Chemical derivatization of therapeutic proteins.

Despite major advances in redesigning and producing proteins through recombinant DNA technology, many therapeutic proteins are still produced by extraction from biological tissues or fluids, or from nonrecombinant microorganisms. Modification of such proteins, to improve potency and bioavailability and reduce immunogenicity, can only be carried out post-translationally by chemical-derivatization methods. Genetic- and chemical-modification methods are not mutually exclusive, however, and may be combined to optimize protein-engineering strategies, because chemical modification can introduce structural changes that are not encoded by DNA into both recombinant, and nonrecombinant proteins.

The effect of compactin, a potent inhibitor of 3-hydroxy-3-methylglutaryl coenzyme-A reductase activity, on cholesterogenesis and serum cholesterol levels in rats and chicks.

Compactin, [7-(1,2,6,7,8,8a-hexahydro-2-methyl-8-(2-methylbutyrylox)naphthyl)-3-hydroxyheptan-5-olide], a potent competitive inhibitor of the rate-determining step in cholesterol biosynthesis, was used to study the influence of changes in cholesterogenesis on serum cholesterol levels. Up to 3 h after a single oral dose (20 or 50 mg/kg) or after the last of a series of daily oral doses (50 mg/kg for 7 or 28 days) to young, male normolipidaemic rats, compactin consistently inhibited cholesterogenesis measured using 3H20 in liver, ileum and other extrahepatic tissues without affecting fatty acid synthesis. Compactin did not reduce serum or tissue cholesterol nor affect the serum concentration of other lipids nor the ratio between lipoprotein classes. A diurnal variation in the effect of compactin on cholesterogenesis was observed. For example, by 12--20 h after dosing, cholesterogenesis at all sites was increased above the comparable control value, indicating the induction of enzyme synthesis and overall there was little effect on the mass of cholesterol synthesized per day. Similar results were obtained using male chicks. Inhibition of cholesterogenesis by compactin was also observed in cholestyramine-treated rats, in which cholesterol turnover was markedly increased, and even in cholesterol-fed rats, in which cholesterogenesis already was repressed. In neither case, however, was inhibition of cholesterogenesis accompanied by a hypocholesterolaemic effect. It is concluded that a more persistent suppression of cholesterogenesis, than that observed with compactin in the rat, may be required in order to affect serum cholesterol concentrations.

Decrease in LDL and increase in HDL concentrations in type II hyperlipoproteinaemic patients on low-dose combination therapy of cholestyramine and Complamin.

A low-dose combination of Complamin retard (1 g t.i.d.) and cholestyramine (4 g b.i.d.) was compared with each agent alone in 2 serial open trials without dietary restriction using type IIa and IIb hyperlipoproteinaemic patients. Complamin alone produced decreases in LDL and VLDL cholesterol concentrations (up to 20%) whereas cholestyramine alone produced only a modest reduction in LDL (up to 15%). The combination produced marked, progressive reductions in total cholesterol (up to 35%) and LDL (up to 40%); reductions in VLDL (up to 45%), total triglyceride (up to 60%) and free fatty acids (up to 60%) were found only in type IIb patients. The average increase in HDL-cholesterol from the 2 studies for combination therapy was 35%. No side-effects were reported or measured and compliance was excellent. The results demonstrate the potential of a method of achieving beneficial actions on lipoprotein levels with a well-tolerated therapy.

The role of cholesterol 7 alpha-hydroxylase in the hypobetalipoproteinaemic activity of SKF-525A in rats.

Administration of SKF-525A to rats fed on a stock diet specifically decreased the serum concentration of low-density lipoprotein. SKF-525A and cholestryamine also reversed the rise in circulating concentration of both very-low density and low-density lipoprotein that was observed in rats given a sucrose-based, cholesterol-supplemented diet. The enhancement of hepatic cholesterol 7 alpha-hydroxylase by SKF-525A or by cholestyramine is accompanied by homeostatic responses by the liver which include induction of low-density lipoprotein clearance and increased cholesterogenesis to attempt to replenish sterol pools. These compensatory mechanisms are separately controlled.

Studies on the response of cholesterol biogenesis in feeding in rats: evidence against the existence of diurnal rhythms.

1. The biosynthesis of cholesterol was studied, by using various precursors, in rats subjected to several dietary regimes. 2. The use of 3H2O as a substrate to demonstrate differences in cholesterogenesis under various conditions was validated by using rats fed on cholesterol or cholestyramine. Cholesterol feeding resulted in decreased cholesterogenesis, whereas cholestyramine caused an increase. 3. With acetate as precursor, the biosynthesis of both digitonin-precipitable sterol and fatty acids was increased in vitro in response to a meal. 4. In rats fed ad libitum, hepatic cholesterogenesis was increased at midnight relative to mid-morning as measured by using acetate precursor in vitro. However, no such difference was found by using 3H2O in vivo. 5. The lipogenic response was measured in meal-fed rats by using 3H2O or octanoate in vivo. In contrast with findings with acetate in vitro, no postprandial stimulation of cholesterogenesis was seen with either 3H2O or octanoate as precursor, whereas fatty acid biosynthesis from either substrate was increased. 6. These findings are discussed with respect to current theories about the circadian rhythm of cholesterogenesis. Such theories are based on experiments using isolated enzyme measurements or non-physiological precursors such as acetate. 7. It is considered that results obtained with 3H2O give an accurate representation of cholesterogenesis under various conditions, and it is therefore suggested that hepatic cholesterogenesis in rats is not subjected to the same degree of diurnal rhythm as has previously been believed.