Comparison of synthetic saponin cholesterol absorption inhibitors in rabbits: evidence for a non-stoichiometric, intestinal mechanism of action.

The hypocholesterolemic activities of pamaqueside and tiqueside, two structurally similar saponins, were evaluated in cholesterol-fed rabbits. The pharmacological profiles of the saponins were virtually identical: both dose-dependently decreased the intestinal absorption of labeled cholesterol 25-75%, increased fecal neutral sterol excretion up to 2.5-fold, and decreased hepatic cholesterol content 10-55%. High doses of pamaqueside (>5 mg/kg) or tiqueside (>125 mg/kg) completely prevented hypercholesterolemia. Decreases in plasma and hepatic cholesterol levels were strongly correlated with increased neutral sterol excretion. Ratios of neutral sterol excreted to pamaqueside administered were greater than 1:1 at all doses, in opposition to the formation of a stoichiometric complex previously suggested for tiqueside and other saponins. Ratios in tiqueside-treated rabbits were less than unity, a reflection of its lower potency. Pamaqueside-treated rabbits exhibited a more rapid decline in plasma cholesterol concentrations than control animals fed a cholesterol-free diet, indicating that the compound also inhibited the absorption of biliary cholesterol. Intravenous administration of pamaqueside had no effect on plasma cholesterol levels despite plasma levels twice those observed in rabbits given pamaqueside orally. These data indicate that pamaqueside and tiqueside induce hypocholesterolemia by blocking lumenal cholesterol absorption via a mechanism that apparently differs from the stoichiometric complexation of cholesterol hypothesized for other saponins.

Steroidal glycoside cholesterol absorption inhibitors.

We have explored the use of steroidal glycosides as cholesterol absorption inhibitors which act through an unknown mechanism. The lead for this program was tigogenin cellobioside (1, tiqueside) which is a weak inhibitor (ED50 = 60 mg/kg) as measured in an acute hamster cholesterol absorption assay. Modification of the steroid portion of the molecule led to the discovery of 11-ketotigogenin cellobioside (5, pamaqueside) which has an ED50 of 2 mg/kg. Replacement of the cellobiose with other sugars failed to provide more potent analogs. However, large improvements in potency were realized through modification of the hydroxyl groups on the cellobiose. This strategy ultimately led to the 4", 6"-bis[(2-fluorophenyl)carbamoyl]-beta-D-cellobiosyl derivative of 11-ketotigogenin (51) with an ED50 of 0.025 mg/kg in the hamster assay, as well as the corresponding hecogenin analog 64 (ED50 = 0.07 mg/kg).

Cellular cholesterol efflux mediated by cyclodextrins.

In this study, we compared cholesterol efflux mediated by either high density lipoproteins (HDL3) or beta-cyclodextrins, cyclic oligosaccharides that are able to dissolve lipids in their hydrophobic core. beta-Cyclodextrin, 2-hydroxypropyl-beta-cyclodextrin, and methyl-beta-cyclodextrin at 10 mM induced the release of 50-90% of L-cell [3H]cholesterol after 8 h of incubation, with a major portion of this cholesterol being released in the first 1-2 h of incubation. The cholesterol efflux kinetics are different if cells are incubated with HDL3, which induces a relatively constant rate of release of cholesterol throughout an 8-h incubation. Cholesterol efflux to cyclodextrins was much greater than phospholipid release. To test the hypothesis that maximal efflux rate constants for a particular cell are independent of the type of acceptor, we estimated the maximal rate constants for efflux (Vmax) of cellular cholesterol from L-cells, Fu5AH cells, and GM3468A fibroblasts. The rate constant for HDL3-mediated efflux varied among cell lines in the order Fu5AH > L-cells > fibroblasts. However, these differences were not evident when cyclodextrins were used as cholesterol acceptors. The estimated Vmax values for cyclodextrin-mediated efflux were 3.5-70-fold greater than for HDL3 for the three cell lines. The very high efficiency of cyclodextrins in stimulating cell cholesterol efflux suggests that these compounds can be used in two general ways for studies of atherosclerosis: 1) as research tools to probe mechanisms of cholesterol transport and aspects of membrane structure or 2) as potential pharmacological agents that could modify in vivo cholesterol metabolism and influence the development of the atherosclerotic plaque.

Pharmacologic consequences of cholesterol absorption inhibition: alteration in cholesterol metabolism and reduction in plasma cholesterol concentration induced by the synthetic saponin beta-tigogenin cellobioside (CP-88818; tiqueside).

Natural and synthetic saponins inhibit cholesterol absorption and reduce plasma cholesterol levels in experimental animals and are therefore of potential pharmacologic utility in the treatment of hypercholesterolemia. To determine the effects of this class of compounds on cholesterol absorption and metabolism, we evaluated the effects of the synthetic saponin, beta-tigogenin cellobioside (tiqueside; CP-88818), on male golden Syrian hamsters. When administered as either a single oral bolus or as a dietary supplement for up to 2 weeks, tiqueside inhibited cholesterol absorption in a dose-dependent manner in both the presence and absence of dietary cholesterol. Administration of tiqueside to chow-fed hamsters as a 0.2% dietary supplement (150 mg/kg per day) for 4 days resulted in a 68% decrease in intestinal cholesterol absorption with no change in either bile absorption or cholesterol 7 alpha-hydroxylase activity, suggesting that tiqueside inhibits cholesterol absorption without interfering with enterohepatic bile acid recirculation. Under these conditions, hepatic cholesterol levels were also reduced in a dose-dependent manner. Hepatic cholesterol reduction was highly correlated with cholesterol absorption inhibition, and induced compensatory increases in both hepatic HMG-CoA reductase activity and hepatic low density lipoprotein (LDL) receptor levels. Compensatory increases in intestinal HMG-CoA reductase activity were also noted after tiqueside administration, and are consistent with a luminal mechanism for tiqueside action. As a consequence of these changes to cholesterol metabolism, tiqueside administration induced plasma cholesterol reductions that were highly correlated with both hepatic cholesterol reduction and cholesterol absorption inhibition. Tiqueside also produced comparable plasma cholesterol lowering in a variety of other species fed either cholesterol-free diets (hamster, rat, mouse, dog) or cholesterol-containing diets (hamster, rat, rabbit, mouse, cynomolgus monkey, rhesus monkey, SEA quail) indicating the ubiquity of tiqueside action. For all species evaluated except the dog, the reduction in plasma cholesterol was due primarily to a reduction in circulating non-HDL cholesterol levels with little or no change in HDL cholesterol levels. Taken together, these results indicate that inhibition of cholesterol absorption by tiqueside produces profound effects on cholesterol metabolism without affecting bile acid metabolism, and that these changes lead to reductions primarily in plasma non-HDL cholesterol concentrations. The synthetic saponin, tiqueside, may thus represent a prototypical form of therapy for the treatment of hypercholesterolemia.

The inactivation of gamma-aminobutyric acid transaminase in dissociated neuronal cultures from spinal cord.

It had previously been shown that dissociated cell cultures from chick embryo spinal cord have a high affinity uptake system for the neurotransmitter gamma-aminobutyric acid (GABA) and make functional inhibitory synaptic contacts as determined by electrophysiology (Farb et al., 1979). It is shown here that these cultures can synthesize GABA from added glutamate in a glutamate decarboxylase-dependent reaction. Furthermore, these cultures have a functional GABA transaminase that degrades the neurotransmitter. This enzyme can be specifically and irreversibly blocked with gabaculine. A 15 min incubation with 10(-6) M-gabaculine completely inactivates the enzyme. The inactivation of the enzyme leads to an increase in GABA levels. Long-term incubation (16 days) of gabaculine in the medium does not appear to alter high affinity GABA transport, suggesting that the drug is not toxic to cells capable of accumulating GABA.

Functional incorporation of synthetic glycolipids into cells.

Synthetic glycolipids containing an alpha-mannoside group linked by a hydrophilic spacer arm to cholesterol were incorporated into bovine erythrocytes by exchange from glycolipid-containing liposomes. When the distance between the sugar and the cholesterol moieties was approximately 26 A, functional incorporation of these glycolipids could be easily detected, as revealed by the concanavalin A-mediated agglutination of these cells. Bovine erythrocytes are not themselves susceptible to concanavalin A-mediated agglutination. The minimal concentration of concanavalin A required for agglutination of modified erythrocytes, containing 9.15 x 10(6) glycolipid molecules per cell, was 4 microgram/ml. Under these conditions, only approximately 4% of the membrane-bound cholesterol had been exchanged for the synthetic glycolipid. The observed aggregation was reversible in the presence of alpha-methyl mannoside and did not occur when beta-galactosyl-containing glycolipids were used in place of their alpha-mannoside isomers. These studies demonstrate a technique of sugar incorporation into cell membranes which should be of great advantage in studies on the roles of cell surface sugars in biological recognition. Furthermore, they demonstrate that the sugars need only be a short distance (26 A) from the membrane in order to functionally bind concanavalin A.

Threshold effects on the concanavalin A-mediated agglutination of modified erythrocytes.

Bovine erythrocytes, which are not concanavalin A (ConA)-agglutinable, can be rendered so by attaching alpha-D-mannose residues to their outer membrane. The sugars are incorporated by mildly oxidizing the cells with periodate followed by coupling the liberated aldehyde groups with an alpha-thiomannosyl containing hydrazide (I). The rate and extent of ConA-mediated aggregation of the modified cells are not linearly dependent on the amount of sugar incorporated. For example, treatment of the erythrocytes with 0.075 mM periodate for 5 min followed by I led to the introduction of 1.05 x 10(6) mannosyl residues/erythrocyte. Binding studies with 125I-ConA demonstrated the presence of 66,525 ConA receptors/cell with an average KA = 4.9 X 10(6) M-1 yet the cells failed to aggregate with ConA at concentrations up to 500 microgram ml-1. Treating the cells with 0.1 mM periodate followed by I led to the introduction of 1.42 x 10(6) mannosyl residues/erythrocyte. Binding studies with 125I-ConA indicated the presence of 78,780 binding sites/cell (KA = 5.9 X 10(6) M-1). These cells were readily aggregated by ConA at concentrations greater than or equal to 64 microgram ml-1. We show here that the sugar incorporation technique is random and that no functional differences were detected in the receptors introduced at the different periodate concentrations. Therefore, the ConA-mediated aggregation of these modified erythrocytes is exquisitely sensitive to small changes in functionally identical receptor densities.

Synthetic glycolipids and the lectin-mediated aggregation of liposomes.

Synthetic mannose-containing glycolipids utilizing the cholesterol nucleus as a lipid anchor, and either the 6-aminohexyl- or the 6-(6-aminohexanamido)hexyl-1-thio-alpha-D-mannopyranosides as the carbohydrate ligands, have been synthesized and incorporated into small unilamellar liposomes. Incorporation of these cholesterol-mannoside derivatives at concentrations up to 14 mol% apparently does not affect the physical characteristics of the liposomes. Addition of concanavalin A to a suspension of liposomes containing the long chain cholesterol-mannose derivative causes an increase in light-scattering at 360 nm. As the increase in absorbance is completely reversed by the addition of alpha-methylmannoside, aggregation rather than fusion of the liposomes appears to be occurring. Liposomes containing 14 mol % of the short chain (6-aminohexyl-) derivative are aggregated by concanavalin A indicating that the lectin can approach to within 10 A of the lipid bilayer. Preliminary results suggest that the aggregation of vesicles containing either the long or short chain derivatives is highly dependent on the density of the sugar in the membrane.

Threshold effects on the lectin-mediated aggregation of synthetic glycolipid-containing liposomes.

Cholesterol analogs containing sugar residues linked by spacer groups to the cholesterol O can be incorporated into egg yolk lecithin small unilamellar liposomes. The synthetic glycolipid analogs distribute evenly on both sides of the bilayer. These liposomes are aggregated by the appropriate lectin. For example, when the sugar residue is a beta-galactoside the liposomes are aggregated by ricin and when it is an alpha-mannoside they are aggregated by Con A. The lectin-mediated aggregation of these liposomes is reversed by the addition of the appropriate sugar. The rates but not the extents of aggregation of these liposomes are highly sensitive to the amount of glycolipid incorporated. Below approximately 5% glycolipid incorporation the rate of the lectin-mediated aggregation of these liposomes is exceedingly slow, whereas above this level rapid aggregation proceeds. At all concentrations studied the synthetic glycolipids are incorporated in a unimodal fashion so that the observed threshold effects cannot be based on possible differences in the manner in which the glycolipids are incorporated at different concentrations. This conclusion is based on 1) studies with galactose oxidase that show that the percentage of galactose oxidation in a liposome prepared from a galactosyl-containing glycolipid is independent of glycolipid concentration, and 2) studies on the aggregation of liposomes containing mixed glycolipids in which the glycolipids are shown to behave independently. The importance of a critical density of membrane-bound receptors in order for aggregation to occur is discussed.