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Transporter-targeted cholic acid-cytarabine conjugates for improved oral absorption
International Journal of Pharmaceutics 511 (2016) 161–169
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Transporter-targeted cholic acid-cytarabine conjugates for improved oral absorption Dong Zhanga,1, Dongpo Lia,b,1, Lei Shangc,**, Zhonggui Hea , Jin Suna,* a
Department of Pharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University, No. 103, Wenhua Road, Shenyang, 110016, China State Key Lab of New-tech for Chinese Medicine Pharmaceutical Processes, Lianyungang, 222001, China c School of Pharmacy, China Medical University, Shenyang 110122, China b
A R T I C L E I N F O
Article history: Received 22 March 2016 Received in revised form 17 June 2016 Accepted 30 June 2016 Available online 1 July 2016 Keywords: Cytarabine Bile acid transporters Prodrugs Oral bioavailability
A B S T R A C T
Cytarabine has a poor oral absorption due to its rapid deamination and poor membrane permeability. Bile acid transporters are highly expressed both in enterocytes and hepatocytes and to increase the oral bioavailability and investigate the potential application of cytarabine for liver cancers, a transporterrecognizing prodrug strategy was applied to design and synthesize four conjugates of cytarabine with cholic acid (CA), chenodeoxycholic acid (CDCA), hyodeoxycholic acid (HDCA) and ursodeoxycholic acid (UDCA). The anticancer activities against HepG2 cells were evaluated by MTT assay and the role of bile acid transporters during cellular transport was investigated in a competitive inhibition experiment. The in vitro and in vivo metabolic stabilities of these conjugates were studied in rat plasma and liver homogenates. Finally, an oral bioavailability study was conducted in rats. All the cholic acid-cytarabine conjugates (40 mM) showed potent antiproliferative activities (up to 70%) against HepG2 cells after incubation for 48 h. The addition of bile acids could markedly reduce the antitumor activities of these conjugates. The N4-ursodeoxycholic acid conjugate of cytarabine (compound 5) exhibited optimal stability (t1/2 = 90 min) in vitro and a 3.9-fold prolonged half-life of cytarabine in vivo. More importantly, compound 5 increased the oral bioavailability 2-fold compared with cytarabine. The results of the present study suggest that the prodrug strategy based on the bile acid transporters is suitable for improving the oral absorption and the clinical application of cytarabine. ã 2016 Elsevier B.V. All rights reserved.
1. Introduction Cytarabine (1-b-D-arabinofuranosylcytosine, ara-C, 1), a pyrimidine nucleoside analog, is widely used in the treatment of both acute and chronic myeloblastic leukemias (Pallavicini, 1984; Rustum and Raymakers, 1992). Cytarabine exhibits a cytotoxic effect in its triphosphate form (ara-CTP), which interferes with the synthesis of DNA by becoming incorporated into the DNA chain and inhibiting DNA polymerase during the S-phase of dividing cells (Chhikara and Parang, 2010). Therefore, the formation and retention of intracellular ara-CTP are closely related to its biological activities. However, it is very difficult to produce an
* Corresponding author at: No. 59 Mailbox, Department of Biopharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University, No. 103 of Wenhua Road, Shenyang 110016, China. ** Corresponding author. E-mail addresses: [email protected] (L. Shang), [email protected] (J. Sun). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ijpharm.2016.06.139 0378-5173/ã 2016 Elsevier B.V. All rights reserved.
adequate amount of ara-CTP inside the target cells following oral administration. Before being converted into its active metabolite (ara-CTP), cytarabine has to face three key challenges: (i) the ubiquitous cytidine deaminase which rapidly convert cytarabine into an inactive form (1-b-D-arabinofuranosyluracil, ara-U) (Aoshima et al., 1976); (ii) the poor membrane permeability due to the hydrophilicity and low affinity for nucleoside transporter (Huber-Ruano and Pastor-Anglada, 2009); and (iii) the intracellular deoxycytidine, which competitively inhibits the phosphorylation of cytarabine to cytarabine mononucleotide (ara-CMP) by deoxycytidine kinase (dCK) (Pallavicini, 1984; Stegmann et al., 1993). Thus, the clinical administration of cytarabine has been limited to intravenous infusion (Sun et al., 2009). A complex and precise dosage schedule of cytarabine is also required to obtain the optimal effectiveness against leukemia (Aoshima et al., 1976; Momparler, 2013). In addition, unlike leukemia cells, solid tumor cells do not have an abundance of nucleoside transporters, which could partly explain why cytarabine exhibits limited anticancer efficacy against solid tumors (Tobias and Borch, 2004).
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Various prodrug approaches have been investigated to address the issues mentioned above (Chhikara and Parang, 2010). Due to the rapid enzymatic hydrolysis of 4-amino group (i.e., N4), most cytarabine derivatives are substituted at this position to prevent the metabolism of cytarabine to ara-U. N4-substituted leucine and isoleucine of cytarabine have been shown to have better metabolic stability compared with cytarabine (Jin et al., 2008). When fatty acid was conjugated to the N4-position of cytarabine, the prodrug exhibited potent antitumor activity against mouse leukemia, resulting from the inhibition of deamination as well as the improved lipophilicity (Aoshima et al., 1976). The 50 -hydroxyl (50 OH) of cytarabine is another site widely used to design cytarabine prodrugs. The 50 -lipophilic phosphate prodrugs of cytarabine has been synthesized to produce ara-CMP directly independent of the level or activity of dCK, thus bypassing the rate-limiting phosphorylation process (Rosowsky et al., 1982). In addition, 50 fatty acid derivatives of cytarabine have also been evaluated. Among such prodrugs, CP-4055 (ara-C-50 -elaidic acid ester) is an outstanding candidate for clinical application, with increased cytotoxicity in both leukemia and solid tumors (Bergman et al., 2004). The results of a Phase I clinical study demonstrated that CP4055 was active against solid tumors (Dueland et al., 2009) and further studies showed that the increased cytotoxicity of cytarabine was primarily helped by the increased cellular uptake of CP-4055 (Breistol et al., 1999). Considering the marked anticancer activity of CP-4055, it has been speculated that altering and/or adding the transmembrane pathway of cytarabine might be an efficient strategy to generate a sufficient level of intracellular cytarabine to combat solid tumors. Hepatocellular carcinoma (HCC), similar to other solid tumors, is insensitive to cytarabine, and is strongly associated with the deficient expression of nucleoside transporters (Galmarini et al., 2001; Huber-Ruano and Pastor-Anglada, 2009; Galmarini et al., 2010). Fortunately, hepatocytes also express other transporters such as bile salt transporters (Kullak-Ublick et al., 2004), providing a potential novel approach for increased uptake of cytarabine via cholic acid-cytarabine conjugates. Among these transporters, Na+dependent taurocholate cotransporting polypeptide (NTCP) and Na+-independent organic anion transporting polypeptides (OATPs), are two key hepatocellular basolateral transporters, responsible for the transport of bile salts to hepatocytes (Alrefai and Gill, 2007). Due to the multi-substrate specificity of NTCP and
OATPs, some liver-specific cholic acid-drug conjugates based on bile salt transporters have been developed for the successful treatment of liver disease (Gonzalez-Carmona et al., 2013; Vivian and Polli, 2014; Dong et al., 2015). Although a slight downregulation of these transporters on HCC cells has been reported (von Dippe and Levy, 1990), cholic acid-cytostatic agent (cisplatin (Larena et al., 2001; Briz et al., 2002) or chlorambucil (KullakUblick et al., 1997)) can enter HCC cells effectively, showing that the expression of bile salt transporters is suitable for drug targeting delivery. Apart from hepatocytes, ileal enterocytes express the apical Na+-dependent bile acid transporter (ASBT) which is involved in the enterohepatic circulation of bile acids. It is also responsible for the intestinal reabsoption of bile acids (Kolhatkar and Polli, 2012). Due to its high transport capacity and broad substrate specificity, ASBT has been used as a potential prodrug target to increase oral drug absorption (Dawson, 2011). For instance, a bile acid-acyclovir conjugate increased the oral bioavailability of acyclovir 2-fold in rats (Tolle-Sander et al., 2004). Also, when low molecular weight heparin (LMWH) was conjugated with deoxycholic acids, its oral bioavailability was increased 5-fold compared with LMWH itself in rats (Al-Hilal et al., 2014). These results show that cholic acid-drug conjugates are able to improve the oral absorption of poorly permeable drugs. Numerous bile acids have been identified in humans, such as cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA) and ursodeoxycholic acid (UDCA) (de Aguiar Vallim et al., 2013). These bile acids are metabolites of cholesterol and could be transported by bile acid transporters. Their structural differences involve the number and position of the hydroxyl groups on the steroid backbone. Although bile acid transporters can transport natural bile acids efficiently, the structural requirements for bile acid prodrugs need to be taken into account. It has been reported that the C-7 hydroxyl group on the cholestane ring plays a critical role during the transport process (Kolhatkar and Polli, 2012). The recommended conjugation positions are the C-3 hydroxyl group and the C-24 region (Rais et al., 2010; Gonzalez et al., 2014). A previous report showed that an amide-bond prodrug of cholic acid and cytarabine exhibited superior plasma stability compared with ester-bond conjugates (Chen et al., 2011). Therefore, in the present study, four cholic acid-cytarabine conjugates (see Fig. 1) were synthesized using the amide bond between cytarabine (4-NH2)
Fig. 1. Chemical structures of cytarabine (compound 1) and four cholic acid-cytarabine conjugates (compounds 2–5).
D. Zhang et al. / International Journal of Pharmaceutics 511 (2016) 161–169
and bile acid (24-COOH) to increase the oral absorption of cytarabine. The successful synthesis of compounds 2–5 was confirmed by 1H NMR and 13C NMR spectra. Then their antitumor activities against HepG2 cells were tested in vitro and a cellular uptake mechanism study was also conducted in a competitive inhibition experiment. The in vitro metabolic stability of the four prodrugs was investigated in rat plasma and liver homogenates. It should be pointed out that, to date, there are no published reports of the effects of bile acids on the metabolic stability and anticancer ability of cholic acid-cytarabine conjugates against HCC. Finally, the oral bioavailability of compound 5 was also evaluated in rats, in comparison with cytarabine. 2. Materials and methods 2.1. Materials Cytarabine was purchased from Surui Chemical Corp. (Suzhou, China), while cholic acid (CA), chenodeoxycholic acid (CDCA), hyodeoxycholic acid (HDCA) and ursodeoxycholic acid (UDCA) were obtained from Zhixin Chemicals Inc (Shanghai, China). Lamivudine was provided by the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Isobutyl chloroformate was obtained from Gade Chemicals Inc (Shanghai, China). Reagents of analytical grade were purchased from Tianjin bodi Chemicals Inc (Tianjin, China) and those of high-performance liquid chromatography grade were obtained from Merck (Darmstadt, Germany). Deionized-distilled water was used throughout the study. A human leukemia cell line (HL-60) and a human hepatocellular liver carcinoma (HepG2) cell line were provided by American Type Culture Collection (ATCC). Fetal bovine serum and RPMI-1640 medium were obtained from Hyclone Biochemical Product Co., Ltd. (Beijing, China). Trypan blue and methyl thiazolyl tetrazolium (MTT) were supplied by Sigma-Aldrich (St. Louis, MO, USA). 2.2. Synthesis of cholic acid-cytarabine conjugates 2.2.1. (R)-N-(1-((3S,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl) tetrahydro-furan-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-(R)-4((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl) pentanamide (2) To a stirred solution of cholic acid (0.49 g, 1.2 mmol) in anhydrous N, N-dimethylformamide (DMF, 10 mL), triethylamine (0.20 mL, 1.5 mmol) was added under nitrogen atmosphere. The mixture was cooled to 15 C and a solution of isobutyl chloroformate (0.20 mL, 1.5 mmol) was added dropwise. The mixture was stirred at 15 C for 15 min and a solution of cytarabine (0.24 g, 1.0 mmol) and triethylamine (0.20 mL, 1.5 mmol) in 10 mL anhydrous DMF was added dropwise to the reaction mixture at the same temperature. The reaction mixture was continually stirred for 30 min at room temperature. After the reaction had been completed, the solvent was removed by RE-52A rotary evaporation (Shanghai Yarong Biochemical Instrument Plant, China). The residue was then purified by chromatography on silica gel eluting with dichloromethane/methanol (10:1) to obtain pure compound 2 as an amorphous white solid (0.50 g, 78%). 1 H NMR(300 MHz, d6-DMSO, d ppm): 10.8 (s, 1H), 8.04 (d, 1H, J = 7.5 Hz), 7.20 (d, 1H, J = 7.5 Hz), 6.05 (d, 1H, J = 3.9 Hz), 5.47(d, 2H, J = 3.0), 5.05 (t, 2H), 4.31 (d, 1H, J = 3.9 Hz), 4.09 (d, 1H, J = 3.3), 3.98 (d, 1H, J = 3.3), 3.78 (s, 1H), 3.61 (s, 1H), 3.18 (t, 1H), 2.51-1.21 (m, 24H), 1.15 (d, 2H, J = 7.2 Hz) 0.93 (d, 3H, J = 6.2 Hz), 0.80 (s, 3H), 0.59 (s, 3H). 13C NMR (75 MHz, d6-DMSO, d ppm): 174.4, 162.3, 154.6, 146.7, 94.5, 87.0, 85.8, 76.2, 74.7, 71.1, 70.5, 66.4, 61.0, 46.3, 45.8, 41.6, 41.4, 35.4, 35.3, 34.9, 34.5, 33.8, 31.0, 30.5, 28.6, 27.3, 26.3,
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22.9, 22.7, 17.1, 12.4. MS (ESI) (m/z): calcd for C33H51N3O9: m/z 634.2 [M+H]+, 656.2 [M+Na]+. 2.2.2. (4R)-N-(1-((3S,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl) tetrahydro-furan-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-(R)-4((3R,5S,7R,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl) pentanamid (3) Compound 3 was synthesized from chenodeoxycholic acid (CDCA) and cytarabine, then purified as described for compound 2 to obtain an amorphous white solid (0.48 g, 78%). 1H NMR (300 MHz, d6-DMSO, d ppm): 10.9 (s, 1H), 8.14 (d, 1H, J = 7.5 Hz), 7.21 (d, 1H, J = 7.5 Hz), 6.18 (d, 1H, J = 4.8 Hz), 5.89 (d, 2H, J = 5.1), 5.11 (t, 2H), 4.28 (d, 1H, J = 4.5 Hz), 4.08 (d, 1H, J = 3.3), 3.63 (s, 1H), 3.18 (t, 1H), 2.26-0.94 (m, 28H), 0.87 (d, 4H, J = 6.6 Hz), 0.84 (s, 4H), 0.61 (s, 3H) 13C NMR (75 MHz, d6-DMSO, d ppm): 174.4, 162.3, 154.6, 146.7, 94.4, 87.0, 85.8, 76.2, 74.7, 70.4, 66.2, 61.1, 55.6, 50.1, 42.0, 41.5, 35.4, 35.2, 35.0, 34.8, 33.5, 32.4, 30.9, 30.6, 27.9, 23.2, 22.8, 20.3, 17.1, 12.4. MS (ESI) (m/z): calcd for C33H51N3O8 m/z 618.2 [M+H]+, 640.2 [M +Na]+. 2.2.3. (4R)-N-(1-((3S,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl) tetrahydro-furan-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-(R)4((5R,8S,10R,13R,17R)-3,6-dihydroxy-10,13-dimethyl2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a] phenanthren-17-yl)pentanamid (4) Compound 4 was synthesized from hyodeoxycholic acid (HDCA) and cytarabine and was purified as described for compound 2 to obtain an amorphous white solid (0.49 g, 80%). 1 H NMR(300 MHz, d6-DMSO, d ppm): 10.8 (s, 1H), 8.02 (d, 1H, J = 7.5 Hz), 7.17 (d, 1H, J = 7.5 Hz), 6.04 (d, 1H, J = 3.3 Hz), 5.44 (d, 2H, J = 3.5), 5.03 (q, 1H), 4.27 (d, 1H, J = 3.9 Hz), 4.05 (d, 3H, J = 5.4 Hz), 3.92 (s, 1H), 3.82 (s, 1H), 3.59 (d, 3H, J = 5.4), 3.15 (d, 1H, J = 4.8 Hz), 2.49-0.97 (m, 25H), 0.88 (d, 3H, J = 6.0 Hz), 0.82 (s, 3H), 0.59 (s, 3H). 13 C NMR (75 MHz, d6-DMSO, d ppm): 174.5, 162.3, 154.6, 146.7, 94.4, 87.0, 85.8, 76.2, 74.7, 71.1, 70.5, 66.3, 61.1, 46.3, 45.8, 41.6, 41.5, 35.4, 35.2, 35.0, 34.5, 33.8, 31.0, 30.5, 28.6, 27.3, 26.3, 22.9, 22.7, 17.1, 12.4. MS (ESI) (m/z): calcd for C33H51N3O8: m/z 618.2 [M+H]+, 640.2 [M+Na]+. 2.2.4. (4R)-N-(1-((3S,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl) tetrahydro-furan-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-(R)-4((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl) pentanamid (5) Compound 5 was synthesized from ursodeoxycholic acid (UDCA) and cytarabine, and was purified as described for compound 2 to obtain an amorphous white solid (0.54 g, 87%). 1 H NMR(300 MHz, d6-DMSO, d ppm): 10.79 (s, 1H), 8.03 (d, 1H, J = 7.5 Hz), 7.19 (d, 1H, J = 7.5 Hz), 6.05 (d, 1H, J = 3.9 Hz), 5.46 (d, 2H, J = 4.5), 5.04 (q, 1H), 4.41 (d, 1H, J = 4.5 Hz),4.04 (s, 1H), 3.85 (s, 1H), 3.82 (d, 2H, J = 3.0), 3.62 (q, 1H), 2.51–0.96 (m, 29H), 0.88 (d, 3H, J = 6.0 Hz), 0.82 (s, 3H), 0.59 (s, 3H). 13C NMR (75 MHz, d6-DMSO, d ppm): 174.4, 162.3, 154.6, 146.7, 94.4, 87.0, 85.8, 76.2, 74.7, 69.8, 69.5, 61.1, 56.0, 54.8, 43.2, 43.1, 42.3, 38.8, 37.8, 37.4, 35.0, 34.9, 33.8, 33.6, 31.0, 30.3, 28.2, 26.8, 22.4, 17.1, 12.4. MS (ESI) (m/z): calcd for C33H51N3O8: m/z 618.2 [M+H]+, 640.2 [M+Na]+. 2.3. HPLC of cholic acid-cytarabine conjugates Four cholic acid-cytarabine conjugates were determined by HPLC (Hitachi, Japan). A C18 analytical column (4.6 mm 250 mm) was maintained at 40 C. In brief, chromatographic separation was carried out using isocratic elution with a mobile phase composed of methanol (70%) and water containing 10 mM ammonium acetate (30%). The detection wavelength was 248 nm and samples
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were delivered at a flow rate of 1.0 mL/min. The auto-sampler was conditioned at 4 C and the sample volume injected was 20 mL. 2.4. UPLC–MS/MS of cytarabine Cytarabine was quantified by UPLC–MS/MS carried out on an ACQUITY UPLC system (Waters Co., Ltd., Milford, MA, USA). A C18 analytical column (50 mm 2.1 mm) was used and maintained at 40 C. A gradient elution program was adopted for chromatographic separation with mobile phase A consisting of methanol containing 0.1% formic acid and mobile phase B consisting of 2% methanol in water. A Waters Tandem Quadrupole (TQ) Detector with an ESI source operating in positive ion mode was used for the determination of cytarabine and lamivudine (IS). Under UPLC–MS/ MS conditions, the precursor/product ion transitions were m/z 244/112 for cytarabine (collision energy 15 eV) and m/z 230/112 for lamivudine (collision energy 15 eV). The autosampler was conditioned at 4 C and the sample volume injected was 5 mL. Injection wash solvents were methanol-water (10:90, v/v) and methanolwater (90:10, v/v) for the weak and strong wash, respectively. 2.5. Cell proliferation assays The human leukemic cell line (HL-60) and human hepatocellular carcinoma cell line (HepG2) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin. All the cells were maintained in an atmosphere of 5% CO2 and 90% relative humidity at 37 C. The HL-60 cells were suspended in their respective media to give 2 mL volumes of cell suspension at a final density of 5 104 cells/mL. Appropriate volumes of drug solution were transferred to the cell suspensions, and incubation was continued for 72 h. Cell viability was determined by staining with trypan blue. Trypan blue-stained (nonviable) cells and the total cell number were measured using a hematocytometer. The growth rates of cells after treatments with these cholic acid-cytarabine conjugates at different concentrations were compared with that of control cells. The data obtained were analyzed using a Logit model, and the results were expressed as the IC50 values (the drug concentration that inhibits cell growth by 50% of the control value). HepG2 cells were seeded into 96-well plates (5 104 cells/mL) and cultured at 37 C in an incubator for 24 h. The cells were then incubated with the same concentration (40 mM) of cytarabine, cholic acidcytarabine conjugates (2–5) and compounds 2–5 with the same amount of CA, CDCA, HDCA and UDCA respectively for 24 h and 48 h, and then 100 mL of a solution of MTT was added to each well, and the plates were returned to the incubator for 3 h. The data were obtained using ELISAs. 2.6. Stability of cholic acid-cytarabine conjugates The stability of cholic acid-cytarabine conjugates was studied at 37 C by incubating a drug solution (3.5 mM) in phosphate buffer solution (PBS) at pH 7.4, fresh plasma or liver homogenates. For the first medium, the samples were detected by HPLC directly. For the latter two media, Sprague–Dawley (SD) rats were first anesthetized with ether, and then the whole blood and livers were collected. All samples were placed in heparinized tubes. The rat plasma samples were centrifuged at 10,000 rpm for 10 min, collected, and diluted by isovolumetric 0.05 M PBS. The tissue was homogenized in a tissue homogenizer and diluted with isovolumetric 0.05 M PBS. At predetermined time, the metabolic reaction was quenched by adding a 4-fold volume of methanol followed by vortexing. The mixture was then centrifuged at 10000 rpm for 10 min and the supernatant was passed through a membrane filter (0.22 mm) and analyzed by HPLC.
2.7. Release of cytarabine in rat plasma The cytarabine released from the conjugates was evaluated following incubation in rat plasma diluted with 50 mM PBS at 37 C. The initial drug concentration was 7.0 mM calculated as cytarabine. After a 2 h incubation, the plasma samples were collected for further analysis. To an aliquot of each plasma sample (30 mL), internal standard solution (lamivudine, 200 ng/mL) (30 mL) and water (400 mL) were added. Each sample was shaken and centrifuged, and then the supernatant was transferred to a solid-phase extraction cartridge. The eluent solution was determined by UPLC/MS/MS. 2.8. Pharmacokinetics in rats Male SD rats weighing 210–240 g were divided randomly into two groups. All animal experiments were performed by following institutional guidelines and were approved by the University Committee on Use and Care of Animals, Shenyang Pharmaceutical University. The aqueous solutions of cytarabine and compound 5 were prepared with 0.1% Tween-80 to give a final concentration of 0.5 mg/mL. Two groups of rats were given cytarabine and prodrug (compound 5) solutions (30 mg/kg, calculated as cytarabine) by oral gavage. Serial blood samples (0.2 mL) were obtained at 2, 5, 15, 30, 45 min and 1, 1.5, 2, 3, 4, 6, 8, 12 and 24 h after administration. During sampling, rats were anesthetized with ether. All samples were collected in heparinized tubes. The rat plasma samples were centrifuged at 10,000 rpm for 10 min, and then frozen at 80 C. After using the solid-phase extraction cartridge as described in the section “2.7. Release of cytarabine in rat plasma”, the samples were analyzed by UPLC/MS/MS. The maximum plasma concentration of cytarabine (Cmax) and the time to reach Cmax (Tmax) were obtained directly from the plasma concentration time profiles. The area under the curve (AUC) and the half-life (t1/2) of cytarabine were calculated using DAS 2.0 software. 2.9. Statistical analysis All statistical analyses in this paper were performed using Student’s t-test. The differences were considered significant at p < 0.05 and markedly significant at p < 0.01. 3. Results and discussion 3.1. Characterization of cholic acid-cytarabine conjugates The 1H NMR spectra confirmed that the acylation of cytarabine was at the position of the amino group without any involvement of the sugar OH group. In addition, due to acylation at the N4 position, a distinct blue shift (272 nm to 248 nm) of lmax could also be observed in the UV spectra of these cytarabine conjugates, consistent with a previous report (Schiavon et al., 2004). These results confirmed the successful synthesis of cytarabine prodrugs. The purity of cholic acid-cytarabine conjugates was examined by HPLC. As shown in Fig. 2, compounds 2–5 with similar structures had different retention times (tR) of 18.1 min, 10.4 min, 7.70 min and 10.3 min, respectively. It was clear that compounds 2– 5 could be separated well by the HPLC method used, with the purities of compounds 2–5 being higher than 98%. 3.2. In vitro cytotoxicity of cholic acid-cytarabine conjugates The antiproliferative activity of cytarabine and its conjugates was evaluated using leukemia HL-60 cells and solid tumor HepG2 cells. In the case of HL-60 cells, all these conjugates had higher IC50
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Fig. 2. The typical HPLC chromatogram of compound 2 (a), compound 3 (b), compound 4 (c) and compound 5 (d) in mobile phase.
Table 1 The antiproliferative effects of compound 1 and the conjugates of cytarabine (compounds 2–5) in HL-60 cells after 72 h (mean S.D., n = 3). compound
IC50 (nM)
1 2 3 4 5
14.4 1.6 110.0 13.7 ** 76.9 8.4 ** 90.5 9.3 66.0 5.2
p < 0.01 (**) versus compound 1 as the control.
values than cytarabine (Table 1), indicating that the structure modification with bile acids reduced the intrinsic cytotoxicity of cytarabine against HL-60 cells. Due to its hydrophilic nature, the membrane transport of cytarabine was strongly dependent on nucleoside transporters, such as the equilibrative nucleoside transporter 1 (hENT1) (Tang et al., 2012; Yamauchi et al., 2014). After conjugation with bile acids, the affinity of cytarabine for nucleoside transporter might be reduced, resulting in lower transport efficiency. Among these cytarabine prodrugs, compound 2 had the highest IC50 (110.0 13.7 nM), which might be due to the high polarity of CA (see Fig. 1), which did not help in the recognition by nucleoside transporters. Compound 5 had the highest cytotoxicity, probably due to the lower influence on the affinity for the prodrug and transporter. Considering the important role of nucleoside transporters in the cellular uptake, insufficient expression of transporters or a
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lower affinity for transporters would markedly reduce the antiproliferative activity of cytarabine. As shown in Fig. 3, HepG2 cells, lacking nucleoside transporters (MacKenna et al., 2009), were insensitive to cytarabine compared with HL-60 cells. In contrast, these cholic acid-cytarabine conjugates exhibited efficient inhibition (over 60%) of HepG2 cell growth after 48 h treatment. The N4-hyodeoxycholic acid of cytarabine (compound 4) had the most significant antitumor activity (over 70% inhibition). It has been established that HepG2 cells express bile salt transporters (OATPs, not NTCP) and produce rapid uptake of bile acids via OATP- mediated transport (Kullak-Ublick et al., 1996; Lee et al., 2001). Moreover, benefiting from the broad substrate specificity of OATPs, a wide variety of drug-bile acid conjugates can also be transported via such a pathway (Kullak-Ublick et al., 1994). Therefore, we speculated that cholic acid-cytarabine conjugates can also be taken up via multispecific OATPs, producing an increased intracellular accumulation of cytarabine against HepG2. To confirm this hypothesis, competitive inhibition experiments were performed to investigate the effects of corresponding bile acids on the cellular uptake of compounds 2–4. Before that, the cell viability of HepG2 treated with four bile acids was determined at 37 C for 48 h. These bile acids produced no cytotoxicity (data not shown), indicating the safety of these endogenous molecules. As shown in Fig. 4C and D, simultaneous treatment with conjugates and corresponding bile acids markedly reduced the cytotoxicity. Also, no effects of bile acids on the cytotoxicity induced by cytarabine were found (Fig. 4A and B). These results demonstrated that cholic acid-cytarabine conjugates altered the transport pathway of cytarabine and the OATPs-mediated uptake was indeed involved in the cellular internalization of these conjugates in HepG2 cells. 3.3. Stability and release of cholic acid-cytarabine conjugates Cholic acid-cytarabine conjugates were developed to protect cytarabine from deamination, and are also able to target the bile acid transporters expressed in HCC cells. In this case, the cholic acid-cytarabine conjugates have a dual functionality, i.e., increased stability via N4-acidylated modification and improved membrane permeability. As the structural integrity of these conjugates directly affects these functional implementations, the stability of these conjugates before intestinal absorption was first investigated in pH 7.4 PBS. Then their metabolic stability was evaluated in rat plasma and liver homogenates. As shown in Fig. 5A, these
Fig. 3. The inhibition of HepG2 cells proliferation by the compounds 1-5 (a-e) at 40 mM after (A) 24 h and (B) 48 h. The results are shown as the percentage of the control containing 1% DMSO (mean + S.D., n = 3). p < 0.05 (*) and p < 0.01 (**) versus compound 1 as the control.
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Fig. 4. The inhibition of HepG2 cells proliferation by compound 1(a), compound 1 with the corresponding amount of CA (b), CDCA (c), HDCA (d) and UDCA (e) at 100 mM after (A) 24 h and (B) 48 h. The inhibition of HepG2 cells proliferation by compound 1 (a), compounds 2–5 with CA, CDCA, HDCA and UDCA (b-e), respectively, at 40 mM after (C) 24 h and (D) 48 h. The results are shown as the percentage of the control containing 1% DMSO (mean + S.D., n = 3).
conjugates effectively resisted hydrolysis except for compound 2, demonstrating the stability of the amide bonds. The concentrationtime profiles of these conjugates in rat plasma are shown in Fig. 5B. The half-lives of compounds 2–5 were 20 min, 20 min, 30 min and 90 min in rat plasma, respectively. Compound 5, the N4-ursodeoxycholic acid of cytarabine, was the most stable and was resistant to
degradation by deaminase. Contrary to our expectation, degradation of the conjugates was slower in liver homogenates. The percentage of compounds 2–5 remaining after incubation in liver homogenates is shown in Fig. 5C. Among these conjugates, compound 5 exhibited the longest half-life (147 min), followed by compound 2 (91 min) and the other two compounds showed a
Fig. 5. Stability of compounds 2–5 was studied in (A) phosphate buffer solution (PBS) at pH 7.4, (B) SD rat plasma and (C) rat liver homogenates.
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deaminase for a shorter period, resulting in the better metabolic stability of cytarabine. So, compound 5 deserved further investigation. 3.4. Pharmacokinetics in rats
Fig. 6. Cytarabine released from compounds 2–5 (a–d) was evaluated after incubation for 2 h in SD rat plasma, and the plasma concentration was determined by UPLC/MS/MS. The results are shown as the percentage of initial concentration (all calculated as cytarabine, mean + S.D., n = 3).
Fig. 7. Mean plasma concentration-time profiles of cytarabine following oral administration of compounds 1 and 5 at 30 mg/kg (calculated as cytarabine) in SD rats (mean + S.E. n = 6).
half-life less 60 min in liver homogenates. Collectively, compound 5 was identified as the optimal cytarabine prodrug with desired metabolic stability. The release of free cytarabine from the conjugates was also studied in rat plasma. As shown in Fig. 6, all the conjugates could be converted into cytarabine and compound 5 exhibited the highest release rate of cytarabine (70.2%) within 2 h. The conversion and deamination of cytarabine occurred simultaneously in plasma. Benefiting from the slow degradation, the sustainedreleased cytarabine from compound 5 was exposed to cytidine
The oral pharmacokinetic study of compound 5 and cytarabine was conducted in rats to evaluate the feasibility of cholic acid-drug conjugates for improving the oral absorption of cytarabine in vivo. As the concentration of compound 5 in plasma was not high enough to be accurately determined, we mainly focused on the determination of cytarabine after oral administration. The reasons for the low level of the conjugate in plasma might include the increased hepatic uptake due to bile salt transporter-mediated transport (Oatps and Ntcp) (Kullak-Ublick et al., 2004; Alrefai and Gill, 2007), and the higher metabolic enzyme activity in vivo. The mean cytarabine blood concentrationtime profiles of cytarabine after administration of cytarabine and conjugate (compound 5) are shown in Fig. 7, and the main pharmacokinetic parameters of cytarabine are summarized in Table 2. The Cmax in the prodrug group was less than that in the cytarabine group, while the Tmax in the prodrug group was extended from 2.5 0.6 h to 3.6 1.1 h. In addition, compound 5 showed a greater t1/2 for cytarabine (15.62 10.30 h) than that of cytarabine itself (4.00 0.89 h). This steady plasma concentration of compound 5 could be ascribed to the improved stability of the cholic acid-cytarabine conjugate and the sustained release of cytarabine (Zheng and Polli, 2010). More importantly, the AUC0-1 of compound 5 (34857.0 7578.0 ng h/mL) was significantly higher than that of cytarabine (16603.7 2627.2 ng h/mL). Similar to other ASBT targeting prodrugs (Han et al., 2015), compound 5 can be recognized by ASBT and actively transported across the enterocytes. Also, the conjugation of UDCA with cytarabine would also increase the lipophilicity of cytarabine, resulting in improved cellular uptake via a passive transport process. These two factors could be responsible for the 2-fold increase in the oral bioavailability of cytarabine provided by compound 5. As cytarabine is a cell cycle-dependent drug (S phase), the extended high drug concentration would produce higher cytotoxic activity. Compound 5 could maintain a stable and high cytarabine concentration for 24 h, while cytarabine itself showed an obvious plasma concentration fluctuation within the initial 8 h. This demonstrated that compound 5 was more effective with reduced side effects. Apart from the bile acid prodrugs of cytarabine developed here, amino acid derivatives, fatty acid derivatives and phosphate derivatives of cytarabine have also been investigated widely. Among these prodrugs, L-valine-cytarabine has been synthesized in two ways using N4-L-valyl- cytarabine and 50 -L-valyl- cytarabine. The former had only a 4% oral bioavailability of cytarabine in rats due to the limited drug release from the prodrug (Cheon and Han, 2007). While the latter provided a 1.75-fold increased oral bioavailability of cytarabine with a suitable conversion rate in vivo. The results of our study demonstrate that 50 -L-valylcytarabine is absorbed via a (oligopeptide transporter 1) PepT1mediated uptake pathway (Sun et al., 2009; Liu and Liu, 2013). In the present study, compound 5, with good metabolic stability and
Table 2 Pharmacokinetic parameters of cytarabine, following oral administration of compounds 1 and 5 at 30 mg/kg (calculated as cytarabine) to SD rats (mean S.E., n = 6). PK parameters
Tmax (h)
Cmax (h)
t1/2 (h)
AUC0-24 (ng h/mL)
AUC0-1 (ng h/mL)
Frel%
Compound 1 Compound 5
2.5 0.6 3.6 1.1
2103.4 251.8 1706.1 125.0
4.00 0.89 15.62 10.30*
16306.9 2695.5 20223.7 1516.0
16603.7 2627.2 34857.0 7578.0*
100 209.9 45.6
p < 0.05 (*) versus compound 1 as the control.
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bile acid transporter-mediated uptake, exhibited a 2-fold increased oral bioavailability of cytarabine. In addition, the good metabolic stability of compound 5 would contribute to the bile acid transporter-targeted delivery of cytarabine and broaden the potential scope of cytarabine applications, such as HCC. The distribution of cytarabine in liver has also been investigated, but, the rapid hepatic degradation of cytarabine resulted in undetectable drug levels. Therefore, a more sensitive method needs to be developed for the determination of cytarabine distributed in hepatic tissues. Moreover, our future studies will also focus on the detailed mechanism of bile acid transporter-mediated cellular uptake, liver targeting ability and the in vivo antitumor efficacy of cytarabine conjugates against HCC. 4. Conclusions In this study, a series of cholic acid-cytarabine conjugates were designed and synthesized to improve the oral absorption of cytarabine and to test the potential antitumor efficacy against HCC. All the conjugates showed potent antiproliferative activities in HepG2 cells, as a result of an increased intracellular drug level achieved by transporter-mediated uptake. Stability tests in rat plasma and liver homogenates demonstrated that the N4ursodeoxycholic acid of cytarabine (compound 5) exhibited optimal stability in vitro. The pharmacokinetic study in rats demonstrated that compound 5 could increase the oral bioavailability of cytarabine over 2-fold, with the benefit of ASBTmediated transmembrane transport. Compared with cytarabine, compound 5 produced a smoother plasma concentration because of the improved metabolic stability of the cholic acid-cytarabine conjugate. In conclusion, the prodrug strategy applying bile salt as the modified moiety is a promising method to improve the oral absorption and metabolic stability of cytarabine. In addition, the cholic acid-cytarabine conjugate could also give cytarabine a novel curative effect such as for the treatment of HCC. Acknowledgments This work received financial support from the National Nature Science Foundation of China (No. 81373336, 81473164), from the National Basic Research Program of China (973 Program), No. 2015CB932100, and from Program for New Century Excellent Talents in University (No. NCET-12-1015). References Al-Hilal, T.A., Park, J., Alam, F., Chung, S.W., Park, J.W., Kim, K., Kwon, I.C., Kim, I.S., Kim, S.Y., Byun, Y., 2014. Oligomeric bile acid-mediated oral delivery of low molecular weight heparin. J. Control. Release 175, 17–24. Alrefai, W.A., Gill, R.K., 2007. Bile acid transporters: structure, function, regulation and pathophysiological implications. Pharm. Res. 24, 1803–1823. Aoshima, M., Tsukagoshi, S., Sakurai, Y., Oh-ishi, J., Ishida, T., 1976. Antitumor activities of newly synthesized N4-acyl-1-beta-D-arabinofuranosylcytosine. Cancer Res. 36, 2726–2732. Bergman, A.M., Kuiper, C.M., Myhren, F., Sandvold, M.L., Hendriks, H.R., Peters, G.J., 2004. Antiproliferative activity and mechanism of action of fatty acid derivatives of arabinosylcytosine (ara-C) in leukemia and solid tumor cell lines. Nucleosides Nucleotides Nucl. Acids 23, 1523–1526. Breistol, K., Balzarini, J., Sandvold, M.L., Myhren, F., Martinsen, M., De Clercq, E., Fodstad, O., 1999. Antitumor activity of P-4055 (elaidic acid-cytarabine) compared to cytarabine in metastatic and s c. human tumor xenograft models. Cancer Res. 59, 2944–2949. Briz, O., Serrano, M.A., Rebollo, N., Hagenbuch, B., Meier, P.J., Koepsell, H., Marin, J.J., 2002. Carriers involved in targeting the cytostatic bile acid-cisplatin derivatives cis-diammine-chloro-cholylglycinate-platinum(II) and cis-diamminebisursodeoxycholate-platinum(II) toward liver cells. Mol. Pharmacol. 61, 853– 860. Chen, D.Q., Wang, X., Chen, L., He, J.X., Miao, Z.H., Shen, J.K., 2011. Novel liver-specific cholic acid-cytarabine conjugates with potent antitumor activities: Synthesis and biological characterization. Acta Pharmacol. Sin. 32, 664–672. Cheon, E.P., Han, H.K., 2007. Pharmacokinetic characteristics of L-valyl-ara-C and its implication on the oral delivery of ara-C. Acta Pharmacol. Sin. 28, 268–272.
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Transporter-targeted cholic acid-cytarabine conjugates for improved oral absorption Dong Zhanga,1, Dongpo Lia,b,1, Lei Shangc,**, Zhonggui Hea , Jin Suna,* a
Department of Pharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University, No. 103, Wenhua Road, Shenyang, 110016, China State Key Lab of New-tech for Chinese Medicine Pharmaceutical Processes, Lianyungang, 222001, China c School of Pharmacy, China Medical University, Shenyang 110122, China b
A R T I C L E I N F O
Article history: Received 22 March 2016 Received in revised form 17 June 2016 Accepted 30 June 2016 Available online 1 July 2016 Keywords: Cytarabine Bile acid transporters Prodrugs Oral bioavailability
A B S T R A C T
Cytarabine has a poor oral absorption due to its rapid deamination and poor membrane permeability. Bile acid transporters are highly expressed both in enterocytes and hepatocytes and to increase the oral bioavailability and investigate the potential application of cytarabine for liver cancers, a transporterrecognizing prodrug strategy was applied to design and synthesize four conjugates of cytarabine with cholic acid (CA), chenodeoxycholic acid (CDCA), hyodeoxycholic acid (HDCA) and ursodeoxycholic acid (UDCA). The anticancer activities against HepG2 cells were evaluated by MTT assay and the role of bile acid transporters during cellular transport was investigated in a competitive inhibition experiment. The in vitro and in vivo metabolic stabilities of these conjugates were studied in rat plasma and liver homogenates. Finally, an oral bioavailability study was conducted in rats. All the cholic acid-cytarabine conjugates (40 mM) showed potent antiproliferative activities (up to 70%) against HepG2 cells after incubation for 48 h. The addition of bile acids could markedly reduce the antitumor activities of these conjugates. The N4-ursodeoxycholic acid conjugate of cytarabine (compound 5) exhibited optimal stability (t1/2 = 90 min) in vitro and a 3.9-fold prolonged half-life of cytarabine in vivo. More importantly, compound 5 increased the oral bioavailability 2-fold compared with cytarabine. The results of the present study suggest that the prodrug strategy based on the bile acid transporters is suitable for improving the oral absorption and the clinical application of cytarabine. ã 2016 Elsevier B.V. All rights reserved.
1. Introduction Cytarabine (1-b-D-arabinofuranosylcytosine, ara-C, 1), a pyrimidine nucleoside analog, is widely used in the treatment of both acute and chronic myeloblastic leukemias (Pallavicini, 1984; Rustum and Raymakers, 1992). Cytarabine exhibits a cytotoxic effect in its triphosphate form (ara-CTP), which interferes with the synthesis of DNA by becoming incorporated into the DNA chain and inhibiting DNA polymerase during the S-phase of dividing cells (Chhikara and Parang, 2010). Therefore, the formation and retention of intracellular ara-CTP are closely related to its biological activities. However, it is very difficult to produce an
* Corresponding author at: No. 59 Mailbox, Department of Biopharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University, No. 103 of Wenhua Road, Shenyang 110016, China. ** Corresponding author. E-mail addresses: [email protected] (L. Shang), [email protected] (J. Sun). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ijpharm.2016.06.139 0378-5173/ã 2016 Elsevier B.V. All rights reserved.
adequate amount of ara-CTP inside the target cells following oral administration. Before being converted into its active metabolite (ara-CTP), cytarabine has to face three key challenges: (i) the ubiquitous cytidine deaminase which rapidly convert cytarabine into an inactive form (1-b-D-arabinofuranosyluracil, ara-U) (Aoshima et al., 1976); (ii) the poor membrane permeability due to the hydrophilicity and low affinity for nucleoside transporter (Huber-Ruano and Pastor-Anglada, 2009); and (iii) the intracellular deoxycytidine, which competitively inhibits the phosphorylation of cytarabine to cytarabine mononucleotide (ara-CMP) by deoxycytidine kinase (dCK) (Pallavicini, 1984; Stegmann et al., 1993). Thus, the clinical administration of cytarabine has been limited to intravenous infusion (Sun et al., 2009). A complex and precise dosage schedule of cytarabine is also required to obtain the optimal effectiveness against leukemia (Aoshima et al., 1976; Momparler, 2013). In addition, unlike leukemia cells, solid tumor cells do not have an abundance of nucleoside transporters, which could partly explain why cytarabine exhibits limited anticancer efficacy against solid tumors (Tobias and Borch, 2004).
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Various prodrug approaches have been investigated to address the issues mentioned above (Chhikara and Parang, 2010). Due to the rapid enzymatic hydrolysis of 4-amino group (i.e., N4), most cytarabine derivatives are substituted at this position to prevent the metabolism of cytarabine to ara-U. N4-substituted leucine and isoleucine of cytarabine have been shown to have better metabolic stability compared with cytarabine (Jin et al., 2008). When fatty acid was conjugated to the N4-position of cytarabine, the prodrug exhibited potent antitumor activity against mouse leukemia, resulting from the inhibition of deamination as well as the improved lipophilicity (Aoshima et al., 1976). The 50 -hydroxyl (50 OH) of cytarabine is another site widely used to design cytarabine prodrugs. The 50 -lipophilic phosphate prodrugs of cytarabine has been synthesized to produce ara-CMP directly independent of the level or activity of dCK, thus bypassing the rate-limiting phosphorylation process (Rosowsky et al., 1982). In addition, 50 fatty acid derivatives of cytarabine have also been evaluated. Among such prodrugs, CP-4055 (ara-C-50 -elaidic acid ester) is an outstanding candidate for clinical application, with increased cytotoxicity in both leukemia and solid tumors (Bergman et al., 2004). The results of a Phase I clinical study demonstrated that CP4055 was active against solid tumors (Dueland et al., 2009) and further studies showed that the increased cytotoxicity of cytarabine was primarily helped by the increased cellular uptake of CP-4055 (Breistol et al., 1999). Considering the marked anticancer activity of CP-4055, it has been speculated that altering and/or adding the transmembrane pathway of cytarabine might be an efficient strategy to generate a sufficient level of intracellular cytarabine to combat solid tumors. Hepatocellular carcinoma (HCC), similar to other solid tumors, is insensitive to cytarabine, and is strongly associated with the deficient expression of nucleoside transporters (Galmarini et al., 2001; Huber-Ruano and Pastor-Anglada, 2009; Galmarini et al., 2010). Fortunately, hepatocytes also express other transporters such as bile salt transporters (Kullak-Ublick et al., 2004), providing a potential novel approach for increased uptake of cytarabine via cholic acid-cytarabine conjugates. Among these transporters, Na+dependent taurocholate cotransporting polypeptide (NTCP) and Na+-independent organic anion transporting polypeptides (OATPs), are two key hepatocellular basolateral transporters, responsible for the transport of bile salts to hepatocytes (Alrefai and Gill, 2007). Due to the multi-substrate specificity of NTCP and
OATPs, some liver-specific cholic acid-drug conjugates based on bile salt transporters have been developed for the successful treatment of liver disease (Gonzalez-Carmona et al., 2013; Vivian and Polli, 2014; Dong et al., 2015). Although a slight downregulation of these transporters on HCC cells has been reported (von Dippe and Levy, 1990), cholic acid-cytostatic agent (cisplatin (Larena et al., 2001; Briz et al., 2002) or chlorambucil (KullakUblick et al., 1997)) can enter HCC cells effectively, showing that the expression of bile salt transporters is suitable for drug targeting delivery. Apart from hepatocytes, ileal enterocytes express the apical Na+-dependent bile acid transporter (ASBT) which is involved in the enterohepatic circulation of bile acids. It is also responsible for the intestinal reabsoption of bile acids (Kolhatkar and Polli, 2012). Due to its high transport capacity and broad substrate specificity, ASBT has been used as a potential prodrug target to increase oral drug absorption (Dawson, 2011). For instance, a bile acid-acyclovir conjugate increased the oral bioavailability of acyclovir 2-fold in rats (Tolle-Sander et al., 2004). Also, when low molecular weight heparin (LMWH) was conjugated with deoxycholic acids, its oral bioavailability was increased 5-fold compared with LMWH itself in rats (Al-Hilal et al., 2014). These results show that cholic acid-drug conjugates are able to improve the oral absorption of poorly permeable drugs. Numerous bile acids have been identified in humans, such as cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA) and ursodeoxycholic acid (UDCA) (de Aguiar Vallim et al., 2013). These bile acids are metabolites of cholesterol and could be transported by bile acid transporters. Their structural differences involve the number and position of the hydroxyl groups on the steroid backbone. Although bile acid transporters can transport natural bile acids efficiently, the structural requirements for bile acid prodrugs need to be taken into account. It has been reported that the C-7 hydroxyl group on the cholestane ring plays a critical role during the transport process (Kolhatkar and Polli, 2012). The recommended conjugation positions are the C-3 hydroxyl group and the C-24 region (Rais et al., 2010; Gonzalez et al., 2014). A previous report showed that an amide-bond prodrug of cholic acid and cytarabine exhibited superior plasma stability compared with ester-bond conjugates (Chen et al., 2011). Therefore, in the present study, four cholic acid-cytarabine conjugates (see Fig. 1) were synthesized using the amide bond between cytarabine (4-NH2)
Fig. 1. Chemical structures of cytarabine (compound 1) and four cholic acid-cytarabine conjugates (compounds 2–5).
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and bile acid (24-COOH) to increase the oral absorption of cytarabine. The successful synthesis of compounds 2–5 was confirmed by 1H NMR and 13C NMR spectra. Then their antitumor activities against HepG2 cells were tested in vitro and a cellular uptake mechanism study was also conducted in a competitive inhibition experiment. The in vitro metabolic stability of the four prodrugs was investigated in rat plasma and liver homogenates. It should be pointed out that, to date, there are no published reports of the effects of bile acids on the metabolic stability and anticancer ability of cholic acid-cytarabine conjugates against HCC. Finally, the oral bioavailability of compound 5 was also evaluated in rats, in comparison with cytarabine. 2. Materials and methods 2.1. Materials Cytarabine was purchased from Surui Chemical Corp. (Suzhou, China), while cholic acid (CA), chenodeoxycholic acid (CDCA), hyodeoxycholic acid (HDCA) and ursodeoxycholic acid (UDCA) were obtained from Zhixin Chemicals Inc (Shanghai, China). Lamivudine was provided by the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Isobutyl chloroformate was obtained from Gade Chemicals Inc (Shanghai, China). Reagents of analytical grade were purchased from Tianjin bodi Chemicals Inc (Tianjin, China) and those of high-performance liquid chromatography grade were obtained from Merck (Darmstadt, Germany). Deionized-distilled water was used throughout the study. A human leukemia cell line (HL-60) and a human hepatocellular liver carcinoma (HepG2) cell line were provided by American Type Culture Collection (ATCC). Fetal bovine serum and RPMI-1640 medium were obtained from Hyclone Biochemical Product Co., Ltd. (Beijing, China). Trypan blue and methyl thiazolyl tetrazolium (MTT) were supplied by Sigma-Aldrich (St. Louis, MO, USA). 2.2. Synthesis of cholic acid-cytarabine conjugates 2.2.1. (R)-N-(1-((3S,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl) tetrahydro-furan-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-(R)-4((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl) pentanamide (2) To a stirred solution of cholic acid (0.49 g, 1.2 mmol) in anhydrous N, N-dimethylformamide (DMF, 10 mL), triethylamine (0.20 mL, 1.5 mmol) was added under nitrogen atmosphere. The mixture was cooled to 15 C and a solution of isobutyl chloroformate (0.20 mL, 1.5 mmol) was added dropwise. The mixture was stirred at 15 C for 15 min and a solution of cytarabine (0.24 g, 1.0 mmol) and triethylamine (0.20 mL, 1.5 mmol) in 10 mL anhydrous DMF was added dropwise to the reaction mixture at the same temperature. The reaction mixture was continually stirred for 30 min at room temperature. After the reaction had been completed, the solvent was removed by RE-52A rotary evaporation (Shanghai Yarong Biochemical Instrument Plant, China). The residue was then purified by chromatography on silica gel eluting with dichloromethane/methanol (10:1) to obtain pure compound 2 as an amorphous white solid (0.50 g, 78%). 1 H NMR(300 MHz, d6-DMSO, d ppm): 10.8 (s, 1H), 8.04 (d, 1H, J = 7.5 Hz), 7.20 (d, 1H, J = 7.5 Hz), 6.05 (d, 1H, J = 3.9 Hz), 5.47(d, 2H, J = 3.0), 5.05 (t, 2H), 4.31 (d, 1H, J = 3.9 Hz), 4.09 (d, 1H, J = 3.3), 3.98 (d, 1H, J = 3.3), 3.78 (s, 1H), 3.61 (s, 1H), 3.18 (t, 1H), 2.51-1.21 (m, 24H), 1.15 (d, 2H, J = 7.2 Hz) 0.93 (d, 3H, J = 6.2 Hz), 0.80 (s, 3H), 0.59 (s, 3H). 13C NMR (75 MHz, d6-DMSO, d ppm): 174.4, 162.3, 154.6, 146.7, 94.5, 87.0, 85.8, 76.2, 74.7, 71.1, 70.5, 66.4, 61.0, 46.3, 45.8, 41.6, 41.4, 35.4, 35.3, 34.9, 34.5, 33.8, 31.0, 30.5, 28.6, 27.3, 26.3,
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22.9, 22.7, 17.1, 12.4. MS (ESI) (m/z): calcd for C33H51N3O9: m/z 634.2 [M+H]+, 656.2 [M+Na]+. 2.2.2. (4R)-N-(1-((3S,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl) tetrahydro-furan-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-(R)-4((3R,5S,7R,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl) pentanamid (3) Compound 3 was synthesized from chenodeoxycholic acid (CDCA) and cytarabine, then purified as described for compound 2 to obtain an amorphous white solid (0.48 g, 78%). 1H NMR (300 MHz, d6-DMSO, d ppm): 10.9 (s, 1H), 8.14 (d, 1H, J = 7.5 Hz), 7.21 (d, 1H, J = 7.5 Hz), 6.18 (d, 1H, J = 4.8 Hz), 5.89 (d, 2H, J = 5.1), 5.11 (t, 2H), 4.28 (d, 1H, J = 4.5 Hz), 4.08 (d, 1H, J = 3.3), 3.63 (s, 1H), 3.18 (t, 1H), 2.26-0.94 (m, 28H), 0.87 (d, 4H, J = 6.6 Hz), 0.84 (s, 4H), 0.61 (s, 3H) 13C NMR (75 MHz, d6-DMSO, d ppm): 174.4, 162.3, 154.6, 146.7, 94.4, 87.0, 85.8, 76.2, 74.7, 70.4, 66.2, 61.1, 55.6, 50.1, 42.0, 41.5, 35.4, 35.2, 35.0, 34.8, 33.5, 32.4, 30.9, 30.6, 27.9, 23.2, 22.8, 20.3, 17.1, 12.4. MS (ESI) (m/z): calcd for C33H51N3O8 m/z 618.2 [M+H]+, 640.2 [M +Na]+. 2.2.3. (4R)-N-(1-((3S,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl) tetrahydro-furan-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-(R)4((5R,8S,10R,13R,17R)-3,6-dihydroxy-10,13-dimethyl2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a] phenanthren-17-yl)pentanamid (4) Compound 4 was synthesized from hyodeoxycholic acid (HDCA) and cytarabine and was purified as described for compound 2 to obtain an amorphous white solid (0.49 g, 80%). 1 H NMR(300 MHz, d6-DMSO, d ppm): 10.8 (s, 1H), 8.02 (d, 1H, J = 7.5 Hz), 7.17 (d, 1H, J = 7.5 Hz), 6.04 (d, 1H, J = 3.3 Hz), 5.44 (d, 2H, J = 3.5), 5.03 (q, 1H), 4.27 (d, 1H, J = 3.9 Hz), 4.05 (d, 3H, J = 5.4 Hz), 3.92 (s, 1H), 3.82 (s, 1H), 3.59 (d, 3H, J = 5.4), 3.15 (d, 1H, J = 4.8 Hz), 2.49-0.97 (m, 25H), 0.88 (d, 3H, J = 6.0 Hz), 0.82 (s, 3H), 0.59 (s, 3H). 13 C NMR (75 MHz, d6-DMSO, d ppm): 174.5, 162.3, 154.6, 146.7, 94.4, 87.0, 85.8, 76.2, 74.7, 71.1, 70.5, 66.3, 61.1, 46.3, 45.8, 41.6, 41.5, 35.4, 35.2, 35.0, 34.5, 33.8, 31.0, 30.5, 28.6, 27.3, 26.3, 22.9, 22.7, 17.1, 12.4. MS (ESI) (m/z): calcd for C33H51N3O8: m/z 618.2 [M+H]+, 640.2 [M+Na]+. 2.2.4. (4R)-N-(1-((3S,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl) tetrahydro-furan-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)-(R)-4((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl) pentanamid (5) Compound 5 was synthesized from ursodeoxycholic acid (UDCA) and cytarabine, and was purified as described for compound 2 to obtain an amorphous white solid (0.54 g, 87%). 1 H NMR(300 MHz, d6-DMSO, d ppm): 10.79 (s, 1H), 8.03 (d, 1H, J = 7.5 Hz), 7.19 (d, 1H, J = 7.5 Hz), 6.05 (d, 1H, J = 3.9 Hz), 5.46 (d, 2H, J = 4.5), 5.04 (q, 1H), 4.41 (d, 1H, J = 4.5 Hz),4.04 (s, 1H), 3.85 (s, 1H), 3.82 (d, 2H, J = 3.0), 3.62 (q, 1H), 2.51–0.96 (m, 29H), 0.88 (d, 3H, J = 6.0 Hz), 0.82 (s, 3H), 0.59 (s, 3H). 13C NMR (75 MHz, d6-DMSO, d ppm): 174.4, 162.3, 154.6, 146.7, 94.4, 87.0, 85.8, 76.2, 74.7, 69.8, 69.5, 61.1, 56.0, 54.8, 43.2, 43.1, 42.3, 38.8, 37.8, 37.4, 35.0, 34.9, 33.8, 33.6, 31.0, 30.3, 28.2, 26.8, 22.4, 17.1, 12.4. MS (ESI) (m/z): calcd for C33H51N3O8: m/z 618.2 [M+H]+, 640.2 [M+Na]+. 2.3. HPLC of cholic acid-cytarabine conjugates Four cholic acid-cytarabine conjugates were determined by HPLC (Hitachi, Japan). A C18 analytical column (4.6 mm 250 mm) was maintained at 40 C. In brief, chromatographic separation was carried out using isocratic elution with a mobile phase composed of methanol (70%) and water containing 10 mM ammonium acetate (30%). The detection wavelength was 248 nm and samples
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were delivered at a flow rate of 1.0 mL/min. The auto-sampler was conditioned at 4 C and the sample volume injected was 20 mL. 2.4. UPLC–MS/MS of cytarabine Cytarabine was quantified by UPLC–MS/MS carried out on an ACQUITY UPLC system (Waters Co., Ltd., Milford, MA, USA). A C18 analytical column (50 mm 2.1 mm) was used and maintained at 40 C. A gradient elution program was adopted for chromatographic separation with mobile phase A consisting of methanol containing 0.1% formic acid and mobile phase B consisting of 2% methanol in water. A Waters Tandem Quadrupole (TQ) Detector with an ESI source operating in positive ion mode was used for the determination of cytarabine and lamivudine (IS). Under UPLC–MS/ MS conditions, the precursor/product ion transitions were m/z 244/112 for cytarabine (collision energy 15 eV) and m/z 230/112 for lamivudine (collision energy 15 eV). The autosampler was conditioned at 4 C and the sample volume injected was 5 mL. Injection wash solvents were methanol-water (10:90, v/v) and methanolwater (90:10, v/v) for the weak and strong wash, respectively. 2.5. Cell proliferation assays The human leukemic cell line (HL-60) and human hepatocellular carcinoma cell line (HepG2) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin. All the cells were maintained in an atmosphere of 5% CO2 and 90% relative humidity at 37 C. The HL-60 cells were suspended in their respective media to give 2 mL volumes of cell suspension at a final density of 5 104 cells/mL. Appropriate volumes of drug solution were transferred to the cell suspensions, and incubation was continued for 72 h. Cell viability was determined by staining with trypan blue. Trypan blue-stained (nonviable) cells and the total cell number were measured using a hematocytometer. The growth rates of cells after treatments with these cholic acid-cytarabine conjugates at different concentrations were compared with that of control cells. The data obtained were analyzed using a Logit model, and the results were expressed as the IC50 values (the drug concentration that inhibits cell growth by 50% of the control value). HepG2 cells were seeded into 96-well plates (5 104 cells/mL) and cultured at 37 C in an incubator for 24 h. The cells were then incubated with the same concentration (40 mM) of cytarabine, cholic acidcytarabine conjugates (2–5) and compounds 2–5 with the same amount of CA, CDCA, HDCA and UDCA respectively for 24 h and 48 h, and then 100 mL of a solution of MTT was added to each well, and the plates were returned to the incubator for 3 h. The data were obtained using ELISAs. 2.6. Stability of cholic acid-cytarabine conjugates The stability of cholic acid-cytarabine conjugates was studied at 37 C by incubating a drug solution (3.5 mM) in phosphate buffer solution (PBS) at pH 7.4, fresh plasma or liver homogenates. For the first medium, the samples were detected by HPLC directly. For the latter two media, Sprague–Dawley (SD) rats were first anesthetized with ether, and then the whole blood and livers were collected. All samples were placed in heparinized tubes. The rat plasma samples were centrifuged at 10,000 rpm for 10 min, collected, and diluted by isovolumetric 0.05 M PBS. The tissue was homogenized in a tissue homogenizer and diluted with isovolumetric 0.05 M PBS. At predetermined time, the metabolic reaction was quenched by adding a 4-fold volume of methanol followed by vortexing. The mixture was then centrifuged at 10000 rpm for 10 min and the supernatant was passed through a membrane filter (0.22 mm) and analyzed by HPLC.
2.7. Release of cytarabine in rat plasma The cytarabine released from the conjugates was evaluated following incubation in rat plasma diluted with 50 mM PBS at 37 C. The initial drug concentration was 7.0 mM calculated as cytarabine. After a 2 h incubation, the plasma samples were collected for further analysis. To an aliquot of each plasma sample (30 mL), internal standard solution (lamivudine, 200 ng/mL) (30 mL) and water (400 mL) were added. Each sample was shaken and centrifuged, and then the supernatant was transferred to a solid-phase extraction cartridge. The eluent solution was determined by UPLC/MS/MS. 2.8. Pharmacokinetics in rats Male SD rats weighing 210–240 g were divided randomly into two groups. All animal experiments were performed by following institutional guidelines and were approved by the University Committee on Use and Care of Animals, Shenyang Pharmaceutical University. The aqueous solutions of cytarabine and compound 5 were prepared with 0.1% Tween-80 to give a final concentration of 0.5 mg/mL. Two groups of rats were given cytarabine and prodrug (compound 5) solutions (30 mg/kg, calculated as cytarabine) by oral gavage. Serial blood samples (0.2 mL) were obtained at 2, 5, 15, 30, 45 min and 1, 1.5, 2, 3, 4, 6, 8, 12 and 24 h after administration. During sampling, rats were anesthetized with ether. All samples were collected in heparinized tubes. The rat plasma samples were centrifuged at 10,000 rpm for 10 min, and then frozen at 80 C. After using the solid-phase extraction cartridge as described in the section “2.7. Release of cytarabine in rat plasma”, the samples were analyzed by UPLC/MS/MS. The maximum plasma concentration of cytarabine (Cmax) and the time to reach Cmax (Tmax) were obtained directly from the plasma concentration time profiles. The area under the curve (AUC) and the half-life (t1/2) of cytarabine were calculated using DAS 2.0 software. 2.9. Statistical analysis All statistical analyses in this paper were performed using Student’s t-test. The differences were considered significant at p < 0.05 and markedly significant at p < 0.01. 3. Results and discussion 3.1. Characterization of cholic acid-cytarabine conjugates The 1H NMR spectra confirmed that the acylation of cytarabine was at the position of the amino group without any involvement of the sugar OH group. In addition, due to acylation at the N4 position, a distinct blue shift (272 nm to 248 nm) of lmax could also be observed in the UV spectra of these cytarabine conjugates, consistent with a previous report (Schiavon et al., 2004). These results confirmed the successful synthesis of cytarabine prodrugs. The purity of cholic acid-cytarabine conjugates was examined by HPLC. As shown in Fig. 2, compounds 2–5 with similar structures had different retention times (tR) of 18.1 min, 10.4 min, 7.70 min and 10.3 min, respectively. It was clear that compounds 2– 5 could be separated well by the HPLC method used, with the purities of compounds 2–5 being higher than 98%. 3.2. In vitro cytotoxicity of cholic acid-cytarabine conjugates The antiproliferative activity of cytarabine and its conjugates was evaluated using leukemia HL-60 cells and solid tumor HepG2 cells. In the case of HL-60 cells, all these conjugates had higher IC50
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Fig. 2. The typical HPLC chromatogram of compound 2 (a), compound 3 (b), compound 4 (c) and compound 5 (d) in mobile phase.
Table 1 The antiproliferative effects of compound 1 and the conjugates of cytarabine (compounds 2–5) in HL-60 cells after 72 h (mean S.D., n = 3). compound
IC50 (nM)
1 2 3 4 5
14.4 1.6 110.0 13.7 ** 76.9 8.4 ** 90.5 9.3 66.0 5.2
p < 0.01 (**) versus compound 1 as the control.
values than cytarabine (Table 1), indicating that the structure modification with bile acids reduced the intrinsic cytotoxicity of cytarabine against HL-60 cells. Due to its hydrophilic nature, the membrane transport of cytarabine was strongly dependent on nucleoside transporters, such as the equilibrative nucleoside transporter 1 (hENT1) (Tang et al., 2012; Yamauchi et al., 2014). After conjugation with bile acids, the affinity of cytarabine for nucleoside transporter might be reduced, resulting in lower transport efficiency. Among these cytarabine prodrugs, compound 2 had the highest IC50 (110.0 13.7 nM), which might be due to the high polarity of CA (see Fig. 1), which did not help in the recognition by nucleoside transporters. Compound 5 had the highest cytotoxicity, probably due to the lower influence on the affinity for the prodrug and transporter. Considering the important role of nucleoside transporters in the cellular uptake, insufficient expression of transporters or a
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lower affinity for transporters would markedly reduce the antiproliferative activity of cytarabine. As shown in Fig. 3, HepG2 cells, lacking nucleoside transporters (MacKenna et al., 2009), were insensitive to cytarabine compared with HL-60 cells. In contrast, these cholic acid-cytarabine conjugates exhibited efficient inhibition (over 60%) of HepG2 cell growth after 48 h treatment. The N4-hyodeoxycholic acid of cytarabine (compound 4) had the most significant antitumor activity (over 70% inhibition). It has been established that HepG2 cells express bile salt transporters (OATPs, not NTCP) and produce rapid uptake of bile acids via OATP- mediated transport (Kullak-Ublick et al., 1996; Lee et al., 2001). Moreover, benefiting from the broad substrate specificity of OATPs, a wide variety of drug-bile acid conjugates can also be transported via such a pathway (Kullak-Ublick et al., 1994). Therefore, we speculated that cholic acid-cytarabine conjugates can also be taken up via multispecific OATPs, producing an increased intracellular accumulation of cytarabine against HepG2. To confirm this hypothesis, competitive inhibition experiments were performed to investigate the effects of corresponding bile acids on the cellular uptake of compounds 2–4. Before that, the cell viability of HepG2 treated with four bile acids was determined at 37 C for 48 h. These bile acids produced no cytotoxicity (data not shown), indicating the safety of these endogenous molecules. As shown in Fig. 4C and D, simultaneous treatment with conjugates and corresponding bile acids markedly reduced the cytotoxicity. Also, no effects of bile acids on the cytotoxicity induced by cytarabine were found (Fig. 4A and B). These results demonstrated that cholic acid-cytarabine conjugates altered the transport pathway of cytarabine and the OATPs-mediated uptake was indeed involved in the cellular internalization of these conjugates in HepG2 cells. 3.3. Stability and release of cholic acid-cytarabine conjugates Cholic acid-cytarabine conjugates were developed to protect cytarabine from deamination, and are also able to target the bile acid transporters expressed in HCC cells. In this case, the cholic acid-cytarabine conjugates have a dual functionality, i.e., increased stability via N4-acidylated modification and improved membrane permeability. As the structural integrity of these conjugates directly affects these functional implementations, the stability of these conjugates before intestinal absorption was first investigated in pH 7.4 PBS. Then their metabolic stability was evaluated in rat plasma and liver homogenates. As shown in Fig. 5A, these
Fig. 3. The inhibition of HepG2 cells proliferation by the compounds 1-5 (a-e) at 40 mM after (A) 24 h and (B) 48 h. The results are shown as the percentage of the control containing 1% DMSO (mean + S.D., n = 3). p < 0.05 (*) and p < 0.01 (**) versus compound 1 as the control.
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Fig. 4. The inhibition of HepG2 cells proliferation by compound 1(a), compound 1 with the corresponding amount of CA (b), CDCA (c), HDCA (d) and UDCA (e) at 100 mM after (A) 24 h and (B) 48 h. The inhibition of HepG2 cells proliferation by compound 1 (a), compounds 2–5 with CA, CDCA, HDCA and UDCA (b-e), respectively, at 40 mM after (C) 24 h and (D) 48 h. The results are shown as the percentage of the control containing 1% DMSO (mean + S.D., n = 3).
conjugates effectively resisted hydrolysis except for compound 2, demonstrating the stability of the amide bonds. The concentrationtime profiles of these conjugates in rat plasma are shown in Fig. 5B. The half-lives of compounds 2–5 were 20 min, 20 min, 30 min and 90 min in rat plasma, respectively. Compound 5, the N4-ursodeoxycholic acid of cytarabine, was the most stable and was resistant to
degradation by deaminase. Contrary to our expectation, degradation of the conjugates was slower in liver homogenates. The percentage of compounds 2–5 remaining after incubation in liver homogenates is shown in Fig. 5C. Among these conjugates, compound 5 exhibited the longest half-life (147 min), followed by compound 2 (91 min) and the other two compounds showed a
Fig. 5. Stability of compounds 2–5 was studied in (A) phosphate buffer solution (PBS) at pH 7.4, (B) SD rat plasma and (C) rat liver homogenates.
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deaminase for a shorter period, resulting in the better metabolic stability of cytarabine. So, compound 5 deserved further investigation. 3.4. Pharmacokinetics in rats
Fig. 6. Cytarabine released from compounds 2–5 (a–d) was evaluated after incubation for 2 h in SD rat plasma, and the plasma concentration was determined by UPLC/MS/MS. The results are shown as the percentage of initial concentration (all calculated as cytarabine, mean + S.D., n = 3).
Fig. 7. Mean plasma concentration-time profiles of cytarabine following oral administration of compounds 1 and 5 at 30 mg/kg (calculated as cytarabine) in SD rats (mean + S.E. n = 6).
half-life less 60 min in liver homogenates. Collectively, compound 5 was identified as the optimal cytarabine prodrug with desired metabolic stability. The release of free cytarabine from the conjugates was also studied in rat plasma. As shown in Fig. 6, all the conjugates could be converted into cytarabine and compound 5 exhibited the highest release rate of cytarabine (70.2%) within 2 h. The conversion and deamination of cytarabine occurred simultaneously in plasma. Benefiting from the slow degradation, the sustainedreleased cytarabine from compound 5 was exposed to cytidine
The oral pharmacokinetic study of compound 5 and cytarabine was conducted in rats to evaluate the feasibility of cholic acid-drug conjugates for improving the oral absorption of cytarabine in vivo. As the concentration of compound 5 in plasma was not high enough to be accurately determined, we mainly focused on the determination of cytarabine after oral administration. The reasons for the low level of the conjugate in plasma might include the increased hepatic uptake due to bile salt transporter-mediated transport (Oatps and Ntcp) (Kullak-Ublick et al., 2004; Alrefai and Gill, 2007), and the higher metabolic enzyme activity in vivo. The mean cytarabine blood concentrationtime profiles of cytarabine after administration of cytarabine and conjugate (compound 5) are shown in Fig. 7, and the main pharmacokinetic parameters of cytarabine are summarized in Table 2. The Cmax in the prodrug group was less than that in the cytarabine group, while the Tmax in the prodrug group was extended from 2.5 0.6 h to 3.6 1.1 h. In addition, compound 5 showed a greater t1/2 for cytarabine (15.62 10.30 h) than that of cytarabine itself (4.00 0.89 h). This steady plasma concentration of compound 5 could be ascribed to the improved stability of the cholic acid-cytarabine conjugate and the sustained release of cytarabine (Zheng and Polli, 2010). More importantly, the AUC0-1 of compound 5 (34857.0 7578.0 ng h/mL) was significantly higher than that of cytarabine (16603.7 2627.2 ng h/mL). Similar to other ASBT targeting prodrugs (Han et al., 2015), compound 5 can be recognized by ASBT and actively transported across the enterocytes. Also, the conjugation of UDCA with cytarabine would also increase the lipophilicity of cytarabine, resulting in improved cellular uptake via a passive transport process. These two factors could be responsible for the 2-fold increase in the oral bioavailability of cytarabine provided by compound 5. As cytarabine is a cell cycle-dependent drug (S phase), the extended high drug concentration would produce higher cytotoxic activity. Compound 5 could maintain a stable and high cytarabine concentration for 24 h, while cytarabine itself showed an obvious plasma concentration fluctuation within the initial 8 h. This demonstrated that compound 5 was more effective with reduced side effects. Apart from the bile acid prodrugs of cytarabine developed here, amino acid derivatives, fatty acid derivatives and phosphate derivatives of cytarabine have also been investigated widely. Among these prodrugs, L-valine-cytarabine has been synthesized in two ways using N4-L-valyl- cytarabine and 50 -L-valyl- cytarabine. The former had only a 4% oral bioavailability of cytarabine in rats due to the limited drug release from the prodrug (Cheon and Han, 2007). While the latter provided a 1.75-fold increased oral bioavailability of cytarabine with a suitable conversion rate in vivo. The results of our study demonstrate that 50 -L-valylcytarabine is absorbed via a (oligopeptide transporter 1) PepT1mediated uptake pathway (Sun et al., 2009; Liu and Liu, 2013). In the present study, compound 5, with good metabolic stability and
Table 2 Pharmacokinetic parameters of cytarabine, following oral administration of compounds 1 and 5 at 30 mg/kg (calculated as cytarabine) to SD rats (mean S.E., n = 6). PK parameters
Tmax (h)
Cmax (h)
t1/2 (h)
AUC0-24 (ng h/mL)
AUC0-1 (ng h/mL)
Frel%
Compound 1 Compound 5
2.5 0.6 3.6 1.1
2103.4 251.8 1706.1 125.0
4.00 0.89 15.62 10.30*
16306.9 2695.5 20223.7 1516.0
16603.7 2627.2 34857.0 7578.0*
100 209.9 45.6
p < 0.05 (*) versus compound 1 as the control.
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bile acid transporter-mediated uptake, exhibited a 2-fold increased oral bioavailability of cytarabine. In addition, the good metabolic stability of compound 5 would contribute to the bile acid transporter-targeted delivery of cytarabine and broaden the potential scope of cytarabine applications, such as HCC. The distribution of cytarabine in liver has also been investigated, but, the rapid hepatic degradation of cytarabine resulted in undetectable drug levels. Therefore, a more sensitive method needs to be developed for the determination of cytarabine distributed in hepatic tissues. Moreover, our future studies will also focus on the detailed mechanism of bile acid transporter-mediated cellular uptake, liver targeting ability and the in vivo antitumor efficacy of cytarabine conjugates against HCC. 4. Conclusions In this study, a series of cholic acid-cytarabine conjugates were designed and synthesized to improve the oral absorption of cytarabine and to test the potential antitumor efficacy against HCC. All the conjugates showed potent antiproliferative activities in HepG2 cells, as a result of an increased intracellular drug level achieved by transporter-mediated uptake. Stability tests in rat plasma and liver homogenates demonstrated that the N4ursodeoxycholic acid of cytarabine (compound 5) exhibited optimal stability in vitro. The pharmacokinetic study in rats demonstrated that compound 5 could increase the oral bioavailability of cytarabine over 2-fold, with the benefit of ASBTmediated transmembrane transport. Compared with cytarabine, compound 5 produced a smoother plasma concentration because of the improved metabolic stability of the cholic acid-cytarabine conjugate. In conclusion, the prodrug strategy applying bile salt as the modified moiety is a promising method to improve the oral absorption and metabolic stability of cytarabine. In addition, the cholic acid-cytarabine conjugate could also give cytarabine a novel curative effect such as for the treatment of HCC. Acknowledgments This work received financial support from the National Nature Science Foundation of China (No. 81373336, 81473164), from the National Basic Research Program of China (973 Program), No. 2015CB932100, and from Program for New Century Excellent Talents in University (No. NCET-12-1015). References Al-Hilal, T.A., Park, J., Alam, F., Chung, S.W., Park, J.W., Kim, K., Kwon, I.C., Kim, I.S., Kim, S.Y., Byun, Y., 2014. Oligomeric bile acid-mediated oral delivery of low molecular weight heparin. J. Control. Release 175, 17–24. Alrefai, W.A., Gill, R.K., 2007. Bile acid transporters: structure, function, regulation and pathophysiological implications. Pharm. Res. 24, 1803–1823. Aoshima, M., Tsukagoshi, S., Sakurai, Y., Oh-ishi, J., Ishida, T., 1976. Antitumor activities of newly synthesized N4-acyl-1-beta-D-arabinofuranosylcytosine. Cancer Res. 36, 2726–2732. Bergman, A.M., Kuiper, C.M., Myhren, F., Sandvold, M.L., Hendriks, H.R., Peters, G.J., 2004. Antiproliferative activity and mechanism of action of fatty acid derivatives of arabinosylcytosine (ara-C) in leukemia and solid tumor cell lines. Nucleosides Nucleotides Nucl. Acids 23, 1523–1526. Breistol, K., Balzarini, J., Sandvold, M.L., Myhren, F., Martinsen, M., De Clercq, E., Fodstad, O., 1999. Antitumor activity of P-4055 (elaidic acid-cytarabine) compared to cytarabine in metastatic and s c. human tumor xenograft models. Cancer Res. 59, 2944–2949. Briz, O., Serrano, M.A., Rebollo, N., Hagenbuch, B., Meier, P.J., Koepsell, H., Marin, J.J., 2002. Carriers involved in targeting the cytostatic bile acid-cisplatin derivatives cis-diammine-chloro-cholylglycinate-platinum(II) and cis-diamminebisursodeoxycholate-platinum(II) toward liver cells. Mol. Pharmacol. 61, 853– 860. Chen, D.Q., Wang, X., Chen, L., He, J.X., Miao, Z.H., Shen, J.K., 2011. Novel liver-specific cholic acid-cytarabine conjugates with potent antitumor activities: Synthesis and biological characterization. Acta Pharmacol. Sin. 32, 664–672. Cheon, E.P., Han, H.K., 2007. Pharmacokinetic characteristics of L-valyl-ara-C and its implication on the oral delivery of ara-C. Acta Pharmacol. Sin. 28, 268–272.
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