Structural Characterization, Kinetic Studies, and in Vitro Biological

Bioconjugate Chem. 2000, 11, 167−174

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Structural Characterization, Kinetic Studies, and in Vitro Biological Activity of New cis-Diamminebis-cholylglycinate(O,O′) Pt(II) and cis-Diamminebis-ursodeoxycholate(O,O′) Pt(II) Complexes Julio J. Criado,*,† Marı´a F. Domı´nguez,‡ Manuel Medarde,§ Emilio R. Ferna´ndez,† Rocı´o I. R. Macı´as,‡ and Jose´ J. G. Marı´n‡ Departamento de Quı´mica Inorga´nica, Departamento de Fisiologı´a y Farmacologı´a, and Departamento de Quı´mica Orga´nica, Campus Miguel de Unamuno, Universidad de Salamanca, 37007-Salamanca, Spain. Received August 20, 1999; Revised Manuscript Received November 25, 1999

The complexes cis-diamminebis-cholylglycinate (O,O′) [Pt(II) C52H90N4O12Pt, for convenience referred to as Bamet-R1] and cis-diamminebis-ursodeoxycholate (O,O′) Pt(II) (C48H84N2O8Pt, Bamet-UD2) were prepared. The structural integrity of the compounds was confirmed by elemental analysis, FT-IR, NMR, FAB-MS, and UV spectroscopies. The kinetic study of both compounds was accomplished by combining the conductivity measurement and those of the analysis of the electronic spectra in aqueous solution for NaCl concentrations of 4 mM (similar to cytoplasmatic concentration), 150 mM (similar to plasmatic concentration), and 500 mM. In water, the compound Bamet-R1 showed a half-life, t1/2, of 3.0 h. This compound forms the chelate species through loss of a ligand, and the other one acts as a bidentate ligand. Ring opening in the presence of chloride ion was produced with a kCl-of 0.25 M-1 h-1. The half-life of Bamet-UD2 in aqueous solution was 3.2 h. However, since this species is not able to chelate and has a lower degree of solubility in the presence of chloride ion, its kinetic behavior was very different from that of the other compound. We consider this to be of great interest with regards to its cytostatic activity. All kinetic measurements were performed under pseudo-first-order conditions, and a pseudo-first-order behavior was found. The antitumoral effect of Bamet-UD2 on several cell lines derived from rat hepatoma, human hepatoma, mouse leukemia, and human colon carcinoma was found to be, in general, similar to that of cisplatin, but higher than that observed for Bamet-R1.

INTRODUCTION

Since its introduction in clinical essays in 1972, the wide success of cis-diamminedichloroplatinum(II) (cisplatin) and its analogues in the treatment of a variety of solid tumors (1) has encouraged the search for new cisplatin derivatives in order to improve the therapeutic index of this compound, which is reduced by dose-limiting toxicity, namely, nephrotoxicity, myelotoxicity, neurotoxicity, nausea, and vomiting (2). Despite many different cisplatin analogues that have been synthesized to date, few of them are actually used in clinical practice. This is probably due to the fact that circumvention of side effects is only partial and is often accompanied by loss of tumoricidal activity. The ability to interact with DNA and to inhibit tumor growth (3, 4) as well as the liver organotropic characteristics (5, 6) of several platinum-containing bile acid derivatives such as Bamet-R2 [[Pt(NH3)2CGCl], cisdiammineplatinum(II)chlorocholylglycinate] and BametH2 [Na[Pt(CG-O,N) (CG-O)Cl], sodium platinum(II)chloro-bischolylglycinate], which were synthesized by our group (7, 8), have been reported previously. These compounds were obtained by using cholylglycinate as the bile acid moiety. In later studies, several alternatives to this choice have been explored. One of them is described in the present paper, in which two molecules of ursode* To whom correspondence should be addressed. Fax: 34-923 29 45 15. E-mail: [email protected]. † Departamento de Quı´mica Inorga ´ nica. ‡ Departamento de Fisiologı´a y Farmacologı´a. § Departamento de Quı´mica Orga ´ nica.

oxycholic (UDCA, 3R,7β-dihydroxy-5β-cholanoic acid) were bound to cisplatin to obtain the new complex named Bamet-UD2. The structural characterization of the compound, kinetic studies of hydrolysis, and determination of its cytostatic activity “in vitro” were performed, comparing the results to those obtained with another new complex named Bamet-R1, similar to Bamet-UD2, except that, in the former, two UDCA moieties were replaced by two cholylglycinate moieties. Because of its peculiar physical-chemical and biological characteristics (9), much work has been devoted to investigating UDCA during the past decade. Owing to the β position of the hydroxyl group located on C7, the behavior of UDCA differs from that of most 7R-hydroxylated bile acids, such as cholic acid and chenodeoxycholic acids, with regards to detergent activity (10, 11) and the pKa′ value of the carboxylate on the side chain (12). These differences are probably related to the very low toxicity of this bile acid as well as in its biological effects, among them its ability to induce hypercholeresis (13), to stimulate bile bicarbonate output (14), and to have a beneficial effect in several pathological conditions characterized by hypercholanemia (15). Accordingly, Bamet-UD2 was synthesized with the aim of maintaining or improving the cytostatic effect of previously described members of Bamet’s family using a complex whose leaving groups (UDCA) present hepatoprotective activity (16). EXPERIMENTAL SECTION

Chemicals. cis-diammineplatinum(II)dichloride Pt(NH3)2Cl2 was purchased from Fluka AG. Sodium cholylglycinate (NaCG; >95%, “glycocholic acid sodium salt”)

10.1021/bc9901088 CCC: $19.00 © 2000 American Chemical Society Published on Web 02/24/2000

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and ursodeoxycholic acid (UDCA; 99%) were obtained from Sigma. All other reagents were of high purity and were used as purchased without any further purification. Analytical Methods. Chemical analysis for C, H, and N were performed on a Perkin-Elmer 2400 elemental analyzer. Platinum was determined by atomic absorption on a Hitachi Z-8100 flameless graphite furnace spectrophotometer set at a wavelength of 265.9 nm, using the following temperature program: 90 °C (20 s), 100 °C (20 s), 800 °C (20 s), 1600 °C (30 s), 2800 °C (5 s), and 3000 °C (4 s). Infrared (IR) spectra were recorded in the 4000200 cm-1 range on a Perkin-Elmer FT-IR 1730 instrument coupled to a Perkin-Elmer Data Station. KBr pellets and spectrophotometric-grade Nujol (Fluka AG) or polyethylene disks were used to record the spectra above and below 400 cm-1, respectively. Mass spectrometry studies were carried out on a VG-Autospec (Universidad Autonoma, Madrid, Spain), using L-SIMS ionization in the FAB+ mode (Cs ion emission) and m-nitrobenzyl alcohol (m-NBA) as matrix. Electrical conductivity in solution was measured using a CDM2e conductimetry Radiometer, with a CDC104 immersion cell. Temperature was controlled in a Unitherm water bath with a precision of (0.01 °C. 1H (400 MHz), 13C (102.6 MHz), and 195Pt (64.5 MHz) NMR spectra were obtained in methanol-d4 and DMSO-d6 solutions on a Bruker DX400 instrument. Carbon resonances were distinguished in DEPT-90 and DEPT-135 experiments. TMS was used as an internal standard for 1H and 13C spectra. Using K2PtCl6 (δ ) 0 ppm) as external standard, 300 000 scans, 2 s relaxation delay, and 48 h acquisition time were used to obtain the 195 Pt spectra. Synthesis and Purification. The platinum complex, named Bamet-R1 Pt(NH3)2 (CG)2, was obtained (8) by the following procedure: a 1.5 mM Pt(NH3)2Cl2 solution in water (250 mL) was prepared at 40 °C and filtered onto paper. Then, an aqueous solution of NaCG, sodium cholylglycinate (0.75 mM, 250 mL), was added. To prevent physicochemical effects due to the presence of bile acid micelles in the reaction mixture, attempts were made to keep the concentrations of free NaCG in the reaction mixture below the critical micellar concentration (CMC) for this bile acid (17). Thus, the NaCG solution was added dropwise (1 mL/min) by means of a peristaltic pump, to the continuously stirred Pt(NH3)2Cl2 solution, which was maintained in the dark at 40 °C. This procedure took about 3 h. The final pH was 5.6. The reaction mixture was allowed to reach room temperature for approximately 3 h, before undergoing solid-liquid extraction. The reaction products were separated from the excess of unreacted platinum by solid-liquid extraction in octadecylsilane cartridges (C18, Sep-Pak, Waters) following a classical procedure (18). The compounds retained were recovered from the cartridges with methanol. Yield was 40% from the starting NaCG. The extract was then concentrated for thin-layer chromatography (TLC) on silica gel plates (60 F254) using butyl acetate/methanol 30/70 (v/v) as the solvent system. Two major bands, one of them corresponding to unreacted CG (Rf ) 0.71) and the other corresponding to Bamet-R1 (Rf ) 0.27), were obtained. The latter was scraped off and extracted with methanol. The resulting solution was further purified by semipreparative highperformance liquid chromatography (HPLC) in reversedphase, using a Waters C18 RCM column (5 µm, 10 mm × 25 cm) with a gradient pump module and a photodiodearray detector set simultaneously at 205 and 250 nm. The system was controlled by an IBM computer using System Gold software from Beckman. The column was

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equilibrated with 10 mM KH2PO4/methanol 25/75 (v/v), pH 7.02 (solvent A), and eluted with an isocratic system with solvent A for 10 min and then with a linear gradient from 100% A to 20% A and 80% methanol in 15 min. The solvent rate was 10 mL/min. In this HPLC system, the retention time for CG was 4.8 min, and that for BametR1 was 17.5 min. During the semipreparative HPLC, 0.5 min fractions were collected automatically. Those corresponding to the Bamet-R1 elution time, taking into account 1-min time lapse between the detector and the fraction collector, were pooled together and dried. The resulting powder was desalted by methanolic extraction in C18 cartridges and dried again. To obtain the platinum complex named Bamet-UD2 Pt(NH3)2(UDC)2, it was necessary to change the synthesis procedure due to the difficulty of purification by chromatographic techniques. AgUDC was synthesized (19) by adding 0.50 mmol of AgNO3 to a stirring solution of 0.50 mmol of UDCA with 0.50 mmol of Na2CO3 in 50 mL of water. The reaction was carried out at room temperature over 30 min. Then, the solid AgUDC was filtered onto paper and washed with water. The white precipitate was desiccated at 50 °C to give AgUDC in 54% yield. The platinum reagent Pt(NH3)2I2 was synthesized (20) as follows: 2 mmol of KI was added under a N2 atmosphere to a stirred 16.5 mM cisplatin aqueous solution at 60 °C. The reaction was carried out at 60 °C for 2 h. The yellow solid was filtered, washed with water, and desiccated at 50 °C to produce Pt(NH3)2I2 (49% yield). Finally, 0.13 mmol of Pt(NH3)2I2 was added to 0.27 mmol of AgUDC in 45 mL of water at 65 °C. The reaction was kept at 65 °C for 3 days, stirring for 8 h/day, up to a final pH 6. When the reaction had reached room temperature, the AgI formed was filtered and washed with water. It was then washed with MeOH and dried to produce crude product. Following this, liquid-liquid extraction was carried out, the solvents being benzene and water at pH 8. The supernatant was dissolved in MeOH and was then evaporated to afford Bamet-UD2 in 38% yield. All processes were carried out in the dark. Bamet-R1 was obtained as a slightly yellowish solid in a 6% yield. mp: 165 ( 2 °C. Solubility in water: 352 mM. EA C52H90N4O12Pt: %C, 53.26 (53.90 theor.); %H, 7.75 (7.84 theor.); %N, 4.70 (4.84 theor.); %Pt, 16.21 (16.84 theor.). IR (ν, cm-1): 1636, 1378, 532. 1H NMR (δ, ppm): 0.69 (s, 3H, Me18), 0.89 (s, 3H, Me19), 1.01 (d, 3H, Me21), 3.38 (m, 1H, H3), 3.76 (bs, 1H, H7), 3.95 (bs, 1H, H12), 3.82 (s, 2H, H25). 13C NMR (ppm): C3 (72.9), C7 (69.1), C12 (74.0), C17 (48.1), C18 (13.0), C19 (23.2), C20 (36.9), C21 (17.6), C22 (33.9), C23 (33.1), C24 (177.0), C25 (42.5), C26 (182.7). 195Pt NMR: -2100 ppm. MS: m/z 1180.8 [M + Na]+, 1158.8 [M + 1]+, 693.3 [M - L]+. Bamet-UD2 a yellowish solid, mp: 175 ( 2 °C. Solubility in water: 288 mM. EA. C48H84N2O8Pt: %C, 56.37 (56.94 theor.); %H, 8.16 (8.37 theor.); %N, 2.34 (2.77 theor.); %Pt, 18.96 (19.27 theor.). IR (ν, cm-1): 1617, 1378, 536. 1H NMR (δ, ppm): 0.60 (s, 3H, Me18), 0.86 (s, 3H, Me19), 0.83 (d, 3H, Me21), 3.30 (m, 1H, H3), 3.30 (bs, 1H, H7). 13C NMR (ppm): C3 (69.7), C7 (69.4), C17 (54.6), C18 (12.6), C19 (23.9), C20 (35.0), C21 (18.6), C22 (32.6), C23 (32.0), C24 (180.6). 195Pt NMR: -1554 ppm. MS: m/z 1034.4 [M + Na]+, 1012.4, [M + 1]+), 620.2 [M - L]+. In Vitro Cytostatic Studies. The complexes were evaluated for in vitro cytostatic activity against rat hepatoma McA-RH 7777, human hepatoma HepG2, and murine lymphocytic leukemia L1210, and human colon adenocarcinoma LS174T cells were obtained and cultured as recommended by the American Type Culture Collec-

Platinum(II) Complexes with Bile Acids

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Figure 1. (A) FAB+/MS spectrum of a freshly prepared sample of Bamet-R1. (B) Zoom region corresponding to the [M + 1]+ ion. (C) Calculated isotopic mass distribution for the [M + 1]+ ion.

tion (ATCC, Rockville, MD). Cells were grown in a humidified atmosphere of 95% air/5% CO2 at 37 °C. As culture media, the following were used: minimum essential medium Eagle (MEM, Sigma, for LS174T cells) or Dubelcco’s modified Eagle’s medium (DMEM, Sigma, for McA-RH 7777 and L1210 cells) supplemented with 2 mM glutamine, 26.2 mM NaHCO3, 25 mM Hepes, and 10% horse (L1210 cells), fetal bovine (LS174T cells) serum, or 4 mM glutamine, 26.2 mM NaHCO3, 25 mM Hepes, with 20% horse and 5% fetal bovine serum (McARH 7777). F-12 Coon’s modification with L-glutamine and zinc sulfate supplemented with 26 mM NaHCO3 and 5% fetal bovine serum (HepG2). On the basis of preliminary studies on the time course of cell growth, the following protocols were carried out for testing purposes. McA-RH 7777, HepG2, L1210, and LS174T cells were harvested during the exponential growth phase, diluted with culture medium, seeded at a cell density of 5700 cells/well into 96-well plates, and incubated with the tested compound for 72 h. Studies comparing the cytostatic activity of cisplatin, Bamet-R1, and Bamet-UD2 were carried out using seven different concentrations (from 0 to 100 µM) of the desired compound in the culture medium and 3 wells/concentration. Similar experiments were carried out in three different cultures. At the end of the culture period, tetrazonium was added to the culture and after incubation for 2 h at 37 °C the absorbance at 490 nm due to formazan was measured. Statistical Analysis. The equations for kinetic studies were adjusted to the experimental data by means of a nonlinear regression program available in the SIMFIT in the statitics package (Bardsley 1992 SIMFIT package 3.2., Department of Obstretics and Gynecology at St. Mary’s Hospital, University of Manchester, U.K.). The results from biological activity measurements are expressed as means ( SE. To calculate the statistical significance of differences among the groups, the Bonferroni method of multiple-range testing was used. IC50 values were calculated from nonlinear regression analysis using Ultrafit software (Biosoft, Cambridge, U.K.). Statistical analysis was performed on a Macintosh LC-III computer (Apple Computer, Inc., Cupertino, CA) with programs supplied by Apple Computer, Inc.

RESULTS AND DISCUSSION

Chemistry. The synthesis and purification of the Platinum complexes were carried out as described in the Experimental Section, by reaction of a solution of the starting platinum complex with sodium cholylglycinate (NaCG) or silver ursodeoxycholate (AgUDC) solutions, followed by chromatographic separations, according to slightly modified previously described procedures (7, 8). The products isolated were named Bamet-R1 (from NaCG) and Bamet-UD2 (from AgUDC), indicating their origin (Ba-bile acid, met-metal complex; R1 or UD2-from NaCG or AgUDC). The elemental chemical analysis of these complexes are in agreement with the molecular formulas C52H90N4O12Pt for Bamet-R1 and C48H84N2O8Pt for Bamet-UD2. Platinum concentrations were measured in the same solution by flameless atomic absorption. From these data, the depicted structures can be proposed for Bamet-R1 and Bamet-UD2.

The complexes were characterized by a combination of spectroscopic techniques, which allowed the confirmation of the proposed structures in the absence of X-ray diffraction studies. The latter were not carried out because it was not possible to crystallize the compounds under any of a large list of assayed conditions.

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Figure 2. (A) FAB+/MS spectrum of a freshly prepared sample of Bamet-UD2. (B) Zoom region corresponding to the [M + 1]+ ion. (C) Calculated isotopic mass distribution for the [M + 1]+ ion.

The MS of Bamet-R1, Figure 1, shows representative peaks at m/z 1180.8, 1158.8, and 693.3 (25% real abundance), which can be respectively assigned to Na+ addition [M + Na]+ (theor. 1180.6), molecular ion [M + 1]+ (calculated 1158.6), and loss of a cholylglycinate ligand [M-CG]+ (calculated 693.3). Similarly, in the MS of Bamet-UD2, Figure 2, related representative peaks appear at m/z 1034.4, 1012.4, and 620.2 (30% real abundance), which can be respectively assigned to Na+ addition [M + Na]+ (calculated 1034.5), molecular ion [M + 1]+ (calculated 1012.6), and loss of a ursodeoxycholate ligand [M - UDC]+ (calculated 620.3). The remaining signals in both spectra, with lower m/z values, correspond to fragmentations of the bile acid moieties (21, 22). Owing to the existence of six different isotopic masses for the metal (190, 192, 194, 195, 196, and 198), it was possible to recognize the presence of the metal in the fragmentation peaks of the MS. An identical isotopic distribution was observed in both cases when the experimental data and previously calculated distribution for high m/z peaks were compared (Figure 1, panels B and C, and Figure 2, panels B and C). Small differences between the IR spectra of the complexes and the ligands (NaCG and AgUDC) were also observed for Bamet-R1 and Bamet-UD2 in their IR spectra. The most significant are νas and νs of the COOgroup, which appear at 1602 and 1399 cm-1 in the NaCG ligand and 1636 and 1378 cm-1 in the Bamet-R1 complex, respectively, and are observed at 1593 and 1380 cm-1 in the AgUDC ligand and 1617 and 1387 cm-1 in the BametUD2 complex (23, 24). These data point to a monodentate union of these ligands in both complexes. An additional absorption band ν(Pt-N) at 532 cm-1 in Bamet-R1 and 536 cm-1 in Bamet-UD2 appears in both complexes (25, 26). The 1H NMR and 13C NMR data confirm the presence of unaltered bile acid moieties in the complexes, and only small differences are observed in comparison with the free ligands. The greatest, though still small, differences were observed in the signals of the C17 chain, in agreement with their close proximity to the metal, mainly affecting C22-C26 in Bamet-R1 and C20-C24 in BametUD2.

The 1H NMR spectrum of the cholylglycinate complex was recorded in methanol-d4. Comparison with the NaCG spectrum only revealed a minor deshielding of the H25 in the complex. The same deshielding effect was observed in the 13C NMR, because shifts of 6.2 ppm for C26 and 2.1 ppm for C25 were detected for the signals of these carbon atoms in the complex. The spectra of Bamet-UD2 were obtained in DMSO-d6 because of solubility problems with this complex. The differences between the complex and the free lignad in this solvent were not appreciable in their 1H NMR spectra (100 µM; HepG2, IC50 ) 61.2 µM) and higher, although in the same order, than that measured for Bamet-UD2 (LS 174T, IC50 ) 23.7 µM; L1210, IC50 ) 33.7 µM; McA-RH 7777, IC50 ) 42.5 µM; HepG2, IC50 ) 50 µM). The higher cytostatic activity observed for Bamet-UD2 as compared with Bamet-R1 in certain cell lines is probably related to differences in the dissociation kinetic of this compound. However, these results reveal clearly cell line-dependent effects for both Bamet-UD2 and Bamet-R1. This suggests that factors others than just disociation rate are involved in determining the cytostatic activity of these compounds. Moreover, differences in the ability of these cells lines to repair Bamet-DNA adducts or to neutralize or extrude the complexes cannot be ruled out.

Figure 5. Effect of cisplatin (open triangles), Bamet-R1 (open squares) and Bamet-UD2 (open circles), on cell viability as measured by mitochondrial capacity of metabolize the tretazolium salt to formazan. (Rat hepatoma McA-RH 7777, human hepatoma HepG2, murine lymphocytic leucemia L1210 and human colon adenocarcinoma LS 174T cells). Values are means ( SE of three different cultures. Carried out using three differents wells per concentration point. (*) p < 0.05 as compared with cultures incubated with cisplatin by the Bonferroni method of multiple-range testing.

Platinum(II) Complexes with Bile Acids CONCLUSION

The application of synthetic methodology for the preparation of bile acids derivatives of cis-diammine platinum(II) gave two new agents endowed with interesting cytostatic activity. The higher cytostatic activity observed for Bamet-UD2 as compared with that seen for BametR1 in certain cell lines is probably related to differences in the dissociation kinetics of this compound. ACKNOWLEDGMENT

This study was supported in part by the Junta de Castilla y Leon (Grant SA36/99) and the CICYT (Grants SAL9410693 and SAF9610146). The authors thank Dr. F. J. Burguillo for his assistance in the kinetic studies, Prof. J. Casado and Prof. V. Rives for fruitful discussions and Dr. M. A. Serrano for her valuable help with cell culture. LITERATURE CITED (1) Loeher, P. J., and Einhorn, L. H. (1984) Cisplatin. Ann. Int. Med. 100, 704-713. (2) Kelland, L. R. (1999) In Cisplatin-based anticancer agents (N. P. Farrell, Ed.) pp 113-120, Royal Society of Chemistry, Cambridge, U.K. (3) Marin, J. J. G., Palomero, M. F., Herrera, M. C., Macias, R. I. R., Criado, J. J., and Serrano, M. A. (1998) In vitro cytostatic activity and DNA-interaction of the new liver organotropic complex chloro-bis-cholylglicinate-platinum(II). Anticancer Res. 18, 1641-1648. (4) Marin, J. J. G., Macias, R. I. R., Criado, J. J., Bueno, A., Monte, M. J., and Serrano, M. A. (1998) DNA interaction and cytostatic activity of the new liver organotropic complex of cisplatin with glycocholic acid: Bamet-R2. Int. J. Cancer. 78, 346-352. (5) Marin, J. J. G., Herrera, M. C., Palomero, M. R., Macias, R. I. R., Monte, M. J., Elmir, M. Y., and Villanueva, G. R. (1998) Rat liver transport and biotransformation of cytostatic complex of bis-cholylglycinate and platinum (II). J. Hepatol. 28, 417-425. (6) Macias, R. I. R., Monte, M. J., El-Mir, M. Y., Villanueva, G. R., and Marin, J. J. G. (1998) Transport and biotransformation of the new cytostatic complex cis-diammineplatinum(II)chlorocholylglycinate (Bamet-R2) by the rat liver. J. Lipid Res. 39, 1792-1798. (7) Criado, J. J., Herrea, M. C., Palomero, M. F., Medarde, M., Rodriguez, E., and Marin, J. J. G. (1997) Synthesis and characterization of a new bile acid and platinum(II) complex with cytostatic activity. J. Lipid Res. 38, 1022-1032. (8) Criado, J. J., Macias, R. I. R., Medarde, M., Monte, M. J., Serrano, M. A., and Marin, J. J. G. (1997) Synthesis and characterization of the new cytostatic complex cis-diammineplatinum(II) chlorocholylglycinate. Bioconjugate Chem. 8, 453- 458. (9) Erlinger, S. (1985) Ursodeoxycholic acid: a very special bile acid. Hepatology, 311-317. (10) Gildutuna, S., Deisinger, B., Weiss, A., Freisleben, H. J., Zimmer, G., Sipos, P., and Leuschner, U. (1997) Ursodeoxycholate stabilizes phospholipid rich membranes and mimics the effect of cholesterol: investigations on large unilamellar vesicles. Biochim. Biophys. Acta Biomembr. 1326, 265274. (11) Heuman, D. M., and Bajaj, R. (1994) Ursodeoxycholate conjugates protect against disruption of cholesterol-rich membranes by bile salts. Gastroenterology 106, 1333-1341. (12) Carey, M. C. (1984) Bile acids and bile salts: ionization and solubility properties. Hepatology 4, 66S-71S. (13) Dumont, M., Erlinger, S., and Uchman, S. (1980) Hypercholeresis induced by ursodeoxycholic acid and 7-ketolitocholic acid in rat: possible role of bicarbonate transport. Gastroenterology 79, 82-89.

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