Pharmacokinetics in Vitro and in Vivo of Two Novel Prodrugs of

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Pharmacokinetics in vitro and in vivo of two novel prodrugs of oleanolic acid in rats and its hepatoprotective effects against liver injury induced by CCl4 Zongjiang Yu, Weizhi Sun, Weibing Peng, Rilei Yu, Guoqiang Li, and Tao Jiang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00129 • Publication Date (Web): 28 Mar 2016 Downloaded from http://pubs.acs.org on March 30, 2016

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Molecular Pharmaceutics

Pharmacokinetics in vitro and in vivo of two novel prodrugs of oleanolic acid in rats and its hepatoprotective effects against liver injury induced by CCl4 Zongjiang Yua, 1, Weizhi Sun b, 1, Weibing Penga, 1, Rilei Yua, Guoqiang Li a,*

, Tao Jiang a,*

a

Key Laboratory of Marine Drugs, Ministry of Education of China, School of Pharmacy, Ocean University of China, Qingdao 266003, P R China b CAS Key Laboratory of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, China *Corresponding author E-mail address: [email protected] (T. Jiang)

Abstract Oleanolic acid (OA) is a well-known pentacyclictriterpenoid compound, which has been used as a dietary supplement and is supplied as an over-the-counter drug for the treatment of human liver diseases. These are reasons for the low bioavailability of OA which have restricted its wider application. In this study, two OA prodrugs (1, 3-cyclic propanyl phosphate esters of OA) were designed and synthesised. The hepatoprotective effects of these prodrugs were evaluated against carbon tetrachloride (CCl4)-induced liver injury in mice, the levels of alanine aminotransferase (ALT), lactic dehydrogenase (LDH), and aspartateaminotransferase (AST) were significantly increased and the levels of the hepatic malondialdehyde (MDA) was increased. The metabolism, in vitro, of the prodrugs was studied by incubation in rat liver microsome; the plasma pharmacokinetics and the biodistribution in vivo after intravenous (i.v.) injection to six rats were investigated, respectively. The prodrugs diminished

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gradually with the time and most of the parent drugs were released within 30 min in vitro, and the presumed mechanism of the in vitro metabolism was confirmed. The plasma-concentration data in vivo was analysed by a compartmental method: both the prodrugs and the corresponding released parent drugs existed at up to 48 h in rats. The t1/2 improved after intravenous administration in rats compared with direct injection of the parent drugs. All analyte concentrations were highest in the liver and most of the prodrugs were excreted in faeces (> 47.11%). Therefore, 1, 3-cyclic propanyl phosphate esters of OA can serve as a promising lead candidate for drugs.

Keywords: Oleanolic acid (OA); Prodrug, Hepatoprotective; Pharmacokinetics; Biodistribution

1 Introduction Oleanolic acid (3β-hydroxyolean-12-en-28-oic acid, OA) is a well-known pentacyclictriterpenoid compound that exists widely in the plant kingdom, in medicinal herbs, and in food products.1 This compound is reported to have numerous pharmacological activities related to human health including being hepatoprotective, anti-tumor, gastroprotective, anti-inflammatory, anti-hypertensive, anti-diabetic, antioxidant, and immunomodulatory.2-4 The most important pharmacological property attributed to OA is the hepatoprotective effect for both acute chemically induced hepatic damage and chronic liver fibrosis and cirrhosis.5 Indeed in countries such as China, OA has been used as a dietary supplement and is supplied as an over-the-counter drug for the treatment of human liver diseases.6 Thus, OA is also a


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good lead compound for novel and potent drugs; however, in previous pharmacokinetic studies it was shown that the elimination half-life of OA in human serum was only 3.65 h after oral administration at a dose of 30 mg and the plasma concentration of OA was low in mice after oral administration.7, 8 The shortcoming in first pass effect, and the low bioavailability in the body, of this compound have limited its development and application.4, 9

So many efforts have been made to improve the defects with chemically modified derivatives. In our previous studies we have studied two novel HepDirect-prodrugs (1, 3-cyclic propanyl phosphate esterprodrugs) of 18β-glycyrrhetic acid, their pharmacokinetic parameters in vivo and pharmacodynamics in vitro have improved over those of their parent drug (glycyrrhetic acid).10, 11 HepDirect-prodrug is a new class of prodrugs, consisting of a 1, 3-cyclic propanyl phosphate ester with a C4-aryl ring substituent, which combines properties of rapid liver cleavage with high plasma, and tissue, stability to achieve increased drug levels in the liver.12 The HepDirect-prodrug can deliver the original drug gradually, and indirectly delay the elimination of the original drug.

The sustained-release property of the prodrug provides a new strategy to improve OA metabolism. In this work, two OA prodrugs, 1, 3-cyclic propanyl phosphate esters of OA (Fig. 1) were designed and synthesised for the first time. To investigate the potential druggability, we also studied the hepatoprotective effects in CCl4-injured mice, the pharmacokinetics in vitro and in vivo in rats, and the biodistribution in



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organs, urine, and faeces in rats given the prodrugs. The presumed mechanism of the metabolism and pharmacokinetics in rat liver microsome and plasma were studied.

Fig. 1. Structures of OA and the prodrugs of OA

2 Materials and methods 2.1 Animals, chemicals, instrumentation and conditions

Male Wistar rats (mass, 200-220 g) were purchased from the Experimental Animal Care Centre, Institute of Food and Drug Control of Qingdao (SCXK2008010), Qingdao, China. Oleanolic acid (OA) was purchased from Nanjing Zelang Yi Yao Ltd, China. Tris, Coomassie brilliant blue G-250, albumin, and NADHP were purchased from Sigma Ltd (USA). The other chemicals were all commercially available and used without further purification. Refrigerated centrifuge Eppendorf 5804R, a vortex G-560E (Germany), and a Beckman-Coulter ultracentrifuge optima LE80K (USA) were used. 1H NMR and

13

C NMR spectra were taken on a Jeol JNM-ECP 600

spectrometer with tetramethylsilane (Me4Si) as the internal standard, and chemical shifts were recorded as δ values. Mass spectra were recorded on a Q-TOF Global


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mass spectrometer. Optical rotations were recorded at 20 ºC on a Jasco P-1020 polarimeter.

High performance liquid chromatography (HPLC) was performed using an Agilent Technologies Series 1100 system, equipped with a diode array detector (G1315B), an autosampler (G1313A), a quaternary pump (G1311A), a 100 µl syringe, a column oven (G1316A), and degasser. A Kromasil C18 analytical column (4.6 mm × 250 mm, 5 µm) was used in the separation and quantification of the flavonols in rat plasma and liver microsome at 25 ºC. The data acquisition and processing were undertaken with Agilent Chemstation Version B.02.01-SR1. The mobile phase, consisting of acetonitrile (85%) and 0.5% acetic acid (15%), was pumped at a flow rate of 1.0 mL/min. The injection volume was 20 µl and the detection wavelength was set at 210 nm.

2.2 Synthesis of 5R or5S

Synthesis of cis-3-O-[4-(R)-(3-Chlorophenyl)-2-oxo-1, 3, 2-dioxaphosphorinan -2-yl]-oleanolic

acid

(5R)

or

cis-3-O-[4-(S)-(3-Chlorophenyl)-2-oxo-1,3,2-

dioxaphosphorinan-2-yl]-oleanolic acid (5S)

A solution of oleanolic acid (1.26 g, 2.77 mmol) in THF (30 mL) was treated with a THF solution of 2 M LDA (6.7 mL, 13.4 mmol) and stirred at room temperature. After 2 h, 4R or 4S (1.5 g, 4.06 mmol) was added in one portion. The mixture was stirred for 48 h at room temperature and then quenched with saturated NH4Cl (10 mL).



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THF was removed in vacuo, 1 M HCl was added, drop-wise, to the mixture at 0 ºC until pH < 3. The mixture was extracted with CH2Cl2 (3 × 50 mL). The organic layer was dried and concentrated. The residue was purified by chromatography to obtain 0.88 g (46.3%) of 5R or 0.86 g (45.2%) of 5S.

5R: mp > 200 ºC; [α]D +54.4º (c 1.0，CHCl3); 1H NMR (600MHz，CDCl3) δ: 7.41-7.26(m, 4H, Ar-H), 5.37(d, J = 11.4Hz, 1H, 4’-H), 5.26(t, J = 3.6Hz, 1H, 12-H), 4.53-4.39(m, 2H, 6’-H), 4.24-4.17(m, 1H, 3-H), 2.82-2.79(m, 1H), 2.29-2.20(m, 1H, 5’-H),1.12(s, 3H, Me-H), 1.08(s, 3H, Me-H), 0.94(s, 3H, Me-H), 0.92(s, 3H, Me-H), 0.90(s, 3H, Me-H), 0.89(s, 3H, Me-H), 0.74(s, 3H, Me-H).

13

C NMR (150MHz，CDCl3) δ: 184.2, 143.7, 141.2, 134.8, 130.2, 128.8, 125.8,

123.6, 122.5, 87.1, 79.8, 67.6, 55.2, 47.6, 46.6, 45.9, 41.6, 40.9, 39.3, 38.9, 38.0, 36.9, 34.2, 33.9, 33.1, 32.6, 32.5, 30.7, 28.3, 27.7, 26.0, 25.3, 23.7, 23.4, 23.0, 18.4, 17.2, 16.5, 15.4.

31

P NMR (243MHz，CDCl3) δ: -7.39.

ESI-MS m/z: 709.3[M+Na] +; HRMS (ESI): calculated for C39H56ClO6PNa: 709.3401 found 709.3371.

5S: mp > 200 ºC; [α]D-2.0º (c 1.0，CHCl3); 1H NMR (600MHz，CDCl3) δ: 7.39(s, 1H), 7.33-7.31(m, 2H), 7.28-7.25(m, 1H), 5.41(d, J = 11.4Hz, 1H), 5.27(t, J = 3.6Hz, 1H), 4.50-4.39(m, 2H), 4.21-4.18(m, 1H), 2.82-2.79(m, 1H),2.33-2.20(m, 1H), 1.12(s, 3H), 1.03(s, 3H), 0.95(s, 3H), 0.92(s, 3H), 0.90(s, 3H), 0.88(s, 3H), 0.75(s, 3H)。


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13

C NMR (150MHz，CDCl3) δ: 184.2, 143.8, 141.2, 134.8, 130.2, 128.8, 125.7,

123.6, 122.4, 87.1, 80.1, 67.3, 55.1, 47.6, 46.6, 45.9, 41.6, 41.0, 39.3, 38.9, 38.1, 36.9, 34.2, 33.9, 33.1, 32.6, 32.5, 30.8, 28.3, 27.7, 26.0, 25.3, 23.7, 23.5, 23.0, 18.4, 17.2, 16.5, 15.5.

31

P NMR (243MHz，CDCl3) δ: -7.39.

ESI-MS m/z: 709.3[M+Na] +; HRMS (ESI): calculated for C39H56ClO6PNa: 709.3401 found 709.3413.

2.3 Hepatoprotective experiments of 5R or 5S in CCl4-injured mice

Kun-Ming mice, weighing 18-22 g, were randomly divided into five groups (positive control group, normal control group, model group, 5R group, and 5S group) with 12 animals each group. The mice were treated with Bifendatatum (150 mg·kg-1, positive control) or distilled water (20 mL/kg, normal control and model group) by gavage for 4 days consecutively; 5R or 5S (15 mg·kg-1) injected to the caudal vein of mice (i.v. 5R group or 5S group) for four consecutive days. One hour after the last dose, the mice (besides those in the model group) were intraperitoneally injected (i.p.) with CCl4 (0.3%, v/v, dissolved in soybean oil, 10 mL·kg-1 body mass) to induce acute liver injury, while the mice in the normal group were administered with an equal volume of soybean oil.13

All the groups were subject to fasting for 16 h after CCl4 intoxication, but allowed to drink water ad libitum. At the end of the experimental period, livers and blood were



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obtained immediately after the animals were sacrificed. Blood samples were collected from all animals from the retro-orbital venous plexus and were centrifuged (3000 rpm × 3 min, 4 °C) for serum collection. The activities of serum alanine aminotransferase (ALT), lactic dehydrogenase (LDH), and aspartateaminotransferase (AST) were determined using commercial reagent kits obtained from the Institute of Biological Engineering of Nanjing Jiancheng (Nanjing, China) and used according to their instruction manuals. Portions of the liver were fixed in 10% formalin in 10 mmol phosphate-buffered saline (PBS) for histological examination, while the remaining parts were homogenised in nine volumes of phosphate buffered saline. The homogenate was centrifuged at 4 ºC (3000 rpm × 15 min) and the supernatant was stored at –20 °C until analysis.14 Malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX) levels were measured colorimetrically according to the instructions supplied with commercial assay kits (Nanjing Jiancheng Biological Technology, Inc. Nanjing, China).

2.4 In vitro metabolism study of 5R or 5Sin rat liver microsome

2.4.1 Preparation of stock solutions

The 200 µg·mL-1 parent stock solutions of OA, 5R, and 5S were prepared with methanol. All working standard solutions were individually prepared at concentrations of: 0.1, 0.5, 1.0, 2.0, 5.0, 25.0, and 50.0 µg·mL-1 by appropriate dilution of the parent stock solutions with methanol. For the studies in rats, 5R or 5S were prepared as sodium salts (5R or 5S was converted to the relevant sodium salt (5R-Na or 5S-Na)


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by reaction with sodium hydroxide in ethanol) and dissolved in physiological saline solution and filtered through a 0.45 µm sterile cellulose membrane.

The 1.0 mg/mL prodrugsolutions of 5R-Na or 5S-Na were prepared with ultra-pure water. Each stock solution was stored immediately at -20 ºC.

2.4.2 Animal treatment and preparation of rat liver microsomes

Rats were used in the study after 1 week in an ambient temperature and humidity controlled room (room temperature 23 ± 2 ºC, humidity 55 ± 10%) with free access to water and food. The light cycle consisted of 12 h of light and 12 h of darkness. The prodrugs were i.v. at a dose of 15 mg·kg-1 in these trials. Before the experiment, all rats were fasted for 24 h but given free access to water.15 Five rats were free to collect blank rat liver microsome and plasma.

The rats were sacrificed by decapitation, and the livers were removed and placed in ice-cold Tris buffer (pH 7.4), which contained 88 mmol/L CaCl2. Then, the livers were homogenised and centrifuged for 15 min at 12,500g at 4 ºC. The resulting supernatant was ultra-centrifuged using a Beckman-Coulter ultra-centrifuge Optima LE80K (USA) for 15 min at 27,000g at 4 ºC to obtain the microsomal pellet. The final microsomal pellets were re-suspended in 100 mM Tris buffer (pH 7.4) and stored at -80 ºC until use. The protein concentrations of the microsomal protion, and total CYP450, content were determined using the methods of Konno et al. and Xin Wang et al., respectively, bovine serum albumin was used as standard.16



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2.4.3 Microsomal incubation conditions

5R or 5S was determined in isolated rat liver microsome. A typical incubation mixture, in a final volume of 4.0 ml, contained 3.0 ml microsomal protein (the concentration was evaluated from 0.5 to 2.0 mg·ml-1 of incubation medium) 400 µL of 0.1 mol·L-1Tris-HCl buffer (pH 7.4), and 100 µL prodrug solutions (1.0 mol·L-1). Methanol concentration in the incubation mixture was 1%, or less. The reactions were initiated by the addition of a NADPH-regenerating system (1.15 mmol NADP, 56.25 mmol KCl, 12.5 mmol isocitric acid, 12.5 mmol MgCl2, 187.5 mmol Tris-HCl, 0.0125 mmol MnCl2, and 0.77 ml·ml-1 isocitric acid dehydrogenase, pH 7.4) following 2 min at 37 ºC in a water bath. Liver microsome was replaced with the same volume of water as blank solution.

Incubation time of the microsomal protein ranged from zero to 60 min depending on the experiment. The samples were withdrawn at appropriate intervals and stopped by the addition of the same amount of ice-cold acetonitrile as the sample. The samples were kept on ice, centrifuged at 12,000g for 15 min and the supernatant was analysed by HPLC.

2.4.4 Preparation of calibration standard

The calibration standard (CS) of OA, 5R, 5S, and the quality control (QC) in rat liver microsomes were prepared by adding 200 µL of working standard solutions to Eppendorf tubes. Next, the samples were vortexed to mix the residue with 200 µL of


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rat liver microsome and 400 µL acetonitrile for 1 min. The supernatant was evaporated to dryness under a gentle flow of nitrogen at 40 ºC and the residue was re-dissolved in 200 µL of the mobile phase, then the samples were injected into the chromatography system.15 The calibration curve of OA was constructed by plotting the peak-area of OA versus analyte concentrations.

The method validation assays were carried out according to the currently accepted U.S. Food and Drug Administration (FDA) bioanalytical method validation guidance (US DHHS, FDA, CDER, 2001).

2.4.5 Assay validation

The assay was validated for linearity, functional sensitivity (limit of quantification), precision and accuracy, extraction recovery, and stability.17

The linearity was assessed by analysing the eight calibration standards of OA, 5R, and 5S prepared above. Blank liver microsome samples were analysed to confirm the absence of interference effects.

The functional sensitivity was defined as the lowest concentration of the calibration curve which could be quantified with an accuracy of within 20% of the nominal concentration, and a precision not exceeding 20% CV. The limit of detection (LOD) was defined as the amount that could be detected with a signal/noise ratio of three. The specificity of the method was evaluated by analysing blank liver microsome from five rats.



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The precision of the assays was determined from the QC liver microsome samples by replicate analysis of three concentration levels of OA, 5R, and 5S (as prepared above). Intra-batch precision and accuracy (each with n = 3) determined by repeated analysis of the samples at different times during the same day. Inter-batch precision and accuracy were determined by repeated analysis of the samples over five consecutive days. All concentrations were determined using the calibration curve prepared and analysed on the same batch.

The extraction recovery of OA, 5R, and 5S were determined at low, medium, and high concentrations. Recovery was calculated by comparing the analyte peak area ratios obtained from liver microsome samples with those from the same concentration standard solutions. Acceptance criterion was a mean recovery between 80% and 120%.

For the freeze and thaw stability, QC liver microsome at three concentration levels (0.5, 5.0, and 50.0 µg·mL-1) were stored at -20 ºC for 24 h and thawed at room temperature. When completely thawed, all samples were refrozen for 24 h under the same conditions. The freeze-thaw cycles were repeated three times, and then the samples were analysed. For the short-term stability, QC samples at three concentration levels (0.5, 5.0, and 50.0 µg·mL-1) were kept at room temperature for 5 h, which exceeded the routine preparation time of the samples. For the long-term stability, stability of QC samples at three concentration levels (0.5, 5.0, and 50.0 µg·mL-1) were kept at a low temperature (-20 ºC) and studied over a 14 day


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period.

2.5 The pharmacokinetic in vivo and biodistribution studies

2.5.1 Preparation of stock solutions

A procedure similar to that outlined in Section 2.4.1.was used to obtain stock solutions.

2.5.2 Animal treatment

Male Wistar rats (200-220 g) were used in the study after at least six days of acclimatisation. The rats were randomly divided into two groups (5R or 5S) with six animals per group. The developed HPLC assay method was used in the pharmacokinetic study after i.v. 15 mg·kg-1 and i.p. 45 mg·kg-1 of the 1.0 mg·mL-1 prodrug solutions. Before the experiment, all rats were fasted for 24 h but given free access to water.

2.5.3 Preparation of plasma samples

About 0.5 mL of blood was collected via the post-orbital venous plexus veins at appropriate intervals (0.08, 0.25, 0.50, 0.75, 1.00, 1.50, 2.00, 4.00, 8.00, 12.00, 24.00, 36.00, 48.00, and 96.00 h) after intravenous administration while at the intervals (0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 8.0, 12.0, 24.0, 32.0, 36.0, and 48.0 h) after intraperitoneal administration. Next, the blood samples were vortexed to mix the residue with 200 µL of blood samples, and 400 µL acetonitrile, for 2 min. The samples were centrifuged at



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3000 rpm for 10 min and the supernatant was evaporated to dryness under a gentle flow of nitrogen at 40 ºC.18 The residue was re-dissolved in 200 µL of mobile phase and the samples were injected into the chromatography system.

2.5.4 Preparation of tissue samples

After intravenous administration, the rats were sacrificed by decapitation at appropriate intervals (0.08, 0.25, 0.50, 0.75, 1.00, 1.50, 2.00, 4.00, 8.00, 12.00, 24.00, 36.00, and 48.00 h) and tissue samples were taken from the internal organs. Each sample of the heart, liver, spleen, lungs, kidneys, and brain was rinsed with saline, weighed, and immediately frozen at -80 °C until use. The tissue samples were homogenised (500 mg of tissue in 1500 mL of saline) for 10 min. Next, the tissue samples were vortexed to mix the residue with 200 µL of tissue samples, and 400 µL acetonitrile, for 2 min. The samples were centrifuged at 1200 rpm for 10 min and the supernatant was evaporated to dryness under a gentle flow of nitrogen at 40 °C.19 The residue was re-dissolved in 200 µL of mobile phase, then the samples were injected into the chromatography system.

2.5.5 Preparation of urine and faecal samples

Urine and faeces were collected at appropriate intervals (2, 4, 8, 12, 24, 30, 36, and 48 h) after dosing, weighed, and immediately frozen at −80°C until use. Dry faecal samples were ground and weighed before being homogenised in a three-fold volume of saline. The faecal samples were obtained by vortexing 200 µL of faecal sample and


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400 µL acetonitrile for 2 min. The samples were centrifuged at 1200 rpm for 10 min and the supernatant was evaporated to dryness under a gentle flow of nitrogen at 40 °C.20 The residue was re-dissolved in 200 µL of mobile phase, then the samples were injected into the chromatography system.

The urine (1 mL) was centrifuged at 1200 rpm for 10 min. The urine sample was obtained by vortexing 200 µL of urine sample and 400µL acetonitrile for 2 min. The samples were centrifuged at 1200 rpm for 10 min and the supernatant was evaporated to dryness under a gentle flow of nitrogen at 40 °C.21 The residue was re-dissolved in 200 µL of mobile phase, then the samples were injected into the chromatography system.

2.5.6 Preparation of calibration standard and assay validation

A procedure similar to that outlined in Section 2.5.4 was used to obtain the calibration standard.

A procedure similar to that outlined in Section 2.5.5 was used for assay validaton.

2.6 Statistical analysis

Results are expressed as mean ± SD, all statistical analysis was performed using a one-way analysis of variance (ANOVA). Statistical significance was achieved if probability values were less than 0.05. The DAS Software (Version 2.1.1, Medical College of Wannan, China) was used to determine the pharmacokinetic parameters of



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5R or 5S.

3 Results and discussion 3.1 Synthesis and characterisation of 5R or 5S

5R or 5S was synthesised by reacting OA with 1,3-cyclic propanyl phosphate ester through an ester exchange reaction. OA can be purchased and 1, 3-cyclic propanyl phosphate were synthesised.11 The synthesis process of 5R, or 5S, is shown in Scheme 1.

Scheme 1: Synthesis process of cyclic phosphates. (a) OA, lithium diisopropylamide, room temperature.

In this reaction, several bases, such as tert-BuMgCl, t-BuOK, n-BuLi, and LDA were used. It was found that only LDA was feasible for this reaction. To increase the yields, a five-fold excess of LDA (Table 1) was used in the process. To increase the solubility of 5R or 5S, sodium salts were prepared (5R-Na and 5S-Na). The aqueous solubility of these sodium salts exceeded 5 mg·mL-1, which was measured by dissolving prodrugs at 5 mg·mL-1 in PBS at pH 7.4 and HPLC analysis.


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Table 1The effects of amount of LDA on the yield Entry

LDA

Yield (%)

1

1 eq.

—

2

4 eq.

28.8

3

5 eq.

46.4

4

6 eq.

38.7

5

10 eq.

—

3.2 Hepatoprotective effects of 5R or 5Sin CCl4-injured mice

Carbon tetrachloride (CCl4) induced hepatic injury is widely used as an established experimental model for the investigation of hepatoprotective agents.22 It is generally accepted that CCl4 is catalysed by cytochrome P4502E1 (CYP2E1) to produce the unstable free radicals of proxyltrichloromethyl (·OOCCl3), trichloro-methyl radical (·CCl3), and reactive oxygen species (ROS). These radicals and ROS can bind to lipids, which subtracts a hydrogen atom from the fatty acid to form a radical leading to chain lipid peroxidation.23 As a result of hepatic injury, in serum hepatic enzymes (such as ALT, AST, and LDH), oxidant parameter (such as GSH and MDA) levels showed a marked increase after CCl4 injection. The increased serum enzyme levels are indicators of cellular damage and the functional integrity of the liver cell membrane.

We evaluated liver function parameters in serum of 5R or 5S against CCl4-induced liver damage in mice by performing an analysis of serum ALT, AST, and LDH with a commercial assay kit. Bifendatatum, a liver-protection drug, was used as a positive control. As shown in Table 2, the activities of the ALT, AST, and LDH were



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significantly increased in the model group compared to the normal group (P < 0.01), reflecting tissue damage in the liver. Treatment with 5R or 5S at doses of 15 mg·kg-1 in different mice groups and the positive group caused a significant decrease in serum level of ALT, AST, and LDH enzymes compared to the model group (P < 0.05). Table 2 Effects of 5R or 5S on the activities of serum ALT, AST, and LDH in CCl4-induced liver damage mice. ( x ± s , n = 12) ALT (U·L-1)

AST (U·L -1)

LDH (U·L -1)

Normal

6.21 ± 1.32

7.88 ± 4.77

7896.21 ± 823.14

Model

371.12 ± 48.96△△

399.75 ± 86.48△△

14986.41 ± 1231.96△△

Group

Dose (mg·kg-1)

Positive

150

33.41 ± 16.19**

58.51 ± 21.63**

10893.62 ± 998.53**

5R

15

96.21 ± 22.34*

102.57 ± 21.69*

11137.21 ± 769.44*

5S

15

90.39 ± 18.25*

110.34 ± 31.66*

10998.31 ± 664.23*

△△P

< 0.01 versus normal group,

*

P < 0.05 versus model group,

**

P < 0.01 versus model group

It is reported that hepatic tissue damage induced by CCl4 causes lipid peroxidation and increases the level of MDA.24 To resist the oxidative stress in liver disease and pathology, antioxidants might prevent hepatic damage through scavenger activity and increase the activity of antioxidant enzymes (such as SOD and GSH-PX). Hence, SOD and GSH-PX levels are also considered as important indicators when evaluating functional recovery of acutely injured liver. In the present study, we evaluated the levels of MDA, SOD, and GSH-PX as biomarkers of lipid peroxidation. Data in the present study are shown in Fig. 1 and Table 3. Compared to the normal group, the model group had a significantly increased MDA content and a decreased activity of SOD and GSH-PX, suggesting stronger oxidative stress and lipid peroxidation in liver


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tissue (P < 0.01). After 5R or 5S treatment in mice with CCl4 and the positive group, the hepatic MDA levels were significantly lower than in the model group (P < 0.01), and the activities of SOD and GSH-PX were enhanced markedly compared to the model group (P < 0.05). Table 3 Effects of 5R or 5S on SOD and GSH-PX levels in liver tissue of CCl4-induced liver damage mice ( x ± s , n = 12) Group

SOD (U·mg-1)

GSH-PX (mol·L-1·mg -1)

Normal

4.21 ± 0.27

90.37 ± 10.62

Model

1.89 ± 0.34△△

70.59 ± 8.77△△

Positive

2.59 ± 0.63*

81.37 ± 9.07*

5R

2.03 ± 0.17*

82.34 ± 4.21*

5S

2.16 ± 0.21*

81.73 ± 3.97*

△△

P < 0.01 versus normal group, * P < 0.05 versus model group

In the present work, histopathological studies of the liver were performed to support the biochemical analysis evidence. As shown in Fig. 2a, the histology of the liver sections of the normal group exhibited normal architectures with well-preserved cytoplasm, prominent nucleus and nucleolus, thin sinusoids, and visible central veins. In contrast, the model group revealed the most severe damage with massive fatty changes, and in which the liver sections showed necrosis, ballooning degeneration, and the loss of cellular boundaries (Fig. 2b). Histopathological changes were remarkably rehabilitated in the positive group compared to the model group (Fig. 2c). However, treatment with 5R (Fig. 2g) or 5S (Fig. 2f) improved the state of steatosis, ballooning degeneration, and necrosis to varying extents as compared with the model



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group.

Fig. 2. Histopathological examination of liver sections (original magnification: ×400) (a)：normal group

(b)：model group

(c)：positive group

(f)：5R group (g)：5S group

Based on the evidence provided, it could be concluded that 5R and 5S show a potent protective effect against CCl4-induced liver injury and there were no significant differences between 5R and 5S treatments. Pretreatment with 5R or 5S in different mice groups caused a significant decrease in the level of ALT, AST, LDH, and MDA, and increased the activities of SOD and GSH-PX, indicating that their hepatoprotective activity was probably boosted by the activities of antioxidant enzymes.

3.3 The metabolism of 5R or 5S in rat liver microsomes 3.3.1 Method validation The calibration curves of OA, 5R, and 5S were constructed by plotting the analyte peak-area ratio against concentration. A good linear correlation between the peak area ratio and OA, 5R, and 5S concentrations were established in the range of 0.2-50.0 µg mL-1 in liver microsome with a coefficient of determination R2 > 0.99 (Table 4). As can be seen from Fig. S1, blank liver microsome had no effect on the


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elution of all of the drugs, indicating that the method was specific to the determination of OA, 5R, and 5S.The functional sensitivity for all three analytes was 0.20 µg ml-1. Table 4 Calibration curves for OA, 5R, and 5S in rat liver microsome

Compounds OA 5R 5S

Standard curves y = 0.0347x + 0.0107 y = 0.0216x + 0.0029 y = 0.0118x + 0.0423

R2 0.9987 0.9962 0.9993

Test range (µg/mL) 0.2-50 0.2-50 0.2-50

LOQ (µg/mL) 0.20 0.20 0.20

The precision and accuracy of the assays were estimated in liver microsome by performing replicate analysis of spiked samples against calibration standards, data from which were expressed as a relative standard deviation (RSD) and mean concentration. Intra-day, and inter-day, precision and accuracy values are summarised in Table 5. The precision (RSD) ranged from 1.2 to 2.3%, respectively. The accuracy ranged from 95.00 ± 0.99 to 102.00 ± 1.09%, respectively. The results were within an acceptable range so the method was proved to be both precise and accurate.

Table 5 Intra-day and inter-day accuracy, precision and recovery for OA, 5R, and 5S in rat liver microsome (n = 5) Spiked concentration (µg/mL)

Intra-day (n = 5) Measured RSD (%) concentration a (µg/mL)

Accuracy b (%)

Inter-day (n = 5) Measured RSD (%) concentration a (µg/mL)

Accuracy b (%)

OA 0.6

0.61±0.14

1.9

101.67±1.51

0.57±0.21

2.0

95.00±0.99

5

4.9±0.01

1.2

98.00±1.23

4.92±0.24

2.1

98.40±1.56

50

50.12±1.23

1.7

100.241.32

49.89±0.18

2.3

99.7±1.31

5R 0.6

0.59±0.17

2.1

98.33±0.98

0.58±0.26

1.6

96.67±0.99

5

5.10±0.12

1.7

102.00±1.09

4.98±0.31

2.1

99.60±1.23

50

49.99±0.98

1.9

99.98±1.13

49.93±1.04

1.8

99.869±1.46

0.7

0.71±0.12

1.4

101.43±1.12

0.68±0.11

1.8

97.14±1.11

5

5.01±0.25

1.6

100.20±1.01

4.98±0.21

1.4

99.60±1.21

50

49.92±0.78

2.0

99.84±1.12

49.89±0.67

1.9

99.78±0.98

5S

a

Mean ± SD; b accuracy = mean of measured concentration / nominal concentration.

The extraction recoveries were determined by five replicates of rat liver microsome



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spiked with high, medium, and low concentrations of the aforementioned drugs. The extraction recoveries of OA, 5R, and 5S in rat liver microsome are shown in Table 6. The recoveries of the samples were all above 94.23%. The results showed that the extraction recoveries of all the analytes in the liver microsome were acceptable. Table 6 Freeze-thaw stability data for OA, 5R, and 5S in rat liver microsome (n = 5) Measured concentration (µg mL-1)

Sample concentration

Before

-1

(µg mL )

Recovery

After

storage

storage

0.5

0.48

5

4.96

50

a

Measured concentration (µg mL-1) b

Before

Recovery

After a

b

(%)

freeze-thaw

freeze-thaw

(%)

0.52 ± 0.04

108.33 ± 0.78

0.49

0.53 ± 0.07

108.16 ± 1.21

4.99 ± 0.54

100.60 ± 1.02

5.03

4.98 ± 0.12

99.01 ± 0.94

50.12

50.07 ± 1.01

99.90 ± 1.14

50.05

49.96 ± 0.11

99.82 ± 1.32

0.5

0.48

0.49 ± 0.02

102.08 ± 0.82

0.48

0.51 ± 0.04

106.25 ± 1.14

5

4.95

4.97 ± 0.25

100.40 ± 1.13

5.02

5.03 ± 0.12

100.20 ± 1.23

50

50.05

50.01 ± 1.02

99.92 ± 1.21

50.05

0.53 ± 0.11

99.96 ± 0.91

0.5

0.52

0.49 ± 0.05

94.23 ± 0.91

0.49

0.53 ± 0.13

108.16 ± 0.81

5

5.01

4.98 ± 0.13

99.40 ± 1.04

4.99

5.04 ± 0.12

101.00 ± 1.03

50.05

50.03 ± 0.21

99.96 ± 1.15

49.97

49.99 ± 0.08

100.04 ± 1.11

OA

5R

5S

50 a

b

Mean ± SD; Recovery = 1–[ ( before concentration–after concentration ) / spiked concentration ] × 100.

Stability studies are one of the most important items evaluated in the analysis of drug efficacy. In this study, the stability of OA, 5R, and 5S during sample handling (freeze-thaw, short-term, long-term) is shown in Table 6. The analytes were stable after three freeze-thaw cycles and stable at room temperature in liver microsome.

In summary, the current study developed, and validated, an HPLC assay method for the determination of OA, 5R, and 5S. This method shows satisfactory sensitivity, precision and accuracy, and provides a reliable method for the evaluation of the metabolism of 5R and 5S in rat liver microsomes.


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3.3.2 Metabolites of 5R or 5S detected in rat liver microsomes

The incubation trials of liver microsomes are one of the most commonly used methods for drug metabolism assessment in vitro: it presents good reproducibility and the preparation is straightforward.25, 26 The method is recommended by the relevant guidelines used in the USA.27 As shown in Fig. 3, the conditions of microsomal protein concentration and incubation time were dependent on the experimental conditions. Based on these results, 1.0 mg mL−1 was chosen as the microsomal protein concentration, and 30 min as the incubation time. Besides, as the parameters of rat liver microsome cytochrome P450 (CYP450), and protein concentration, played an important function in the metabolism and ultimate clearance of many drugs. It can be seen from Table 7 that, the parameters of rat liver microsome were all consistent with the in vitro metabolism. Above all, a simple, reproducible, sensitive and accurate analytical method was developed for the assay of 5R or 5S in rat liver microsome.

Table 7 The parameters of rat liver microsome (n = 5)

Liver mass

Content (Mean ± SD)

P

4.89 ± 0.34

0.09

13.04 ± 3.21

0.23

0.32 ± 0.07

0.41

(g) Total protein (mg/g liver) Total P450 (nmol/mg protein)



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Fig. 3. Effect of time and concentration of liver microsomes on drug incubation (A): Effect of time to drug incubation (B): The incubation of 5R at different concentrations of liver microsomes (C): The incubation of 5S at different concentrations of liver microsomes

As shown in Fig. 4A, the concentration of prodrugs diminished gradually with time. The mean releases of 5R or 5S in rat liver microsome were all above 87% within 30 min after incubation in vitro. Compared with 5S, 5R had higher metabolic rates in the same time period. As for the parent drugs (Fig. 4B), the concentrations of released parent drugs were lower than those of the prodrug and most of them were released in 30 min. From the linear nature of logarithmic prodrug concentrations versus incubation time plots (5R: y = -0.0985x + 3.0527, r = 0.9922; 5S: y = -0.0658x + 3.1382 r = 0.9916), it was found that the half-lives of 5R and 5S in in vitro incubation of rat liver microsome were 5.35 ± 0.15 min and 9.31 ± 0.11 min, respectively. These results indicated that these two prodrugs could be easily metabolised in rat liver microsome, and have the potential to be targeting drugs to the


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liver, especially 5R. The half-lives are given as reference values for in vivo experiments because the rat liver microsome was an independent system.

Fig. 4. The concentration-time profiles after incubation of 5R or 5S (A): Mean concentration-time profiles of 5R and 5S after incubation (B): Mean concentration-time profiles of releasing parent drugs after incubation of 5R and 5S

It can be seen from Fig. S2 that, besides OA (2), 5R and 5S were all transformed to a new metabolite (1). We have identified the metabolite by ESI-MS (Fig. S3) and NMR, as the structure (OA-PA) shown in Fig. 5. These results confirmed that the presumed mechanism of the metabolism of 5R or 5S was that the prodrugs were slowly converted to a ring-opened intermediate, which was subsequently transformed by a β-elimination reaction to a free phosphate (OA-PA, Fig. 5). The free phosphate was further dephosphorylated by microsomal phosphatases, releasing the parent molecule with a free hydroxyl group.

In total, drug-metabolism studies in vitro are key stages in early drug development. In our study, we illustrated the metabolic rates of prodrugs and the parent drugs in rat liver microsome. In addition, we also demonstrated the presumed mechanism of the metabolism of produgs. These data indicate that 5R or 5S are considered as promising



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drug candidates, and in particular 5R.

Fig. 5. The metabolism mechanism of the modifiers

3.4 The pharmacokinetics in the plasma and the biodistribution studies of 5R or 5S 3.4.1 Pharmacokinetics of produgs in the plasma In this study, an HPLC assay method was developed and validated for the pharmacokinetic

study

after

intraperitoneal

administration

and

intravenous

administration of the prodrugs (5R or 5S). Due to the lack of an appropriate internal standard, the standard curve was established by external standard method. The pharmacokinetic parameters are listed in Table 8 and Table 9, and there were no significant differences between 5R and 5S. The pharmacokinetic profiles of prodrugs and released parent drugs (OA) are shown in Figure 7. The areas under concentration-time curves (AUC0–t) of 5R, 5R-OA, and 5S, 5S-OA were 345.359 ± 12.315, 83.014 ± 0.805, and 420.775 ± 20.663, 75.314 ± 2.516 µg h/mL after intraperitoneal administration, while they were 216.878 ± 5.289, 51.913 ± 2.612, 254.09 ± 6.546, and 58.806 ± 1.486 µg h/mL after intravenous administration, respectively. The in vivo behaviour of prodrugs and released parent drugs could be described by a two-compartment model (weight coefficient 1/cm3).


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The absorption and elimination of the prodrugs in the plasma after intraperitoneal administration are shown in Fig. 6A: after reaching the maximum level the prodrug concentration gradually decreased. As shown in Fig. 6B, the concentrations of the prodrugs were depressed after intravenous administration. In this study, 5R, 5S, and OA were almost eliminated within 48 h after intraperitoneal administration or intravenous administration. Compared with the prodrugs, the concentration of OA (Figs 6B and 6C) were much lower in plasma, which maybe related to the metabolic rate of prodrugs and the transformation of OA.

All the aforementioned evidence suggests that 5R or 5S could directly increase the half-life of parent drug administration. These results indicate that 5R or 5S formed a successful sustained-release system.

Table 8 Pharmacokinetic parameters of drugs after i.p. (45 mg kg-1) Pharmacokinetic

OA17R

OA17R-OA

OA17S

OA17S-OA

t1/2α (h)

8.015±6.394

16.436±4.898

9.071±3.967

15.162±7.788

t1/2β (h)

12.152±13.272

35.053±26.6

9.41±3.552

15.594±7.07

AUC(0-t) (µg/L*h)

345.359±12.315

83.014±0.805

420.775±20.663

75.314±2.516

AUC(0-∞) (µg/L*h)

369.469±14.9

104.417±6.533

435.208±20.727

92.877±8.785

Tmax (h)

2.0±0.015

2.0±0.012

3.0±0.023

1.667±0.577

Cmax (µg/L)

22.37±1.205

4.263±0.095

25.477±0.14

3.783±0.207

parameters



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Table 9 Pharmacokinetic parameters of drugs after i.v. (15 mg kg-1) Pharmacokinetic

OA17R

OA17R-OA

OA17S

OA17S-OA

t1/2α (h)

0.541±0.041

1.706±0.218

0.605±0.104

7.946±8.578

t1/2β (h)

11.487±0.435

28.527±1.211

11.087±0.803

21.933±3.545

AUC(0-t) (µg/L*h)

216.878±5.289

51.913±2.612

254.09±6.546

58.806±1.486

AUC(0-∞) (µg/L*h)

232.026±5.031

74.471±7.307

269.397±9.204

73.715±5.837

Tmax (h)

0.083±0.001

0.5±0.002

0.083±0.001

0.75±0.002

Cmax (µg/L)

35.607±0.577

3.513±0.144

33.257±1.052

3.753±0.255

parameters

Fig. 6. Plasma concentration-time profiles after i.p. (45 mg kg-1) or after i.v. (15 mg kg-1) (A): Mean plasma concentration-time profiles of modifiers after i.p. (45 mg kg-1) (B): Mean plasma concentration-time profiles of modifiers after i.v. (15 mg kg-1) (C): Mean plasma concentration-time profiles of releasing parent drugs (OA) after i.p. (45 mg kg-1) (D): Mean plasma concentration-time profiles of releasing parent drugs (OA) after i.v. (15 mg kg-1)

3.4.2 Biodistribution of 5R and 5S and composition changes in organs, urine, and faeces

Besides their pharmacokinetic properties, the differences in the biodistributions of 5R, 5S, and their parent drugs (OA) were also taken into account. After intravenous


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administration of 5R or 5S in rats the biodistributions in heart, liver, spleen, lungs, kidneys, brain, urine, and faeces were investigated. As shown in Fig. 8, the concentration in the main organs was ranked as liver > lungs > spleen > heart > kidneys > brain. After reaching the highest level at different times in different organs, all analyte levels were gradually decreased in all organs. This indicates that 5R, 5S, and OA could be eliminated over time, and there was no long-term accumulation, which was in accordance with the variation seen in the plasma concentration.

It can be seen that all analyte concentrations were highest in the liver, and there were no significant differences between 5R and 5S (Fig. 8A). The maximum amounts of 5R, 5S, and OA were higher in liver than in plasma, and all prolonged the mean retention time of their parent drug (OA) in liver (36 h for 5R, 48 h for 5S). Both 5R and 5S was still detectable in brain which showed that they could also pass through the blood–brain barrier.

Similar to plasma and tissue data, 5R, 5S, and OA were found in the urine and faeces samples. The results (Figs 8A and 8C) showed that the urinary and faecal excretion of 5R or 5S reached a steady level at 12 h after dosing. There were no significant differences between 5R and 5S. Approximately 11.43% (5R) and 11.83% (5S) were observed to be eliminated from the urine within 48 h after injection, while 47.11% (5R) and 48.11% (5S) were observed in the faecal samples. A small amount of 5R or 5S was excreted in urine in accordance with the lower observed distribution and they were eliminated rapidly in the kidneys (Fig. 7E). The excretion data in faeces



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indicated that 5R or 5S was mainly passed into the small intestine with bile, then excreted into the faeces after intravenous administration. A small amount of the parent drug in both urine and faeces samples (Figs 8B and 8D) suggested that OA was metabolized in the liver: this was consistent with previous research into OA.


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Fig. 7. The tissue distribution of 5R and 5S after i.v. (15mg kg-1) (A):Mean liver concentration-time profiles (B): Mean heart concentration-time profiles (C): Mean spleen concentration-time profiles (D): Mean lung concentration-time profiles (E): Mean kidney concentration-time profiles (F): Mean heart concentration-time profiles

Fig. 8. Plot of accumulation of 5R, 5S, and OA excretion rate in different times after i.v. (15mg kg-1) (A): The mean accumulative excretory rate in urine of 5R and 5S (B): Mean urine concentration-time profiles of OA (C): The mean accumulative excretory rate in faeces of 5R and 5S (D): Mean faecal concentration-time profiles of OA

4 Conclusions In this study, two novel prodrugs of oleanolic acid were designed and synthesised to change OA metabolism by the sustained-release properties of 1,3-cyclic propanyl phosphate esters. Our findings suggested that the prodrugs possess strong antioxidant activities and offered significant protective effect against liver injury induced by CCl4. Based on the results from both in vitro and in vivo metabolism, the prodrugs could increase the half-life and significantly changed the pharmacokinetic parameters after



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direct administration. Based on the structures of the metabolites, the metabolic mechanism of the prodrugs was proposed. Further biodistribution study showed that the prodrugs and OA concentrations were highest in the liver and most them were excreted in faeces. In summary, these two novel 1, 3-cyclic propanyl phosphate esters of OA would potentially improve the efficacy and the safety profile of the parent drugs: they are, as such, promising candidates for liver-targeted drugs.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI： Chromatographic profiles, ESI-MS spectra of OA-PA, 1H NMR of 5R and 5S, 13

C of 5R and 5S, NMR 31P NMR of 5R and 5S

AUTHOR INFORMATION Corresponding Author * (Tao Jiang) Phone: +86-0532-82033054. Fax: +86-0532-82033054. E-mail: [email protected]. * (Guoqiang Li) Phone: +86-0532-82032323. Fax: +86-0532-82032323. E-mail: [email protected]. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS ACS Paragon Plus Environment

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We also gratefully acknowledge Ms Xiu-li Zhang and Su-mei Ren (Key Laboratory of Marine Drugs, Ministry of Education, Marine Drug and Food Institute, Ocean University of China) for the measurement of ESI-TOF-MS and NMR spectra, respectively. This research was supported by the Natural Science Foundation of China (Grant No. 81373322), the Key International Cooperation Project of CMST (Grant No. 2013DFG32160) and NSFC-Shandong Joint Fund (U1406402).

ABBREVIATIONS USED OA, Oleanolic acid; CCl4, Carbon tetrachloride; ALT, alanine aminotransferase; LDH, lactic dehydrogenase; AST, aspartateaminotransferase; MDA, malondialdehyde; i.v., intravenous; THF, tetrahydrofuran; CS, calibration standard; QC, quality control; LOD, limit of detection; ROS, reactive oxygen species

REFERENCES (1) Li, T.X.; Chu, C.S.; Zhu, J.Y.; Yang, T.Y.; Zhang, J.; Hu, Y.T.; Yang, X.T. A rapid UPLC-MS/MS method for the determination of oleanolic acid in rat plasma and liver tissue: application to plasma and liver pharmacokinetics. Biomed. Chromatogr. 2015.

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Table of Contents /Abstract Graphic:

1.36 inches high × 2.52 inches wide


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Abstract Graphic 69x36mm (150 x 150 DPI)



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Fig. 1. Structures of OA and the prodrugs of OA 184x96mm (96 x 96 DPI)


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Fig. 2. Histopathological examination of liver sections 98x48mm (150 x 150 DPI)



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Fig. 3. Effect of time and concentration of liver microsomes on drug incubation 210x141mm (96 x 96 DPI)


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Fig. 4. The concentration-time profiles after incubation of 5R or 5S 221x72mm (96 x 96 DPI)



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Fig. 5. The metabolism mechanism of the modifiers 227x38mm (96 x 96 DPI)


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Fig. 6. Plasma concentration-time profiles after i.p. (45 mg kg-1) or after i.v. (15 mg kg-1) 221x129mm (96 x 96 DPI)



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Fig. 7. The tissue distribution of 5R and 5S after i.v. (15mg kg-1) 168x225mm (96 x 96 DPI)


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Fig. 8. Plot of accumulation of 5R, 5S, and OA excretion rate in different times after i.v. (15mg kg-1) 220x149mm (96 x 96 DPI)


Pharmacokinetics in Vitro and in Vivo of Two Novel Prodrugs of

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