1. In vivo molecular imaging of the Brain Type 1 Cannabinoid Receptor in Eating Disorders 25/06/2009Nathalie Gérard - MIRC seminar1 Nathalie Gérard Division of Nuclear Medicine UZ Leuven – KU Leuven Promotor : Prof. Dr. Koen Van Laere work in progress seminar

2. 25/06/2009Nathalie Gérard - MIRC seminar2 Outline 1.Introduction Anorexia nervosa / Bulimia nervosa Activity-based anorexia rat model The endocannabinoid system CB1 receptor imaging 2.Hypothesis and aims 3.CB1 receptor characterization in an animal model for anorexia nervosa Cross-sectional design Longitudinal design Pharmacological intervention 4.CB1 receptor characterization in anorexia nervosa and bulimia nervosa patients 5.Additional and future work

3. 25/06/2009Nathalie Gérard - MIRC seminar3 Introduction Anorexia nervosaBulimia nervosa refusal to maintain normal body weight intense fear of gaining weight distorted body perception amenorrhea excessive physical activity restrictive / binge - purge 0.7% in young women 47% full recovery 5 to 10% mortality rate binge eating with loss of control inappropriate compensatory behaviour excessive emphasis on body shape / weight twice a week for min. 3 months purging / (non-purging) 2% in young women 50% full recovery over 10 years 0 to 3% mortality rate Fairburn, Lancet 2003 ; Keel, Am J Psychiatry 1997

4. 25/06/2009Nathalie Gérard - MIRC seminar4 Introduction Routtenberg, J Comp Physiol Psychol 1968 activity-based anorexia model : well-characterized and validated model for anorexia nervosa in the rat limited feeding period (1.5h / day) running wheel + induction of hyperactivity and spontaneous food restriction

5. 25/06/2009Nathalie Gérard - MIRC seminar5 Introduction the endocannabinoid system : important in brain neuromodulation Guzman Nature Reviews 2003

6. 25/06/2009Nathalie Gérard - MIRC seminar6 Introduction the role of the endocannabinoid system in eating behaviour Van den Eynde Child Adolesc Psychiatric Clin N Am 2008 homeostatic hedonic somaticmetabolicwantingliking dopamine neurotransmission opiates cannabinoid system

7. 25/06/2009Nathalie Gérard - MIRC seminar7 Introduction the role of the endocannabinoid system in eating behaviour Cannabinoid agonists stimulate food intake e.g. - cannabis-induced hunger (Abel Behav Biol 1975) - agonist therapy in AIDS-related anorexia (Beal et al. J Pain Symptom Manage 1997) ; however one trial with ∆⁹-THC in AN patients failed (Gross et al. J Clin Psychopharmacol 1983) - orexigenic effect in animals (Arias Horcajadas Mol Neurobiol 2007) Cannabinoid inverse agonists / antagonists as anti-obesity medication (Rimonabant, Taranabant) (Pi-Sunyer et al. JAMA 2006, Addy et al. Cell Metabolism 2008) Different CB1R alleles associated with Anorexia Nervosa-restrictive and Anorexia Nervosa-bingeing purging subtypes (Siegfried et al. Am J Med Genet 2004) Endocannabinoid anandamide increased in plasma of AN patients, not in BN (Monteleone et al. Neuropsychopharmacology 2005)

8. 25/06/2009Nathalie Gérard - MIRC seminar8 Introduction Imaging of the type 1 cannabinoid receptor [18F]MK-9470 High-affinity, highly selective CB 1 receptor tracer Preclinical validation / clinical application Burns et al. PNAS 2007 Receptor K i (nM) hCB 1 0.7 hCB 2 44

9. 25/06/2009Nathalie Gérard - MIRC seminar9 Hypothesis and aims PET characterization of in vivo cerebral CB1R availability in activity-based anorexia rats / female patients with anorexia or bulimia nervosa compared to controls Is the endocannabinoid system hypoactive in anorectic conditions ? Endocannabinoid augmenting therapy in ABA

10. 25/06/2009Nathalie Gérard - MIRC seminar10 ANIMAL RESEARCH CB1R microPET in the activity-based anorexia model

11. 25/06/2009Nathalie Gérard - MIRC seminar11 Cross-sectional design CB1R microPET ABA, n = 14 CONTROL, n = 10 DIET, n = 10 WHEEL, n = 10 n = 44 time (days) arrival randomisationIN ABA model

12. 25/06/2009Nathalie Gérard - MIRC seminar12 Longitudinal design randomisationIN ABA modelOUT ABA model c recovery CB1R microPET time (days) arrival body weight

13. 25/06/2009Nathalie Gérard - MIRC seminar13 Methods microPET : 500 µCi 18 F-MK9470 20’ 1 hour post injection image processing : spatial normalization on 18 F-MK9470 binding template oriented in standardized Paxinos space mSUV = Activity conc * ((body weight + 250 g) / 2) Injected dose parametric images based on mSUV quantification

14. 25/06/2009Nathalie Gérard - MIRC seminar14 Results relative mean body weight mean food intake CSLG ABA DIET WHEEL CON

15. 25/06/2009Nathalie Gérard - MIRC seminar15 Results wheel rotations weight during recovery CSLG ABA DIET WHEEL CON

16. Nathalie Gérard - MIRC seminar mSUV *** p < 0,001 mean CB1R binding (mSUV) + 51% + 53% 25/06/200916 Results : cross-sectional CB1R ABADIETWHEELCON

17. Nathalie Gérard - MIRC seminar25/06/200917 Results : longitudinal CB1R BASELINE CHRONICRECOVERY ABA CON mSUV 0.7 2.3 median CB1R binding (mSUV) ** p < 0,01

18. Nathalie Gérard - MIRC seminar25/06/200918 Results : regional CB1R binding in ABA Cross-sectionalLongitudinal %P-value% Hippocampus+ 4,6P < 0,0003+ 2,1P < 0,004 ABA > control Most pronounced regional increase in CB1R binding was consistently found in the hippocampus stress ECS + _ HPA axis Hypercortisolism, Affects especially hippocampal neurons due to large amounts of glucocorticoid receptors ↓ Hillebrand et al. Peptides 2005

19. Nathalie Gérard - MIRC seminar25/06/200919 Conclusions Global significant increase in CB1R binding in ABA ; confirmed in a follow-up experiment CB1R upregulation suggests a reversible compensation mechanism for a hypoactive ECS CB1R upregulation in ABA normalizes with recovery Could endocannabinoid augmenting therapy be beneficial in ABA?

20. Nathalie Gérard - MIRC seminar25/06/200920 EC augmenting therapy in ABA 4x 7 ABA rats, 1 week i.p. injection twice a day with : Exogenous CB1R agonist : WIN 55,212-2 ; low dose 0.25 mg/kg or higher dose 1 mg/kg Fatty acid amide hydrolase inhibitor : URB-597 ; 0.3 mg/kg Vehicle : 1% Tween-80 in aqua

21. 25/06/200921 Preliminary results ∆ food intake (g) WIN L URBVEH WIN H ∆ body weight (g) WIN L URBVEH WIN H ∆ running activity (km) WIN L URBVEH WIN H mean mean ± SE min-max Nathalie Gérard - MIRC seminar

22. 25/06/200922 Conclusions No significant differences between groups in changes of body weight, food intake and running activity after therapy compared to before therapy CAVE suboptimal low dosage vs. more rapid tolerance with higher dosage? Effect of endogenous 2-AG increase with monoamine glycerol lipase inhibitors?

23. 25/06/2009Nathalie Gérard - MIRC seminar23 HUMAN RESEARCH CB1R PET in anorexia and bulimia nervosa

24. 25/06/200924Nathalie Gérard - MIRC seminar Study aim To characterize the in vivo cerebral CB1R availability in female patients with anorexia and bulimia nervosa compared to healthy women Is the in vivo cerebral CB1R availability correlated with eating disorder-related symptoms in AN or BN?

25. 25 Subjects ParameterANBNControls N14 F (7 AN-BP/7 AN-R)16 F (16 BN-P)19 F Age (yrs) 20.5 ± 3.6 23.8 ± 7.125.2 ± 8.5 Weight (kg) 42.5 ± 6.2 * # 61.2 ± 10.3 *66.4 ± 13.1 # BMI (kg/m²) 15.5 ± 1.3 * # 21.8 ± 2.5 * 23.1 ± 4.3 # Disease dur. (yrs) 4.2 ± 4.5 6.3 ± 6.3 - Handedness (L/AMB/R) 2/0/122/0/140/2/17 EDES 36.7 ± 9.5 # 37.4 ± 9.5 § 69.5 ± 6.2 # § EDI87.3 ± 19.4 # 83.4 ± 30.1 § 13.7 ± 9.6 # § p < 0.05 : * AN vs. BN, # AN vs. controls, § BN vs. controls EDES : eating disorder evaluation scale (Vandereycken 1993) EDI : eating disorder inventory (Garner 2007) Nathalie Gérard - MIRC seminar25/06/2009

26. 26 Methods (1) [ 18 F]MK-9470 ~ 306 MBq 90 min. 30 min. … Modified Standard Uptake Values PET scan 25/06/2009Nathalie Gérard - MIRC seminar

27. 25/06/200927Nathalie Gérard - MIRC seminar Methods (2) mSUV = activity concentration x (body mass +70)/2 injected dose quantification of [ 18 F]MK-9470 PET Sanabria et al. 2009 (submitted) based on the macroparameter mSUV as index of CB1R availability mSUV is correlated with irreversible uptake rate constant K i which is a measure for V T, in healthy subjects and AN/BN patients, and is independent of blood flow no arterial sampling -> better clinical feasibility

28. 28 Methods (3) PET – MRI coregistration Normalization on CB1R binding template - Voxel-by-voxel : Statistical Parametric Mapping (SPM2) - Volumes-of-interest (Pmod) Statistical analyses 25/06/2009Nathalie Gérard - MIRC seminar

29. 29 Results (1) Global cerebral increase of CB1R binding in AN patients : compared to BN patients on average + 14.7 % ; p < 0.05 compared to controls on average + 24.5 % ; p < 0.001 Nathalie Gérard - MIRC seminar25/06/2009

30. 30 Results (2) Regional increases of CB1R binding in AN and BN patients compared to controls (mSUV normalized on global cerebral uptake) p height < 0.001 ; p cluster < 0.05 : AN > CON Insular cortexInferior frontal cortexInferior temporal cortex Nathalie Gérard - MIRC seminar25/06/2009

31. 31 Results (3) Regional increases of CB1R binding in AN and BN patients compared to controls (mSUV normalized on global cerebral uptake) p height < 0.001 ; p cluster < 0.05 : BN > CON Insular cortex No significant differences between AN and BN patients No significant differences between AN subtypes Nathalie Gérard - MIRC seminar25/06/2009

32. 32Nathalie Gérard - MIRC seminar Results (4) Positive correlation in the superior temporal brain area with the EDI subscale ‘drive for thinness’ Regional CB1R binding correlates with disease parameters in AN, not in BN patients p height < 0.001 ; p cluster < 0.05 :

33. 25/06/200933Nathalie Gérard - MIRC seminar Discussion (1) Global cerebral increase of in vivo CB1R availability in AN, not in BN - Reducing the endocannabinoid tone in anorectic conditions could reduce appetite and motivation to eat (Fride et al. Exp Biol Med 2005) - CB1R upregulation as a compensatory response ? BN : overeating (Klein et al. Physiol Behav 2004) hormonal effects : e.g. FAAH inhibition by estrogen absent in AN patients (due to amenorrhea), anandamide ↓ (Waleh et al. Gene 2002)

34. 25/06/200934Nathalie Gérard - MIRC seminar Discussion (2) Common feature in AN and in BN : increased insular CB1R availability - Insula serves an integrative function for brain processes relevant to eating disorders : hunger and food intake, reward and emotion processing, interoceptive awareness -Insular dysfunction in eating disorders : µ-opioid receptor decrease in BN (Bencherif et al J Nucl Med 2005) altered activity with symptom provocation or taste stimuli (fMRI) (e.g. Oberndorfer et al Biol Psychiatry 2008)

35. 25/06/200935 Nathalie Gérard - MIRC seminar Conclusion ECS seems to be a relevant player in AN, to a lesser extent in BN Importance of insular dysfunction in AN / BN Trait- or state- related changes? Follow-up study in subgroup fulfilling criteria indicating recovery

36. 25/06/2009Nathalie Gérard - MIRC seminar36 ADDITIONAL AND FUTURE WORK

37. Nathalie Gérard - MIRC seminar25/06/200937 Results : metabolic imaging in AN and BN AN < HV BN < HV parietal cortex cingulate brain area central brain area Common metabolic signatures in anorectics and bulimics :

38. Nathalie Gérard - MIRC seminar25/06/200938 AN > HV BN > HV occipital cortex orbitofrontal cortex mesiotemporal cortex Results : metabolic imaging in AN and BN

39. Nathalie Gérard - MIRC seminar25/06/200939 Human studies : ongoing CB1R PET in eating disorders : repeat study in recovering anorectics and bulimics CB1R PET in obesity : what about the opposite site of the eating disorder spectrum?

40. 40 Acknowledgements All study participants University Hospitals Leuven : Laboratory for Experimental and Functional Neurosurgery Merck & Co, Inc. for the tracer precursor Research Council K.U. Leuven (OT/05/58) Nuclear Medicine team Centre for Eating Disorders PET Radiopharmacy team Nathalie Gérard - MIRC seminar25/06/2009