In the search for specific inhibitors of human 11beta-hydroxysteroid-dehydrogenases (11beta-HSDs): chenodeoxycholic acid selectively inhibits 11beta-HSD-I

Abstract

Objective: Selective inhibitors of 11b-hydroxysteroid-dehydrogenase type I may be of therapeutical interest for two reasons: i) 9a-Fluorinated 11-dehydrosteroids like 11-dehydro-dexamethasone (DH-D) are rapidly activated by human kidney 11b-hydroxysteroid-dehydrogenase type II (11b-HSD-II) to dexamethasone (D). If the same reaction by hepatic 11b-HSD-I could be selectively inhibited, DH-D could be used for selective renal immunosuppressive therapy. ii) Reduction of cortisone to cortisol in the liver may increase insulin resistance in type 2 diabetes mellitus, and inhibition of the enzyme may lead to a decrease in gluconeogenesis.Therefore, we characterized the metabolism of DH-D by human hepatic 11b-HSD-I and tried to find a selective inhibitor of this isoenzyme. Methods: For kinetic analysis of 11b-HSD-I, we used microsomes prepared from unaffected parts of liver segments, resected because of hepatocarcinoma or metastatic disease. For inhibition experiments, we also tested 11b-HSD-II activity with human kidney cortex microsomes. The inhibitory potency of several compounds was evaluated for oxidation and reduction in concentrations from 10 ¹9 to 10 ¹5 mol/l. Results: Whereas D was not oxidized by human liver microsomes at all, cortisol was oxidized to cortisone with a maximum velocity (V max ) of 95 pmol/mg per min. The reduction of DH-D to D (V max = 742 pmol/ mg per min, Michaelis-Menten constant (K m ) = 1.6 mmol/l) was faster than that of cortisone to cortisol (V max =187 pmol/mg per min). All reactions tested in liver microsomes showed the characteristics of 11b-HSD-I: K m values in the micromolar range, preferred cosubstrate NADP(H), no product inhibition. Of the substances tested for inhibition of 11b-HSD-I and -II, chenodeoxycholic acid was the only one that selectively inhibited 11b-HSD-I (IC 50 for reduction: 2.8 × 10 ¹6 mol/l, IC 50 for oxidation: 4.4 × 10 ¹6 mol/l), whereas ketoconazole preferentially inhibited oxidation and reduction reactions catalyzed by 11b-HSD-II. Metyrapone, which is reduced to metyrapol by hepatic 11b-HSD-I, inhibited steroid reductase activity of 11b-HSD-I and -II and oxidative activity of 11b-HSD-II. These findings can be explained by substrate competition for reductase reactions and by product inhibition of the oxidation, which is a well-known characteristic of 11b-HSD-II. Conclusions: Our in vitro results may offer a new concept for renal glucocorticoid targeting. Oral administration of small amounts of DH-D (low substrate affinity for 11b-HSD-I) in combination with chenodeoxycholic acid (selective inhibition of 11b-HSD-I) may prevent hepatic first pass reduction of DH-D, thus allowing selective activation of DH-D to D by the high affinity 11b-HSD-II in the kidney. Moreover, selective inhibitors of the hepatic 11b-HSD-I, like chenodeoxycholic acid, may become useful in the therapy of patients with hepatic insulin resistance including diabetes mellitus type II, because cortisol enhances gluconeogenesis.

Comparative enzymology of 11β-hydroxysteroid dehydrogenase type 1 from six species

“…There is controversy about the inhibitory effect of the bile acid CDCA on 11 -HSDs. Diederich et al (2000) suggested that CDCA acts as a selective 11 -HSD1 inhibitor, with an IC 50 of 2·8 µM obtained with human hepatic microsomes. Morris et al (2004) reported inhibition of the dehydrogenase activity of 11 -HSD1 with IC 50 values of 0·2-7 µM, but only 37-200 µM for the oxoreductase activity in rat liver microsomes.…”

Functional Expression, Characterization, and Purification of the Catalytic Domain of Human 11-β-Hydroxysteroid Dehydrogenase Type 1

“…Activity Assays-Cleared lysates and other enzyme preparations (typically 1-10 l) were incubated in 0.5 ml of phosphate buffer (0.1 M, pH 7.6) containing 50,000 cpm [ 3 H]cortisol, 100 nM unlabeled cortisol, and 200 M NADP for 30 min at 37°C to assess dehydrogenase activity or [ 3 H]cortisone (generated as reported previously (19)), 100 nM unlabeled cortisone, 200 M NADPH, and a regeneration system (10 mM MgCl 2 , 5 mM glucose-6-phosphate, and 10 units glucose-6-phosphate dehydrogenase) (20) to assess reductase activity. Steroids were partitioned into 10 volumes of dichloromethane and separated by TLC using ethanol/chloroform (8:92) as the mobile phase.…”

Clinical implications of glucocorticoid metabolism by 11beta-hydroxysteroid dehydrogenases in target tissues

“…The consequence is a conversion of an inactive 9-fluorinated 11-dehydrosteroid (e.g. 11-dehydrodexamethasone (11-DH-D)) to an active 11-hydroxysteroid (dexamethasone) in the kidney (51,56,122). These observations explain the greatly enhanced MC effect of 9a-fluorocortisol and the strong GC effect of dexamethasone.…”