta-D-galactopyranosyl)methyl]methyl]-N alpha-[1-(6- methyladipyl)]glycinamide (4a), a cluster galactoside lacking the spacer. It is concluded that vicinal galactosyl moieties within a cluster galactoside are more optimal recognized by the galactose binding sites of the asialoglycoprotein receptor upon proper spacing. The most potent galactoside, TG(20A), may constitute an attractive targeting device for the specific delivery of drugs and/or genes to the parenchymal liver cell.

Design and synthesis of novel amphiphilic dendritic galactosides for selective targeting of liposomes to the hepatic asialoglycoprotein receptor.

A series of glycolipids have been prepared which contain a cluster galactoside moiety with high affinity for the hepatic asialoglycoprotein receptor and a bile acid ester moiety which mediates stable incorporation into liposomes. Loading of liposomes with these glycolipids at a ratio of 5% (w/w) resulted in efficient recognition and uptake of the liposomes by the liver. Preinjection with asialofetuin almost completely inhibited the uptake, establishing that the liposomes were selectively recognized and processed by the asialoglycoprotein receptor on liver parenchymal cells. In contrast, a glycolipid content of 50% (w/w) led to a liver uptake that could not be inhibited by preinjection with asialofetuin, indicating that the liposomes were now processed by the Gal/Fuc-recognizing receptor on liver macrophages. The results presented in this study are important for future targeting of water-soluble and amphiphilic drugs, enveloped in these glycolipid-laden liposomes, to parenchymal liver cells.

Evidence for the existence of at least two different binding sites for 5HT-reuptake inhibitors within the 5HT-reuptake system from human platelets.

Chemical modification procedures have been used to study the interaction of tricyclic and non-tricyclic 5HT-reuptake inhibitors with the [3H]imipramine binding site (IBS). N-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) induced a pronounced loss in [3H]imipramine binding due to a reduction in Bmax. Preincubation with reuptake inhibitors and subsequent inactivation by EEDQ revealed that imipramine and 5HT prevented the EEDQ-induced inhibition, but citalopram and fluoxetine did not. Thiol modification studies demonstrated that reduction by dithiothreitol (DTT) enhanced the binding of [3H]imipramine by increasing the Bmax. The thioselective reagents 1,1-diazobis- (N,N-dimethylformamide) (diamide), phenyl-arsineoxide (PAO) and N-ethylmaleimide (NEM) attenuated the binding capacity by lowering the Bmax. PAO, a reversible thiol reagent, prevented NEM alkylation indicating that dithiols are involved in the NEM-induced inactivation. Binding of tricyclics or non-tricyclics prior to PAO inactivation revealed that tricyclics provide complete protection against thiol modification, while the non-tricyclics do not. The results support the hypothesis that the 5HT-reuptake system of human platelets possesses at least two distinguishable binding sites.