# Presentation: 621199

## Bivalent Mu-Delta Opioid Ligands: Potential for Pain Control Without Tolerance or Dependence

- David J Daniels
- Department of Medicinal Chemistry
- University of Minnesota

## Opioid Analgesics

- From the poppy Papaver Somniferum
- One of the oldest recorded medications
- Long history of use and abuse
- Morphine: Isolated from opium poppy seeds in the early 1800s.
- Morpheus (latin) = Greek God of Dreams

**Opium**

## Diacetylmorphine - Heroin

- 1st Synthesized in 1874 by C.R. Alder Wright
- 1897 - Bayer Pharmaceutical company “re-invented” diacetylmorphine
- named it **Heroin** after the German word heroisch  “heroic”
- 1898 – 1910 Bayer marketed Heroin as a non-addictive morphine substitute
- 1914 Harrison Narcotics Tax Act
- 1924 US banned all use of Heroin

## Opioid Analgesics – The Holy Grail

- Countless synthetic opiate compounds have been synthesized over the last century in an attempt to create the “perfect” analgesic

## History and Background

- G protein-coupled receptors
- 3 Opioid Receptor Subtypes:  Mu, Delta, Kappa
- Approximately 60 % homology
- 372 to 398 amino acid residues

## Opioid Receptor Subtypes

- Dimerization/Oligomerization?

- Pharmacology predicts greater receptor subtypes than receptor cloning reveals!
- Single gene-deleted mice lack all subtypes for deleted receptor

- Mu1, Mu2, Mu3
- Delta1, Delta2
- Kappa1, Kappa2

## GPCR Dimerization

## Opioid Receptor Dimerization

- Homodimers:
- Mu-Mu
- Delta-Delta
- Kappa-Kappa
- Heterodimers:
- Mu-Delta
- Delta-Kappa
- Mu-Kappa

- Evidence for dimerization

- 1. Pharmacological
- 2. Coimmunoprecipitation
- 3. BRET and FRET
- 4. Confocal microscopy
- 5. Atomic-force microscopy

## Bivalent Ligands

- Pharmacological Probes Selective for Dimerized Opioid Receptors

##  Portoghese, P.S. et. al.  Journal of Medicinal Chemistry, 1986, 29, 1855-1861 

-  Portoghese, P.S. et. al.  *Journal of Medicinal Chemistry*, **1986**, 29, 1855-1861

## Purpose of our Projects

- Develop selective bivalent ligands to probe opioid receptor organization
- See if we can characterize putative opioid receptor subtypes
- Gain additional insight into the molecular organization of opioid receptors
**Develop novel analgesics devoid of tolerance and physical dependence**

## Early Evidence For Interactions Between Mu and Delta Opioid Receptors

- Potentiation of morphine with delta agonists
- Vaught, J.; Takemori, A. Differential effects of leucine enkephalin and methionine enkephalin on morphine-induced analgesia, acute tolerance and dependence. *J. Pharm. Exp. Ther.*, **1979**, 208, 86-94
- Rothman,R.B.; Westfall,T.C. Allosteric coupling between morphine and enkephalin receptors in vitro. *Mol Pharmacol*., **1982**, 21, 548-557

## Pharmacological Evidence for Tolerance and Dependence

- Abdelhamid, E.E.; Sultana, M.; Portoghese, P.S.; Takemori, A.E.  Selective blockage of delta opioid receptors prevents the development of morphine tolerance and dependence in mice. *J. Pharm. Exp. Ther.,* **1991**, 258, 299-303
- Zhu, Y.; King, M.A.; Schuller, A.G.; Nitsche, J.F.; Reidl, M.; Elde, R.P.; Unterwald, E.; Pasternak, G.W.; Pintar, J.E.  Retention of supraspinal morphine-like analgesia and loss of morphine tolerance in delta opioid receptor knockout mice.  *Neuron,* **1999 **24, 243-252

## Recent Evidence For Interactions Between Mu and Delta Opioid Receptors

- Cultured cells
- George, S.R.; Fan, T.; Xie Z.; Tse R.; Tam, V.; Varghese, G.; O’Dowd, B.F.  Oligomerization of mu and delta-Opioid Receptors.  *Journal of Biological Chemistry, ***2000***,* 275, 26128-26135
- Gomes, I.; Jordan, B.A.; Gupta, A.; Trapaidze, N.; Nagy, V.; Devi, L.A.  Heterodimerization of mu and delta Opioid Receptors: A Role in Opiate Synergy, *The Journal of Neuroscience*, **2000**, 20, 1-5
- Living Cells - BRET
- Mu luciferase and delta YFP cotransfected into living cells
- Spinal cord membranes
- Coimmunoprecipitation experiments with mouse abs to mouse mu and mouse delta opioid receptors in WT and delta “knockout” mice

- Gomes, I.; Gupta, A.; Filipovska, J.; Szeto, H. H.; Pintar, J. E.; Devi, L. A. A role for heterodimerization of mu and delta opiate receptors in enhancing morphine analgesia. *Proc. Natl. Acad. Sci. U.S.A. ***2004**, 101, 5135-5139.** **

##  M  D A N  -   #

** ****M  D A N  -   #**

**MU**

**Delta**

- Agonist

- Antagonist

- # of Atoms of
- the spacer

- Agonist

- Antagonist

- Daniels, D.J.; Lenard, N.R.; Etienne, C.L.; Law, P.Y.; Roerig, S.C.; Portoghese, P.S.  Opioid-induced tolerance and dependence in mice is modulated by the distance between pharmacophores in a bivalent ligand series.  *Proc. Natl. Acad. Sci. U.S.A*. **2005, **102, 19208-19213**.**

## Model of a Mu-Delta Heterodimer

**Mu Receptor**

**Delta Receptor**

**Approximate Distance Between recognition sites = 17 ****Å**

- MDAN-19 through MDAN-21 Spacer Distance = 23 -26 Å

## Mu-Delta Bivalent Ligand Project

**MDAN Series**
**n = 2 - 7**

**MA Series**
**n = 2 - 7**

**DN-20**

## Measure of analgesia/antinociception

**Mouse Tail Flick Assay**

**Rat**

## Antinociceptive activity in the mouse tail-flick assay after acute I.C.V administration

## Model for Testing Tolerance and Physical Dependence in Mice

- Osmotic minipump infuses constant rate of ligand via cannula i.c.v. for 3 days
- Day 4, naloxone (1 mg/kg s.c.) injected; the number of jumps in ten minutes is recorded
- 4 hours laters: “chronic” ED50 value with tail-flick assay
- Tolerance determined by comparing saline infusion vs. chronic infusion

## Effect of Spacer Length on Tolerance and Dependence in Mice 

| Compound | Saline ED50 nmol
(95% C.I.) | Chronic ED50 nmol
(95% C.I.) | Fold Diff  | # of Jumps (SEM) |
| --- | --- | --- | --- | --- |
| MDAN - 16 | 1.62 (1.35 – 1.89) | 4.72 (3.47 – 5.91) | 2.8 | 30 (23) |
| MDAN - 17 | 1.54 (0.89 – 2.20) | 5.61 (4.39 – 6.83) | 3.6 | 0.9 (0.7) |
| MDAN - 18 | 1.29 (0.97 – 1.61) | 4.75 (3.50 – 6.00) | 3.7 | 8.9 (3.0) |
| MDAN - 19 | 0.42 (0.37 – 0.47) | 0.40 (0.33 – 0.47) | 1.0 | 3.6 (1.7) |
| MDAN - 20 | 0.17 (0.15 – 0.20) | 0.17 (0.13 – 0.21) | 1.0 | 0.4 (0.4) |
| MDAN - 21 | 0.10 (0.09 – 0.11) | 0.10 (0.09 – 0.11) | 1.0 | 3.5 (1.7) |
| MA - 19 | 0.04 (0.03 – 0.05) | 0.22 (0.19 – 0.26) | 5.5 | 83 (13) |
| MA-19 + DN-19 | 0.04 (0.03 – 0.04) | 0.03 (0.02 – 0.05) | 9.4 | 29 (8) |
| Morphine | 4.54 (3.51 – 5.56) | 26.8 (20.8 – 32.8) | 6.0  | 100 (15) |

**Tolerance**

**Dependence**

## Model for the role of  heterodimers in 

- Model for the role of  heterodimers in
- 	   tolerance and dependence

## Model for the role of  heterodimers in 

- Model for the role of  heterodimers in
- 	   tolerance and dependence

**Tolerance &**
**Dependence**

**CAMKII ??**

**Analgesia**

**Analgesia**

**Analgesia**

**Opioid receptor organization may be responsible for separate mechanisms leading to tolerance, dependence, and other unwanted side effects **

## Conditioned Place PreferenceA Model of Addiction

- Conditioned Place PreferenceA Model of Addiction

- Adapted from Feldman, R.S.; Meyer, J.S. and Quenzer, L.F.  Principles of Neuropsychopharmacology
- Sunderland, MA, Sinauer Associates, 1997.

- Lenard, N.R.; Daniels, D.J.; Portoghese, P.S.; Roerig, S.C.  Absence of conditioned place
- preference or reinstatement with bivalent ligands containing mu-opioid agonist and delta-opioid
- receptor antagonist pharmacophores.  *Eur. J. Pharmacol.* **2007**, Accepted and in press.

## Acquisition of Place Preference

- Day 1: Exposure to novel environment (15 min)
- Day 2: Preconditioning
- The time spent in each side of the box in 15 min recorded
- Days 3 – 5: Injected with saline and confined to one side of box (30 min); Later injected with ligand and confined to other side of box (30 min)
- Day 6: Determine place preference (15 min)
- Place Preference = Percent change in time spent on drug-paired side
- Positive = Place Preference
- Negative = Place Aversion

## Conditioned

**MA-19**

**MA-19 + **
**DN-20**

**DN-20**

**Saline**

**Saline**

**MDAN-16**

**MDAN-19**

**MDAN-21**

**Conditioned**
**Place Preference**
**Results**

**Monovalent**
**Ligands**

**Bivalent**
**Ligands**

## Primining injection-induced reinstatement of morphine CPP

## Parenteral Bioavailability

**Comparison of Intravenous to Intracerebral Ventricular **
**Administration Potencies for MDAN-21, MA-19 and Morphine**

| Ligand | i.c.v. ED50 (95% C.I.) nmol | i.v. ED50 (95% C.I.) nmol | i.v. / i.c.v. ratio |
| --- | --- | --- | --- |
| MA-19 | 0.04 (0.03 – 0.05) | 1.61 (1.29 – 1.92) | 40.3 |
| MDAN-21 | 0.08 (0.06 – 0.10) | 3.3 (3.0 – 3.6) | 41.3 |
| Morphine | 4.1 (3.7 – 4.8) | 168 (146 – 178) | 41.0 |

## Project Summary

- Clinical Implications
- Potent, efficacious analgesics
- No tolerance
- No physical dependence
- No drug seeking behavior
- Molecular Implications
- Tolerance and dependence mediated through associated mu-delta opioid receptors??

## Future Directions

- University of Minnesota is in the process of patenting the MDAN series
- Several pharmaceutical companies have expressed interest
- Other laboratories are in the process of designing compounds with mixed -agonist/-antagontist activity

## Acknowledgements

- Philip Portoghese
- Sandy Roerig

- Natalie Lenard
- Ping Law
- Chris Etienne

**Work Supported by NIH project Grant DA1509**
**NIDA Postdoctoral Fellowship DA18028 (to N.L.)**

**Funding:**

## Slide 32

## Slide 33

## Distance between recognition sites in Mu-Mu opioid receptor models using two TM helices as the interface between dimers

| Dimer Interface | Distance Å |  | Dimer Interface | Distance Å |
| --- | --- | --- | --- | --- |
| TM1,2 – TM2,1 | 41.6 |  | TM3,4 –TM5,4 | 37.5 |
| TM1,2 – TM3,2 | 41.0 |  | TM3,4 –TM6,5 | 29.5 |
| TM1,2 – TM4,3 | 38.6 |  | TM3,4 –TM7,6 | 26.8 |
| TM1,2 – TM5,4 | 39.8 |  | TM3,4 –TM1,7 | 29.5 |
| TM1,2 – TM6,5 | 31.8 |  | TM4,5 –TM5,4 | 33.8 |
| TM1,2 – TM7,6 | 28.2 |  | TM4,5 –TM6,5 | 29.2 |
| TM1,2 – TM1,7 | 38.3 |  | TM4,5 –TM7,6 | 30.8 |
| TM2,3 –TM3,2 | 36.7 |  | TM4,5 –TM1,7 | 30.2 |
| TM2,3 –TM4,3 | 36.1 |  | TM5,6 –TM6,5 | 22.2 |
| TM2,3 –TM5,4 | 38.7 |  | TM5,6 –TM7,6 | 19.2 |
| TM2,3 –TM6,5 | 30.0 |  | TM5,6 –TM1,7 | 18.7 |
| TM2,3 –TM7,6 | 27.0 |  | TM6,7 –TM7,6 | 17.4 |
| TM2,3 –TM1,7 | 38.0 |  | TM6,7 –TM1,7 | 24.2 |
| TM3,4 –TM4,3 | 36.4 |  | TM7,1 –TM1,7 | 32.4 |

## Antinociceptive activity in the mouse tail-flick assay after acute I.C.V administration

| Ligand | Spacer Length (Å)a | ED50b (95% C.I.) nmol |
| --- | --- | --- |
| MA-16 | 19.1 | 0.039 (0.032 –  0.046) |
| MA-17 | 20.4 | 0.040 (0.033 – 0.046) |
| MA-19 | 22.9 | 0.040 (0.023 – 0.050) |
| MA-20 | 24.1 | 0.037 (0.029 – 0.045) |
| MA-21 | 25.4 | 0.044 (0.039 – 0.048) |
| MDAN-16 | 19.1 | 1.79 (1.54 - 2.04) |
| MDAN-17 | 20.4 | 1.49 (1.04 - 1.95) |
| MDAN-18 | 21.6 | 0.95 (0.68 - 1.23) |
| MDAN-19 | 22.9 | 0.43 (0.36 - 0.50) |
| MDAN-20 | 24.1 | 0.17 (0.15 - 0.19) |
| MDAN-21 | 25.4 | 0.08 (0.06 - 0.10) |
| MA-19 + DN-20 |  | 0.037 (0.031 – 0.043) |
| Oxymorphone | 25.4 | 0.043 (0.034 – 0.052) |

## NTI Pretreatment

- Effect of Pretreatment with Naltrindole on Acute Antinociceptive
- Potency of Selected - Bivalent Ligands Administered i.c.v.

|  | ED50 (95% C.I.) nmol |  |
| --- | --- | --- |
| Ligand | No Pretreatment | NTI (50 pmol) Pretreatment |
| MDAN-16 | 1.70 (1.50 – 2.00) | 0.30 (0.19 – 0.43) |
| MDAN-19 | 0.43 (0.36 – 0.50) | 0.05 (0.05 – 0.07) |
| MDAN-21 | 0.08 (0.06 – 0.10) | 0.06 (0.05 – 0.07) |
| MA-19 | 0.04 (0.023 – 0.045) | 0.08 (0.065 – 0.085) |

## Model for Negative Modulation of Antinociception

## Other Approaches to Avoid Tolerance and Dependence

- 	Ananthan, S.; Khare, N.K.; Saini, S.K.; Seitz, L.E.; Bartlett, J.L.; Davis, P.; Dersch, C.M.; Porreca, F.; Rothman, R.B.; Bilsky, E.J.  Identification of opioid ligands possessing mixed mu agonist/delta antagonist activity among pyridomorphinans derived from naloxone, oxymorphone, and hydromorphone. *J. Med. Chem.* **2004**, 47, 1400-1412

**DIPP-NH2[********]**

**SoRI 9409**

- Schiller, P.W.; Fundytus, M.E.; Merovitz, L.; Weltorski, G.; Nguyen, T.M.D.; Lemieux, C.; Chung, N.N.; Coderre, T.  The opioid mu agonist/delta antagonist DIPP-NH(2)[Psi] produces a potent analgesic effect, no physical dependence, and less tolerance than morphine in rats. *J. Med. Chem. ***1999**, 42, 3520-3526.** **