LA CANAPA E LO STIMOLO DELLA FAME: STORIA DI AGONISMI ED ANTAGONISMI

1. LA CANAPA E LO STIMOLO DELLA FAME: STORIA DI AGONISMI ED ANTAGONISMI Dr. Uberto Pagotto Unità Operativa di Endocrinologia Dip. Medicina Int. e Gastroenterologia Ospedale S.Orsola-Malpighi Bologna

2. The first written evidence of medical use of cannabis sativa comes from China some 5000 years ago The drug was introduced in Europe by Napoleon Bonaparte’s troops when returning from Egypt Nowadays marijuana and its derivatives have become the most widely consumed illegal drugs in western countries

3. 1990 cloning of cannabinoid receptor 1 (Cb1) (Matsuda et al.) Both receptors belong to the G protein coupled receptor superfamily 1993 cloning of a second cannabinoid receptor (Cb2) (Munro et al.) Ion channels Intracellular signal-regulated kinases Adenylate-cyclase 1992 purification of anandamide, the first endogenous cannabinoid (Devane et al.) The discovery of Cb receptors and their endogenous ligands suggested the existence of an Endogenous Cannabinoid System 1964 isolation of 9-THC (Gaoni and Mechoulam)

4. 9-THC 8-THC Classical cannabinoids Cannabinol Cannabidiol HU 210 Non classical cannabinoids CP-55,940 Aminoalkylindols WIN-55,212 Cb1 ligands Anandamide Endocannabinoids 2-Arachidonylglycerol SR 141716A Cb1 selective antagonists AM 630

5. 1990 CB1 1992 1964 Hormones modulation Feeding Anandamide D9-Tetrahydrocannabinol Learning & Memory Antitumoral activity Rewarding Neuroprotection Pain perception Blood pressure Locomotion Sleep Thermoregulation

6. Orexigenic peptides • Neuropeptide Y (NPY) Anorexigenic peptides • Agouti-related protein (AgRP) • Melanin-concentrating hormone (MCH) • Orexins (Hypocretins) • Ghrelin • Corticotrophin-releasing hormone (CRH) • -melanocyte-stimulating hormone (-MSH) • Cocaine-and amphetamine regulated transcripts (CART)

7. Role of Reward System in feeding behaviorin the mesocorticolimbic pathaways Substantia Nigra Hippocampus Amygdala GABA D2-R Reward DOPA DOPA enkephalin GABA enkephalin 5-HT DOPA Reward DOPA D2-R Ventral tegmental region Nucleus Accumbens Hypothalamus Blue: excitatory neurons Red: inhibitory neurons

8. IN 1985, THE USE OF 9-THC (DRONABINOL) WAS APPROVED BY FDA FOR TREATMENT OF CANCER-CHEMOTHERAPY INDUCED NAUSEA AND VOMITING REFRACTORY TO OTHER AGENTS Average weight loss or gain during placebo and treatment periods with 9-THC (2.5 mg or 5.0 mg per os, twice per day, for 2 weeks) Average gain during Marijuana week Average loss during Placebo week Number of subjects 8 Marijuana- placebo 0.69 2.10 9 Placebo- marijuana 0.39 1.11 Regelson et al.

9. Dronabinol Placebo Dronabinol Placebo 1.5 0.3 1.0 Mean Weight change (Kg) 0.2 0.1 0.5 Mean Weight change (Kg) 0.0 -0.1 0.0 -0.2 -0.3 -0.5 Day 0 -0.4 Day 14 Day 28 Day 42 -0.5 Day 0 Day 14 Day 28 Day 42 Mean change in weight from baseline, evaluable AIDS patients. Excludes patients with moderate, severe, or life-threatening drug- nonrelated adverse events Mean change in weight from baseline, evaluable AIDS patients Beal et al, 1995 IN 1992, DRONABINOL WAS THE FIRST DRUG APPROVED BY FDA FOR THE TREATMENT OF ANOREXIA AND WEIGHT LOSS IN AIDS PATIENTS...

10. * 40 30 % patients with at least 2Kg BW gain 20 10 Dronabinol 2.5mg os Placebo 1 6 12 month STUDIES SUPPORT LONG-TERM, SAFE USE OF DRONABINOL FOR ANOREXIA AND CACHEXIA IN AIDS PATIENTS (Beal et al., 1997)

11. DRONABINOL WAS TESTED ALSO IN ALZHEIMER PATIENTS 15 PATIENTS WERE TREATED FOR 6 WEEKS WITH PLACEBO AND 6 WEEKS WITH DRONABINOL THE RESULTS WERE….. ”BODY WEIGHT OF STUDY SUBJECTS INCREASED MORE DURING THE DRONABINOL TREATMENT THAN DURING THE PLACEBO PERIODS..” Volicer L et al. 1997

12. Hyperphagia in pre-fed rats following oral 9-THC (Williams et al, 1998) Reversal of 9-THC hyperphagia in pre-fed rats by CB1 selective antagonist SR141716 (Williams et al, 2002) 10 SR141716 9-THC +SR 8 8 ††† * * * 6 2-hours food intake (g) * 6 *** *** *** 4 2-hours food intake (g) 4 2 2 0 0.05 0.1 0.5 1 SR141716 mg/Kg s.c. 0.5 0.25 1 2 0.125 0.063 0 9-THC mg/Kg

13. The selective Cb1 antagonist, SR 141716A, reduces dose-dependently sucrose and ethanol intake in rodents SR 141716 Sucrose Sucrose NPY - Ethanol pellets solution induced solution sucrose (rats) (rats) drinking (mice) (rats) ± ± ± ± vehicle 3.1 0.4 28.3 5.0 8.1 1.7 1.9 0.1 † ± ± ± ± 0.3 mg/Kg 2.1 0.2 16.1 3.9 3.6 1.2 1.3 0.2 †† ± ± ± ± 1 mg/Kg 1.6 0.2** 7.4 2.8** 1.2 0.4 1.1 0.2** ± ± ± 3 n.d. mg/Kg 1.0 0.4** 8.6 2.9** 1.0 0.2** Arnone et al, 1997

14. If you want a knockout... The conditional mutagenesis Breeding CB1 Null-mutation Feeding behavior CRE/lox P system Gene targeting

15. CB1-/- mice show decreased body weight and reduced fat mass CB1+/+ * 30 * T * CB1-/- * T * * * 25 * B * 20 body weight (g) 15 * 10 CB1+/+ 5 2 4 6 8 10 12 14 16 Age (week) T T B CB1+/+ CB1-/- 75 15 * ** 70 10 % of body weight % of body weight CB1-/- 65 5 60 Fat mass Lean mass Cota D. et al., J Clin Invest 2003

16. * b a CART/PVN CRH/PVN c d * MCH/LHA Pre-pro-orexin/LHA CB1 transcripts are co-localized with mRNAs of hypothalamic neuropeptides Cota D. et al., J Clin Invest 2003

17. CB1-/- lean phenotype is principally related to the decreased caloric intake Adult 30 15 Young 25 20 10 Caloric intake (kcal/day) Caloric intake (kcal/day) 15 10 5 5 6 20 21 22 3 4 5 Age (week) Age (week) CB1+/+ 45 25 CB1-/- * * * * * * * * * * * * * * * * CB1+/+ pair-fed * * * * * * * * 40 * * * † † * 20 † † † * † * 35 † † * † * Body weight (g) † † * Body weight (g) † * 30 † 15 * * CB1+/+ CB1+/+ * 25 * CB1-/- CB1-/- 10 20 15 5 21 20 22 3 4 5 6 Age (week) Age (week) Cota D. et al., J Clin Invest 2003

18. H2O NC Hippo NC CB1+/+ CB1-/- 400 M * 300 1.0 LPL activity (% of control) 200 0.5 CB1 100  Actin 0 CT WIN WIN+SR SR White adipose tissue may represent a novel peripheral target for cannabinoid action Cota D. et al., J Clin Invest 2003

19. We confirm that the cannabinoid system is strongly involved in the body weight regulation I conclusions • CB1ko mice are lighter and leaner than their wt controls • Pairfeeding studies using young animals indicate a main role of central orexigenic drive in body weight regulation, however in adults also a food-intake-independent mechanism contributes to the lean phenotype • CB1ko mice are not different in energy expenditure, body temperature and locomotor activity from their wt controls

20. We confirm that the cannabinoid system is strongly involved in the body weight regulation II conclusions • CRH, CART, Pre-pro-orexin, MCH co-expression and CB1 • have been found in hypothalamus of wt fed ad libitum • Higher expression of CRH mRNA in PVN of CB1ko mice • fed ad libitum,lower expression of CART mRNA • in LHA and DMN of CB1ko mice fed ad libitum • CB1 is expressed in adipose tissue • and stimulates lipogenesis

21. Hypothalamic action of CB1 antagonists may modulate neuropeptide network controlling food intake CB1 antagonists acting in the limbic system may decrease the rewarding property of food Endogenous Cannabinoid System seems to be an ideal target for the treatment of eating disorders and obesity Blockade of gastroenteric CB1 might contribute to the therapy by modulating feeding-related peripheral signals Anti-obesity action of CB1 antagonists could involve metabolic processes and directly target adipose tissue

22. “…..FEEL LESS PAIN, CONTROL YOUR MOVEMENT, RELAX, EAT, FORGET, SLEEP AND PROTECT.” Di Marzo et al. Trends Neurosc, 1998 The cannabinoid system is involved in several physiological functions and might be related to a general stress-recovery system. Such a variety of effects was concisely summarized by a recent statement by Di Marzo: