PEGylated triacontanol substantially enhanced the pharmacokinetics

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Bioactive Constituents, Metabolites, and Functions

PEGylated triacontanol substantially enhanced the pharmacokinetics of triacontanol in rats ning Li, Xiaoyu Lu, Min Fang, Zhixia Qiu, Xijing Chen, Lili Ren, Pingkai Ouyang, and Guoguang Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02684 • Publication Date (Web): 23 Jul 2018 Downloaded from http://pubs.acs.org on July 24, 2018

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Journal of Agricultural and Food Chemistry

PEGylated triacontanol substantially enhanced the pharmacokinetics of triacontanol in rats

Ning Li1,2, Xiaoyu Lu3, Min Fang3, Zhixia Qiu4, Xijing Chen3, LiLi Ren2, Pingkai Ouyang1*, Guoguang Chen2* 1

School of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University,

Nanjing, China 2

School of Pharmaceutical Sciences, Nanjing Tech University, Nanjing, China

3

Clinical Pharmacokinetics Research Laboratory, China Pharmaceutical University,

Nanjing, China 4

School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing,

China

*Corresponding author: Guoguang Chen ([email protected], School of Pharmaceutical Sciences, Nanjing Tech University, Nanjing, Jiangsu, 211816, China) and Pingkai Ouyang ([email protected], School of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, Jiangsu, 211816, China) Tel: 86-(25)-86185379 Fax: 86-(25)- 86185379

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ACS Paragon Plus Environment


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Abstract

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Triacontanol (TA), a natural compound with various health benefits, is extensively

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used as a nutritional supplement. The therapeutic and nutraceutical applications of TA

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are limited due to its poor aqueous solubility. PEGylated triacontanol (PEGylated TA)

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was designed to improve the solubility and pharmacokinetics of TA. After PEGylation,

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the solubility (~250 g•L-1 versus 9×10-14 g•L-1), body residence (MRT, 9.40±2.03 h

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versus 2.59±0.705 h, p99%) was purchased from Aladdin

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(Shanghai, China). Ammonium acetate buffer solution (10 M) was prepared using

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purified water, generated from a Milli-Q water purification system.

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Dosing formulation preparation

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The PEGylated TA (mPEG2K-SA-TA) was readily soluble in physiological saline, 6


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with the solubility at about 250 g•L-1(depicted in Supporting information), which was

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classified as the freely soluble compound18. Prior to drug administration, the stability

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of PEGylated TA in saline was examined. At room temperature (25 oC), the

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PEGylated TA remained stable with only a portion of 5.4% PEGylated TA hydrolysis

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up to 4 h and11.7% to 24 h in saline (pH7.4). When spiked with plasma and placed

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under room temperature, the measured amounts in stock for 4h were close to those of

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freshly prepared samples. All these data indicated the, degradation of PEGylated TA

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was restricted minor. The in vitro stability could guarantee the intact form of

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PEGylated TA in dosing solution. In spite of the reliable stability of PEGylated TA,

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the dosing samples were freshly prepared in saline and administrated at the doses of

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6.85, 22.8 and 68.5 µmol •kg-1 (3, 10 and 30 mg •kg-1, TA equivalent).

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Experimental animals

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Sprague-Dawley rats (male and female, 240±20 g) were supplied by Shanghai

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SIPPR/BK Experimental Animal Co., Ltd (Shanghai, China). The rats were kept

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under humanized conditions with suitable temperature (20±2oC) and humidity

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(relative humidity, 50±20%) with free access to water and standard diet for 1-week 7



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(acclimatized period) prior to experiment. The experiment protocol was approved by

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China Pharmaceutical University (Nanjing, China).

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Before drug administration, the rats (3 male and 3 females in each group) were fasted

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overnight with free access to water. TA solutions at the dosage of 6.85 or 240 µmol•

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kg-1 were administrated intravenously or intragastrically to the rats. For single

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administration study, the rats were intravenously administrated mPEG2K-SA-TA in

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three escalated doses [6.85, 22.8 and 68.5 µmol•kg-1 (3, 10 and 30 mg•kg-1, TA

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equivalent)] to the groups 1, 2 and 3 for investigating the effect of dose escalation on

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the pharmacokinetics of both TA and PEGylated TA. The rats of group 4 were

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intravenously administered with PEGylated TA in physiological saline at the dose of

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22.8 µmol•kg-1 (10 mg•kg-1, TA equivalent) for seven successive days. A complete

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pharmacokinetic profile of the drug was evaluated to investigate the impact of

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multi-dosing. In another study, the PEGylated TA was intragastrically (p.o)

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administrated at 274 µmol•kg-1 (120 mg•kg-1, TA equivalent).

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An aliquot of 100 µL blood was collected from the jugular veins of the rats into

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heparinized tubes before and after the drug administration at different time-points (0, 8


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0.083, 0.167, 0.5, 0.75, 1, 2, 4, 8, 12, 24, 36 and 48 h). The blood samples were

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immediately centrifuged at 12,000 rpm for 2 min for harvesting plasma. The plasma

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samples were stored at -80 oC till GC-MS/MS analysis.

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Sample preparation

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A modified liquid-liquid extraction (LLE) was employed to separate the aqueous and

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organic phase using high concentration acetate buffer19,20. An aliquot of 50 µL of rat

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plasma sample was mixed with an isometric volume of ammonium acetate solution

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(10 M), and the mixture was continuously vortexed for 3 min. About 150 µL of

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acetonitrile-methanol (9:1, v/v) was added to the previous mixture and vortexed for 5

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min. The resultant mixture was centrifuged (4 oC) at 5,000 rpm for 10 min, and then

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the top (mainly PEGylated TA) and the bottom layers (TA and bonded TA) were

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transferred into separate 10 mL glass tubes, and 50 µL of internal standard

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(octacosanol used as internal standard, 100 ng•mL-1) and 1 mL of ethanolic NaOH

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solution (1 M) were added for saponifying the content and incubated on the water

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bath at 80 oC for 1 h; then 2 mL of heptane was added to the saponified mixture for

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extracting TA. The extraction process was repeated thrice. The combined extract was 9



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evaporated by Labconco CentriVap (CentriVap) to dryness. An aliquot of 50 µL of

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derivatization reagent (BSTFA) was added to the dry extract and incubated at 80 °C

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for 20 min to ensure complete derivatization. Finally, the derivatized samples were

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condensed and reconstituted with 25 µL of heptane. An aliquot of 1 µL was directly

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injected into the GC-MS/MS system.

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Samples determination by GC-MS/MS

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The GC-MS/MS system included a gas chromatograph (Agilent, 6890N, USA) and a

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Quattro Micro Mass spectrometer (Waters, Micromass, USA) with an electron impact

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ion (EI) source. A CD-5MS capillary column (30 m × 0.25 mm × 0.25 µm, CNW)

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coated with 5% diphenyl and 95% dimenthyl polysiloxane was used as the stationary

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phase. The carrier gas, helium (He, 99.999% pure) at a flow rate at 1.0 mL/min, and

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argon as the collision gas (Ar, 99.999% pure) were employed during the analysis.

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Split-less injection mode was employed maintaining the injection port temperature at

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300 oC (Back injector). The initial temperature of the system was maintained at 100 oC

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for 1 min, and increased to 200 oC at a rate at of 40 oC •min-1, which was subsequently

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increased to 300 oC (rate 18.52 oC •min-1), and then maintained at 300 oC for 9.5 min. 10


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The total run-time for each analysis was 18.4 min. The period of solvent delay was

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maintained for 3 min. The purge time was 1.5 min with purge flow at 50 mL•min-1.

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The ion source (EI) temperature was maintained at 200 oC, and the ion voltage was set

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at 70 eV. The multiple reaction monitoring (MRM) mode was employed to monitor

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m/z 495.6→96.9 for triacontanol and m/z 467.5→96.9 for octacosanol (internal

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standard). The MassLynx (V.4.1) was used for data acquisition and analysis.

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Pharmacokinetic analysis of both PEGylated TA and TA

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The pharmacokinetic parameters were calculated by non-compartmental analysis

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using WinNonlin (version 6.4, Pharsight Corporation Inc, Mountain View, CA, USA).

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Specifically, the maximum plasma concentration (Cmax) and the time to reach peak

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concentration (Tmax) were directly determined by the observed data. The systemic

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exposure and area under curve (AUC0–t and AUC0–∞) were estimated by the

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trapezoidal rule. The total plasma clearance (CL) was calculated as dose/AUC0–∞, and

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the mean residence time (MRT) was calculated as AUMC0–∞/AUC0–∞, where AUMC

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was the first moment of the plasma concentration-time curve. The potential risk of

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accumulation was evaluated by comparing the major pharmacokinetic parameters

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between multi-dose and single dose, including AUC0–t, AUC0–∞, Cmax, Tmax, t1/2 etc.

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A compartmental model simulation was also carried out to evaluate whether there

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existed the possible accumulation upon repeated dosing, by means of comparison

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between the observed data and predicted data via compartmental model fitting. Model

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description was depicted in Supporting Information.

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Statistical analysis

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The normality of distribution was examined by Kolmogorov–Smirnov test (IBM

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SPSS Statistics 22.0). The major pharmacokinetic parameters largely followed the

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Gaussian distribution, including Cmax, AUC0-t, AUC0-inf, V and CL. As the observed

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Tmax was not continuous, the nonparametric test was applied to analyze Tmax. The

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experimental parameters were presented as means ± SD. Two-tailed t test was used to

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compare the differences between the pharmacokinetic parameters of PEGylated TA

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and pristine TA, and p

PEGylated triacontanol substantially enhanced the pharmacokinetics

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