“Anti-” properties of the positive allosteric modulators

of the GABAB

Giancarlo Colombo CNR Neuroscience Institute Cagliari, Italy [email protected] “Anti-alcohol” properties of

GABA, Baclofen Baclofen-induced suppression of alcohol self- administration in sP rats

160 Alcohol

140

120 100 80 60 Cryan & Kaupmann, 2005 40

Total responses * Lever responses Lever 20 Non-sedative doses of baclofen suppress: 0 0 1.7 3

• Regular alcohol drinking behavior in rats 0,8 • Relapse-like drinking in rats 0,7 • Binge-like drinking in rats and mice 0,6

0,5 administered • Operant alcohol self-administration in rats and - 0,4 mice (reinforcing and motivational properties) 0,3 • Reinstatement of alcohol-seeking behavior in rats (g/kg) alcohol 0,2 *

0,1 Amount self of Amount • Development of conditioned place preference to (g/kg)intakealcohol Total 0,0 alcohol in mice 0 1.7 3 • Alcohol-stimulated motor activity in rats and mice Baclofen Extension to the positive allosteric modulators (PAMs) of the

GABAB receptor of the “anti-alcohol” properties of baclofen

GABA, Baclofen

PAMs

Cryan & Kaupmann, 2005

GABAB PAMs: a new generation of GABAB receptor ligands

• The discovery of a positive allosteric modulatory binding site in the structure of the

GABAB receptor, together with the synthesis of in vivo effective ligands, offer a novel mechanism for pharmacological manipulation of the GABAB receptor

• GABAB PAMs bind to a distinct site from the orthosteric agonist binding site, and are devoid of intrinsic agonistic activity in the absence of GABA

• Thus, PAMs activate the GABAB receptor in vivo in a more “physiological” way than agonists, as they do not perturb receptor signaling on their own, but potentiate the effect of GABA only where and when it is endogenously released Presently available, in vivo effective positive allosteric modulators of the GABAB receptor

CGP7930 2,6-Di-tert-butyl-4-(3-hydroxy-2,2-dimethyl-propyl)-

GS39783 N,N'-dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine

BHF177 N-[(1R,2R,4S)-bicyclo[2.2.1]hept-2-yl]-2-methyl-5-[4-(trifluoromethyl)phenyl]-4- pyrimidinamine

rac-BHFF (R,S)-5,7-di-tert-butyl-3-hydroxy-3-trifluoromethyl-3H-benzofuran-2-one

ADX71441 Chemical structure currently unravelled Operant, oral alcohol self-administration under the Fixed Ratio (FR) schedule of reinforcement

alcohol dispenser

FIXED RATIO 4 (FR4) SCHEDULE OF REINFORCEMENT: -Fixed number (4) of lever-responses to access alcohol -0.1 ml alcohol/reinforcement -30 min/day session -[Alcohol]: 15% (v/v) -Measure of alcohol REINFORCING properties Reducing effect of acutely administered GS39783 on alcohol reinforcing properties in Sardinian alcohol-preferring (sP) rats

200 M&M: AlcoholAlcohol 200 SucroseSucrose

- Adult, male sP rats

150 150 - Self-administration * sessions: 30 min/day *

- [Alcohol]: 15% (v/v) 100 ** 100

responses responses -

- [Sucrose]: 0.3% (w/v) - Total Total responses

- FR4 schedule 50 responses Total 50

Lever Lever

0 0

0 25 50 100 0 25 50 100

1,0 5,0

0,8

4,0

0,6 * administered

administered 3,0 - * - **

0,4 2,0

alcohol(g/kg)

Total Total amount of

Total amount of amount Total sucrose (ml/kg) sucrose

0,2 1,0

administered administered alcohol (g/kg)

-

administered sucrose (ml/kg) sucrose administered

-

self

Amount of selfAmount Amount Amount of self 0,0 self 0,0 0 25 50 100 0 25 50 100 GS39783GS39783 (mg/kg; (mg/kg) i.g.) GS39783GS39783 (mg/kg; (mg/kg) i.g.) Reducing effect of repeatedly administered GS39783 on alcohol reinforcing properties in sP rats

M&M: - Adult, male sP rats - Self-administration sessions: 30 min/day & FR4 schedule - 10 consecutive sessions - [Alcohol]: 15% (v/v); [Sucrose]: 1% (w/v) Alcohol Sucrose

         

          Reducing effect of acutely administered rac-BHFF on alcohol reinforcing properties in Sardinian alcohol-preferring (sP) rats

M&M: Alcohol Sucrose 250 250

- Adult, male sP rats

- Self-administration 200 200

 sessions: 30 min/day 150 150 

- [Alcohol]: 15% (v/v)

responses

responses -  -

- [Sucrose]: 0.7% (w/v) 100 100 Total responses Total

- FR4 schedule responses Total Lever 50  Lever 50

0 0 0 50 100 200

0 50 100 200

1,2 12

1,0 10 

0,8 8

administered

administered - - 0,6   6

0,4 4

alcohol(g/kg) sucrose (ml/kg) sucrose 0,2 

Total alcohol intake (g/kg) intake alcohol Total 2 Total sucrose intake (ml/kg) intake sucrose Total

0,0 0 Amount Amount of self Amount Amount of self 0 50 100 200 0 50 100 200 rac-BHFF (mg/kg; i.g.) rac-BHFF (mg/kg; i.g.) Reducing effect of repeatedly administered rac-BHFF on alcohol reinforcing properties in sP rats

M&M: - Adult, male sP rats - Self-administration sessions: 30 min/day & FR4 schedule - 5 consecutive sessions - [Alcohol]: 15% (v/v); [Sucrose]: 1% (w/v)

Alcohol Sucrose

+     +  

+    +   Operant, oral alcohol self-administration under the Progressive Ratio (PR) schedule of reinforcement

alcohol dispenser

PR SCHEDULE OF REINFORCEMENT: -RR: 4, 9, 12, 15, 20, 25, 32, 40, 50, 62, 77, 95, etc. -0.1 ml alcohol/reinforcement -30 min/day session -[Alcohol]: 15% (v/v) -Measure of alcohol MOTIVATIONAL properties Reducing effect of acutely administered GS39783 on alcohol motivational properties in Sardinian alcohol-preferring (sP) rats

M&M: 250 AlcoholAlcohol 250 SucroseSucrose

- Adult, male sP rats

200 200

- Breakpoint sessions: 60 min - [Alcohol]: 15% (v/v) 150   150 - [Sucrose]: 3% (w/v)  100 100

- PR schedule Total responses Total

Total responses Total 50 50

Lever responses Lever Lever responses Lever

0 0 0 25 50 100 0 25 50 100 70 70

60 60

50

50

40    40

30 30

Break point Break

Break point Break Breakpoint Breakpoint 20 20

10 10

0 0 0 2525 50 100 0 GS39783 25 50 (mg/kg) 100 GS39783 (mg/kg) GS39783 (mg/kg; i.g.) GS39783 (mg/kg; i.g.) Comparative study on the reducing effect of acute GS39783 on alcohol reinforcing and motivational properties in alcohol-preferring P, sP, and AA rats

Rat line Acronym Foundation stock/ Location Selection procedure

 ALKO Alcohol AA Wistar/Outbreeding Helsinki, Finland

 Alcohol-preferring P Wistar/Outbreeding Indianapolis, IN, USA

 Sardinian alcohol-preferring sP Wistar/Outbreeding Cagliari, Italy

FIXED RATIO 4 (FR4) SCHEDULE OF REINFORCEMENT: - 4 lever-responses to access alcohol - 0.1 ml alcohol/reinforcement - 30 min/day session - [Alcohol]: 15% (v/v) - Measure of alcohol REINFORCING properties alcohol dispenser PROGRESSIVE RATIO SCHEDULE OF REINFORCEMENT: - RR: 4, 9, 12, 15, 20, 25, 32, 40, 50, 62, 77, 95, etc. - 0.1 ml alcohol/reinforcement - 30 min/day session - [Alcohol]: 15% (v/v) - Measure of alcohol MOTIVATIONAL properties Differential effect of acute GS39783 on alcohol reinforcing properties in alcohol-preferring P, sP, and AA rats FR4 procedure P rats sP rats AA rats

250 250 250

200 200 * 200 * § * * * * + 150 * * 150 * * * 150 + * * * * * * 100 * 100 * 100 * *

* * *

on on the alcohol lever

on the alcohol lever alcohol the on

on the alcohol lever alcohol the on Numberresponses of

50 50 responses of Number 50 Number of responses of Number

0 0 0 0 25 50 100 0 25 50 100 0 25 50 100

GS39783 (mg/kg) GS39783 (mg/kg) GS39783 (mg/kg)

1,6 1,6 1,6 1,4 1,4 1,4

1,2 1,2 1,2

1,0 * 1,0 1,0 + * * * 0,8 * 0,8 * * * 0,8

* * * * * * Amount of Amount 0,6 * of Amount * * * * * 0,6 * of Amount 0,6 * *

0,4 * 0,4 0,4 administered alcohol (g/kg) alcohol administered

* (g/kg) alcohol administered

-

- administered alcohol (g/kg) alcohol administered

0,2 0,2 - 0,2

self self 0,0 0,0 self 0,0 0 25 50 100 0 25 50 100 0 25 50 100 GS39783 (mg/kg) GS39783 (mg/kg) GS39783 (mg/kg) Order of potency and efficacy of GS39783: P > sP > AA rats Differential effect of acute baclofen on alcohol motivational properties in alcohol-preferring P, sP, and AA rats PR procedure

P rats sP rats AA rats

80 80 80

60 60 60

+ § + 40 * 40 * 40 + * * * * * * * *

20 20 20

Breakpoint alcohol for Breakpoint

Breakpoint alcohol for Breakpoint Breakpoint alcohol for Breakpoint

0 0 0 0 25 50 100 0 25 50 100 0 25 50 100 GS39783 (mg/kg) GS39783 (mg/kg) GS39783 (mg/kg)

Order of potency and efficacy of GS39783: P > sP >> AA rats Potentiating effect of the acute combination of per se ineffective doses of GS39783 and baclofen on alcohol self-administration in sP rats Alcohol Sucrose M&M: - Adult, male sP rats - Self-administration sessions: 30 min/day - [Alcohol]: 15% (v/v)  - [Sucrose]: 1% (w/v) - FR4 schedule

 Potentiating effect of the acute combination of per se ineffective doses of rac-BHFF and baclofen on alcohol self-administration in sP rats Alcohol Sucrose M&M: - Adult, male sP rats - Self-administration sessions: 30 min/day  - [Alcohol]: 15% (v/v) - [Sucrose]: 1% (w/v) - FR4 schedule

 - - - - M&M: Reducing effect of acutely administered CGP7930 and BHF177 Liang et al., 2006

Adult, male Indiana alcohol FR3 schedule of reinforcement [Alcohol]:10% (v/v) Self Lever responses - administration sessions: 40 min/day

0 CGP7930 (mg/kg; i.p.) (mg/kg;CGP7930

in alcohol on alcohol reinforcing properties 10 -

preferring (P) rats

20  -

preferring P and sP rats

- - - - M&M:

Adult, male Sardinian alcohol FR4 schedule of reinforcement [Alcohol]:15% (v/v) Self

Total number of responses - administration sessions: 30 min/day

100 150 200 250

50 0 BHF177 (mg/kg; i.g.) BHF177 (mg/kg; 0

12.5

- preferring (sP) rats

25 

 50

Reducing effect of acutely administered GS39783 on alcohol binge-like drinking in Sardinian alcohol-preferring (sP) rats

M&M: - Adult, male sP rats 2,5 - Daily drinking sessions of 1 hour

during the dark phase with unpredictable time schedule 2,0 - 4-Bottle choice regimen (0%, 10%, 20%, and 30% alcohol) 1,5  

1,0 10% 20% 30% H2O  EtOH EtOH EtOH

0,5 Alcohol intakeAlcohol (g/kg)

0,0 0 25 50 100 GS39783 (mg/kg; i.g.) Reducing effect of acutely administered GS39783 and ADX71441 on alcohol binge-like drinking in alcohol-consuming C57BL/6J mice

M&M: - Adult, male C57BL/6J mice - Drinking-in-the-Dark procedure: daily sessions of 2 hours, starting 3 hours into the dark phase - [Alcohol]: 20%

GS39783 (30 mg/kg) Vehicle

Time (h) Linsenbardt & Boehm II, 2013 Hwa et al., 2014 Possible mechanism of GABAB PAM action / 1

Inhibition of alcohol-induced stimulation of the mesolimbic dopamine system (the brain “reward” circuit)

VTA NAc

DOPAMINE

Alcohol

GABAB PAM GLUTAMATE GABAB receptor Possible mechanism of GABAB PAM action / 2

VTA NAc

DOPAMINE

Alcohol

GABAB PAM GLUTAMATE GABAB receptor

The intra-VTA administration of GABAB PAMs suppressed: - Alcohol-stimulated dopamine release in the rat NAc - Oral self-administration of alcohol in rats - Extinction responding for alcohol in rats

(Leite-Morris et al., in preparation) Summary / 1

• Non-sedative doses of GABAB PAMs selectively reduced alcohol reinforcing and motivational properties in alcohol-preferring rats

• This reduction is maintained after repeated treatment, suggesting

the lack of tolerance to the reducing effect of GABAB PAMs on alcohol self-administration

• In the “comparative” study, the rank of order of GS39783 potency and efficacy paralleled that of the strength of the reinforcing and motivational properties of alcohol among P, sP, and AA rats

• The heterogeneity in sensitivity to GS39783 of alcohol self- administration in P, sP, and AA rats may resemble the differential effectiveness of pharmacotherapies among the different typologies of human alcoholics

• Pretreatment with GABAB PAMs greatly potentiated the reducing effect of a per se ineffective dose of baclofen on alcohol self-

administration  GABAB PAMs are indeed capable of facilitating in vivo the pharmacological activation of the GABAB receptor Summary / 2

• The selectivity of GABAB-PAM effect on alcohol self-administration was complete  GABAB PAMs apparently meet the ideal condition of decreasing alcohol reinforcing and motivational properties (or craving for alcohol, if theoretically transposed to humans) without exerting any influence on other appetitive or consummatory behaviors

• GABAB PAMs suppressed alcohol intake in recently established rodent models of binge-like drinking

• GABAB PAMs retain baclofen capacity to suppress different alcohol- related behaviors

• These data support the hypothesis of the involvement of the GABAB receptor in the neural substrate controlling alcohol-drinking and - seeking behaviors

• Because of the predictive validity of the experimental procedures

used in these studies, GABAB PAMs may represent a novel, potentially effective pharmacotherapy for alcohol use disorder Contributors

CNR Neuroscience Institute Novartis Pharma AG Cagliari, Italy Basel, Switzerland Mauro A.M. Carai Klemens Kaupmann Gian Luigi Gessa Sébastien Guery Barbara Loi Carla Lobina AC Immune SA Paola Maccioni Lausanne, Switzerland Alessandro Zaru Wolfgang Froestl Indiana University Indianapolis, IN, USA Hoffmann-La Roche Ltd Basel, Richard L. Bell Switzerland Lawrence Lumeng Pari Malherbe Andrew W. Thomas Natl Inst for Health & Welfare Helsinki, Finland Petri Hyytiä University of Siena Siena, Italy Inst Psychiatry & Neurology Federico Corelli Warsaw, Poland Claudia Mugnaini Przemyslaw Bienkowski Serena Pasquini