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

David J DanielsDavid J DanielsDepartment of Medicinal ChemistryDepartment of Medicinal ChemistryUniversity of MinnesotaUniversity of Minnesota

Opioid AnalgesicsOpioid Analgesics

From the poppy Papaver SomniferumFrom the poppy Papaver Somniferum One of the oldest recorded medicationsOne of the oldest recorded medications Long history of use and abuseLong history of use and abuse

Morphine: Isolated from opium poppy seeds in Morphine: Isolated from opium poppy seeds in the early 1800s. the early 1800s.

Morpheus (latin) = Greek God of DreamsMorpheus (latin) = Greek God of Dreams

HO O OH

NH

CH3

(-)-MorphineOpium

Diacetylmorphine - HeroinDiacetylmorphine - Heroin

11stst Synthesized in 1874 by C.R. Alder Wright Synthesized in 1874 by C.R. Alder Wright 1897 - Bayer Pharmaceutical company 1897 - Bayer Pharmaceutical company

“re-invented” diacetylmorphine“re-invented” diacetylmorphine named it named it HeroinHeroin after the German word after the German word

heroisch heroisch “heroic” “heroic” 1898 – 1910 Bayer marketed Heroin as a 1898 – 1910 Bayer marketed Heroin as a

non-addictive morphine substitute non-addictive morphine substitute 1914 Harrison Narcotics Tax Act1914 Harrison Narcotics Tax Act 1924 US banned all use of Heroin1924 US banned all use of Heroin

Opioid Analgesics – The Holy GrailOpioid Analgesics – The Holy Grail Countless synthetic opiate compounds have Countless synthetic opiate compounds have

been synthesized over the last century in an been synthesized over the last century in an attempt to create the “perfect” analgesicattempt 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 Opioid Receptor Dimerization/Oligomerization?Dimerization/Oligomerization?

Opioid Receptor SubtypesOpioid Receptor Subtypes

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 DimerizationGPCR DimerizationContact DimerMonomer

4

6

25

7

3

1

1

23

4

5

67

4

6

25

7

3

1

1

23

4

5

67

4

6

25

7

3

1

4

6

25

7

3

1

1

23

4

5

674

6

2

7

3

1

1

2

3

4

5

67

+5

Heterodimer

4

6

25

7

3

1

1

2

3

4

5

67 4

6

25

7

3

1

1

23

4

5

67

+Homodimer

Oligomer

Opioid Receptor DimerizationOpioid Receptor Dimerization

Homodimers:Homodimers: Mu-MuMu-Mu Delta-DeltaDelta-Delta Kappa-KappaKappa-Kappa

Heterodimers:Heterodimers: Mu-DeltaMu-Delta Delta-KappaDelta-Kappa Mu-KappaMu-Kappa

Evidence for dimerization1. Pharmacological2. Coimmunoprecipitation3. BRET and FRET4. Confocal microscopy5. Atomic-force microscopy

Bivalent LigandsBivalent Ligands Pharmacological Probes Selective for Pharmacological Probes Selective for

Dimerized Opioid ReceptorsDimerized Opioid Receptors

1 2 3

4

1 2 3

4

1.066.8 ± 11.75

2.925.1 ± 5.229.434

18.93.9 ± 0.621.723

2.122.0 ± 5.014.012

2.430.2 ± 8.76.301

Rel potencybIC50 nMSpacer Lengtha ÅnCmpd no.

Table 1. Opioid Agonist Potencies in the GPI. aDistance from the -nitrogen of one oxymorphamine to the other -nitrogen. bBivalent potency ratio dived by monovalent potency ratio.

O HN

O O

HN

O

NH O

OH

N

OH

O

OH

NH

N

HOn n

Spacer Length

O

OH

NH

N

HO HN

O

ONH

O

CH3

5

1.066.8 ± 11.75

2.925.1 ± 5.229.434

18.93.9 ± 0.621.723

2.122.0 ± 5.014.012

2.430.2 ± 8.76.301

Rel potencybIC50 nMSpacer Lengtha ÅnCmpd no.

Table 1. Opioid Agonist Potencies in the GPI. aDistance from the -nitrogen of one oxymorphamine to the other -nitrogen. bBivalent potency ratio dived by monovalent potency ratio.

O HN

O O

HN

O

NH O

OH

N

OH

O

OH

NH

N

HOn n

Spacer Length

O

OH

NH

N

HO HN

O

ONH

O

CH3

5

0

5

10

15

20

5 15 25 35

Spacer Length (Å)

Rela

tive

pote

ncy

0

5

10

15

5 15 25 35

Spacer Length (Å)

Rela

tive

Affin

ity

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

Purpose of our ProjectsPurpose of our Projects

Develop selective bivalent ligands to probe opioid Develop selective bivalent ligands to probe opioid receptor organizationreceptor organization

See if we can characterize putative opioid receptor See if we can characterize putative opioid receptor subtypessubtypes

Gain additional insight into the molecular Gain additional insight into the molecular organization of opioid receptorsorganization of opioid receptors

Develop novel analgesics devoid of tolerance Develop novel analgesics devoid of tolerance and physical dependenceand physical dependence

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

Potentiation of morphine with delta agonists Potentiation of morphine with delta agonists Vaught, J.; Takemori, A. Differential effects of leucine Vaught, J.; Takemori, A. Differential effects of leucine

enkephalin and methionine enkephalin on morphine-induced enkephalin and methionine enkephalin on morphine-induced analgesia, acute tolerance and dependence. analgesia, acute tolerance and dependence. J. Pharm. Exp. J. Pharm. Exp. Ther.Ther., , 19791979, 208, 86-94, 208, 86-94

Rothman,R.B.; Westfall,T.C. Allosteric coupling between Rothman,R.B.; Westfall,T.C. Allosteric coupling between morphine and enkephalin receptors in vitro. morphine and enkephalin receptors in vitro. Mol PharmacolMol Pharmacol., ., 19821982, 21, 548-557, 21, 548-557

Pharmacological Evidence for Tolerance Pharmacological Evidence for Tolerance and Dependenceand Dependence

Abdelhamid, E.E.; Sultana, M.; Portoghese, P.S.; Takemori, Abdelhamid, E.E.; Sultana, M.; Portoghese, P.S.; Takemori, A.E. Selective blockage of delta opioid receptors prevents A.E. Selective blockage of delta opioid receptors prevents the development of morphine tolerance and dependence in the development of morphine tolerance and dependence in mice. mice. J. Pharm. Exp. Ther.,J. Pharm. Exp. Ther., 19911991, 258, 299-303 , 258, 299-303

Zhu, Y.; King, M.A.; Schuller, A.G.; Nitsche, J.F.; Reidl, M.; Zhu, Y.; King, M.A.; Schuller, A.G.; Nitsche, J.F.; Reidl, M.; Elde, R.P.; Unterwald, E.; Pasternak, G.W.; Pintar, J.E. Elde, R.P.; Unterwald, E.; Pasternak, G.W.; Pintar, J.E. Retention of supraspinal morphine-like analgesia and loss of Retention of supraspinal morphine-like analgesia and loss of morphine tolerance in delta opioid receptor knockout mice. morphine tolerance in delta opioid receptor knockout mice. Neuron,Neuron, 1999 1999 24, 243-252 24, 243-252

Recent Evidence For Interactions Recent Evidence For Interactions Between Mu and Delta Opioid ReceptorsBetween Mu and Delta Opioid Receptors Cultured cellsCultured cells

George, S.R.; Fan, T.; Xie Z.; Tse R.; Tam, V.; Varghese, G.; O’Dowd, George, S.R.; Fan, T.; Xie Z.; Tse R.; Tam, V.; Varghese, G.; O’Dowd, B.F. Oligomerization of mu and delta-Opioid Receptors. B.F. Oligomerization of mu and delta-Opioid Receptors. Journal of Journal of Biological Chemistry, Biological Chemistry, 20002000,, 275, 26128-26135 275, 26128-26135

Gomes, I.; Jordan, B.A.; Gupta, A.; Trapaidze, N.; Nagy, V.; Devi, L.A. 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 Heterodimerization of mu and delta Opioid Receptors: A Role in Opiate Synergy, Synergy, The Journal of NeuroscienceThe Journal of Neuroscience, , 20002000, 20, 1-5, 20, 1-5

Living Cells - BRETLiving Cells - BRET Mu luciferase and delta YFP cotransfected into living cellsMu luciferase and delta YFP cotransfected into living cells

Spinal cord membranesSpinal cord membranes Coimmunoprecipitation experiments with mouse abs to mouse mu and Coimmunoprecipitation experiments with mouse abs to mouse mu and

mouse delta opioid receptors in WT and delta “knockout” mice 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. 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 A role for heterodimerization of mu and delta opiate receptors in enhancing morphine analgesia. enhancing morphine analgesia. Proc. Natl. Acad. Sci. U.S.A. Proc. Natl. Acad. Sci. U.S.A. 20042004, 101, , 101, 5135-5139.5135-5139.

Mu-Delta Bivalent Ligand ProjectMu-Delta Bivalent Ligand Project

M D A N - #

MU

Delta

Agonist

Antagonist

# of Atoms ofthe 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.

Mu Receptor Delta Receptor

Approximate Distance Between recognition sites = 17 Å

Model of a Mu-Delta Heterodimer

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

Mu-Delta Bivalent Ligand ProjectMu-Delta Bivalent Ligand Project

MDAN Seriesn = 2 - 7

MA Seriesn = 2 - 7

DN-20

Measure of analgesia/antinociceptionMeasure of analgesia/antinociception

Mouse Tail Flick Assay

Rat

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

10 100 10000

25

50

75

100

MA-16MA-17

MA-20MA-21

MA-19

Oxymorphone

Drug ED50 (95% CI) pmolOxy 43 (34-52)MA-16 39 (32-47)MA-17 40 (33-46)MA-19 40 (29-50)MA-20 37 (29-45)MA-21 43 (39-48)

Drug Dose (pmol)

0.01 0.1 1 100

25

50

75

100

MDAN-18MDAN-17

MDAN-19

MDAN-21MDAN-20

MDAN-16

Drug Dose (nmol)

Model for Testing Tolerance and Model for Testing Tolerance and Physical Dependence in MicePhysical Dependence in Mice

Osmotic minipump infuses constant rate of Osmotic minipump infuses constant rate of ligand via cannula i.c.v. for 3 daysligand via cannula i.c.v. for 3 days

Day 4, naloxone (1 mg/kg s.c.) injected; Day 4, naloxone (1 mg/kg s.c.) injected; the number of jumps in ten minutes is the number of jumps in ten minutes is recordedrecorded

4 hours laters: “chronic” ED50 value with 4 hours laters: “chronic” ED50 value with tail-flick assaytail-flick assay

Tolerance determined by comparing saline Tolerance determined by comparing saline infusion vs. chronic infusioninfusion vs. chronic infusion

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

CompoundSaline 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 tolerance and dependence

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 PreferenceConditioned Place PreferenceA Model of AddictionA Model of Addiction

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

Lenard, N.R.; Daniels, D.J.; Portoghese, P.S.; Roerig, S.C. Absence of conditioned placepreference 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 PreferenceAcquisition of Place Preference Day 1: Exposure to novel environment (15 min)Day 1: Exposure to novel environment (15 min) Day 2: PreconditioningDay 2: Preconditioning

The time spent in each side of the box in 15 min recordedThe time spent in each side of the box in 15 min recorded Days 3 – 5: Injected with saline and confined to one Days 3 – 5: Injected with saline and confined to one

side of box (30 min); Later injected with ligand and side of box (30 min); Later injected with ligand and confined to other side of box (30 min)confined to other side of box (30 min)

Day 6: Determine place preference (15 min)Day 6: Determine place preference (15 min) Place Preference = Percent change in time spent on Place Preference = Percent change in time spent on

drug-paired sidedrug-paired side Positive = Place PreferencePositive = Place Preference Negative = Place AversionNegative = Place Aversion

MA-19MA-19 + DN-20

DN-20Saline

Saline

MDAN-16

MDAN-19

MDAN-21

ConditionedPlace PreferenceResults

MonovalentLigands

BivalentLigands

Primining injection-induced Primining injection-induced reinstatement of morphine CPPreinstatement of morphine CPP

Parenteral BioavailabilityParenteral Bioavailability

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

Ligandi.c.v. ED50 (95%

C.I.) nmoli.v. ED50 (95% C.I.)

nmoli.v. / i.c.v.

ratio

MA-19 0.04 (0.03 – 0.05) 1.61 (1.29 – 1.92) 40.3MDAN-21 0.08 (0.06 – 0.10) 3.3 (3.0 – 3.6) 41.3Morphine 4.1 (3.7 – 4.8) 168 (146 – 178) 41.0

Project SummaryProject Summary

Clinical ImplicationsClinical Implications Potent, efficacious analgesicsPotent, efficacious analgesics No toleranceNo tolerance No physical dependenceNo physical dependence No drug seeking behaviorNo drug seeking behavior

Molecular ImplicationsMolecular Implications Tolerance and dependence mediated through Tolerance and dependence mediated through

associated mu-delta opioid receptors??associated mu-delta opioid receptors??

Future DirectionsFuture Directions University of Minnesota is in the process of University of Minnesota is in the process of

patenting the MDAN seriespatenting the MDAN series Several pharmaceutical companies have Several pharmaceutical companies have

expressed interestexpressed interest

Other laboratories are in the process of Other laboratories are in the process of designing compounds with mixed designing compounds with mixed -agonist/-agonist/--antagontist activityantagontist activity

AcknowledgementsAcknowledgements

Philip PortoghesePhilip Portoghese Sandy RoerigSandy Roerig

Natalie LenardNatalie Lenard Ping LawPing Law Chris EtienneChris Etienne

1. Work Supported by NIH project Grant DA15092. NIDA Postdoctoral Fellowship DA18028 (to N.L.)

Funding:

Distance between recognition sites in Mu-Mu Distance between recognition sites in Mu-Mu opioid receptor models using two TM helices as opioid receptor models using two TM helices as

the interface between dimersthe interface between dimersDimer 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 22.2TM2,3 –TM5,4 38.7 TM5,6 –TM7,6 19.2TM2,3 –TM6,5 30.0 TM5,6 –TM1,7 18.7TM2,3 –TM7,6 27.0 TM6,7 –TM7,6 17.4TM2,3 –TM1,7 38.0 TM6,7 –TM1,7 24.2

TM3,4 –TM4,3 36.4 TM7,1 –TM1,7 32.4

LigandSpacer 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)

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

NTI PretreatmentNTI PretreatmentEffect of Pretreatment with Naltrindole on Acute Antinociceptive Potency of Selected - Bivalent Ligands Administered i.c.v.

ED50 (95% C.I.) nmol

Ligand No PretreatmentNTI (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 AntinociceptionModel for Negative Modulation of Antinociception

Other Approaches to Avoid Tolerance Other Approaches to Avoid Tolerance and Dependenceand Dependence

Ananthan, S.; Khare, N.K.; Saini, S.K.; Seitz, L.E.; Bartlett, J.L.; Davis, P.; Dersch, C.M.; 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 Porreca, F.; Rothman, R.B.; Bilsky, E.J. Identification of opioid ligands possessing mixed mu agonist/delta antagonist activity among pyridomorphinans derived from mixed mu agonist/delta antagonist activity among pyridomorphinans derived from naloxone, oxymorphone, and hydromorphone. naloxone, oxymorphone, and hydromorphone. J. Med. Chem.J. Med. Chem. 20042004, 47, 1400-1412 , 47, 1400-1412

DIPP-NH2[]

1

1

2

2SoRI 9409

Schiller, P.W.; Fundytus, M.E.; Merovitz, L.; Weltorski, G.; Nguyen, T.M.D.; Lemieux, C.; 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] 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 produces a potent analgesic effect, no physical dependence, and less tolerance than morphine in rats. morphine in rats. J. Med. Chem. J. Med. Chem. 19991999, 42, 3520-3526., 42, 3520-3526.