Bile acid derived HMG-CoA reductase inhibitors

Bile acid derived HMG-CoA reductase inhibitors

BB ELSEVIER Biochimica et Biophysica Acta 1227 (1994) 137-154 Biochi~ic~a et Biophysica~ta Bile acid derived HMG-CoA reductase inhibitors Wemer Kr...

2MB Sizes 0 Downloads 71 Views

BB

ELSEVIER

Biochimica et Biophysica Acta 1227 (1994) 137-154

Biochi~ic~a et Biophysica~ta

Bile acid derived HMG-CoA reductase inhibitors Wemer Kramer a,*, Giinther Wess a, Alfons Enhsen a, Klaus Bock a, Eugen Falk a, Axel Hoffmann a, Georg Neckermann a, Dietrich Gantz a, Siegfried Schulz b, Lutz Nickau b, Ernst Petzinger b, Stephen Turley c, John M. Dietschy c a HOECHSTAktiengesellschaft, D-65926 Frankfurt am Main, Germany b Institutf~r Pharmakologie und Toxikologie, Frankfurter Strafle 107, D-35392 Giessen, Germany ¢ The Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-8887, USA

Received 16 February 1994; revised 10 June 1994

Abstract 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~-(to-aminoalkoxy)-7a, 12a-dihydroxy-5fl-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 /zM for the most potent compound S 2467 and 6 /zM 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 ICso-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 ICso-value of 16 /xM compared to 55 /xM 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. Measurement of inhibition of cholesterol biosynthesis by incorporation of [14C]octanoate or [3H]H 2° into the digitonin-precipitable sterol fraction in the liver and extrahepatic organs demonstrated a significant increase of liver selectivity of HMG-CoA reductase inhibitors such as HR 780 by coupling to bile acids. Taken together, these studies demonstrate that the liver selectivity of an HMG-CoA reductase inhibitor can be increased by combining with bile acid structural elements and making use of the specific bile acid transport pathways of the liver. Keywords: Bile acid transport; HMG-CoA reductase inhibitor; Liver specificity; Drug delivery; Drug targeting; Photoaffinity labeling

Abbreviations: EDTA, ethylenediamine tetraacetic acid; HMGCoA reductase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (Mevalonate, NADP+ oxidoreductase (CoA acylating), EC 1.1.1.34); Hepes, 4-(2-Hydroxyethyl)-l-piperazinethanesulfonic acid; HPLC, high pressure liquid chromatography; HPTLC, high performance thin-layer chromatography; SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoreses. * Corresponding author. Fax: (+ 49) 69 305 13333. 0925-4439/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0925-4439(94)00046-S

1. Introduction Hypercholesterolemia is a primary risk factor for arteriosclerosis and coronary heart disease which are major causes of death in western countries [1]. A key regulatory enzyme in the biosynthesis of cholesterol is 3-hydroxy-3methylglutaryl coenzyme A reductase ( H M G - C o A reduc-

138

w. Kramer et al. /Biochimica et Biophysica Acta 1227 (1994) 137-154

tase) [2,3] and therefore a primary target for intervention in the pathogenesis of arteriosclerosis. With the discovery of specific inhibitors for HMG-CoA reductase [4] and the succesful development of drugs like lovastatin [5], simvastatin [6] and pravastatin [7], it was unequivocally shown that inhibition of HMG-CoA-reductase is a very effective way for lowering total and low density lipoprotein cholesterol levels in hypercholesterolemic patients [8]. The target organ for inhibitors of HMG-CoA reductase is the liver since the liver is the major site of both lipoprotein production and low density lipoprotein catabolism. Inhibition of hepatic HMG-CoA reductase results in an increase in hepatic LDL receptor activity which in turn increases the removal of LDL from plasma [9]. The clinically hitherto used HMG-CoA reductase inhibitors are well-tolerated in man [10], but in 2-5% of patients adverse side effects such as elevated liver enzymes [11], sleep disturbances [12], severe myositis and rhabdomyolysis [13,14] as well as a decrease of cardiac ubiquinone levels [15,16] are observed, caused by inhibition of HMG-CoA-reductase in tissues other than the liver. Since the liver is the target organ for inhibitors of HMG-CoA reductase, the ideal inhibitor should show little or no inhibition of cholesterol biosynthesis in non-hepatic tissues, such as lens, which rely upon endogenous synthesis of cholesterol [17]. Physiological compounds with a specific organotropism for the liver are bile acids which undergo a biological recycling during enterohepatic circulation [18-21] involving the liver and the terminal ileum under physiological conditions. This organotropism for bile acids is explained by the existence of specific transport systems for bile acids with high transport capacity in the sinusoidal membrane of hepatocytes [22,23] and the brush-border membrane of ileal enterocytes [19,20,24,25]. Consequently, this organotropism recommends to make use of the bile acid transport pathway to achieve a liverspecific drug action of HMG-CoA-reductase inhibitors. In previous papers we could show that drugs such as chlorambucil or peptides can be specifically delivered to the liver or the ileum by covalent coupling to modified bile acid molecules [26,27]. In the present manuscript we applied this concept to HMG-CoA-reductase inhibitors by combining with bile acid structural elements, either by using bile acid molecules as shuttles or by synthesis of hybrid-molecules containing the structural elements essential for inhibition of HMG-CoA-reductase and recognition by the hepatic bile acid transport systems.

2. Materials and m e t h o d s

2.1. Materials

HR 780 was synthesized as described [28]. The synthesis of conjugates of HMG-CoA-reductase inhibitors with modified bile acids and of bile acids with a 3,5-dihydroxy-

heptanoic side chain is described elsewhere [29-32]. As the 5-hydroxy group has been generated unselectively, the hybrid molecules were obtained as a pair of diastereomeric syn-3,5-dihydroxy acids, which could be separated during synthesis. Since absolute stereochemistry has not been determined unambigously, the respective compounds are characterized as polar or unpolar diastereomer. [14C]labeled S 3554 (specific radioactivity 19.38 mCi/mMol) was synthesized at HOECHST Aktiengesellschafl starting from [14C]HR-780 and coupling to 7a, 12a-dihydroxy3fl-(2-aminoethoxy)-5fl-cholan-24-oic acid [29]. (7,7azo3 a, 12 a-dihydroxy-5/3 [3/3- 3H]cholan-24-oyl)-2-aminoethanesulfonic acid (specific radioactivity 20 Ci/mMol) was synthesized as described [33,34]. [G-3H]Taurocholic acid (specific radioactivity 2.1 Ci/mMol) was purchased from New England Nuclear (Dreieich, Germany). [14C]HMGCoA (specific radioactivity 58 mCi/mMol), [2-14C]acetic acid (sodium salt, 0.1 GBq/mmol) and [1,2-3 H]cholesterol, (2294 GBq/mmol) were obtained from NEN, (Dreieich, Germany). RPMI 1640 Medium and fetal calf serum were from Flow (Meckenheim, Germany). Natural bile acids were from Sigma (Miinchen, Germany) and cellulose nitrate filters (diameter 25 mm, 0.45 /xm pore size) from Schleicher and Schiill (Dassel, Germany). Scintillators Quickszint 501, 361, 212 and Unisolve I for liquid scintillation counting were obtained from Zinsser Analytic GmbH (Frankfurt, Germany). HPTLC-plates and solvents for HPLC were obtained from Merck (Darmstadt, Germany). 2.2. Methods Cell culture

Hep G2 cells were obtained from American Type Culture Collection, Rockville, MD USA. Stock cultures were maintained in RPMI 1640 medium containing 10% fetal calf serum. 2.3. Animals

Male Wistar rats (Tierzucht Hoechst AG, Kastengrund, Frankfurt am Main, Germany) weighing 400-500 g were maintained on a standard diet (Altromin ®) with free access to water. Food was withdrawn 18 h prior to the studies. For measurement of cholesterol biosynthesis in vivo rats adapted to an inverse dark-light cycle were used [35]. 2.4. Enzymatic activity o f HMG-CoA reductase

For investigations of the effects of HMG-CoA-reductase inhibitors on the enzymatic activity of HMGCoA-reductase, microsomes from rat liver were prepared as follows: Male Wistar rats (weighing 200-220 g) were fed a standard diet (Altromin ®) containing 2% cholestyramine for 10 days under reversed light cycle. The animals were sacrificed by decapitation on the following day. The livers were removed and chilled on ice. The minced livers

W. Krameret al./ Biochiraicaet BiophysicaActa 1227 (1994) 137-154 were homogenized in buffer A, (2 g liver in 3 ml buffer containing 100 mM sucrose, 50 mM KC1 and 40 mM KH2PO4 and adjusted to pH 7.2 by addition of a solution of 0.23 mM EDTA in 1 M KOH). The supernatant after centrifugation at 10 000 X g was collected. Centrifugation was repeated after homogenization of the pellet in the same volume of buffer A. The combined supernatants were then subjected to ultracentrifugation at 100000 X g for 1 h. The supernatant was discarded and centrifugation was repeated with the pellet homogenized in buffer A containing 10 mM DTr. The resulting pellet was resuspended in buffer B and was kept frozen at -80°C in 200-/zl portions. For the determination of enzyme activity [36], the complete assay medium contained the following in a total volume of 100 /zl at pH 7.4: K x Hy PO4:100 mM; KCI: 50 mM; EDTA: 2 mM, DDT: 5 mM; NADP: 3 mM; glucose-6-phosphate: 32 mM; glucose-6-phosphate dehydrogenase: 0.3 units and rat liver microsomes (40 /xg of protein). After 10 min preincubation at 37°C, a solution of non-radioactive HMG-CoA and 36 nCi [14C]HMG-CoA were added to give a final substrate concentration of 30 /xM in the assay. Test compounds were added to the assay system in 5 ~1 volumes at multiconcentration levels. The complete assay was incubated at 37°C with shaking for further 15 rain and the reaction was stopped by addition of 400/.~1 of Dowex WX4 cationic exchange resin suspended in 1 M HC1. After 30 min shaking at room temperature, the whole sample volume was applicated to an 0.6 X 8.0 cm column containing 100-200 mesh AG1-X8, formiate form (Bio Rad). The column was pre-washed with 500 /xl water, and the mevalonolactone was eluted with additional 1.75 ml of water. After addition of 15 ml of Quickszint 212 (Zinsser), samples were measured in a scintillation counter. The assay was carried out in triplicate, the average of six values was calculated for the percentage inhibition. IC50 values were obtained by plotting the percentage inhibition against the logarithm of the test compound concentration. 2.5. Liver perfusion experiments Rats starved for 18 h were unaesthetized with urethane (25%, 5 m l / k g i.m.) and the common bile duct was cannulated. After an initial bile collection period of 30 min, 500 ~1 of a solution of the respective compounds (concentration usually 0.5 mM) dissolved in 10 mM of Tris/Hepes buffer (pH 7.4/300 mM mannitol, ethanol content < 5%) were injected as bolus into a peripheral mesenteric vein and bile was collected after 2, 4, 6, 8, 10, 15, 20 min and subsequently in 10-min steps until 120 min after the initial injection. 2.6. Isolation of hepatocytes Rat liver hepatocytes were isolated by perfusion of the liver with 0.05% collagenase in Krebs-Henseleit-buffer

139

according to Berry and Friend [37] as described [38]. The rats were anaesthetized with urethane by i.p. injection (1 m g / g body weight) and received 1000 U heparin (Liquemin®) via the femoral vein. The vena portae and vena cava caudalis (pars thoracalis) were cannulated and after perfusion with Ca2+-free Krebs-Henseleit-buffer (pH 7.4) at 37°C to remove blood, the liver was transferred to a temperature-controlled hood and perfused with 0.05% collagenase solution for 15 min under O2/CO 2 (95%/5%) atmosphere. Then, the liver was sliced and the cells were dissociated by gas-bubbles for 2 min. The cell suspension was centrifuged twice at 40 X g for 3 min to discard non-parenchymal cells and cell detritus. The pellets were resuspended and freshly isolated hepatocytes were equilibrated for 30 min in Tyrode buffer (pH 7.4) at 37°C under a water-saturated O2/CO 2 atmosphere (95% O2/5%CO2). 2.7. Uptake measurements in isolated hepatocytes 2 . 1 0 6 hepatocytes suspended in 1 ml of buffer were preincubated for 30 s with the respective compounds (concentrations given in the legends to figures and tables) and the radioactively labelled substrates, 40 nM [3H]taurocholate adjusted to 10/zM, 0.55 ~M [14C]cholate adjusted to 10 /zM or 3 /zM [3H]serine adjusted to 10 /xM. The putative inhibitors were added dissolved in dimethylsulfoxide (final concentration < 1%, v / v ) and controls were exposed to identical concentrations of dimethylsulfoxide. Samples of 100 /~1 cell suspension were withdrawn after 15, 45, 75, 105, 135 s and during the following 20 min. The probes were immediately centrifuged at 10000 X g through a silicone oil layer. Radioactivity was measured in the separated cell pellet and the supematant by liquid scintillation counting. Contamination of the cell pellet with extracellular volume according to [3H]inulin measurement was below 0.2%. 2.8. Analysis of bile samples by thin-layer chromatography For the determination of the hepatobiliary clearance of [14C]S-3554 or [14C]HR 780 aliquots of bile were analysed by liquid scintillation counting after addition of 4 ml of scintillator Quickszint 361. From the bile samples collected during the liver perfusion experiments, 10-/zl aliquots were applied onto HPTLC-thin-layer plates (20 X 10 cm) together with the respective reference standards. The chromatograms were developed using the following solvent systems: I: Chloroform/methanol 3 / 1 (v/v) II: n-Butanol/water/acetic acid 5 / 3 / 2 ( v / v / v ) III: n-Butanol/water/acetic acid 9 / 2 / 1 ( v / v / v ) IV: n-Butanol/water/acetic acid 1 0 / 1 / 1 ( v / v / v ) Subsequently the chromatograms were analyzed by radiothin-layer chromatography using a Berthold radiochromatogram scanner (Berthold AG, Calw, Germany).

140

W. Kramer et at/Biochimica et BiophysicaActa 1227 (1994) 137-154

of [14C]S3554

Metabolic stability of HR-780-bile acid conjugates For the determination of the stability of HR-780-bile acid conjugates against metabolic hydrolysis, 100 /xg of protein, either ileal brush-border membrane vesicles, rat or rabbit small intestinal homogenate, rat liver homogenate or rat serum (20 ~1) were mixed with 180 /zl of a 2 mM solution of the corresponding compound in 10 mM TrisHepes buffer (pH 7.4)/300 mM mannitol. After 0, 5, 10, 20, 30, 60 and 120 min of incubation at 20°C, 20 /xl aliquots were removed and immediately mixed with 40 ~1 of dioxane to stop enzymatic reactions. After centrifugation the supernatants were applied to HPTLC thin-layer plates (10 × 20 cm) and chromatograms were developed in the above-mentioned solvent systems. Afterwards the individual tracks were scanned either at 260 nm or after staining with molybdatophosphoric acid at 595 nm.

2.10. In vivo distribution

2.9. Measurement of cholesterol biosynthesis in HepG2 cells

2.11. Determination of rates of sterol synthesis in vivo

Cholesterol biosynthesis was determined by measuring the incorporation of [14C]acetate into total cholesterol. Subconfluent HepG2 cells were trypsinized and seeded at a density of 5 . 1 0 4 cells/cm 2 on 6-well-tissue culture plates. The cells were maintained in RPMI 1640-medium containing 10% fetal calf serum for 24 h. Thereafter this medium was replaced by RPMI 1640-medium supplemented with 10% LPDS (lipoprotein deficient serum). After 24 h in LPDS-containing medium, the cells were fed RPMI 1640-medium containing 10% LPDS and the various test compounds. Stock solutions of the drugs were prepared in dimethylsulfoxide, the final concentration being 1% in both controls and all the test samples. After a preincubation for 1 h with the inhibitors, [14C]acetate was added (0.5 mM final concentration, 4.8- 106 dpm/mmol) and the cells were pulsed for 17 h. Then the medium was removed, the cell layer was rinsed four times with ice-cold 0.9% NaC1 and the cells were scraped from the wells into 0.9% NaC1. Lipids were saponified with ethanol/KOH for 1 h at 80°C and extracted with chloroform/methanol (2:1). This organic extract was evaporated under a stream of nitrogen and the lipids redissolved in chloroform and spotted on TLC-plates (silica, Merck, Darmstadt, F 254). The chromatograms were developed in chloroform/acetone (1/10, v / v ) for 1 h. The cholesterol band was stained with iodine vapor and scraped into scintillation vials for quantitation. [3H]Cholesterol, which was added to the cell suspension prior to saponification was used for quantitation of recovery and for the identification of the cholesterol band on chromatograms. For visualization of the cholesterol band on TLC plates, unlabeled cholesterol was applied on a separate lane. Cholesterol biosynthesis was expressed as nmol acetate incorporated into total cholesterol/mg cell protein. Protein content of cell layers was determined from aliquots of the starting cell suspension according to the procedure of Lowry [39].

and [14C]HR 780

Male Wistar rats (240-260 g) were anaesthetized by intraperitoneal injection of 1.7 ml of a 20% solution of urethane. After cannulation of the common bile duct, the compound solution (dissolved in 10 /zl ethanol/500 /xl saline) was injected during 60 s into a peripheral mesenteric vein and bile was fractionated. At the end of the experiment blood was collected from the vena cava abdominalis and the bladder was punctured to collect urine. Organs were removed and after weighing definite amounts of the respective organs were kept in 400 /~1 of 4 M potassium hydroxide for 3 h at 70°C and subsequently 50 /zl of hydrogenperoxide solution (30%, w / v ) were added. After addition of scintillator radioactivity was measured by liquid scintillation counting.

Male Sprague-Dawley rats, in the weight range of 180-230 g were fed a regular Chow diet and subjected to light cycling for at least 7 days before use. At the day of the study the animals were given an intravenous application of 20/xCi [1-14C]octanoate in saline into the tail vein between 07.30 and 08.00 h. Together with this application the animals received intravenously equimolar doses of HR 780, S 3554 or mevindin. One hour after the application, at the mid-dark phase of the light cycle, the animals were killed. 700-800 mg of liver tissue was saponified in ethanolic KOH in triplicate, and the digitonin precipitable sterol fraction (DPS) was isolated as described [40]. The resulting sterol/digitonin pellet was resolved in 2 ml of methanol and counted in a liquid scintillation counter. The rate of sterol synthesis was expressed as c p m / g liver. For studies in the hamster, female Golden Syrian hamsters (4 animals per group) obtained 2 /~mol/kg of the respective compounds p.o. as suspension in 2% methylcellulose. After 1 h, 70 mCi [3H]H20 were injected into a femoral vein and one hour later the animals were killed. Cholesterol biosynthesis was determined by measurement of inorporation of tritium into the digitonin precipitable sterol fraction (DPS) and expressed as nmol [3H]H20 incorporated into DPS per hour and gram of liver tissue. Preparation of brush-border membrane vesicles Brush-border membrane vesicles from the ileum of male white New Zealand rabbits (weighing 4-5 kg) were prepared by the Mg 2÷ precipitation method as described previously [24,25,41,42]. The entire small intestine was removed and divided into 10 segments of equal length, numbered 1-10, proximal to distal. Segments 8-10 were used for the preparation of ileal brush-border membrane vesicles. The brush-border membranes were enriched 19 ___ 4-fold with regard to aminopeptidase N (EC 3.4.11.2) and 16 + 7-fold for y-glutamyltransferase (EC 2.3.2.2) and free of contamination by other cell compartments as shown by

W. Kramer et al. / Biochiraica et Biophysica Acta 1227 (1994) 137-154

enzymatic and immunological methods [25]. Immediately after preparation, the vesicles were stored in liquid nitrogen without loss of transport and enzymatic activity for at least 4 weeks. The intactness of the vesicles was determined by measuring Na+-dependent D-glucose uptake after 15 s of incubation; usually the overshoot uptake at 15 s was greater than 20-fold. The enzymatic acitivities of aminopeptidase N and y-glutamyltransferase were determined with Merckotest kits and protein was determined according to Bradford [43] using the Bio-Rad assay (BioRad, Miinchen, Germany).

Transport measurements in ileal brush-border membrane vesicles Uptake of radiolabeled substrates by brush-border membrane vesicles was determined by the membrane filtration method as described previously [24,25,41,42,44]. Typically, the transport reaction was initiated by adding 10 ~1 of the vesicle suspension (50-100 /zg of protein) equilibrated with 10 mM Tris-Hepes buffer (pH 7.4), 300 mM mannitol to 90 /~1 of incubation medium containing the radioactively labeled substrate kept at 30°C. The composition of the incubation medium for measurements in the presence of a Na ÷ gradient usually was 10 mM Tris-HC1 (pH 7.4), 100 mM NaCI, 100 mM mannitol and in the absence of a Na ÷ gradient, 10 mM Tris-HC1 (pH 7.4), 100 mM KCI, 100 mM mannitol. For measurement of taurocholate uptake, these media contained 50 /zM (0.75 /.tCi) [3H]taurocholate. At desired time points, the transport reaction was terminated by the addition of 1 ml of ice-cold stop solution (10 mM Tris-Hepes buffer (pH 7.4), 150 mM KC1). The entire content was pipetted onto the middle of a prewashed, prechilled filter kept under suction with the aid of a vacuum controller. The filter was rinsed immediately with 5 ml of ice-cold stop solution and then solubilized in scintillator Quickszint 361. The radioactivity remaining on the filter was counted with standard liquid scintillation techniques. After correction of medium radioactivity bound to the filter in the absence of membrane vesicles and eventual chemiluminescence, absolute solute uptake was calculated and expressed as nmol/mg protein. All experiments were performed in triplicate and uptake values are given as mean ___S.D. Photoaffinity labeling Photoaffinity labeling with photoreactive bile acids was performed as described previously [25,33,34,44,45]. Typically, 15 /zl of brush-border membrane vesicles (150 /zg of protein) equilibrated with 10 mM Tris-Hepes buffer (pH 7.4), 300 mM mannitol, were added in the dark to 185 /zl of 10 mM Tris-Hepes buffer (pH 7.4), 100 mM NaC1, 100 mM mannitol containing the radiolabeled photoreactive derivatives of taurocholic acid and non-radioactively labeled putative inhibitors. After 5 min of preincubation, the suspensions were irradiated for 10 min at 350 nm in a Rayonet photochemical reactor RPR-100 (The Southern

141

Ultraviolet Co., Hamden CT) equipped with 16 RPR 3500,~ lamps. Afterwards, the suspensions were diluted with 1 ml of ice-cold buffer (10 mM Tris-Hepes buffer (pH 7.4), 300 mM mannitol) and centrifuged for 30 min at 48 000 X g. The superuatant was carefully removed and membrane proteins were precipitated [46]. The dried membrane proteins were solubilized in 50 /xl of 62.5 mM Tris-HC1 buffer (pH 6.8), 2% SDS (w/v), 10% glycerol (v/v), 5% 2-mercaptoethanol (v/v), 0.001 bromphenol blue (w/v), and submitted to SDS-PAGE. For photoaffinity labeling of hepatocytes, freshly prepared rat hepatocytes (5 • 105 cells) suspended in Tyrode buffer were incubated in the absence or presence of the respective inhibitors for 5 min at 30°C in the dark and subsequently irradiated at 350 nm for 6 min. After washing the hepatocytes were suspended in 10 mM sodium phosphate buffer (pH 7.4) and after centrifugation at 15 000 X g, the resulting pellets containing cell organelles were submitted to SDS-PAGE.

Gel electrophoresis SDS-PAGE was carried out in vertical slab gels (20 X 17 X 0,15 cm) using an electrophoresis System LE 2 / 4 (LKB Pharmacia Biotechnologie, Freiburg, Germany) as described [25,45,47]. After staining with Serva Blue R 250, the gels were scanned with a densitometer CD 50 (DESAGA) and the individual lanes were cut into slices of 2 mm thickness. Each slice was solubilized with 250/xl of tissue solubilizer Biolute S overnight and after addition ot 4 ml of scintillator Quickszint 501 the samples were counted for radioactivity. Subsequently, radioactivity was detected by fluorography as described elsewhere [25,47].

3. Results

3.1. Design of liver-specific HMG-CoA-reductase hibitors

in-

In order to meet the requirements of exclusive action of an HMG-CoA-reductase inhibitor in the hepatocyte, the respective drug should be taken up into hepatocytes by a mechanism which is unique to the parenchymal liver cell. Bile acids are physiological compounds showing a nearly exclusive organotropism for the liver and the small intestine under normal physiological conditions [18,21]. Furthermore, the liver and the intestine are the only tissues where inhibition of cholesterol biosynthesis should occur during treatment of hyperlipidemea. Therefore, a HMGCoA-reductase-inhibitor is expected to be hepatotropic, if it shares the hepatic uptake system(s) for bile acids. For optimal recognition by the Na+-dependent bile acid uptake systems in the hepatocyte and the ileocyte, the respective compound should contain a steroid moiety with a cisorientation of rings A and B, a negative charge in the side chain at position 17 and at least one hydroxyl group in a-orientation at position 3,7 or 12 of the steroid nucleus

142

W. Kramer et a l . / Biochimica et Biophysica Acta 1227 (1994) 137-154

[19,20,25-27]. As a consequence, the structural requirements of a bile acid molecule necessary for optimal interaction with organotropic bile acid transporters may be combined with the structural elements essential for molecular recognition of a compound as a specific inhibitor of HMG-CoA-reductase. These requirements can in principle be fulfilled by two approaches (Fig. 1): 1. Synthesis of hybrid bile acid: HMG-CoA-reductase inhibitors by replacement of the side chain of a bile acid molecule at position 17 against the 3,5-dihydroxyheptanoic acid moiety essential for inhibitory action on HMG-CoA-reductase. After uptake by the hepatic bile acid transport systems, the intact hybrid molecule should act as an inhibitor of HMG-CoA reductase. 2. Synthesis of prodrugs of HMG-CoA-reductase inhibitors by covalent coupling to modified bile acids yielding molecules of the general formula D-X-B, where D represents the HMG-CoA-reductase inhibitor, X a linker moiety and B a bile acid molecule. After uptake by the hepatic bile acid transport systems the active HMG-CoA-reductase inhibitor should be released within the hepatocyte.

A variety of such new HMG-CoA-reductase inhibitors have been synthesized (Fig. 2, [29-32]). 3.2. Effect of bile acid-derived HMG-CoA-reductase inhibitors on rat liver microsomal HMG-CoA-reductase

In a first series of experiments the effect of both classes of bile acid derived HMG-CoA-reductase inhibitors was investigated. Fig. 3, upper panel, and Table 1 show the effect of hybrid bile acid: HMG-CoA reductase inhibitors on the enzymatic activity of HMG-CoA reductase using a microsomal fraction isolated from rat liver. The inhibitory potency strongly depended on the substituents at positions 3, 7 and 12 of the steroid nucleus. S 1202 and S 1203 with hydroxy groups in a-position at C-3, C-7 and C-12 did not show any inhibition up to a concentration of 10 -3 M in contrast to the sodium salt of mevinolin with an IC50 value of 12 nM. By acetylation in position 7 and 12 a weak inhibition with IC50 values of 400 /zM and 1500 /zM for the two diastereomeric syn-diols S 1323 and S 1324 was found. The corresponding lactones of S 1202, S 1203, S 1323 and S 1324 did not, as expected, inhibit HMG-CoA

0

HO"~"'OH H Exchangeof / side chain at7 18chmanl of an HMG-CoA-reductase r Io bile acids as shuttles :lio.

~.,i

: "~

:

- - -

"COOH:

~,,/o,

i

:

o

i

It

Bileacid- HMG-CoA-reduclase Inhibitorhybrids

Direct action u HMG-CoAreductase

Inhibitor

Pro-Drug

ll Specific Uptake by hepalocytas and Intrecelluar release of acUve drug

Fig. 1. Concept for liver-specific HMG-CoA reductase inhibitors by combining with bile acid structural elements.

143

W. Kramer et al. / Biochimica et Biophysica Acta 1227 (1994) 137-154

Bile Acid - HMG-CoA-Reductase Inhibitor Hybrids

.o~~., o o

"

R1

Rz

Code

OH

OH

unpolar

S 1200

OH

OH

polar

S 1201

OAc

OAc

unpolw

S 1321

OAc

OAC

polar

S 1322

unpolar

S 2485

polar

S 2466

o

H

,~o

140~'

O

."~

H

~

o~

o

I

OH

OH

unpolar

$1202

OH

OH

polar

S 1203

OAc

OAc

unpolar

S 1323

OAc

OAc

polar

S 1324

unpolar

S 2487

polar

S 2468

o

HO~'-" " v ~m' +~~H"

H

~ o

H

~o

o

Bile Acid - HMG-CoA-Reductase Inhibitor Prodrugs

B

HR 780

X

©F +

SY OH o x'

0

Y

Code

HN~o~

OCH3

S 2426

HN~O4

OH

S 3554

HN~o4

H N ~ COOH

S 3898

HN~o~'

HN~ S O I H

$4193

HN',~O4

'"''HH

OH

S 1592

HN4

OH

S 1011

HN~O

4

OH

S 0630

mi~O

"

OH

S 0631

4

OH

S 5262

HN~O~o

o "°-~o ° o

Mevinolin

O

S 2887

Fig. 2. Chemical structures of hybrid bile acid: HMG-CoA-reductase inhibitors (A) and bile acid-HMG-CoA-reductase inhibitor prodrugs (B).

144

W. Kramer et al. / Biochimica et Biophysica Acta 1227 (1994) 137-154

were inactive. Bile acid conjugates of these HMG-CoA reductase inhibitors (Fig. 2) where the free 3,5-dihydroxy heptanoic acid moiety was covalently linked via an amide bond to 3-~-(to-aminoalkoxy)-7a,12a-dihydroxy-5flcholan-24-oic acids such as S 3554 did not result, as expected in a significant inhibition of HMG-CoA reductase, up to concentrations of 10 -6 M, because the free 3,5-dihydroxy heptanoic side chain necessary for enzyme inhibition was blocked by conjugation with the modified bile acid. The ICs0 value for S 3554 was determined as 5 /zM. (A strong inhibition by these conjugates in the submicromolar range found in some experiments could be ascribed to a small contamination of the bile acid conjugates with free HR 780 or mevinolin.)

100

O Z

o

50

o ,

B

i

7

6

5

t.

3

2

100

3.3. Interaction of bile acid derived HMG-CoA-reductase inhibitors with hepatic uptake of [3H]taurocholate

0

~

50

12

11

10

9 8 7 - log [INHIBITOR]

6

5

t+

Fig. 3. Effect of bile acid-derived HMG-CoA reductase inhibitors on microsomal HMG-CoA reductase from rat liver. The enzymatic activity of rat liver microsomal HMG-CoA reductase activity was measured as described in Materials and Methods in the absence and in the presence of the indicated concentrations of the respective inhibitors. Upper panel: • : S 1202, ~: S 1203, • : S 1323, n : S 1324, O: S 2467, O: S 2468, Lower panel: O: HR 780, Na÷-salt, ©: Mevinolin, Na+-salt, • : S 3554, [] : Chenodeoxycholate.

reductase, because the free 3,5-dihydroxy heptanoic acid side chain is necessary for HMG-CoA reductase inhibition (data not shown). The deoxycholate-derived compounds S 2467 and S 2468 carrying a methylbutanoyl residue at the 12a-hydroxy group thus being most closely related by structure to mevinolin, showed an ICs0-value of 0.7 /xM for S 2467 and 6 /xM for its diastereomere S 2468. This profound stereospecificity of HMG-CoA reductase inhibition strongly argues for a specific effect of the hybrid bile acid: HMG-CoA reductase inhibitors with their target enzyme. During preparation of this manuscript we obtained knowledge of the attempts of two other groups to achieve live-specificity of HMG-CoA-reductase inhibitors by synthesis of hybrid bile acid: HMG-CoA-reductase inhibitors by replacement of the natural side chain of bile acids against the 3,5-dihydroxyheptanoic acid side chain [48,49]. Fig. 3 lower panel shows that the potent HMG-CoA reductase inhibitors mevinolin and HR 780 in their open ring form concentration-dependently inhibited rat liver HMG-CoA reductase with ICs0-values of 12 nM and 2 nM respectively, whereas bile acids such as chenodeoxycholate

A prerequisite for liver-selective drug action is an efficient and specific uptake by the liver. Therefore, we investigated the interaction of bile-acid derived HMGCoA-reductase inhibitors and the HMG-CoA-reductase inhibitors HR 780 and mevinolin with the Na+-dependent uptake of [3H]taurocholate by freshly isolated hepatocytes. Fig. 4 shows the effect of the hybrid bile acid: HMGCoA-reductase inhibitors S 2465, S 2466, S 2467 and S 2468 on [3H]taurocholate uptake; all compounds significantly inhibited [3H]taurocholate uptake with the following ranking: S 2467, S 2468 > S 2465 > S 2466, i.e., the open ring forms with a free carboxylic group in the side chain at C-17 were more potent than the corresponding lactones, in accordance with the structure/activity relationships [18,21] of bile acids for hepatic uptake. Furthermore, we investigated the effect of bile acid conjugates of HR-780 and mevinolin in comparison with the parent drugs on [3H]taurocholate uptake by isolated rat hepatocytes. The inhibitory effect of these conjugates was greater than by the bile-acid hybrids shown above. The

Table 1 Effect of bile acid-derived HMG-CoA reductase inhibitors on microsomal HMG-CoA reductase from rat liver Compound

IC50 value

Prodrugs HR 780-Na ÷ mevinolin-Na ÷ S 3554 and HR 780-bile acid prodrugs

2.10 -9 M 12.10 -9 M > 10 -6 M

Hybrids S S S S S S

1202 1203 1323 1324 2467 2468

> 10 -3 M > 10 3 M 4.10 -4 M 1.5-10 -3 M 0.7-10 -6 M 6-10 -6 M

The effect of the indicated compounds on rat microsonal HMG-CoA reductase was determined as described in Section 2.

W. Kramer et al. / Biochimica et Biophysica Acta 1227 (1994) 137-154

ICs0-value for HR 780-1acton was determined to 100/zM, that of the open ring form to 25 /xM, whereas mevinolin showed an IC5o-value of > 100 /xM compared to 75 /zM for the open ring form. This increased affinity of HR 780 acid towards the hepatic bile acid uptake system may explain the higher liver-specificity of HR 780 compared to mevinolin in animal studies [50]. By coupling of HR 780 to modified bile acids, the ICs0-value was decreased to 5 /xM for S 3554, 7 /xM for S 3898 and S 4193, that of mevinolin in the conjugate S 2887 to 9 /xM. This inhibitory effect of HMG-CoA-reductase inhibitor bile acid prodrugs was specific for the uptake of bile acids, both for [3H]taurocholate and [3H]cholate, whereas no significant effect on the Na+-dependent uptake of [3H] serine was observed. The specific interaction of bile acid derived HMG-CoA reductase inhibitors with the hepatic uptake systems for bile acids was further confirmed by photoaffinity labeling experiments of freshly prepared rat hepatocytes using photolabile 7-azi- or 3-azi-derivatives of taurocholate. Fig. 4 shows that the labeling of the bile acid binding proteins of M r 48000 and 54000 presumably involved in hepatic uptake of bile acids [51-53] in the particulate fraction of hepatocytes was greatly decreased, if the labeling was performed in the presence of bile acid derived HMG-CoA reductase inhibitors as shown for the hybrid bile acid: HMG-CoA reductase inhibitors (Fig. 5). Similar results were obtained with the bile acid prodrugs, which showed an even greater inhibitory effect on photoaffinity labeling of the hepatic bile acid binding proteins in accordance with

A

1.5

~ 1.0

~ 0.5 1

I

I

I

0.5

1

1.5

2

TIME

(min)

Fig. 4. Inhibition of [3H]taurocholate uptake into rat hepatocytes by hybrid bile acid: HMG-CoA reductase inhibitors. Freshly isolated rat hepatocytes (2.106 cells) suspended in Tyrode butter were incubated with 100 o,M of the compounds S 2465, S 2466, S 2467 and S 2468 at 30°C for 30 s. After addition of 10/~M [3H]taurocholate, bile acid uptake was measured over a period of 2.5 min, and the amount of [3H]taurocholate taken up by the cells was measured at the indicated time points. X: control; O: + S 2467; O: + S 2468; I1: + S 2465; [2: + S 2466.

145

their greater inhibitory effect on [3H]taurocholate uptake by hepatocytes (data not shown).

3.4. Interaction of bile acid derived HMG-CoA-reductase inhibitors with the Na +-dependent ileal uptake system for bile acids The uptake of conjugated and unconjugated bile acids in the terminal ileum of mammals occurs by means of an active Na+-dependent uptake system in the ileocyte brush-border membrane [19,20,24,25]. An integral 93 kDa and a peripheral 14 kDa membrane protein have been identified as essential protein components of this transporter in the rabbit, whereas an integral 87 kDa protein present in the whole small intestine is thought to be involved in passive bile acid uptake [25]. Therefore, the effect of bile acid derived HMG-CoA reductase inhibitors on the Na+-dependent uptake of [3H]taurocholate into rabbit ileal brush-border membrane vesicles as well as on photoaffinity labeling of the respective bile acid binding proteins was investigated. Fig. 6 shows that both types of bile acid derived HMG-reductase inhibitors, hybrids and prodrugs, concentration-dependently inhibited the uptake of [3H]taurocholate into ileal brush-border membrane vesicles in contrast to convential HMG-CoA-reductase inhibitors such as mevinolin or HR 780. The methylester of the HR-780-bile acid prodrug S 3354, S 2426, showed only a very weak inhibition of ileal [3H]taurocholate uptake in contrast to the other prodrugs, which carry a free carboxylgroup at carbon atom C-24 of the bile acid moiety (S 3354, S 0630, S 0631, S 1011, S 1592) or which are conjugated with the amino acids taurine (S 4193) or glycine (S 3898). These findings are in accordance with the structure/activity relationships of bile acids for recognition by the ileal Na+/bile acid cotransporter [19,20,2527]. Photoaffinity labeling of rabbit ileal brush-border membrane vesicles with 7-azi or 3-azi-derivatives of taurocholate revealed, that the bile acid derived HMG-CoA reductase inhibitors had profound inhibitory effects on photoaffinity labeling of the bile acid binding proteins of Mr 93000, 87000 and 14000, whereas convential HMGCoA reductase inhibitors like HR 780 or mevinolin had no effect as is evident from Fig. 7. The inhibitory effect of the HMG-CoA-reductase inhibitor bile acid prodrugs, both on the uptake of [3H]taurocholate as well as on the labeling of the bile acid binding proteins, is significantly greater than that of the hybrid derivatives, indicating a considerably higher affinity of the prodrugs to the ileal bile acid transporter. 3.5. Uptake and metabolism of HMG-CoA-reductase in-

hibitor bile acid prodrugs by the in sita perfused liver The conjugates of HMG-CoA-reductase inhibitors with modified bile acids do not as expected act as direct in-

146

w. Kramer et al. /Biochimica et Biophysica Acta 1227 (1994) 137-154

hibitors o f H M G - C o A - r e d u c t a s e as s h o w n above. For action as H M G - C o A - r e d u c t a s e inhibitors the respective conjugates have to be taken up by the hepatocyte and intracellularly the active drug must be released. A prerequisite for a liver specific action o f such conjugates is that they

Mr" 10"3

1

2

3

1

2

3

4

are transported in intact f o r m to the liver and c l e a v e d into the active drug and the bile acid shuttle m o l e c u l e exclusively within the hepatocyte. Thus, these c o m p o u n d s must be stable in s e r u m and during their transport to the liver. The stability o f these c o m p o u n d s was e x a m i n e d by incuba-

5

6

7

8

9

10

11

u

205 116 97

m

66

m

m

43 36 29

---

24

20

--

14

--

M r • 10-3

4

5

6

7

8

9

10

11

m

2O5 116 97 66

m

m

,54 "-".~

48'--~ 43 36 29

m

D

i

24

20~ 14--

Fig. 5. Photoaffinity labeling of freshly isolated rat hepatocytes with (7,7-azo- 3 ct,12 ot-dihydroxy-5/3[3/3-3H]cholan-24-oyl)-2-aminoethanesulfonic acid in the presence of hybrid bile acid: HMG-CoA reductase inhibitors. Freshly isolated rat hepatocytes (5 • 105 cells) suspended in 1 ml of buffer were incubated at 37°C for 2 min with 0,099 ~M (2 /xCi) (7,7-azo-3 ot,12 ot-dihydroxy-5fl[3/3-3H]cholan-24-oyl)-2-arninoethanesulfonic acid in the absence or presence of 200 /xM of hybrid bile acid: HMG-CoA reductase inhibitors and subsequently photolyzed at 350 nm for 6 min. After washing, the hepatocytes were suspended in 10 mM phosphate buffer (pH 7.4) and after centrifugation at 15 000 x g, the resulting pellets containing cell organelles were submitted to SDS-PAGE using 12% gels and radioactivity was detected by fluorography. A. Serva Blue R-250 stained polypeptides. B. Fluorogram. lane 1, control; lane 2, control; lane 3, +200 ~M S1202; lane 4, +200 txM S1203; lane 5, +200/xM S1323; lane 6, +200 p~M $1324; lane 7, +200 ~M $2465; lane 8, +200 IxM $2466; lane 9, +200 /~M $2467; lane 10, +200 /.LM $2468; lane 11, +200 /~M taurocholate.

W. Kramer et a l . / Biochimica et Biophysica Acta 1227 (1994) 137-154

tion with intestinal brush-border membranes, freshly prepared homogenates from intestine and liver or rat serum and analysis of the medium by thin-layer chromatography over an incubation period of 2 h. During this incubation no detectable hydrolysis of the conjugates S 3554, S 3898 and S 4193 could be observed by rat serum or brush-border membrane vesicles from rabbit small intestine. Homogenates prepared from rat or rabbit small intestine led to a slow hydrolysis of the HR-780 bile acid conjugates (data not shown). The suitability of modified bile acids as shuttles for a specific delivery of HMG-CoA-reductase inhibitors was demonstrated by liver perfusion experiments in situ. In a first series of experiments, [aac] S 3554 being labelled in the HR 780 moiety, and [14C]HR 780 were injected into a peripheral mesenteric vein of anaesthesized rats and subsequently the appearance of the respective compounds and their metabolites in bile was determined. Fig. 8 shows the secretion profile measured as radioactivity/5 /xl bile of

3 A

o~e

¢

0

I

I

I

I

I

50

100

150

200

250

[INHIBITOR]

(~M)

Fig. 6. Interaction of bile acid derived HMG-CoA reductase inhibitors with Na÷-dependent [3H]taurocholate uptake by rabbit ileal brush-border membrane vesicles. Ileal brush border membrane vesicles (10 #1, 100/zg of protein) equilibrated with 10 mM Tris-Hepes buffer (pH 7.4), 300 mM mannitol were incubated at 30°C with 90/.tl of 10 mM Tris-Hepes buffer (pH 7.4), 100 mM mannitol, 100 mM NaC1 containing 50 /xM (0.75 /.~Ci) [3H]taurocholate and the indicated concentrations of competing compounds. Subsequently, [3H]taurocholate uptake was measured for 1 min by a rapid membrane filtration method. Upper panel: hybrid bile acid: HMG-CoA reductase inhibitors; O: S 2467, O: S 2468 • S1323, r-l: S1324, 0 : Cholate, O: Taurochenodeoxycholate. Lower panel: bile acid HMG-CoA reductase inhibitor prodrugs. Q: $3554, Q: $4193, • : S0630, rq: S0631, 0 : S1011, half-shaded square: S1592, half-shaded circle: $2426, ~ Tanrochenodeoxycholate, Q: HR 780.

147

labelled S 3554 ( 0 ) and the parent drug [14C] HR 780 (O). The secretion maximum of S 3554 and its metabolites was after 20 min and secretion was nearly complete after 100 min. In contrast, the secretion profile of HR 780 did not show such a profound secretion maximum and secretion was not completed after 100 min continuing beyond the collection period of 120 min. The cumulative biliary excretion profiles over 120 min for the bile acid conjugate S 3554 and the parent compound HR 780 are shown in the inset of Fig. 8. 62% of the injected dose of S 3554 were excreted into bile within 60 min compared to 32% for HR 780. After 2 h 71% of S 3554 and 48% of HR 780 had appeared in bile. Radiothin-layer chromatograms of the collected bile obtained after 15 min, showed that besides S 3554 a polar metabolite (M 1) was detectable, whereas in the perfusion experiments with HR 780 besides the parent compound at least 3 further metabolites Mr, M 2 and M 3 could be detected. The time-dependent secretion profile of the respective compounds and their metabolites is shown in Fig. 9. The intact HR 780 bile acid conjugate S 3554 appeared in bile with a maximum of secretion at 8-10 min compared with 4 min for the natural bile acid taurocholate [34] (Fig. 9, upper panel). The polar metabolite M 1 was excreted with a maximum at about 25 min. HR 780 was excreted into bile predominantly as the polar metabolite M 1 and only to a low extent as the original compound. By cochromatography of bile samples obtained by perfusion with [14C]S 3554 and [14C]HR 780, it was shown that the metabolite M t from S 3554 and M x from HR 780 cochromatographed in different solvent systems, indicating the formation of identical metabolites from HR 780 and its bile acid conjugate S 3554. Radiothin-layer chromatography in further solvent systems indicated that the metabolite peak M a from S 3554 contains several different compounds, presumably also to a significant extent the taurine-conjugate S 4193 of S 3554. The bile samples obtained after perfusion of liver in situ with S 3554 strongly inhibited microsomal HMG-CoA reductase in contrast to S 3554 and S 4193, thus indicating a release of active drug from the inactive bile acid prodrug within the hepatocyte. [14C]

3.6. Effect of bile-acid derived HMG-CoA-reductase inhibitors on cholesterol biosynthesis in cell culture and in vivo For the evaluation of the potential of bile-acid derived HMG-CoA-reductase inhibitors, we investigated their effect on cholesterol biosynthesis in Hep G2 cell culture by measuring the incorporation of [14C]acetate into total cholesterol. Hep G2 cells were preincubated with [taC]acetate in an LPDS-containing medium for 1 h and then pulsed with the drugs for 17 h. Among the hybrid bile acid: HMG-CoA-reductase inhibitor molecules, only the derivatives mostly related to mevinolin with an methylbutanoyl substitution of the 12-hydroxy group of the bile acid

148

W. Kramer et al. /Biochimica et Biophysica Acta 1227 (1994) 137-154

molecule showed a c o n c e n t r a t i o n - d e p e n d e n t inhibition of cholesterol biosynthesis (Fig. 10, lower panel), whereas all other derivatives had no clear inhibitory effect (Table 2). The lacton S 2465 and its open ring for S 2467 exhibited ICs0-values of 1.5 • 10 -5 M and 1.6 • 10 s M respectively, compared to m e v i n o l i n with 3 - 10 -8 M. The correspond-

Mr.lO-3

1

2O5 116 97

2

3

4

ing diastereomeres S 2466 and S 2468 also led to a concentration-dependent inhibition of cholesterol b i o s y n thesis. The ICso-value for the lacton S 2466 was determ i n e d to 3 . 1 0 -5 M compared to 5 . 5 . 1 0 -5 M for its open ring form S 2468. The open ring c o m p o u n d S 2467 and its lacton S 2465 showed with IC50 values of 16 /xM

5

6

7

8

9

10

11

B

66

43

--

36

--

29

--

24

--

20

--

14

--

M r • 10"3

1

2

3

4

5

6

7

8

9

10

11

205 - 116 - -

93._,.. 87--""

9766--

43-36__

29_ 24--

20--

1 4 - - , ' - 14 - -

Fig. 7. Photoaffinity labeling of rabbit ileal brush-border membrane vesicles with (7,7-azo,3a,12 a-dihydroxy-5fl[3/3-3H]cholan-24-oyl)-2-aminoethanesulfonic acid in the presence of the HMG-CoA-reductase inhibitor HR 780 and its bile acid prodrugs. Ileal brush-border membrane vesicles (15 /zl, 150 /xg of protein) were mixed for 5 min in the dark with 185 /zl of 10 mM Tris-Hepes buffer (pH 7.4), 100 mM mannitol, 100 mM NaC1 containing, 0,25 p,M (2 /zCi) (7,7-azo-3a,12a-dihydroxy-5/313/3-3H]cholan-24-oyl)-2-aminoethanesulfonic acid without or with 200 /zM of the indicated compounds. Subsequently, the vesicle suspension was irradiated at 350 nm for 10 min. After washing the vesicles, membrane proteins were separated by SDS-PAGE (12% gels) and radioacitivy was detected by fluorography. A, Serva Blue R 250 stained polypeptides. B, Fluorogram. lane 1, control; lane 2, control; lane 3, +200 /zM HR 780 (K--salt); lane 4, +200 /xM $3554; lane 5, +200 ~M $2887; lane 6, +200 /zM $4193; lane 7, +200 /zM S0630; lane 8, +200 /zM S0631; lane 9, +200 /zM S1011; lane 10, +200 /xM S1592; lane 11, +200 /zM S 5262.

149

W. Kramer et al. /Biochimica et Biophysica Acta 1227 (1994) 137-154

0ii

o ::L tt~

i 100t

S

e~

&

¥

;5

5O

o,,

e~

'

dl,

~o

r~ 0 Z

20

6-0

60

80

100

120

8

7

6

5

4-

6 5 lag IlNItlBITORI

t~

"~~d 100 U Z t~

O Z 20

t*O

60

TIME

80

100

x

120

~

(min)

Fig. 8. Biliary excretion of the HMG-CoA reductase inhibitor [14C]HR780 and its bile acid conjugate [14C]S-3554. [14C]HR-780 (3 /.tCi) ((3) and [14C]S 3554 (3 /xCi) (Q), were adjusted to a concentration of 0.5 mM with the respective unlabeled compounds in 0.5 ml of 10 mM Tris/Hepes buffer (pH 7.4), 300 mM mannitol, 5% ethanol and injected as a bolus into a peripheral mesenteric vein of anaesthetized rats. Bile was collected at the indicated time points and 5-/xl aliquots were used for the determination of radioactivity in the individual fractions. The inset shows the cumulative secretion of S 3554 and HR 780 into bile.

15

so

o o

z I

I

7 -

Fig. 10. Inhibition of cholesterol biosynthesis in Hep G2 cells by bile acid-derived HMG-CoA reductase inhibitors. Cultivated Hep G2 cells were inculated for 1 h with the indicated concentrations of the respective compounds. Subsequently [14C]acetate was added to a concentration of 0.5 mM and the cells were pulsed for 17 h. After extraction and thin-layer chromatography of lipids, cholesterol biosynthesis was calculated as nmol acetate incorporated into total cholesterol/mg cell protein. Inhibition is expressed as percentage of the respective controls. Upper panel: HR-780 bile acid prodrugs. 0 : S 3554, ©: S 0631, I : S 2426, D: S 1011, ©: S 1592, × : Mevinolin. Lower panel: hybrid bile acid: HMG-CoA reductase inhibitors. O: S 2465, O: S 2467, I3: S 2466, I1: S 2468, 0: S 1321, ~: S 1322, ×: Mevinolin.

0,.,,i 1(]

z Z

eL

20

t.O

60

TIME F i g . 9. T i m e - d e p e n d e n t

i

i

I

80

100

120

(min)

s e c r e t i o n p r o f i l e o f the H M G - C o A

zeductase

inhibitor [14C]HR 780, its bile acid conjugate [14C]S-3554 and their metabolites into bile. The bile samples obtained from the experiment described in Fig. 8 were submitted to thin-layer chromatography and the distribution of radioactivity labelled compounds was determined with a radiochromatogram scanner. Upper panel: Biliary excretion of [14C]S 3554 (91) and its metabolite M 1 (O). Lower panel: Biliary excretion of [14C]HR-780 (9t) and its metabolites M 1 (O), M 2 ( I ) and M 3 (17).

and 15 /xM the highest activity a m o n g the bile acid H M G - C o A reductase inhibitor hybrid m o l e c u l e s , corres p o n d i n g to the findings with m i c r o s o m a l H M G - C o A reductase. The significant higher concentrations necessary for H M G - C o A reductase inhibition in v i v o c o m p a r e d to m i c r o s o m a l fractions m a y be explained by a lack o f bile acid transport proteins in H e p G 2 cells and an uptake of bile acid derived H M G - C o A reductase inhibitors preferentially by diffusional processes. The investigation of bile acid prodrugs o f H M G - C o A reductase inhibitors in H e p G 2 cell culture revealed a concentration-dependent inhibition o f cholesterol biosynthesis with most derivatives. Fig. 10, upper panel, shows the results with derivatives o f H R 780. If the bile acid m o i e t y carried a free c a r b o x y l i c acid function at carbon C-24 o f the bile acid m o i e t y a l l o w i n g access o f the respective derivatives to the e n d o p l a s m i c reticulum during intracellular trafficking o f bile acids, cholesterol biosynthesis was c o n c e n t r a t i o n - d e p e n d e n t l y inhibited. S 3554 inhibited cholesterol biosynthesis with an ICs0 v a l u e o f 68 n M c o m p a r e d to 30 n M for m e v i n o l i n and 3 n M for H R 780;

W. Kramer et a l . / Biochimica et Biophysica Acta 1227 (1994) 137-154

150

the methylester S 2426 of S 3554 showed an IC50 value of 350 nM, whereas S 0631 where HR 780 was conjugated to 7a, 12 a-dihydroxy-3 c~-(3-aminopropyloxy)-5/3-cholan-24oic acid inhibited cholesterol biosynthesis with an IC50 value of 270 nM. The lower inhibitory activity of S 0631 compared to S 3554 correlates with a much slower biliary excretion of polar metabolites after in situ perfusion of livers with S 0631 compared to S 3554. In contrast, if the bile acid derivative was conjugated with taurine or glycine at carbon C-24 of the bile acid moiety, no inhibition of cholesterol biosynthesis was observed. Next we investigated the effect of the HR 780-bile acid conjugate S 3554 on cholesterol biosynthesis in vivo. One

hour after intravenous application of equimolar doses either of S 3554 or HR 780 the incorporation of [14C]octanoate into cholesterol was measured in different organs. Table 3 shows the ratios of inhibition of cholesterol biosynthesis in the liver/inhibition of cholesterol biosynthesis in extrahepatic organs as a parameter for liver selectivity. With the exception of the spleen, the ratios were significantly higher after application of the HR 780 bile acid conjugate compared to the parent drug, most profoundly seen in the adrenal gland, where the ratio was increased more than threefold. After intravenous injection either of []4C]S 3554 or [14C]HR 780 into a peripheral mesenteric vein of anaes-

Organ distribution 10 minutes after application of the drug A

HR 780

S 3554 7%

Douy 83 %

' ~ " t 98%

testicle 0,14% testicle 0,3%

.

other organs 52,7%

other organs 60,9% liver 43,9%

liver 31,

~

adrenal gland 0,05% heart 0,07%

heart 0,4%

serum 2,7%

adrenal gland 0,07%

serum 4,8%

Organ distribution 2 hours after application of the drug E~

HR 780

S 3554

O%

$ adrenal gland 0,05%, udne 0,09%

,=v,.,g 62%

serum 3,8%

liver S.3%

heart 0,31% heart 0,04%

e O,06*/q se~um 2 other organs 41,0%

testicle 0,6%

J

other organs 1,5%

adrenal gland 0,02%, urine 0,01%

Fig. 11. Distribution of [14C]S 3554 and [14C]HR 780 after intravenous injection into rats. Male Wistar rats were given intravenously either 40 nmol [14C]$3554 or 40 nmol [14C]HR 780 after anaesthesia and cannulation of the common bile duct. After 10 min (A) or 2 h (B) the animals were killed and the distribution of radioactivity in bile, serum, urine and tissues was determined.

W. Kramer et aL / Biochimica et Biophysica Acta 1227 (1994) 137-154 Table 2 Effect of bile acid-acid-derived HMG-CoA reductase inhibitors on cholesterol biosynthesis in Hep G2 ceils Compound

IC50-value

HR 780-Na + Mevinolin-Na +

0.3.10 -8 M 3.10 -8 M

Prodrugs S S S S S S S S

3554 2426 0631 3898 4193 1592 1011 2887

6.8.10 -8 M 35.10 -8 M 27.10 -8 M no inhibition no inhibition 43.10 -8 M 60.10 -8 M 25.10 -8 M

Hybrids S S S S S S S S

1202 1203 1323 1324 2465 2466 2467 2468

> 10 -5 M > 10 -5 M > 10 -5 M > 10 -5 M 1.5.10 -5 M 3.10 -5 M 1.6.10 -5 M 5.5.10 -5 M

The effect of the indicated compounds on cholesterol biosynthesis in Hep G2 ceils was determined as described in Section 2.

thetized male Wistar rats the distribution of radioactivity in bile, serum, urine and organs was determined. After 10 min 17% of S 3554 and only 2% of HR 780 were secreted into bile and the organ distribution also showed a preference of S 3554 for the liver. Two hours after application of the drugs, 90% of S 3554 and 38% of HR 780 were found in bile (Fig. 11). The organ distribution also shows that S 3554 predominantly was found in bile, liver and serum. The concentration of S 3554 in organs like testes, heart or adrenal glands was significantly lower - up to a factor of 10 - than for the parent drug HR 780 which itself is a liver-selective HMG-CoA-reductase inhibitor [50]. After 10 min the ratio of radioactively labeled compound in liver and bile/serum was 19.6 for S 3554 and 7.06 for HR 780. After 2 h this ratio increased to 32.9 for S 3554 and 14,4 for HR 780. These results clearly demonstrate that the liver selectivity of a drug can be tremendously increased by making use of the hepatocellular bile acid transport pathways.

151

Table 3 Effect of HR 780 and its bile acid prodrug S 3554 on in vivo sterol synthesis in different organs of the rat HR 780

S 3554

0.98

3.22

0.98

0.96

0.92

1.21

1.43

0.93

1.22

1.61

1.13

1.45

Liver Adrenals Liver Spleen Liver Kidney Liver proximal small intestine Liver distal small intestine Liver

Blood

Male Sprague-Dawley rats were given intravenously 20 /zCi [1-14C]octanoate together with 2 ~ M o l / k g either HR 780 or its bile acid conjugate S 3554. Sterol synthesis in the different organs was determined by liquid scintillation counting of the digitonin-precipitable sterol fraction. As an indicator of tissue-selectivity the values are expressed as the ratio inhibition of cholesterol biosynthesis in the liver (in % compared to control) inhibition of cholesterol biosynthesis in extrahepatic " tissues (in % compared to control)

In order to see whether the bile-acid derived HMGCoA-reductase inhibitors are also orally active, preliminary experiments were performed where the respective compounds were applied by gavage to rats or hamsters. Two hours later, [1-14 C]octanoate was given intravenously and 1 h later the animals were killed and cholesterol biosynthesis was determined in the liver. None of the hybrid bile acid: HMG-CoA-reductase inhibitors was able to produce a significant inhibition of cholesterol biosynthesis. After oral application of S 3554 to hamsters however, a significant inhibition of liver cholesterol biosynthesis could be observed (Table 4), indicating a certain degree of intestinal absorption; this is in context with a high affinity of these compounds to the ileal bile acid transport system. In further experiments in the rat, the time course of inhibition of cholesterol biosynthesis by S 3554 in comparison to mevinolin was measured. Fig. 12, upper panel, shows that inhibition of cholesterol biosynthesis by mevinolin in the

Table 4 In vivo sterol biosynthesis in the hamster after oral application of HMG-CoA reductase inhibitors and the bile acid conjugate S 3554 Compound

DPS ( n m o l / g / h )

% sterol biosynthesis

% inhibition

Methylcellulose HR 780 Mevinolin S 3554

541.4 74.6 130.1 388.3

100 _+ 16 14 + 2 24 + 4 72 + 27

0 86 76 28

+ 88.2 + 10.3 + 22.9 _+ 147

Female Golden Syrian Hamsters were given 2 /xmol/kg of the respective compounds p.o. in 2% methylcellulose and 60 min later 70 mCi [3H]H20 were injected into the femoral vein. After a further 60 min the animals were killed and the incorporation of tritium into the digitonin precipitable sterol fraction was measured. Values are given as /zmol [3H]H20 incorporated into the digitonin-precipitable sterol fraction per hour and of liver tissue (DPS).

152

W. Kramer et aL /Biochimica et Biophysica Acta 1227 (1994) 137-154

4. Discussion

100

~

",

50

~

",,

i

I

~ 50

I

!

,,/

~



Ii I

I

i

I

I

0

2

t,

6

TIME (h)

Fig. 12. Time dependence of inhibition of hepatic and intestinal cholesterol biosynthesis by S 3554 (0) and mevinolin (©). Malc SpragueDawley rats were given orally Tylose (control), 5 mg/kg mevinolin or 0.1 mg/kg S 3554. After 1 h, 3 h or 5 h 20 /zCi [1-Z4C]octanoatewere applied intravenously and 1 h later the animals were killed and cholesterol biosynthesis in the liver and the small intestine was determined. Cholesterol biosynthesis was expressed as percentage of the respective controls+ S.E. The upper panel shows the time dependenceof cholesterol biosynthesis inhibition in the liver by S 3554 (0) or mevinolin(O), the lower panel in the small intestine.

liver was maximal 2 h after application of the drug and decreased after 4 h and 6 h. In contrast, a clear inhibitory effect of S 3554 on liver cholesterol biosynthesis was seen 4 h and 6 h after drug application increasing with time. This different pharmacokinetic behaviour of S 3544 and mevinolin may be explained by an intestinal absorption of S 3554 predominantly by the Na+/bile acid transporter in the ileum. Therefore, the intestinal passage time of S 3554 in the small intestine has a significant influence on the onset of pharmacological action in the liver. Inhibition of liver cholesterol biosynthesis by S 3554 is presumably caused by a release of HR 780 from the prodrug S 3554 in the liver, since cholesterol biosynthesis in the small intestine was not inhibited by S 3554 in these experiments. In contrast, mevinolin showed a time-dependent inhibition of cholesterol biosynthesis in the small intestine similar to that in the liver (Fig. 12, lower panel). These findings further confirm the hypothesis that a liver-specific drug action of HMG-CoA reductase inhibitors can be achieved by combining with bile acid structural elements.

A major medicinal breakthrough for the treatment of hypercholesterolemia was the development of specific inhibitors for HMG-CoA-reductase. Since the liver is responsible for cholesterol homeostasis and since the liver does not synthesize the major mass of endogenous cholesterol in humans [54], there is a great potential for enzyme inhibitors exclusively acting in the liver and the small intestine without any effect in extrahepatic tissues. Bile acids are acidic sterols that undergo a biological recycling during enterohepatic circulation involving exclusively the liver and the small intestine under normal physiological conditions. Consequently, we investigated whether this organotropism for bile acids can be applied to HMGCoA-reductase inhibitors by combining structural elements of HMG-CoA reductase inhibitors necessary for a specific inhibition of HMG-CoA-reductase with structural elements of bile acids essential for specific recognition by hepatic and intestinal bile acid transport systems. Such an approach appears very attractive for several reasons: a: The possibility to deliver the HMG-CoA reductase inhibitor specifically to the liver and the small intestine, i.e., to those organs where an HMG-CoA reductase inhibitor should exclusively exert its pharmacological action. b: Owing to an enterohepatic circulation of a bile-acid derived HMG-CoA reductase inhibitor, a low drug load to attain pharmacological drug levels in the liver seems principly possible. To evaluate the potential of bile acid organotropism for a liver-specific drug action of HMG-CoA-reductase inhibitors, we have followed two ways: 1. To use modified bile acids as a prodrug form and as a natural drug delivery system to direct drugs such as HMG-CoA reductase inhibitors to the liver by covalent coupling of HMG-CoA reductase inhibitors to linkermodified bile acids, an approach whose success has been shown recently for peptides, cytostatics and thyroid hormones [26,27,55,56]. 2. To combine the essential structural element of an HMG-CoA-reductase inhibitor - the 3,5-dihydroxyheptanoic side chain - with the hydroxylated steroid nucleus of bile acid by replacement of the side chain at carbon C-17 against 3,5-dihydroxyheptanoic acid. Both classes of bile-acid-derived HMG-CoA reductase inhibitors specifically interacted with the active Na÷-depen dent bile acid transport systems of the liver and the small intestine. The hybrid bile acid: HMG-CoA-reductase inhibitors were able to inhibit microsomal HMG-CoA-reductase, the best compound S 2467 with an IC50 value of 0.7/zM compared to 6 /xM for its diastereomere S 2468. This remarkable stereospecificity indicates a specific interaction of these compounds with HMG-CoA reductase. In contrast, the prodrugs formed between HMG-CoA reductase inhibitors and 7c~,12a-dihydroxy-3~-(co-

w. Kramer et al. / Biochimica et Biophysica Acta 1227 (1994) 137-154

a m i n o a l k o x y ) - 5 f l - c h o l a n - 2 4 - o i c acids were inactive as inhibitors of H M G - C o A - r e d u c t a s e because the 3,5 -dihyd r o x y h e p t a n o i c side chain of the H M G - C o A reductase inhibitor was m a s k e d by covalent linkage to the bile acid. For a p h a r m a c o l o g i c a l action of such a bile acid H M G C o A - r e d u c t a s e inhibitor, the active drug has to be released within the hepatocyte after specific targeting of the inactive prodrug to the liver. Both classes of bile-acid derived H M G - C o A - r e d u c t a s e inhibitors were able to inhibit cholesterol b i o s y n t h e s i s in Hep G2 cells and the s t r u c t u r e / a c t i v i t y relationships found in e n z y m a t i c and perfusion experiments were confirmed. After oral application to rats or hamsters, however, only the bile acid prodrugs were able to inhibit cholesterol biosynthesis, whereas the hybrid bile acid: H M G - C o A reductase inhibitors did not s h o w a significant effect on cholesterol biosynthesis, p r e s u m a b l y due to a poor intestinal absorption. Preliminary animal experiments indicated a liverselective inhibition of cholesterol biosynthesis after oral application of H M G - C o A reductase inhibitor-bile acid prodrugs. Taken together, the present studies indicate that the selectivity of H M G - C o A - r e d u c t a s e inhibitors for hepatocytes can be significantly increased by c o m b i n i n g with structural elements of bile acids m a k i n g use of the natural o r g a n o t r o p i s m of bile acids. M o r e extended animal studies and later on clinical trials are necessary to evaluate the medicinal potential of such liver-specific H M G - C o A - r e ductase inhibitors.

Acknowledgements W e thank Susanne W i n k l e r and Silke Elsesser for preparing the manuscript and excellent secretarial assistance. W e are greatly indebted to Frank Girbig, Ulrike Gutjahr, S i m o n e K o w a l e w s k i , R o s e m a r i e KaulfuB v.d. Heyden, M a n f r e d Schmalz, H e i n z - J o a c h i m Th/Snges, T a m a r a W e i g e l t and Gert W e i s e r for their help in biological experiments.

References [1] [2] [3] [4]

Endo, A. (1985) J. Med. Chem. 28, 401-405. Brown, M.S. and Goldstein, J.L. (1986) Science 232, 34-47. Brown, M.S. and Goldstein, J.L. (1980) J. Lipid Res. 21, 515-517. Endo, A., Kuroda, M. and Tanzawa, K. (1976) FEBS Lett. 72, 323-326. [5] Alberts, A.W., Chen, J., Kuron, G., Hunt, V., Huff, J., Hoffman, C., Rothrock, J., Lopez, M., Joshua, H., Harris, E., Patchett, A., Monaghan, R., Currie, S., Stapley, E., Albers-Schonberg, G., Hensens, O., Hirschfield, J., Hoogsteen, K., Liesch, J. and Springer, J. (1980) Proc. Natl. Acad. Sci. USA 77, 3957-3961. [6] Hoffman, W.F., Alberts, A.W., Anderson, P.S., Chen, J.S., Smith, R.L., and Willard A.K. (1986) J. Med. Chem. 29, 849-852. [7] Tsujita, Y., Kuroda, M., Shimada, Y., Tanzawa, K., Arai, M.,

153

Kaneko, I., Tanaka, M., Masuda, H., Tarumi, C., Watanabe, Y. and Fujii, S. (1986) Biochim. Biophys. Acta 877, 50-60. [8] Grundy, S.M. (1988) N. Engl. J. Med. 319, 24-32. [9] Brown, M.S. and Goldstein, J.L. (1981) N. Engl. J. Meal. 305, 515-517. [10] Tobert, J.A., (1988) Am. J. Cardiol. 62, 28 J-34 J. [11] Henwood, J.M. and Heel, R.C. (1988) Drugs 36, 429-454. [12] Schaefer, E. (1988) N. Engl. J. Meal. 319, 1222. [13] Israeli, A. Raveh, D., Amon, R. Eisenberg, S., and Stein, Y. (1989) Lancet 1989, 725. [14] Walravens, P.A. Greene, C. and Frerman, F.E. (1989) Lancet 1989, 1097-1098. [15] Willis, R.A., Folkers, K., Tucker, J.-H., Ye, C.-Q., Xia, L.-J., and Tamagawa, M. (1990), Proc. Natl. Acad. Sci, USA 87, 8928-8930. [16] Folkers, K., Langsjoen, P., Willis, R., Richardson, P. Xia, L.-J., Ye, C.-Q. and Tamagawa, M. (1990), Proc. Natl. Acad. Sci. USA 87, 8931-8934. [17] Mosley, S.T., Kalinowski, S.S., Schafer, B.L. and Tanaka, R.D. (1989) J. Lipid Res. 30, 1411-1420. [18] Carey, MC. and Cahalane, M.J. (1988) In: The Liver: Biology and Pathobiology, 2nd edn. (I.M. Arias, W.B. Jakoby, H. Popper, D. Schachter and D.A. Shafritz, eds.), pp. 576-616, Raven Press, New York. [19] Wilson, F.A. (1981) Am. J. Physiol. 241, 683-692. [20] Lack, L. (1979) Environ. Health Perspect. 33, 79-90. [21] Vlahcevic, Z.R., Heuman, D.M. and Hylemon, P.B. (1980). In: Hepatology (Zakim, D. and Boyer, T.D. eds.), W.B. Saunders, Philadelphia, pp. 341-377. [22] Anwer, M.S., Kroker, R. and Hegner, D. (1976) Hoppe-Seyler's Z. Physiol. Chem. 375, 1477-1486. [23] Schwarz, L.R., Burr, R., Schwenk, M. Pfaff, E. and Greim, H. (1975) Eur. J. Biochem. 55, 617-623. [24] Burckhardt, G., Kramer, W., Kurz, G. and Wilson, F.A. (1983) J. Biol. Chem. 258, 3618-3622. [25] Kramer, W., Girbig, F., Gutjahr, U., Kowalewski, S., Jouvenal, K., Miiller, G., Tripier, D. and Wess, G. (1993) J. Biol. Chem. 268, 18035-18046. [26] Kramer, W., Wess, G., Schubert, G., Bickel, M., Girbig, F., Gutjahr, U., Kowalewski, S., Bariughaus, K.-H., Enhsen, A., Glombik, H., Miillner, S., Neckermann, G., Schulz, S. and Petzinger, E. (1992) J. Biol. Chem., 267, 18594-18604. [27] Kramer, W., Wess, G., Neckermann, G., Schubert, G., Fink, J., Girbig, F., Gutjahr, U., Kowalewski, S., Bariughaus, K.-H., B6ger, G., Enhseu, A., Falk, E., Friedrich, M., Glombik, H., Hoffmann, A., Pittius, C. and Urmann, M. (1994) J. Biol. Chem 268, 10621-10627. [28] Wess, G., Kesseler, K., Baader, E., Bartmann, W., Beck, G., Bergmann, A., Jendralla, H., Bock, K., Holstein, G., Kleiue, H. and Schnierer, M. (1990) Tetrahedron Lett. 31, 2545-2548. [29] Wess, G., Kramer, W., Bartmann, W., Enhsen, A., Glombik, H., Miillner, S., Bock, K., Dries, A., Kleine, H. and Schmitt, W. (1992) Tetrahedron Lett. 33, 195-198. [30] Wess, G., Kramer, W., Hun, X.B., Bock, K., Enhsen, A., Glombik, H., Baringhaus, K.-H., B6ger, G., Urmann, M., Hoffmann, A. and Falk, E. (1994) J. Med. Chem., in press. [31] Wess, G., Kramer, W., Enhsen, A., Glombik, H., Miillner, S., Bock, K., Dries, A., Kleine, H. and Schmitt, W. (1973) Tetrahedron Lett. 34, 817-818. [32] Wess, G., Kramer, W., Schubert, G., Enhsen, A., Baringhaus, K.-H., Glombik, H., Miillner, S., Bock, K., Kleine, H., John, M., Neckermann, G. and Hoffmann, A. (1993) Tetrahedron Lett. 34, 819-822. [33] Kramer, W. and Kurz, G. (1983) J. Lipid Res. 24, 910-923. [34] Kramer, W. and Schneider, S. (1988) J. Lipid Res. 30, 1281-1288. [35] Edwards, P.A., Muroya, H. and Gadd, R.G. (1972) J. Lipid Res. 13, 396-401. [36] Ong, K.K., Khor, H.T. and Tan, D.T.S. (1991) Anal. Biochem. 196, 211-214. [37] Berry, M.N. and Friend, D.S. (1969) J. Cell. Biol. 43, 506-520.

154

W. Kramer et aL /Biochimica et Biophysica Acta 1227 (1994) 137-154

[38] Petzinger, E. and Seeger, R. (1976) Naunyn-Schmiedeberg's Arch. Pharmacol. 295, 211-213. [39] Lowry, O.H., Roseborough, N.J., Farr, A.L. and Randall, R.J. (1951), J. Biol. Chem. 193, 265-275. [40] Jeske, D.J. and Dietschy, J.M. (1980), J. Lipid Res. 21, 364-376. [41] Kramer, W. (1987) Biochim. Biophys. Acta 905, 65-74. [42] Kramer, W., Girbig, F., Leipe, I. and Petzold, E (1988) Biochem. Pharmacol. 37, 2427-2435. [43] Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. [44] Kramer, W., Nicol, S.-B., Girbig, F., Gutjahr, U., Kowalewski, S. and Fasold, H. (1992) Biochim. Biophys. Acta 1111, 93-102. [45] Kramer, W., Burckhardt, G., Wilson, F.A. and Kurz, G. (1983) J. Biol. Chem. 258, 3623-3627. [46] Wessel, D. and Fli~gge, U.J. (1984) Anal. Biochem. 138, 141-143. [47] Kramer, W., Girbig, F., Gutjahr, U. and Leipe, I. (1990) J. Chromatogr. 521, 199-210. [48] Menear, K.A., Patel, D., Clay, V., Howes, C. and Taylor, P.W. (1992) Biorg. Med. Chem. Lett. 2, 285-290. [49] McGarry, J.D., Volz, F.A., Regan, J.R. and Chang, M.M. US-Patent US 5, 216 015 (1993).

[50] Krause, R., Neubauer, H., Leven, M. and Kesseler, K. (1990) J. Drug Der. 3, (Suppl. 1) 255-257. [51] Kramer, W., Bickel, U., Buscher, H.-P., Gerok, W. and Kurz, G. (1982) Eur. J. Biochem. 129, 13-24. [52] Wieland, T., Nassal, M. Kramer, W., Fricker, G., Bickel, U. and Kurz, G. (1984) Proc. Natl. Acad. Sci. USA 81, 5232-5236. [53] Abberger, H., Buscher, H.-P., Fuchte, K., Gerok, W., Giese, U., Kramer, W., Kurz, G., and Zanger, U. (1983) In: Bile Acids and Cholesterol in Health and Disease (Paumgartner, G., Stiehl, A. and Gerok, W. eds.) pp. 77-87, MTP Press, Lancaster, UK. [54] Turley, S.D. and Dietschy, J.M. (1988) In: The Liver: Biology and Pathobiology (I.M. Arias, W.B. Jakoby, M. Popper, D. Schachter and D.A. Shafritz, eds.) Raven Press, New York, pp. 617-641. [55] Stephan, Z.F., Yurachek, E.C., Sharif, R., Waswary, J.M., Steele, R.E. and Howes, C. (1992) Biochem. Pharmacol. 43, 1969-1974. [56] Kramer, W., Wess, G., Schubert, G., Bickel, M., Hoffmann, A., Baringhaus, K.-H., Enhsen, A., Glombik, H., Miillner, S., Neckermann, G., Schulz, S. and Petzinger, E. (1993) In: Bile Acids and the Hepatobiliary System (Paumgartner, G., Stiehl, A. and Gerok, W., eds.) pp. 161-176, Kluwer, Dordrecht.