New treatment options for sleep disorders Chairs: Gabriella Gobbi and Anton Y. Bespalov

. Novel selective melatonin MT2 receptor agonist in the treatment of insomnia . Gabriella Gobbi, Canada

. Orexin receptor antagonists and insomnia treatment: state of the art . Anthony Gotter, US

. Development of piromelatine, a novel multimodal sleep medicine . Nava Zisapel, Israel

. The brain H3-receptor as a novel therapeutic target for vigilance and sleep-wake disorders Abstract . Jian Sheng Lin , France Prevalence insomnia in Canada and Europe

Canadian survey1 European survey2 on sleep and related factors on insomnia and sleep symptoms (N=2000) (N=25,579) 50 50

40.4% 40 40 34.5%

30 30

Respondents(%) Respondents(%) 20 20 13.4% 9.8% 10 10

0 0 Insomnia symptoms Insomnia disorder Insomnia symptoms Insomnia disorder

1. Morin et al. Can J Psychiatry. 2011;56:540-548; 2. Ohayon et al. Sleep Med. 2009;10:952-960. Prevalence in the American Insomnia Survey

. 10,094 members of a managed health care plan . Prevalence 23.6%* . Insomnia symptoms: about 45% . Risk of insomnia higher in: . Women vs men . Older age vs younger groups (18-64 vs >65 y) . Disabled or retired vs employed . Shift workers vs day workers . Obese vs normal BMI

Diagnosis based on any one of 3 systems: DSM-IV-TR, ICD-10, RDC/ICSD-2. *Insomnia assessed using Brief Insomnia Questionnaire (BIQ).

Roth et al. Biol Psychiatry. 2011;69:592-600. Insomnia and Societal Burden

•Increased risks of depression and alcohol Psychiatric dependence1,2 •Increased risks of hypertension, metabolic Health syndrome, and coronary heart disease2 •Decreased productivity and increased Occupational absenteeism1,2

Economic •Increased health care costs1,2

Public safety •Increased risks of accidents1

1. Léger, Bayon. Sleep Med Rev. 2010;14:379-389; 2. Buysse. JAMA. 2013;309:706-716. DRUG DISCOVERY FOR SLEEP DISORDERS

2014 2005 1986 Orexin antagonists 1960 MLT agonist Benzodiazepine- Ramelteon (US) derivates 1903 Benzodiazepines (zopiclone) Circadin (EU) (chlordiazepoxide) 1869 Barbiturates Chloral Hydrate

600 BC: Alcohol as therapeutics

Important need of novel targets and novel drug discovery Novel selective melatonin MT2 partial agonists for the treatment of insomnia

Gabriella Gobbi, MD, PhD Neurobiological Psychiatry Unit Department of Psychiatry, McGill University Montréal, Canada Sleep Stages: EEG Features1,2 NREM REM

Wakefulness Light sleep Light sleep Deep SWS REM stage 1 stage 2 stage 3 & 4

• Rapid eye movement • Transitional stage • Light sleep (REM) • Similar to drowsy waking • Low muscle tone, slow • Slow-wave sleep (SWS) • Muscle atonia • High muscle tone rolling eye movements • Active dreams EEG activity: EEG activity: •  Rate & variability EEG activity: EEG activity: • Frequency begins • Strong delta of autonomic nervous • Low voltage fast activity to slow • Sleep spindles (0.5–4 Hz) activity system function in the beta range (7–14 Hz) and (15–30 Hz) • Alpha (8–14 Hz) activity K-complexes • Large amplitude EEG activity: mostly at posterior sites increases • Frequency slows to the • Returns to profile similar theta range (4–8 Hz) to wakefulness (low voltage fast activity)

1. Iber et al. AASM manual for the scoring of sleep and associated events. http://www.aasmnet.org/scoringmanual/default.aspx; 2. Brown et al. Physiol Rev. 2012;92:1087-1187. Visualizing Sleep Quantity and Quality Hypnograms showing distribution of normal sleep pattern1 Schematic representation Normal subject

Over the course of normal sleep, SWS and REM sleep alternate cyclically2

1. Gander. Sleep in the 24-Hour Society. Lower Hutt, NZ: The Open Polytechnic of New Zealand, 2003; 2. Colten, Altevogt, eds. Sleep Disorders and Sleep Deprivation. 2006; www.nap.edu. Common Wisdom: Melatonin and sleep Melatonin & sleep: Clinical

 Melatonin (N-acetyl-5-methoxytryptamine, MLT), synthesized by the pineal gland, has been reported to have hypnotic effects in humans, although results are still controversial (Brzezinski et al., 2005; Buscemi et al., 2006).

 Controversial studies on the role of melatonin in sleep, even if there is a concordance on the soporific effect and sleep-inducing effects of melatonin. Melatonin & sleep: Animal

 Animal studies have demonstrated that MLT reduces time to sleep onset and increases both NREMS and REMS (Holmes and Sugden, 1982; Mirmiran and Pevet, 1986), both effects being blocked by the GABAA receptor antagonists flumazenil and picrotoxin (Wang et al., 2003a).

 Other studies showed little or no effect (Mailliet et al., 2001; Wang et al., 2003). Melatonin and its receptors

c-AMP MT1 receptor PIP-2 MLT1a

c-AMP PIP-2 MT2 receptor

MLT1b Guanylyl cyclase

Dubocovich et al., Pharm Reviews , 2010 MT1 and MT2 in sleep

 Which receptor is involved in sleep?

 Lack of sleep studies using selective MT1 or MT2 ligands

 Lack of sleep studies in MT1KO and MT2KO. Ligand-model provides 3D-QSAR and SAR analysis based on 100s of compounds

3D-QSAR Agonist model pharmacophore

Purple: rhodopsin Rivara S. et al., J. Med. Chem. 2005, 48, 4049 Green: MT2 Spadoni G.et al. J. Med. Chem. 1997, 40, 1990 Mor M. et al. J. Med. Chem. 1998, 41, 3831 Orange MT2 TM5 UCM765 and UCM924: novel MT2 selective partial agonists Pro-Lead Compound Lead Compound

UCM 765 UCM 924 N-{2-[(3-methoxyphenyl)phenylamino]ethyl}acetamide N-{2-[(3-bromophenyl)-(4-fluorophenyl)amino]ethyl}acetamide

N-(Substituted-anilino-ethyl)amides

Rivara et al., JMC, 2007 Rivara et al., CMC, 2009 UCM765 and UCM924: novel MT2 selective partial agonists

PRO-LEAD LEAD

UCM-765 UCM-924 8.38 6.75 MT1 (pKi) Partial agonist Antagonist 10.18 10.2 MT2 (pKi) Partial agonist Partial agonist 6.3 (23.7% F% (p.o) 0.9 oral formulation) 44 IV 39 IV T1/2 (min) 68 PO

Rivara et al., 2007, 2009 UCM765 SLEEP RECORDING Effects of UCM765 on sleep parameters NREM NREM Latency Total time +48% 30 450 * * * 25 400 350 20 -59% 300 250 15 * * 200 10 150 100 Light phase 5 50 Dark phase 0 0 Veh 20 40 60 Veh 20 40 60 UCM765 (mg/kg, s.c.) UCM765 (mg/kg, s.c.)

REM Wakefulness Total time Total time 100 600 90 500 80 70 400 60 * 50 300 * * * 40 200 30 Light phase Light phase 20 100 Dark phase Dark phase 10 0 0 Veh 20 40 60 Veh 20 40 60 UCM765 (mg/kg, s.c.) UCM765 (mg/kg, s.c.) Ochoa-Sanchez et al., J.Neuroscience, 2011 UCM765 (40 mg/kg) on NREM sleep 24 hours analysis

Ochoa-Sanchez et al., J.Neuroscience, 2011 LEAD COMPOUND UCM924 SLEEP RECORDING COMPARISON WITH MELATONIN AND NON- SELECTIVE MT1-MT2 UCM924, MLT and UCM793 (40 mg/kg) on NREM sleep 24 hours analysis

VEH UCM924 (40 mg/kg) MELATONIN (40 mg/kg) UCM793 (40 mg/kg)

Ochoa-Sanchez et al, Neuroscience Letters 201421 MLT, UCM793, UCM924, 24 hours analysis (total Time)

Latency NREM REM Wake to sleep fullness

MLT  δ  

Non-     selective MT1-MT2

MT2  δ   selective

Ochoa-Sanchez et al, Neuroscience Letters 2014 Immunocytochemical localization of MT2 receptors Reticular Thalamus

Ochoa-Sanchez et al., J.Neuroscience, 2011 Reticular thalamic neurons NREM/SWS State-dependent activity in thalamic and cortical neurones (from Steriade et al., 1993) Hypothesis

If MT2 receptors are located in the Rt nuclei, the UCM765 should be able to activate these neurons Electrophysiological in vivo recording in reticular thalamic neurons DIFFERENTIAL AMPLITUDE DISCRIMINATOR UCM765 increases firing and burst activity of reticular thalamic neurons MT2 ANTAGONIST 4P-PDOT

The effect of the partial agonist is blocked by the antagonist

H3CH2COCHN

MT2 receptors (pKi = 8.8)

MT1 receptors (pKi = 6.3)

(Dubocovic et al., 1997) Microinfusion into the reticular thalamic neurons of

the MT2 antagonist 4P-PDOT. 4P-PDOT prevents the UCM 765 from increasing firing and burst activity Conclusion 1

 The pharmacological activation of MT2 receptors promotes sleep, in particular NREM or SWS and decrease the latency to sleep  Melatonin decreases the latency to sleep but not sleep duration

 The MT2 receptors are localized in the reticular thalamus, and they promote NREM through the rhythmic and burst activation of reticular thalamic neurons, which is the hallmark of NREM/SWS.  The MT2 receptor represents a novel target for hypnotics Sleep recording in MT1, MT2 and MT1-MT2 knockout mice

C3H Sleep recording in MT1, MT2 and MT1-MT2knockout mice

24 h sleep-wake analysis

Comai et al., Behavior Brain Res, 2013 NREM NREM NREM

Time (min) 4 second period analysis 1 1 2 2 3 3 4 4 5 0 5 0 5 0 5 0 5 0 0 0 0 0 0 0 0 0 0 W T decreased M T L 1 - i / - g M h T t 2 - / - M T 1 - / T - M o T t 2 - a / - l t i m e W T in MT2KO ,but M T D 1 - / - a r M k T 2 - / - N M T 1 - R / - M T E 2 Comai - / - M s l e e et al., Behavior Brain Res, 2013 Res, Brain Behavior al., et p

Time (min) 10 second period analysis 1 1 2 2 3 3 4 4 5 0 5 0 5 0 5 0 5 0 0 0 0 0 0 0 0 0 0 W T M increased L T 1 i - / g - h M T t 2 - / - M T 1 - T / - M o T t 2 a - / - l t i m W e T M D T 1 in MT1KO - a / - r M k T 2 - / - M T 1 - / - M T 2 - / - NREM sleep in MT1, MT2 and MT1- MT2knockout mice: NREM decreases in MT2 KO (light)

Comai et al., Behavior Brain Res, 2013 Opposite effects of MT1 and MT2 receptors in NREM

MT2

MT1

NREM REM REM decreases only in MT1 KO Blunted light-dark difference in MT1 KO

4 second period analysis 10 second period analysis

REM sleep

120 Total time 120 Total time

100 no light/dark difference 100

80 80

) ) n

n no light/dark difference

i i

m m (

60 ( 60

e e

m m

i i T 40 T 40

20 20

0 0 -/- -/- -/- -/- -/- -/- -/- -/- -/- -/- -/- -/- -/- -/- -/- -/- WT MT1 MT2 MT1 MT2 WT MT1 MT2 MT1 MT2 WT MT1 MT2 MT1 MT2 WT MT1 MT2 MT1 MT2 Light Dark Light Dark

Comai et al., Behavior Brain Res, 2013 REM sleep in MT1, MT2 and MT1- MT2 knockout mice: REM decreases only in MT1 KO

Comai et al., Behavior Brain Res, 2013 Differential role of receptors

NREM REM WAKEFULNESS

MT1 knockout *  

MT2 knockout   

MT1-MT2    knockout (24)

* 10 sec analysis Comai et al., 2013 Conclusion 2

 The MT1 and MT2 receptor have a differential role in sleep regulation, this role could explain the relative low effect of non-selective MT1-MT2 agonist, including melatonin, and the lack of significant sleep impairment in double MT1- MT2 knockout

 Genetic and pharmacological manipulation of MT2 receptor has confirmed the selective implication of this receptor in NREM Pharmacological activation of

melatonin MT2 receptors

NREM Acknowledgments

Gabriella Gobbi’s lab Pharmacology and antibodies, Immunohisto EEG/EMG & Electrophysiology, • Franco Fraschini (Univ. of Milan, Italy) Behaviour, Pain • Deborah Angeloni (Scuola Superiore Sant’Anna, Pisa, Italy) • Laurent Descarries (Univ. of Montreal, Canada) • Dr Rafael Ochoa Sanchez • Dr Stefano Comai Modeling and Synthesis UCM 765 • Dr Sergio Dominguez-Lopez • Giorgio Tarzia (Univ. of Urbino, Italy) • Gilberto Spadoni (Univ. of Urbino, Italy) • Dr Francis Rodriguez Bambico • Annalida Bedini (Univ. of Urbino, Italy) • Dr Quentin Rainer • Marco Mor (Univ. of Parma, Italy) • Silvia Rivara (Univ. of Parma, Italy) • Marta Lopez-Canul Knockout-mice • David Weaver (Univ. of Massachusset, US) • Steven Reppert (Univ. of Massachusset, US)

Pain models • Sabatino Maione (Second Univ. of Naples, Italy) • Enza Palazzo (Second University of Naples, Italy) • Vinicio Granados Soto (CINVESTAV, Mexico City, Mexico) Immunohistochemistry • Dr Baptiste Lacoste Grants support: CIHR, FRSQ, CFI, MSBiV, MDEIE