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Investigation of the interaction between antagonists and the Cannabinoid receptor type 1: from cell membrane to in vivo experiments

Thursday, 10 June, 2010 - 09:45
Campus: Brussels Humanities, Sciences & Engineering campus
Marie Wennerberg
phd defence

Samenvatting

De cannabinoide receptor 1 (CB1r) antagonisten zijn in de belangstelling gekomen omwille van hun afslankend vermogen. In deze studie werd de interactie van drie antagonisten (AZ12491187, rimonabant en taranabant) met deze receptor in intacte recombinante cellen en in membranen hiervan met hun in vivo effecten (gewichtsafname en terugkeer) in muizen vergeleken. Aandacht werd gewijd aan hun potentie alsook aan hun dissociatiekinetiek en hun vermogen om herbinding te omdergaan. Deze laatste aspekten genieten toenemende belangstelling in de farmaceutische sector. 

CP55940-gestimuleerde [35S]GTPγS bindingstudies (een maat voor G eiwit activatie) werden op membranen uitgevoerd. Rimonabant inhibeerde deze respons met de laagste potentie. Co-incubatie experimenten wezen op de competitiviteit van alle antagonisten. AZ12491187 was de meest onoverbrugbare onder hen in pre-incubatie experimenten (duidend op trage dissociatie) in de afwezigheid van BSA. Doch in aanwezigheid van BSA was taranabant even onovebrugbaar. Radioligand binding wees op de zeer snelle dissociatie van [3H]-taranabant.

Alle antagonisten ondergingen specifieke CB1r binding in intacte cellen en [3H]-rimonabant was hier ook het minst potent. [3H]-Rimonabant- en [3H]-AZ12491187-CB1r complexen dissocieerden trager in intacte cellen dan in membranen en, in vergelijking met [3H]-taranabant, ondergingen beiden duidelijk meer incorporatie in het membraan (bijdragend tot hun niet-specifieke binding). De drie antagonisten ondergingen uitgesproken herbinding. Dit werd gedeeltelijk voorkomen door toevoeging van BSA of een overmaat ongemerkt ligand maar de aanwezigheid van beiden in het wasmedium was noodzakelijk voor volledige preventie. De “zuivere” dissociatie van [3H]-rimonabant (zoals in deze laatste conditie gemeten) was hier opnieuw het snelst. Tenslotte, en vooral voor [3H]-rimonabant, moest rekening gehouden worden met het vermogen van net gedissocieerde radioligand moleculen om in het membraan te incorporeren en om tot plastic te binden.

Herbinding draagt mogelijk bij tot lange effectieve in vivo CB1r bezetting door deze antagonisten. In overeenstemming met de in vitro gegevens, was de CB1r-bezetting en het afslankend vermogen in muizen het minst uitgesproken voor rimonabant. AZ12491187 was het langst werkzaan, zoals aangetoond door de traagste gewichtstoename na stopzetting van de behandeling. AZ12491187 en taranabant vertoonden even lange in vitro CB1r bezetting onder in vivo nabootsende condities (d.i. in de afwezigheid van albumine). Het is dus mogelijk dat de herbinding en membraan incorportatie op synergetische wijze bijdragen tot de langdurige clinische werking van AZ12491187. Een andere slotbevinding is dat, omwille van de meer fysiologische condities en grotere flexibiliteit, experimenten op intacte celllen de voorkeur genieten t.o.v. deze op membraanbereidingen.

 

Summary

Cannabinoid receptor 1 (CB1r) antagonists have cought attention because of their weight-reducing properties. Here, we compared the interaction of three antagonists (AZ12491187, rimonabant and taranabant) with this receptor in intact recombinant cells and in membranes thereof with their in vivo effects (weight reduction and regain) in mice. Besides potency, attention was devoted to their dissociation kinetics and their ability to undergo rebinding. These latter issues have recently sparked great interest in the pharmaceutical sector. 

In membranes, CP55940 (an agonist)- stimulated [35S]GTPγS-binding assays (reflecting G protein activation) were performed. Rimonabant decreased this response with the lowest potency. All antagonists acted competitively as seen in co-incubation experiments and AZ12491187 was the most insurmountable (suggestive for slow dissociation) as seen in pre-incubation experiments in the absence of BSA. Yet, in the presence thereof it was comparable to taranabant. Radioligand binding revealed very fast dissociationof  [3H]-taranabant.

In intact cells, all antagonists showed specific binding at the CB1 receptor. [3H]-Rimonabant was here again less potent. [3H]-Rimonabant and [3H]-AZ12491187 dissociated more slowly than from membranes and, compared to [3H]-taranabant, both underwent appreciably more membrane partitioning (showing up as part of the non-specific binding). The three antagonists experienced considerable rebinding. This was in part prevented by BSA or an excess of unlabelled ligand in the wash-out medium but both were needed for complete prevention. The genuine dissociation as measured under these latter conditions was, here again, fastest for  [3H]-rimonabant. Finally, especially for [3H]-rimonabant, care had to be taken to membrane partitioning and plastic binding of molecules that freshly dissociated from the receptor.

Rebinding phenomena likely contribute to increase the in vivo “residence time” of these anagonists at the CB1r.  In agreement with the in vitro data, both receptor occupancy and weight reduction studies in mice showed that rimonabant was the least efficacious one. AZ12491187 displayed the longest clinical efficacy as evidenced by the slowest regain of weight after stopping the treatment. AZ12491187 and taranabant had similar in vitro residence time under in vivo like conditions (i.e. in the absence of albumin) it is possible that rebinding and membrane partitioning work hand in hand to increase the duration of the clinical action of AZ12491187. It is also suggested that, due to the more physiological conditions and experimental flexibility,  intact cells are to be preferred over those based on membrane preparations.