Synthesis of a highly water-soluble acacetin prodrug for treating experimental atrial fibrillation in beagle dogs

We previously reported that duodenal administration of the natural flavone acacetin can effectively prevent the induction of experimental atrial fibrillation (AF) in canines; however, it may not be used intravenously to terminate AF due to its poor water-solubility. The present study was to design a water-soluble prodrug of acacetin and investigate its anti-AF effect in beagle dogs. Acacetin prodrug was synthesized by a three-step procedure. Aqueous solubility, bioconversion and anti-AF efficacy of acacetin prodrug were determined with different methodologies. Our results demonstrated that the synthesized phosphate sodium salt of acacetin prodrug had a remarkable increase of aqueous solubility in H2O and clinically acceptable solution (5% glucose or 0.9% NaCl). The acacetin prodrug was effectively converted into acacetin in ex vivo rat plasma and liver microsome, and in vivo beagle dogs. Intravenous infusion of acacetin prodrug (3, 6 and 12 mg/kg) terminated experimental AF without increasing ECG QTc interval in beagle dogs. The intravenous LD50 of acacetin prodrug was 721 mg/kg in mice. Our preclinical study indicates that the synthesized acacetin prodrug is highly water-soluble and safe; it effectively terminates experimental AF in beagle dogs and therefore may be a promising drug candidate for clinical trial to treat patients with acute AF.

mobile phase was composed of a mixture of water and methanol (v/v, 3:7, with 0.2% phosphate acid) using an isocratic elution (Hitachi L2130 pump, L2200 Autosampler and L2400 Detector, Hitachi, Japan). The flow rate was 1.0 mL/min. The UV absorbance detector was set at 330 nm.

Effects of acacetin prodrug on cardiac potassium currents
Whole-cell currents of hKv1. 5  were pulled with a Brown/Flaming puller (model P-97, Sutter Instrument, Nato, CA) and had resistances of 1.5~2.5 M when filled with the pipette solution. A 3-M KCl agar bridge was used as the reference electrode. The tip potential was zeroed before the patch pipette contacted the cell. After a giga-Ohm seal was obtained, the cell membrane was ruptured by applying gentle pressure to establish a whole-cell configuration. Series resistance (Rs) was 3~5 M and was compensated by 50-70% to minimize voltage errors. The liquid junction potential (14.7 mV) calculated with the software Clampex was not corrected in the experiment and data analysis.
Whole-cell current of IKACh was determined in isolated rat atrial myocytes. The myocytes were dissociated from male Sprague-Dawley rats (230-300 g, n = 5), were obtained from Laboratory Animal Unit of University of Hong Kong. The protocol was approved by Ethic Committee of Animal Use for Teaching and Research of University of Hong Kong. Atrial myocytes from rat hearts were enzymatically dissociated by the procedure described previously. 3 The isolated myocytes were kept in a high potassium medium at room temperature for 1 h before electrophysiological recording. 4 After whole-cell membrane current reached stable, 5 M carbachol was added to elicit IKACh in rat atrial myocytes. Current and voltage signals were low-pass filtered at 5 kHz and stored in the hard disk of an IBM compatible computer. All experiments were conducted at room temperature.

Bioconversion of acacetin prodrug in rat plasma and hepatic microsomes
Hydrolysis of acacetin prodrug was initially determined in rat plasma. The 100 L rat plasma samples containing 5 g/mL prodrug was prepared at 4 o C and then incubated in a water bath at 37 o C and continuously shaken at 60 rpm. The plasma samples were collected at 0, 5, 15, 30, 60, 120, 180, 240 and 360 min, respectively. The reaction was terminated by adding 1 mL cold methanol containing the IS solution (50 ng/mL pentamethylquercetin). The plasma treatment was described below.
Bioconversion of acacetin prodrug was also determined in rat hepatic microsomes containing high content alkaline phosphatase, 5 as described previously. 6  Linearity range with 1/x/x weighting was from 1~500 ng/mL. The lower limit of quantification was 0.5 ng/mL, and the limit of detection was 0.1 ng/mL for acacetin. Assay accuracy at low, medium, and high QCs was 100 ± 3.2%, 103.6 ± 2.9% and 97.5 ± 5.1% respectively (n = 5). Samples were stable within 4 h on the auto-sampler. The peak concentration (Cmax) and time to reach Cmax (Tmax) were read directly from individual acacetin plasma concentration-time profiles. The mean values of plasma concentrations were calculated and plotted against the time points.

Pre-pharmacokinetics of acacetin in beagle dogs
The pharmacokinetic parameters were calculated using non-compartmental analysis and compartmental analysis respectively using WinNonlin®1.

Vagal nerve stimulation-induced atrial fibrillation in Beagle dogs
Beagle dogs (either gender, 10~12 kg, Wuhan Anlu Experimental Animal Laboratory, Wuhan, by weight and randomly assigned to six groups: (n = 10 in each group, 5 male and 5 female).
Acacetin prodrug dissolved in 5% glucose was intravenously administered at 900, 810, 765, 720, 630 and 540 mg/kg through tail vein, respectively. The animals were closely observed for symptoms and mortality in 24 h. All of the surviving animals were euthanized at the end of the study, and their vital organs were individually observed for gross pathology by necropsy, and the LD50 was calculated by the Bliss method. 11,12 Supplemental figures Figure S1 1 H NMR spectra of compound 2.