Title:  Tobacco cembranoids block the expression of the behavioral sensitization to nicotine and inhibit neuronal acetylcholine receptors

United States Patent:  6,489,357

Issued:  December 3, 2002

Inventors:  Eterovic; Vesna A. (Rio Piedras, PR); Ferchmin; Pedro A. (Rio Piedras, PR); Hann; Richard M. (Guaynabo, PR); Pagan; One R. (Ithaca, NY); Rodriguez; Abimael D. (Rio Piedras, PR); Rosario; Osvaldo (Rio Piedras, PR)

Assignee:  University of Puerto Rico (San Juan, PR); Universidad Central del Caribe Bayamon (PR)

Appl. No.:  693431

Filed:  October 21, 2000

The subject invention relates the use of cembranoids to inhibit nicotine addiction at the behavioral level. The subject host is sensitized to nicotine. Cembranoids are then administered to reduce such sensitization.

DETAILED DESCRIPTION OF THE INVENTION

We have discovered that cembranoids inhibit nicotine addiction at the behavioral level. Thus, the administration of cembranoids may be used to block the behavioral sensitization to nicotine in living organisms. The administration of such compounds does not cause any immediate adverse health consequences to the subject host.

In one group of tests, rats were exposed and sensitized to nicotine. Specifically, the rats were injected with 0.3 mg/kg or 0.4 mg/kg of nicotine and tested on the Greek cross maze. The Greek cross maze is a maze shaped like a cross that contains a central gray compartment which connects to two black compartments on opposite sides and two white compartments on the remaining sides.

After the rats were sensitized, they were injected with one of the following: saline, nicotine, or a preinjection of at least one cembranoid 30 minutes before injecting them with nicotine, chosen as a result of which experimental group each rat belonged to. Immediately after one of the injections was administered to the rats, they were placed in the Greek cross maze for observation and analysis of locomotor activity. The rats were placed in the gray area and the times each rat fully entered or partially (both front paws and head) entered the black and white compartments were recorded for 10 minutes.

The rats that were injected with nicotine, for the first time, showed loss of control of voluntary muscle movement. One group of rats was injected with nicotine once a day for 6 days, and on the seventh day they were injected with a saline control solution. Another group had the rats injected with at least on cembranoid before being injected with the nicotine. Those injected with the saline solution did not express sensitization to nicotine. Those that were preinjected with at least one cembranoid showed a response similar to that observed after saline, thus no expression.

The specific test results will now be described in further detail.

EXAMPLE 1

First, cembranoids, when administered alone, do not effect the behavior of rats as measured on the Greek Cross maze. To show this we injected rats with DMSO and with cembranoids dissolved in DMSO. The activity of both groups of rats did not exhibit any significant differences. The specific results for a five-minute period are shown in the following table (Table I):

 

The above table lists the results for five trials, as identified by the column titled "Exp. #." The column tilted "Treatment" identifies the type and dose of a cembranoid that was administered to the rats. EUAC is Eupalmerin Acetate. The column titled "Total Entries" lists the mean and .+-.s.e.m. of entries for each group. The column titled "N" lists the number of rats used. EXAMPLE 2

After sensitizing rats to nicotine as discussed above, the locomotor activity in the Greek cross maze was again measured, this time for a ten minute period. Marine cembranoids were administered to decrease the expression of sensitization to nicotine. The following table (Table II) illustrates the results:

 

The above table lists the results for three different trials, as identified by the column titled "Exp. #." The locomotor activity was measured at various days after the initiation of the sensitization treatment as indicated in the column titled "Days." The column titled "Drug Treatment" identifies the drug(s) injected before measuring the locomotor activity. The doses used were: nicotine, 0.2 mg/Kg in exp. 1 and 0.3 mg/Kg in experiments 2 and 3; eunicin and eupalmerin acetate, 6 mg/Kg. The column titled "Total Entries" lists the mean and .+-.s.e.m. of entries measured on the Greek cross maze for each group. In experiments 1 and 2, the activity observed after injecting saline solution provides a negative control (where sensitization is not expressed) to which the values from the other treatments are compared; in experiment 3, the activity observed after injection of nicotine (day 13) provides a positive control (100% expression of sensitization) for comparison with the other treatments.

EXAMPLE 3

Again, after sensitizing rats to nicotine as discussed above, the locomotor activity in the Greek cross maze was measured. A tobacco cembranoid was administered to reduce the sensitization to nicotine. Mecamylamine (MCA) was also administered and its effect measured. The following table (Table III) illustrates the results:

 

For the above table, the locomotor activity was measured at various days after the initiation of the sensitization treatment as indicated in the column titled "Days." The column titled "Treatment" identifies the drug(s) injected before measuring the locomotor activity. The dose of 4R-cembratriene, also termed (1S,2E,4R,6R,7E)-CEMBRA-2,7,11-TRIENE-4,6-DIOL, was 6 mg/kg. The activity of day 19, after saline injection, provides a control measurement in which expression of sensitization is absent.

The following references are hereby expressly incorporated by reference in their entirety.

U.S. patent application Ser. No. 09/092,407, filed Jun. 4, 1998; now U.S. Pat. No. 6,204,289;

U.S. Provisional Patent Application No. 60/224,659, filed Aug. 11, 2000;

Biello M. Pagan O. R., Cortes S., Rosario O., Ferchmin P. A. and Eterovic V. A. (1999). Determination of cembranoids in cigarette smoke. Eight Puerto Rico Neuroscience Conference (San Juan, Puerto Rico);

Clementi F., Fornasari D., Gotti C., 2000 Neuronal nicotinic acetylcholine receptors: from structure to therapeutics. TiPS 21:35-37;

Hann R. M., Pagan O. R., Gregory L., Jacome T., Rodriguez A. D., Ferchmin P. A., Lu R., and Eterovic V. A. (1998) Characterization of cembranoid interaction with the nicotinic acetylcholine receptor. J.Pharmac.Exp. Ther. 287:253-260;

Pagan O. R. (1998) Effect of cembranoids and anesthetic agents on [³ H]-tenocyclidine binding to the nicotinic acetylcholine receptor from Torpedo californica. M.S. Thesis, University of Puerto Rico Medical Sciences Campus, San Juan, Puerto Rico;

Picciofto M., Zoli M., Rimondini R., Lena C., Marubio L. M., Merlo Pich E., Fuxe K. and Changeux, J.-P. (1998) Acetylcholine receptors containing the .beta.2 subunit are involved in the reinforcing properties of nicotine. Nature, 391:173-177; and

Ferchmin P. A., Lukas R. J., Hann R. M., Fryer J. D., Eaton J. B., Pagan O. R., Rodriguez A. D., Nicolau Y., Rosado M., Cortes S. and Eterovic V. A., (2000) Tobacco Cembranoids Block the Expression of the Behavioral Sensitization to Nicotine and Inhibit Neuronal Acetylcholine Receptors. Submitted for publication to Journal of Neuronal Science Research (copy attached as Appendix A.)

The above general and detailed descriptions are exemplerary only and are intended to provide further explanation of the invention as claimed. Of course those skilled in the art will appreciate many modifications that may be made without departing from the scope and spirit of the invention. The following claims are intended to encompass all such modifications.

Claim 1 of 16 Claims

We claim:

1. A method of reducing sensitization to nicotine in a living host by:

administering nicotine to the living host so that the living host becomes sensitized to nicotine; and

administering at least one cembranoid to the living host to reduce the sensitization to nicotine.
 

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