La potentialisation des opioides endogènes (enképhalines): une nouvelle voie dans le traitement des douleurs aigües ou chroniques et son extension à d’autres domaines.

Bernard P. Roques Professeur Emerite

Pharmaleads SAS, 11 rue Watt, 75013 Paris, France

U 266 INSERM, Université Paris Descartes, 4 avenue de l’Observatoire, 75006 Paris, France

ATHS (Colloque International Addictions Toxicomanies Hépatites SIDA) Biarritz 17-20 Octobre 2017 1 2 Neuropathic Pain : an unmet medical need Current treatments

 Do not satisfactorily treat neuropathic pain

 Are mainly secondary indication (e.g. , anti-convulsants)

 Have strong precautions of use with regard to side effects and interactions

Other drugs under development

 Most of them have old mechanisms of action

 Very few innovative approaches, still in early development stage

 Pharmaleads’ PL37 now in Phase 2, PL265 in Phase 1

3 Challenges in development

As many as 50 millions people in USA suffer from chronic pain for which there is no really efficient drugs.

Regarding severe acute pain from various deseases or post-operative events, remains the most efficient drugs but its long-term use leads to severe side effects (sedation, nausea, constipation, tolerance …) which limits its use.

Unfortunately despite considerable efforts no really efficient novel analgesic have been designed since several decades.

New Approaches : • Potentialisation of the endogenous system of pain control with main interest for peripheral targeting (Pharmaleads). • Association of drugs leading to very efficient synergy resulting in reduction of the adverse effects of each main analgesic (morphine, THC …). • Use of homo-, hetero-dimerization of RCPGs receptors expand the repertoire of GPCRs ( and/or ) signaling, opening the way to novel classes of with compounds targeting selectivity the dimeric form.

4 Three levels of pain control by endogenous opioid system (EOS)

EOS

EOS

Attacking pain at EOS its source

 Relieving or reducing pain at its source  More than 50% of MO effects are attributable to peripheral neurons (nociceptors)

Roques, B.P., Fournié-Zaluski, M.C. and Wurm, M., Nature Reviews Drug Discovery, 2012

5 Endorphins and their receptors.

Endomorphin-1 Tyr – Pro – Trp – Phe – NH2

Endomorphin-2 Tyr – Pro – Phe – Phe – NH2

6 6 Signalisation opioïde endogène

Le système limbique et les structures cérébrales impliquées dans la mémoire, la Central sensory-discriminative network peur, l’anxiété, etc, prennent en compte l’état (quality, intensity, location of the noxious émotionnel et le souvenir des sensations stimulus, emotional context) antérieures

EOS

PAG

EOS Integrates information from limbic system

EOS

Descending systems C’est le système nerveux périphérique qui génère le signal initial qui sera (serotonine, nor-adrenaline) perçu par le cerveau comme douloureux. Il représente donc une cible particulièrement intéressante pour le développement de nouveaux analgésiques. 7 Signalisation intracellulaire induite par stimulation des récepteurs opioides RCPG (i)

(From Hyman S.E., 2006 Annu. Rev. Neurosci.) 8 DENKIs: mechanism of action

The endogenous opioid system (EOS) is present at all levels of physiological-nociceptive control i.e. periphery, spinal cord and brain

Elements of the EOS are opioid receptors, and their inactivating enzymes

Dual Inhibitors of (DENKIs) potentiate physiological functions of DENKI enkephalins (e.g. pain control) only on those pathways where they are tonically released

Enkephalinases APN NEP No adverse effects Enkephalins Y G G F M(L) (endogenous)

Opioid receptors  

Morphine indiscriminatingly overstimulates all opioid receptors including those not involved in (Morphine) pain control (exogenous)

Adverse effects

EWCBR – Mars 2017 9 Distribution of MOR, DOR and NEP and APN in rat brain. Effect rhizotomy (right) on MOR, DOR and NEP. NEP Mu Delta APN [3H]HACBOGly* [3H]DAMGO* [3H]DTLET* [125I]RB 129**

* Waksman et al., Proc. Natl. Acad. Sci. (1986) Waksman et al., EMBO J. (1986) ** Noble et al., Neuroscience (2001) Waksman et al., C. R. Acad. Sci. (1984). 10 DENKIs: mechanism of action

The endogenous opioid system (EOS) is present at all levels of physiological-nociceptive control i.e. periphery, spinal cord and brain

Elements of the EOS are opioid receptors, enkephalins and their inactivating enzymes

Dual Inhibitors of ENKephalinases (DENKIs) potentiate physiological functions of DENKI enkephalins (e.g. pain control) only on those pathways where they are tonically released

Enkephalinases APN NEP No adverse effects Enkephalins Y G G F M(L) (endogenous)

Opioid receptors  

Morphine indiscriminatingly overstimulates all opioid receptors including those not involved in (Morphine) pain control (exogenous)

Adverse effects

11 Les démonstrations initiales de l’efficacité de l’approche DENKI chez l’homme

 En 1988, l’injection intrathécale du mélange de selective NEP inhibiteur (2 nM) et de bestatine, faible et non spécifique, inhibiteur d’APN

(IC50 - 4000 nM).  Effets prolongés d’inhibition totale de la douleur (>18 heures) chez les 5 patients traités (J. Meynadier et al., J. Pain Clinic, 1988) devenus complètement résistants à la morphine et/ou ne la supportant plus.

 Un effet analgésique encore plus important a été observé avec le même protocole chez le même type de patient (J. Meynadier, non publié) avec Kélatorphan, premier DENKI, NEP (4 nM), APN (360 nM).

 Pour des raisons pharmacocinétiques, le Kélatorphan ne peut être transformé en médicament mais le résultat a ouvert la voie aux DENKIs.

 Comme attendu le thiorphan i.v. ne possède pas d’action analgésique chez l’homme (J.C. Willer et al. Neuropharmacol. 1986).

ProjectCONFIDENTIAL Epione – Confidential Scientific Presentation – April 2012 12 Structure inhibitory potencies of three families of dual enkephalinases (NEP/APN) inhibitors DENKIs.

• Rapid cleavage of the cascade ester followed by in vivo reduction of the disulfide bond.

• Both active moieties inhibiting APN and NEP act independently

LogP 4.13 LogP 2.79 LogP 1.73

• Rapid cleavage of the ester. No reduction required

• The same molecule inhibits both enzymes

13 The two leading families of DENKIs

Cascade PL 265 -9 PL 37 Cascade ester (*) -9 8x10 M carbamate (*) 3x10 M PL800 PL804 -9 Ki = 8 nM -9 4x10 M Ki = 22x10 nM M Ki = 3 nM Ki = 4 nM PL PL800 PL804 PL 254 254

APN NEP APN NEP

Tyr-Gly-Gly-Phe-Met(Leu) Tyr-Gly-Gly-Phe-Met(Leu)

Enkephalins Enkephalins

(*) Increases bio -availability (*) Increases bio-availability

+ PL254 : R1 = CH3 ; R2 = CH3 ; NH3

+ PL265 : R1 = CH3 ; R2 = CH3 ; NH3 = -NH-COO-CH(CH3)-OCOIPr

PL37 : R1 = H ; R2 = -CH(CH3)OCOOEt

14 Potentiation of the action of Met- (ME) on locus coeruleus neurons by (20 µM). Lack of proper effect of kelatorphan.

Locus coeruleus : brain structure involved in central control of respiration and cardiac rythm and at the source of physical symptoms of addiction. 250 * Wild type 200   ) KO preproenK 150

100 * * Exogenous ME (or MO) applied by * Jump latency (s latency Jump 50 * pressure pulse () on slice containing the LC, leads to hyperpolarization. The 0 RB101 response is amplified by kelatorphan (J.T. 0 20 0 20 (20 mg/kg i.v.) Williams et al., J. Pharmacol. Exp. Ther., 243(1), 397-401, 1987). * P < 0.05 ; ** P < 0.01 as compared to wild-type control mice Kelatorphan alone is inactive suggesting a No change in morphine-induced very low basal release of ENKs in this antinociceptive effetc in PENK-KO mice. structure. DENKIs’ antinociceptive effects are specifically related to enkephalin protection DENKIs have no detrimental effect on respiration and cardiac frequency Noble F., Psychopharmacol. (2008) (E. Boudinot et al., Pain 90, 7-13 (2001).

15 Potentiation of the action of Met-enkephalin (ME) on locus coeruleus neurons by kelatorphan (20 µM). Lack of proper effect of kelatorphan

Locus coeruleus : brain structure involved in central control of respiration and cardiac rhythm and at the source of physical symptoms of addiction

  

Exogenous ME (or MO) applied by pressure pulse () on slice containing the LC, leads to hyperpolarization. The response is amplified by kelatorphan (J.T. Williams et al., J. Pharmacol. Exp. Ther., 243(1), 397-401, 1987) Kelatorphan alone is inactive suggesting a very low basal release of ENKs in this structure

DENKIs have no detrimental effect on respiration and cardiac frequency (E. Boudinot et al., Pain 90, 7-13 (2001)

16 Effect of DENKIs on the extracellular efflux of Met-enkephalin in the nucleus accumbens, periaqueductal grey (PAG) and cerebrospinal fluid (CSF) Microdialysis or superfusion in awake and freely moving rats

Subarachnoidal

Bourgoin S. et al., JPET, 1986. 17 Pharmacological effects of pre- (PENK) gene KO in mice i.c.v. ENKs : transient MO-like antinociceptive effect at high doses due to rapid metabolism

i.c.v. in mice ED50 MO : 0.63 nmol/mouse; ED50 Met-E : 330 nmol/mouse (Interussi, PNAS 1980) ED50 Met-E+kelatorphan (50 µg) : 1,2 nmol/mouse (Fournié-Zaluski, EJP 1984) 100 250 * 80 Hot plate test

200

  ) Wild type 60 150 KO preproenK 40 100 * Baseline jump latency (s) Baseline jump latency * * 20 (s latency Jump 50 *

0 0 +/+ -/- +/+ 0 20 0 20 RB101 (20 mg/kg i.v.) * P < 0.05 ; ** P < 0.01 as compared to wild-type control mice *** P < 0.001 No change in morphine-induced Cutt-off 240 s antinociceptive effetc in PENK-KO mice. A physiological enkephalin tone DENKIs’ antinociceptive effects are is active in pain control specifically related to enkephalin protection König et al. Nature (1996) Noble F., Psychopharmacol. (2008)

18 Pharmacological effects of pre-proenkephalin (PENK) gene KO in mice i.c.v. ENKs : transient MO-like antinociceptive effect at high doses due to rapid metabolism

i.c.v. in mice ED50 MO : 0.63 nmol/mouse; ED50 Met-E : 330 nmol/mouse (Interussi, PNAS 1980) ED50 Met-E+kelatorphan (50 µg) : 1,2 nmol/mouse (Fournié-Zaluski, EJP 1984) 100 250 * 80 Hot plate test

200

  ) Wild type 60 150 KO preproenK 40 100 * Baseline jump latency (s) Baseline jump latency * * 20 (s latency Jump 50 *

0 0 +/+ -/- +/+ 0 20 0 20 RB101 (20 mg/kg i.v.) Naloxone * P < 0.05 ; ** P < 0.01 as compared to wild-type control mice *** P < 0.001 No change in morphine-induced Cutt-off 240 s antinociceptive effetc in PENK-KO mice. A physiological enkephalin tone DENKIs’ antinociceptive effects are is active in pain control specifically related to enkephalin protection König et al. Nature (1996) Noble F., Psychopharmacol. (2008)

19 Puissance analgésique similaire de la morphine et des enképhalines par voie i.v. dans les modèles des douleurs aiguës.

i.c.v. in mice ED50 MO : 0.63 nmol/mouse; ED50 Met-E : 330 nmol/mouse (Interussi, PNAS 1980) ED50 Met-E+kelatorphan (50 µg) : 1.2 nmol/mouse (Fournié-Zaluski, EJP 1984)

Comparison between i.v. morphine and i.v. PL37 in the Hot Plate Test (HPT)

vehicle (EtOH/cremophor/H2O) improving BBB crossing 100 *** PL37 ED par Toutes les 50 remplacement du réponses sont *** chrémophor ou du tween inhibées par pré- 80 par une cyclodextrine administration de *** utilisable chez l’homme naloxone 75 i.v. perf douleur aigüe ?

MO, ED 1.6 mg/kg

% analgesia % 50 PL37, ED 1.8 mg/kg 50 ** 50 *** % analgesia = 100 x (test latency – control ** p<0.01 ** vs. vehicle latency) / (cut-off time *** p<0.001 – control latency) Dunnett’s t test 25 1 mg/kg 2 mg/kg 3 mg/kg Morphine PL37

Analgesic effects of morphine or PL37 injected i.v. 10 min before the test in the hot plate test in mice (jump response). Points represent the percentage of analgesia for n = 8 mice Control group corresponds to i.v. administration of vehicle.

20 20 No tolerance after repeated administration of PL37 and no cross- tolerance between morphine and PL37 in nociceptive pain.

21 Trafficking of bound opioid receptors Differences in the traffic and reinitialisation of active opioid receptors bound to ENKs or MO

bound receptors dimerisation

MO ENK

very slow active internalisation active form form

very rapid internalisation

• MO-induced tolerance contrasting to its lack for DENKIs-protected ENKs. • Decrease in MO-tolerance induced by DENKIs. • Increase of analgeic response produced by combination of MO and ENKs

22 22 Very efficient potentiation of morphine antinociceptive effect induced by dual inhibitor RB101 in the hot-plate test and tail-flick test (not shown here)

5 10 20 0,5 0,5/5 0,5/10 0,5/20 mg/kg

RB 101 Morphine Morphine/RB 101

same effect with PL37 23 Peripheral control of inflammatory and neuropathic pain by DENKI- protected enkephalins diffusing in an extended synaptic area

 Pharmaleads has developed orally active Peripheral opioid control of NP and IP DENKIs which do not enter the CNS,  and are hence devoid of behavioural effects

CRF

CRFR SP

DOR MOR Noxious stimulation of specific targets on NEP APN peripheral nociceptor terminal DENKI Ca2+,K+

YGG

TRPV Peripheral mediators released in tissue SV 1 damage (inflammatory pain) or in nerve DENKI AEA injury (neuropathic pain). NGF, BK, 5HT, ATP, H+, TNF, IL6, histamine, ET 1 ORs strongly enhanced on APN noxious inputs each sides of nerve injury to spinal cord (Truong et al. 2000) From A.T. Basbaum et al. Cell (2009). Roques, B.P., Fournié-Zaluski, M.C. and Wurm, M., NRDD, 2012

24 Action des opioïdes exogènes (ex MO) et endogènes (ENKs) dans la douleur neuropathique

• In NP, oral act centrally and facilitate pain by activating  In models of neuropathic (CCI in rodents) and inflammatory pain, descending pathways release and concentration of enkephalins increase constantly during a week or two before plateauing for as long as the injury • In NP, oral DENKIs have no access to the CNS and therefore ENKs lasts (Zollner et al. 2008) cannot activate descending pathways • Moreover, tonic and phasic release of ENKs is weak in these NOCICEPTORS structures AREA

opioid receptors « On » cell (facilitatory) « Off » cell (inhibitory) CRF

CRFR SP

DOR MOR NEP

APN RVM 2+ + DENKI Ca ,K RM LC YGG duloxetin gabapentinoids

TRPV SV 1 5HT NA DENKI AEA

ORs strongly enhanced on Aδ APN noxious inputs eachsides of nerve injury to spinal cord (Truong et al. 2000)

25 BP. Roques et al., NRDD, 2012 ; A. Schreiter et al. FASEB J., 2012) 25 Structure and inhibitory potencies of orally active DENKIs PL37 and PL265

• Rapid cleavage of the cascade ester followed by in vivo PL800 PL804 ( ) reduction of the disulfide bond.

• Both active moieties inhibiting APN and NEP act independently

, LogP 4.13 , LogP 2.79 , LogP 1.73

• Rapid cleavage of the ester. No reduction required

• The same molecule inhibits both enzymes

26 Radioactivity distribution of radio-labeled PL37 200 mg/kg p.o. in rat Correlation with brain NEP/APN enzymatic activities

Time dependant distribution of radio-labeled PL37 on APN or NEP inhibiting moieties

Expressed in % of PL37 oral administration

NEP and APN activities in rat brain membrane before and after 200 mg/kg oral PL37

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t 10000

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a 3000

a 7500

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p

p

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P 1000 N 2500

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0 0 Saline PL37 Saline PL37

27 PL37 reduction of neuropathic pain (allodynia and thermal hyperalgesia) induced by partial ligation of sciatic nerve in mice

10 mg/kg/po 20 mg/kg/po 40 mg/kg/po

IPSILATERAL, sham-operated 1.3

IPSILATERAL, sciatic nerve injury 0.6

***p<0.001 versus baseline values; ###p<0.001, #p<0.05 versus vehicle (post injection). 2-way ANOVA followed by Bonferroni’s multiple comparison test. Seltzer’s model 7 # # # vehicle   6 PL37, 10 mg/kg/po Plantar test 5 4 3 von Frey Test

2 pawwithdrawal(s) 1 0 Before admin. 20-40 mn of PL37 after gavage

**p<0.001; *p<0.05 versus baseline values; ##p<0.01 ; #p<0.05 versus vehicle (post injection). 2 way ANOVA followed by Bonferroni’s multiple comparison test.

The effect of PL37 is reversed by prior administration of naloxone methiodide (not shown here) 28 Synergistic combinations of the dual PL265 given orally with various analgesic compounds acting on different targets, in a murine model of bone cancer-induced pain.

CONFIDENTIAL 29 Effect induced by the oral administration of PL 265 (12.5-37.5 mg/kg 30 min before testing) in mice intratibially inoculated with B16-F10 cells

Antihyperalgesic effects of PL265 Antialgesic effects

30 Effect of opioid antagonists on the antihyperalgesic responses induced by PL265 in mice intratibially inoculated with B16-F10 cells

 Antihyperalgesic effects induced by PL265-protected ENKs depends on peripheral MORs stimulation  No effects on DOR and KOR 31 Potentiation of the antihyperalgesic effect induced by by the simultaneous per os of PL265 in mice intratibially inoculated with B16-F10 cells

 Synergistic effects involve MORs stimulation with NO released by Gabapentin 32 Potentiation of the antihyperalgesic effect induced by A-317491 (P2X3 antagonist) by the simultaneous per os of PL265 in mice

 Synergistic effects involve peripheral ENKs released by P2X3 antagonist, as demonstrated with an anti-ENK antibody (Neurosci Lett, 2009) 33 Potentiation of the antihyperalgesic effect induced by ACEA (CB1 agonist) by the simultaneous per os of PL265 in mice intratibially inoculated with B16-F10 cells

 Synergistic effects involve interaction between cannabinoid and opioid receptors sytems 34 Potentiation of the antihyperalgesic effect induced by AM1241 (CB2 agonist) by the simultaneous per os of PL265 in mice

 Synergistic effects involve interaction with beta-endorphin released by the CB2 agonist, AM1241 35 Potentiation of the antihyperalgesic effect induced by URB-937 (FAAH inhibitor) by the simultaneous per os of PL265 in mice

 Synergistic effects involve interaction between PL265-protected-ENKs and URB937- protected-AEA degradation by FAAH 36 Potentiation of the antihyperalgesic effect induced by NAV26 (Nav 1.7 blocker) by the simultaneous per os of PL265 in mice

 Synergistic effects is due to the increase of PENK by blockade of Nav 1.7 channel, adding to PL265-protected-ENKs, Nat Commun 2015 37 Effects of the administration of PL265 alone and combined drugs in mice intratibially inoculated with B16-F10 measured by the unilateral hot plate test.

ED50: Effective dose resulting in a 50% of the antihyperalgesic effect. Data are the mean ± S.E.M. of the estimate. * Significantly different from the theoretical combination data (p<0.05, Student’s t test) CONFIDENTIAL 38 Synergistic combinations of the dual enkephalinase inhibitor (DENKI) PL265 given orally with various analgesic compounds acting on different targets, in a murine model of bone cancer-induced pain.

Conclusions : These multi-target-based antinociceptive strategies using combinations with dual inhibitors of enkephalin degrading enzymes of various compounds with direct or indirect mechanism of action with opioid system provide therapeutic advantages in terms of efficacy and safety by allowing the reduction of doses of one of the compounds or of both, which is of the utmost interest in the chronic treatment of cancer pain.

39 Plurality of pain relief by dual enkephalinase inhibitors

PL37 is active after oral administration on :

1°) model of inflammatory pain (carageenan)

2°) model of neuropathic pain, sciatic nerve ligation (Coll. R. Maldonado).

3°) Streptozotocin-induced model of diabetic neuropathy (coll. A. Eschalier).

4°) Osteosarcona-induced thermal hyperalgesia (Coll. A. Baamonde).

Synergetic effects controlled by isobolographic analysis for all the models with different molecule acting on different opioid target.

40 High doses of RB101 administered by i.p or i.v. routes are unable to induce:

1° A psychic dependence measured by conditionnel place preference / test (PMT) in contrast to morphine. Unlike morphine the endogenous enkephalins protected by RB101 are unable to establish a conditioned place preference in mice. Noble F. et al., Eur. J. Pharmacol. 230 (1993) 139-149.

2° In contrast to morphine repeated systemic administration of RB101 do / not produce physical dependence.

Lack of physical dependence in mice after repeated systemic administration of the mixed inhibitor prodrug of enkephalin-degrading enzymes, RB101. Noble F. et al. Eur. J Pharmacol. 223 (1992) 91-96.

Similar responses were obtained by using PL37 in place of RB101.

Coll. : F. Noble et al. R. Maldonado et al.

41 Clinical results : POC in addition to pregalalin in patient with diuretic neuropathy

Currently available data on this phase 2a trial confirm that use of PL37 for the treatment of PDN is safe and causes no major side effects in patients insufficiently relieved by their gabapentin or treatment.

Patients treated with pregabalin+PL37 showed pain score decreased with time treatment

Evolution of pain score for patients with pregabalin+PL37

PL37 orally administered three times a day in addition to a stable dose of either pregabalin or gabapentin.

Results presented at the last IASP Congress in Japan Oct. 2016 (M. Wurm, B. Frank, B.P. Roques, M.C. Fournié-Zaluski, H. Poras, T. Ouiimet) 42 Extension des effets antineuropathiques du PL37 à d’autres modèles animaux de NP

 Des agressions pharmacochimiques (antitumoraux, antiviraux) altérant directement le nerf (modèle de la vincristine chez le rat (coll. B. Calvino)

 Des maladies organiques tel que le diabète. Modèle du rat diabétique par traitement à la streptotozocine (coll. A. Eschalier)

 Diminution de l’allodynie (Von Frey test) et de l’hyperalgésie mécanique ou thermique (plantar test).

ProjectCONFIDENTIAL Epione – Confidential Scientific Presentation – April 2012 43 Les DENKIs n’induisent pas les effets indésirables produits par la morphine chez l’animal et chez l’homme

 Pas d’effet de dépression respiratoire

 Pas d’effet cardiovasculaire mesurable

 Pas de constipation, même après administration chronique

 Pas de tolérance après administration répétée

 Pas d’effet nauséeux chez le furet et le chien Les résultats obtenus chez les rongeurs ont été retrouvés dans l’étude clinique de phase 1 (pharmacologie de sécurité). 136 sujets ont reçu PL37 orale dose unique à 6.25-1000 mg et multiples doses de 200-1000 mg 4 fois/jour pendant 5 jours.

ProjectCONFIDENTIAL Epione – Confidential Scientific Presentation – April 2012 44 CONFIDENTIAL 45 CONFIDENTIAL 46 Drug Discovery : designing the ideal opioid

(From B.L. Kieffer, Nature (2016), 537, 170-171) 47 Différences entre l’action analgésique des opioïdes exogènes (ex. MO) et les opioïdes endogènes enképhalines protégées par les DENKIs

Le mauvais exemple : la Met-E, modifiée pour résister aux enképhalinases, développée par SANDOZ - essai chez l’homme – analgésie similaire à MO … mais tous ses effets indésirables !!

Les avantages de l’utilisation des DENKIs Les inconvénients du contrôle opioïde exogène

Les opioïdes endogènes (i.e. enképhalines) ont été démontrés responsables des actions analgésiques obtenues en clinique par stimulation transcraniale électrique ou Les opioïdes exogènes (i.e. MO) sur-stimulent magnétique, acupuncture et autres manipulations tous les récepteurs opioïdes y compris ceux non impliqués dans le contrôle de la douleur et Par rapport à MO, les ENKs se comportent in vivo comme dépourvus de rôle physiologique des agonistes purs avec une “efficacité pharmacologique” supérieure

Les DENKIs n’augmentent la concentration locale des ENKs que là où ils sont phasiquement secrétés (ex. circuit de la La majorité des effets indésirables douleur) (sédation, dépression respiratoire, nausée, constipation, accoutumance … sont dus à Les ENKs même lorsqu’elles sont protégées par un cette stimulation ubiquitaire DENKI n’atteignent pas des concentrations suffisantes pour stimuler efficacement les ORs responsables des effets secondaires de la morphine

A l’inverse de MO, les ENKs induisent un trafic rapide des ORs stimulés avec re-initialisation à la surface cellulaire sous forme active.

NO ADVERSE EFFECTS ADVERSE EFFECTS

48 effects of RB101 protecting endogenous opioid peptides acting on delta receptors and direct activation by delta agonists such as SNC80

The effects of RB101 (i.v.) in the forced wim test in Sprague-Dawley rats (n=6/dose). A single dose of RB101 was injected 30 min before the forced swim test. The colums and vertical lines represent the mean and S.E.M. for immobility (open columns), swimming counts (single-hatched columns), and climbing counts (double-hatched columns). **P<0.001 compared with vehicle as determined by Dunnett’s post hoc test.

E.M. Jutkiewicz et al. / European Journal of 531 (2006) 151–159 157

Representative traces by electroencephalogram showing the effects of i.v. administration of 3.2 mg/kg SNC80 (A) and 32 mg/kg RB101 (B) on electroencephalographic recordings. The asterisks indicate the time of injection, and the time lines indicate 2-min intervals. Baseline amplitude is 150- 200 µV.

Fig. 5. Representativetraces showingContrairementtheeffects of i.v. administratioàn ofl’agoniste3.2 mg/kg SNC80 (A)nonand 32 mg/kgpeptidiqueRB101 (B) on electroencephadelta lographicsélectifrecordingSNCs. The 80, la potentialisation des asterisks indicate the time of injection,opioïdesand the time linesendogènesindicate 2-min intervals.(enképhalinesBaseline amplitude is 150–200parV. l’inhibiteur mixte RB101 produit des effets

Finally, a dose of 32 mg/antidépresseurskg RB101 increased ACTH (pto 327.ex. receptor-preferringdans le testneurotderansmitterla nagemet-enkephforcée)alin (Daugémaiset n’induit pas d’activité (±113.3) pg/ml as compaépileptiformered to 125.5 (±102.3)démontrantpg/ml in al.,le1996potentiel). This antidepresthérapeutiquesant activity was blockeddes récepteurswith the delta activés par voie vehicle-treated rats at 15 minendogènepost-injection.. failed selective delta- antagonist naltrindole, but not to block the RB101-induced increase in ACTH (274±89.6 pg/ with(frommu-selectJutkiewiczive dosesEM etof al. Eur. J. Pharmacolnor the kappa-s., 2006,elective 531, 151-159) 49 ml) (data not shown). , nor-BNI. These findings confirm previous

4. Discussion

The enkephalinase inhibitor RB101 (i.v.) demonstrated antidepressant-like effects in the forced swim test in rats, presumably by increasing endogenous levels of delta-opioid

Fig. 7. The effect of 32 mg/kg RB101 (i.v.) ( ), 10 mg/kg naltrindole (s.c.) ( ), and 10 mg/kg naltrindole given 15 min prior to 32 mg/kg RB101 ( ) on BDNF mRNA expression, in comparison to vehicle controls ( ). Quantification of BDNF mRNA signal was obtained from photomicrographs of X-ray films exposed for 14 day s after in situ hybridization with the antisense cRNA probe to rat BDNF mRNA. BDNF Fig. 6. The effects of RB101 (i.v.) administered 10 min prior to a single doseof mRNA levels were obtained from frontal cortex, and three hippocampal SNC80 (i.v.) on the convulsive (A) and antidepressant-like effects (B) in subfields, the CA1, CA3, and dentate gyrus regions (panel A), and from the Sprague–Dawley rats. For measure of antidepressant-like effects, data are basolateral amygdale (BLA), endopiriform nucleus (EN), and olfactory cortex represented as counts of immobility only. (Olf. Ctx.) (panel B). Data are expressed as mean±S.E.M. Extension des propriétés analgésiques des inhibiteurs mixtes des opioïdes endogènes à la douleur oculaire et à l’inflammation de la cornée

Les récepteurs opioïdes « delta » et leurs ligands sont présents en quantité dans les cellules épithéliales et sont impliqués dans leur protection.

Ces études effectuées en collaboration avec les chercheurs de l’Institut de la Vision (Hôp. Saint Antoine). L’Unité INSERM a montrée que :

- Le DENKI PL265 est capable de diminuer la douleur dans diverses lésions de la cornée. - Le PL265 local réduit très significativement l’inflammation de la cornée.

Tous ces effets sont obtenus par voie locale et n’impliquent pas le système nerveux central.

Présenté au congrès ARVO, Baltimore 7-11 May 2017

50 ARVO 2017 Meeting (Baltimore 7-11 May)

Dual enkephalinase inhibitor (DENKI) PL265: a novel topical treatment for ocular pain ? REAUX-LE GOAZIGO Annabelle1, PORAS Hervé2, OUIMET Tanja2, BAUDOUIN Christophe1,3, MELIK PARSADANIANTZ Stéphane1, WURM Michel2 1Therapeutic Department, INSERM UMR S968, Vision Institute, PARIS, FRANCE, 2Pharmaleads SA, PARIS, FRANCE – 3INSERM-DHOS CIC 1423, Quinze-Vingts National Ophthalmology Hospital, PARIS, FRANCE INTRODUCTION METHODS EFFECTS OF TOPICAL PL265 ON AN INFLAMMATORY PAIN MODEL Adult male C57BL/6 mice (8-week old) were used. Animal procedures were The peripheral endogenous opioid system is critically involved in IVCM Corneal Behavioral Mechanical corneal sensitivity (von Frey filaments) carried out by authorized investigators in accordance with institutional LPS neuropathic and inflammatory pain (1). Enkephalins, the main Injury + LPS analysis endogenous , play a key role at all levels of pain control and guidelines for the care and use of experimental animals approved by the PBS instillation Corneal sensitivity to (palpebral closure time) European Communities Council Directive 2010/63/UE). 1 2 3 4 5 exhibit an analgesic efficacy comparable to morphine without the days Corneal integrity and inflammation adverse effects. Corneal injury model PL265 instillation 10 mM Under anesthesia, a corneal scraping was performed in one eye with a 1.5 mm Dual ENKephalinases Inhibitors (DENKIs) are specific and selective • Topical PL265 elicits significant antinociceptive effects • Topical PL265 decreases corneal inflammation inhibitors of aminopeptidase N (APN) and (NEP), trephine (Beaver Visitec) at day 0 (D0). Non-operated and operated mice were Inflammatory cells protecting enkephalins from enzyme degradation. DENKIs, thereby treated twice a day either with a drop of PL265(ONa)2 (10 mM) or with PBS LPS + PBS nerve LPS + PL265 (10 mM) potentiate physiological functions of enkephalins (e.g. pain control). (control animals) in the right eye for 5 days. Topical PL265 shows potent The DENKI PL265 – Mechanism of action and pharmacology Inflammatory pain antinociceptive effects. Mice treated with (*) Increases bioavailability After corneal injury, some operated animals received a drop of LPS (0111:B4, PL265 exhibited an increased mechanical DENKI Sigma, 50 µg/10 µl). Non-operated and operated mice were treated twice a day threshold 61.00 ± 14.70 mg vs 24.00 ± Enkephalinases APN NEP either with a drop of PL265(ONa)2 or with PBS (control animals) in the right eye 4.00 mg, n=5 at D5 Enkephalins Y G G F M(L) (endogenous) for 5 days. In a second set of experiments, some animals received a drop of naloxone Opioid receptors methiodide (100 µM, Sigma), a non selective opioid receptor antagonist, which In addition, the palpebral closure time does not cross the blood brain barrier, 15 minutes before a topical instillation of (Morphine) induced by topical capsaicin (100 µM) was (exogenous) PL265(ONa) (10 mM). 2 significantly decreased (70 vs 170 seconds, The corneal integrity and inflammation were evaluated by in vivo confocal n= 5 per group) after PL265 treatment. • PL265 is a small non-peptide molecule (MW: 576 Da). microscopy (IVCM, Heidelberg Retina Tomography (HRT)II/Rostock Cornea The corneal stroma contained numerous hyper • PL265 is active orally in all rodent models of neuropathic pain Module Heidelberg) at D5. We observed a decreased inflammation in reflective cells. High inflammation in the corneal the corneal stroma tested with a strictly peripheral effect (2). Behavioral tests stroma • PL265 has been given orally to rats (up to 1200 mg/kg) and dogs Mechanical and chemical corneal sensitivities were evaluated using von Frey • Reversion of antinociceptive up to 800 mg/kg) without toxicity. filaments (Bioseb) and capsaicin (100 µM, Sigma) instillation at D5. • Dose-response of PL265 • Time-course of action of PL265 effects by naloxone methiodide • Oral PL265 has safely completed Phase 1 single ascending dose Von Frey filaments were applied onto the center of the cornea and the LPS Behavioral Corneal Behavioral Corneal Behavioral Corneal LPS LPS analysis (100-800 mg) and is now in Phase 1 multiple ascending dose (up mechanical threshold was determined by blinking response 15 minutes after the Injury + LPS analysis Injury + LPS analysis Injury + LPS PBS instillation PBS instillation PBS instillation to 300 mg b.i.d.) for 4 days, before entering Phase 2 in 2018 in last instillation. ± Naloxone methiodide 1 2 3 4 5 1 2 3 1 2 3 4 5 TM 10, 20, painful diabetic neuropathy. Data are presented as means ± SEM and analyzed using Prism software. days days days 40,240 min PL265 instillation PL265 instillation PL265 instillation 10 mM 100 µM, 1mM, 10 mM 10 mM ± Naloxone methiodide 100 µM PURPOSE RESULTS • All elements of the enkephalinergic system (opioid receptors, In non-operated mice (without corneal injury), topical instillation of PL265 for 5 enkephalins and their degrading enzymes) are also expressed in days does not alter ocular surface (slit lamp and IVCM examinations) or corneal ocular surface tissues. Topical morphine has actually been shown mechanical sensitivity compared to PBS-treated mice. to relieve pain associated with corneal lesions in rat and dog pain Corneal injury model models (3,4). • To date, no topical ocular analgesics are available for the Corneal Behavioral Injury analysis treatment of acute or chronic pain, only anesthetics which have Mechanical corneal sensitivity PBS PL265 PBS PL265 PBS instillation (von Frey filaments) many shortcomings. 1 2 3 4 5 PBS Nal. Methiod. • Here, we display the antinociceptive and anti-inflammatory effects days Corneal sensitivity to capsaicin Dose response of PL265 eye-drops. This confirms that Time course of action of PL265(10 mM) eye-drops PL265 antinociceptive effects are purely peripheral PL265 instillation 10 mM (palpebral closure time) the 10 mM dose was relevant shows that the effects on LPS-induced inflammation and are mediated by the opioid receptors on the of a highly effective DENKI prodrug, PL265, using experimental last for at least 4 hours corneal surface murine models of ocular nociception and inflammation. CONCLUSIONS Corneal nociceptive signal pathways Topical instillation of PL265 (10 mM) significantly increased corneal mechanical Brainstem • This study provides the first evidence that PL265, a prodrug from a new therapeutic How PL265-protected enkephalins block Second order Spinal trinegeminalurons threshold at D5 compared to PBS-treated complex class, DENKI, is highly effective in decreasing corneal nociception after various nociceptive signal pathways mice (49.00 ± 4.58 mg vs 29.60 ± 4.43, experimental corneal lesions. n=10 per group). Nociceptive signal Trigeminal ganglion • PL265 also significantly reduces corneal inflammation in a model of LPS-induced pathways PBS PL265 inflammatory pain. Topical DENKI Spinal trigeminal • The antinociceptive effects of PL265 are mediated by opioid receptors on ocular complex Corneal nerve ending surface nerve endings, without involvement of the central nervous system. Opioid In addition, the palpebral closure time receptors • PL265 appears as a promising topical medication for safely and effectively Trigeminal ganglion induced by topical capsaicin was -Nociceptive signal pathways - Inflammation Enkephalins significantly decreased (97 vs 210 alleviating ocular pain and inflammation. Interruption seconds, n= 10 per group) after PL265 References: APN 1 ✂️ of nociceptive stimuli treatment. Roques B.P., Fournie-Zaluski M.C., and Wurm M. Inhibiting the breakdown of endogenous opioids and to alleviate pain. Nature Rev. Drug Discov., 2012, 11, 292-310. NEP 2 Bonnard E., Poras H., Nadal X., Maldonado R., Fournié-Zaluski M.C., Roques B.P. Long-lasting oral analgesic effects of N-protected aminophosphinic Dual ENKephalinase Inhibitors (DENKis) in ✂️ PBS PL265 peripherally controlled pain. Pharmacol. Res. Persp., 2015, 3(2), e00116, doi: 10.1002/prp2.116. DENKIs 3 Stiles J., Honda CN., Krohne SG., Kazacos EA. Effect of topical administration of 1% morphine sulfate solution on signs of pain and corneal wound healing in dogs. Am. J. Vet. Res., 2003, 64, 813- 818. 4 Wenk HN., Nannenga MN., Honda CN. Effect of morphine sulfate eye drops on hyperalgesia in the rat cornea. Pain, 2003, 105, 455-465. 51 Synergistic combinations of the dual enkephalinase inhibitor PL265 given orally with various analgesic compounds acting on different targets in a murine model of bone cancer-induced pain.

52 Les dangers du versus ceux de l’alcool: où en est-on à la suite du Rapport sur la Dangerosité des Drogues ?

Bernard P. Roques Professeur Emerite

Pharmaleads SAS, 11 rue Watt, 75013 Paris, France

U 266 INSERM, Université Paris Descartes, 4 avenue de l’Observatoire, 75006 Paris, France

ATHS (Colloque International Addictions Toxicomanies Hépatites SIDA) Biarritz 17-20 Octobre 2017 53 Facteurs de dangerosité des « drogues »

Aucune de ces substances n’est complètement dépourvue de danger. Toutes sont hédoniques, le tabac à un degré nettement moins important, toutes activent le système dopaminergique, toutes sont susceptibles d’entraîner des effets plus ou moins accentués de dépendance psychique. Rapport sur La Dangerosité des Drogues, Bernard Roques, Eds O. Jacob, 1999 54 CONFIDENTIAL 55 La « loi Evin » Article L3323-2 du Code de la Santé Publique – 10 Janvier 1991.

La propagande ou la publicité, directe ou brochures, dès lors que ces documents ne indirecte, en faveur des boissons comportent que les mentions prévues à alcooliques dont la fabrication et la vente ne l’article L. 18 et les conditions de vente des sont pas interdites, sont autorisées produits qu’ils proposent ; exclusivement : 5°) Par inscription sur les véhicules utilisés 1°) Dans la presse écrite à l’exclusion des pour les opérations normales de livraison publications destinées à la jeunesse, des boissons, dès lors que cette inscription définies au premier alinéa de l’article 1er de ne comporte que la désignation des produits la loi n° 49-956 du 16 Juillet 1949 sur les ainsi que le nom et l’adresse du fabricant, publications destinées à la jeunesse ; des agents ou dépositaires, à l’exclusion de 2°) Par voie de radiodiffusion sonore pour toute autre indication ; les catégories de radios et dans les 6°) En faveur des fêtes et foires tranches horaires déterminées par décret en traditionnelles consacrées à des boissons Conseil d’Etat ; alcooliques locales et à l’intérieur de celles- 3°) Sous forme d’affiches et d’enseignes ci, dans des conditions définies par décret ; dans les zones de production, sous forme 7°) En faveur des musées, universités, d’affichettes et d’objets à l’intérieur des lieux confréries ou stages d’initiation œnologique de vente à caractère spécialisé, dans des à caractère traditionnel ainsi qu’en faveur de conditions définies par décret en Conseil présentations, de dégustations, dans des d’Etat ; conditions définies par décret. 4°) Sous forme d’envoie par les produc- Toute opération de parrainage est interdite teurs, les fabricants, les importateurs, les lorsqu’elle a pour objet ou pour effet la négociants, les concessionnaires ou les propagande ou la publicité, directe ou entreprises, de messages, de circulaires indirecte, en faveur des boissons commerciales, de catalogues et de alcooliques.

56 Article 62 ter de la « loi Macron »

« Après l’article L. 3323-3 du code de la santé publique, il est inséré un article L. 3323-3-1 ainsi rédigé :

« Ne sont pas considérés comme une publicité ou une propagande au sens du présent chapitre les contenus, images, représentations, descriptions, commentaires ou références relatifs à une région de production, à une potonymie, à une référence ou une indication géographique, à un terroir, à un itinéraire, à une zone de production, au savoir faire, à l’histoire ou au patrimoine culturel, gastro- nomique ou paysager liés à une boisson alcoolique disposant d’une identification de la qualité ou de l’origine, ou protégée au titre de l’article L. 665-6 du code rural et de la pêche maritime. »

57 Antinociceptive effects of THC given in Effects induced by THC in combination with RB 101 combination with RB 101 in the hot plate test in on the suppression of conditioned suppression of mice motility test in mice, antagonism by SR-141,716A

% of analgesia

Counts

Valverde et al., Eur. J. Neurosci. (2001)

CONFIDENTIAL 58 Traitements des addictions à l’héroïne ou morphine:

Amélioration par combinaison avec un antagoniste DA, D2, l’amisulpride et un DENKI RB101

Maintenance des traitements avec la méthadone ou la buprénorphine sont plus ou moins efficaces pour aider les héroïnomanes à à stopper leur consommation ou au moins la réduire.

Le processus addictif se caractérise par certains comportements tels que la sensibilisation du système dopaminergique via le récepteur D2 (coll. E. Borelli ; KO, D2) qui s’exprime chez le rat par une augmentation de l’activité locomotrice.

Les résultats montrent que utilisés seuls, l’amisulpride, la buprénorphine, la méthadone ou le RB101 sont incapables de supprimer l’expression de la sensibilisation comportementale au traitement chronique à la morphine.

Au contraire comme espéré, l’augmentation des enképhalines induites par le RB101 en association avec l’amisulpride supprime les signes comportementaux de l’addiction. Ces effets sont dus à l’implication des récepteurs delta. Ceci montre que c’est l’action sur différents systèmes neuronaux qui est important.

« Blockade of morphine-induced behavioral sensitization by a combination of Amisulpride and RB101, comparaison with classical opioid maintenance treatments. » L. Cordonnier, M. Sanchez, B.P. Roques, F. Noble. Br. J. PharmacoL (2007) 151, 75-83. 59 Effets d’un traitement addictogène à la morphine et l’évolution au cours du syndrome de manque sur l’expression des gènes dans les cellules sanguines (monocytes) du rat

Evolution des cibles de manque, 12h, 18h et 36h après la prise de morphine.

Expression (by RTPCR) of various genes.

Dans certaines maladies impliquant le CNS (dépression bipolaire, schizophrénie, parkinson), une corrélation entre l’expression de certains gènes et ceux détectés dans les cellules sanguines (cellules mononucléaires) a été trouvée à 12h, 18h et 26h après l’arrêt du traitement à MO. Cynthia MARIE-CARE et ses collaborateurs ont entrepris de rechercher si une telle corrélation existait chez le rat (avec extension possible chez l’homme) au cours du syndrome de manque. Ceci pourrait servir de marqueurs pour suivre les traitements du manque et de l’addiction. (from S. Desjardins et al., Neuropharmacology, 2008, 55, 1347-1354) 60 CONFIDENTIAL 61 61