Keith E. Suckling

Cholesterol lowering and bile acid excretion in the hamster with cholestyramine treatment

Cholestyramine was administered to hamsters at 6 doses in the diet for 1 week. Plasma cholesterol, LDL + VLDL cholesterol and HDL cholesterol were measured after this period. Bile acid excretion was measured in faeces collected over the final 24 h of the experiment. A dose-response curve for each parameter measured was constructed using data from individual hamsters. For the bile acid and the cholesterol measurements a maximum response was observed at the highest doses. A correlation between the bile acids excreted over 24 h and the LDL + VLDL cholesterol showed that the maximum effect of cholestyramine on lowering plasma and lipoprotein cholesterol occurred at a submaximal excretion level of bile acids. Comparison of the efficiency of cholestyramine in reducing plasma cholesterol in the hamster with limited data in the dog and in man suggest that a greater lowering of plasma cholesterol is achieved in the dog and in man for an equivalent increase in bile acid excretion caused by the sequestrant. As is already known, cholestyramine treatment caused an increase in hepatic cholesterol 7 alpha-hydroxylase and HMG-CoA reductase activity. Interestingly in this study the novel observation was made that the bile acid sequestrant reduced the activity of hepatic acyl-CoA: cholesterol acyltransferase.

Effect of membrane environment on inhibition of acyl-CoA:Cholesterol acyltransferase by a range of synthetic inhibitors

The effect of the membrane environment of acyl-CoA:cholesterol acyl transferase (ACAT), an important intracellular enzyme of cholesterol metabolism, on the properties of a range of inhibitors of varying potencies was studied. ACAT activity from rat liver was solubilised with 3% deoxycholate (97% solubilised activity). After dilution into cholesterol/phosphatidylcholine liposomes (molar ratio 0.35), the assay of this reconstituted system showed linearity with protein and time. Saturation with oleoyl-CoA was achieved at 10 microM. Comparison of the potency of the ACAT inhibitors in the reconstituted assay and in a microsomal assay revealed a relationship between the lipid content of the assay and the inhibitory activity for potent inhibitors of ACAT (CI976, CL277,082, YMI7E and DuP128). This relationship was unrelated to lipophilicity of the drugs. Octimibate, lovastatin and progesterone, none of which is a potent ACAT inhibitor but which have all been described as ACAT inhibitors in the literature, all had low potencies in both assay systems. These results suggest that the lipid concentration must be taken into account when comparing potencies of ACAT inhibitors. The present data also indicate that some compounds which inhibit cholesterol esterification may do so by an indirect mechanism.

SR-12813 lowers plasma cholesterol in beagle dogs by decreasing cholesterol biosynthesis

SR-12813 inhibits cholesterol biosynthesis in Hep G2 cells via an enhanced degradation of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. Here we also show that SR-12813 inhibits cholesterol biosynthesis in vivo. A sterol balance study was performed in normolipemic beagle dogs. The dogs were given SR-12813 orally at dosages of 10 and 25 mg/kg/day for a period of 9 days. After 7 days plasma cholesterol was decreased by 15% in the 10 mg/kg/day group and by 19% in the 25 mg/kg/day group. Using a dual isotope technique no effects on intestinal cholesterol absorption were observed. The sterol balance indicated that endogenous synthesis of cholesterol was reduced by 23% in the 10 mg/kg/day group and by 37% in the 25 mg/kg/day group. Plasma lathosterol-cholesterol levels in dogs treated with 25 mg/kg/day SR-12813 were reduced by 56%, confirming a reduction of the cholesterol biosynthesis. Treatment with SR-12813 or the HMG-CoA reductase inhibitor lovastatin resulted in a large decrease in low density lipoprotein (LDL) cholesterol. It is concluded that SR-12813 reduces cholesterol biosynthesis in the dog model which results in a decrease of bile acid excretion, cholesterol excretion and plasma cholesterol level. The in vivo profile of SR-12813 is very similar to that of direct HMG-CoA reductase inhibitors, although the mode of action of the compound is unique.

SK&F 97426-A a more potent bile acid sequestrant and hypocholesterolaemic agent than cholestyramine in the hamster

SK&F 97426-A is a novel bile acid sequestrant which was selected for comparison with cholestyramine in vivo because of its superior in vitro bile acid binding properties. The effects of the two sequestrants on faecal bile acid excretion, plasma total cholesterol, VLDL + LDL and HDL cholesterol and triglyceride concentrations and on liver enzymes involved in the synthesis and metabolism of cholesterol were investigated in normocholesterolaemic hamsters. Four studies were conducted to determine the relative potencies of the two resins using a range of doses of the sequestrants over treatment periods of up to 2 weeks. Curves fitted to the resulting data allowed common maximum responses and separate ED50s to be calculated for each sequestrant. The maximum response of both sequestrants was to increase bile acid excretion by 352% and lower plasma total cholesterol by 37-58%. LDL + VLDL and HDL cholesterol were reduced by 56-75% and 25-41%, respectively. SK&F 97426-A was 3 times more potent than cholestyramine at increasing the excretion of bile acids in the faeces and 2.1-3.4-fold and 2.3-3.2-fold more potent at lowering total plasma cholesterol and LDL plus VLDL cholesterol, respectively. In some of the experiments SK&F 97426-A was also more potent than cholestyramine at lowering HDL cholesterol. Plasma triglycerides were also lowered by both sequestrants by up to 31% after 1 week but the relative potency could not be determined. These HDL cholesterol and total triglyceride lowering effects of bile acid sequestrants in the hamster are known not to occur in people treated with cholestyramine. There were minimal differences between hamsters treated for 1 or 2 weeks in the relative potencies or ED50s calculated for the total plasma cholesterol, LDL + VLDL and HDL cholesterol. Both sequestrants may have been slightly more efficacious on these parameters after 2 weeks of treatment. Liver weights were reduced by about 15% by both sequestrants at 2% (w/w) in the diet for 1 week. The activities of the liver HMG-CoA reductase and cholesterol 7 alpha-hydroxylase were increased as expected, whilst the activity of the acyl-CoA:cholesterol acyltransferase was reduced by both sequestrants at this dose. SK&F 97426-A was, therefore, 2-3-fold more potent as a bile acid sequestrant and hypocholesterolaemic agent than cholestyramine when tested in the hamster.

Evaluation of cultured hamster hepatocytes as an experimental model for the study of very low density lipoprotein secretion

The secretion of triacylglycerol, cholesterol and cholesteryl ester in very low density lipoprotein (VLDL) by cultured hamster hepatocytes was studied, and the results compared with those obtained previously using cultured rat hepatocytes and the human hepatoma cell line HepG2. The hamster cells secreted apolipoprotein B and VLDL triacylglycerol, cholesterol and cholesteryl ester linearly during 24 h in culture, and this time period was used in all experiments. Addition of oleate (1 mM) to the culture medium resulted in increased secretion of triacylglycerol, but cholesterol ester output were unchanged. Triacylglycerol secretion was also increased in the presence of lipogenic substrates (10 mM lactate + 1 mM pyruvate) plus dexamethasone (1 microM), but not with either of these agents alone. Inhibition of cholesterol synthesis in the hamster cells by incubation with mevinolin (2 micrograms/ml) did not change VLDL lipid secretion, but stimulation using mevalonate lactone resulted in decreased triacylglycerol output. Manipulation of the rate of cholesterol esterification in the hepatocytes by inhibiting or stimulating the activity of acyl coenzyme A cholesterol:acyl transferase using the inhibitor Dup128 (25 microM) and 25-hydroxycholesterol (50 microM), respectively, had no effect on the secretion of VLDL lipid. In the presence of 1 mM oleate plus 25-hydroxycholesterol, however, a rise in the output of triacylglycerol and cholesteryl ester was observed. Hepatocytes prepared from hamsters fed 2% cholestyramine secreted significantly less triacylglycerol than those from animals given the control diet, but cholesterol and cholesteryl ester output were unchanged, despite a decrease of about 40% in the total cholesterol content of the cells. These results show that the secretion of lipid in VLDL in hamster hepatocytes differs from that in rat and human liver in its response to dietary cholestyramine, and from rat hepatocytes and HepG2 cells in its response to changes in the rate of lipogenesis and cholesterol synthesis and esterification. Overall, hamster hepatocytes appear to be less susceptible to modification the rate of hepatic VLDL secretion, and should provide a useful additional tool for the investigation of this process.

Effects of novel synthetic sterol probes on enzymes of cholesterol metabolism in cell-free and cellular systems.

A series of novel sterols was synthesised as probes for the enzymatic and cellular functions of two important enzymes of intracellular cholesterol metabolism, acyl-CoA:cholesterol acyltransferase (ACAT) and cholesterol 7 alpha-hydroxylase. The compounds were 6-fluoro-5-cholesten-3 beta-ol (6-fluorocholesterol), 7-cholesten-3 beta-ol (7-cholestenol), 6 beta-fluorocholestan-3 beta-ol (6 beta-fluorocholestanol), 3-acetoxy-6-fluorocholestan-3 beta-ol (3-acetoxy-6-fluorocholestanol) and 7-methoxy-5-cholesten-3 beta-ol (7-methoxycholesterol). They were designed to reveal the effect of small changes in sterol structure, particularly reactivity of certain parts of the ring structure and polarity, on enzyme activity and intracellular cholesterol metabolism. The 3 beta-hydroxy group was essential for interaction with both enzymes since 3-acetoxy-6-fluorocholestanol did not affect any of the enzyme-catalysed reactions. 6-Fluorocholesterol and 7-cholestenol had no effect on cholesterol esterification but did inhibit the hydroxylation of cholesterol, as did the other compounds with groups that could influence the 7 position, namely 6 beta-fluorocholestanol and 7-methoxycholesterol. The fluorocholestanols were all competitive substrates for cholesterol esterification in cell-free and cellular assays of ACAT activity. 7-Methoxycholesterol was a surprisingly effective inhibitor of ACAT for a simple sterol. However, 6-fluorocholesterol did not have any effect on ACAT, suggesting that interactions between the enzyme and the region around C-6 and C-7 of the sterol are important. These results show that it is possible to dissect components of cholesterol metabolism using simple, specifically substituted sterols and thus define a new approach to studying the relationships between the various enzymes that catalyse intracellular cholesterol metabolism.

What is the future for drug development in atherosclerosis and dyslipidaemia

Major companies have recently announced a reduction or termination of their work in cardiovascular disease, specifically atherosclerosis and dyslipidaemia. This editorial considers the background to these developments, relating it to the continuing development of the lipoprotein-associated phospholipase A2 inhibitor darapladib and the potential for the field in the future.

The effect of cyclic AMP analogues and glucagon on cholesteryl ester synthesis and hydrolysis in cultured hamster hepatocytes

Two cyclic AMP analogues, 8‐chloro cyclic AMP and 8‐(4 ehlorophenylthio) cyclic AMP, were found to increase the incorporation of [3H]oleate into cholereryl ester in cultured hamster hapatocytes (30–40%), while incorporation into triacylglycerol was unaffected. An increase of a similar magnitude was observed in the presence of glucagon and the phosphodiesterase inhibitor, theophylline. The cyclic AMP analogues also stimulated the activity of neutral cholesteryl ester hydrolase in the cells, and this effect was mimicked by glucagon and theophylline. These results show that cyclic AMP can affect the cholesteryl ester cycle in hamster hepatocytes, and support the idea that the enzymes involved may be co‐ordinately regulated.

The landscape of drug discovery in atherosclerosis and dyslipidaemia: a survey from patenting activity

Background/objective: Patenting activity now and over the past decade in the fields of atherosclerosis and dyslipidaemia was used to assess targets and mechanisms of active interest and to identify trends in current scientific and clinical research. Patents can be considered an index of the perception in a field of the potential value and feasibility of an approach. Methods: Two hundred patents published over 11 months claiming an indication of atherosclerosis or dyslipidaemia were analysed in detail. Two major groups were considered, lipid/metabolic and vascular/inflammation, reflecting scientific and clinical activity in the field. Mechanistic subgroups within these were identified. The analysis was amplified by reviewing the trends for the two groups as a whole over the past 10 years, and also for specific targets within groups. Results/conclusions: Patenting activity in both groups increased over the first half of the past decade and has remained at peak level for lipid/metabolic targets. However, activity for vascular/inflammation targets has significantly decreased. Overall, mechanisms that raise plasma high density lipoprotein predominated. The trends are assessed for their relationship to current scientific and clinical work.

Cholesterol and plasma lipid modifying patents

Cholesterol lowering by the 3-hydroxy-3-methylglutaryl co-enzyme A (HMG-CoA) reductase inhibitors (statins) has been shown to have significant effects on mortality due to coronary heart disease (CHD) in several trials reported over the last four or so years. However, it has long been recognised that hypercholesterolaemia is not the only dyslipidaemia which can contribute to CHD. This article reviews patenting activity over the past few years directed towards normalisation of the plasma lipid profile by mechanisms which do not involve modulation of cholesterol synthesis. Such mechanisms might include lowering plasma triglyceride or lipoprotein (a) [Lp(a)] or raising plasma high-density lipoprotein (HDL). Many claimed compounds appear to have effects in animal or cell models, but do not have a defined molecular target. Of the molecular targets defined, the peroxisome proliferator-activated receptors (PPARs) have great promise. Gene therapy is well-represented in the patent claims, but many technical hurdles...