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.

Insulin antagonises the growth hormone-mediated increase in the activity of phosphatidate phosphohydrolase in isolated rat hepatocytes.

Rat hepatocytes were incubated in monolayer culture, under serum-free conditions for 8 h. Rat growth hormone (up to 100 nM) increased the activity of phosphatidate phosphohydrolase by up to 47%. Insulin (500 pM or 35 nM), cycloheximide or actinomycin D reversed this effect. The ability of growth hormone to modify the effects of insulin is discussed in relation to the control of the phosphohydrolase activity and glycerolipid synthesis.

Spermine antagonises the effects of dexamethasone, glucagon and cyclic AMP in increasing the activity of phosphatidate phosphohydrolase in isolated rat hepatocytes.

Rat hepatocytes were incubated in monolayer culture, under serum free conditions, for 8 h. Glucagon (10 nM), 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (100 microM) and dexamethasone (100 nM) increased the activity of phosphatidate phosphohydrolase by approx. 2-, 3.6- and 3.3-fold, respectively. Spermine alone had no significant effect. Spermine (2.5 mM) almost completely inhibited the glucagon induced increase in phosphohydrolase activity. It only partially inhibited the dexamethasone and cyclic AMP mediated inductions. Spermidine had no significant effect in this respect. The results are discussed in relation to the known effects of polyamines on glycerolipid synthesis, in particular, and on intermediary metabolism.

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.

Hypolipidemic analogues of ethyl 4-benzyloxybenzoate.

A series of compounds related to ethyl 4-benzyloxybenzoate was synthesized and evaluated for potential hypolipidemic activity in rats. Structure--activity relationships are discussed in terms of cholesterol-lowering activity together with effects on weight gain and liver lipids. A number of the compounds inhibited cholesterol and free fatty acid biosynthesis from [1-14C]acetate in rat liver slices in vitro. Ethyl 4-benzyloxybenzoate, ethyl-4-benzyloxybenzoic acid, ethyl 4-p-bromobenzyloxybenzoates, and 4-o-methoxybenzyloxyphenyl acetate exhibited the most favorable spectrum of activity.

Isolation and preliminary characterisation of active B-chain of recombinant tissue-type plasminogen activator.

Purified 2-chain recombinant tissue-type plasminogen activator (t-PA) was reduced under mild conditions - 10 mM dithiothreitol/5 degrees C/1.5 h - and the two chains were separated by chromatography on lysine Sepharose. The t-PA B chain was fully active as determined by its activity towards the chromogenic substrate S-2288 (H-D-ile-pro-arg p-nitroanilide). Analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis under reducing or non-reducing conditions revealed a single polypeptide at Mr = 35,000 or 29,000 respectively. In addition, under non-reducing conditions a fibrinolytic band at apparent Mr = 29,000 was present after fibrin zymography. The N-terminal sequence was confirmed as ile-lys-gly. The t-PA B chain had a specific amidolytic activity, using S-2288, of 170,000 to 210,000 SU/mg protein. (This compares to a specific activity of the native 2-chain t-PA of 170,000 SU/mg). It resembles urokinase-type plasminogen activator in its inability to be stimulated by fibrin and its dose response on human fibrin plates. However, t-PA B-chain was stimulated to almost the same extent as t-PA by poly-D-lysine. The isoelectric points, at pH 5.6 and 5.7, fall outside the range generally quoted for t-PA preparations (pH 7.8-8.8).

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.

Effects of novel bile salts on cholesterol metabolism in rats and guinea-pigs.

Novel bile salts (quaternary ammonium conjugates) inhibited cholic acid binding and transport in everted ileal sacs in vitro. The cationic piperazine conjugate of lithocholic acid (di-iodide salt, compound 8, BRL 39924A) appeared most active, inhibiting binding by 29% and transport by 59% in guinea-pig ileum (200 microM). BRL 39924A also inhibited taurocholate uptake into guinea-pig ileal sacs and cholate uptake into rat ileal sacs and was selected for further study in vivo. In hyperlipidaemic rats, BRL 39924A significantly raised cholesterol 7 alpha-hydroxylase activity and decreased hepatic accumulation of exogenous cholic acid. HDL cholesterol concentration in the serum increased and the level of VLDL plus LDL cholesterol decreased. In hyperlipidaemic guinea-pigs. BRL 39924A lowered serum total cholesterol and triglyceride levels. Although metabolic changes were less than those achieved with the bile acid sequestrant, cholestyramine, the doses of BRL 39924A used were much lower (100-500 mg/kg body wt). Selective inhibition of receptor mediated bile acid uptake may be associated with local side-effects but these novel bile salts are useful pharmacological tools to examine the effects of receptor blockade on lipoprotein metabolism.

Hyperalphalipoproteinaemic activity of BRL 26314--I. Enhanced cholesterol turnover in rats.

Administration of BRL 26314 [N-(4-chlorobenzyl)-L-phenylalanine] raises circulating high-density lipoprotein (HDL) cholesterol and lowers total triglyceride levels in rats whether maintained on stock or semi-synthetic diets. HDL is also elevated by BRL 26314 in hypothyroid rats and in rats with pre-existing hyperlipidaemia where aortic total cholesterol concentration is decreased. BRL 26314 promotes the excretion of a dose of radiolabelled cholesterol as faecal sterols and bile acids, and decreases the extent of cholesterol-radiolabelling in tissue pools, particularly the aorta and adipose tissue. The increase in cholesterol and bile acid (cholic acid) turnover distinguishes BRL 26314 from a cholestatic agent such as 1-naphthyl isothiocyanate where a superficially similar change in HDL concentration disguises an impaired cholesterol transport. BRL 26314 is not a general protein inducer but part of the mechanism of action may involve enhancement of white adipose tissue lipoprotein lipase activity.

Hyperalphalipoproteinaemic activity of BRL 26314--II. Inhibition of atherosclerosis in rabbits.

Optimization of a combination of balloon catheter-induced aortic de-endothelialization with provision of a palatable atherogenic diet to rabbits leads to hyperbetalipoproteinaemia and atherosclerosis rather than to the cholesterol-storage disease which characterized earlier models. Administration of BRL 26314 [N-(4-chlorobenzyl)-L-phenylalanine] during the induction of atherosclerosis specifically raised high-density lipoprotein (HDL) and decreased the arterial content of cholesterol and collagen in association with reduction in severity of thoracic sudanophilic lesions and intimal-thickening. This anti-atherosclerotic activity was superior to that observed for various standard compounds, and the present studies, using BRL 26314 as a pharmacological tool, provide evidence in vivo for an association between the elevation of HDL and reduction of arterial disease.

Comparison of the effects of streptokinase, t-PA and APSAC on human platelet aggregation in vitro in the absence and presence of aspirin.

SK, t-PA or APSAC were incubated in human plasma (adjusted to 300,000 platelets/mm3), in vitro, for up to 90 minutes using concentrations which were equivalent to those achieved in the treatment of AMI patients. Aggregation was measured in response to ADP and collagen. SK inhibited platelet aggregation after a 60 minute incubation. t-PA was less inhibitory and significant effects were only achieved on extended incubation with a higher concentration of activator. APSAC markedly inhibited platelet aggregation in response to both ADP and collagen and the inhibition was achieved earlier than with SK. The difference in temporal response between APSAC and SK was not attributed to differences in systemic plasminogen activation. There was no influence of anti-SK antibody (IgG) on the platelet function response to APSAC or SK. Aspirin inhibited second phase aggregation induced by ADP but even in the presence of aspirin, the net inhibition of platelet aggregation was greater for APSAC than for SK. This marked effect of APSAC on platelet aggregation helps to explain the high initial patency and low re-occlusion rates seen when APSAC is administered to AMI patients.

Induction of a sustained fibrinolytic response by BRL 26921 in vitro.

The role of thrombus-binding in the fibrinolytic response to the acylated streptokinase.plasminogen activator complex, BRL 26921, has been examined using human plasma clots, radiolabelled with 125I-fibrin, in vitro. When clots were briefly exposed to BRL 26921, washed and returned to homologous plasma, lysis continued for up to 3 hours and attained approximately 25% of that lysis achieved by incubating with BRL 26921 for 5 hours. This continuing lysis was potentiated by return of exposed clots to alpha 2-antiplasmin-depleted plasma, or buffer and is attributed to an initial uptake of BRL 26921 rather than the binding of exogenous plasmin that was observed for streptokinase and high concentrations of urokinase. The sustained lysis is not explained by transfer of loosely-associated surface material or by dissociation of agent from the clot with reuptake from a dilute systemic pool. The response can be attributed, at least in part, to specific fibrin binding, mediated by kringles 1-4, for a low-molecular weight plasminogen (Val442) variant was less active.

Clinical effects and kinetic properties of intravenous APSAC--anisoylated plasminogen-streptokinase activator complex (BRL 26921) in acute myocardial infarction.

Fifty patients with a first myocardial infarction presenting within 4 hours of the onset of symptoms were treated with intravenous anisoylated plasminogen-streptokinase activator complex (APSAC-BRL 26921). Vessel patency with good flow was documented in 88%. The left ventricular ejection fraction declined with the duration of symptoms before treatment (r = -0.53, P less than 0.001). The correlation persisted for the group with anterior infarction (r = -0.46, P less than 0.05) where the mean left ventricular ejection fraction prior to discharge from hospital was 36 +/- 9% compared to 49 +/- 7% for the group with inferior infarction. Reinfarction developed in 12% and mortality at 6 months for the whole group was 6%. A degree of systemic fibrinolysis did occur with a fall in mean plasma fibrinogen from 3.20 g/l to 1.08 g/l. A pharmacokinetic study was performed in six patients demonstrating a clearance half-life of fibrinolytic activity of 87.5 +/- 5.0 min. APSAC is an effective intravenous thrombolytic agent with a relatively long half-life of fibrinolytic activity.

Articulating the value of health research.

Previous contributions to Quarterly Journal of Medicine have drawn attention to the work of FEAM, the Federation of European Academies of Medicine, in collaboration with others, in exploring and explaining the issues that will ensure an appropriate European Union (EU) policy framework for health research and innovation. In this article, we present a proposal for an archive of important research conducted in the EU that will act as a resource for illustrating and guiding the development of the necessary regulatory framework.