Synthesis and Characterization of the New Cytostatic Complex cis

Molecular Biology, University of Salamanca, 37007 Salamanca, Spain. Received October 30, 1996X. Owing to the high efficiency of hepatocytes to take up...
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JULY/AUGUST 1997 Volume 8, Number 4 © Copyright 1997 by the American Chemical Society

ARTICLES Synthesis and Characterization of the New Cytostatic Complex cis-Diammineplatinum(II)-Chlorocholylglycinate Julio J. Criado,*,† Rocio I. R. Macias,‡ Manuel Medarde,§ Maria J. Monte,‡ Maria A. Serrano,| and Jose J. G. Marin*,‡ Departments of Inorganic Chemistry, Physiology and Pharmacology, Organic Chemistry, and Biochemistry and Molecular Biology, University of Salamanca, 37007 Salamanca, Spain. Received October 30, 1996X

Owing to the high efficiency of hepatocytes to take up bile acids, these endogenous compounds or their analogues can be considered as potential shuttles for delivering drugs to the liver. With the aim of using this strategy to target platinum(II)-related cytostatic drugs toward the hepatobiliary system, a cholylglycinate (CG) derivative of cis-diammineplatinum(II) has been synthesized by treatment of cis-diammineplatinum(II) dichloride with sodium cholylglycinate. The complex, named Bamet-R2, was characterized by spectroscopy and elemental analysis. Results obtained in these studies together with conductivity measurements, which pointed to nonelectrolyte behavior, allowed the structure of the complex to be identified as C26H48N3O6ClPt. The compound was found to be soluble (up to 3 mM) in water and was highly soluble (more than 10 mM) in ethanol, methanol, and dimethyl sulfoxide. Its stability in solution was monitored by HPLC analysis. In deionized water, the compound remains >90% pure in solution for up to 7 days and >80% for up to 28 days. However, in 150 mM NaCl it remains as >90% pure compound in solution for only 1 day. By contrast with the parent compound CG, Bamet-R2 was found to significantly inhibit the growth of rat hepatocytes in primary culture and L1210 murine leukemia cells, although in a less marked way than that observed for cisplatin. The cytostatic effect of Bamet-R2 was particularly strong against human colon adenocarcinoma LS174T cells. The results point to the potential usefulness of Bamet-R2 in the antitumoral therapy of enterohepatic-derived neoplasias.

INTRODUCTION

Considerable efforts have been devoted to the search for new chemotherapeutic agents with antitumoral activ* Address correspondence to these authors at Universidad de Salamanca Campus Miguel de Unamuno Edificio Departamental, Room S-09 37007, Salamanca, Spain (telephone 34-23294674; fax 34-23-294669; e-mail [email protected]). † Department of Inorganic Chemistry. ‡ Department of Physiology and Pharmacology. § Department of Organic Chemistry. | Department of Biochemistry and Molecular Biology. X Abstract published in Advance ACS Abstracts, June 1, 1997.

S1043-1802(97)00061-X CCC: $14.00

ity. However, for several reasons such efforts have been only partially successful. The main drawbacks of currently available chemotherapy are toxicity for extratumoral tissues and the pre-existing or developed resistance of cancer cells to cytostatic agents. Several attempts have been made to circumvent resistance (1) and to improve the vectoriality of such drugs (2). In this sense, the marked organotropism of bile acids toward the hepatobiliary system has been proposed as an interesting characteristic for use of these endogenous compounds or their analogues as shuttles for delivering drugs to the liver. Some examples of this approach are the binding © 1997 American Chemical Society

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of inhibitors of hydroxymethylglutaryl CoA (HMG-CoA) reductase (3) or the cytostatic compound chlorambucyl (4) to bile acids. Since its introduction into clinical trials in 1972, the widespread success of cis-diamminedichloroplatinum(II) (cisplatin) and its analogues in the treatment of a variety of solid tumors (5) has encouraged the search for new cisplatin derivatives with a view to reducing its doselimiting side effects, namely nephrotoxicity, myelotoxycity, neurotoxicity, nausea, and vomiting (6). Although many different cisplatin analogues have been synthesized, few of them are currently used in clinical practice (6). This is probably because the achieved reductions in extratumoral toxicity are only partial and are often accompanied by a loss of tumoricidal activity. Two interesting second-generation cisplatin analogues are cisdiammine(1,1-cyclobutanedicarboxylate)platinum(II) (carboplatin) and 1,2-cyclohexanediammineplatinum(II) (DACH-Pt) and their derivatives. These are not organotropic compounds, although changes in the physicalchemical properties of the resulting molecules do enhance their excretion. They are less nephrotoxic than cisplatin, although other side effects, such as myelotoxicity and neurotoxicity, have been reported to be similar or even greater than that of the parent drug (7). On the basis of the amphipathic properties of these steroids (8), previous DACH-Pt complexes with bile acids have been synthesized in attempts to obtain derivatives with both lipophilicity and water miscibility (9, 10). However, although promising results regarding the antitumoral activity of these complexes have emerged, no further investigations in this direction have been undertaken. Perhaps the large size of the complexes, containing a DACH-Pt moiety together with two bile acid molecules, excludes the possibility of their being transported by carrier proteins located in the basolateral and canalicular membrane of hepatocytes. These proteins are responsible for the selective and efficient clearance of bile acids from systemic blood, where these compounds are maintained at very low concentrations (11). The rationale of the present work was that cisplatin analogues with molecular structures closer to that of a natural bile acid might be recognized by transmembrane bile acid carrier proteins, which would enhance their vectoriality toward the hepatobiliary system. With this aim, cis-diammineplatinum(II) chlorocholylglycinate was synthesized and characterized. Preliminary results on the liver organotropism and in vivo antitumoral activity of this compoundsnamed Bamet-R2shave been reported elsewhere (12, 13). MATERIALS AND METHODS

Chemicals. cis-Diammineplatinum(II) dichloride [Pt(NH3)2Cl2] was purchased from Fluka Quimica (Madrid, Spain). Sodium cholylglycinate (NaCG; >95% by thinlayer chromatography) and 3R-hydroxysteroid dehydrogenase were obtained from Sigma Quimica (Madrid, Spain). All other reagents were of high purity and were used as purchased without any further purification. Analytical Methods. 3R-Hydroxysteroid concentrations were measured according to the classic method of Talalay (14). Chemical analyses for C, H, and N were performed on a Perkin-Elmer 2400 elemental analyzer (Perkin-Elmer Hispania SA, Madrid, Spain). Platinum was determined by atomic absorption in a flameless graphite furnace spectrophotometer Z-8100 Hitachi (Hitachi, Tokyo, Japan), set at a wavelength of 265.9 nm and 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

Criado et al.

in the 4000-200 cm-1 range on a Perkin-Elmer FT-IR 17300 instrument coupled to a Perkin-Elmer 3600 data station. KBr pellets and spectrophotometric grade Nujol (Fluka, Quimica) or polyethylene (Aldrich, Madrid, Spain) disks were used to record 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-NBA as matrix. Electrical conductivity in solution was measured using a CDM2e conductimeter (Radiometer, Copenhagen, Denmark), with a CDC104 immersion cell. Temperature was controlled in a Unitherm water bath (Selecta, Barcelona, Spain) with a precision of (0.01 °C. Nuclear magnetic resonance (NMR) spectra of 1H (400 MHz) and 13C (102.6 MHz) were obtained in CD3OD solutions, with TMS as internal standard using a Bruker DX400 instrument (Karlsruhe, Germany). Carbon resonances were distinguished in DEPT-90 and DEPT-135 experiments. Synthesis. The platinum complex, named Bamet-R2 [Pt(NH3)2CGCl], was obtained according to the following procedure: A 3 mM Pt(NH3)2Cl2 solution in water (100 mL) was prepared at room temperature and filtered onto paper. Then, an aqueous solution of NaCG (1.5 mM, 100 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. The NaCG solution was therefore 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 80 °C. This procedure takes about 1.5 h. The final pH was 6.0. The reaction mixture was allowed to reach room temperature for approximately 2 h, before undergoing solid-liquid extraction. The final product was obtained as a yellow solid after evaporation to dryness in a desiccator containing P4O10. Purification. Reaction products were separated from the excess of unreacted platinum by solid-liquid extraction in octadecylsilane cartridges (C18, Sep-Pak, Waters Cromatografia SA, Madrid, Spain) following a classical procedure (15). The retained compounds were recovered from the cartridges with methanol. The extract was then concentrated for thin-layer chromatography (TLC) on silica gel plates (60 F254, Merk, Darmstadt, Germany) 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-R2 (Rf ) 0.36) were obtained. The latter band was scraped off and extracted with methanol. The resulting solution was further purified by semipreparative high-performance liquid chromatography (HPLC) in reversed phase, using a Waters C18 RCM column (5 µm, 10 mm × 25 cm) with a gradient pump module (Model 126, Beckman, Madrid, Spain) and a photodiode array detector (Model 168, Beckman) set simultaneously at 205 and 250 nm. The system was controlled by an IBM computer (Model 30286, IBM Corp., Portsmouth, U.K.) using System Gold software from Beckman. The column was 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 5 min and then with a linear gradient from 100% solvent A to 20% solvent A and 80% methanol over 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 Bamet-R2 was 11.2 min. During the semipreparative HPLC, fractions were collected automatically every 0.5 min. Those corresponding to the Bamet-R2 elution time, taking into account a 1 min time

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Figure 2. Time-course of Bamet-R2 stability in water or saline (150 mM NaCl) solution. Bamet-R2 solution (100 µM) was maintained in the dark at 4 °C. After vigorous stirring, samples were collected at the indicated times for immediate HPLC analysis. Bamet-R2 concentrations were calculated from the height of the absorbance peak (250 nm wavelength) eluted at 9 min.

Figure 1. Reversed phase HPLC of purified Bamet-R2 as recorded by using photodiode array detector in scan mode from 190 to 500 nm wavelength (upper panel) and at 250 nm wavelength (lower panel). Inset represents the results of flameless atomic absorption spectroscopy measurement of platinum contents in samples collected every 0.5 min during HPLC of pure Bamet-R2. The delay from the detector to the fraction collector was approximately 1 min.

lapse between the detector and the fraction collector, were pooled together and dried. The result was desalted by methanolic extraction in C18 cartridges and dried again. With this procedure, highly pure (>95%) BametR2 was obtained as a yellowish solid in 40% yield from the starting NaCG. The results of purity control by analytical HPLC (column, Beckman Ultrasphere C18 ODS, 5 µm, 4.6 mm × 250 mm; flow, 1 mL/min) carried out at the end of the purification process are shown in Figure 1. In this HPLC system the Bamet-R2 retention time was 9 min. Stability in Solution. Bamet-R2 was dissolved (100 µM) in ultrapure deionized water or in saline solution (150 mM NaCl). These solutions were maintained in the dark at 4 °C. After vigorous stirring, samples were collected periodically for immediate analysis by HPLC according to the analytical adaptation of the semipreparative procedure described above. The concentration of Bamet-R2 in solution was calculated from the height of the absorbance peak (250 nm wavelength) eluted at 9 min (Figure 2). In Vitro Cytostatic Studies. The complex was evaluated for in vitro cytostatic activity against murine lymphocytic leukemia L1210 and human colon adenocarcinoma LS174T cells, obtained from the American Type Culture Collection (ATCC, Rockville, MD), and rat hepatocytes in primary culture, obtained by an adaptation of the two-step collagenase perfusion method (16). Cells were grown in a humidified atmosphere of 95% air/ 5% CO2 at 37 °C using minimum essential medium Eagle (MEM, Sigma, for LS174T cells) or Dulbecco’s modified Eagle’s medium (DMEM, Sigma, for L1210 cells) supplemented with 2 mM glutamine, 26.2 mM NaHCO3, 25 mM

Hepes, and 10% horse (L1210 cells) or fetal bovine (LS174T cells) serum in the culture of cell lines and serum-free Williams’ Medium E (Sigma) supplemented with 26.2 mM NaHCO3, 10 mM Hepes, 100 nM Na2SeO3, 30 nM dexamethasone, 100 nM insulin, 5 nM EGF, 11.1 mM galactose, 1 µM ethanolamine, 5 µg/mL transferrin, 5 µg/mL linoleic acid, 5 µg/mL albumin, 10 mM nicotinamide, and 6 mM ornithine in the culture of rat hepatocytes. On the basis of preliminary studies on the time course of cell growth, the following protocols were carried out for testing purposes. LS174T and L1210 cells were harvested during the exponential growth phase, diluted 1/20 with culture medium, seeded into 3 cm diameter cell culture dishes, and exposed to the tested compound for 72 h. Hepatocytes were allowed to become attached to the dish and start proliferation. The compounds were added to the culture at 24 h, and the liver cells were exposed for up to 72 h of culture. Studies comparing the cytostatic activity of cisplatin and Bamet-R2 were carried out using eight different concentrations (from 0.063 to 100 or 200 µM) of the desired compound in the culture medium. At the end of the culture period, the culture medium and cell debris were removed and attached cells were washed and digested using Lowry solution (100 mM NaOH and 189 mM Na2CO3), after which DNA was determined fluorometrically using Hoechst-33258 (17). The trypan blue exclusion test performed on some dishes that were not subjected to digestion confirmed that the viability of remaining cells in the culture dishes was close to 100%. Statistical Analysis. Results are expressed as means ( SE. To calculate the statistical significance of differences among the groups, the Bonferroni method of multiple-range testing was used. Comparison between two means was carried out by Student t-test. Values for IC50 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

Chemical Characterization. The pure complex [named Bamet-R2 (Ba stands for bile acid and -met stands for metal)] is a solid that has a melting point with

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decomposition at 189 °C. The compound was found to be soluble (up to 3 mM) in water and was highly soluble (more than 10 mM) in ethanol, methanol, and dimethyl sulfoxide (DMSO). Its stability in solution was measured by HPLC analysis. In deionized water the compound remained >90% pure in solution for up to 7 days and >80% for up to 28 days. However, in 150 mM NaCl it remains in solution as >90% pure compound for only 1 day (Figure 2). Bamet-R2 elemental analysis (EA) revealed a 1:1 ratio between the cholylglycinate residue and platinum in the complex, in agreement with the molecular formula C26H48N3O6ClPt, ruling out other combinations containing higher or lower numbers of nitrogen atoms and cholylglycine residues (calcd: C, 42.82; H, 6.63; N, 5.76; Pt, 26.75. Found by EA: C, 43.01; H, 6.59; N, 5.80; Pt, 26.68). The stoichiometry of the bile acid moieties and platinum atoms was also assessed by enzymatically measuring concentrations of 3R-hydroxysteroid groups (3R-HS) in a pure solution of Bamet-R2 using the 3R-hydroxysteroid dehydrogenase assay. Platinum concentrations were measured in the same solution by flameless atomic absorption. The result also indicated a 3R-HS-toplatinum ratio of 1:1. The complex was characterized by a combination of spectroscopic methods. These allowed us to propose its structure in the absence of X-ray diffraction studies. The latter were not carried out because it was not possible to crystallize the compound under any of the large list of solvents and conditions assayed (data not shown). In the IR spectrum there are no great differences between the cholylglycine ligand and the complex. Both showed the stretching vibrational modes of NH and OH bonds as a broad band with a maximum at 3408 cm-1 in the ligand and as a split band in the complex, with a maximum at 3434 cm-1, a small peak at 3521 cm-1, and a shoulder at 3311 cm-1. Major differences are expected to appear in the part of the ligand nearest to the metal or directly bonded to it. Thus, the carboxylate in the free CG shows characteristic bands at 1603 cm-1 (υas) and 1400 cm-1 (υas). By contrast, in the complex, the asymmetric vibration band appears at 1638 cm-1, with an increase of 35 cm-1 (18, 19). The remaining absorption peaks appear without changes in the complex because they are related to unmodified skeletal vibrations (20, 21). Finally, in the far infrared the pronounced sharp absorption at 320 cm-1 of the vibration ν(Pt-Cl) appears at the same wavelength, although with much lower intensity than that observed in the case of pure cisplatin (22). In the mass spectrum the molecular ion was not observed. Instead, a group of ions around m/z 693.3 had the highest value. These can be interpreted as [M - Cl]+ produced by the ions containing different platinum isotopes, as suggested by their intensities, which match the natural abundance of platinum ions and the pattern displayed in a computer simulation of [M - Cl]+ (m/z 693.3 calcd). The loss of Cl- from this type of complex when it is analyzed by this technique is well documented (23). Other ions found at m/z 413, 289, and 176 can be considered as the result of typical fragmentation of the cholylglycinate moiety (24, 25). Nuclear magnetic resonance (NMR) spectra of the complex confirmed the presence of only one type of cholylglycinate residue, which in comparison with the spectra of the free ligand is slightly modified in some of its signals by the effect of the association around the metal ion. The presence of the metal, two ammonia ligands, and the chlorine produces variations in the

Criado et al. Table 1. NMR Analysis of Bamet-R2 in CD3ODa C

NaCG

Bamet-R2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

36.5 31.3 72.9 40.5 43.0 35.9 69.1 41.1 27.7 35.9 29.6 74.0 47.5 43.2 24.2 28.7 48.1 13.0 23.2 37.0 17.6 34.2 33.1 176.3 44.6 176.5

36.5 31.3 72.9 40.6 43.0 35.9 69.1 41.1 27.7 35.9 29.6 74.1 47.6 43.3 24.2 28.7 47.7 13.0 23.2 37.3 18.0 * 33.1 182.7 56.0 189.4

13C

H

and 1H for NaCG and NaCG

Bamet-R2

18

0.71 s

0.71 s

19

0.92 s

0.91 s

21

1.03 d

1.04 s

other

1.30-2.35 m

1.25-2.35 m

3

3.38 m

3.38 m

25

3.74 s

4.05 s

7

3.80 bs

3.79 bs

12

3.95 bs

3.94 bs

a Numbering of the positions for C is the usual one for a bile acid structure of cholanoic acid type. Numbering of H is that of the carbon to which H is bound. *, no signal found masked by others in the spectrum.

signals of closer atoms: hydrogen atoms at position 25 and the glycinate methylene, in the 1H NMR spectrum, and C-24 to C-26 in the 13C NMR spectrum. The observed values are shown in Table 1 in comparison with the chemical shifts of the NaCG free ligand (26-28). All these data are in agreement with the presence of only one type of cholylglycinate ligand in the complex, because the simultaneous existence of a monodentate and a bidentate ligand would produce duplicate sets of signals in the NMR spectra. Furthermore, the elemental analysis and mass spectrum clearly support the presence of two amino, one chloride, and one cholylglycinate ligand in the complex. The conductivity measurements carried out in water and methanol are consistent with the rest of the data obtained from Bamet-R2. In water, the Bamet-R2 concentration used was 0.87 mM and the observed conductivity was ΛM ) 50.5 Ω-1 cm2 mol-1. In methanol, the conductivity displayed by a 0.85 mM Bamet-R2 solution was ΛM )33.17 Ω-1 cm2 mol-1. These results confirmed the absence of ions in both solutions, further supporting the structure proposed for this complex. Therefore, the isolated Bamet-R2 is assumed to be a neutral complex, the platinum charge being compensated by both a cholylglycinate and a chloride, carrying two additional ammonia ligands to complete the coordination sphere, as represented below. The geometry of the complex

presumably is of the planar-quadrate type usually displayed by this kind of Pt(II) derivative. Cytostatic Activity. According to the results of previous in vivo studies (29), we have reported that unlike the taurine-amidated cholic acid and similarly to

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Conversely, hepatocytes were more sensitive to BametR2 (IC50 ) 131 ( 20 µM) than L1210 (IC50 ) 170 ( 20 µM). This difference makes sense because hepatocytes, but not L1210 cells, were expected to be able to take up Bamet-R2 more efficiently than cisplatin, provided that this bile acid derivative is indeed transported across the plasma membrane by the specific carrier proteins responsible for liver bile acid uptake (11). Efficient bile acid intestinal absorption, mainly in the ileum, also occurs via both passive and active transport processes, the latter involving specific carrier proteins located at the basal and apical membranes of ileocytes. Although there are no available data on the ability of LS174T cells to take up these compounds, this possibility cannot be ruled out and thus, very interestingly, we found that this human colon cancer-derived cell line was highly sensitive to Bamet-R2 (IC50 ) 17 ( 2 µM). In summary, these results show a strong cytostatic ability of Bamet-R2 on rat hepatocytes in primary culture and L1210 cells, which was significantly higher (p < 0.05) against LS174T cells. Moreover, they suggest that the potential interest of this new complex would be based on a combination of its cytostatic ability, which is lower than that of cisplatin, and the markedly enhanced enterohepatic organotropism as compared with cisplatin, which has been reported in preliminary studies (12, 13). ACKNOWLEDGMENT

This study was supported in part by the Ministerio de Educacion y Ciencia (Grants SAF94-0693 and SAF960146), Spain. We thank Ms. M. I. Hernandez Rodriguez for secretarial help, Mr. M. Fernandez Gutierrez for technical assistance, and Mr. J. F. Martin Martin for caring for the animals. We also thank Nicholas Skinner for revising the English version of the manuscript. LITERATURE CITED

Figure 3. Effect of CG, cisplatin, and Bamet-R2 on cell growth as revealed by measurement of the amount of DNA in primary culture of rat hepatocytes, mouse lymphocytic leukemia L1210, and human colon adenocarcinoma LS174T cells. Cultures involved incubation with one of the above-mentioned compounds at the indicated concentrations from the beginning of the culture, in the case of cell lines, or after replacing the initial culture medium at 24 h from seeding in the case of rat hepatocytes to the end of the culture at 72 h, when the viability of the cells was tested and DNA contents in the dishes measured. Values are means ( SE of three different cultures carried out in triplicate. *, p < 0.05 as compared with cultures incubated with Bamet-R2 by the Bonferroni method of multiplerange testing.

other different glycine-conjugated bile acids, NaCG does not seem to be able to affect the rate of liver cell proliferation. The present work confirms this point for rat hepatocytes in primary cultures (Figure 3). A similar absence of effect of NaGC on L1210 and LS174T cells was found (Figure 3). Cisplatin, which presumably enters the cells through a nonspecific pathway (probably by diffusion through the plasma membrane), was found to exert cytostatic activity against all of the cell types studied (Figure 3). Growth of the culture, as measured by the amount of DNA in the dishes at the end of the exposure period (72 h), was significantly reduced by incubation in the presence of cisplatin. Calculation of the concentration required to reduce the cell population by 50%, or inhibitory concentration 50 (IC50), indicated that the sensitivity to cisplatin was higher for tumoral cell lines (L1210, IC50 ) 18 ( 3 µM; LS174T, IC50 ) 3.3 ( 0.5 µM) than for rat hepatocytes (IC50 ) 56 ( 7 µM).

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