Actions of Octocoral and Tobacco Cembranoids on Nicotinic Receptors

Nicotinic acetylcholine receptors (AChRs) are pentameric proteins that form agonist-gated cation channels through the plasma membrane. AChR agonists and antagonists are potential candidates for the treatment of neurodegenerative diseases. Cembranoids are naturally occurring diterpenoids that contain a 14-carbon ring. These diterpenoids interact with AChRs in complex ways: as irreversible inhibitors at the agonist sites, as noncompetitive inhibitors, or as positive modulators, but no cembranoid was ever shown to have agonistic activity on AChRs. The cembranoid eupalmerin acetate displays positive modulation of agonist-induced currents in the muscle-type AChR and in the related gamma-aminobutyric acid (GABA) type A receptor. Moreover, cembranoids display important biological effects, many of them mediated by nicotinic receptors. Cembranoids from tobacco are neuroprotective through a nicotinic anti-apoptotic mechanism preventing excitotoxic neuronal death which in part could result from anti-inflammatory properties of cembranoids. Moreover, tobacco cembranoids also have anti-inflammatory properties which could enhance their neuroprotective properties. Cembranoids from tobacco affect nicotine-related behavior: they increase the transient initial ataxia caused by first nicotine injection into naive rats and inhibit the expression of locomotor sensitization to repeated injections of nicotine. In addition, cembranoids are known to act as antitumor compounds. In conclusion, cembranoids provide a promising source of lead drugs for many clinical areas, including neuroprotection, smoking-cessation, and anti-cancer therapies

A cembranoid from tobacco prevents the expression of nicotineinduced withdrawal behavior in planarian worms

Using an adaptation of published behavioral protocols, we determined that acute exposure to the cholinergic compounds nicotine and carbamylcholine decreased planarian motility in a concentration-dependent manner. A tobacco cembranoid (1S,2E,4R,6R,7E,11E)-cembra-2,7,11- triene-4,6-diol (4R-cembranoid), also decreased planarian motility. Experiments in the presence of 1 μM 4R-cembranoid did increase the IC50 for nicotine- but not carbamylcholine-induced decrease in planarian motility. When planarians were exposed for 24 h to either nicotine or carbamylcholine at concentrations near their respective IC50 values and then transferred to plain media, nicotineexposed, but not carbamylcholine- or cembranoid-exposed worms displayed withdrawal-like distress behaviors. In experiments where planarians were pre-exposed to 100 μM nicotine for 24 h in the presence of 1 μM 4R-cembranoid, the withdrawal-like effects were significantly reduced. These results indicate that the 4R-cembranoid might have valuable applications for tobacco abuse research. This experimental approach using planarians is useful for the initial screening of compounds relevant to drug abuse and dependence

Kinin-B2 Receptor Mediated Neuroprotection after NMDA Excitotoxicity Is Reversed in the Presence of Kinin-B1 Receptor Agonists

Background: Kinins, with bradykinin and des-Arg 9-bradykinin being the most important ones, are pro-inflammatory peptides released after tissue injury including stroke. Although the actions of bradykinin are in general well characterized; it remains controversial whether the effects of bradykinin are beneficial or not. Kinin-B2 receptor activation participates in various physiological processes including hypotension, neurotransmission and neuronal differentiation. The bradykinin metabolite des-Arg 9-bradykinin as well as Lys-des-Arg 9-bradykinin activates the kinin-B1 receptor known to be expressed under inflammatory conditions. We have investigated the effects of kinin-B1 and B2 receptor activation on N-methyl-Daspartate (NMDA)-induced excitotoxicity measured as decreased capacity to produce synaptically evoked population spikes in the CA1 area of rat hippocampal slices. Principal Findings: Bradykinin at 10 nM and 1 mM concentrations triggered a neuroprotective cascade via kinin-B2 receptor activation which conferred protection against NMDA-induced excitotoxicity. Recovery of population spikes induced by 10 nM bradykinin was completely abolished when the peptide was co-applied with the selective kinin-B2 receptor antagonist HOE-140. Kinin-B2 receptor activation promoted survival of hippocampal neurons via phosphatidylinositol 3-kinase, while MEK/MAPK signaling was not involved in protection against NMDA-evoked excitotoxic effects. However, 100 nM Lys-des-Arg 9-bradykinin, a potent kinin-B1 receptor agonist, reversed bradykinin-induced population spik

In Vivo Evaluation of the Acute Systemic Toxicity of (1S,2E,4R,6R,7E,11E)-Cembratriene-4,6-diol (4R) in Sprague Dawley Rats

The tobacco cembranoid (1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4,6-diol (4R) interacts with nicotinic acetylcholine receptors, which results in neuroprotection against organophosphate toxicity, brain ischemia, and Parkinson’s disease. The present study is a continuation of our previous research in which we applied a single dose of 4R 1 h before or 24 h after exposure to diisopropylfluorophosphate (DFP) (analog of the nerve agent sarin). The 4R dose robustly decreased neuroinflammation and neuronal death at both timepoints. Here, we investigated the toxicity of a single dose of 4R in male and female Sprague Dawley (SD) rats after a subcutaneous (s.c.) injection of 6, 24, or 98 mg/kg. Body weight was not affected by 4R during the 7-day observation period. No histopathologic changes in the organs were attributed to 4R. Minor hematological and blood composition variations were detected on Day 3 in the mid- and the high-dose males, but these were resolved by Day 8. At the area of the s.c. injection site, alopecia and dry skin were detected in both the 4R-treated males and females and in the female controls

Pharmacokinetics and Metabolism of 4R-Cembranoid.

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

Metabolites of 4R detected in 10 and 50 μM samples incubated with human and rat liver microsomes. Samples from human or rat liver microsomes incubated with 10 or 50 μM 4R generated the same metabolite species.

<p>a. RT = retention time.</p><p>b. 4R metabolite masses were detected by LC-MS/MS in multiple reaction monitoring (MRM) mode using a range of precursor masses with a common product ion mass of 95 m/z, and a collision energy of 38 eV.</p><p>Metabolites of 4R detected in 10 and 50 μM samples incubated with human and rat liver microsomes. Samples from human or rat liver microsomes incubated with 10 or 50 μM 4R generated the same metabolite species.</p