Pharmacokinetics and Metabolism of 4R-Cembranoid

Abstract

4R-cembranoid (4R) is a natural cyclic diterpenoid found in tobacco leaves that displays neuroprotective activity. 4R protects against NMDA, paraoxon (POX), and diisopropylfluorophosphate (DFP) damage in rat hippocampal slices and against DFP in rats in vivo. The purpose of this study was to examine the metabolism and pharmacokinetics of 4R as part of its preclinical development as a neuroprotective drug. 10 µM 4R was found to be very stable in plasma for up to 1 hr incubation. 4R metabolism in human microsomes was faster than in the rat. Ten metabolites of 4R were detected in the microsomal samples; 6 dihydroxy and 4 monohydroxy forms of 4R. Male rats received a single dose of 4R at 6 mg/kg i.v., i.m., or s.c. The i.v. group had the highest plasma concentration of 1017 ng/mL. The t1/2 was 36 min and reached the brain within 10 min. The brain peak concentration was 6516 ng/g. The peak plasma concentration in the i.m. group was 163 ng/mL compared to 138 ng/mL in the s.c. group. The t1/2 of 4R after i.m. and s.c. administration was approximately 1.5 hr. The brain peak concentration was 329 ng/g in the i.m. group and 323 ng/g for the s.c. group. The brain to plasma ratio in the i.v. group was 6.4, reached 10 min after dose, whereas in the i.m. and s.c. groups was 2.49 and 2.48, respectively, at 90 min after dose. Our data show that 4R crosses the BBB and concentrates in the brain where it exerts its neuroprotective effect.

Fig 1. Structural formula of 4R.

Fig 2. In vitro metabolic stability of 4R.

10 μM 4R was incubated with human and rat liver microsomes at selected time points (0, 5, 15, 30 and 60 min). As positive control, 10 μM midazolam was incubated with human and rat microsomes at 0, 15, 30 and 60 min. 4R levels were determined by LC-MS/MS. All samples were assayed in duplicate.

Fig 3. Relative percent of 4R metabolites in human (A) and rat (B) microsomal samples.

50 μM 4R microsomal human and rat samples were incubated for 5, 15, and 30 min. 4R metabolites levels were determined by LC-MS/MS. The symbols represent the relative percent peak area of each metabolite, which was calculated as the peak area of each metabolite per sample / total peak area of the 10 metabolites per sample * 100.

Fig 4. Plasma and brain 4R levels in male rats.

Rats received 6 mg/kg of 4R by (A) i.v., (B) i.m., or (C) s.c. routes. Blood was collected at 2, 5, 10, 20, 45, 60, 90 min and 8 hr after administration. Brains were collected at 10 and 60 min after i.v. administration and at 10, 90 min and 8 hr after i.m. and s.c. administration. Samples were analyzed by LC-MS/MS for determination of 4R concentration. Values represent the mean ± SD of 3 rats.