Azalanstat (RS-21607), a lanosterol 14 alpha-demethylase inhibitor with cholesterol-lowering activity.

Agents that inhibit hepatic cholesterol biosynthesis reduce circulating cholesterol levels in experimental animals and humans, and may be of pharmacological importance in the prevention of atherosclerosis. Azalanstat (RS-21607), a synthetic imidazole, has been shown to inhibit cholesterol synthesis in HepG2 cells, human fibroblasts, hamster hepatocytes and hamster liver, by inhibiting the cytochrome P450 enzyme lanosterol 14 alpha-demethylase. When administered orally to hamsters fed regular chow, RS-21607 (50 mg/kg/day) lowered serum cholesterol in a dose-dependent manner (ED50 = 62 mg/kg) in a period of 1 week. It preferentially lowered low density lipoprotein (LDL) cholesterol and apo B relative to high density lipoprotein (HDL) cholesterol and apo A-1. It also lowered plasma cholesterol levels in hamsters fed a high saturated fat and cholesterol diet. RS-21607 inhibited hepatic microsomal hydroxymethylglutaryl-CoA (HMG-CoA) reductase activity in hamsters in a dose-dependent manner (ED50 = 31 mg/kg), and this was highly correlated with serum cholesterol lowering (r = 0.97). Cholesterol lowering by azalanstat and cholestyramine was additive, and the increase in HMG-CoA reductase brought about by cholestyramine was attenuated significantly by azalanstat. In vitro studies with HepG2 cells indicated that this modulation of reductase activity was indirect, occurring at a post-transcriptional step, and it is proposed that a regulatory oxysterol derived from dihydrolanosterol (or lanosterol) may be responsible for this regulation. Azalanstat does not appear to lower circulating cholesterol in the hamster by up-regulation of the hepatic LDL receptor, suggesting that other mechanisms are involved. Orally administered azalanstat (50-75 mg/kg) stimulated hepatic microsomal cholesterol 7 alpha-hydroxylase activity by 50-400% in hamsters, and it is postulated that this may result from modified cholesterol absorption and bile acid synthesis.

Delayed primary repair of intrathoracic esophageal perforation: is it safe?

The management of intrathoracic esophageal perforation with delayed diagnosis is a subject of controversy. Because of the obvious advantages of primary repair as a simple single-stage operation, this technique was preferentially used to treat 18 of 22 consecutive patients with esophageal perforation. These patients were stratified into three groups according to the time interval between perforation and repair: group A, less than 6 hours, five patients (28%); group B, 6 to 24 hours, six patients (33%); and group C, more than 24 hours, seven patients (39%). Group A patients were older (p < 0.05) and group B had fewer iatrogenic perforations (B, 17%; A, 80%; C, 57%, p < 0.1). Additional tissue was used to buttress the repair site in all three groups (A, 3/5 patients, 60%; B, 4/6 patients, 67%; C, 6/7 patients, 86%; p = not significant). In seven patients (39%), a fundic wrap was used to reinforce the site of primary repair. The outcomes of the three groups were analyzed. Group A had the lowest proportion of postoperative leaks (A, 0/4 patients, 0%; B, 4/6 patients, 67%; C, 5/6 patients, 83%; p < 0.05) and postoperative morbidity (A, 2/5 patients, 40%; B, 6/6 patients, 100%; C, 6/7 patients, 86%; p < 0.1). However the increased incidence of leak and morbidity did not lead to an increase in mortality. One death occurred in each group, with an overall mortality of 17% (A, 1/5 patients, 20%; B, 1/6 patients, 17%; C, 1/7 patients, 14%; p = not significant). We conclude that in the era of advanced intensive care capabilities, primary repair of intrathoracic esophageal perforation can be safely accomplished in most patients regardless of the time interval between perforation and operation. Leakage at the suture site is common unless primary repair is carried out without delay. Postoperative leakage, however, is usually inconsequential and does not necessarily result in an adverse outcome.

Isolation, characterization and quantification of apolipoproteins A-1 and B of the Golden Syrian hamster (Mesocricetus auratus) and modification of their levels by dietary cholesterol.

1. Apolipoprotein A-1, isolated from hamster high density lipoprotein, possessed a molecular weight of approximately 27,000. 2. Its amino acid composition differed from human apo A-1 and it contained a higher threonine to serine ratio and a higher methionine and leucine content. 3. The concentration in normal serum was 126.0 +/- 1.9 mg/dl. 4. Apolipoprotein B, isolated from hamster low density lipoprotein consisted of three major components when analyzed by SDS-polyacrylamide gel electrophoresis with Mrs of 635 Kd, 460 Kd and 305 Kd respectively. 5. Hamster apo B possessed a higher aspartic acid to glutamic acid ratio and a higher methionine and valine content than human apo B. 6. The concentration in normal serum was 20.9 +/- 1.0 mg/dl. 7. The apolipoprotein and lipoprotein profile of hamsters fed a high cholesterol diet for 30 days changed considerably. 8. Total serum cholesterol levels increased 7 fold; LDL levels increased 14 fold; HDL levels doubled and total serum triglyceride increased 3 fold. 9. Apo A-1 levels increased by 45% and apo B levels increased 5 fold.

The active centre of triose phosphate isomerase.

The molecular weight and amino acid composition of triose phosphate isomerase have been determined. The molecular weight (43000) is lower and the molecular activity (500000) higher than those of most other glycolytic enzymes. Reaction with iodoacetate (studied with radioactive reagent) takes place in two phases: in the first phase, at pH6.3, cysteine and methionine groups react and enzymic activity is unimpaired; in the second phase, histidine reacts and enzymic activity is lost. Photo-oxidation leads to inactivation, with loss of cysteine, of histidine and of tryptophan, but little loss of tyrosine. The mechanism postulated for the action of the enzyme demands the intervention of a group functioning as a base, and the results obtained are consistent with histidine's being the basic group in the active centre.

Studies on the sub-units of triose phosphate isomerase.

The sub-unit structure of rabbit muscle triose phosphate isomerase was studied by determination of the number of unique cysteine peptides. Alkylation of the thiol groups with radioactive iodoacetate in the presence of guanidine hydrochloride gave the S-carboxy[(14)C]methyl derivative of the protein. This was digested with trypsin, and the radioactive peptides were fractionated by ion-exchange chromatography; four main radioactive peaks were obtained, one of which contained two radioactive peptides. Peptide ;maps' of the tryptic digest showed five main spots. The relationship between the members of both sets of five peptides was established. The radioactive peptides were characterized, and the results indicated the presence of five unique cysteine residues in the protein. Since there are approximately ten thiol groups/molecule, there are two closely related or identical sub-units. Studies of the terminal residues bear out this suggestion; only one kind of N-terminal residue (alanine) and one kind of C-terminal residue (glutamine) were detected. These results are in accord with the evidence from crystallography.

The effects of homogeneous human prealbumin on in vitro and in vivo immune responses in the mouse.

A highly purified preparation of human prealbumin was shown to potentiate the sensitivity of rosette spleen forming cells of adult thymectomized mice to azathioprine in vitro and in vivo and to induce the appearance of the Thy 1, 2 antigen in vitro on spleen cells of adult thymectomized mice. Prealbumin also enhanced IgM antibody synthesis to sheep red blood cells (SRBC) in vitro in 12 week old mice and in vivo in aged (45 - 58 week old) and nude (nu/nu) mice. In vivo administration, to mice that had been pre-treated with hydrocortisone, resulted in a decrease in the specific activity of thymocyte terminal deoxynucleotidyl transferase. The data indicate that the prealbumin molecule possesses immunopotentiating properties in a number of in vitro and in vivo immunocompromised murine models and that the immuno-enhancing properties of the partially purified preparation previously described were in fact due to the prealbumin component and not to other contaminating proteins.

Immuno-enhancing activity of the amino-terminal domain of human prealbumin: isolation, characterization and synthesis.

A decapeptide isolated from highly purified preparations of human prealbumin was able to restore azathioprine (Az) sensitivity, a property of a sub-class of T-lymphocytes, to the spleen rosette-forming cells (RFC) of adult thymectomized (ATx) mice in vitro. The peptide was sequenced by the Edman method and shown to correspond to the ten amino-terminal residues of prealbumin, Gly-Pro-Thr-Gly-Thr-Gly-Glu-Ser-Lys-Cys. Synthesis of this peptide by solid phase methodology confirmed its activity both in vitro and in vivo. Synthesis of a number of structural analogues indicated that the amino-terminal deca, undeca and dodecapeptides of prealbumin as well as some of their derivatives were also able to restore Az sensitivity to RFC in vitro and in vivo. The Cys10 residue and the Glu7 residues both contributed significantly to potency in vitro. Removal of up to three amino acids from the N-terminus of the decapeptide led to a progressive loss of activity. The data indicates that the ability of human prealbumin to restore the Az sensitivity to the RFC of adult Tx mice is intrinsic to the protein and resides in the amino-terminal domain of the molecule.

Selective inhibition of mammalian lanosterol 14 alpha-demethylase by RS-21607 in vitro and in vivo.

The discovery of selective lanosterol 14 alpha-demethylase inhibitors may lead to novel hypolipidemic drugs. RS-21607, (2S,4S)-cis-2[1H-imidazol-1-yl)methyl]-2-[2-(4-chlorophenyl)ethyl]-4- [[(4-aminophenyl)thio]methyl]-1,3-dioxolane, was characterized as a tight-binding, competitive inhibitor of lanosterol 14 alpha-demethylase purified from rat liver. The apparent Ki was determined to be 840 pM and found to be similar in hepatic microsomes from human, rat, and hamster. RS-21607, which contains two chiral centers, was a more effective lanosterol 14 alpha-demethylase inhibitor than its three stereoisomers. In vitro, RS-21607 had a greater affinity for lanosterol 14 alpha-demethylase than the other cytochromes P450 evaluated: CYP7, CYP27, CYP11A1, CYP19, CYP17, CYP11B1, CYP21, CYP3A4, CYP4A, CYP2D6, CYP1A2, CYP2C9, and 27-hydroxycholesterol 7 alpha-hydroxylase. The other stereoisomers were not as selective as RS-21607. Doses of 3-30 mg/kg RS-21607 given orally to hamsters caused a dose-dependent decrease in cholesterol biosynthesis with a corresponding accumulation of 24,25-dihydrolanosterol. RS-21607 inhibited the enzyme and cholesterol biosynthesis in hamster liver by 50% at 18 h following a 30 mg/kg oral dose. This was interpreted to indicate that RS-21607 is able to distribute to the site of action in hamsters and inhibit the target enzyme. In the same dose range, the plasma concentrations of testosterone, corticosterone, and progesterone, the endpoints for the cytochromes P450 involved in steroid biosynthesis, were relatively unaffected. These data show RS-21607 to be an effective and selective inhibitor of lanosterol 14 alpha-demethylase, both in vivo and in vitro. RS-21607 interacted with the purified enzyme to produce a type II binding spectrum, consistent with an interaction between the imidazole moiety and the heme. The electrostatic contribution of the imidazole binding was investigated using the desimidazole analog of RS-21607. The apparent Ki for the desimidazole compound (65 microM) was similar to the apparent Km for the substrate DHL (79 microM). Together, these data confirm that the ligand attached to the imidazole in RS-21607 is a good non-sterol substitute for DHL, i.e., binding to the enzyme with similar affinity, and that the coordination of the imidazole to the heme provides a major electrostatic contribution for the inhibition of lanosterol 14 alpha-demethylase by RS-21607. RS-21607 was also observed to increase the accumulation of 3 beta-hydroxy-24,25-dihydrolanost-8-en-32-al, the second intermediate in the multistep oxidation, but not the first intermediate. 24,25-dihydrolanost-8-ene-3 beta,32-diol.(ABSTRACT TRUNCATED AT 400 WORDS)