Efficacy of once-daily extended-release lovastatin as compared to immediate-release lovastatin in patients with hypercholesterolemia.

OBJECTIVE: to compare the efficacy and safety of 20 mg of lovastatin when administered once daily as an extended-release (ER) tablet or as an immediate-release (IR) tablet. RESEARCH DESIGN AND METHODS: Male or female patients aged 21-70 years with hypercholesterolemia who provided written informed consent and met the inclusion criteria were screened. A total of 179 patients were enrolled: 100 male and 79 female; 153 were Caucasian, eight Black and 18 other races; the mean age was 56 years. Patients were generally in good health as evidenced by medical history, physical and laboratory examination. Patients were required to not exceed specific low-density lipoprotein cholesterol (LDL-C) levels depending on their risk category. The trial was conducted as a multi-center, randomized, double-blind, positive-controlled, double-dummy, two-way crossover study. Patients were washed-out of any prior lipid-lowering medications (period 1) and then received one ER or one IR lovastatin tablet for 12 weeks (period 2) and then washed out with placebo for 6 weeks (period 3). They then received the alternate treatment for an additional 12 weeks (period 4). MAIN OUTCOME MEASURES: The primary efficacy variable was the combined mean percent change in LDL-C from baseline to endpoint for periods 2 and 4. Secondary variables included the mean percent change from baseline in high-density lipoprotein cholesterol (HDL-C), total cholesterol (TC), and triglycerides (TG) for periods 2 and 4 combined. Least-square mean differences between ER and IR treated groups were estimated at both treatments. All tests were two-sided and a p-value of < 0.05 was considered statistically significant. RESULTS: Both ER and IR lovastatin tablets produced statistically significant changes in the lipid profile from baseline. Differences in HDL-C (4.1% and 4.3% for ER and IR, respectively) and TG (7.4% and 10.4% for ER and IR, respectively) were not significant between treatments. TC (19.1% and 17.2% for ER and IR, respectively) and LDL-C (26.4% and 23.1% for ER and IR, respectively) were also reduced significantly from baseline by both treatments. However the ER lovastatin reduced TC by an additional 1.9% (p = 0.0355) and LDL-C by a further 3.3% decrease (p = 0.0028) as compared to the IR formulation. The increase in LDL-C efficacy is equivalent to an increase of 50% in the dose of IR lovastatin, suggesting that 20 mg ER is equivalent to about 30 mg IR in LDL-C-lowering capacity. No apparent difference in the safety profile between the two formulations was noted. CONCLUSIONS: The data show that 20 mg of ER lovastatin was about one and one-half times as effective at lowering LDL-C than the same dose of IR lovastatin. Both regimens were tolerated well.

Treatment with controlled-release lovastatin decreases serum concentrations of human beta-amyloid (A beta) peptide.

The deposition of beta-amyloid (A beta) in neuronal plaques is believed to be crucial for the initiation and progression of Alzheimer's disease (AD). Studies in vitro have shown that inhibiting cholesterol metabolism with lovastatin, or its active metabolite lovastatin acid, lowers A beta production. To examine the effects of lovastatin on A beta in vivo, human subjects who had elevated low-density lipoprotein cholesterol were treated during a double-blind, randomized, placebo-controlled study with 10, 20, 40 or 60 mg once-daily doses of a controlled-release formulation of lovastatin, or matching placebo. Serum A beta concentrations were measured before and after up to 3 months of treatment. Mean and median changes from baseline in serum A beta concentrations showed a dose-dependent decrease, and analysis of variance indicated that treatment was statistically significant (p < 0.0348). Differences between the 40- and 60-mg dose groups and placebo were statistically significant (Dunnett's p < or = 0.05).

Posttranslational processing of transfected mouse pro-adrenocorticotropin/endorphin in rat growth hormone-secreting tumor cells.

Pro-ACTH/endorphin (PAE; also POMC) is a hormone precursor that contains potential sites for endoproteolytic cleavage, glycosylation, phosphorylation, acetylation, sulfation, and amidation. Different sets of these sites are used in different neuroendocrine tissues. To learn more about the factors influencing the posttranslational processing of peptide hormone precursors, the cDNA for mouse PAE was cloned into a metallothionein expression vector. Two lines of rat GH-secreting tumor cells (GH3 and GC) were transfected with the resulting expression plasmid, and stable PAE-producing clones were isolated. Both GH3 and GC cells gave rise to clones in which about 75% of PAE was endoproteolytically processed. Mol wt distributions of PAE peptides were similar in cell extracts and medium, indicating that processing was not the result of extracellular proteases. The identity of immunoprecipitated molecules was confirmed by analysis of tryptic digests by reverse phase HPLC. Amidated forms of joining peptide were detected in the transfected cells by immunocytochemistry, adding evidence that GH-producing tumor cells can perform endoproteolysis, exoproteolysis, and alpha-amidation of the PAE precursor. One of the two forms of joining peptide from cell extracts was also shown to comigrate during reverse phase HPLC with authentic alpha-amidated mouse joining peptide. Comparisons of Northern analyses and peptide synthetic rates suggested that PAE mRNA was used for synthesis of PAE more efficiently in corticotropes than in the transfected GC cells.

Membrane-associated forms of peptidylglycine alpha-amidating monooxygenase activity in rat pituitary. Tissue specificity.

Membrane-associated peptidylglycine alpha-amidating monooxygenase (PAM) activity was investigated in rat anterior and neurointermediate pituitary tissues and in pituitary AtT-20/D-16v and GH3 cell lines. A substantial fraction of total pituitary PAM activity was found to be membrane-associated. Triton X-100, N-octyl-beta-D-glucopyranoside, and Zwittergent were effective in solubilizing PAM activity from crude pituitary membranes. The distribution of enzyme activity between soluble and membrane-associated forms was tissue-specific. In the anterior pituitary lobe and pituitary cell lines, 40-60% of total PAM activity was membrane-associated while only 10% of the alpha-amidating activity in the neurointermediate lobe was membrane-associated. Soluble and membrane-associated forms of PAM shared nearly identical characteristics with respect to copper and ascorbate requirements, pH optima, and Km values. Upon subcellular fractionation of anterior and neurointermediate pituitary lobe homogenates on Percoll gradients, 12-18% of total PAM activity was found in the rough endoplasmic reticulum/Golgi fractions and 42-60% was localized to secretory granule fractions. For both tissues, membrane-associated PAM activity was enriched in the rough endoplasmic reticulum/Golgi pool, whereas most of the secretory granule-associated enzyme activity was soluble.

Biosynthesis of amidated joining peptide from pro-adrenocorticotropin-endorphin.

Joining peptide is the major alpha-amidated product of pro-ACTH/endorphin (PAE) in AtT-20 corticotropic tumor cells. To study intracellular joining peptide synthesis, affinity purified antibodies directed against gamma-MSH, joining peptide, and ACTH were used to immunoprecipitate extracts from biosynthetically labeled AtT-20 cells. Immunoprecipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by tryptic peptide mapping on HPLC. In steady labeling experiments, radioactivity in amidated joining peptide (JP) increased roughly linearly with time, in the manner of a final product, whereas radioactivity associated with PAE (1-94)NH2 reached a constant value after 2-4 h, indicating that PAE(1-94)NH2 is an intermediate in the biosynthesis of JP. Radioactivity appeared in ACTH(1-39) well before JP, consistent with a cleavage order in which ACTH is cleaved from PAE(1-95) before JP sequences are cleaved from PAE(1-74). This conclusion was supported by tryptic peptide analyses of immunoprecipitates, which indicated that less than 5% of JP-related material is cleaved from PAE(1-74) before being cleaved from ACTH-related sequences. After a pulse label, radioactivity in PAE(1-94)NH2 reached a peak value after 1 h of chase and declined with a half-life of less than 1 h. Amidated JP increased to a constant level after 2 h of chase. Enough radiolabeled PAE(1-94)NH2 was detected to account for about half of the radioactivity found in amidated JP, indicating that about half of JP-related material is first cleaved from PAE(1-95) before being amidated. This result was corroborated using HPLC purification to determine both amidated and glycine-extended forms of JP.

The role of secretory granules in peptide biosynthesis.

There are many events in the posttranslational processing of bioactive peptides that occur in secretory granules and not to any great extent in other cellular organelles and that do not appear as modifications of the structure of many conventional neurotransmitters. In addition, at least two very important steps are unique to peptide-containing granules: (1) the peptides must begin their trek to the secretory granule in the RER as a larger precursor, rather than being taken up as a finished or nearly finished product into a mature granule; (2) there is at least one crucial sorting step on the way from the RER to the secretory granule that must occur faithfully before the peptide correctly appears in the granule. As for small molecules such as the catecholamines, the posttranslational processing enzymes and any required cofactors must also be put into the granules if the final events of processing are to occur with fidelity. Many of the posttranslational processing enzymes are only beginning to be identified. It is clear from these studies on purified PAM and peptide alpha-amidation as it occurs in cells that correlating test tube studies with the functioning of secretory granules is a worthwhile, if difficult, pursuit. The unique milieu inside the granule is difficult to mimic in a test tube. Transfection of peptide-producing cells with cDNAs encoding precursors with specific alterations in processing sites offers perhaps the best way to interface the studies of secretory granules and the posttranslational processing enzymes that mediate those functions.

Transport and stability of ascorbic acid in pituitary cultures.

Ascorbic acid uptake in AtT-20 tumor cells and primary cultures of rat anterior and intermediate pituitary was sodium-dependent and showed half-maximal saturation between 9 and 18 microM ascorbate. When incubated in [14C]ascorbic acid at concentrations similar to those in serum (50 microM), all of the cells concentrated ascorbate 20- to 40-fold, producing intracellular ascorbate concentrations of 1-2 mM. HPLC analyses showed that over 90% of the intracellular label comigrated with authentic ascorbic acid. Although ascorbate was rapidly oxidized in culture medium in the absence of cells, incubation of ascorbate in culture medium in the presence of cells stabilized the ascorbate substantially. Unlike systems that transport dehydroascorbic acid, the ascorbate transport systems in all three preparations were not inhibited by glucose. Thus all three systems possess similar saturable, high-affinity, sodium-dependent active transport systems for ascorbic acid.

An artifactual component of drug-protein binding generated in vitro.

In the course of investigating the binding of imipramine to soluble cellular fractions from brain and leukocytes using equilibrium dialysis, an artifactual binding component was produced. On Scatchard and double-reciprocal plots of the data, the component appeared as a homogenous population of sites displaying a binding affinity of 170 nM. The obtained pattern of biphasic interaction bore a marked resemblance to reported Scatchard plots representing the interaction of drugs with bovine serum albumin, and depicting two components of widely differing binding affinity and capacity. The artifact occurred when solutions were transferred after dialysis and before quantitation to intermediate containers, and resulted from binding of 3H-imipramine to the walls of these containers. The latter interaction decreased the concentration of radiolabeled drug in the dialysate but not in the dialyzed solution, and thus mimicked increased imipramine binding to the biological material under study. The effect was particularly pronounced at low drug concentrations, and was particularly pronounced at low drug concentrations, and was prevented by the presence of either proteinaceous material, or of an excess of another basic compound such as methadone. The concentration dependence of the phenomenon led to its appearance as a discrete binding component. The artifact was eliminated either by applying an appropriate correction factor, or by transferring the dialyzed solutions directly into scintillation vials for counting.