Category: Preclinical Development
Purpose: Antitumor B (ATB), also known as Zeng Sheng Ping, is a Chinese herbal mixture composed of six plants: Sophora tonkinensis, Polygonum bistorta, Prunella vulgaris, Sonchus brachyotus, Dictamnus dasycarpus, and Dioscorea bulbifera. Recent in vitro, in vivo and clinical studies showed that ATB is active against oral cancer. Maackiain (Maac), matrine (Mat), dictamnine (Dic), and fraxinellone (Frax) were identified as key active components of Antitumor B. Therefore, the aim of this study is to characterize the pharmacokinetics profile of those components in C57BL/6 mouse blood and saliva.
Methods: ATB drug solution for intraperitoneal injection was prepared by sonicating ATB powder in saline. The drug solution was centrifugated at 15000 rpm in 10 minutes. The supernatant was aseptically filtrated through 0.2 µM membrane filter. ATB drug solution was administrated via intraperitoneal injection at a dose of 500 mg/kg once every 24 hours in 7 days. Blood (about 20 µL) and saliva (about 1.5 mg) samples were collected at each time point. Blood samples was collected using mouse tail sectioning method. Salimetric® foam swab method was use to collect saliva samples. The samples collected at at 5, 15, 30, 60, 120, 240, 360, 480, and 1440 min in the first day and the last day (day 7th) of experiment. In between days, samples were collected before and 30 mins (Tmax) after drug administration. The blood and saliva samples were stored at -80 °C until analysis.
Sample processing method: 10 µL blood and saliva (about 1.5 mg each) were extracted by 400 uL of ethyl acetate with 100 nM of Bauhoside I (internal standard). The extracted solution was evaporated under nitrogen flow and reconstituted in 50% MeOH. Chromatographic separation was achieved in 6.5 minutes using gradient elution with 2 mM amonium acetate in water/acetonitrile. The detections in multiple reaction-monitoring (MRM) mode for Maac, Dic, Frax and Mat were m/z at 283.12→ 254.1, 200.4 → 129.0, 233.4 → 129.0 and 249.0 à 148.0, respectively.
The method was developed and validated on an AB Sciex QTrap 5500 LCMS machine with the column maintained at 45o C. The column was Acquity UPLC BEH C18 50 x 2.1 mm I.D, 1.7 µm.
Results: Table 1. Pharmacokinetic parameters of ATB components after I.P administration with 500 mg ATB/kg (n = 5)
14.05 ± 11.20
13.41 ± 7.67
0.21 ± 0.17
0.77 ± 0.26
3.81 ± 0.90
6.55 ± 2.11
0.18 ± 0.18
3.65 ± 0.13
13.57 ± 2.19
105.16 ± 53.73
0.38 ± 0.14
2.47 ± 0.07
809.16 ± 225.22
1629.94 ± 360.54
0.29 ± 0.20
5.12 ± 1.09
54.55 ± 25.92
415.91 ± 177.33
Rashim Singh– Post-doc, University of Houston, Houston, Texas
Ming You– Director, Medical College of Wisconsin Cancer Center Senior Associate Dean for Cancer Research, Medical College of Wisconsin, Milwaukee, Wisconsin
Ming Hu– Professor of Pharmaceutics, University of Houston, Houston, Texas