Dipeptide Diester Prodrugs of

Jan 22, 2013 - State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, ..... and 90% relative humidity at 37 °C. ...
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Propylene Glycol-Linked Amino Acid/Dipeptide Diester Prodrugs of Oleanolic Acid for PepT1-Mediated Transport: Synthesis, Intestinal Permeability, and Pharmacokinetics Feng Cao,† Yahan Gao,† Meng Wang,† Lei Fang,*,†,‡ and Qineng Ping*,† †

State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, P. R. China Pharmaceutical Research Center, Southeast University, Nanjing 211189, P. R. China

‡

S Supporting Information *

ABSTRACT: In our previous studies, ethylene glycol-linked amino acid diester prodrugs of oleanolic acid (OA), a Biopharmaceutics Classification System (BCS) class IV drug, designed to target peptide transporter 1 (PepT1) have been synthesized and evaluated. Unlike ethylene glycol, propylene glycol is of very low toxicity in vivo. In this study, propylene glycol was used as a linker to further compare the effect of the type of linker on the stability, permeability, affinity, and bioavailability of the prodrugs of OA. Seven diester prodrugs with amino acid/dipeptide promoieties containing L-Val ester (7a), L-Phe ester (7b), L-Ile ester (7c), D-Val-L-Val ester (9a), LVal-L-Val ester (9b), L-Ala-L-Val ester (9c), and L-Ala-L-Ile ester (9d) were designed and successfully synthesized. In situ rat single-pass intestinal perfusion (SPIP) model was performed to screen the effective permeability (Peff) of the prodrugs. Peff of 7a, 7b, 7c, 9a, 9b, 9c, and 9d (6.7-fold, 2.4-fold, 1.24-fold, 1.22-fold, 4.15-fold, 2.2-fold, and 1.4-fold, respectively) in 2-(Nmorpholino)ethanesulfonic acid buffer (MES) with pH 6.0 showed significant increase compared to that of OA (p < 0.01). In hydroxyethyl piperazine ethanesulfonic acid buffer (HEPES) of pH 7.4, except for 7c, 9a, and 9d, Peff of the other prodrugs containing 7a (5.2-fold), 7b (2.0-fold), 9b (3.1-fold), and 9c (1.7-fold) exhibited significantly higher values than that of OA (p < 0.01). In inhibition studies with glycyl-sarcosine (Gly-Sar, a typical substrate of PepT1), Peff of 7a (5.2-fold), 7b (2.0-fold), 9b (3.1-fold), and 9c (2.3-fold) had significantly reduced values (p < 0.01). Compared to the apparent permeability coefficient (Papp) of OA with Caco-2 cell monolayer, significant enhancement of the Papp of 7a (5.27-fold), 9b (3.31-fold), 9a (2.26-fold), 7b (2.10-fold), 7c (2.03-fold), 9c (1.87-fold), and 9d (1.39-fold) was also observed (p < 0.01). Inhibition studies with Gly-Sar (1 mM) showed that Papp of 7a, 9b, and 9c significantly reduced by 1.3-fold, 1.6-fold, and 1.4-fold (p < 0.01), respectively. These results may be attributed to PepT1-mediated transport and their differential affinity toward PepT1. According to the permeability and affinity, 7a and 9b were selected in the pharmacokinetic studies in rats. Compared with group OA, Cmax for group 7a and 9b was enhanced to 3.04-fold (p < 0.01) and 2.62-fold (p < 0.01), respectively. AUC0→24 was improved to 3.55-fold (p < 0.01) and 3.39-fold (p < 0.01), respectively. Compared to the ethylene glycol-linked amino acid diester prodrugs of OA in our previous work, results from this study revealed that part of the propylene glycol-linked amino acid/dipeptide diester prodrugs showed better stability, permeability, affinity, and bioavailability. In conclusion, propylene glycol-linked amino acid/dipeptide diester prodrugs of OA may be suitable for PepT1-targeted prodrugs of OA to improve the oral bioavailability of OA. KEYWORDS: oleanolic acid, peptide transporter 1, propylene glycol linker, ester prodrug, in situ single-pass intestinal perfusion, Caco-2, pharmacokinetics, bioavailability

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past two decades.1,2 Recent developments in molecular biology have revealed a lot of information about the functional and structural characteristics of transporters. Prodrugs can be designed by coupling amino acids or peptides to compounds such that they resemble the intestinal nutrients structurally and are absorbed by specific carrier proteins. Many transporters present at the intestinal epithelium may be selected as the

INTRODUCTION Recently, various strategies have been employed to improve oral absorption of parent drugs which have undesirable biopharmaceutical characteristics, such as low aqueous solubility and low permeability. Prodrugs are bioreversible derivatives of drug molecules that could be used to alter the physicochemical properties of drugs. In vivo prodrugs must be converted to the active parent compounds as soon as the goal is achieved to exert therapeutic effect. This may be achieved by different mechanisms such as transporter−substrate specificity or enzyme−substrate specificity. The transporter targeted prodrug derivatization approach is a common tool over the © 2013 American Chemical Society

Received: Revised: Accepted: Published: 1378

November 10, 2012 January 13, 2013 January 22, 2013 January 22, 2013 dx.doi.org/10.1021/mp300647m | Mol. Pharmaceutics 2013, 10, 1378−1387

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prodrugs’ target, including peptide transporter 1(PepT1),3 human apical sodium-dependent bile acid transporter (hASBT),4 monocarboxylate transporter 1(MCT1),5 organicanion transporting polypeptides (OATPs),6 glucose transporter 1(GluT1),7 sodium dependent vitamin C transporter (SVCT),8 etc. Overall membrane transporter-targeted prodrugs play an important role in drug delivery and will achieve important advances. Peptide transporters, especially PepT1, have captured the greatest attention in prodrug design to improve oral absorption among various membrane transporters, mainly due to high capacity, broad substrate specificity, high level of expression in the intestinal epithelium, and low occurrence of functional polymorphisms. Furthermore many drugs have been chemically modified for targeting delivery via peptide transporters, including modifications to amine, hydroxyl, or carboxylic acid functionality in the parent drug. The promoiety is either a single amino acid or a peptide. There are many examples of targeting PepT1 to improve the oral bioavailability of a parent drug. Valacyclovir (Valtrex; GlaxoSmithKline) and valganciclovir (Valcyte; Roche) are two outstanding examples of marketed prodrugs in exploiting PepT1-mediated transport with the aim to improve oral bioavailability of acyclovir and ganciclovir, respectively.9,10 The Amidon lab is committed to peptide transporter study and prodrug targeting/delivery strategies. Their findings are of great significance to the development of PepT1-targeted prodrug.10−12 Recently, it has been reported that prostate cancer cells express functionally active peptide transporters on the cytoplasmic membrane,13 indicating that peptide transporters are overexpressed in prostate cancer cells and can be adopted as a promising target for tumor-specific drug delivery.14 Oleanolic acid (OA), (3β)-3-hydroxyolean-12-en-28-oic acid, belongs to a triterpenoid compound that is extracted from many Asian herbs, such as Fructus ligustri lucidi, Fructus forsythiae, Radix ginseng, and Akebia trifoliate. OA has been shown to have many important biological actions including hepatoprotective effect, anti-inflammatory effect, antinociceptive potential, antitumor effect, antioxidant effect, and antiglycative effect.15 OA is a BCS class IV category drug because of its low aqueous solubility (1.2 × 10−2 μg/mL),16 low apparent permeability through Caco-2 cell monolayer model (1.1−1.3 × 10−6 cm/s),17 and low oral bioavailability in rats (approximately 0.7%).17 Moreover, various formulation strategies have been attempted to improve oral bioavailability of OA, such as nanosuspension,18 self-nanoemulsified system,19 and solid dispersion.20 However, these formulation approaches still have limitations in terms of bioavailability enhancement. In our previous research, with the aim to further extend the kind of structure of parent drugs selected for PepT1-targeted prodrug design and improve the oral bioavailability of OA, the carboxylic group of OA was esterified with the carboxylic acid group of amino acid to form the conjugated diester prodrug through an ethoxy linker. Results from in situ single-pass intestinal perfusion (SPIP) and oral absorption in vivo with rats showed that this strategy was feasible for PepT1-targeted prodrug design.21 In this work, the effect of the type of linker on the stability, permeability, affinity, and bioavailability of the amino acid diester prodrugs of OA was researched. Unlike ethylene glycol, propylene glycol was chosen as it is a commonly used excipient in pharmaceutical formulations with no known toxic effects. With linker of propylene glycol, the amino acid diester prodrugs containing L-Val ester (7a), L-Phe

ester (7b), and L-Ile ester (7c) were synthesized. Moreover, the dipeptide promoiety prodrugs may have better stability and higher affinity for PepT1 than amino acid ester prodrugs.22−25 Therefore, dipeptide promoiety prodrugs containing D-Val-LVal ester (9a), L-Val-L-Val ester (9b), L-Ala-L-Val ester (9c), and L-Ala-L-Ile ester (9d) are researched. Permeability studies are carried out with SPIP model in rat and the Caco-2 model, which both are excellent predictive tools for permeability. In order to evaluate affinity of the prodrugs to PepT1, inhibition studies were conducted by the addition of Gly-Sar, a PepT1 inhibitor. Finally, pharmacokinetic studies in rats are conducted after oral administration of OA, L-Val ester (7a), and L-Val-LVal ester (9b) of OA, respectively.

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EXPERIMENTAL SECTION Materials. OA was purchased from Nanjing Zelang Medical Technological Limited Company. Gly-Sar was obtained from Sigma-Aldrich (St. Louis, MO, USA). Hydroxypropyl-betacyclodextrin (HP-β-CD) was purchased from Taixing YiMing Biological Products limited company. Cremophor ER was supplied by BASF. Hydroxyethyl piperazine ethanesulfonic acid (HEPES) and 2-(4-morpholino)ethanesulfonic acid (MES) were purchased from Bio basic Inc. and Nanjing Robiot limited company, respectively. Glycyrrhetinic acid (GA, lot No: 110723-200612) was purchased from National Institute for the Control of Pharmaceutical and Biological Products. Caco-2 cells were obtained from Chinese Academy of Science, Shanghai. Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), and nonessential amino acid came from Gibco. Trypsin was obtained from Sangon. Dimethyl sulfoxide (DMSO) was gained from Shanghai Lingfeng Chemical Reagent Limited Company. All other chemicals were of analytical grade or HPLC (high performance liquid chromatography) grade. General Method. Synthesis of Diester Prodrugs of Oleanolic Acid (7a−7e, 9a−9d). To a solution of oleanolic acid (1 equiv) in N,N- dimethylformamide (DMF) were added chloroacetone (1.5 equiv) and K2CO3 (catalytic amount). The mixture was stirred for 3.5 h at 60 °C and then cooled to room temperature. The crude product was filtrated and purified by column chromatography. Compound 2 (1 equiv) was reacted with 3,4-dihydro-2H-pyran (5 equiv) in CH2Cl2 in the presence of a catalytic amount of p-toluenesulfonate at room temperature for 3 h to give ether product 3. Product 3 (1 equiv) was reduced by NaBH4 (10 equiv) in methanol, offering compound 4. Compound 4 (1 equiv), N,N′-dicyclohexylcarbodiimide (DCC) (1.2 equiv), and a catalytic amount of 4(dimethylamino)pyridine (DMAP) were dissolved in CH2Cl2 and stirred overnight at room temperature to give a crude product, which was purified by column chromatography to yield 5a−5e. 5a−5e was treated with catalytic amount of ptoluenesulfonate at room temperature for 3 h to offer 6a−6e. Then dry hydrogen chloride gas was bubbled into a solution of 6 in dry ether for 2.5 h. The precipitate was collected by filtration and washed with cold dry ether to give 7a−7e. Finally compound 7 (1 equiv), DCC (1.2 equiv), and a catalytic amount of DMAP were dissolved in CH2Cl2 and stirred overnight at room temperature to give 8a−8d, which were further treated with dry hydrogen chloride gas for 3 h to yield 9a−9e. More detailed information is summarized in the Supporting Information. HPLC Analysis. The concentration of prodrugs and OA was determined by a validated HPLC method. The HPLC system 1379

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literature.28 Following the guide for the care and use of laboratory animals of China Pharmaceutical University, animal experiments were implemented. Male albino Sprague−Dawley rats (weighing 200−250 g) were fasted for 14−20 h, prior to the perfusion experiment, with tap water. Briefly, male rats were anesthetized with urethane (1.5 g/kg) by intraperitoneal injection. The animals were placed on a table lamp as a source of heat to maintained body temperature at 37 °C. A midline longitudinal incision was made on the abdomen. Approximately 10 cm of the jejunal segment was located. Two treated PVC tubings (4 mm o.d., inlet tube 40 cm, outlet tube 5 cm) were inserted at the proximal and distal ends of the segment. A cotton pad soaked in normal saline covered the exposed segment, and normal saline flushed the intestinal segment until the outlet solution appeared clear. Thereafter, using a peristaltic pump (BT100-2J, Baoding Longer Precision Pump Limited Company, China), a perfusion solution perfused the jejunum segment at a constant flow rate (Qin) of 0.2 mL/min with perfusion solution for 30 min. When the steady state (the inlet over outlet concentration ratio of phenol red approaches 1 at steady state) had been reached, the outflow samples were collected at 20, 40, 60, 80, 100, and 120 min and centrifuged at 12000 rpm for 10 min. The concentration of the test compound was determined by HPLC, and the levels of phenol red were analyzed by UV−vis spectrometer (UV-9600, Beijing Rayleigh Instrument Limited Company, China). The length and radius of each perfused jejunal segment were measured at the end of the experiment, and finally the animals were euthanized. The same perfusion procedure was followed to determine the permeability of compounds with and without Gly-Sar. The data analysis method was as indicated below. Caco-2 Permeability Experiments. Permeability Experiments. Caco-2 cells were seeded on permeable filter supports at a density of 1,000,000 cells/mL and cultured in DMEM containing 10% FBS, 1% nonessential amino acids, 1% Lglutamine, and 25 mM HEPES. Cells were grown in 5% CO2 and 90% relative humidity at 37 °C. The culture medium was replaced once a day. 0.3 mL of cell suspension with a density of 200,000 cells/mL was inoculated to the apical side of Millicell at a density of 100,000 cells/mL for 21 days. The culture medium was replaced every other day for the first week and then replaced every day. Caco-2 monolayers with a transendothelial electrical resistance (TEER) of exceeding 350 Ω·cm2 were used for the transepithelial transport studies. The cell monolayers were incubated at 37 °C with HEPES buffer (pH 7.4, containing 5.4 mM KCl, 140 mM NaCl, 1.8 mM CaCl2, 0.8 mM MgSO2, 5 mM D-glucose, 25 mM HEPES)11 for 15 min, then the TEER was measured, and then the HEPES was removed from both compartments. 400 μL of MES buffer (pH 6.0, 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO2, 5 mM D-glucose, 25 mM MES)11 containing test drug (10 μM) or test drug (10 μM) and Gly-Sar (1 mM) was applied to the apical side as donor buffer, and 600 μL of HEPES buffer (pH 7.4) was applied to the basolateral side as receiver buffer. The samples of 200 μL were taken from the basolateral side at 30, 60, 90, and 120 min. The same volume (200 μL) of HEPES buffer was replenished to the basolateral side at the sampling time. TEER was measured on all monolayers after transepithelial transport studies. The sample was stored at −80 °C immediately until further treatment following the method below. HPLC/MS/MS Analysis. The concentration of the drugs was measured by the HPLC/MS method. The HPLC separation

consisted of a pump (model LC-10AT, Shimadzu, Japan), a Inertsil SIL-100A column (150 mm × 4.6 mm i.d., GL) maintained at 30 °C, a UV detector (model SPD-10A, Shimadzu), an autosampler (model SIL-10AD, Shimadzu, Japan), and a system controller (model SCL-10A, Shimadzu). The mobile phase used for all samples was water−methanol− TEA (12:88:0.01). The flow rate was 1 mL/min, and the detection wavelength was set at 210 nm. The retention time of OA, 7a, 7b, 7c, 9a, 9b, 9c, and 9d was about 11.5 min, 17 min, 20 min, 12.5 min, 12.7 min, 20 min, 11.9 min, and 10.84 min, respectively. Solubility. According to Higuchi and Connor’s method,26 the equilibrium solubility of the prodrugs and OA was determined (n = 3). Excess amounts (∼10 mg) of the samples were added in 1 mL of pure water and kept in a thermostatted oscillator (Z82, Changzhou Guohua Instrument Co. Ltd., China) maintained at 37 °C for 24 h. Samples were centrifuged at 10000 rpm for 10 min with a high speed centrifuge (TGL16GB, Shanghai Anting Scientific Instrument Plant, China). The supernatant was diluted, and the concentration of the drug was measured with HPLC. In Situ Rat Single-Pass Intestinal Perfusion (SPIP) Experiments. Perfusion Solutions. The blank perfusion solutions comprise MES buffer (pH 6.0, 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaC12, 0.8 mM MgSO4, 5 mM D-glucose, 25 mM MES, and 0.05% Cremophor EL) and HEPES buffer (pH 7.4, 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4, 5 mM D-glucose, 25 mM HEPES, and 0.05% Cremophor EL).11 The solubility of the test compounds in the perfusion solution was very low; 0.05% (w/v) Cremophor ER was added to improve solubility of the compounds. As the volume of perfusion solutions may be changed when the perfusion solutions passed the gastrointestinal tract, phenol red was used as a nonabsorbable marker for calculating the intestinal net water flux. Phenol red (80 μg/mL) was directly added to perfusion solutions containing 0.1 mM test compound. In inhibition studies, 50 mM Gly-Sar, a PepT1 inhibitor, was additionally dissolved in the perfusion solutions. Stability in Perfusion Buffers. The nonenzymatic stability of test compounds in both MES and HEPES buffers was determined using the procedure below. The test solutions were prepared by diluting DMSO (8 mg/mL) stock solutions containing the tested compounds with 3 mL of blank perfusion buffer. The test solutions were placed in a shaker bath set at 37 °C. At various time points, aliquot samples (0.1 mL) were removed and centrifuged at 12000 rpm for 10 min. The supernatants were immediately analyzed by HPLC to monitor degradation of the test compounds. All experiments were carried out at least in triplicate. Physical Absorption. In order to further ensure that the loss of drug from the perfusion is due to absorption, the nonspecific binding of the drug to the tubing is necessarily studied before the SPIP studies. The MES and HEPES buffers were selected in the binding studies. The absorption of prodrugs was tested in the tubing used for perfusion. According to the method of Cook,27 the tubings were treated with boiling water incubation for 2 h. Then, the solution of test compounds (1 mM) in buffer was separately incubated in the PVC tubing including treated and untreated for 2 h at 37 °C. Samples were removed at 0, 15, 30, 45, 60, 75, 90, and 120 min and centrifuged at 12000 rpm for 10 min. The supernatants were analyzed by HPLC. Single-Pass Intestinal Perfusion. SPIP studies were performed using established methods adapted from the 1380

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was performed on an ODS-SP (Inertsil, 150 × 4.6 mm, 5 μm, GL, Japan) with the following gradient at a flow rate of 1.0 mL/ min. The column temperature was set at 30 °C. The mobile phase A is methanol, and the mobile phase B is water (0.1% formic acid and 0.2% ammonium acetate). The gradient began with 70% A in 2 min and was changed to 97% A in about 4−5 min. Finally, it was changed back to 70% A. According to the structure of different prodrugs, the elution time was different. The tR of 7a (8.5 min), 7b (9 min), 7c (9 min), 9a (8.6 min), 9b (8 min), 9c (8 min), 9d (8 min), and OA (11.5 min) were measured in preliminary experiments. The mass spectrometer was operated according to the method in previous work.21 Quantification was performed with multiple selected reaction monitoring (MRM) of the transitions of m/z 455.4 → 455.4 for oleanolic acid, 600.4 → 483.7 for 7a, 648.8 → 484.4 for 7b, 614.5 → 483.7 for 7c, 713.5 → 497.7 for 9a and 9b, 685.4 → 497.4 for 9c, and 699.5 → 497.7 for 9d with a scan time of 0.2 s per transition. Data analysis method was as indicated below. Bioavailability Experiments in Rats. Pharmacokinetic Studies. The experiments were carried out using male Sprague−Dawley rats (weighing from 220 to 250 g). Rats were fasted for 12 h prior to drug administration with tap water. Perfusions were prepared by dissolving the prodrug 7a or 9b in water solution containing 20% HP-β-CD, and OA was suspended in the same solution. OA (300 mg/kg body weight) and prodrugs (300 mg/kg body weight, calculated as OA), respectively, were given to rats by the oral gavage method. Eighteen rats were divided randomly into three groups as follows: six rats of group OA were administered OA suspension; six rats of group 7a were administered 7a solution; six rats of group 9b were administered 9b solution. A blood sample of 0.4 mL was collected from the orbital plexus into heparin-treated tubes at 5, 15, and 30 min and 1, 2, 4, 6, 8, 12, and 24 h. These samples were immediately centrifuged at 5000 rpm for 10 min, and the plasma was collected and stored at −80 °C immediately until further treatment following the method described previously.29 HPLC/MS/MS Analysis. The sample was analyzed by the LC/MS/MS system as described previously.29 This method was verified in our previous research.21 Data analysis method was as indicated below. Data Analysis. SPIP Experiments. The intestinal effective permeability (Peff, cm/s) was calculated according to the following equation:30 Peff = [ − Q in ln Cout(corr)/C in]/2πrL

Caco-2 Permeability Experiments. The apparent permeability (Papp, cm/s) was calculated using the following equation:31 Papp = dQ /dt /A /C0

where dQ/dt is the initial permeation rate across the Caco-2 cell monolayer (micromoles per second), A is the surface area of the filter membrane (0.6 cm2), and C0 is the initial concentration (mg/mL) of tested compounds in the donor solution. The concentration of OA and its prodrug were analyzed using LC−MS. Pharmacokinetic Studies. The mean plasma concentration vs time profile was obtained. The peak plasma concentration (Cmax) and the time to reach peak concentration levels (tmax) were obtained. To estimate absorption profile the standard noncompartmental analysis was performed (Kinetica, Ver 4.4, Innaphase, Corp., Philadelphia, PA, USA). These pharmacokinetic parameters were calculated including the area under the plasma concentration−time curve (AUC) from time zero to the last measured concentration (AUC0−t) and the apparent bioavailabilities (Fapp) of OA which following administration of prodrug was calculated as OA. Statistically significant differences between two groups were evaluated by Student’s test. Significance is reported as **p < 0.01 for all tests.

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(1)

where Qin is the flow rate (mL/min) of inlet solution, Cin is the concentration (mg/mL) of drug in the entering solution, and Cout(corr) is the concentration (mg/mL) of drug in the exiting solution corrected for water flux. L is the length of the segment (cm), r is the radius of the intestinal lumen (cm), and the Cout(corr) was calculated according to the following equation: Cout(corr) = CoutCphenolred(in)/Cphenolred(out)

RESULTS AND DISCUSSION

Chemistry. For the synthesis of the prodrugs, we first tried our former reported protocol by means of directly connecting 1,2-propanediol to the 28-carboxyl group of oleanolic acid to form the ester intermediate.21 However, the results turned out that two isomers had been formed after the reaction, which were very difficult to separate. The isomers probably result from the poor reaction selectivity between the two hydroxyl groups of 1,2-propanediol, though the reactivity of the primary hydroxyl group may be higher than that of the second hydroxyl group. Furthermore, we also tried using propylene oxide to introduce the 1,2-propanediol linker, but again we got a mixture of the two ester products. Thus, we turned to chloroacetone, which is a suitable substrate readily reacting with oleanolic acid to form the ester product 2; then reducing the carbonyl group with NaBH4 offered the desired intermediate 3′. Then our task is to connect the amino acid or dipeptide moiety to the hydroxyl group of 1,2-propanediol linker. Again we first tried directly treating 3′ with the corresponding Boc-amino acid or Boc-dipeptide to form the ester bond. Surprisingly two products were obtained after the reactions. The MS spectroscopy showed they had the same molecular weight, indicating the 3-position hydroxyl group of oleanolic acid, besides the secondary hydroxyl group of the 1,2-propanediol linkers, probably also took part in the esterification reaction. It was out of our expectation because our former experience in preparing ethylene glycol-linked oleanolic acid prodrugs suggested the reactivity of 3-position hydroxyl group was too low to participate in the reaction. Therefore, in order to avoid the 3-OH ester side products, 3,4-dihydro-2H-pyran was employed as a protective agent. Generally, compound 2 first reacted with 3,4-dihydro-2H-pyran to give ether intermediate 3, which was then reduced by NaBH4 to yield 4. Treating 4 with a different Boc-amino acid in the presence of DCC and a catalytic amount of DMAP offered 5. Thereafter, the tetrahydropyran group was successfully removed by treating 5 with ptoluenesulfonic acid, resulting in 6a−e, respectively. After the

(2)

where Cout is the concentration (mg/mL) of outlet drug, Cphenolred (in) is the concentration (mg/mL) of phenol red in the entering solution, and Cphenolred (out) is concentration (mg/mL) of phenol red in the exiting solution. Values are expressed as means ± standard deviation (SD) for at least 3 measurements. Statistically significant differences between two groups were evaluated by Student’s test. A p < 0.05 was considered significant for all tests. 1381

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Scheme 1. Synthesis of the Target Compounds 7a−c and 9a−da

Reaction conditions: (a) ClCH2COCH3, DMF, K2CO3, 60 °C. (b) CH2Cl2, rt. (c) NaBH4, CH3OH, 0 °C. (d) Boc-amino acid, DCC, DMAP, CH2Cl2, rt. (e) p-CH3C6H4SO3H, CH3OH, rt. (f) (C2H5)2O, dry HCl gas, 0 °C, then NaHCO3 aqueous solution. (g) Boc-amino acid, DCC, DMAP, CH2Cl2, rt. (h) (C2H5)2O, dry HCl gas, 0 °C. a

Water Solubility. Solubility studies were carried out in water. The equilibrium solubility values of all the test prodrugs are significantly higher relative to OA, which is practically insoluble in water, about 0.012 μg/mL.16 The water solubility of 7a, 7b, 7c, 9a, 9b, and 9d is 35.3, 23.4, 12.3, 69.6, 58.2, and 83.2 μg/mL, respectively, and the water solubility of 9c is more than 1 mg/mL. Our previous research showed that the water solubility of L-Val diester and L-Phe diester with ethylene glycol linker was only 25 ± 6 and 3.7 ± 1.0 μg/mL, respectively.21 The prodrugs with propylene glycol linker (7a, 7b) have higher

removal of the Boc group by means of treating with HCl gas in ether, the amino acid derivatives 7a−e were successfully obtained. For the synthesis of dipeptide derivatives 9a−d, we had made attempts at treating commercially available Bocdipeptides with 4 in the presence of DCC and a catalytic amount of DMAP. However, few products were formed after the reactions. Thus, we had to prepare them by introducing two amino acid units one by one. Compounds 7a, 7d, and 7e then further reacted with the corresponding Boc-amino acid to form 8a−d, respectively. Finally, treating 8a−d with HCl gas in ether yielded the desired dipeptide derivatives 9a−d (Scheme 1). 1382

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permeability because it has been shown to be predictive of in vivo absorption in humans.33 Studies demonstrated that Cremophor EL showed higher solubilizing capacity of OA and the prodrugs than PEG 200, PEG 400, and HP-β-CD (data not shown). It was easy to get perfusion solutions (containing 0.05% Cremophor EL) of OA or the prodrugs with the concentration of 0.1 mg/mL OA or the prodrugs, so 0.05% Cremophor EL was used to increase the solubility in SPIP studies. In this work, since PepT1 employed a proton gradient as the driving force, the effective permeability (Peff) for OA and the prodrugs was compared in two perfusion solutions with different pH (6.0 and 7.4).34,35 0.1 mM OA or seven prodrugs in the buffers was separately prepared for perfusion studies. The results are listed in Figure 1 and show that there was no difference of the Peff of OA in the two buffers.

water solubility, which may offer some formulation-related advantages. Stability Studies. In order to verify that loss of drug during the perfusion is due to permeation of the intestine, chemical stability of the diester prodrugs was performed at 37 °C in MES and HEPES buffers for 2 h. In the MES buffer, the remaining percentage of OA, 7a, 7b, 7c, 9a, 9b, 9c, and 9d was 97.25%, 98.52%, 94.80%, 97.58%, 100.37%, 95.87%, 100.20%, and 97.46%, respectively. The remaining percentage of OA, 7a, 7b, 7c, 9a, 9b, 9c, and 9d in HEPES buffer was 100.42%, 100.20%, 98.33%, 99.51%, 97.06%, 95.15%, 97.13%, and 96.85%, respectively. Both OA and the prodrugs showed little degradation in two kinds of perfusion buffers at 37 °C for 2 h. These results demonstrated that the loss of drug during the perfusion was not due to the drug degradation in the buffer and guaranteed the test substances were sufficiently stable for the duration of SPIP experiments. Our previous studies revealed that the remaining percentage of L-Val diester and L-Phe diester with ethylene glycol linker was 97.75% 93.40% in the MES buffer and 93.28% and 99.81% in the HEPES buffer, respectively.21 The results show that in two buffers chemical stability of the prodrugs with different linker is good and show little difference. Additionally, the stability of the L-valine ester prodrugs with propylene glycol linker (7a) was increased in both buffers. The reason may be that the propylene glycol linker changes the stability of the ester bond. An electron-donating methyl is introduced which decreases the electropositivity of carbonyl carbon and increases the stability of the ester bond. The Amidon group has reported stability profiles for amino acid ester prodrugs with various acyloxy linkers and has shown that the prodrugs with propylene glycol and ethoxy linker are more stable than the prodrugs with methoxy linker.32 Our result is similar to Amidon’s results. Physical Absorption of Test Compounds. The perfusion tube is the PVC tubing, so physical adsorption probably happens during the experiments. The physical adsorption of test compounds with different tubing including the untreated PVC tubing and the treated PVC tubing, which was boiled in boiling water for 2 h, was compared. For the untreated PVC tubing, the remaining percentage of OA, 7a, 7b, 7c, 9a, 9b, 9c, and 9d was 74.07%, 62.37%, 63.94%, 76.35%, 90.22%, 90.31%, 80.66%, and 83.21%, respectively. The results show that OA, 7a, 7b, and 7c exhibited obvious binding to the untreated PVC tubing at 37 °C for 2 h and 9c and 9d exhibited little adsorption. For pretreatment of the tubing with boiling water, the remaining percentage of OA, 7a, 7b, 7c, 9a, 9b, 9c, and 9d was 101.13%, 95.16%, 93.16%, 96.34%, 96.08%, 95.78%, 94.58%, and 97.87%, respectively. This study revealed the nonspecific binding of the test compounds was effectively decreased in the treated tubing. Moreover, the change of the concentration of all test drugs was very small during 30 min. Based on the binding results, the pretreated PVC tubing with boiling water for 2 h was used in the SPIP studies and the tubing was perfused with perfusion solutions for 30 min to attain stable test compounds before the SPIP studies. The combined results of the binding and stability studies showed that the loss of drug from the perfusion was owing to absorption only but not due to other losses (e.g., chemical degradation or nonspecific binding to the tubing).27 SPIP of the Test Compounds in Rat Jejunums. For permeability determinations, a variety of experimental methods can be used, such as SPIP in rats or Caco-2 cell culture models. The SPIP model was used to screen drug membrane

Figure 1. The effective permeability (Peff) for 0.1 mM OA and its prodrugs in MES buffer (pH 6.0) and HEPES buffer (pH 7.4) obtained from in situ rat single-pass intestinal perfusion (SPIP) experiments (mean ± SD, n ≥ 3). **p < 0.01 as compared to that in pH 7.4. *p < 0.05 as compared to that in pH 7.4. †p < 0.01 as compared to that of OA in pH 6.0.

In HEPES buffer of pH 7.4, compared to OA, 5.2-fold, 2.0fold, 3.1-fold, and 1.7-fold enhancement of Peff was observed for 7a, 7b, 9b and 9c, respectively. The enhancement of Peff was observed with the reported prodrugs L-Val diester (1.31-fold) and L-Phe diester (1.85-fold) with ethylene glycol linker, compared to that with OA.21 In MES buffer of pH 6.0, Peff of 7a, 7b, 9b, and 9c were 6.7-fold, 2.4-fold, 4.2-fold, and 2.2-fold higher than that of OA (p < 0.01), respectively. Compared with Peff of OA, Peff was 2.3-fold and 2.5-fold increased with the reported prodrugs with ethylene glycol linker of L-Val ester and 21 L-Phe ester, respectively. Except 7b, all prodrugs showed significantly increased permeability through jejunum in pH 6.0 which had high H+ concentration than that in pH 7.4 (p < 0.01). This result showed that H+ concentration of perfusion solution had a great effect on the transport of the prodrugs across the intestinal wall, indicating that this may be attributed to PepT1-mediated transport. 7a, 9b, and 9c showed significantly increased permeability through jejunum compared to OA (p < 0.01). The differences of the prodrugs’ permeability may be owing to their differential affinity toward the PepT1. Above all, prodrugs with propylene glycol linker have shown higher permeability in rat jejunums than prodrugs with ethylene glycol linker. The possible reason is that the propylene glycol linker promotes the affinity of the prodrugs for PepT1. The results above also have 1383

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is bigger than the prodrugs with ethylene glycol linker, indicating stronger affinity to PepT1 of the prodrugs with propylene glycol linker. Moreover, the dipeptide diester prodrugs 9b showed preferences for PepT1 transport than 9c. Caco-2 Permeability Experiments. To further confirm the superiority of permeability and affinity of these prodrugs, the Caco-2 cell model, which has been recognized as an excellent model in vitro for intestinal absorption, was employed to investigate the affinity for PepT1.38 During the transport and inhibition experiments, TEER on all monolayers was little changed, indicating that the tight junctions were not opened and the cell function was normal. The rank order of Papp of all prodrugs’ transport across the Caco-2 cell monolayer (n = 3) was as follows: 7a (5.27-fold), 9b (3.31-fold), 9a (2.26-fold), 7b (2.10-fold), 7c (2.03-fold), 9c (1.87-fold), 9d (1.39-fold). All prodrugs showed significantly increased permeability through the Caco-2 cell monolayer compared to OA (p < 0.01) (Figure 3). Inhibition studies were carried out for 7a, 9a, 9b, and 9c in

demonstrated that the phenylalanine ester prodrugs have less permeability and affinity of PepT1 than the corresponding valine counterpart. Our results are also consistent with previous observations from some researchers that have proved that compounds with valine terminal residue of 1-beta-D-arabinofuranosylcytosine and didanosine exhibited higher permeability and affinity of PepT1.36,37 The reason may be that L-valine has the optimal combination of chain length and branch at the β-C of the amino acid for PepT1 recognition and binding affinity. Among the dipeptide promoieties prodrugs, L-Val-L-Val ester (9b) showed the highest permeability. Compared with D-Val-LVal ester (9a) and L-Ala-L-Val ester (9c), attachment of an Lvaline to the middle of the prodrugs showed a relatively higher permeability and affinity to PepT1, which is in agreement with the previous report of dipeptide prodrugs of saquinavir.28 Compared with L-Ala-L-Val ester (9c) and L-Ala-L-Ile ester (9d), attachment of an L-valinyl promoiety to the N-terminus of L-alaninyl OA resulted in permeability and affinity enhancement. SPIP of OA and Its Prodrugs in the Presence of GlySar. For the further evaluation affinity of the prodrugs to PepT1, inhibition studies in MES buffer containing 0.1 mM prodrug were carried out in the presence of 50 mM Gly-Sar. It turned out that Peff of 7a (5.2-fold), 7b (2.0-fold), 9b (3.1-fold), and 9c (2.3-fold) had significantly reduced (p < 0.01) in the presence of Gly-Sar (Figure 2).

Figure 3. The apparent permeability coefficient (Papp) for 10 μM OA and its prodrugs in MES buffer (pH 6.0) transport across Caco-2 cell monolayers (n = 3). **p < 0.01 as compared to that of OA.

the presence of Gly-Sar (1 mM). Papp of 7a, 9b, and 9c reduced by 1.3-fold, 1.6-fold, and 1.4-fold (p < 0.01), respectively, but for that of 9a no significant difference was detected (p < 0.05) across the Caco-2 cell monolayer (Figure 4). First the same results from the SPIP model and the Caco-2 model showed that 7a and 9b showed higher permeability than

Figure 2. The effective permeability (Peff) for 0.1 mM prodrugs in MES buffer (pH 6.0) obtained from in situ rat single-pass intestinal perfusion (SPIP) experiments in the presence and absence of 50 mM Gly-Sar (mean ± SD, n ≥ 3). **p < 0.01 as compared to that in the absence of Gly-Sar.

We supposed that it was attributable to PepT1-mediated transport of these prodrugs. Certainly, the amino acid promoieties also influenced the affinity of the prodrugs to PepT1. The permeability of 7a and 9b was reduced significantly (p < 0.01) in the presence of Gly-Sar from 21.110 ± 0.953 × 10−5 and 13.150 ± 1.940 × 10−5 cm/s to 4.010 ± 1.068 × 10−5 and 4.189 ± 0.183 × 10−5 cm/s, respectively. These results suggest that prodrug with valinyl counterpart has stronger affinity to PepT1. The same conclusion has been observed in our previous studies.21 Peff of the reported prodrugs with ethylene glycol linker of L-Val ester and L-Phe ester was reduced in the presence of Gly-Sar from 7.3589 ± 1.5537 × 10−5 cm/s and 7.9734 ± 1.3748 × 10−5 cm/s to 4.2722 ± 1.3509 × 10−5 cm/s and 4.2191 ± 1.1103 × 10−5 cm/s, respectively.21 The extent of reduction of the prodrugs with propylene glycol linker

Figure 4. The apparent permeability coefficient (Papp) for 10 μM OA and its prodrugs in MES buffer (pH 6.0) transport across Caco-2 cell monolayers in the presence of 1 mM Gly-Sar (n = 3). **p < 0.01 as compared to that in the absence of Gly-Sar. *p < 0.05 as compared to that in the absence of Gly-Sar. 1384

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Figure 5. Mean plasma concentration−time profiles of oleanolic acid for group OA, group 7a, group 9b following intragastric administration of OA (300 mg/kg body weight) or prodrugs (300 mg/kg body weight, calculated as OA) in water solution containing 20% HP-β-CD to rats (mean ± SD, n = 6).

prodrugs with propylene glycol linker have a longer time to activate to the parent compound following transport across intestinal membrane and have a higher activity. Values of MRT for OA, 7a, and 9b were 15.52 ± 3.40 h, 25.20 ± 1.16 h (p < 0.01), and 63.87 ± 3.17 h (p < 0.01), respectively. Compared to the reported group L-Val ester with ethylene glycol linker with MRT of 26.85 ± 4.19 h (p < 0.01),21 MRT of the dipeptide diester prodrugs 9b was significantly prolonged. The reason may be that the peptidases hydrolyze the dipeptide bond generating the amino acid ester of OA which will be further hydrolyzed into OA. With the same L-valine counterpart the effect of two linkers on MRT was similar. Fapp of groups 7a and 9b was 355% (3.55-fold) and 339% (3.99-fold). Finally, Figure 5 indicates that the plasma concentration of OA declined rapidly over the first stage and was followed by another peak, which was probably due to the enterohepatic recirculation.39 These results demonstrated that amino acid/dipeptide diester prodrugs of OA with the propylene glycol linker targeted to PepT1 is an effective strategy to improve the oral bioavailability of this parent drug. Moreover, it was shown that the phenylalanine prodrugs have less stability, permeability, and affinity than the valine prodrugs. This conclusion verifies our previous results.21

other prodrugs. In inhibition studies, permeability of 7a and 9b showed significant decrease in the presence of Gly-Sar. Overall 7a and 9b had stronger affinity for PepT1 than other prodrugs, so 7a and 9b were used in the pharmacokinetic studies. Pharmacokinetic Studies. Based on the permeability and affinity to PepT1 in both SPIP and Caco-2 models, 7a and 9b were selected to conduct the pharmacokinetic studies with OA as comparison. As the prodrugs were rapidly hydrolyzed into OA and the concentration of the prodrugs in plasma was very low (data not shown), therefore, we mainly focused on the pharmacokinetic performances of OA after oral administration of compounds 7a and 9b. The concentration of OA in rat plasma was determined by the HPLC/MS/MS method. The accuracy of analysis method has been confirmed in our previous research.21 According to the results in vivo, plasma concentration−time profiles of OA in 24 h are shown in Figure 5. Pharmacokinetic parameters have been summarized in Table 1. Table 1. Main Pharmacokinetic Parameters and Apparent Bioavailability (Fapp) of Oleanolic Acid, Following Intragastric Administration of 300 mg/kg (Calculated as OA) 7a, 9b, and OA to Rats, Respectively (Mean ± SD, n = 6) Cmax (μg/mL) Tmax (h) AUC0→24h (μg·h/mL)b MRT (h) Fapp (%) a

group OA

group 7a

group 9b

0.47 ± 0.034 0.50 4.98 ± 0.42

1.43 ± 0.17**a 1.25 ± 1.37 17.68 ± 3.07**

1.23 ± 0.24** 1.67 ± 1.81 16.88 ± 2.84**

15.52 ± 3.40 100

25.20 ± 1.16** 355

63.87 ± 3.17** 339

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CONCLUSIONS To further evaluate effect of the type of linker on stability, permeability, affinity, and bioavailability, seven amino acid/ dipeptide diester prodrugs with propylene glycol linker of OA were successfully synthesized. The water solubility of all the test prodrugs was significantly greater than that of OA. The SPIP model was performed to screen the Peff of the prodrugs. Peff of all prodrugs in MES of pH 6.0 is greater than that of OA. In HEPES of pH 7.4, except for 7c, 9a, and 9d, Peff of the other prodrugs exhibited better than that of OA (p < 0.01). In inhibition studies in the presence of Gly-Sar, it turned out that Peff of 7a, 7b, 9b, and 9c was significantly reduced (p < 0.01). Compared to Papp of OA with Caco-2 cell monolayer, significant enhancement of the Papp of all test prodrugs also was observed (p < 0.01). Inhibition studies with Gly-Sar (1 mM) showed that Papp of 7a, 9b, and 9c was reduced by 1.3fold, 1.6-fold, and 1.4-fold (p < 0.01), respectively. Furthermore, 7a and 9b exhibited enhanced oral bioavailability of OA in rats with Fapp of 3.55- and 3.39-fold increase, respectively. These results demonstrated that amino acid/ dipeptide diester prodrugs of OA with the propylene glycol

p < 0.01 as compared to group OA. bAUC is for oleanolic acid.

Following oral gavage administration of 300 mg/kg (calculated as OA), values of Tmax of groups 7a and 9b were about 1.25 and 1.67 h, respectively. Values of AUC0→24 and Cmax of both groups 7a and 9b had significant increases, compared to group OA (p < 0.01). Values of Cmax for 7a and 9b were 1.43 ± 0.17 μg/mL (p < 0.01) and 1.23 ± 0.237 μg/mL (p < 0.01), respectively; AUC0→24 was improved to 3.55-fold (p < 0.01) and 3.39-fold (p < 0.01), respectively. These results suggested that 7a and 9b had better oral absorption than OA. Moreover, the reported value of Cmax of the group L-Val ester, the prodrugs with ethylene glycol linker, was 0.73 μg/mL and Tmax of about 0.83 h.21 Hence, we can conclude that the 1385

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linker targeted to PepT1 is an effective approach to improve the oral bioavailability of OA, a BCS class IV drug. Combining our previous studies with this study, we can conclude that prodrugs of OA with propylene glycol linker might possess advantages of solubility, stability, permeability, affinity, and bioavailability over ethylene glycol linker for designing diester prodrugs of poorly absorbed carboxylic acid drug of OA. Certainly, some research may be further conducted. We have selected two models, namely, the SPIP model and the Caco-2 model, which both are traditional methods to evaluate membrane permeability. Inhibition studies just preliminarily demonstrated the PepT1 targetability of the prodrugs. PEPT1expressing Xenopus laevis oocytes, HeLa cells, or MDCK cells can be used to directly evaluate the target of the prodrugs to PepT1.

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ASSOCIATED CONTENT

S Supporting Information *

Additional experimental details. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*L.F.: Pharmaceutical Research Center, Southeast University, No. 2 Southeast-University Road, Nanjing 211189, P. R. China; tel/fax, +86-25-83272381; e-mail, [email protected]. Q.P.: Department of Pharmaceutics, School of Pharmacy, China Pharmaceutical University, No. 24, Tongjia Road, Nanjing 210009, P. R. China; tel/fax, +86-25-83271092; e-mail, [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful for financial support from the National Natural Science Foundation of China (No. 81001413), Specialized Research Fund for the Doctoral Program of Higher Education of China (No.20090096120002) and the Open Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University (No. G120005).

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ABBREVIATIONS USED PepT1, peptide transporter 1; hPepT1, human peptide transporter 1; HP-β-CD, hydroxypropy-beta-cyclodextrin; BCS, Biopharmaceutics Classification System; OA, oleanolic acid; Peff, effective permeability; Papp, apparent permeability; SPIP, single-pass intestinal perfusion; Gly-Sar, glycyl-sarcosine; HPLC/MS/MS, high-performance liquid chromatography tandem mass spectrometry; MES, 2-(4-morpholino)ethanesulfonic acid; HEPES, hydroxyethyl piperazine ethanesulfonic acid; DMF, N,N-dimethylformamide; DCC, N,N′dicyclohexylcarbodiimide; DMAP, 4-(dimethylamino)pyridine; DMSO, dimethyl sulfoxide; DMEM, Dulbecco’s modified Eagle medium; FBS, fetal bovine serum; TEER, transendothelial electrical resistance; AUC, area under the plasma concentration time curve; Cmax, peak plasma concentration; MRT, mean residence time; Fapp, apparent bioavailability

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