Agents for preventing and treating disorders connected to impaired neurotransmission

#This application is the US national phase of international application PCT/CH2005/000621 filed 21 Oct. 2005 which designated the U.S. and claims benefit of EP 04025165.4, dated 22 Oct. 2004, the entire content of which is hereby incorporated by reference.

#The present invention refers to coumarin ethers for use as medicaments, especially for the treatment of disorders connected to impaired neurotransmission, as well as to dietary and pharmaceutical compositions and plant materials and extracts containing such coumarin ethers and their uses.

#It is well known that impaired neurotransmission, e.g. low neurotransmitter levels, is connected to mental diseases such as depression.

#In example 1 a racemic mixture of obliquin obtained from MicroSource Discovery Systems Inc, 21 George Washington Plaza, Gaylordsville, Conn. 06755 USA was used. It had a purity of ≧96%. In examples 2 and 3 a racemic mixture of obliquin synthesized according to J. Chem. Soc. (C) 1969, 526-531 was used. Xanthyletin were obtained from Microsource as well. 7-geranyloxycoumarin, 6′-dehydromarmin, geiparvarin, (S)—O-acetylcolumbianetin and suberosin were obtained from APIN Chemicals, 43d Milton Park, Abingdon, Oxon, OX14 4RU, United Kingdom. And the compound of the formula VII was obtained from InterBioScreen Ltd., Institutsky Prospect, 7a 142432 Chernogolovka, Russia.

#HEK-293 cells stably expressing the human serotonin re-uptake transporter (hSERT) were obtained from R. Blakely, Vanderbilt University, USA. The cells were routinely grown in Dulbeco's Modified Eagles Medium, purchased from Bioconcept, Allschwil, Switzerland containing 10% fetal calf serum, penicillin, streptomycin, L-glutamine and the antibiotic G418 and passaged by trypsinisation. 1 day prior to the assay cells were seeded in the above mentioned medium. Immediately prior to the assay the medium was replaced by Krebs Ringers bicarbonate buffer, purchased from Sigma Chemicals Ltd., supplemented with 35 μM pargyline, 2.2 mM CaCl2, 1 mM ascorbic acid and 5 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (buffer called “Hepes”). Serotonin uptake into the cells was determined by addition of radio-labeled (3H) serotonin (Amersham Biosciences GE Healthcare, Slough, UK) to a concentration of 20 nM, and incubation for 30 minutes at room temperature. Following removal of unincorporated label by gentle washing three times with the above buffer, incorporated serotonin was quantified by liquid scintillation counting.

#Membranes containing hSERT were prepared from the above cell line according to Galli et al, Journal of Experimental Biology 1995, 198, 2197-2212. 3 μg of membrane protein and 0.8 mg of wheat germ aglutinin coated SPA® (scintillation proximity assay) beads (Amersham Biosciences GE Healthcare, Slough, UK) per data point were mixed with radio-labeled (3H) citalopram (Amersham Biosciences GE Healthcare, Slough, UK) until a final concentration of 3.3 nM in 50 mM Tris-HCl, 300 mM NaCl (pH 7.4). After incubation for 18 hours at room temperature, the bound citalopram was quantified by liquid scintillation counting.