Improvement of pharmacokinetics behavior of apocynin by nitrone derivatization: comparative pharmacokinetics of nitrone-apocynin and its parent apocynin in rats

Abstract

Apocynin, a potent inhibitor of NADPH-oxidase, was widely studied for activities in diseases such as inflammation-mediated disorders, asthma and cardiovascular diseases. In our recent study, a novel nitrone derivative of apocynin, AN-1, demonstrated potent inhibition to oxidative injury and to high expression of gp91(phox) subunit of NADPH-oxidase induced by tert-butyl hydroperoxide (t-BHP) in RAW 264.7 macrophage cells, and displayed promising preclinical protective effect against lipopolysaccharide (LPS)-induced acute lung injury in rats. In this work, the pharmacokinetic behaviors of AN-1 in Sprague-Dawley rats with single intravenous and intragastric doses were investigated for further development. Furthermore, apocynin's pharmacokinetics remain lacking, even though its pharmacological action has been extensively evaluated. The pharmacokinetics of parent apocynin were also comparatively characterized. A simple HPLC method was developed and validated to determine both AN-1 and apocynin in rat plasma. The chromatographic separation was achieved on an Agilent HC-C18 column (250 mm×4.6 mm, 5 µm) at an isocratic flow rate of 1.0 mL/min, with the mobile phase of methanol and water (53∶47, v/v) and the UV detection set at 279 nm. Good linearity was established over the concentration range of 0.1-500 µg/mL for AN-1 and 0.2-100 µg/mL for apocynin. The absolute recovery, precision and accuracy were satisfactory. Compared with the parent compound apocynin, AN-1 yielded a much longer T1/2 (AN-1 179.8 min, apocynin 6.1 min) and higher AUC0-t (AN-1 61.89 mmol/L·min, apocynin 2.49 mmol/L·min) after equimolar intravenous dosing (0.302 mmol/kg). The absolute bioavailability of oral AN-1 was 78%, but that of apocynin was only 2.8%. The significant improvement of pharmacokinetic behavior might be accounted for the effective pharmacodynamic results we documented for the novel nitrone derivative AN-1.

Figure 1. Chemical Structures of Apocynin and AN-1.

Figure 2. Typical Chromatograms for Selectivity of HPLC-UV Method.

Chromatograms of (A) blank plasma; (B) blank plasma spiked with apocynin (10 µg/mL); (C) blank plasma spiked with AN-1 (8 µg/mL); (D) blank plasma spiked with IS (80 µg/mL); (E) blank plasma spiked with apocynin (10 µg/mL), AN-1 (20 µg/mL) and IS (80 µg/mL); (F) plasma sample collected at 6 min after intravenous injection of apocynin (50 mg/kg) spiked with IS (8 µg/mL); (G) plasma sample collected at 4 min after intragastric administration of apocynin (50 mg/kg) spiked with IS (8 µg/mL): (H) plasma sample collected at 1 h after intravenous injection of AN-1 (80 mg/kg) spiked with IS (80 µg/mL); (I) plasma sample collected at 30 min after intragastric administration of AN-1 (40 mg/kg) spiked with IS (80 µg/mL). Peaks of 1, apocynin; 2, AN-1; 3, IS (carbamazepine); 4, proposed metabolite.

Figure 3. Mean Plasma Concentration-Time Profiles of AN-1 and Apocynin in Sprague-Dawley Rats.

Profiles of (A) AN-1 after a single intravenous dose at 20 mg/kg (n = 8), 40 mg/kg (n = 6) and 80 mg/kg (n = 8); (B) AN-1 after a single intragastric dose at 40 mg/kg (n = 6); (C) apocynin after a single intravenous dose at 50 mg/kg (n = 8); (D) apocynin after a single intragastric dose at 50 mg/kg (n = 6). Each point represented as mean ± SD.

Figure 4. Individual Plasma Concentration-time Profiles of AN-1 in Sprague-Dawley Rats.

Profiles of (A) AN-1 after a single intravenous dose at 80 mg/kg (n = 8); (B) AN-1 after a single intravenous dose at 40 mg/kg (n = 6); (C) AN-1 after a single intravenous dose at 20 mg/kg (n = 8); (D) AN-1 after a single intragastric dose at 40 mg/kg (n = 6).

Figure 5. Representative Chromatogram of Plasma Sample Collected at 1 h after Intravenous Injection of AN-1 (80 mg/kg) Containing the Unknown Metabolite Spiked with IS (80 µg/mL) and Apocynin (10 µg/mL).

Peaks of 1, apocynin; 2, AN-1; 3, IS (carbamazepine); 4, proposed metabolite.