Morphine Indiscriminatingly Overstimulates All Opioid Receptors Including Those Not Involved in (Morphine) Pain Control (Exogenous)

Home , Enkephalinase inhibitor, Kelatorphan

Extensions des propriétés des inhibiteurs mixtes des enképhalinases aux douleurs de la sphère cranio-faciale. Nouvelles applications à la migraine et aux douleurs de la cornée.

Bernard P. Roques

Professeur Emérite, Université Paris Descartes Unité 1267 Inserm, 4 avenue de l’Observatoire, 75006 Paris.

ATHS Biarritz, 1-4 Octobre 2019

CONFIDENTIAL 1 Endorphins and their receptors.

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

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

2 2 Drug Discovery : designing the ideal opioid

(From B.L. Kieffer, Nature (2016), 537, 170-171) 3 Three levels of pain control by endogenous opioid system (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

4 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

5 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.

6 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). 7 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) jump Baseline 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)

8 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) jumplatency Baseline * * 20 (s latency Jump 50 *

9 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. 10 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)

11 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.

12 12 Very efficient potentiation of morphine antinociceptive effect induced by dual enkephalinase 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 13 Mechanism of action for peripheral analgesic effects of DENKIs- protected enkephalins in chronic pain and Ors hypersensitivity

 In models of neuropathic (CCI in rodents) and inflammatory pain, release and concentration of enkephalins increase constantly during a week or two before plateauing for as long as the injury lasts (Zollner et al. 2008)

CFA 24 h–96 h CRF

CRFR SP 12 h

DOR MOR NEP 6 h

APN DENKI Ca2+,K+

YGG untreated paw TRPV SV 1 DENKI AEA

ORs strongly enhanced on APN noxious inputs each sides of nerve injury CFA, ipl injection day -6 to spinal cord (Truong et al. 2000) Roques, B.P., Fournié-Zaluski, M.C. and Wurm, M., NRDD, 2012 Roques, B.P., Fournié-Zaluski, M.C. and Wurm, M., NRDD Schafer, M. et al., Eur J Pharmacol (1995) (2012)

NP, sciatic nerve ligation

Pertovaara, A. and Wei, H., Eur J Pharmacol (2001) 14 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 paw withdrawalpaw(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) 15 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 16 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.

17 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 18 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 19 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 20 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 21 CONFIDENTIAL 22 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.

23 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 24 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

25 Mecanism of migraine crisis

Wheter the initiating event is central or peripheral, CGRP can act on its receptors and beighboring terminals of trigemical afferent myelinated A∂ fibers, leading to sensitization of these sensory neurons. Moreover, acting on endothelial CGRP receptors, released CGRP can produce direct vasodilation and activate signaling cascades resulting in activation of eNOS and NO production. NO causes vasodilation and diffuses to the trigeminal nerve terminal, where it can enhance the further release of CGRP. (From Lyengar S. et al. J. Head and Face Pain, 2019) ATHS 201926 Absence of reduction in the effect of PL37 after repeated oral administration of PL37 (50 mg/kg). No tolerance.

(From Descheemaeker A. and Dallel R., Assocation for Research in Vision and Ophtalmology 2017meeting in Baltimore) ATHS 201927 Model of long duration duration of migraine induced by systemic administration of dinitrate d’isosorbide a donor of NO triggering migraine crisis. The mechanical sensitivity is measured by a head withdrawal movement measured by the Von Frey test characterized by the use of filaments of increasing force express in « g » applied on the periorbitral area.

(From Descheemaeker A. and Dallel R., Assocation for Research in Vision and Ophtalmology 2017meeting in Baltimore) ATHS 201928 Increased time effect of chronic PL37 suggesting a preventive action of the DENKi on the induced migraine crisis

(From Descheemaeker A. and Dallel R., Assocation for Research in Vision and Ophtalmology 2017meeting in Baltimore) ATHS 201929 The various types of pain which were shown to be treated by exogenous and endogenous opioids.

- Transient acute pain (or not reversible) - Hyperalgesic pain (post-operating or not) - Chronic pain - Neuropathic pain (complication of coma, diabetic, antiviral or antitumor side effects, treatments (chemiotherapy) - Fibromyalgia (non identified cause) - Migraine - Unescape based pain occurring in - Lower back disorders - Inflammation linked pain. (See Roques et al., Inhibiting the breakdown of endogenous opioids and cannabinoids to alleviate pain, NRDD, 11, 292, 2012)

For almost all these different pains, the drug of reference remains morphine ! In spite of its numerous and severe side effects (except in well controlled use), respiratory depression, constipation, sedation, hyperalgesia, possible tolerance and dependence. These effects were also produced by morphinans, which are related morphine derivatives (codeine, oxycodone, methadone, fentanyl, etc). These molecules have been prescripted in USA during the 3 last years in patients for often a pain which did not requested morphine treatment leading to 64.000 overdoses with numerous subsequent deaths.

ATHS 201930