Cholesterol Derivative of a New Triantennary Cluster Galactoside Lowers Serum Cholesterol Levels and Enhances Secretion of Bile Acids in the Rat
Background Previous studies have demonstrated that cholesterol-derivatized galactosides exert a hypocholesterolemic effect by inducing hepatic uptake of atherogenic lipoproteins by means of galactose-recognizing receptors in the liver. However, a prolonged infusion of high concentrations of these compounds was required for this effect, possibly because of low affinity for the galactose-recognizing asialoglycoprotein receptor on the parenchymal liver cell.
Methods and Results We have designed a new series of triantennary galactosides to optimize the affinity and specificity for this receptor. The affinity of a triantennary galactoside for the asialoglycoprotein receptor appeared to be dramatically enhanced by proper spacing of the three terminal galactose groups. In rats, a single injection of N-[tris-O-(3,6,9-trioxaundecanyl-β-d-galactopyranosyl)methoxymethyl]methyl-Nα-[1-(6-(5-cholesten-3β-yloxy)glycyl)adipyl]glycinamide [TG(20Å)C], the cholesterol derivative of the most selective galactoside, causes a dose-dependent decrease of ≤45% in the serum cholesterol concentration (P<.001). This decrease is mainly attributed to a decrease in the level of serum HDL (P=.0066) and, to a lesser extent, serum LDL (P=.036). In addition, TG(20Å)C strongly enhances the bile-acid secretion in rats during the first 2 hours after administration, which indicates that TG(20Å)C-induced clearance of cholesterol from the bloodstream is efficiently coupled to hepatic bile-acid secretion.
Conclusions We conclude that TG(20Å)C efficiently directs lipoproteins that contain cholesterol to the liver at a 30-fold-lower concentration than previously developed cholesterol-derivatized cluster galactosides. This newly developed approach to lower cholesterol levels may prove valuable for familial hypercholesterolemic patients or those with familial defective apolipoprotein B-100 who do not respond or who respond insufficiently, respectively, to conventional therapies.
High levels of serum LDL, the major vehicle for cholesterol transport in humans, are correlated with an increased occurrence of arteriosclerosis.1 2 Therapy for hypercholesterolemia is mainly focused on the induction of LDL receptors in the liver, which leads to an enhanced clearance and thus catabolism of LDL from the bloodstream. This process is accomplished through inhibition of cholesterol synthesis with 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors or by stimulation of biliary cholesterol excretion with bile-acid sequestrants.3 4 However, patients with the homozygous form of familial hypercholesterolemia, who lack functional LDL receptors, do not respond to such therapy,5 whereas in patients with familial defective apolipoprotein (apo) B-100,6 7 the above therapy may be of limited value.8 Furthermore, blockade of cholesterol synthesis may influence the level of intermediates of the cholesterol synthesis pathway that are essentially involved in various cellular functions.9 These unwanted side effects are circumvented by a direct removal of LDL through an alternative high-capacity receptor on the parenchymal liver cell, the asialoglycoprotein receptor. This receptor is involved in hepatic uptake and subsequent lysosomal processing of galactose-terminated substrates.10 11 Theoretically, a bifunctional compound that consists of a lipophilic moiety for anchorage to LDL and a second moiety displaying a high affinity for the asialoglycoprotein receptor should enhance the catabolism of LDL. Earlier studies from our department12 13 14 15 indicated that use of this concept to lower serum lipoprotein levels is valid. In 1984, a cholesterylated cluster galactoside with hypocholesterolemic activity, N-[tris-O-(β-d-galactopyranosyl)methyl]methyl-Nα-[(5-cholesten-3β-yloxy)succinyl]glycinamide [TG(4Å)C], was synthesized.16 However, continuous intravenous infusion of high doses of TG(4Å)C was required to accomplish a detectable therapeutic effect.12 Subsequently, monogalactosylated cholesterol derivative, which displayed a comparable hypocholesterolemic activity,15 was synthesized.17 Both compounds stimulated the hepatic uptake of LDL by directing LDL to the galactose-recognizing receptor on the Kupffer cell.13 14 15 Both the low level of lipid-lowering activity and the lack of specificity for targeting lipoproteins to the asialoglycoprotein receptor were argued to be caused by the moderate affinity and specificity of the cholesterol-derivatized cluster galactoside for this receptor.
In the present study, we show that both the affinity and specificity of a triantennary cluster galactoside for the asialoglycoprotein receptor could be significantly improved by elongation of the spacer that connects the terminal galactose moieties of a cluster galactoside with the branching point of the dendrite from 4 to 20Å. In view of its high affinity, the most selective compound, N-[tris-O-(3,6,9-trioxaundecanyl-β-d-galactopyranosyl)methoxymethyl]methyl-Nα-[1-(6-methyl)adipyl]glycinamide [TG(20Å)], may offer a new tool for the development of a more potent hypocholesterolemic therapeutic treatment. Therefore, we synthesized the cholesterol derivative of this cluster galactoside, N-[tris-O-(3,6,9-trioxaundecanyl-β-d-galactopyranosyl)methoxymethyl]methyl-Nα-[1-(6-(5-cholesten-3β-yloxy)glycyl)adipyl]glycinamide [TG(20Å)C], and tested its cholesterol-lowering activity and its effect on the biliary secretion of bile acids.
Carrier-free sodium 125I was supplied by Amersham International. Collagenase type I, N-acetyl-d-galactosamine, and BSA (fraction V) were obtained from Sigma Chemical Co. All other chemicals were analytical grade. N-[tris-O-(β-d-glucopyranosyl)methyl]methyl-Nα-[4-O-(5-cholesten-3β-yl)succinyl]glycinamide [TGlc(4Å)C] was synthesized according to the procedure described by Kempen et al.16 The synthesis of N-[tris-O-(β-d-galactopyranosyl)methyl]methyl-Nα-[6-(1-O-methyl)adipyl]glycinamide [TG(4Å)], N-[tris-O-(ethyl-β-d-galactopyranosyl)methoxymethyl]ethyl-Nα-[1-(6-methyl)adipyl]glycinamide [TG(9Å)], N-[tris-O-(propyl-β-d-galactopyranosyl)methoxymethyl]methyl-Nα-[1-(6-methyl)adipyl]glycinamide [TG(10Å)], N-[tris-O-(3-oxapentyl-β-d-galactopyranosyl)methoxymethyl]methyl-Nα-[1-(6-methyl)adipyl]glycinamide [TG(13Å)], and N-[tris-O-(3,6,9-trioxaundecanyl-β-d-galactopyranosyl)methoxymethyl]methyl-Nα-[1-(6-methyl)adipyl]glycinamide [TG(20Å)] will be described in detail elsewhere.17A TG(20Å)C was synthesized, as will be described in detail elsewhere (E.A.L.B., H.B., J.H.V.B., T.J.C.V.B, unpublished results).
Isolation of Kupffer and Parenchymal Cells
Male Wistar rats (≈250 g body wt each) were anesthetized by peritoneal injection of 20 mg sodium pentobarbital. Parenchymal liver cells were isolated after a 20-minute perfusion of the liver with 0.05% collagenase type IV at 37°C according to the method of Seglen,18 modified as previously described.19 After perfusion, parenchymal and Kupffer cells were purified by differential centrifugation and counterflow elutriation, as described in detail elsewhere.20 The purity of the Kupffer cells was >95%, as judged by peroxidase staining (0.1% 3,3′-diaminobenzidine in 0.05 mol/L Tris-HCl, 7% sucrose, 0.1% (vol/vol) 30% H2O2, pH=7.4) for 20 minutes at 37°C.
Isolation and Radioiodination of Asialo-orosomucoid and LDL
Human orosomucoid was isolated and subsequently desialylated enzymatically, as described previously.21 Human LDL (1.024<d<1.063) was isolated by density-gradient ultracentrifugation, according to Redgrave et al22 (see also Bakkeren et al23 ). The purity of LDL was monitored by PAGE analysis, by agarose-gel electrophoresis (0.8%), and by particle-size electrophoresis. LDL contained <0.06% apoE and >99% apoB. The mean diameter was 23±1 nm, whereas <1% of the mass was recovered in particles >40 nm. LDL was lactosylated by reductive alkylation of the lysines, as described previously.24 Both lipoproteins were radiolabeled with carrier-free [125I]NaI by the iodine monochloride method of McFarlane as modified by Bilheimer et al.25
In Vitro Binding Studies
Displacement of 125I-asialo-orosomucoid (ASOR) binding to parenchymal liver cells by unlabeled ASOR or by cluster galactosides [TG(4Å), TG(9Å), TG(10Å), TG(13Å), and TG(20Å)] was determined as follows. Rat parenchymal liver cells (1 to 1.5×106 cells, viability >90%) were incubated in 1 mL DMEM containing 2% BSA with 5 nmol/L 125I-ASOR in the presence or absence of cluster galactosides in eight concentrations ranging from 1 nmol/L to 1 mmol/L. After the liver cells were incubated for 2 hours at 4°C under gentle agitation, the medium was removed by aspiration and the cells were washed twice in 2 mL ice-cold medium containing 0.2% BSA and once in medium without BSA. Nonspecific binding was measured in the presence of 100 mmol/L N-acetyl galactosamine (GalNAc).
Displacement of binding of 125I-lactosylated LDL (125I-Lac-LDL) to Kupffer cells by unlabeled Lac-LDL or by the newly synthesized cluster galactosides [TG(4Å), TG(9Å), TG(10Å), TG(13Å), and TG(20Å)] was determined analogously. Rat Kupffer cells (1 to 1.5×106 cells, viability >90%) were incubated in 1 mL DMEM containing 2% BSA with 5 nmol/L 125I-Lac-LDL in the presence or absence of cluster galactosides at eight concentrations ranging from 1 nmol/L to 1 mmol/L. After the Kupffer cells were incubated for 2 hours at 4°C under gentle agitation, the medium was removed by aspiration and the cells were washed twice with 2 mL ice-cold medium containing 0.2% BSA and once with medium without BSA. Nonspecific binding was measured in the presence of 100 mmol/L GalNAc.
Protein contents were determined according to the method of Lowry, with BSA as the standard. Displacement binding data were analyzed according to a single-site model with a computerized nonlinear fitting program (Graph-Pad) to calculate the inhibition constant, Ki.24
Male Ry-Wistar rats (250 to 300 g each) were anesthetized with ether, and a 300-μL blood sample was collected by orbital puncture. Subsequently, 500 μL NaPi buffer (10 mmol/L, pH 7.4) containing 150 mmol/L NaCl (PBS) or PBS containing TGlc(4Å)C (560 μg) or TG(20Å)C (56, 180, or 560 μg) was injected in the vena penis, and blood samples (300 μL) were taken at the indicated times by orbital puncture (three animals per treatment). After sampling, blood samples were centrifuged for 5 minutes at 1500g; the serum was collected and stored for further analysis. After the last puncture, the rats were killed and exsanguinated. The serum was assayed for the concentration of total cholesterol with a CHOD-PAP kit (Boehringer Mannheim). The sera obtained at t=24 hours were subjected to density-gradient ultracentrifugation in NaCl-KBr buffer for 22 hours at 150 000g. The gradient was subsequently subdivided according to density by aspiration of 300-μL fractions, starting from the bottom of the tube; very-low-density lipoprotein (VLDL, d<1.006), intermediate-density lipoprotein (IDL, 1.006<d<1.019), LDL (1.024<d<1.055), and HDL (1.063<d<1.21 mg/mL) fractions were isolated from the density gradient. Since the densities of rat lipoproteins differ slightly from those of human lipoproteins, the isolation procedure of Redgrave was slightly adapted according to Bakkeren et al.23 The purity of the isolated lipoprotein fractions was verified by PAGE analysis and was always >95%. The lipoprotein fractions were assayed for total cholesterol content, as described below. TG(20Å)C did not interfere with the cholesterol assay in the absence or the presence of cholesterol esterase.
Liver Uptake of Lipoprotein-TG(20Å)C Complexes
Male Wistar rats (≈250 to 300 g body wt each) were anesthetized by injection of 15 to 20 mg IP sodium pentobarbital. The abdomen was opened, and the complexes of 125I-LDL or 125I-HDL (50 μg apolipoprotein in 500 μL PBS) and TG(20Å)C, which were prepared by incubation of the lipoprotein with 50 μg TG(20Å)C for 30 minutes at 20°C, were injected into the inferior vena cava. Five minutes after injection, a liver lobule was tied off, excised, weighed, and counted for radioactivity. The excised liver tissue amounted to <15% of the total liver mass. The liver uptake of the injected compound was corrected for radioactivity in serum assumed to be entrapped in the tissue at the time of sampling (85 μL/g fresh wt).26
Bile was collected from unrestrained 3-month-old Wistar rats, as reported previously.27 Rats received tap water and standard chow ad libitum and were equipped with permanent catheters in the bile duct, duodenum, and heart. To maintain an intact enterohepatic circulation, the bile duct and duodenum catheters were connected immediately after surgery. Rats were allowed to recover for 1 week. At the start of the experiment, 560 μg TG(20Å)C (dissolved in 500 μL PBS) or 500 μL PBS was introduced within 1 minute through the heart catheter. The bile-duct catheter was then connected to a fraction collector, and the bile was collected for 48 hours. After the bile acids were extracted from the cholesterol–cholesterol ester fraction at pH 7.0, according to Bligh and Dyer,28 the aqueous layer was analyzed for bile-acid content and the organic phase was analyzed for total cholesterol content as described below.
Bile-Acid and Total Cholesterol Contents
Total cholesterol content of the sera was determined colorimetrically, in duplicate, with a CHOD-PAP kit (Boehringer Mannheim). The content of bile acids in the bile was determined colorimetrically with a Sterognost-3α PHO kit from Nycomed with sodium taurocholate (Sigma) as the standard.
The time curves for the effect of TG(20Å)C administration on the serum cholesterol level were analyzed statistically on the basis of the significance of the difference between four treatment groups and of a potential effect of time on the cholesterol content for the treatment groups by repeated-measures ANOVA after correction for missing values (SPSS/PC+). The effect of TG(20Å)C on the cholesterol content of the various lipoprotein fractions from serum (Fig 4⇓) was evaluated statistically by one-way ANOVA (SPSS/PC+), whereas the significance of the differences between means was tested by unpaired two-way Student’s t test.
Affinity and Specificity of Galactose-Terminated Triantennary Cluster Galactosides for the Hepatic Asialoglycoprotein Receptor
The affinity of the synthesized cluster galactosides (see Fig 1A⇓ for structures) for the hepatic asialoglycoprotein receptor was determined by use of competition studies of 125I-ASOR binding to parenchymal liver cells. The affinity of the cluster galactoside for the asialoglycoprotein receptor increased dramatically with elongation of the spacer connecting the terminal galactosyls with the branching point of the dendrite (Fig 2A⇓). TG(20Å), provided with a 20-Å spacer, displayed an Ki of 190 nmol/L, whereas TG(4Å), with a 4Å spacer, was only marginally capable of inhibiting 125I-ASOR binding (Ki=390 μmol/L). The cluster galactosides with intermediate spacer length, TG(9Å), TG(10Å), and TG(13Å), exhibited intermediate affinities (Ki=19, 1.2, and 10 μmol/L, respectively) for the asialoglycoprotein receptor. As a control, competition studies of 125I-ASOR binding by unlabeled ASOR were performed. In agreement with previous studies, the affinity of ASOR for the asialoglycoprotein protein receptor was in the low nanomolar range, with an Ki of 6.46±1.75 nmol/L.32 33
In addition to the asialoglycoprotein receptor on the parenchymal liver cell, the liver contains a second galactose-recognizing receptor: the galactose-fucose receptor on the Kupffer cell.24 29 30 To assess the cellular specificity of the synthesized cluster galactosides, we have determined the affinity of the galactosides for the competing galactose-fucose receptor on Kupffer cells by use of competition studies of 125I-Lac-LDL binding to this receptor. Lac-LDL has been established to be specifically recognized by the fucose-galactose receptor on Kupffer cells.30 125I-Lac-LDL binding to Kupffer cells could be inhibited up to 78% by excess unlabeled Lac-LDL at an Ki of 1.15±0.30 nmol/L (Fig 2B⇑). In contrast, none of the compounds was capable of displacing 125I-Lac-LDL binding from Kupffer cells at concentrations of up to 400 nmol/L.
Effect of TG(20Å)C on the Cholesterol Concentration of Rat Serum
The galactoside with the highest affinity for the asialoglycoprotein receptor TG(20Å) has been derivatized with a cholesterol moiety, yielding TG(20Å)C (Fig 1B⇑) (E.A.L.B., H.B., J.H.V.B., T.J.C.V.B., unpublished results), and the biological activity of the compound has been evaluated in the rat. Although the rat is not the most appropriate model for evaluating the hypocholesterolemic activity of TG(20Å)C in terms of serum lipoprotein profile and cholesterol metabolism, utilization of the rat enables a direct comparison with earlier studies on the hypocholesterolemic activity of TG(4Å)C and mono-gal-chol.12 13 14 15 16 17 The effect of an intravenous bolus injection of TG(20Å)C on the total serum cholesterol content in rats is shown in Fig 3⇓. A dose-dependent decrease of cholesterol level was observed after injection of TG(20Å)C. A slight initial decrease of the cholesterol level was induced, even at a dose of 56 μg. A dose of 560 μg TG(20Å)C 11 hours after injection reduced the serum cholesterol level by 45%. Repeated-measures ANOVA of the concentration-time curves for the four treatment groups revealed that there were statistically significant differences between these curves (P<.001). In subsequent analyses, it was found that the curves for the groups of rats treated with 180 and 560 μg TG(20Å)C differed significantly from the control group (P=.002 and P=.004, respectively), whereas the curve for 56 μg TG(20Å)C differed only marginally from that of the control group (P=.076). In addition, a clear-cut and comparable effect of time on the serum cholesterol levels was noticed for all treatment groups (P<.01). The persistence of this reduction was remarkable. Twenty-four hours after administration of the agent, serum levels still had not reached control values. The effect of TGlc(4Å)C, the cholesterol derivative of a triantennary cluster glucoside, was studied as a control for a potential nonspecific effect of cholesterylated cluster galactosides. However, a bolus injection of 560 μg TGlc(4Å)C did not affect the level of total serum cholesterol over a 24-hour period after injection (2.28±0.2 mmol/L).
The levels of the various individual lipoprotein fractions were measured 24 hours after administration of PBS or TG(20Å)C. In view of the predominant contribution of HDL cholesterol to the total serum cholesterol level in rats (≈65%), it was anticipated that the level of HDL must be affected by administration of TG(20Å)C. Indeed, the HDL level was reduced in a dose-dependent manner to a maximum of 35% at a dose of 560 μg (P=.0066 by one-way ANOVA) (Fig 4⇓). The LDL level tended to decrease by 25% to 30% (P=.036). Surprisingly, administration of TG(20Å)C tends to enhance the VLDL level (P=.07). Serum samples of the TG(20Å)C-treated animals did not exhibit any sign of hemolysis.
Effect of TG(20Å)C on Biliary Secretion in Rats
Subsequently, we investigated whether administration of TG(20Å)C influenced biliary secretion. TG(20Å)C (560 μg) was injected into unrestrained rats that were equipped with catheters in the bile duct, duodenum, and heart; bile was collected for 48 hours, and the bile flow and amount of bile acid–cholesterol secretion were determined. The biliary secretion of cholesterol in the TG(20Å)C-treated rats was identical to that in the control rats (0.41 versus 0.37 μmol/h, respectively) (Fig 5⇓). In contrast, the secretion of bile acids in the bile was significantly accelerated, from 72.9±23 to 152±22 μmol per 2 hours during the first 2 hours after injection of TG(20Å)C (n=3, P<.05 by Student’s t test, Fig 5⇓). After 2 hours, the rate of bile-acid secretion in the TG(20Å)C-treated rats stabilized at control values, ie, 9.5±2.8 and 7.7±2.0 μmol/h, respectively. The biliary flow (Fig 5⇓) was not affected by injection of TG(20Å)C (0.75 versus 0.70 mL/h, n=3).
Effect of Loading of 125I-HDL and 125I-LDL With TG(20Å)C on the Liver Uptake of Lipoproteins
The liver uptake of native 125I-HDL 5 minutes after injection was low (1.9±1.4% of the injected dose; see Table⇓). However, incubation of 50 μg 125I-HDL with 15 μg TG(20Å)C markedly stimulated the hepatic uptake of the lipoprotein to 33.6±1.3% of the injected dose (P<.001 by Student’s t test). Preincubation of 50 μg 125I-LDL with 15 μg TG(20Å)C also caused a significant increase in liver association of the lipoprotein from 1.7±0.4% to 20.1±1.2% of the injected dose (P<.002 by Student’s t test). To verify whether hepatic galactose-recognizing receptors are involved in the increased up-take of TG(20Å)C-lipoprotein complexes, we studied the effect of preinjection of GalNAc on the liver uptake of these complexes. The hepatic uptake of both TG(20Å)C–125I-HDL and TG(20Å)C–125I-LDL appeared to be almost completely inhibited by an excess of GalNAc (89% and 81% inhibition, respectively; P<.002 by Student’s t test).
We previously reported that lipoprotein catabolism can be enhanced by administration of lipophilized galactose-terminated galactosides, which induce uptake of lipoproteins by hepatic galactose-recognizing receptors.13 14 15 However, prolonged infusion of relatively high doses of these galactosides was required to lower the blood cholesterol content significantly. We rationalized that the low potency may be caused by the low affinity and specificity of the galactose-terminated cluster galactoside for the hepatic asialoglycoprotein receptor. On the basis of previous studies of the prerequisites for high-affinity recognition by this receptor,31 32 33 and by use of molecular modeling, new triantennary cluster galactosides have been designed and synthesized.17A In the present study, we demonstrate that the affinity of these new galactosides for the asialoglycoprotein receptor is markedly increased as a result of proper spacing of the terminal galactose units within a triantennary cluster galactoside. TG(20Å), in which the terminal galactose moieties were spaced 20 Å from the branching point of the dendrite, possessed a 2000-fold higher affinity (Ki=190 nmol/L) than a previously used cluster galactoside, TG(4Å)12 13 14 (Ki=390 μmol/L). TG(9Å), TG(10Å), and TG(13Å) displayed intermediate affinities for the asialoglycoprotein receptor. Remarkably, TG(10Å) had a slightly higher affinity for the asialoglycoprotein receptor than TG(9Å) or TG(13Å). This difference probably results from the higher hydrophobicity of the propyl moiety of TG(10Å) compared with the ethylene glycol units TG(9Å) and TG(13Å), as has been suggested previously for other triantennary galactoside substrates of the asialoglycoprotein receptor.33
In contrast, elongation of the spacers within a cluster galactoside from 4 to 20Å did not influence the affinity for the galactose-fucose receptor on the Kupffer cell, which also recognizes galactose-terminated galactosides. Hence, it can be concluded that not only the affinity but also the specificity for the asialoglycoprotein receptor compared with the galactose-fucose receptor is dramatically enhanced upon elongation of the spacer. In view of the affinity of ASOR for the asialoglycoprotein receptor, an additional 25-fold gain in affinity and specificity may be achieved for synthetic cluster galactosides. However, the affinity of TG(20Å) for the asialoglycoprotein receptor, which is 2000-fold higher than that of TG(4Å), may indicate that the cholesterol-lowering activity of a cholesterylated galactoside correlates with an increase in the affinity of the galactoside for the asialoglycoprotein receptor; further improvement in affinity may lead to direct clearance of the compound before the compound can accumulate in the lipoproteins. Thus, we have derivatized the most selective galactoside, TG(20Å), with cholesterol. The resulting compound, TG(20Å)C, is an amphiphilic compound. Previous study has demonstrated that TG(20Å)C incorporates spontaneously into lipoproteins in incubation with serum17A and with isolated lipoproteins.34 Subsequently, we evaluated the physiological activity of TG(20Å)C in the rat. Intravenous bolus injection of TG(20Å)C into rats resulted in a significant dose-dependent decrease of the serum cholesterol concentration. A maximal decrease (45%) of the serum cholesterol concentration was observed after a single injection of only 560 μg TG(20Å)C; its hypocholesterolemic potency was at least 30-fold higher than that of the previously developed compound TG(4Å)C.12 In contrast to TG(4Å)C, application of TG(20Å)C did not require an infusion protocol and did not lead to hemolysis at therapeutic doses.12 Even intravenous injection of 6 mg TG(20Å)C/kg into rats was tolerated well. The decrease in the serum cholesterol level persisted for at least 24 hours, possibly reflecting the low rate of de novo synthesis of HDL in the rat. Alternatively, this decrease may arise from storage of TG(20Å)C in and sustained release from a hydrophobic compartment in the rat (ie, cell membranes).
Further analysis of the serum lipoprotein profile of both TG(20Å)C-treated and untreated control rats showed that the decrease in the level of total serum cholesterol can be attributed mainly to an interaction of TG(20Å)C with HDL and, to a lesser extent, with LDL. However, before extrapolating these in vivo results in rats to the human situation, one should realize that the ratio of LDL-cholesterol to HDL-cholesterol in the rat is approximately 15-fold lower than in humans (0.2 and 3.0, respectively). The rat evidently is not the most appropriate species for studying the in vivo LDL-lowering activity of TG(20Å)C. Further studies must therefore be performed to investigate the hypocholesterolemic activity of TG(20Å)C in an animal more comparable to the human species in terms of lipoprotein profile and cholesterol metabolism (ie, the Watanabe heritable hyperlipidemic rabbit or cholesterol-fed hamster). At first glance, accelerating the catabolism of HDL seems to be undesirable because it might result in a more atherogenic plasma lipoprotein profile. However, a compound that selectively enhances the hepatic uptake of HDL may concomitantly stimulate the HDL-mediated reverse cholesterol transport from the periphery to the liver, which, of course, is beneficial. In this respect it needs to be verified whether long-term administration of TG(20Å)C will affect the reverse cholesterol transport of HDL, the rate of de novo synthesis of HDL and, consequently, the lipoprotein profile.
The high potency of the compound to direct lipoproteins to the liver is illustrated by the amount of TG(20Å)C molecules that is required for the removal of one molecule of HDL from the bloodstream. If we assume that the total HDL pool of the rat is ≈80 nmol, the highest dose of TG(20Å)C (560 μg=0.3 μmol per rat) corresponds to 4 moles TG(20Å)C per mole HDL. Incorporation of 4 moles of TG(20Å)C per HDL apparently suffices to induce efficient removal of the lipoprotein from the bloodstream. Further study has demonstrated that the hepatic uptake of 125I-HDL and 125I-LDL was significantly increased upon incubation of these lipoproteins in TG(20Å)C before injection into the rat. The TG(20Å)C-induced stimulation of the liver uptake of 125I-HDL and 125I-LDL could be prevented by preinjection of an excess of GalNAc, which suggests the involvement of galactose-recognizing receptors in the TG(20Å)C-induced liver uptake of HDL and LDL. Recent studies with premixed TG(20Å)C-LDL and TG(20Å)C-HDL clearly identified the parenchymal liver cell to be responsible for the hepatic uptake of TG(20Å)C–125I-HDL and TG(20Å)C–125I-LDL complexes.34 This is rather surprising, since previously developed cholesterylated galactosides TG(4Å)C and mono-gal-chol induced uptake of LDL by the galactose-fucose receptor24 29 30 on the Kupffer cell.13 15 We therefore suggest that the TG(20Å)C-induced reduction in the serum cholesterol level arises from an increased hepatic uptake of lipoproteins by the asialoglycoprotein receptor on parenchymal liver cells. An additional indication that parenchymal cells are involved in the increased clearance of cholesterol from the circulation was obtained from the effect of TG(20Å)C on biliary secretion in the rat. During the first 2 hours after administration of TG(20Å)C, the secretion of bile acids was two times normal levels (P<.05), whereas the biliary flow and the secretion of cholesterol remained constant. This suggests that TG(20Å)C-induced hepatic uptake of cholesterol is rapidly shunted into the bile-acid pathway, a route morphologically linked to the parenchymal cell. The reduction in serum cholesterol (15 μmol), however, is too small to fully account for the observed increment in the secretion of bile acids. Apparently, TG(20Å)C also mobilizes bile acids from the bile-acid pool. A more direct study of the effect of TG(20Å)C on the biliary secretion profile is required to unravel this phenomenon.
In conclusion, the present data show that TG(20Å)C is a promising and potent serum cholesterol–lowering agent. In rats, TG(20Å)C principally induces hepatic uptake of HDL, thereby stimulating reverse cholesterol transport. Further study of TG(20Å)C or analogues on the lipid metabolism in an animal model that is more comparable with humans in terms of lipoprotein profile and cholesterol metabolism will reveal whether TG(20Å)C is also capable of inducing significant hepatic uptake of LDL. Administration of lipoprotein uptake enhancers, such as TG(20Å)C, involves a completely new approach to treatment of hypercholesterolemia. Its therapeutic activity does not depend on the presence of functional LDL receptors, as do conventional therapies based on HMG-CoA reductase or bile-acid sequestrants. Therefore, we envision use of a therapy involving TG(20Å)C or analogues to be a promising alternative for those patients who do not respond or who respond insufficiently to the aforementioned therapies.
This work was supported by a grant from the Dutch Heart Foundation (42.005). Dr E.A. Van de Velde is gratefully acknowledged for his help in statistical analysis.
Presented in part at the 66th Scientific Sessions of the American Heart Association and in abstract form (Circulation. 1993;88:465).
- Received June 2, 1994.
- Revision received September 8, 1994.
- Accepted October 2, 1994.
- Copyright © 1995 by American Heart Association
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