Regulatory considerations for preclinical development of anticancer drugs.

The entry of new anticancer treatments into phase I clinical trials is ordinarily based on relatively modest preclinical data. This report defines the battery of preclinical tests important for assessing safety under an Investigational New Drug application (IND) and outlines a basis for extrapolating starting doses of investigational anticancer drugs in phase I clinical trials from animal toxicity studies. Types of preclinical studies for the support of marketing of a new anticancer drug are also discussed. This report addresses differences and similarities in the preclinical development of cytotoxic drugs (including photosensitizers and targeted delivery products), drugs used chronically (chemopreventive drugs, hormonal drugs, immunomodulators), and drugs intended to enhance the efficacy (MDR-reversing agents and radiation/chemotherapy sensitizers) or diminish the toxicity of currently used anticancer therapies. Factors to consider in the design of preclinical studies of combination therapies, alternative therapies, and adjuvant therapies in the treatment of cancer, and to support changes in clinical formulations or route of administration, are also discussed.

Long term regulation of glycogen metabolizing enzymes by insulin in H4 hepatoma cells.

Insulin alone at concentrations of less than 1 to 5 uU/ml increased the enzyme activities of glycogen synthase, synthase phosphatase, phosphorylase, and phosphorylase phosphatase in hepatoma H4 cells in culture in the presence and absence of serum. Increase in total and active forms of glycogen synthase and phosphorylase were observed. Cycloheximide blocked the action of insulin on glycogen synthase, glycogen synthase phosphatase and phosphorylase phosphatase. The enzymes with the exception of glycogen synthase phosphatase were expressed with greater hormonal sensitivity in the absence as compared to the presence of serum in terms of hormone concentration required and or time of onset. These results demonstrate that these glycogen metabolizing enzymes are under long term control by insulin, with glycogen synthase being the most sensitive of the enzymes studied to the action of the hormone.

Dichloroacetate inhibits glycolysis and augments insulin-stimulated glycogen synthesis in rat muscle.

The decrease in plasma lactate during dichloroacetate (DCA) treatment is attributed to stimulation of lactate oxidation. To determine whether DCA also inhibits lactate production, we measured glucose metabolism in muscles of fed and fasted rats incubated with DCA and insulin. DCA increased glucose-6-phosphate, an allosteric modifier of glycogen synthase, approximately 50% and increased muscle glycogen synthesis and glycogen content greater than 25%. Lactate release fell; inhibition of glycolysis accounted for greater than 80% of the decrease. This was associated with a decrease in intracellular AMP, but no change in citrate or ATP. When lactate oxidation was increased by raising extracellular lactate, glycolysis decreased (r = - 0.91), suggesting that lactate oxidation regulates glycolysis. When muscle lactate production was greatly stimulated by thermal injury, DCA increased glycogen synthesis, normalized glycogen content, and inhibited glycolysis, thereby reducing lactate release. The major effect of DCA on lactate metabolism in muscle is to inhibit glycolysis.

Specific defects in insulin-mediated muscle metabolism in acute uremia.

The mechanisms underlying the abnormal insulin-mediated muscle glucose metabolism occurring in acute uremia (ARF) have not been identified. To characterize the defects, insulin dose-response curves for glucose uptake, glycogen synthesis, glucose oxidation, glycolysis, and lactate release were measured in incubated rat epitrochlearis muscles. ARF did not affect insulin sensitivity, but decreased the responsiveness to insulin of glucose uptake, glycogen synthesis, and glucose oxidation. Glycogen synthesis was subnormal at all levels of insulin and at the maximal insulin concentration; it was 54% lower in muscles of ARF compared to control rats. This inhibition of glycogen synthesis in ARF could be caused by a 23% decrease in the total activity of muscle glycogen synthase and the percentage of enzyme in the activated form. Glycogen phosphorylase activity was unchanged by ARF. ARF also increased the ratio of muscle lactate release to glucose uptake at concentrations of insulin from 10 to 10(4) microU/ml. In the absence and presence of insulin, muscle protein degradation was increased by ARF. In individual muscles incubated with insulin, the rate of proteolysis was correlated with the ratio of lactate release to glucose uptake (r = + 0.82; P less than 0.01). From the insulin dose-response relationships and changes in enzyme activities, we conclude that ARF increases protein degradation in muscle and causes abnormal insulin-mediated glucose metabolism. The abnormalities in glucose metabolism are caused by changes in post-receptor events.

Precocious development of uridine diphosphate glucuronosyltransferase activity during organ culture of foetal rat liver in the presence of glucocorticoids.

1. Precocious development of mammalian UDP-glucuronosyltransferase (EC 2.4.1.1.7) induced by endogenous compounds of known chemical composition is reported for the first time. 2. This development occurs in cultured explants of foetal rat liver when exposed to corticosteroids possessing a pregn-4'-ene structure and a hydroxy or an oxo group at C-11. 3. Explants from 14-day foetuses cultured for 3 days in a chemically defined medium containing dexamethasone exhibited transferase activities towards o-aminophenol within adult male values. Those liver transferase activities attained in utero by 17 days were still negligible. 4. Evidence from several approaches indicated that the explants required glucocorticoids for expression of the transferase, not for maintenance of viability. 5. Glucocorticoid-dependent stimulation of transferase activity required incorporation of L-[14C]leucine into protein, as judged from the pulsing of cultures with cycloheximide. 6. The relevance of these culture experiments to the situation in vivo is discussed.

Preparation of immobilized baker's-yeast glucose 6-phosphate dehydrogenase attached to modified sepharose and sephadex and a comparison of the properties of these preparations with those of the soluble enzyme.

1. Glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate-NADP+ oxidoreductase, EC 1.1.1.49) from baker's yeast (Saccharomyces cerevisiae) was immobilized on CNBr-activated Sepharose 4B with retention of about 3% of enzyme activity. This uncharged preparation was stable for up to 4 months when stored in borate buffer, pH7.6, at 4 degrees C. 2. Stable enzyme preparations with negative or positive overall charge were made by adding valine or ethylenediamine to the CNBr-activated Sepharose 4B 30min after addition of the enzyme. 3. These three immobilized enzyme preparations retained 40-60% of their activity after 15 min at 50 degrees C. The soluble enzyme is inactivated by these conditions. 4. The soluble enzyme lost 45 and 100% of its activity on incubation for 3h at pH6 and 10 respectively. The three immobilized-enzyme preparations were completely stable over this entire pH range. 5. The pH optimum of the positively and negatively charged immobilized-enzyme preparations were about 8 and 9 respectively. The soluble enzyme and the uncharged immobilized enzyme had an optimum pH at about 8.5 6. Glucose 6-phosphate dehydrogenase immobilized on CNBr-activated Sephadex G-25 was unstable, as was enzyme attached to CNBr-activated Sepharose 4B to which glycine, asparitic acid, valine or ethylenediamine was added at the same time as the enzyme.

Kinetic mechanism from steady-state kinetics of the reaction catalysed by baker's-yeast glucose 6-phosphate dehydrogenase in solution and covalently attached to sepharose.

1. The reaction catalysed by glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate-NADP+ oxidoreductase, EC 1.1.1.49) from baker's yeast was studied in 42mM-glycylglycine buffer, pH7.4 at 25 degrees C, by initial-velocity studies and by the use of NADPH as a product inhibitor. 2. The reactions catalysed by both the soluble enzyme and a stable enzyme covalently attached to CNBr-activated Sepharose 4B probably follow an ordered reaction mechanism with NADP+ and NADPH as the leading reactants. 3. The kinetic constants obtained for the soluble enzyme lere: KNADP+m, 19 muM; KNADP+s, 23 muM; KNADPHs, 15 muM. Similar values were obtained for the immobilized enzyme. 4. The assay of the immobilized enzyme was done by using a micro packed-bed recirculation reactor, and the advantages of this technique are discussed.