Ethylene Glycol-Linked Amino Acid Diester Prodrugs of Oleanolic Acid

Feb 21, 2012 - *Y.L.: State Key Laboratory of Natural Medicines, Center of Drug Discovery, ... Fapp of group 5a and 6f was 2.21- and 2.04-fold increas...
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Ethylene Glycol-Linked Amino Acid Diester Prodrugs of Oleanolic Acid for PepT1-Mediated Transport: Synthesis, Intestinal Permeability and Pharmacokinetics Feng Cao,†,‡ Jinghao Jia,‡,§ Zhi Yin,† Yahan Gao,† Lei Sha,§ Yisheng Lai,*,§ Qineng Ping,*,† and Yihua Zhang§ †

Department of Pharmaceutics and §Center of Drug Discovery, School of Pharmacy, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, P. R. China S Supporting Information *

ABSTRACT: The purposes of this study were to expand the structure of parent drugs selected for peptide transporter 1 (PepT1)-targeted ester prodrug design and to improve oral bioavailability of oleanolic acid (OA), a Biopharmaceutics Classification System (BCS) class IV drug. Through an ethoxy linker the carboxylic acid group of OA was conjugated with the carboxylic acid group of different amino acid promoieties to form six diester prodrugs. The effective permeability (Peff) of prodrugs was screened by in situ rat single-pass intestinal perfusion (SPIP) model in two buffers with different pH (6.0 and 7.4) as PepT1 employs a proton-gradient as the driving force. Compared to OA, 2.5-fold, 2.3-fold, 2.2-fold, 2.1-fold, and 1.9-fold enhancement of Peff in buffer with pH 6.0 was observed for L-Phe ester (5c), L-Val ester (5a), L-Lys ester (5e), D-Phe ester (5d), and D-Val ester (5b), respectively. Furthermore, Peff of 5a, 5c, 5d and 5e in pH 6.0 was significantly higher than that in pH 7.4 (p < 0.01), respectively. These results showed that the H+ concentration of perfusion solution had great effect on the transport of the prodrugs across intestinal membrane. For the further evaluation of affinity to PepT1, inhibition studies were performed by coperfusing 0.1 mM prodrug with 50 mM glycyl-sarcosine (Gly-Sar, a typical substrate of PepT1). It turned out that the Peff of 5a, 5b, 5c and L-Tyr ester (6f) significantly reduced in the presence of Gly-Sar (1.7-fold, 2.2-fold, 1.9-fold, and 1.4-fold, respectively). We supposed that it may be attributed to PepT1 mediated transport of these prodrugs. 5a and 6f were selected as the optimal target prodrugs for oral absorption in vivo. Following intragastric administration of 300 mg/kg (calculated as OA) 5a, 6f and OA in three groups of rats, compared with group OA, Cmax for the group of 5a and 6f was enhanced by 1.56-fold and 1.54fold, respectively. Fapp of group 5a and 6f was 2.21- and 2.04-fold increased, respectively, indicating that 5a and 6f had better oral absorption than OA. The combined results also suggest that diester prodrugs which conjugated two carboxylic acid groups of proper amino acid promoieties and parent drug through a linker can be used for PepT1-targeted prodrug design. With this strategy, oral bioavailability of OA in rats could be improved significantly. KEYWORDS: oleanolic acid, peptide transporter 1, ester prodrug, in situ single-pass intestinal perfusion, pharmacokinetics, bioavailability

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effect.4 However, its water solubility is only 1.2 × 10−2 μg/mL5

INTRODUCTION

Oleanolic acid (OA), (3β)-3-hydroxyolean-12-en-28-oic acid, is a triterpenoid compound that exists widely in many Asian herbs, such as Fructus ligustri lucidi, Fructus forsythiae, Radix ginseng and Akebia trifoliate. OA has been used for several decades in tablet form for its hepatoprotective effects,1 antiinflammatory effects,2 antinociceptive potential3 and antitumor © 2012 American Chemical Society

and its absolute oral bioavailability is approximately 0.7% in Received: Revised: Accepted: Published: 2127

August 31, 2011 February 8, 2012 February 20, 2012 February 21, 2012 dx.doi.org/10.1021/mp200447r | Mol. Pharmaceutics 2012, 9, 2127−2135

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rats.6 Transepithelial transport studies performed in Caco-2 cell monolayer showed that apparent permeability (Papp) value of OA was 1.1−1.3 × 10−6 cm/s in the apical-to-basolateral direction at 10 and 20 μM.6 According to the Biopharmaceutics Classification System (BCS),7 OA can be categorized as a BCS class IV drug. In order to improve oral bioavailability of OA, various formulation strategies have been attempted, such as nanosuspension,8,9 self-nanoemulsified system10 and solid dispersion.11 However, in terms of bioavailability enhancement, these formulation approaches still have limitations, since they do not give a solution of poor intestinal permeability with a BCS class IV compound. Prodrug strategies have been increasingly utilized over the past two decades in order to avoid undesirable biopharmaceutical characteristics of parent drugs, such as low aqueous solubility, low permeability, propensity to be an efflux substrate, and rapid and extensive hepatic metabolism and biliary excretion, and eventually improve their absorption.12 Recent progress in molecular biology has allowed the identification and cloning of nutrient transporters, the elucidation of the functional and structural characteristics and regulation of these proteins. Membrane transporter targeted prodrugs are designed to have structural features that would allow them to be taken up by one of the endogenous transporters present at the intestinal epithelium. Furthermore, lots of prodrugs have been designed for targeting delivery via specific membrane transporters, including peptide transporter 1(PepT1),13 human apical sodium-dependent bile acid transporter (hASBT),14 monocarboxylate transporter 1 (MCT1),15 organic-anion transporting polypeptides (OATPs),16 glucose transporter 1(GluT1),17 sodium dependent vitamin C transporter (SVCT),18 and so on. Peptide transporters appear to be attractive targets in prodrug design for their high capacity, broad substrate specificity, high level of expression in the intestinal epithelium and low occurrence of functional polymorphisms. The Amidon lab is well-known for their findings and studies done on peptide transporters in the intestine and their use in prodrug targeting/ delivery.19−21 Conjugating a specific amino acid with a drug can make them a substrate for PepT1, thus enhancing the absorption of the parent drug. Two excellent examples of marketed prodrugs that exploit carrier-mediated transport are valacyclovir (Valtrex; GlaxoSmithKline) and valganciclovir (Valcyte; Roche). They are L-Val esters (for example, amino acid valine as the promoiety) of acyclovir and ganciclovir, which both have limited and variable oral bioavailability owing to their high polarity. These amino acid prodrugs increased the intestinal permeation of their parent drugs by 3−10-fold, and their membrane transport was mediated predominantly by the di- and tripeptide transporter (hPepT1) expressed in the intestinal epithelial cell.21,22 In recent years, many novel structural variations of prodrugs targeting PepT1 have been reported. Two amide prodrugs have been demonstrated whose transport is mediated by PepT1. The N-linked alanyl derivative LY544344 which was selected from a large number of amino acid derivatives of LY354740 showed high affinity to PepT1 in Caco-2 cells, and a significant increase in the bioavailability of the active drug.23,24 The prodrug midodrine containing a glycine promoiety that is attached to the amine functionality of desglymidodrine is a substrate for hPepT1. This carrier mediated transport raises the bioavailability of midodrine to 93%, compared with 50% for desglymidodrine.25 In prodrug design, amides have been used

only to a limited extent owing to their relatively high enzymatic stability in vivo. The use of the hydroxyimine functionality as a prodrug for ketones targeted to PepT1 has been reported recently. The authors designed thiodipeptide as carrier attached to the desired drug through a hydrolyzable linker.26,27 More recently, PepT1-mediated absorption of peptidomimetic prodrugs of didanosine and cidofovir has been verified in Caco-2 cells and in Xenopus laevis oocytes overexpressing human PepT1, respectively.28,29 According to the data on several hundred PepT1 substrates which have now been published, a N-terminal amino group may be required for the optimal transport of prodrug targeted to PepT1.30 In view of this, almost all new PepT1-targeted ester prodrugs designed were involved in modifications of amine, hydroxyl or carbonyl groups of the parent drugs with carboxylic acid group of amino acids, resulting in a N-terminal amino group. Few carboxylic acid parent drugs have been so far employed for this type of transporter targeted ester prodrug design. To the best of our knowledge, there is only one paper reported by the Amidon group which evaluated the chemical and enzymatic stabilities of prodrugs containing methoxy, ethoxy and propylene glycol linkers of benzoic acid and phenyl acetic acid.31 In order to further extend the kind of structure of parent drugs selected for PepT1-targeted prodrug design and improve the oral bioavailability of OA, a BCS class IV drug, 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. In this work, six amino acid ester prodrugs of OA have been designed and prepared for targeted delivery to the PepT1. Amino acid promoieties with a broad range of physicochemical and structural properties were selected in this study, including the aliphatic amino acids (Lvaline, D-valine), as well as the aromatic amino acids (Lphenylalanine, D-phenylalanine and L-tyrosine), and the polar amino acid L-lysine. Permeability studies were carried out with the rat in situ single-pass intestinal perfusion (SPIP) model and used to screen their membrane permeability because SPIP has been shown to be predictive of in vivo absorption in humans.32 The effect of Gly-Sar, a PepT1 inhibitor, on the permeability was also conducted to preliminarily evaluate PepT1-targeted characteristics of the prodrugs. Finally, pharmacokinetic studies in rats were performed after oral administration of OA, L-valine ester and L-tyrosine ester of OA, respectively.

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EXPERIMENTAL SECTION Materials. OA was purchased from Nanjing Zelang Medical Technological Co. Ltd. Gly-Sar was obtained from SigmaAldrich (St. Louis, MO, USA). HP-β-CD was purchased from Taixing YiMing Biological Products Co., Ltd. Cremophor EL was kindly supplied by BASF. Hydroxyethyl piperazine ethanesulfonic acid (HEPES) and 2-(4-morpholino)ethanesulfonic acid (MES) were purchased from Bio basic Inc. and Nanjing Robiot Co., Ltd., respectively. Glycyrrhetinic acid (GA, lot No.: 110723-200612) was purchased from National Institute for the Control of Pharmaceutical and Biological Products. All other chemicals were of analytical grade or HPLC (high performance liquid chromatography) grade. General Method. Synthesis of Diester Prodrugs of Oleanolic Acid (5a−5e, 6f). To a solution of oleanolic acid (6.6 mmol) in DMF (40 mL) were added 2-bromoethanol (3 equiv) and potassium carbonate (1.5 equiv), and the mixture was stirred at room temperature for 6 h. After filtration, water 2128

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(50 mL) was added into the filtrate and the obtained mixture was stirred for an additional 0.5 h. The precipitate was collected by filtration and washed with water to give oleanolic acid 2hydroxylethyl ester 3. To a solution of 3 (1 equiv) in dry CH2Cl2 (40 mL) were added Boc-amino acid 2a−2f (1.5 equiv), DCC (1.2 equiv), and DMAP (0.5 equiv), and the mixture was stirred at room temperature for 12 h. After filtration, the filtrate was evaporated to give a crude product, which was purified by column chromatography to yield Bocamino acid ethyl esters 4a−e. Dry hydrogen chloride gas was bubbled into a solution of 4a−4e, 5f in dry ether (40 mL) for 0.5 h. The precipitate was collected by filtration and washed with dry ether to give the final product. More detailed information is summarized in the Supporting Information. HPLC Analysis. Samples of the prodrugs and OA were analyzed by HPLC consisting of a pump (model LC-10AT, Shimadzu, Japan), a Insertsil SIL-100A column (150 mm × 4.6 mm i.d., GL) maintained at 30 °C, a UV detector (model SPD10A, Shimadzu), an autosampler (model SIL-10AD, Shimadzu, Japan) and a System Controller (model SCL-10A, Shimadzu). The compounds were eluted with different mobile phases and wavelengths (Table 1). The flow rate was 1 mL/min.

mL. In addition, 50 mM Gly-Sar was dissolved in perfusion solutions to achieve solutions containing PepT1 inhibitor. Stability in Perfusion Buffers. Stability of the test compounds was tested in both MES and HEPES buffers. Stock solutions of the prodrugs (8 mg/mL) were prepared in dimethyl sulfoxide (DMSO) and used immediately. Aliquots (9 mL) of the blank buffer were placed in screw-capped vials and allowed to equilibrate at 37 °C. Prodrug stock solution (60 μL) was subsequently added to the buffer, and the vials were placed in a constant shaker bath set at 37 °C and 60 rpm. Samples (0.5 mL) were collected at appropriate time intervals for up to 2 h and centrifuged at 12000 rpm for 10 min. The supernatants were immediately assayed by HPLC to monitor degradation of the test compounds. All experiments were conducted at least in triplicate. Physical Absorption. The binding studies of the compounds were performed in the tubing used in SPIP experiments. MES and HEPES buffers were used in the binding studies. According to the method of Cook,39 solution of test compounds in buffer (50 μg/mL) was incubated in the PVC tubing at 37 °C. The tubings were left untreated before incubation or pretreated for 12 h with a 1 mg/mL solution of the test compound in methanol. All test compounds were separately incubated in the tubing for 2 h at 37 °C. Samples were collected at 0, 15, 30, 45, 60, 75, 90, and 120 min and centrifuged at 12000 rpm for 10 min. The supernatants were assayed for the test compounds by HPLC. Single-Pass Intestinal Perfusion. Animal experiments were conducted in accordance with the guide for the care and use of laboratory animals of China Pharmaceutical University. Male albino Sprague−Dawley rats, weighing 200−250 g, were used in these studies. Animals were fasted for 14−20 h before the perfusion experiment with free access to tap water only. The surgery for SPIP of the rat jejunum was performed as described in detail previously.35 Briefly, anesthesia was induced by intraperitoneal injection of urethane at a dose of 1.5 g/kg. The abdomen was opened with a midline longitudinal incision. A jejunal segment of approximately 10 cm was carefully exposed and cannulated on two ends with PVC tubing (4 mm o.d., inlet tube 40 cm, outlet tube 5 cm). The animals were maintained at 37 °C throughout the experiments by focusing a table lamp as a source of heat. The exposed segment was covered with a cotton pad soaked in normal saline. Intestinal segment was initially rinsed with normal saline maintained at 37 °C until the outlet solution was visually clear. Thereafter the jejunum segment was perfused with perfusion solution at a constant flow rate (Qin) of 0.2 mL/min with a peristaltic pump (BT100-2J, Baoding Longer Precision Pump Co., Ltd., China). After approximately 40 min, when steady state was achieved (as assessed by the inlet over outlet concentration ratio of nonabsorbable marker which approaches 1 at steady state), the outlet perfused samples were collected at 20 min intervals up to 120 min and centrifuged at 12000 rpm for 10 min. Phenol red levels were assayed by UV−vis spectrometer (UV9600, Beijing Rayleigh Instrument Co. Ltd., China), and test compounds were analyzed by HPLC. Following the termination of the experiments, length and radius of each perfused jejunal segments were accurately measured. Finally, the animals were sacrificed by excess of ether. Data Analysis. Test compounds’ concentrations obtained from the perfusate samples were corrected for changes in the water flux during each time interval. The steady-state intestinal

Table 1. HPLC Conditions of the Prodrugs prodrugs/ OA

wavelength (nm)

OA

210

5a 5b 5c 5d 5e 6f

225

mobile phase water−methanol−TEA (12:88:0.01), the pH adjusted to 8.0 with orthophosphoric acid water−methanol−TEA (10:90:0.01), the pH adjusted to 8.0 with orthophosphoric acid

water−methanol−acetonitrile−TEA (50:13:37:0.05), the pH adjusted to 3.2 with orthophosphoric acid water−methanol−TEA (10:90:0.01), the pH adjusted to 8.0 with orthophosphoric acid

tR (min) 8 16 16 18 18 9 10

Solubility. Water solubility for the prodrugs and OA was determined by the equilibrium solubility method according to Higuchi and Connor’s method.33 Excess amounts (∼10 mg) of the samples were suspended in 1 mL of water in screw-capped glass vials. These vials were kept in a thermostated oscillator (Z82, Changzhou guohua Instrument Co. Ltd., China) maintained at 37 °C. After 24 h, the suspensions were centrifuged at 12000 rpm for 10 min with high speed centrifuge (TGL-16GB, Shanghai Anting Scientific Instrument Plant, China). The supernatants were assayed by HPLC. All experiments were conducted at least in triplicate. In Situ Rat Single-Pass Intestinal Perfusion (SPIP) Experiments. Perfusion Solutions. In order to improve the solubility of the test compounds, 0.05% (w/v) Cremophor EL was used in the perfusion solution. 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) were used as blank perfusion solutions.19 Phenol red was directly added to perfusion solutions containing 0.1 mM test compound as nonabsorbable marker for measuring water flux at a concentration of 24 μg/ 2129

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Scheme 1. Synthesis of the Target Compounds 5a−ea

a

Reagents and conditions: (a) (Boc)2O, t-BuOH, 2.5% NaOH, rt, 6−12 h; (b) 2-bromoethanol, K2CO3, DMF, rt, 6 h; (c) DCC, DMAP, CH2Cl2, rt, 6−12 h; (d) Et2O, HCl (g), rt, 0.5 h.

experiment. Prodrug 5a or 6f was dissolved in water solution containing 20% HP-β-CD, and OA was suspended in the same solution. Eighteen rats were divided randomly into three groups as follows: six rats of group OA were treated with OA suspension; six rats of compound group 5a were treated with 5a solution; six rats of compound group 6f were treated with 6f solution. These groups received, by oral gavage, either OA (300 mg/kg body weight) or prodrugs (300 mg/kg body weight, calculated as OA). At predetermined time points (5, 15, 30 min and 1, 2, 4, 6, 8, 12, 24 h), the rats were anesthetized with ether and blood samples were collected from the orbital plexus into heparin-treated tubes. The blood samples were immediately centrifuged at 2500 rpm for 10 min to isolate the plasma. The collected plasma was stored at −80 °C immediately until further treatment following the method described previously.37 Briefly, a sample of 0.1 mL was spiked with glycyrrhetinic acid as internal standard (20 μL of 2.0 μg/mL methanol solution), mixed briefly, and then extracted with 2.0 mL of ethyl acetate through vigorous vortex mixing for 3 min and centrifugation at 3500 rpm for 10 min. 1.5 mL of the supernatant ethyl acetate layer was separated and evaporated under a gentle stream of nitrogen gas at 40 °C. The residue was reconstituted in 100 μL of the mobile phase. An aliquot of 25 μL was injected into the HPLC column.

effective permeability (Peff, cm/s) was calculated according to the “plug flow” model expressed in the following equation:36 Peff = [−Q in ln Cout(corr)/C in]/2πrL

(1)

where r is the radius of the intestinal lumen (cm), L is the length of the segment (cm), 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. The Cout(corr) was calculated according to the following equation: Cout(corr) = CoutCphenolred(in)/Cphenolred(out)

(2)

where Cout is the outlet drug concentration, 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 t test. A p < 0.05 was considered significant for all tests. Rats Bioavailability Experiments. Pharmacokinetic Studies. Male Sprague−Dawley rats (weighing from 220 to 250 g) were fasted but had free access to water for 12 h before 2130

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Scheme 2. Synthesis of the Target Compound 6fa

a Reagents and conditions: (a) (Boc)2O, i-PrOH, 8 M KOH, rt, 12 h; (b) 3, DCC, DMAP, CH2Cl2, rt, 24 h; (c) piperidine, CH2Cl2, rt, 12 h; (d) Et2O, HCl (g), rt, 0.5 h.

HPLC/MS/MS Analysis. The analytes were determined by the LC/MS/MS system as described previously,37 which consists of a SHIMADZU LC-2010C HT and a TSQ Quantum Ultra AM triple-quadrupole tandem mass spectrometer with an ion max source, Xcalibur 2.1 software for data acquisition and analysis (Thermo Finnigan, San Jose, CA, USA). The HPLC separation was performed on an ODS-SP (Inertsil, 150 × 4.6 mm, 5 μm, GL, Japan) with a mobile phase of methanol− ammonium acetate (32.5 mM) (88:12, v/v) delivered at 1.0 mL/min and 30% of the eluent after the column was injected into the MS system using an electrospray ionization (ESI) source. The mass spectrometer was operated in the negative ion detection mode with the spray voltage set at 4.5 kV. Nitrogen sheath gas was set at 35 psi and auxiliary gas at 7 psi. The heated capillary temperature was set at 350 °C. A collision energy of 38 V was used with argon at a pressure of 1.5 mTorr for collision-induced dissociation (CID). Quantification was performed with multiple selected reaction monitoring (MRM) of the transitions of m/z 455.4 → 455.4 for oleanolic acid and 469.3 → 425.2 for glycyrrhetinic acid (internal standard) with a scan time of 0.2 s per transition. This HPLC/MS/MS method for the determination of OA in rat plasma has been developed and validated over the concentration range from 10 to 1000 ng/mL. The regression equation for OA content (ng/mL) in the plasma range from 10 to 1000 ng/mL was A = 0.056C + 0.6939 (R2 = 0.9939). The plasma assay was validated by analysis of n = 3 quality control samples containing 20, 200, and 600 ng/mL OA in blank plasma. The recoveries were found to be accurate to 98.44%, 107.78%, and 97.05% and precise to ±6.05%, 5.20%, and 8.77% of 20, 200, and 600 ng/mL, respectively. The within-day and between-day precision (RSD %, n = 5) for the OA samples at 20, 200, and 600 ng/mL levels varied between 1.90 and 11.43. Pharmacokinetic Data Analysis. The peak plasma concentration (Cmax) and the time to reach peak concentration levels (tmax) were obtained from the time versus plasma concentration profile. Standard noncompartmental analysis was performed for estimation of absorption profile (Kinetica, Ver 4.4, Innaphase, Corp., Philadelphia, PA, USA). The area under the plasma concentration−time curve (AUC) from the time zero to the last measured concentration (AUC0−t) was calculated according to the linear trapezoidal rule. The apparent bioavailability (Fapp) of OA following administration of prodrug was calculated by

dividing OA AUC0−t following prodrug dosing by that from OA dosing. Statistically significant differences between two groups were evaluated by Student’s t test. Significance is reported as *p < 0.05 and **p < 0.01 for all tests.

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RESULTS AND DISCUSSION Chemistry. The synthesis of the target compounds 5a−e was carried out from commercially available amino acids 1a−e as depicted in Scheme 1. Accordingly, amino acids 1a−e were treated with di-tert-butyl bicarbonate (Boc2O) in a solution of 2.5% NaOH in t-BuOH at room temperature to give N-Boc amino acids (2a−e). 38 The reaction of OA with 2bromoethanol in the presence of potassium carbonate in N,N-dimethylformamide (DMF) at room temperature for 6 h furnished oleanolic acid 2-hydroxyl ethyl ester 3. The following selective esterification of 3 was performed with N-Boc amino acids 2a−e in the presence of N,N′-dicyclohexylcarbodiimide (DCC) and 4-dimethylamino-pyridine (DMAP) in CH2Cl2 to afford the corresponding esters 4a−e. Subsequent deprotection of N-Boc was performed by introducing dry HCl gas to a solution of 4a−e in ethyl ether to obtain hydrochloride salts 5a−e. The preparation of 6f was demonstrated in Scheme 2. Since the phenolic hydroxyl moiety in L-tyrosine 1f is active, the protection of hydroxyl is necessary during the downstream esterification. In this regard, 1f was treated with Boc2O in the solution of 8 M KOH and i-PrOH to furnish N,O-bis-Boc-Ltyrosine 2f, which was reacted with intermediate 3 in the presence of DCC and DMAP to give ester 4f. After removal of the O-Boc protecting group with piperidine,39 the N-Bocdeprotection was carried out in the presence of dry hydrogen chloride gas to afford 6f. Water Solubility. Water solubility of OA is about 0.012 μg/ mL while all the test prodrugs showed significantly greater water solubility than OA. Water solubility of 5a, 5b, 5c, 5d and 6f is 25 ± 6, 33 ± 3, 3.7 ± 1.0, 3.1 ± 0.9, 12 ± 9 μg/mL, respectively. In water self-assembly of 5e into nanosized aggregates or particles has been observed due to the amphiphilicity of 5e. It was found that water solubility of 5e was more than 10 mg/mL. Reasons for water-solubility enhancement of other amino acid ester derivatives may be that all the prodrugs are hydrochlorate and the amino acid promoieties can improve the hydrophilicity of OA. 2131

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Stability and Physical Absorption of Test Compounds. In the SPIP experiments, compounds of interest are monitored in the perfusate only, not in the blood. The SPIP procedure assumes that loss of drug during the perfusion is due to permeation of the intestine. Therefore, determination of the nonabsorptive loss of perfused drug is required. The test compounds’ stability studies were carried out in both MES and HEPES buffers at 37 °C for 2 h. After OA, 5a, 5b, 5c, 5d, 5e or 6f was added to the MES buffer for 2 h, the remaining percentage of it was 97.25%, 97.75%, 95.09%, 95.40%, 97.26%, 97.05% and 97.25%, respectively. The remaining percentage of OA, 5a, 5b, 5c, 5d, 5e or 6f in HEPES buffer at 37 °C for 2 h was 100.42%, 99.28%, 97.25%, 97.81%, 95.25%, 98.39% and 96.10%, respectively. These results demonstrated that both OA and the prodrugs were found to be stable in two kinds of perfusion buffers at 37 °C for 2 h. When comparing the Val and Phe derivatives, compounds 5a and 5b were more stable than their corresponding Phe derivatives 5c and 5d at either pH value, which might be due to destabilization by the electronwithdrawing phenyl ring. In addition, the stereochemistry of the amino acid promoiety did not influence the chemical stability of the diester prodrugs of OA in the buffers. All the test compounds exhibited extensive binding to the untreated PVC tubing at 37 °C for 2 h. When the PVC tubing was incubated in solutions of test compounds (0.1 mM) in MES buffer at 37 °C for 2 h, the remaining percentage of OA, 5a, 5b, 5c, 5d, 5e and 6f was 74.07%, 62.37%, 63.94%, 76.35%, 70.22%, 90.31% and 93.21%, respectively. Pretreatment of the tubing with a 1 mg/mL methanol solution of the test compounds for 12 h effectively decreased the nonspecific binding, and after 2 h the remaining percentage of OA, 5a, 5b, 5c, 5d, 5e and 6f was 101.13%, 102.12%, 98.10%, 96.76%, 95.27%, 96.10% and 98.18%, respectively. In addition, the physical absorption of the test compounds achieved steady state after the pretreated PVC tubing had been perfused with perfusion solutions for 75 min (data not supplied). Taken together, these results ensure that the loss of drug from the perfusion is due to absorption only but not due to other losses (e.g., nonspecific binding to the tubing or chemical degradation).34 SPIP of the Test Compounds in Rat Jejunums. The human permeability database has been used for predicting human absorption and for classifying drugs into the BCS category. However, human permeability determinations are expensive. Thus, alternative models for prediction of drug absorption and permeability have garnered increasing attention. Recently, the in situ rat perfusion model has been proved to be reliable for permeability determination which will more accurately facilitate drug discovery and development as well as regulatory standards for marketed products.40 In this work, SPIP experiments in rat jejunum in different perfusion solutions were undertaken to determine intestinal permeability of the test compounds. PepT1 employs a proton gradient as the driving force, and its activity is pH dependent. Accordingly, it is strongly suggested that the intestinal luminal pH would be important to determine the apparent membrane permeability via PepT1. Therefore, two solutions with different pH (6.0 and 7.4) were used in the perfusion studies.41,42 Equimolar concentrations (0.1 mM) of OA and six prodrugs in the buffers were prepared for perfusion studies. In HEPES buffer of pH 7.4, except for 5e, Peff of the other prodrugs exhibited better than that of OA (Figure 1). Peff of 6f and 5b were roughly 2.6-fold and 2.0-fold higher than that

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

of OA (p < 0.05), respectively. There was no difference of the Peff in the two buffers for OA. Rank order of the Peff in MES buffer of pH 6.0 for OA and its prodrugs was 5c > 5a > 5e > 5d > 5b > 6f > OA. Significant enhancement (p < 0.01) of Peff was observed with 5c, 5a, 5e, 5d, 5b (2.5-fold, 2.3-fold, 2.2-fold, 2.1fold, 1.9-fold), compared to that with OA. Besides, Peff of 5a, 5c, 5d and 5e was significantly enhanced in pH 6.0 compared to that in pH 7.4 (p < 0.01), respectively. These results indicated that the structure of the promoieties influenced the increased permeability of the prodrugs. It was found that Peff of L-amino acid ester prodrug (5a or 5c) was better than that of their D counterparts (5b or 5d), which is in agreement with previous findings with other PepT1-targeted prodrugs.43,44 This may be attributed to PepT1 mediated translocation and to their differential affinity toward the PepT1. SPIP of OA and Its Prodrugs in the Presence of GlySar. To preliminarily demonstrate active transport by PepT1, inhibition studies were performed by coperfusing prodrugs with Gly-Sar, a known substrate of PepT1. PepT1 is a low-affinity, high-capacity transporter with apparent affinities (Km values) ranging from 0.2 to 10 mM, depending upon the substrate, species, tissue cell types, and experimental conditions such as buffer pH. In addition, the jejunal Peff of Gly-Sar in wild-type mice was 2.2 × 10−4 cm/s by SPIP studies.45 Compared to the concentration of the test compounds 100- or 800-fold excess of Gly-Sar has been used in the SPIP in rats in previous reports.13,35 These experiments were carried out in the presence (500-fold excess) of the 50 mM Gly-Sar in MES buffer containing 0.1 mM prodrug, which ensured that large excess of Gly-Sar existed in MES buffer for 2 h. Significant reduction (p < 0.01) of Peff was observed with 5b and 5c (2.2-fold and 1.9-fold lower), compared to that in the absence of Gly-Sar (Figure 2). The permeability of 5a and 6f was reduced significantly (p < 0.05) in the presence of Gly-Sar from 7.4 ± 1.5 × 10−5 and 5.7 ± 1.2 × 10−5 cm/s to 4.2 ± 1.3 × 10−5 and 4.1 ± 0.8 × 10−5 cm/s, respectively. These results suggest that PepT1 might be involved in the uptake of these prodrugs. Furthermore there was no difference in the Peff of the 5d and 5e in the presence and absence of Gly-Sar. These findings indicate that structure of the amino acid promoiety influences the affinity of OA prodrugs for PepT1. Generally, amino acid prodrugs of OA with aliphatic (5a, 5b) or aromatic (5c, 6f) side chains showed higher affinity than that with basic 2132

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Table 2. Main Pharmacokinetic Parameters and Apparent Bioavailability (Fapp) of Oleanolic Acid, Following Intragastric Administration of 300 mg/kg (Calculated as OA) 5a, 6f and OA to Three Groups of Sprague−Dawley Rats, Respectively (Mean ± SD, n = 6) group OA Cmax (μg/mL) Tmax (h) AUC0→24h (μg·h /mL)a MRT (h) Fapp (%)

group 5a

group 6f

0.47 ± 0.034 0.50 4.98 ± 0.42

0.73 ± 0.067*b 0.72 ± 0.070* 0.83 ± 0.22 0.58 ± 0.17 10.99 ± 0.65** 10.14 ± 1.14**

15.52 ± 3.40 100

26.85 ± 4.19** 44.11 ± 10.84** 221 204

AUC is for oleanolic acid. b*p < 0.05 as compared to group OA.**p < 0.01 as compared to group OA.

a

Figure 2. The effective permeability (Peff) for prodrugs obtained from in situ rat single-pass intestinal perfusion (SPIP) experiments in the presence and absence of Gly-Sar (mean ± SD, 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.

parent compound following transport across intestinal membrane. In addition, the average value of Cmax was 0.47 μg/mL after oral administration of OA suspension with a Tmax of about 0.5 h. There are significant differences of Cmax between both group 5a or 6f and group OA (p < 0.05). Values of AUC0→24h were observed to be 10.99 ± 0.65, 10.14 ± 1.14 and 4.98 ± 0.42 μg·h/mL with group 5a, 6f and OA, respectively. AUC0→24h showed significant differences between both group 5a or 6f and group OA (p < 0.01). The mean residence times (MRT) of OA were 26.85 ± 4.19, 44.11 ± 10.84 and 15.52 ± 3.40 h for group 5a, 6f and OA, respectively. This difference in MRT may be due to the difference in the conversion rate of 5a or 6f into the parent drug OA possibly in the gastrointestinal tract, intestinal intracell or in the liver microsome. Compared to group OA, the significant enhancement of MRT for group 5a and 6f indicated the prodrugs 5a and 6f showed sustainedrelease characteristics in vivo (p < 0.01), which had been observed for PepT1-targeted prodrug of D-phenylglycine-Ldopa.47 Fapp of 5a and 6f was 2.21 and 2.04 times higher than that of OA in rats, suggesting that conjugating to proper amino acid promoieties with an ethylene glycol linker is an effective PepT1-targeted prodrug strategy for improving the oral absorption of OA. Finally, Figure 3 indicated that the plasma concentration of OA declined rapidly over the first stage and was followed by a second peak at 12 h, 4 h and 4 h for group 5a, group 6f and group OA, respectively, which was probably due to the enterohepatic recirculation.48

(L-Lys, 5e) side chains, which was similar to the previous finding.44 The stereochemistry of the amino acid promoieties also influenced the affinity of the prodrugs for PepT1. L-Amino acid prodrugs showed preference for PepT1 transport than their D counterparts, which are also consistent with the stereochemical preferences observed with other amino acid prodrugs designed for targeting PepT1.44,46 Pharmacokinetic Studies. In view of water solubility, Peff and preliminary PepT1-targeted studies, 5a and 6f were selected to determine whether ethylene glycol-linked amino acid diester prodrug strategy could improve oral absorption of OA in vivo. As the amino acid prodrugs would rapidly be hydrolyzed into OA and their concentration in plasma was very low (data not shown), therefore, we mainly focused on the pharmacokinetic performances of OA after oral administration of compound 5a and 6f. Three groups of rats were received by oral gavage with the same equivalent to 300 mg/kg body weight of OA of OA suspension, 5a solution and 6f solution, respectively. Plasma concentration−time profiles of OA are shown in Figure 3. Pharmacokinetic parameters have been summarized in Table 2. It was shown that the average value of Cmax was 0.73 μg/mL and 0.72 μg/mL after oral administration of 5a and 6f solution with a Tmax of about 0.83 and 0.58 h, respectively. From the Tmax of group 5a and 6f we can find that diester prodrugs 5a and 6f could be quickly activated to the

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CONCLUSIONS Due to its broad substrate specificity and high capacity, PepT1 is considered as an attractive target for drug delivery strategies aimed at improving the bioavailability of poorly permeable drugs. Six amino acid diester prodrugs of the carboxylic acid drug OA were successfully synthesized by linking the carboxylic acid group of OA and the carboxylic acid group of amino acid promoieties through an ethylene glycol linker. Water solubility of all the test prodrugs was showed to be significantly greater than that of OA. The permeability studies with rats in SPIP model showed that all the prodrugs had higher membrane effective permeability and inhibition studies with Gly-Sar suggested that PepT1 might be involved in the intestinal membrane transport of compound 5a, 5b, 5c and 6f. Furthermore, 5a and 6f exhibited enhanced oral bioavailability of OA in rats with Fapp of 2.21- and 2.04-fold increase, respectively. With a linker the kind of structure of parent drugs selected for PepT1-targeted prodrug design can be extended for parent drugs with carboxylic acid group. The structure and

Figure 3. Mean plasma concentration−time profiles of oleanolic acid following intragastric administration of 300 mg/kg (calculated as OA) 5a, 6f and OA to three groups of Sprague−Dawley rats, respectively (mean ± SD, n = 6). 2133

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(4) Hsu, H. Y.; Yang, J. J.; Lin, C. C. Effects of oleanolic acid and ursolic acid on inhibiting tumor growth and enhancing the recovery of hematopoietic system postirradiation in mice. Cancer Lett. 1997, 111, 7−13. (5) Cheung, H. Y.; Zhang, Q. F. Enhanced analysis of triterpenes, flavonoids and phenolic compounds in Prunella vulgaris L. by capillary zone electrophoresis with the addition of running buffer modifiers. J. Chromatogr., A 2008, 1213 (2), 231−238. (6) Jeong, D. W.; Kim, Y. H.; Kim, H. H.; Ji, H. Y.; Yoo, S. D.; Choi, W. R.; Lee, S. M.; Han, C. K.; Lee, H. S. Dose-linear pharmacokinetics of oleanolic acid after intravenous and oral administration in rats. Biopharm. Drug Dispos. 2007, 28, 51−57. (7) Amidon, G. L.; Lennernas, H.; Shah, V. P.; Crison, J. R. A theoretical basis for a biopharmaceutics drug classification: the correlation of in vitro drug product dissolution and in vivo bioavailability. Pharm. Res. 1995, 12 (3), 413−420. (8) Chen, Y.; Liu, J.; Yang, X.; Zhao, X.; Xu, H. Oleanolic acid nanosuspensions: preparation, in-vitro characterization and enhanced hepatoprotective effect. J. Pharm. Pharmacol. 2005, 57, 259−264. (9) Li, W.; Das, S.; Ng, K. Y.; Heng, P. W. Formulation, biological and pharmacokinetic studies of sucrose ester-stabilized nanosuspensions of oleanolic Acid. Pharm. Res. 2011, 28 (8), 2020−2033. (10) Xi, J.; Chang, Q.; Chan, C. K.; Meng, Z. Y.; Wang, G. N.; Sun, J. B.; Wang, Y. T.; Tong, H. H.; Zheng, Y. Formulation development and bioavailability evaluation of a self-nanoemulsified drug delivery system of oleanolic acid. AAPS PharmSciTech 2009, 10 (1), 172−182. (11) Tong, H. H.; Du, Z.; Wang, G. N.; Chan, H. M.; Chang, Q.; Lai, L. C.; Chow, A. H.; Zheng, Y. Spray freeze drying with polyvinylpyrrolidone and sodium caprate for improved dissolution and oral bioavailability of oleanolic acid, a BCS Class IV compound. Int. J. Pharm. 2011, 404 (1−2), 148−158. (12) Jana, S.; Mandlekar, S.; Marathe, P. Prodrug design to improve pharmacokinetic and drug delivery properties: challenges to the discovery scientists. Curr. Med. Chem. 2010, 17 (32), 3874−3908. (13) Brandsch, M. Transport of drugs by proton-coupled peptide transporters: pearls and pitfalls. Expert Opin. Drug Metab. Toxicol. 2009, 5 (8), 887−905. (14) Rais, R.; Fletcher, S.; Polli, J. E. Synthesis and in vitro evaluation of gabapentin prodrugs that target the human apical sodiumdependent bile acid transporter (hASBT). J. Pharm. Sci. 2011, 100 (3), 1184−1195. (15) Cundy, K. C.; Branch, R.; Chernov-Rogan, T.; Dias, T.; Estrada, T.; Hold, K.; Koller, K.; Liu, X.; Mann, A.; Panuwat, M.; Raillard, S. P.; Upadhyay, S.; Wu, Q. Q.; Xiang, J. N.; Yan, H.; Zerangue, N.; Zhou, C. X.; Barrett, R. W.; Gallop, M. A. XP13512 [(±)-1-([(α-Isobutanoyloxyethoxy) carbonyl] aminomethyl)-1-cyclohexane Acetic Acid], A Novel Gabapentin Prodrug: I. Design, Synthesis, Enzymatic Conversion to Gabapentin, and Transport by Intestinal Solute Transporters. J. Pharmacol. Exp. Ther. 2004, 311 (1), 315−323. (16) Kato, K.; Shirasaka, Y.; Kuraoka, E.; Kikuchi, A.; Iguchi, M.; Suzuki, H.; Shibasaki, S.; Kurosawa, T.; Tamai, I. Intestinal Absorption Mechanism of Tebipenem Pivoxil, a Novel Oral Carbapenem: Involvement of Human OATP Family in Apical Membrane Transport. Mol. Pharmaceutics 2010, 7 (5), 1747−1756. (17) Gynther, M.; Ropponen, J.; Laine, K.; Leppänen, J.; Haapakoski, P.; Peura, L.; Järvinen, T.; Rautio, J. Glucose promoiety enables glucose transporter mediated brain uptake of ketoprofen and indomethacin prodrugs in rats. J. Med. Chem. 2009, 52 (10), 3348− 3353. (18) Luo, S.; Wang, Z.; Patel, M.; Khurana, V.; Zhu, X.; Pal, D.; Mitra, A. K. Targeting SVCT for enhanced drug absorption: synthesis and in vitro evaluation of a novel vitamin C conjugated prodrug of saquinavir. Int. J. Pharm. 2011, 414 (1−2), 77−85. (19) Sun, J.; Dahan, A.; Amidon, G. L. Enhancing the intestinal absorption of molecules containing the polar guanidino functionality: a double-targeted prodrug approach. J. Med. Chem. 2010, 53 (2), 624− 632. (20) Landowski, C. P.; Vig, B. S.; Song, X.; Amidon, G. L. Targeted delivery to PEPT1-overexpressing cells: acidic, basic, and secondary

stereochemistry of the amino acid promoieties influenced PepT1-mediated transport and bioconversion of amino acid diester prodrugs of OA. This approach could improve oral bioavailability of OA, a BCS class IV drug.

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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

*Y.L.: State Key Laboratory of Natural Medicines, Center of Drug Discovery, China Pharmaceutical University, No. 24, Tongjia Road, Nanjing 210009, China; tel/fax, +86-2586635503; e-mail, [email protected]. Q.P.: State Key Laboratory of Natural Medicines, Department of Pharmaceutics, School of Pharmacy, China Pharmaceutical University, No. 24, Tongjia Road, Nanjing 210009, China; tel/fax, +86-2583271092; e-mail, [email protected]. Author Contributions ‡

Contributed equally.

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), Key New Drug Innovation Project from the Ministry of Science and Technology of the People’s Republic of China (No. 2009ZX09310-004) and the Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University.

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ABBREVIATIONS USED PepT1, intestinal oligopeptide transporter 1; hPepT1, human intestinal oligopeptide transporter 1; BCS, Biopharmaceutics Classification System; OA, oleanolic acid; Peff, effective permeability; SPIP, single-pass intestinal perfusion; Gly-Sar, glycyl-sarcosine; HPLC/MS/MS, high-performance liquid chromatography/tandem mass spectrometry; HP-β-CD, hydroxypropyl-beta-cyclodextrin; DMF, N, N-dimethylformamide; THF, tetrahydrofuran; DCC, N,N′-dicyclohexylcarbodiimide; DMAP, 4-dimethylamino-pyridine; EtOAc, ethyl acetate; PE, petroleum ether; EtOH, alcohol; DMSO, dimethyl sulfoxide; (Boc)2O, di-tert-butyl bicarbonate; t-BuOH, t-butyl alcohol; i-PrOH, isopropanol; AUC, area under the plasma concentration time curve; MRT, mean residence time; Fapp, apparent bioavailability

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REFERENCES

(1) Liu, J. Oleanolic acid and ursolic acid: research perspectives. J. Ethnopharmacol. 2005, 100 (1−2), 92−94. (2) Giner-Larza, E. M.; Máñez, S.; Recio, M. C.; Giner, R. M.; Prieto, J. M.; Cerdá-Nicolás, M.; Ríos, J. L. Oleanolic acid, a 3-oxotriterpene from Pistascia, inhibits leukotriene synthesis and has anti-inflammatory activity. Eur. J. Pharmacol. 2001, 428 (1), 137−143. (3) Maia, J. L.; Lima-Júnior, R. C.; David, J. P.; David, J. M.; Santos, F. A.; Rao, V. S. Oleanolic acid, a pentacyclic triterpene attenuates the mustard oil-induced colonic nociception in mice. Biol. Pharm. Bull. 2006, 29 (1), 82−85. 2134

dx.doi.org/10.1021/mp200447r | Mol. Pharmaceutics 2012, 9, 2127−2135

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Article

floxuridine amino acid ester prodrugs. Mol. Cancer Ther. 2005, 4 (4), 659−667. (21) Han, H.; de Vrueh, R. L.; Rhie, J. K.; Covitz, K. M.; Smith, P. L.; Lee, C. P.; Oh, D. M.; Sadée, W.; Amidon, G. L. 5′-Amino acid esters of antiviral nucleosides, acyclovir, and AZT are absorbed by the intestinal PEPT1 peptide transporter. Pharm. Res. 1998, 15 (8), 1154− 1159. (22) Sugawara, M.; Huang, W.; Fei, Y. J.; Leibach, F. H.; Ganapathy, V.; Ganapathy, M. E. Transport of valganciclovir, a ganciclovir prodrug, via peptide transporters PEPT1 and PEPT2. J. Pharm. Sci. 2000, 89, 781−789. (23) Varma, M. V.; Eriksson, A. H.; Sawada, G.; Pak, Y. A.; Perkins, E. J.; Zimmerman, C. L. Transepithelial transport of the group II metabotropic glutamate 2/3 receptor agonist (1S,2S,5R,6S) −2aminobicyclo[3.1.0] hexane-2,6-dicarboxylate (LY354740) and its prodrug (1S,2S,5R,6S) 2- [(2′S) -(2′-amino)propionyl] aminobicyclo[3.1.0]hexane-2,6-dicarboxylate(LY544344). Drug Metab. Dispos. 2009, 37 (1), 211−220. (24) Perkins, E. J.; Abraham, T. Pharmacokinetics, metabolism, and excretion of the intestinal peptide transporter 1 (SLC15A1)-targeted prodrug (1S,2S,5R,6S) −2-[(2′S)-(2-amino)propionyl]aminobicyclo[3.1.0] hexen-2,6-dicarboxylic acid (LY544344) in rats and dogs: assessment of first-pass bioactivation and dose linearity. Drug Metab. Dispos. 2007, 35 (10), 1903−1909. (25) Tsuda, M.; Terada, T.; Irie, M.; Katsura, T.; Niida, A.; Tomita, K.; Fujii, N.; Inui, K. Transport characteristics of a novel peptide transporter 1 substrate, antihypotensive drug midodrine, and its amino acid derivatives. J. Pharmacol. Exp. Ther. 2006, 318 (1), 455−460. (26) Foley, D.; Bailey, P.; Pieri, M.; Meredith, D. Targeting ketone drugs towards transport by the intestinal peptide transporter, PepT1. Org. Biomol. Chem. 2009, 7 (6), 1064−1067. (27) Foley, D.; Pieri, M.; Pettecrew, R.; Price, R.; Miles, S.; Lam, H. K.; Bailey, P.; Meredith, D. The in vitro transport of model thiodipeptide prodrugs designed to target the intestinal oligopeptide transporter, PepT1. Org. Biomol. Chem. 2009, 7 (18), 3652−3656. (28) Yan, Z.; Sun, J.; Chang, Y.; Liu, Y.; Fu, Q.; Xu, Y.; Sun, Y.; Pu, X.; Zhang, Y.; Jing, Y.; Yin, S.; Zhu, M.; Wang, Y.; He, Z. Bifunctional Peptidomimetic Prodrugs of Didanosine for Improved Intestinal Permeability and Enhanced Acidic Stability: Synthesis, Transepithelial Transport, Chemical Stability and Pharmacokinetics. Mol. Pharmaceutics 2011, 8 (2), 319−329. (29) Peterson, L. W.; Sala-Rabanal, M.; Krylov, I. S.; Serpi, M.; Kashemirov, B. A.; McKenna, C. E. Serine side chain-linked peptidomimetic conjugates of cyclic HPMPC and HPMPA: synthesis and interaction with hPEPT1. Mol. Pharmaceutics 2010, 7 (6), 2349− 2361. (30) Bailey, P. D.; Boyd, C. A.; Bronk, J. R.; Collier, I. D.; Meredith, D.; Morgan, K. M.; Temple, C, S. How to Make Drugs Orally Active: A Substrate Template for Peptide Transporter PepT1. Angew. Chem., Int. Ed. 2000, 39 (3), 505−508. (31) Gupta, D.; Gupta, S. V.; Lee, K. D.; Amidon, G. L. Chemical and enzymatic stability of amino acid prodrugs containing methoxy, ethoxy and propylene glycol linkers. Mol. Pharmaceutics 2009, 6 (5), 1604− 1611. (32) Eriksson, A. H.; Varma, M. V.; Perkins, E. J.; Zimmerman, C. L. The intestinal absorption of a prodrug of the mGlu2/3 receptor agonist LY354740 is mediated by PepT1: in situ rat intestinal perfusion studies. J. Pharm. Sci. 2010, 99 (3), 1574−1581. (33) Higuchi, T.; Connors, K. A. Phase-solubility techniques. Adv. Anal. Chem. Instrum. 1965, 4, 117−212. (34) Cook, T. J.; Shenoy, S. S. Intestinal permeability of chlorpyrifos using the single-pass intestinal perfusion method in the rat. Toxicology 2003, 184 (2−3), 125−133. (35) Jain, R.; Duvvuri, S.; Kansara, V.; Mandava, N. K.; Mitra, A. K. Intestinal absorption of novel-dipeptide prodrugs of saquinavir in rats. Int. J. Pharm. 2007, 336 (2), 233−240. (36) Fagerholm, U.; Johansson, M.; Lennernas, H. Comparison Between Permeability Coefficients in Rat and Human Jejunum. Pharm. Res. 1996, 13, 1336−1342.

(37) Song, M.; Hang, T. J.; Wang, Y.; Jiang, L.; Wu, X. L.; Zhang, Z.; Shen, J.; Zhang, Y. Determination of oleanolic acid in human plasma and study of its pharmacokinetics in Chinese healthy male volunteers by HPLC tandem mass spectrometry. J. Pharm. Biomed. Anal. 2006, 40 (1), 190−196. (38) Ling, Y.; Ye, X.; Ji, H.; Zhang, Y.; Lai, Y.; Peng, S.; Tian, J. Synthesis and evaluation of nitric oxide-releasing derivatives of farnesylthiosalicylic acid as anti-tumor agents. Bioorg. Med. Chem. 2010, 18 (10), 3448−3456. (39) Nakamura, K.; Nakajima, T.; Kayahara, H.; Namura, E.; Taniguchi, H. Base-labile tert-butoxycarbonyl (Boc) group on phenols. Tetrahedron Lett. 2004, 45 (3), 495−499. (40) Kim, J. S.; Mitchell, S.; Kijek, P.; Tsume, Y.; Hilfinger, J.; Amidon, G. L. The suitability of an in situ perfusion model for permeability determinations: utility for BCS class I biowaiver requests. Mol. Pharmaceutics 2006, 3 (6), 686−694. (41) Våbenø, J.; Nielsen, C. U.; Ingebrigtsen, T.; Lejon, T.; Steffansen, B.; Luthman, K. Dipeptidomimetic ketomethylene isosteres as pro-moieties for drug transport via the human intestinal di-/ tripeptide transporter hPEPT1: design, synthesis, stability, and biological investigations. J. Med. Chem. 2004, 47 (19), 4755−4765. (42) Zhang, E. Y.; Emerick, R. M.; Pak, Y. A.; Wrighton, S. A.; Hillgren, K. M. Comparison of human and monkey peptide transporters: PEPT1 and PEPT2. Mol. Pharmaceutics 2004, 1 (3), 201−210. (43) Song, X.; Lorenzi, P. L.; Landowski, C. P.; Vig, B. S.; Hilfinger, J. M.; Amidon, G. L. Amino acid ester prodrugs of the anticancer agent gemcitabine: synthesis, bioconversion, metabolic bioevasion, and hPEPT1-mediated transport. Mol. Pharmaceutics 2005, 2 (2), 157− 167. (44) Song, X.; Vig, B. S.; Lorenzi, P. L.; Drach, J. C.; Townsend, L. B.; Amidon, G. L. Amino acid ester prodrugs of the antiviral agent 2bromo-5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole as potential substrates of hPEPT1 transporter. J. Med. Chem. 2005, 48 (4), 1274−1277. (45) Jappar, D.; Wu, S. P.; Hu, Y.; Smith, D. E. Significance and regional dependency of peptide transporter (PEPT) 1 in the intestinal permeability of glycylsarcosine: in situ single-pass perfusion studies in wild-type and Pept1 knockout mice. Drug Metab. Dispos. 2010, 38 (10), 1740−1746. (46) Talluri, R. S.; Samanta, S. K.; Gaudana, R.; Mitra, A. K. Synthesis, metabolism and cellular permeability of enzymatically stable dipeptide prodrugs of acyclovir. Int. J. Pharm. 2008, 361 (1−2), 118− 124. (47) Wang, C. L.; Fan, Y. B.; Lu, H. H.; Tsai, T. H.; Tsai, M. C.; Wang, H. P. Evidence of D-phenylglycine as delivering tool for improving L-dopa absorption. J. Biomed. Sci. 2010, 17, 71. (48) Xu, X. D.; Sun, H. H.; Rong, R.; Wen, B.; Zhang, X. R.; He, Z. G. Pharmacokinetics and relative bioavailability of compound oleanolic acid soft capsules in rats. Chin. J. Pharm. 2008, 6 (6), 392−399.

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