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The target organ for HMG-CoA reductase inhibitors to decrease cholesterol biosynthesis in hypercholesterolemic patients is the liver. Since bile acids undergo an enterohepatic circulation showing a strict organotropism for the liver and the small intestine, the structural elements of an inhibitor for HMG-CoA reductase were combined with those for specific molecular recognition of a bile acid molecule for selective uptake by hepatocytes. Either, the HMG-CoA reductase inhibitors HR 780 and mevinolin were covalently attached to 3 xi-(omega-aminoalkoxy)-7 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oic acids to obtain bile acid prodrugs, or the side chain of bile acids at C-17 was replaced by 3,5-dihydroxy-heptanoic acid--a structural element essential for inhibition of HMG-CoA reductase--to obtain hybrid bile acid: HMG-CoA reductase inhibitors. The prodrugs could, as expected, not inhibit rat liver HMG-CoA reductase to a significant extent, whereas the hybrid inhibitors showed a stereospecific inhibition of HMG-CoA reductase from rat liver microsomes with an IC50-value of 0.7 microM for the most potent compound S 2467 and 6 microM for its diastereomere S 2468. Uptake measurements with isolated rat hepatocytes and ileal brush-border membrane vesicles from rabbit small intestine revealed a specific interaction of both classes of bile acid-derived HMG-CoA reductase inhibitors with the hepatocyte and ileocyte bile acid uptake systems. Photoaffinity labeling studies using 3-azi- or 7-azi-derivatives of taurocholate with freshly isolated rat hepatocytes or rabbit ileal brush-border membrane vesicles revealed a specific interaction of bile acid derived HMG-CoA reductase inhibitors with the respective putative bile acid transporters in the liver and the ileum demonstrating the bile acid character of these derivatives, both for the prodrugs and the hybrids. Cholesterol biosynthesis in Hep G2 cells was inhibited by the bile acid prodrugs with IC50-values in the range of 68 nM to 600 nM compared to 13 nM for HR 780 and 130 nM for mevinolin. Among the hybrid inhibitors, S 2467 was the most active compound with an IC50-value of 16 microM compared to 55 microM for its diastereomere S 2468. Preliminary in vivo experiments showed an inhibition of hepatic cholesterol biosynthesis after oral dosage only with prodrugs such as S 3554, whereas the hybrid molecules were inactive after oral application.(ABSTRACT TRUNCATED AT 400 WORDS)

1. 1. Sulphmyoglobin is known to differ from myoglobin in containing one additional S atom per molecule and in its spectrum which is characterized by a strong band at 617 mμ (g3 mM 17.5). 2. 2. Further evidence is brought showing that the additional S atom of sulphmyoglobin is not attached to the Fe atom: since its presence is associated with the unusual spectrum it must therefore be found so as to affect the porphyrin macroring conjugation. 3. 3. Using [ 35 S]sulphmyoglobin it is shown that acetone-HCl frees the additional S atom from both haem and protein moieties. The freed S or S compound is much more soluble in organic solvents than in water. 4. 4. Crystalline protoporphyrin dimethyl ester was prepared in 44% yield from the haematin of sulphmyoglobin. 5. 5. By a comparison of the spectra of sulphmyoglobin derivatives with those of the corresponding derivatives of ferromesochloringlobin it is concluded that the haem of intact sulphmyoglobin has a chlorin-type conjugation. It is also concluded that this conjugation is produced by the addition of S or a compound containing one atom of S across a pyrrole β-β double bond of the protohaenmatin of myoglobin.

The effect of oxidizable substrates, α-ketoglutarate and isocitrate on 11β-hydroxylation in guinea-pig adrenal mitochondria has been studied. These substrates supported the conversion of Compound S (17α,21-dihydroxy-pregnene-3,20-dione) into cortisol as well as exogenously added NADPH in Ca 2+ -swollen mitochondria. Progesterone was also hydroxylated at the C-11β position but not at the C-21 position. Microsomes, on the other hand, when NADPH was added, converted pregnenolone, progesterone and 17α-hydroxyprogesterone into Compound S, but showed no steroid 11β-hydroxylating activity. Evidence obtained in incubations carried out with α-ketoglutarate and isocitrate in the presence of 2,4-dinitrophenol, oligomycin, amytal and antimycin A indicate that α-ketoglutarate utilization for steroid 11β-hydroxylation is dependent on activity of the classical electron chain. This activity can be related to high energy intermediates possibly needed for NADPH reduction arising from NADH oxidation via the energy-requiring transhydrogenase reaction. These reactions do not appear necessary for isocitrate utilization and isocitrate oxidation probably gives rise to intramitochondrial NADPH reduction as a result of a NADP + -linked isocitrate dehydrogenase. Data obtained in oxygen uptake studies with an antimycin A blocked system supplied with α-ketoglutarate, are in accordance with this conclusion. The high-speed supernatant fraction (103000 × g ) could partially replace α-ketoglutarate, isocitrate or Ca 2+ + NADPH, indicating that it contains a factor(s) (the physiological substrate?) which brings about intramitochondrial NADPH.

1. 1. Reichstein's Compound S, when added to a culture of Curvularia lunata, is hydroxylated mainly at the positions 11β and 14α. The nature of the hydroxylation reactions resulting in the simultaneous production of two major metabolites was studied, using cell-free preparations. 2. 2. A preparative procedure was developed which included a partial purification by (NH4)2SO4 precipitation. Consistent activity in cell-free preparations was found to depend on rather exacting conditions during growth of the microorganism and during preparation of the cell-free extract. The more effective of these were induction and the presence of EDTA, GSH and Tween 80 in the extraction medium. 3. 3. With partially purified preparations hydroxylation was NADPH- and O2- dependent; other characteristics were the instability of the preparations, the absence of properties indicating haem or flavin groups, and the activation by complexing agents. 4. 4. No separation of the 11β- and 14α-hydroxylation activities was observed upon further purification by DEAE-chromatography or upon attempts at differential induction or modification of the activities. Of a number of 11β- or 14α-substituted steroids, two were found to be inductors of both activities.

1. 1. Suspensions of Endamoeba histolytica form CO2 and H2S under anaerobic conditions from sugars in the presence of cysteine (or cystine). 2. 2. Evidence was obtained which indicated that glucose is phosphorylated and HDP is fermented to pyruvate which is then decarboxylated. 3. 3. It was shown that during the fermentation of glucose (or HDP in broken cells) triose phosphate is oxidized by cystine (or another ortho substituted aromatic disulfide). The oxidation of the resulting thiols occurs by way of a “labile S compound” (β-thiopyruvate or a derivative of it) which is enzymically desulfurated and the S reduced to H2S.