British journal of pharmacology 2015 volume 172 part 2

Overexpression and down-regulation of theμ-opioid receptor in cancer cells before injecting them intomice were shown to increase and decrease, respectively,primary tumour growth and meta

Themed Section: Opioids: New Pathways to Functional Selectivity

EDITORIAL

M J Christie1, M Connor2 and J R Traynor3

1Discipline of Pharmacology, University of Sydney, NSW Australia,2Australian School of

Advanced Medicine, Macquarie University, Sydney, NSW, Australia, and3Department of

Pharmacology, University of Michigan, Ann Arbor, MI, USA

Correspondence

M J Christie, Discipline ofPharmacology, University ofSydney, NSW Australia E-mail:mac.christie@sydney.edu.au

LINKED ARTICLES

This article is part of a themed section on Opioids: New Pathways to Functional Selectivity To view the other articles in thissection visit http://dx.doi.org/10.1111/bph.2015.172.issue-2

This is the first themed issue on new developments in opioid

pharmacology published by British Journal of Pharmacology

(BJP) It is a bumper issue, with 39 papers, including 17

topical reviews The issue emerged from invited review

sub-missions by speakers at the International Narcotics Research

Conference (INRC) held in Cairns, Australia from 14–18 July

2013, along with open submissions from attendees, and

arti-cles freely submitted following a call for papers The meeting

was sponsored in part by BJP and the British Pharmacological

Society INRC has been the major international meeting

on opioid research for more than 40 years (see http://www

.inrcworld.org/history.htm) Invited presentations at the

2013 meeting were largely focused on novel mechanisms of

opioid receptor function and systems that are developing

novel therapeutic avenues that could improve the clinical

profile of opioids In their International Union of Basic and

Clinical Pharmacology (IUPHAR) review, Cox et al (2015)

discuss nomenclature recommendations for opioid receptors

Most papers in the themed issue conform to these

recom-mendations

It is very difficult to separate therapeutic actions of

opioids such as analgesia from serious adverse effects

includ-ing (potentially lethal) respiratory depression, constipation,

somnolence, tolerance and addiction because most are

discussed in the themed issue explore current knowledge of

new pharmacological understanding of MOPr and its

inter-actions other opioid receptors that could be exploited in

future drug development to reduce these adverse effects In its

current state, the opioid therapeutic armamentarium has

only just begun to exploit novel pharmacological

mecha-nisms such as hetero-oligomer formation, ligand bias,

allos-tery and synergy with other receptor systems, including other

opioid receptors

The INRC meeting was opened with the traditional

(Henderson, 2015) The Founders Lecture honours the

con-tributions of individuals who have a made a sustained and

substantial contribution to the science upon which the

conference is based Graeme is certainly one of those Graeme

was the first to show in 1980 that opioids directly inhibit CNSneurons via hyperpolarization (Pepper and Henderson, 1980)which was later shown to be due to potassium channel acti-vation At the time of his seminal work, the predominantthinking was that morphine acted much like a local anaes-thetic, simply blocking nerve conduction His review reflectsthe progress made since then and the unanswered questionsfrom an electrophysiologist’s perspective

A potential opportunity to exploit functional selectivity isdevelopment of heteromer selective opioids Since theground-breaking work of Lakshmi Devi suggesting that dif-ferent opioid receptor types can form heteromers in heterolo-gous expression systems there has been an extensive searchfor their presence and function in the central nervous system.The review by Massotte (2015) critically evaluates the evi-

dence required to establish existence of heteromers in vivo.

One of the crucial pieces of evidence is co-expression of thepotential partner GPCRs in the same neuron Massotte (2015)appraises the evidence for this and introduces her ownstudies of co-localization of MOPr and the opioidδ-receptor(DOPr) using knock-in mice that express both MOPr fusedwith a red fluorescent protein (mCherry) and DOPr fusedwith eGFP The restricted colocalization in the CNS suggestspotential for opioid drugs that selectively target MOPr-DOPrheteromers, moreover the general expression in lower brainregions involved in nociception indicates the potential forheteromer selective analgesics Of course co-localization doesnot establish the existence of functional heteromers Mas-

sotte discusses this and the review by Gendron et al (2015) also touches on the question The review by Fujita et al.

(2015) further discusses the evidence for potential heteromerformation among opioid receptors or between opioid recep-tors and other GPCRs, revealing extensive potential for het-eromer formation

Multiple opioid receptors expressed in a single cell mayinteract as heterodimers or, alternatively, modulate thesurface expression and function of the other partner Zhang

et al (2015) review the evidence that MOPr and DOPr

interact in small dorsal root ganglion neurons and that theinteraction is modulated by neuronal activity and morphine

tolerance This may begin to explain the widely published

findings that DOPr antagonists can suppress morphine

antinociceptive tolerance Other reviews (e.g Gendron et al.,

2015) and research papers, e.g Ong et al (2015) also address

this theme

Biased signaling and allostery have emerged as

proper-ties of many GPCRs that may provide opportuniproper-ties to limit

side effects Biased opioid agonists that select for G-protein

signaling in preference toβ-arrestin pathways are in clinical

development as analgesics with reduced side effects Based

on the plenary lecture from Arthur Christopoulos on bias

and allostery, Thompson et al (2015) provide an

introduc-tion to mechanisms of bias at opioid receptors focusing on

MOPr with a detail review of the issues that must be

con-sidered in quantification of bias The DOPr is also a

poten-tial target for pain management, particularly in neuropathic

pain Gendron et al (2015) have comprehensively reviewed

evidence for physiological functions of DOPr, including its

potential for biased signaling, as well as the role of

traffick-ing and surface expression of the receptor and potential

interacting proteins involved in its regulation Charfi et al.

(2015) review different approaches to identify and quantify

types of experimental and analytical confounds in these

analyses

Allosteric modulators have been reported for a range of

GPCRs but until very recently none were known for opioid

receptors Burford et al (2015) review the principles of

posi-tive, negative and ‘silent’ (neutral antagonists at the allosteric

site) allosteric modulation in the context of their exciting

recent discovery of allosteric modulators of MOPr,

particu-larly positive allosteric modulators (PAMs) that enhance the

activity of orthosteric MOPr agonists PAMs of MOPr have the

potential to enhance the effects of endogenously released

opioids or low doses of opioid orthosteric agonists The

authors speculate on the potential advantages that a PAM

approach might bring to the design of novel therapeutics for

pain that may avoid the side effects currently associated with

opioid therapy The further development of PAMs and biased

PAMs has great potential to contribute to pain therapy,

perhaps in ways that have not been considered previously

Analgesics such as tramadol and more recently tapentadol

exploit therapeutic interactions between opioid and other

neurotransmitter systems Synergistic interactions improve

the therapeutic profile of opioids by limiting the degree

of stimulus of the opioid system required to produce pain

relief Chabot-Doré et al (2015) comprehensively review

evidence for the best established interactions between

provide pain relief in animal models Sadeghi et al (2015)

describe additive mechanisms underlying the action of

tap-entadol in brain neurons

Development of tolerance is one of the major limitations

of long-term opioid treatment Understanding the

mecha-nisms of MOPr regulation is thought to be crucial for

under-standing and potentially developing strategies to limit

tolerance Coordinated phosphorylation of C-terminal serine

and threonine residues on MOPr plays a crucial role in the

initial steps and perhaps sustained mechanisms of MOPr

regulation, arrestin binding and endocytosis Stefan Schulz’s

group review their pioneering work (Mann et al., 2015) on

the development and use of phospho-site specific antibodies

to study homologous and heterologous MOPr regulation, thelatter mediated by protein kinase C phosphorylation ofMOPr This could provide an explanation for the proteinkinase C (PKC)-mediated desensitization of MOPr by mor-phine, when PKC has been activated, as observed in a range

of cells (Henderson, 2015) However, the research paper of

Arttamangkul et al (2015) suggests the effects of

PKC-inhibitors on MOPr may be indirect Understanding the evance of different phosphorylation sites and regulation ofMOPr is very important because many splice variants andsome of the polymorphisms of human MOPr involve thisregion of the receptor with potential implications for sensi-tivity to opioid analgesics, tolerance and addiction Knapmanand Connor (2015) comprehensively review the evidence forfunctional implications of human MOPr polymorphisms and

rel-a reserel-arch prel-aper in the themed issue (Cooke et rel-al., 2015)

examines the effects of one of these polymorphisms, L83I, onMOPr endocytosis in detail Several of the submitted researchpapers in the themed issue further address mechanisms ofMOPr regulation after chronic treatment with morphine (e.g

Connor et al., 2015; Macey et al., 2015) The research paper

by Lowe and Bailey (2015) adds to the evidence that themechanisms of MOPr desensitization in nerve terminalsdiffer from those in the soma

The important role of the DOPr in mechanisms of ance and dependence to MOPr agonists and addiction relatedmechanisms is discussed in a number of papers that highlightthe as yet unrealised therapeutic potential of DOPr drugs foraddiction management Comprehensive review and research

toler-papers by Laurent et al (2015a,b) comment on the role of

forebrain MOPr and particularly DOPr in reward and decisionmaking The main finding that long-term translocation ofDOPr to the surface of cholinergic interneurons in thenucleus accumbens shell is associated with the selection andexecution of goal-directed actions is particularly interestingalthough the cellular and molecular mechanisms involved

are not yet understood The review by Klenowski et al (2015)

comprehensively addresses the role of DOPr in addiction to a

range of drugs Baimel et al (2015) discusses the interactions

between the orexin/hypocretin system and opioids in brainregions related to addiction and potential for modulationaddiction to opioids and other drugs

Therapeutic actions and adverse effects of opioids are notlimited to analgesia or other nervous system actions Reviewsand research papers arising from the INRC symposium on

‘Opioids in Non-Neuronal Cells’ provide a perspective

on actions not often considered by those working in theCNS There is considerable interest and some controversyconcerning the influence of opioid therapeutics on tumour

progression Yamamizu et al (2015) discuss evidence and

anti-angiogenic so may have tumour suppressing properties.Morphine is commonly used in cancer pain management butthere have been concerns that the drug may adversely influ-

Afsharimani et al (2015) provide a critical review of the

valid-ity of animal models designed to evaluate the effect of phine on tumour growth and metastasis and suggest ways toimprove current approaches Another noteworthy action ofopioids on non-neuronal cells includes the influence of DOPrBJP M J Christie et al.

mor-248 British Journal of Pharmacology (2015) 172 247–250

receptor agonists on cutaneous wound healing (Bigliardi

et al., 2015).

Ultimately, the value of much of the knowledge of novel

opioid mechanisms in the themed issue will be its translation

into clinical practice Avoiding tolerance and dependence,

severe side effects and improving efficacy in chronic pain

conditions all seem possible but there is a long way to go For

example, careful meta-analyses of weak and strong opioid use

in chronic non-cancer pain (Reinecke et al., 2015) found only

modest trends for efficacy of opioids and no evidence to

support the sole or preferential use of opioids Hopefully

drugs exploiting novel opioid mechanisms will be better

Opioid agonists and antagonists also have an important place

in management of addictions For reviews of opioid

treat-ments for addiction in humans the reader is referred to the

recent issue of British Journal of Clinical Pharmacology on

Addiction (2014, vol 77, Issue 2 Pp 225–400) In particular,

articles by Bell (2014) and Garcia-Portilla et al (2015) on

maintenance treatments for opioid addiction and sustained

release naltrexone for the management of opioid dependence

(Kunøe et al., 2014).

Current opioid therapeutics for chronic pain

manage-ment and addiction are problematic and still largely rely on

drugs developed many decades ago There is hope that

find-ings in this themed issue will lead to the development of new

generation opioid analgesics with improved clinical profiles

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250 British Journal of Pharmacology (2015) 172 247–250

Themed Section: Opioids: New Pathways to Functional Selectivity

REVIEW

Comparison and analysis of

the animal models used to

study the effect of

morphine on tumour

growth and metastasis

B Afsharimani1, C W Doornebal2, P J Cabot1, M W Hollmann2and

pain management of cancer surgery patients The literature presents conflicting and inconclusive in vitro and in vivo data

about the potential effect of opioids, especially morphine, on tumour growth and metastasis To inform clinical practice,appropriate animal models are needed to test whether opioids alter the course of tumour growth and metastasis Here, wereview the literature on animal-based studies testing the effect of morphine on cancer so far, and analyse differences betweenthe models used that may explain the discrepancies in published results Such analysis should elucidate the role of opioids incancer and help define ideal pre-clinical models to provide definitive answers

Morphine and other opioid analgesics are potent

pain-relieving agents that are essential for pain management in

cancer patients (Dalal and Bruera, 2013) Besides being the

standard of care for the treatment of cancer-related pain in

patients with advanced stage disease, opioids – especially

morphine – are also routinely used for anaesthetic procedures

in cancer patients undergoing surgery However, there have

been concerns that they may affect the rate of post-operative

cancer recurrence and metastasis (Afsharimani et al., 2011).

Recent retrospective clinical studies evaluating the effects of

anaesthetic technique on relapse-free survival after cancer

surgery indicated that cancer patients receiving perioperative

morphine-based analgesia had a worse prognosis compared

with those receiving loco-regional anaesthesia (Exadaktylos

et al., 2006; Biki et al., 2008) Based upon these findings,

mor-phine and other opioid analgesics have been postulated topromote cancer progression and relapse (Heaney and Buggy,2012) Although still rather controversial, these studies col-lectively raised the question of whether the anaesthetic tech-nique applied during cancer surgery might affect relapse-freesurvival after surgery (Sessler, 2008; Singleton and Moss,2010)

To resolve this controversy, several randomized clinicaltrials in breast, lung and prostate cancer patients have beeninitiated These clinical studies are designed to directlycompare relapse-free survival after cancer surgery in patientsreceiving either loco-regional anaesthesia or perioperativemorphine-based analgesia Yet, given their design, these

studies will not allow assessment of any potential

tumour-promoting effects of morphine-based analgesia To address

this question, we need to rely upon in vivo studies that

evalu-ate the effects of morphine on tumour progression and

meta-static disease in a well-controlled experimental setting In this

review, we summarize the currently available data from

pre-clinical studies evaluating the effects of morphine on tumour

growth and metastatic disease Interestingly, results from

these studies show discrepant results ranging from

deleteri-ous, null to protective effects for morphine This review

criti-cally evaluates the models that have been used, in an attempt

to elucidate the parameters that may explain these

discrep-ancies and therefore shed some light on the role of morphine

in cancer To support future research, we further discuss some

essential characteristics that should be met by pre-clinical

models in order to address this question in a clinically

rel-evant setting

The tumour models used

To evaluate the effects of morphine on tumour progression

and metastatic disease, a wide variety of pre-clinical models

have been employed As shown in Table 1, most studies are

performed with cancer cell line-based tumour models In

these models, in vitro maintained cancer cell lines are

trans-planted either orthotopically (in the anatomic location of

origin for this specific tumour cell line) or ectopically (in

another organ or location), or injected i.v into hosts

Unfor-tunately, these models present considerable shortcomings, as

they do not faithfully reproduce de novo tumourigenesis and

metastatic disease in humans For example, cancer cell lines,

maintained in vitro, often fail to reflect the original

heteroge-neity of the parental tumour (Keller et al., 2010; Domcke

et al., 2013) As intra-tumour heterogeneity corresponds to a

wide phenotypic variety and at least partially determines

clinically relevant tumour-related features including the

ability to seed and responses to therapy (Marusyk et al.,

2012), data from such studies cannot easily be extrapolated to

the clinical setting

Most studies evaluating the impact of morphine on

meta-static disease have not used orthotopic tumour models Most

of them utilize s.c tumour cell inoculation or tail vein

injec-tion assays Tail vein injecinjec-tions of tumour cells have been

used in rats (Yeager and Colacchio, 1991; Page et al., 1993;

1994; 1998; Colacchio et al., 1994; Bar-Yosef et al., 2001;

Franchi et al., 2007) and mice (Harimaya et al., 2002;

Afsharimani et al., 2014) with measurement of the tumour

burden in the lungs or liver These models attempt to mimic

homing and outgrowth of circulating tumour cells or cells

released during surgery, at distant sites However, cultured,

usually adherent, tumour cells are likely to be different from

the circulating cells that are found in increased numbers in

patients undergoing surgery, as well as spontaneous

circulat-ing tumour cells (Thompson and Haviv, 2011) Furthermore,

these models fail to reproduce the biology of de novo

meta-static disease (Fantozzi and Christofori, 2006; Jonkers and

Derksen, 2007; Valastyan and Weinberg, 2011) These defects

are further complicated by the fact that most inoculated

tumour cells are likely to undergo apoptosis The massive

release of tumour-related antigens may induce acute adaptive

anti-tumour immune responses, which are normally absentdue to the formation of immuno-suppressive networksdriving escape from immune surveillance in spontaneously

arising tumours (Willimsky et al., 2008) Consequently, the

efficacy of immune surveillance may be overestimated incancer cell line-based tumour models

Orthotopic models (Gupta et al., 2002) appear

appropri-ate if the objective of the study is to assess the effect ofmorphine on the growth of a primary tumour Moreover,spontaneously metastasizing models have been proposed topresent the advantage of allowing the study of the effect ofmorphine on metastasis A major factor that needs to betaken into consideration is whether the animals are immuno-competent Immunocompromised mice must be used when

allogeneic tumour cells are implanted (Gupta et al., 2002; Tegeder et al., 2003; Roy et al., 2006), and, while this allows

the study of cancer cells of human origin, the effects ofopioids on the immune response are underestimated in suchmodels This is of paramount importance, as accumulatingevidence indicates that the immune system plays a crucialrole both at the level of the primary tumour and at distant,

metastatic sites (de Visser et al., 2006; Joyce and Pollard,

inflam-metastatic process (Talmadge et al., 1980) The inclusion of

pain or surgical stress into an animal model of tumour celldissemination and growth is thus a major factor that willinfluence the role of opioids and the experimental outcome

Indeed, with few exceptions (Colacchio et al., 1994; Farooqui

et al., 2007), morphine affords protection towards tumour

growth or dissemination in the context of pain and surgicalstress – elicited intentionally by laparotomy or tumour-induced hyperalgesia, or unintentionally, for example, bysurgical insertion of drug-releasing pellets – but not in theabsence of pain (Simon and Arbo, 1986; Yeager and

Colacchio, 1991; Page et al., 1993; 1994; 1998; Bar-Yosef et al., 2001; Sasamura et al., 2002; Franchi et al., 2007) A model

with no pain can specifically reveal the non-analgesic effects

of morphine In contrast, an animal tumour model, whichincludes pain or stress response to surgery, is better suited torepresent the perioperative period in humans but does notallow dissection of the mechanisms (analgesia-mediated or-independent) of morphine’s actions

Designing new animal models to evaluate the effect of morphine on tumour growth and metastasis

Given these considerations, how should models be designed

to study the effects of morphine on tumour growth andBJP B Afsharimani et al.

252 British Journal of Pharmacology (2015) 172 251–259

Increased tumour burden Colacchio et al (1994)

A/J mice are

immuno-competent but present

Increased tumour weightand presence ofmetastases

Yes (laparotomy) 10 mg·kg−1immediately

and 5 h after surgery

Increased lung diffusion oftumour cells in theabsence of surgery

Slightly decreased tumourload (non-statisticallysignificant) in thepresence of surgery

Yes (laparotomy) 20 mg·kg−1morphine s.c

1 day before and 2 daysafter tumour inoculation

Reduced tumour burden Yeager and Colacchio

5–10 mg·kg−1s.c inslow-release suspension

5 h after surgery

Reduced tumour burden

in the presence ofsurgical stress

No effect in the absence

Yes (laparotomy) 10 mg·kg−1i.p 30 min

before surgery and

5 mg·kg−1s.c inslow-release suspensionafter surgery

Reduced tumour burden

in the presence ofsurgical stress,

No effect in the absence

of surgical stress

Page et al (1994)

metastasis? To address this question, pre-clinical tumour

models that most closely mimic the clinical setting must be

carefully designed To study the effect of morphine on

metas-tasis independent of the surgery, one approach may be to

evaluate the effects of morphine on genetically engineered

mouse models of de novo tumourigenesis, which have been

used successfully to study many aspects of tumour biology

(Frese and Tuveson, 2007) These models are generated bytissue-specific manipulation of genes known to be relevant in

a certain subtype of human cancer and allow the study ofspontaneously arising tumours that closely mimic theirhuman counterparts in an orthotopic, immuno-competent

setting However, with some exceptions (Muller et al., 1988; Boggio et al., 1998; Paez-Ribes et al., 2009), employing geneti-

Yes (laparotomy) 8 mg·kg−1i.p 30 min

before surgery and/or

4 mg·kg−1s.c

immediately aftersurgery in a slow-releasesuspension and/or

2 mg·kg−1s.c in aslow-release suspension

5 h after surgery

Reduced lung tumourburden in the presence

of surgery in alltreatment groups

Ectopic (melanoma cells

s.c in hind paw) even

though the authors

claim orthotopic

Yes(tumour-inducedhyperalgesia)

5 and 10 mg·kg−1s.c

daily for 6 days (days16–21 post-inoculation)Analgesia was

50–60μM at 10–25 min,0.9–3.4μM at 1–2 h

Decreased tumour volumefor breast cancer celllines MCF7 andMDA-MB231, no effectfor colon cancer HT-29

the right flank s.c.)

Not intentionally (butsurgical insertion

of the pellets)

Day of tumourinoculation: 75 mgmorphine pelletsimplanted days 7–14

cally engineered mouse models to study metastatic disease is

complicated by asynchroneously arising, rapidly growing,

primary tumours that do not allow sufficient time for the

establishment of (advanced) metastatic disease (Francia et al.,

2011) As a consequence, these models generally show a

rela-tively low incidence of metastatic disease and do not allow

the effects of morphine on advanced metastatic disease to be

analysed Another condition is required to better mimic the

perioperative setting, which is that the animal model should

include a surgical intervention, either primary tumour

resec-tion or a more artificial event, inducing surgical stress, tissue

damage and pain

To circumvent these limitations, and to provide

informa-tion relevant to the context of cancer surgery patients, we

have recently developed a pre-clinical mouse model of de

novo breast cancer metastasis formation (Doornebal et al.,

2013) In this model, small tumour fragments of a de novo

mouse mammary tumour (Derksen et al., 2006) are

orthotopi-cally transplanted into wild-type recipients Once mammary

tumours are established, a mastectomy is performed and the

mammary tumour is surgically resected Following surgery,

these mice spontaneously develop clinically overt metastatic

disease in lungs, liver, spleen and lymph nodes Using a

similar approach to exploit other genetically engineered

mouse models provides a unique opportunity to create

models that not only reproduce the biology of de novo

meta-static disease but also allows the evaluation of the effects of

morphine using clinically defined outcomes – that is,

metastasis-specific survival – in a context that closely mimics

the perioperative setting

The dose and mode of administration

of morphine used

A wide range of morphine doses have been used in the

pre-clinical experiments testing its effect on tumour growth and

metastasis (Table 1), which may contribute to the differences

in outcome of these studies It has been proposed that low,

sub-analgesic doses of morphine have mitogenic and

angio-genic properties (Tegeder and Geisslinger, 2004) Most studies

and very few (Tegeder et al., 2003) verify the resulting

receptors are not critically different between humans and

mice (Kidatabase at http://pdsp.med.unc.edu/) However, as

previously noted (Parat, 2013), rodents metabolize morphine

differently from humans and produce mostly

morphine-3-glucuronide (M3G) (Kuo et al., 1991), which is not analgesic

(Shimomura et al., 1971) In contrast, humans produce not

only M3G but also morphine-6-glucuronide (M6G), which is

a more potent analgesic than morphine (Shimomura et al.,

1971; Osborne et al., 1988; 1990) To achieve analgesia, doses

of morphine (in mg·kg−1) are therefore much higher in mice

than humans The effect of morphine per se can only be

compared between rodents and humans, if the circulating

(and presumably tissue) concentrations of morphine are

similar Furthermore, given that pain influences tumour

growth and metastasis (Page et al., 2001), it is important to

note whether the dose of morphine employed in rodent

models is actually producing analgesia, especially if themodel includes pain In addition, the metabolite M3G, pre-dominantly produced in rodents, might have non-opioidreceptor-mediated activities (see below)

Lastly, the continuity of delivery (i.e osmotic pumps ormorphine-releasing pellets vs injections at time intervals)and the duration of morphine treatment both differ betweenstudies This may be important if the effect of morphine ontumours is mediated by mechanisms subject to tolerance and

withdrawal, such as the immune function (West et al., 1998; Eisenstein et al., 2006) Indeed, in contrast to continuous

administration by constant infusion or slow-release pellets,intermittent administration of morphine (every 12 h for 4days) to rats was characterized as a chronic stressor, inducingwithdrawal-like conditions in each interval and increasingthe hypothalamic–pituitary–adrenal (HPA) axis response to

novel stimuli (Houshyar et al., 2003; 2004) Activation of the

HPA axis is known to facilitate cancer progression and

metas-tasis (Li et al., 2013), via many mechanisms, including pression of cell-mediated immunity (Benish et al., 2008), promotion of angiogenesis (Yang et al., 2009) and direct action on cancer cells (Bernabe et al., 2011) Only a few

sup-studies have tested the effect of continuous administration of

morphine on tumour growth and metastasis Koodie et al used morphine-releasing pellets, and the studies by Page et al.

mention s.c injection of morphine in a slow-release sion They all resulted in anti-tumour ,rather than pro-

suspen-tumour effects (Page et al., 1993; 1994; 1998; Koodie et al.,

2010) Implantation of a 75 mg morphine-releasing pellet

Patient-controlled analgesia with morphine, often used inpost-operative pain management, was suggested, using phar-macokinetic simulation, to result in relatively stable effect-site concentrations of morphine and its metabolites M3G and

M6G in patients (Sam et al., 2011), and animal models should

therefore mimic this continuity Of the animal studies ontumour growth and metastasis that employed doses of mor-phine escalating over the course of the treatment to account

for the development of tolerance (Gupta et al., 2002; Tegeder

et al., 2003; Farooqui et al., 2007; Koodie et al., 2010), only

those using high doses (Tegeder et al., 2003; Koodie et al.,

2010) demonstrated anti-tumour effects of morphine Takentogether, these observations indicate that continuous admin-istration of high doses of morphine that produce analgesia ismore likely to result in prevention of tumour growth andmetastasis, in rodent models

The receptors involved

We have limited inclusion in Table 1 to studies measuringtumour growth and metastasis in animals treated with mor-phine, but it should be noted that further studies using

receptor (nomenclature follows Alexander et al., 2013) have

been carried out Overexpression and down-regulation of theμ-opioid receptor in cancer cells before injecting them intomice were shown to increase and decrease, respectively,primary tumour growth and metastasis in mice expressing

μ-opioid receptors (Biji et al., 2011; Lennon et al., 2012) In

addition, infusion of theμ-opioid receptor antagonist

meth-ylnaltrexone reduced tumour growth and metastasis in

wild-type mice (Biji et al., 2011) Growth of cancer cells expressing

μ-opioid receptors in mice lacking μ-opioid receptors

(knockout mice) was also reduced compared with that in

wild-type mice This indicates thatμ-opioid receptor

activa-tion on both tumour cells and cells of the host may promote

tumour growth and metastasis However, neither of these

studies have included evidence that morphine increases

tumour growth and metastasis in vivo (Biji et al., 2011;

Lennon et al., 2012).

Very little is known about the possible consequences of

μ-opioid receptor dimerization on cancer A role for μ- and

δ-opioid receptor heterodimerization has been suggested in

natural killer cells, in terms of their cytolytic function, with

reciprocal regulation of each receptor homodimerization and

potential consequences on tumour growth (Sarkar et al.,

2012) In addition, activation of opioid receptors other than

μ-receptors may contribute to the role of morphine in cancer,

depending upon the doses of morphine involved Expression

ofμ-, δ- and κ-opioid receptors has been detected in cancer

cell lines and in tumour tissue (Nylund et al., 2008; Tang

et al., 2013; Zhang et al., 2013; Zylla et al., 2013), and some

studies suggest that opioid receptors in tumours are

up-regulated, compared with control tissue (Madar et al.,

2007; Biji et al., 2011; Tang et al., 2013; Zhang et al., 2013) In

situ detection of opioid receptor expression in tumour stroma

is lacking, although endothelial, immune and fibroblast cells

are known to express opioid receptors in non-tumour

con-texts (Stefano et al., 1995; Sharp, 2006; Cheng et al., 2008).

Similarly, endogenous opioids can be produced by cancer

cells and are detected in some tumours (Bostwick et al., 1987;

Krajnik et al., 2010) where they modulate cancer progression

(Boehncke et al., 2011) presumably via regulation of

tumour-associated immune cells (Ohmori et al., 2009; Boehncke et al.,

2011) Lastly, whetherμ-opioid receptor alternative splicing,

which results in multiple variants in both humans and mice,

modulates tumour growth is underexplored

A growing amount of studies looking for

non-GPCR-mediated actions of opioids on immune pathways have

iden-tified that the Toll-like receptor 4 (TLR4), which is activated

by LPS produced by bacteria, may respond to opioids One

group has proposed that opioid receptor ligands produce a

slight but significant activation (morphine) or antagonism

(naloxone) of the TLR4, in a non-stereospecific fashion, i.e

the (+) enantiomers were active at TLR4 receptors but not at

opioid receptors (Wang et al., 2012) In contrast, others have

suggested that morphine produced, by itself, a slight

activa-tion of TLR4, but inhibited TLR4 activaactiva-tion by LPS in a

non-competitive fashion, as did naloxone (Stevens et al.,

2013) Interestingly, M3G, which has limited opioid receptor

activity (Ulens et al., 2001), induced activation of TLR4 (Lewis

et al., 2010; Due et al., 2012) This might be of considerable

importance if TLR4 mediates some of the effects of opioids on

cancer growth and metastasis, as rodent models employ high

doses of morphine that result in high doses of M3G in the

circulation (Zelcer et al., 2005) and, presumably, at tissue

level

Lastly, a variety of mouse strains are used in experiments

testing the effect of morphine on tumour growth and

metas-tasis, and they may respond differently to the drug since it isknown that different mouse strains exhibit polymorphisms

in the 5′ flanking region and 3′ untranslated region of theμ-opioid receptor gene that are associated with differences inopioid sensitivity (measured as locomotor hyperactivity and

antinociception) (Shigeta et al., 2008) The

immunosuppres-sive effects of morphine are also likely to vary between mousestrains This was clearly shown for the direct effect of mor-

phine on mouse spleen cells (Eisenstein et al., 1995).

The cells targeted by morphine

A major question remains whether the putative effects ofmorphine on tumour growth and metastasis might be medi-ated by direct activation of cellular receptors or indirectlymediated by morphine-initiated effects that lead to therelease of secondary factors The cells on which morphine canact directly to modulate the growth and metastasis oftumours include the cancer cells as well as other cell typessuch as immune cells, and cells of the tumour microenviron-ment such as tumour-associated macrophages and endothe-lial cells Experiments employing disruption of theμ-opioidreceptor show that opioid receptor activation on the cancercells injected into the mice as well as the cells of the tumour-bearing animal can interfere with tumour growth and metas-

tasis (Biji et al., 2011; Lennon et al., 2012).

Much of the literature on the effect of morphine on theimmune response has assessed the functions of immune cellscollected from mice or humans after they were given mor-phine, thereby testing potential indirect and direct effects ofmorphine on those cells However, morphine added to

immune cells ex vivo also showed some direct effects (Eisenstein et al., 1995; Condevaux et al., 2001; Malik et al., 2002; Fuggetta et al., 2005) Macrophage phagocytic ability

was inhibited by acute, but not chronic, direct exposure to

morphine in vitro (Casellas et al., 1991; Tomei and Renaud,

1997) This phenomenon occurred via activation of opioid

receptors (Tomassini et al., 2003) and was subject to ‘in vitro

withdrawal’ (Tomei and Renaud, 1997) In co-cultures oftumour cells with macrophages, morphine prevented parac-rine communication through which macrophages couldpromote the production of matrix-degrading enzymes by the

tumour cells (Afsharimani et al., 2014) A direct effect of

mor-phine on endothelial cells has also been proposed (Gupta

et al., 2002; Singleton et al., 2006; Leo et al., 2009) and

sug-gests pro-angiogenic properties for low concentrations ofmorphine All these reports suggest that some of the effects of

morphine in vivo might be mediated by direct action on the

immune or endothelial cells

In line with in vivo data showing that the dose and mode

of administration influenced the effect of morphine ontumours, at the cellular level, responses that may be involved

in tumour progression, such as proliferation or apoptosis, orimmune cell responses, have also been shown to dependupon the concentration of morphine applied, with low dosespromoting cell proliferation and high doses promoting apo-ptosis, and to be susceptible to development of tolerance andreceptor desensitization (see Tegeder and Geisslinger, 2004;

Eisenstein et al., 2006).

BJP B Afsharimani et al.

256 British Journal of Pharmacology (2015) 172 251–259

Conclusion and perspectives

To extrapolate animal experimental data to human patients,

mouse models used to study the effects of morphine on

tumour growth and metastasis should adhere to the

follow-ing criteria The mice should spontaneously develop

ortho-topic primary tumours in an immuno-competent setting In

addition, the tumour models should reproduce the biology of

de novo metastatic disease To relate the animal data to

perio-perative use of morphine in cancer surgery patients, surgical

resection of the primary tumour is desirable as part of the

model The doses of morphine used should be analgesic in

mice and the duration of morphine exposure should match

post-operative analgesia regimens, avoiding unnecessary

withdrawal as much as possible

Overall, the current literature does not provide definitive

evidence for a modulation of tumour growth and metastasis

by morphine Morphine might modulate tumour growth and

metastasis though a combination of direct (on cells) and

indirect (neuroendocrine) responses, central and peripheral

mechanisms and modulation of physiopathological

func-tions key to tumour development, such as inflammation,

stress and pain It is further likely that the effects of morphine

are in addition to the effects of endogenous opioids and are

regulated by tolerance and withdrawal responses The

dis-crepancies found in the literature are thus not surprising, and

refining the animal models that we use, on the basis of all

these criteria, will hopefully provide, in the future, definitive

answers than can be taken into consideration for patient care

Acknowledgements

M.-O P and P J C acknowledge the financial support of the

Australian and New Zealand College of Anaesthetists

Conflict of interest

The authors declare no conflict of interest

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Themed Section: Opioids: New Pathways to Functional Selectivity

mechanisms by which acute and chronic activation ofμ-opioid receptors by morphine and other opioid drugs modify

DAMGO, [D-Ala2,NMe-Phe4,gly-ol5]-enkephalin; GIRK, G protein-activated potassium conductance; GRK,

G protein-coupled receptor kinase; INRC, International Narcotics Research Conference; LC, locus coeruleus; Met Enk,methionine enkephalin; PAG, periaqueductal grey region; VTA, ventral tegmental area

This perspective is based on the author’s Founders’ Lecture

delivered at the 2013 International Narcotics Research

Con-ference (INRC) The aim was to review the contribution that

electrophysiological recording techniques have made over

the past 40 years to elucidating the actions of opioid drugs on

neurones of the CNS This is not intended to be a

conforms to Alexander et al., 2013) pharmacology rather it

reflects somewhat the author’s scientific journey and so

apologies are due to those whose work is not cited

μ-Opioid receptor activation

Interaction with potassium and

calcium channels

In the early 1970s experiments using extracellular recording

from brain neurones in vivo led to reports such as the

follow-ing – ‘Out of 76 neurones studied, morphine [applied by

iontophoresis] increased the firing rate of 33 and depressed

that of 17 The remaining 26 neurones were unaffected’

(Bradley and Dray, 1974, p 48) It was the introduction ofintracellular recording that enabled more sophisticatedanalysis of opioid action, first with the use of sharp electroderecording of membrane potential and single-electrodevoltage clamp then with patch clamp recording of whole-celland single-channel currents In the mid-1970s in Aberdeen,the late Hans Kosterlitz, one of the founders of INRC, withgreat foresight encouraged Alan North and myself to studyopioid action by recording from opioid-sensitive neurones.This led to the observation that activation ofμ-opioid recep-tors resulted in membrane hyperpolarization throughopening of potassium channels in guinea pig myentericplexus neurones (North and Tonini, 1977) and guinea pig andrat locus coeruleus (LC) neurones (Figure 1A; Pepper and

Henderson, 1980; Williams et al., 1982).

The opioid-activated potassium conductance in LC rones was subsequently characterized as inwardly rectifying(North and Williams, 1985) and, as the coupling from recep-tor to channel is through pertussis toxin-sensitive G-proteins,

neu-is now referred to as a G-protein-activated inwardly rectifyingpotassium conductance (GIRK) We now know from studies

in other types of neurones thatμ-opioid receptors can couple

BJP British Journal of

260 British Journal of Pharmacology (2015) 172 260–267 © 2014 The British Pharmacological Society

to a variety potassium channels including calcium-activated,

channels (for review, see Williams et al., 2001) The relative

importance of each opioid-sensitive potassium channel in

behavioural responses to opioids remains to be elucidated

fully

Around the same time as studies on the opioid-activated

GIRK current were progressing, it was reported that in

cul-tured dorsal root ganglion cells opioids reduced the calcium

component of the action potential without activating a

potas-sium conductance (Mudge et al., 1979) However, it was not

until some time later that two groups, using whole-cell patchclamp recording, demonstrated thatμ-opioid receptor activa-tion resulted in G-protein-mediated inhibition of voltage-

activated N-type calcium channels (Schroeder et al., 1991; Seward et al., 1991) P-type and Q/R-types of voltage-activated

calcium channel have also now been shown to be inhibited.Other Gi/o-coupled receptors couple to the same ion chan-

-Figure 1

Milestones in electrophysiological studies ofμ-opioid receptor function in LC neurones over 33 years (1980–2013) (A) The first publishedmembrane hyperpolarization in response to opioid activation of theμ-opioid receptor in an LC neurone Reproduced with permission from Pepperand Henderson (1980) (B) Rapid morphine-induced desensitization of μ-opioid receptor-induced GIRK current in LC neurones requiresconcomitant PKC activation by stimulation of M3muscarinic receptors Reproduced with permission from Bailey et al (2004) (C) Off rate of

agonist binding fromμ-opioid receptors on LC neurones measured by the decrease in each opioid-evoked GIRK current following local, flash

release of naloxone from a caged derivative Traces supplied by J.T Williams, Vollum Institute; experimental details are as in Banghart et al (2013).

The amplitudes of currents in (B) and (C) have been normalized to facilitate comparison

coupled receptor do the different receptor types share pools of

G-protein and ion channels or do they function

indepen-dently? This was a long time before we knew of GPCR

dimeri-zation and scaffolding proteins North and Williams (1985)

observed in LC neurones that the GIRK currents activated by

μ-opioid receptors and α2-adrenoceptors were non-additive

not, however, determine whether it was the pool of G-protein

or the pool of GIRK channels that was limiting More recently,

this question has been re-addressed by John Traynor’s

labora-tory studying inhibition of adenylyl cyclase rather than

potas-sium or calcium channels (Levitt et al., 2011) They used the

‘neuronal’ cell line, SH-SY5Y, in which a number of

endog-enously Gi/o-coupled receptors, includingμ-opioid receptors,

δ-opioid receptors and α2-adrenoceptors, are expressed and

share a common pool of adenylyl cyclase and concluded that

the limiting factor is the G-protein rather than adenylyl

cyclase becauseδ-opioid receptor activation did not increase

GTPγS binding after it had been maximally stimulated

with theμ-opioid receptor agonist [D-Ala2,NMe-Phe4,gly-ol5

]-enkephalin (DAMGO)

Opioid excitation

One might predict that if opioids activate potassium

conduct-ances (which will lead to membrane hyperpolarization) and

inhibit calcium entry during action potential firing, which

would be expected to inhibit neurotransmitter release, then

should be inhibitory Such inhibition is seen at the level of

the dorsal horn of the spinal cord and in the LC However,

Nicoll et al (1977) reported that opioids excite hippocampal

pyramidal neurones This gave rise to a flurry of activity as

well as to several divergent, contradictory hypotheses on how

such excitation was produced (for historical review, see

Henderson, 1983) At the time it was difficult to rationalize

the various theories because the results reported from

differ-ent laboratories were often contradictory, a common theme

in opioid research With the passage of time, however, things

have become clearer and there is now general agreement that

activation results in excitation, not only in the hippocampus

but also in other brain regions important in the analgesic and

euphoric actions of opioids [i.e the periaqueductal grey

region (PAG) and ventral tegmental area (VTA)], is by

disin-hibition whereby opioids act onμ-opioid receptors located on

inhibitory interneurones (usually GABAergic interneurones)

and reduce inhibitory tone resulting in apparent excitation of

the output neurone (Johnson and North, 1992; Vaughan and

Christie, 1997)

Opioid inhibition of transmitter release

μ-Opioid receptor activation results in inhibition of the

release of numerous neurotransmitters from nerve terminals

in both the peripheral and central nervous systems There has

been much debate about the relative importance of

potas-sium channel activation and inhibition of voltage-activated

receptor-mediated inhibition of neurotransmitter release (see e.g Shen

and Surprenant, 1990; Vaughan and Christie, 1997; Vaughan

et al., 1997) It would appear thatμ-opioid receptor activationcan also directly inhibit the neurotransmitter release machin-ery independent of any effect on membrane conductances

(Capogna et al., 1993) given thatμ-opioid receptor activationreduced the miniature GABAergic inhibitory synaptic cur-rents evoked by the calcium ionophore ionomycin, that is, by

direct calcium entry into the nerve terminals (Capogna et al., 1996; Bergevin et al., 2002).

Endogenous opioid peptide activation of μ-opioid receptors

In many areas of the CNS opioid peptide-containing nerveterminals can be seen to form axo-dendritic as well as axo-axonic synapses In the LC methionine enkephalin (MetEnk)-containing nerve terminals form synapses on to tyros-

ine hydroxylase-containing dendrites (Pickel et al., 1979; Van Bockstaele et al., 1995) In addition the LC receives a

β-endorphin-containing input from neurones whose cellbodies lie in the arcuate nucleus Although it has been

reported that stimulation of the arcuate nucleus in vivo

pro-duces a naloxone-sensitive inhibition of neuronal firing in

the LC (Strahlendorf et al., 1980), several investigators

studying synaptic transmission in LC slices have failed toobserve any endogenous opioid-mediated inhibitory postsy-

naptic responses (see e.g Egan et al., 1983) This has been

mirrored in studies of other opioid peptide containing brainregions

In contrast, in a number of brain regions including pocampal CA1 region and dentate gyrus (see Simmons andChavkin, 1996) and the amygdala (E Bagley, pers comm.),endogenous opioid peptides released on nerve stimulationhave been shown to act at presynapticμ-opioid receptors aswell asδ- and κ-opioid receptors to inhibit the release of otherneurotransmitters This is not to say that endogenous opioidsonly mediate presynaptic inhibition but, similar to nicotinicand P2X ligand-gated ion channels in the CNS, it wouldappear that presynaptic effects may predominate (Khakh andHenderson, 2000)

hip-Tolerance and dependence

Following two decades of studying primarily the acuteactions of opioids on brain neurones, the focus of muchelectrophysiological research on opioids moved on to study-ing adaptive responses that occur as a result of long-term

some way to elucidating the adaptive changes that underlieopioid tolerance and physical dependence, but many ques-tions remain unanswered

μ-Opioid receptor desensitization

One advantage of electrophysiological recording is that itprovides real-time readout of receptor–effector couplingduring prolonged agonist application (Figure 1B) Another isthat it allows comparison between different, sometimes smallpopulations of neurones that would be difficult with a tech-nique such as GTPγS binding However, unlike GTPγS bindingassays, changes inμ-opioid receptor coupling to ion channels

262 British Journal of Pharmacology (2015) 172 260–267

can occur at the receptor, G-protein or ion channel level and

care must be taken to determine which of these components

has been altered

Numerous kinases have been implicated in neuronal

μ-opioid receptor desensitization and opioid tolerance

includ-ing G protein-coupled receptor kinases (GRKs), PKC isoforms,

JNK and ERK The exact roles of each kinase and the

mecha-nisms by which they contribute toμ-opioid receptor

desensi-tization have still to be worked out For a detailed discussion

of the evidence for the involvement of each of these kinases in

μ-opioid receptor desensitization and opioid tolerance,

readers are referred to the extensive review by Williams et al.

(2013) Here I will focus primarily on two: PKC and GRK

Although highly effective in producing analgesia and

res-piratory depression, in the whole animal morphine has lower

agonist intrinsic efficacy atμ-opioid receptor than drugs such

as methadone, fentanyl and DAMGO (McPherson et al.,

2010) In LC neurones in vitro morphine induced much less

μ-opioid receptor desensitization than higher efficacy opioid

agonists (Alvarez et al., 2002; Bailey et al., 2003) The level of

morphine-induced desensitization could be enhanced by

concomitant activation of PKC either indirectly by

stimula-tion of Gq-coupled M3muscarinic receptors on LC neurones

(Figure 1B) or directly with a phorbol ester (Bailey et al.,

2004) In both trigeminal and nucleus accumbens neurones

enhanced PKC activity (Chen and Huang, 1991; Martin et al.,

1997), but in LC neurones there was no such directμ-opioid

receptor-mediated enhancement of PKC (Oleskevich et al.,

desensitization PKC activity had to be increased by other

means This may be due to differential expression of PKC

isoforms in different neuronal populations – the isoform

responsible forμ-opioid receptor desensitization in LC

neu-rones is PKCα (Bailey et al., 2009a) – or PKC activity being low

in LC neurones when they are in a brain slice, rather than the

brain in vivo It would be of interest to determine whether

morphine induces a PKC-dependent desensitization of

μ-opioid receptors in trigeminal and nucleus accumbens

neu-rones without the requirement for additional PKC activation

to enhance desensitization or acts indirectly to facilitate some

other desensitization mechanism still needs to be determined

(see Bailey et al., 2006; Johnson et al., 2006).

been a tendency in the literature to conflate the processes of

μ-opioid receptor desensitization and trafficking (both

inter-nalization and reinsertion into the plasma membrane) by

assuming (i) that desensitization and internalization will

occur sequentially by the same mechanism and (ii) that GRK

receptor desensitization as well as internalization Although

there is little doubt that a GRK- and arrestin-dependent

signalling as well as in its trafficking in response to occupancy

by high-efficacy agonists such as DAMGO and Met Enk in

recombinant expression systems, the role of GRKs and

arrestins inμ-opioid receptor desensitization in neurones by

such drugs is still contentious (see Williams et al., 2013) In

electrophysiological studies of CNS neurones, various

experi-mental approaches have been used by different investigators

to inhibit GRK activity (e.g intracellular perfusion withpeptide and small-molecule inhibitors, viral overexpression

of dominant negative mutant GRKs and transgenic cation of specific GRKs to render them sensitive to chemicalinhibition) and these have provided contradictory results.Endomorphin-2, which has similar low-agonist efficacy tomorphine in GTPγS binding assays (McPherson et al., 2010),inducesμ-opioid receptor desensitization in LC neurones in

modifi-the absence of PKC activation (Rivero et al., 2012), suggesting

that agonist efficacy for G-protein activation is not the minant of which desensitization pathway an agonist willinduce That endomorphin-2 is an arrestin-biased opioid

deter-agonist (Rivero et al., 2012) could be taken to indicate that

arrestin binding is involved inμ-opioid receptor tion Unconditional arrestin3 knockout, however, has been

desen-sitization unaffected in sensory neurones (Walwyn et al., 2007) and LC neurones (Arttamangkul et al., 2008), but there

may be other confounding effects of arrestin3 knockout

(Mittal et al., 2012) Also, knockout of only one form of

arrestin may not be sufficient to attenuate desensitization ifboth arrestins can bind to agonist-activatedμ-opioid recep-

tors (Groer et al., 2011) Furthermore, Dang et al (2009) have

suggested that there are in fact two mechanisms underlyinghigh-efficacy agonist-induced μ-opioid receptor desensitiza-tion in LC neurones, a GRK component and an ERK compo-nent Both need to be inhibited concomitantly to reduce MetEnk-induced desensitization (i.e there is redundancy)

Presynaptic μ-opioid receptors

Until very recently, intracellular recordings were invariablymade from relatively large and easily imaged cell somatarather than from small nerve terminals as the somata weremore readily accessible to sharp and patch electrodes This

terminals, the ones important for inhibition of mitter release, more difficult Recording the characteristics ofspontaneous and evoked synaptic responses in the postsyn-aptic cell does, however, give a measure of neurotransmitterrelease, which can be used to study the effect of activatingpresynapticμ-opioid receptors In elegant studies of opioidinhibition of synaptic transmission, several groups have

terminals in the PAG (Fyfe et al., 2010) and VTA (Lowe and

Bailey, 2015) as well as those on the terminals ofβ-endorphin-containing arcuate neurones (Pennock and

Hentges, 2011; Pennock et al., 2012) do not desensitize in

response to acute agonist activation, whereas those on thesomata of the same neurones do desensitize Why this should

be is still unknown but a likely explanation would seem to beexpression of essential components of the acute desensitiza-tion mechanism(s) in the soma but not in nerve terminals

exhibit desensitization on acute exposure to morphine, there

is a loss ofμ-opioid receptor function following chronic phine exposure (i.e tolerance develops) The very recentdescription of a technique by which patch clamp recordingscan be made from nerve terminals of cultured neurones

mor-(Novak et al., 2013) is an exciting development that will

facilitate studies of nerve terminalμ-opioid receptor function

Morphine tolerance

On repeated or prolonged exposure to opioids, tolerance

develops This can be observed not only in intact animals

withμ-opioid receptor-mediated antinociception and

respira-tory depression but also at the level of individual neurones

Building on results from in vivo experiments that had

demonstrated that morphine antinociceptive tolerance was

reversed by PKC inhibition (Smith et al., 2002; 2007), we

observed that cellular tolerance in LC neurones either

follow-ing prolonged exposure of brain slices to morphine in vitro or

in brain slices taken from morphine-treated animals was

reversed by PKC inhibition (Bailey et al., 2009b) Cellular

desensitization (see above), did not require PKC activity to be

enhanced The PKC-mediated cellular tolerance was due to a

loss of μ-opioid receptor function indicating that μ-opioid

receptor desensitization contributes to opioid tolerance

More recently, Levitt and Williams (2012) have shown that

there are in fact two components to cellular tolerance to

morphine in LC neurones, a PKC-mediated, rapidly reversible

component and a second component that does not reverse

rapidly on removal of morphine The second component of

cellular tolerance may be responsible for the tolerance

observed in nerve terminals The mechanism underlying this

second component of tolerance has not yet been elucidated

In a historical context, it is interesting to note that back in

1975 Brian Cox observed that tolerance to the

antinocicep-tive effect of morphine in vivo consisted of a large, rapidly

reversing component and a second, smaller and more

sus-tained component (Cox et al., 1975).

Opioid withdrawal mechanisms

Early in the 1970s the first observations were made of the

μ-opioid receptor being negatively coupled to AC resulting in

decreased production of cAMP Thereupon the late Harry

Collier, another founder of INRC, postulated that opioid

withdrawal might result from the opposite of the acute

response, that is, a rebound increase in the production of

cAMP This was some time before the discovery of forskolin,

and so to increase cAMP levels in the brain, he and his

colleagues used theophylline to inhibit cAMP breakdown by

PDE They observed that in nạve rats (i.e non-morphine

treated) theophylline administration reproduced some of the

symptoms associated with morphine withdrawal (Collier

et al., 1974) Soon after, using an in vitro neurochemical

approach, Sharma et al (1975) demonstrated in NG108-15

cells that morphine withdrawal resulted in a rebound

increase in cAMP production that resulted from

superactiva-tion of AC It might appear churlish to some to point out that

the NG108-15 cells used in this latter study express only

δ-opioid receptors and so the effect observed, which has had

a major impact on our understanding of the mechanisms

underlying physical dependence, was actually made

authors of the original article were not to know that,

however, as theδ-opioid receptor was not discovered until 2

years after the publication of their paper

Over the years it had been observed in vivo that opioid

withdrawal resulted in increased neuronal excitability and

enhanced neurotransmitter release, but it took 30 years for

the link between opioid withdrawal-induced AC

superacti-vation and increased neuronal excitability and enhancedneurotransmitter release to be revealed at the molecularlevel Recording from PAG neurones in brain slices from

mice chronically treated with morphine, Bagley et al (2005)

observed that opioid withdrawal increased the GABAtransporter-1 cation current and that this resulted fromenhanced PKA activity

Is there a future?

Readers may wonder whether after 40 years there are stillscientific questions about μ-opioid receptor function to beanswered using electrophysiological recording techniques.What a stupid idea, of course there are! The combination ofelectrophysiological recording with fluorescence and confo-cal imaging as well as optogenetic techniques provides aneven greater experimental power with which to study opioidresponses on mature neurones Also, being able to recordfrom small neuronal entities such as nerve terminal varicosi-ties and dendritic spines will provide fascinating insights intoreceptor function

Even the LC, which has been very extensively studiedwith regard toμ-opioid receptor function, still has its uses

Recently, Banghart et al (2013) reported on the off rate of

endog-enously expressed on intact LC neurones in an extracellular

environment designed to replicate the brain in vivo

(Figure 1C) No longer do we have to extrapolate from studiesperformed on membrane fragments in Tris buffer lackingsodium ions

An exciting area that I have not even touched on in thisperspective has been the adaptive changes in synaptic effi-cacy that occur in response to drugs of abuse such as stimu-lants and opioids Acute and chronic drug administrationalters both long-term potentiation and long-term depression

in various areas of the brain including the VTA (for review, seeLüscher and Malenka, 2011) Such changes are likely to con-tribute to the intensity of the memory of the drug experience

and relate to craving and relapse (Xia et al., 2011; Van den Oever et al., 2012) There is much still to be understood in

this area

Opioid addicts are notorious polydrug users often takingalcohol, benzodiazepines, cocaine and other drugs in addi-tion to opioids The interaction between opioids and theseother drugs is an under-researched area We have recentlyobserved that relatively low amounts of ethanol can reversetolerance to morphine at both the cellular and whole animal

level (Hull et al., 2013; Llorente et al., 2013), a finding that

may have significance in regard to the frequency with whichethanol and opioids are found in the bloodstream of subjectswho have died from acute overdose

How changes in gene expression induced by chronicopioid exposure alter neuronal function and contribute topsychological and physical dependence as well as to toleranceare subjects that are only beginning to be investigated Adap-tive changes at the level of synaptic remodelling andneurone–glia interactions might be refractory to access byelectrophysiological techniques alone – an important illustra-tion to any budding electrophysiologist of the limitations of

264 British Journal of Pharmacology (2015) 172 260–267

the single experimental technique approach in today’s

science

It was an honour for me to deliver the INRC Founders’

Lecture in 2013 I attended my first opioid scientific meeting

in Aberdeen in 1971 as a young graduate student and had the

pleasure of meeting several of the venerable scientists who

founded the INRC a few years later Perusing the proceedings

of that meeting (Kosterlitz et al., 1971) reminded me of just

how far our understanding of opioid pharmacology has

advanced since that time It would be a brave person who

would try to predict what new discoveries will be made over

the next 40 years

Ultimately, such discoveries need to be harnessed and

used to solve the clinical and social problems surrounding

opioids by facilitating the development of new

thera-peutics Remifentanil, tapentadol, alvimopan and Subutex®,

although useful additions to the pharmacopeia, hardly

rep-resent quantum advances or an adequate return for the time,

effort and resources that has been invested

Heterodimer-selective ligands, biased ligands and allosteric modulators

offer new hope The elucidation of the crystal structures of

the opioid receptors represents a new dawn that will provide

further stimulus for research and drug development

Acknowledgements

The author would like to acknowledge his long-standing

col-laborators, Chris Bailey, Eamonn Kelly and Bill Dewey, for

their influence in developing the views expressed in this

article Work in the author’s laboratory is currently funded by

the MRC

Conflict of interest

The author has no conflicts of interest

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Themed Section: Opioids: New Pathways to Functional Selectivity

Kohei Yamamizu1, Yusuke Hamada2and Minoru Narita2

1Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore,

MD, USA, and2Department of Pharmacology, Hoshi University School of Pharmacy and

Pharmaceutical Sciences, Tokyo, Japan

Correspondence

Dr Kohei Yamamizu, NationalInstitute on Aging, NationalInstitutes of Health, Laboratory

of Genetics, Baltimore, MD, USA.E-mail: kohei.yamamizu@nih.gov

vasculature to sustain homeostasis Disturbance of this balance causes pathogenic angiogenesis and, especially in tumours,several activators such as VEGF are highly expressed in the tumour microenvironment and strongly induce tumour

angiogenesis, the so-called angiogenic switch Recently, we demonstrated thatκ opioid receptor agonists function as

anti-angiogenic factors, which impede the angiogenic switch, in vascular development and tumour angiogenesis by inhibitingthe expression of receptors for VEGF In clinical medicine, angiogenesis inhibitors that target VEGF signalling such as

bevacizumab are used as anti-cancer drugs Although therapies that inhibit tumour angiogenesis have been highly successfulfor tumour therapy, most patients eventually develop resistance to this anti-angiogenic therapy Thus, we must identify noveltargets for anti-angiogenic agents to sustain inhibition of angiogenesis for tumour therapy The regulation of responses toκopioid receptor ligands could be useful for controlling vascular formation under physiological conditions and in cancers, andthus could offer therapeutic benefits beyond the relief of pain

LINKED ARTICLES

This article is part of a themed section on Opioids: New Pathways to Functional Selectivity To view the other articles in thissection visit http://dx.doi.org/10.1111/bph.2015.172.issue-2

Abbreviations

BBB, blood–brain barrier; E, embryonic day; ECs, endothelial cells; ES, embryonic stem; FGF, fibroblast growth factor;

Gi, inhibitory G protein; HIF, hypoxia inducible factor; iPS, induced pluripotent stem; LLC, Lewis lung carcinoma; NRP,neuropilin; TKIs, tyrosine kinase inhibitors; TSP1, thrombospondin1

Introduction

One of the earliest events in organogenesis is the

develop-ment of the vascular system, which contributes to the

forma-tion of most organs in our bodies The vascular system is first

formed as a primitive vascular network by the differentiation

and assembly of vascular progenitor cells derived from

mesodermal cells These progenitor cells undergo a complex

remodelling process, in which growth, migration, sprouting

and pruning lead to the development of a functional

circulatory system Earlier studies have suggested that many

of the events in normal vascular formation during

embryo-genesis are recapitulated during de novo angioembryo-genesis in

adults such as tumour angiogenesis and neovascularizationinduced after tissue damage (Carmeliet, 2003) Furthermore,the disordered vascular function triggers the development oflifestyle-related diseases such as hypertension, diabetes andhyperlipidaemia Thus, a better understanding of vascularbiology may lead to novel strategies for the treatment of avariety of diseases

BJP British Journal of

268 British Journal of Pharmacology (2015) 172 268–276 © 2014 The British Pharmacological Society

It has been recognized for hundreds of years that the

vascular network is closely associated with the neuronal

network throughout development and in adulthood Blood

vessels deliver oxygen and nutrients throughout the body,

and also provide some nerve-related growth factors to guide

neurons to target organs On the other hand, neural tissues

also provide vascular-related growth factors such as VEGF

and members of the Wnt signalling pathways, under

physi-ological conditions, indicating that the vasculature could

communicate with neurons and form complicated

neurons is important for blood vessel ingression, correct

vas-cular density, fine-tuning of the blood vessel pattern and

arterial differentiation (Kutcher et al., 2004; Mukouyama

et al., 2005; James et al., 2009) Members of the Wnt

subfam-ily derived from neurons promote the acquisition of

charac-teristics of the blood–brain barrier (BBB) in intra-neural

vessels (Stenman et al., 2008; Daneman et al., 2009)

Further-more, repulsive axon guidance molecules such as plexin/

semaphorine/neuropilin (NRP) coordinate with VEGF-A

signalling to determine the pattern of blood vessel ingression

in the neural tube (Miao et al., 1999; Bates et al., 2003)

There-fore, vascular–nerve networks play critical roles in vascular

formation in both embryos and adults

Although endogenous opioids were first characterized in

the brain, these transmitters and their receptors (μ κ and δ;

receptor nomenclature follows Alexander et al., 2013a) are

found in both neural (brain and spinal cord) and extraneural

tissues (ganglia, gut, spleen, stomach, lung, pancreas, liver,

heart, blood and blood vessels) Opioids and opioid receptors

are present in blood vessels from the later stages of the rat

embryo [embryonic day (E)-16] through to adulthood (Zagon

et al., 1996; Wu et al., 1998) Treatment with opioid peptides

inhibited both angiogenesis in a chick chorioallantoic

mem-brane model (Blebea et al., 2000) and DNA synthesis in rat

vascular walls (Zagon et al., 1996) In adults, the endogenous

opioid system has been shown to be active in hemodynamic

and cardiovascular responses, such as haemorrhagic shock,

agonist U-50,488H has beneficial effects on vascular injury

after spinal cord trauma by improving vascular permeability

and oedema (Qu et al., 1993) Moreover, morphine, an

agonist atμ opioid receptors, suppresses tumour angiogenesis

through the inhibition of hypoxia-inducible transcription

factors (HIFs), which enhances the expression of VEGF-A and

VEGF receptors (Koodie et al., 2010) These findings suggest

that opioid systems play important roles in vascular

func-tions, although their physiological roles and molecular

mechanisms remain largely unknown

Roles of opioid systems in

vascular development

VEGF signalling in vascular development

Several factors affecting vascular formation, such as VEGF,

NRP, angiopoietins, TGF-β, PDGF, fibroblast growth factor

(FGF), ephrin and notch have been identified over the past

few decades, mainly by the characterization of

vascular-mutant phenotypes in mice Among these factors, VEGF

sig-nalling is a key modulator of vascular development duringembryogenesis and for neovascularization in the adult

(Coultas et al., 2005) In mammals, five VEGF ligands,

VEGF-A, -B, -C, -D and placenta growth factor, have beenidentified and have been shown to bind in an overlappingpattern to three receptor tyrosine kinases, known as VEGFreceptor-1, -2, -3 (VEGFR1–3; receptor nomenclature follows

Alexander et al., 2013b)), as well as to co-receptors such as

heparin sulphate proteoglycans and NRPs VEGF-A gote knockout mice die early in gestation due to failure of the

heterozy-vascular formation (Carmeliet et al., 1996) On the other

hand, the two- to threefold overexpression of VEGF-A fromits endogenous locus results in abnormal heart formation and

lethality at E12.5 to E14.0 (Miquerol et al., 2000), indicating

that strictly balanced VEGF function is important in normalembryogenesis Furthermore, the intensity of VEGF signal-ling is strictly regulated through ligand-receptor interaction.VEGFR2 (also known as Flk1 in mice or KDR in humans) istyrosine-phosphorylated much more efficiently than VEGFR1

(also known as Flt1) upon VEGF binding (Millauer et al., 1993; Waltenberger et al., 1994; Shibuya, 2006) Although

VEGFR1 tyrosine kinase-deficient homozygous mice

devel-oped normal vessels and survived (Hiratsuka et al., 1998),

mice that were homozygous for point mutation at Tyr1173of

without any organized blood vessels or yolk sac blood islands,and haematopoietic progenitors were severely reduced, as

seen with Flk-1 null mice (Sakurai et al., 2005) Interestingly,

VEGFR1-null mice die at midgestation with vascular

over-growth and disorganization (Fong et al., 1995) Taken

together, these findings suggest that VEGFR2 is the majorreceptor in endothelial cells (ECs) for VEGF-inducedresponses, and VEGF signal intensity on VEGFR2 is regulated

by the binding of VEGF to the higher affinity receptor,VEGFR1

Another receptor for VEGF, NRP1, is expressed in ECs ofblood vessels and endocardial cells of the heart (Kitsukawa

et al., 1995; Kawakami et al., 1996; Soker et al., 1998) NRP1 is

also expressed in particular classes of developing neuronsand functions as a receptor for the class 3 semaphorinsthat mediate semaphorin-elicited inhibitory axon guidance

signals to neurons (Kitsukawa et al., 1995; Kawakami et al.,

1996; He and Tessier Lavigne, 1997) NRP1, together withVEGFR2, forms a specific receptor for VEGF165, an isoform of

enhances VEGFR2 signalling (Soker et al., 1998) NRP1-null

mice die midway through gestation at E10.5 to E12.5 andexhibit defects in the heart, vasculature, and nervous system

(Kawakami et al., 1996) Overexpression of NRP1 resulted in

the excess production of blood vessels and malformed hearts

(Kitsukawa et al., 1995) These findings indicated that NRP1

plays a critical function in the formation of blood vessels,along with VEGF

Inhibitory effects of the κ opioid agonists in vascular formation

Many studies on vascular development have focused on geneknockout and gene inhibition using mice and zebra fish.Although these studies have led to the discovery of essentialfactors in vascular development, they could not identify thesufficient conditions required for vascular formation To

clarify the ‘constructive’ mechanisms that underlie vascular

development, we have developed a novel embryonic stem

(ES)/induced pluripotent stem (iPS) cell differentiation

system that exhibits early vascular development using

VEGFR2-positive cells as common progenitors for vascular

cells (Figure 1A) (Yamashita et al., 2000; Narazaki et al., 2008).

In the early embryo and in differentiating ES/iPS cells,

VEGFR2 expression marks a common progenitor for both

blood and endothelium ES/iPS cell-derived VEGFR2+ cells

can differentiate into both ECs and mural cells (vascular

smooth muscle cells and pericytes) and form mature

vascular-like structures in vitro With the use of this system, we had

proposed that vascular formation was accomplished via two

mechanisms (Figure 1B) (Yamamizu et al., 2010; Yamamizu

and Yamashita, 2011): first, a basal mechanism for common

EC differentiation, where VEGF signalling plays a central role,

and second, a vascular diversification mechanism that works

on the basis of common EC differentiation Vascular

diversi-fication such as artery and vein formation can only be

achieved by the action of specific mechanisms in the

pres-ence of the basal EC machinery We have shown that cAMP/

PKA signalling contributes to common EC differentiation

through the upregulation of VEGF-A receptors, VEGFR2 and

NRP1 (Figure 1B) (Yamamizu et al., 2009).

Opioid systems are mainly present in neural tissues andcould be involved in neurogenesis during brain development

(Zhu et al., 1998; Tripathi et al., 2008) The three opioid

recep-tors, μ, δ and κ, mainly act as inhibitory G (Gi) coupled receptors through which endogenous opioids(endorphins, enkephalins and dynorphins) regulate physi-ological functions (Kieffer and Gaveriaux Ruff, 2002) Thesereceptors also activate other G protein-dependent signallingsuch as Gsor Gq, and G protein-independent signalling, for

investigated whether opioid systems (ligands and receptors)were involved in vascular formation with our vascular differ-entiation system using ES cells Interestingly,κ opioid recep-tors, but notμ or δ opioid receptors, were highly expressed invascular progenitors and ECs, and negatively regulated EC

differentiation and in vitro vascular formation via the tion of cAMP/PKA signalling (Yamamizu et al., 2011) Activa-

inhibited the expression of VEGFR2 and NRP1, but not other

recep-tor agonist) showed a significant increase in vascular tion in early embryos (Figure 2) Moreover, ectopic vascularinvasion into somites of E10.5 embryos accompanied by

forma-Figure 1

Vascular differentiation system using ES cells and iPS cells (A) Flk1 (VEGFR2) positive cells derived from ES/iPS cells are vascular progenitors thatcan differentiate into ECs, mural cells and blood cells ECs have specific characters of arterial-venous-lymphatic ECs (B) EC differentiation andvascular diversification in vascular development Vascular formation in embryogenesis is considered to have two main mechanisms: (i) a basalmechanism for common EC differentiation, in which VEGF signalling plays a central role; and (ii) a vascular diversification mechanism working onthe basis of common EC differentiation Vascular diversification, such as artery and vein formation, can be achieved only by activating specificmechanisms in the presence of the basal EC machinery cAMP/PKA signalling contributes to common EC differentiation through up-regulation ofVEGF-A receptors, Flk1 and NRP1 Other protein critically involved in differentiation into arterial, venous or lymphatic vessels are: delta like ligand

4 (Dll 4); hairy and enhancer of split-1 (Hes 1); prospero homeobox 1 (Prox 1); COUP transcription factor 2 (COUP-TFII) (adapted from Yamamizuand Yamashita, 2011)

BJP K Yamamizu et al.

270 British Journal of Pharmacology (2015) 172 268–276

decreased plexinD1 expression in ECs was observed in both

strains of null mice (Yamamizu et al., 2011) Therefore, theκ

opioid receptor system may be a dual inhibitory regulator of

EC differentiation and of vascular angiogenesis

Roles of the κ opioid receptor system

in tumour angiogenesis

Angiogenic switch in tumours

Tumour angiogenesis is required for tumour progression, to

provide nutrients and oxygen and to remove metabolic

wastes and carbon dioxide (Folkman, 1971; Carmeliet andBaes, 2008) The balance between endogenous activation andinhibition of angiogenesis critically maintains a normallyquiescent vasculature to sustain homeostasis One character-istic feature of tumour blood vessels is that they have lost theappropriate balance between positive and negative controlsand fail to become quiescent, leading to the constant pro-gression of tumour angiogenesis (Bergers and Benjamin,2003) Therefore, restoration of the balance between activa-tion and inhibition of angiogenesis is a critical treatmentstrategy for tumours

Growth factors and hypoxia are known to induce VEGF-Agene expression The hypoxia-inducible transcription factor,

Figure 2

Representative results of WT, prodynorphin-null (PDYN KO) andκ opioid receptor-null (KOPr KO) mouse embryos at E10.5 (A) Whole-mountCD31 (red) staining Left panels, WT mice Pn; perineural vascular plexus, Isv; intersomitic vessels Middle panels, PDYN KO mice Right panels,KOPr KO mice Scale bars: 2 mm (B) High-magnification views of CD31-stained Pn region Scale bars: 200μm (C) Higher magnification viewscorresponding to boxed regions in Figure 2B Scale bars: 40μm (D) Quantitative evaluation of CD31+ area in Pn CD31 staining of WT mice was

set as 1.0 (n = 3, **P < 0.01 vs WT) (E) Flow cytometry X-axis: VE-cadherin, Y-axis: CD31 Percentages of CD31+/VE-cadherin+/CD45-ECs in the

embryo are indicated (F) Quantitative evaluation of CD31+/VE-cadherin+/CD45-ECs in the embryo n = 3; *P < 0.05 vs WT (adapted from Yamamizu et al., 2011).

HIF, is a strong inducer of VEGF and contributes to the

for-mation of vascular tubes in embryogenesis as well as in

adults Many studies have shown that HIF is highly expressed

in various types of tumours, thereby enhancing angiogenesis

via VEGF and reproducing tumour cells Mice lacking HIF1a-,

HIF2a- and HIF-related genes exhibit vascular defects and

death at E9.5–E10.5 (Dunwoodie, 2009), indicating that

HIF-related VEGF production regulates vascular formation

Moreover, oncogene signalling molecules such as Ras and

Myc in cancer cells, up-regulate VEGF expression, which

would lead to the formation of vasculature and the

prolifera-tion of tumours (Carmeliet, 2005)

Angiogenesis inhibitors in cancer therapy

The concept of using angiogenesis inhibitors as anticancer

drugs was received with considerable scepticism when first

presented by Dr Folkman in the early 1970s (Folkman, 1971)

Solid tumours cannot grow beyond 2 to 3 mm in diameter

without being able to recruit their own blood supply

Beva-cizumab, a humanized monoclonal antibody that is specific

for human VEGF-A, was the first anti-angiogenic agent

approved by the Food and Drugs Administration (FDA) in

2004 for the treatment of colorectal cancer, renal cell cancer,

non-small cell lung cancer, and glioblastoma (Ferrara et al.,

2004) Furthermore, sunitinib and sorafenib were approved

by the FDA in 2008 as multi-target tyrosine kinase inhibitors

(TKIs) and have demonstrated efficacy against various solid

tumours in clinical trials (Llovet et al., 2008; Ivy et al., 2009;

Huang et al., 2010) TKIs can interact physically with a highly

conserved kinase domain shared by VEGFR1–3, as well as

PDGF receptors, FGF receptors, EGF receptor, Raf kinase and

cKit Although VEGF-targeted therapy for cancer has been

highly successful for the prevention of tumour angiogenesis

so far, most patients eventually acquire resistance to

anti-angiogenic therapy and rapid vascular regrowth in tumours

occurs after the discontinuation of anti-VEGF therapy

Fur-thermore, treatment with VEGF-targeted drugs has side

effects, such as hypertension and proteinuria-related kidney

dysfunction Thus, there is a clear need to identify novel

targets for anti-angiogenic therapeutic agents to achieve a

continuous inhibition of angiogenesis for tumour therapy

To date, approximately 30 endogenous inhibitors of

angiogenesis have been identified (Nyberg et al., 2005) Many

endogenous inhibitors including thrombospondin1 (TSP1),

which was the first protein to be recognized as an

endog-enous angiogenesis inhibitor, are fragments of naturally

occurring extracellular matrix and basement membrane

pro-teins (Cao, 2001) The expression of TSP1 is inversely

corre-lated with tumour progression in melanoma, lung and breast

carcinoma (Zabrenetzky et al., 1994) Suppression of TSP1

augmented tumour angiogenesis through the production of

matrix metalloprotease 9 and the enhancement of VEGFR2

signalling (Zabrenetzky et al., 1994) In contrast, TSP1

over-expression resulted in delayed tumour growth by the

inhibi-tion of tumour angiogenesis (Rodriguez Manzaneque et al.,

2001) Although many studies on these endogenous

angio-genesis inhibitors have shown that they significantly inhibit

tumour angiogenesis and tumour growth, it is still difficult to

accurately control their expression and to apply them in

clinical practice

Potential of κ opioid receptor agonists in cancer therapy

Opioid analgesics such as morphine have been broadly used

to relieve pain from all types of cancer The effect of phine on tumour growth is still controversial Independentstudies have shown that morphine can either decrease or

mor-increase tumour growth in mice (Gupta et al., 2002; Tegeder

et al., 2003; Koodie et al., 2010) A recent study showed that

morphine suppressed tumour angiogenesis through the bition of HIF transcription, which enhances the expression

inhi-of VEGF and VEGF receptors (Koodie et al., 2010) Moreover,

morphine inhibited tumour cell proliferation through

acti-vation of p53 (Tegeder et al., 2003) On the other hand,

morphine stimulated HUVEC proliferation and promotedtumour neovascularization in a human breast tumour,and further potentiated endothelial-pericyte interaction viaPDGF-BB and PDGF receptor-β (PDGFR-β), thereby enhanc-ing coverage of tumour vessels through pericyte recruitment

(Gupta et al., 2002; Luk et al., 2012) Furthermore,

methyln-altrexone, a peripherally restricted antagonist ofμ, exerted asynergistic effect with 5-fluorouracil and bevacizumab oninhibition of VEGF-induced human pulmonary microvascu-

lar EC proliferation and migration (Singleton et al., 2008).

However, naltrexone effectively induced new blood vesselgrowth in the chick chorioallantoic membrane assay (Blebea

et al., 2000) In-depth investigations would be needed in

purified ECs from tumours, but not tumours themselves, forelucidation of regulatory mechanisms of tumour angiogen-esis by opioids

Based on our earlier study of vascular development

(Yamamizu et al., 2011), we investigated whether opioid

ago-nists could act as anti-cancer drugs through the inhibition ofVEGF signalling In HUVEC and in ECs purified from adultmice,κ opioid receptors but not δ or μ opioid receptors, were

ago-nists U50,488H and TRK820 significantly inhibited HUVECmigration and vascular tube formation by suppressing

VEGFR2 expression (Yamamizu et al., 2013) Treatment with

nor-BNI, aκ opioid receptor antagonist, blocked the effects of

κ opioid receptor agonists on HUVEC migration Interestingly,Lewis lung carcinoma (LLC) or B16 melanoma cells grafted inKOPr-knockout mice showed increased proliferation andmarkedly enhanced tumour angiogenesis, compared withthose in wild-type mice In contrast, repeated intraperitonealinjection of TRK820 significantly inhibited tumour growth by

suppressing tumour angiogenesis (Figure 3) (Yamamizu et al.,

receptor system in vivo acts as an anti-angiogenic mediator

only in tumour vasculatures, or if it also is functional in othercell lineages such as pericytes, blood cells and the tumoursthemselves Furthermore, because our studies showed thatLLC or B16 melanoma grafted in prodynorphin-null miceshowed increased proliferation of tumours compared with

those in wild-type mice (unpublished data, Yamamizu et al.,

2013), more careful studies usingκ opioid receptor nists, which inhibit endogenous dynorphin will be needed

which has been clinically approved in Japan for use inhaemodialysis-related uremic pruritus, could be useful fortumour therapy by suppressing tumour angiogenesis, andBJP K Yamamizu et al.

272 British Journal of Pharmacology (2015) 172 268–276

thus could offer therapeutic benefits beyond the relief of

cancer pain Furthermore, because opioids have been used as

analgesics for more than 2000 years, there is considerable

experience of the clinical use of opioids As opioid systems

function through ligand–receptor interaction, it should be

relatively easy to apply opioid agonists to cancer therapy

However, patients also develop tolerance to opioid receptor

agonists, including TRK820, through repeated use (Suzuki

et al., 2004) Our results showed that although a low dose

(0.1–10μg·kg−1, b.i.d.) of TRK820, which is effective for

man-aging itching and pain in mice, inhibited tumour

angiogen-esis and tumour growth, constant treatment with a much

higher dose (150μg·kg−1) had no significant effect on tumour

growth (unpublished data; Yamamizu et al., 2013) These

results suggest that continuous treatment with high doses of

κ opioid receptor agonists might lead to the development of

tolerance to their anti-angiogenic effects on tumours or

might induce biphasic effects for tumour angiogenesis or

ligand off-target effects on tumour vasculatures Therefore,

more precise and careful observations are required to develop

effective tumour therapies with κ opioid receptor agonists

ligands as anti-angiogenic regulators and the ability to inhibit

tumour angiogenesis by manipulating the opioid

ligand-receptor system may lead to an feasible cancer therapy

Summary and future direction

De novo angiogenesis is a critical process both in

embryogen-esis and in many cancers The balance between angiogenic

activators and inhibitors controls sprouting, elongation andstabilization of the blood vessels in most organs and intumours Although we have made significant progress in ourunderstanding of opioid agonists as anti-angiogenic factors invascular development and in tumours (Figure 4), much workremains to be done We still do not fully understand theorigins of opioids that act as angiogenesis inhibitors or theintensity and degree of the contribution of opioids in physi-ological angiogenesis in development and tumours Further-more, although many diseases of the vascular system can also

affect the CNS, and vice versa, we do not fully understand

whether the opioids from neural tissues contribute to lar function and formation under pathological conditions.The multiple roles of VEGF signalling in endothelial devel-opment and function have made it the most popular targetfor therapeutic interventions in angiogenesis A more refinedunderstanding of the complex interaction between opioidsystems and VEGF signalling, which controls all aspects ofvascular formation and remodelling should provide noveland more specific targets for future therapeutic intervention

vascu-Acknowledgements

We thank Dr JK Yamashita for supervising the studies onvascular formation, Dr H Nagase for giving us TRK820 and SKatayama for helping with the figures This study was sup-ported by grants from the Ministry of Education, Science,Sports and Culture of Japan, the Ministry of Health, Labour

Figure 3

Effects of theκ opioid receptor agonist TRL-820 on melanoma (A) Example of a mouse bearing B16 melanoma without treatment (control, left)

or with TRK-820 treatment (right) (B) Quantitative analysis of tumour size among PBS-treated (n= 16) and TRK820 (0.1 μg·kg−1(n= 9), 1 μg·kg−1

(n= 17), 10 μg·kg−1(n = 9)-treated mice at 4, 7, 11, 14 days after tumour transplantation **P < 0.01, *P < 0.05 vs Control (C) Fluorescent staining

for CD31 (red) and VE-cadherin (green) at 14 days after tumour transplantation Nuclei are stained with DAPI (blue) Left panel, PBS treated Rightpanel, TRK820 (1 mg·kg−1)-treated Scale bars: 200μm (adapted from Yamamizu et al., 2013).

and Welfare of Japan, the Project for Realization of

Regenera-tive Medicine, the Japan Society for the Promotion of Science,

and the Intramural Research Program of the NIH, National

Institute on Aging

Conflict of interest

The authors declared that they have no conflict of interest

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276 British Journal of Pharmacology (2015) 172 268–276

Themed Section: Opioids: New Pathways to Functional Selectivity

REVIEW

Positive allosteric

receptor: a novel approach

for future pain medications

N T Burford1, J R Traynor2and A Alt1

1GPCR Lead Discovery & Optimization, Bristol-Myers Squibb Company, Wallingford, CT, USA,

and2Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI, USA

Correspondence

Neil T Burford, Bristol-MyersSquibb Company, 5 ResearchParkway, Wallingford, CT 06492,USA E-mail:

Morphine and other agonists of theμ-opioid receptor are used clinically for acute and chronic pain relief and are considered

to be the gold standard for pain medication However, these opioids also have significant side effects, which are also

mediated via activation of theμ-opioid receptor Since the latter half of the twentieth century, researchers have sought totease apart the mechanisms underlying analgesia, tolerance and dependence, with the hope of designing drugs with fewerside effects These efforts have revolved around the design of orthosteric agonists with differing pharmacokinetic propertiesand/or selectivity profiles for the different opioid receptor types Recently,μ-opioid receptor-positive allosteric modulators(μ-PAMs) were identified, which bind to a (allosteric) site on the μ-opioid receptor separate from the orthosteric site thatbinds an endogenous agonist These allosteric modulators have little or no detectable functional activity when bound to thereceptor in the absence of orthosteric agonist, but can potentiate the activity of bound orthosteric agonist, seen as an

increase in apparent potency and/or efficacy of the orthosteric agonist In this review, we describe the potential advantagesthat aμ-PAM approach might bring to the design of novel therapeutics for pain that may lack the side effects currentlyassociated with opioid therapy

Introduction

Pain and opioid analgesics

Pain is the most common ailment for which people seek

medical attention Chronic pain is a problem for millions of

patients and can be disabling, interfering with day-to-day

functions both at home and in the workplace Costs in the

United States from healthcare expenditure and lost work time

due to pain are estimated at $100 billion/year (Melnikova,

2010)

Opioid receptors are key targets in the management of

pain (Przewlocki and Przewlocka, 2001; Vallejo et al., 2011).

Drug therapies derived from morphine, its derivatives and

other small molecules induce pain relief by acting as agonists

(Alexander et al., 2013) Morphine-induced analgesia is lost

in mice lacking the μ-opioid receptor gene (Matthes et al.,

1996) Opioid drugs can produce serious side effects, ing respiratory suppression, constipation, allodynia, toler-ance, dependence and withdrawal symptoms, as well asrewarding effects and abuse potential (Przewlocki and

includ-Przewlocka, 2001; McNicol et al., 2003) All of these effects are

μ-opioid receptor-knockout animals (Matthes et al., 1996),

showing that they are mediated through activation of theμ-opioid receptor

Since the early 1990s, there has been a significant increase

in the use of opiate analgesics for non-cancer chronic pain,

partly due to the belief that opiate dependence and addiction

liability had previously been overstated (Juurlink and Dhalla,

2012) However, this has led to a substantial increase in

patients with opiate dependence and addiction The

increased presence of opiates in the household has also led to

higher abuse, both accidental and intentional, leading to

increased admissions to hospitals for treatment (Woodcock,

2009) Thus, physicians walk a tightrope balancing act in an

attempt to achieve both effective pain management and drug

safety

The ‘holy grail’ of opioid research has been, and

contin-ues to be, the identification of drugs that can produce the

beneficial analgesic effects of opiates without the

develop-ment of tolerance or other side effects, including their clear

abuse liability Over the past several decades, many opioid

ligands have been synthesized, with varying affinities for the

opioid receptor types, and varying pharmacokinetic

proper-ties Combinations of these ligands have also been used

(Snyder and Pasternak, 2003; Corbett et al., 2006; Lambert,

2008) However, these efforts have not yet yielded dramatic

improvements in the availability of pain medications with

fewer side effects

Opioid receptors

Opioid receptors are categorized within the Class A family of

GPCRs Four opioid receptor types exist;μ-opioid receptors,

κ-opioid receptors, δ-opioid receptors and NOP receptors

(also known as ORL1) (Alexander et al., 2013; Cox et al.,

2015) These receptors were cloned in the 1990s (Evans

et al., 1992; Kieffer et al., 1992; Chen et al., 1993; Yasuda et al.,

1993; Mollereau et al., 1994; Raynor et al., 1994), and their

crystal structures have recently been elucidated (Granier

et al., 2012; Manglik et al., 2012; Thompson et al., 2012; Wu

et al., 2012) The opioid receptors share about 60% amino

acid identity (mainly within the transmembrane domains)

and signal through the Gi/o family of heterotrimeric G

pro-teins, resulting in inhibition of adenylate cyclase (AC),

modu-lation of ion channel activity (via G proteinβγ subunits), and

transcriptional changes in the cell (Waldhoer et al., 2004).

There is also evidence for activation of non-G

protein-mediated pathways viaβ-arrestin (Bohn et al., 1999).

The endogenous ligands for the opioid receptors are

pep-tides derived from large precursors and include the

enkepha-lins, endorphins and dynorphins, which have selective

affinities for each of the three main opioid receptor types

(Janecka et al., 2004) but very low affinity for the NOP

recep-tor The endomorphins (Zadina et al., 1997) are considered

endog-enous peptide for the NOP receptor is nociceptin/orphanin

FQ peptide (Meunier et al., 1995; Reinscheid et al., 1995),

which has no affinity forμ-, κ- or δ-opioid receptors

Opiate physical dependence correlates closely with the

development of opiate tolerance (Way et al., 1969),

suggest-ing that they may share common mechanisms Tolerance can

be defined as a reduced response to repeated administration

of the same dose of drug, or put another way, increased doses

of drug are required to produce the same magnitude of

response There have been considerable studies investigating

the underlying mechanisms that result in opioid tolerance

and dependence, which have been reviewed elsewhere

(Bailey and Connor, 2005; Sadee et al., 2005; Bian et al., 2012;

Whistler, 2012; Williams et al., 2013) Hypotheses include

μ-opioid receptor phosphorylation and desensitization,receptor internalization/down-regulation, and up-regulation

of AC It has been suggested that the intracellularβ-arrestin-2protein is significantly involved in agonist-mediated devel-

knockout mice have enhanced analgesic effects in response tomorphine and lower levels of receptor desensitization, and

other unwanted side effects (Bohn et al., 1999) However,

despite several decades of research, the mechanistic standing of how tolerance develops is still relatively poorlyunderstood

under-Orthosteric and allosteric ligands

Before we introduce the concept of allosteric modulators, it isbeneficial to start with orthosteric ligand interactions withGPCRs Orthosteric ligands bind to the same site on thereceptor that recognizes an endogenous agonist – in the case

of the opioid receptors these are the opioid peptides GPCRsexist in multiple conformational states, but for simplicity wewill only refer to two, an inactive (R) conformation and anactive (R*) conformation Orthosteric agonists bind withhigher affinity to R*, thus driving the receptor equilibriumfrom R towards R* to give a high R*/R ratio Based on theintrinsic activity of a given agonist, the agonist can be full(eliciting a maximal achievable response in that system) orpartial (where the elicited response is less than that of a fullagonist despite full occupancy of all the available receptorbinding sites) This can be explained by a reduced ability ofpartial agonists to differentiate R and R*, thereby producing alesser equilibrium shift towards R* than full agonists and/or

an ability to induce a different active conformation of thereceptor (R+), which produces less activation of effectors (e.g

G proteins) compared with R* (Tota and Schimerlik, 1990).The phenomenon of biased agonism (Kenakin, 2011) con-firms the existence of multiple active conformations of thereceptor, but the simple R and R* model is clearly usefulbecause it leads to predictions that are supported by experi-mental evidence For example, high-efficacy agonists show agreater binding affinity shift (from high affinity to low affin-ity) in the presence of guanine nucleotides, compared with

lower efficacy agonists (Evans et al., 1985; Emmerson et al.,

1996)

The demonstration of constitutive GPCR activity (Costaand Herz, 1989) indicated that receptors could form the R*state and activate G proteins even in the absence of ligand.Ligands termed ‘inverse agonists’ bind with higher affinity tothe R conformation of the receptor, thus driving the receptorequilibrium from R* towards R and inhibiting constitutiveactivity of the receptor Neutral antagonists show no pre-ference for binding to the R or R* state and therefore donot affect the equilibrium of receptor conformations, butcompete with orthosteric agonists for the orthosteric bindingsite The ability to detect constitutive activity in recombinantsystems expressing high levels of receptors suggests that mostcompounds thought to be neutral antagonists may showsome preference for R or R*, and are either very weak efficacyagonists or weak efficacy inverse agonists

BJP N T Burford et al.

278 British Journal of Pharmacology (2015) 172 277–286

It has become increasingly evident that certain ligands

can bind to sites on GPCRs that are separate (allosteric) from

the orthosteric site The term ‘allosteric’ from the Greek

‘other site’ was first coined in a journal title 50 years ago by

Monod, Changeux and Jacob (Monod et al., 1963), followed 2

years later by the Monod, Wyman and Changeux model

(Monod et al., 1965) which describes a two-state model where

proteins can exist spontaneously in two conformations, an

active and inactive state Orthosteric and allosteric ligands

binding to their respective (non-overlapping) binding sites

can stabilize one receptor state at the expense of the other

The effects observed from interactions between the

orthos-teric and allosorthos-teric ligands, binding to the protein, were

termed the ‘allosteric interactions’

The concept of allostery was first applied to GPCRs with

the development of the ternary complex model (De Lean

et al., 1980), which described the interactions between

agonist, receptor and G protein, where the G protein can be

considered as the allosteric modulator, binding at the

intra-cellular side of the receptor At around the same time, an

introduction to the allosteric ternary complex model for

GPCRs was also described based on the observed effects of

gallamine on muscarinic receptors, which led to the

conclu-sion that gallamine binds to a site distinct from other

mus-carinic agonists and antagonists (Clark and Mitchelson, 1976;

Stockton et al., 1983) Further modifications to these models

to account for receptor constitutive activity led to the

extended ternary complex model (Samama et al., 1993) and

complex model (Weiss et al., 1996), which applies specifically

to two states of the receptor and their interactions with G

proteins The allosteric two-state model (Hall, 2000) looks

very similar to the cubic ternary complex model but

substi-tutes G protein (G) with allosteric ligand (B), and applies

more directly to orthosteric and allosteric ligands interacting

with active and inactive conformations of the receptor For a

comprehensive review, see Christopoulos and Kenakin, 2002

From a drug discovery perspective, the aim is to first

identify and then to monitor the structure activity

relation-ship of allosteric compounds using functional assays An

operational model has been developed based on the allosteric

binding models of Ehlert (Ehlert, 1988) and the Black & Leff

operational model of agonism (Black and Leff, 1983) that

tracks the allosteric cooperativity factors (αβ) The final

deri-vation of this operational model is shown in Scheme 1 as

presented by Leach and colleagues (Leach et al., 2007).

⎛

(1)

Within this model, E is the pharmacological effect, KAand KB

denote the equilibrium binding constants for the orthosteric

ligand, A, and the allosteric ligand, B, at the receptor The

binding cooperativity factor, α, represents the effect of the

allosteric ligand on orthosteric agonist binding affinity, and

vice versa An activation cooperativity factor,β, denotes the

effect the allosteric ligand has on orthosteric agonist efficacy

Agonism constantsτAandτBrepresent the intrinsic activity of

the orthosteric agonist and any intrinsic activity of the

allos-teric ligand, respectively, which is dependent on the cell

context and receptor expression level of the cell system, and

intrinsic efficacy of the ligands used The remaining eters, Em and n, denote the maximal response of the system,and the slope, respectively A simplified cartoon representingcomponents of the operational model and how they apply tothe various modes of allosteric modulation observed is shown

param-in Figure 1

These parameters lead to the multiple ‘flavours’ of teric ligands that can be observed Allosteric agonists that canactivate the receptor even in the absence of an orthostericagonist, have τBactivity, leading to functional efficacy thatappears similar to an orthosteric agonist Allosteric inverseagonists bind to an allosteric site and inhibit the constitutiveactivity of the receptor in the absence of orthosteric ligand.However, allosteric modulators may have very weak or unde-tectable intrinsic efficacy when they bind to the receptor, but

binding affinity and/or efficacy of the orthosteric agonistwhen it binds to the receptor Compounds with combinedcooperativity factor (αβ) values > 1 are considered positiveallosteric modulators (PAMs) and result in increased apparentpotency and/or efficacy of the orthosteric agonist response

concentration-response curve for the orthosteric agonist inthe presence of the PAM Systems with spare receptors ‘recep-tor reserve’ exhibit leftward shifts in the orthosteric agonist

from the functional assay, but only the combined tivity effect (αβ) The magnitude of these leftward shiftsincreases with increasing PAM concentration, until the PAMeffect saturates when the allosteric sites are fully occupied.Therefore, beyond this concentration of PAM there is nofurther leftward shift in the agonist concentration-responsecurve The maximal ‘fold-shift’ in agonist potency is equal tothe cooperativity factor (αβ), and the concentration of PAMwhich induces a half-maximal leftward fold-shift of the

Figure 1

Modes of allosteric modulation Allosteric ligands (B) bind to a graphically distinct site on the receptor compared with the orthos-teric agonist (A), and can modulate orthosteric agonist bindingaffinity (α), orthosteric agonist efficacy (β), and may have intrinsicagonist activity (τB) Cartoon is a modified figure from Conn et al.,

topo-2009a

(equilibrium binding constant for the PAM), or more

collo-quially the ‘shifty50’ (Hendricson et al., 2012) It is fairly

common for allosteric ligands to have a combination of both

allosteric agonist (τB) and PAM (αβ) activities depending on

the cellular system, and the assay used to monitor functional

activity In these cases, direct agonism is typically seen at

significantly higher concentrations of the allosteric ligand

than are required for PAM activity (Burford et al., 2011).

values <1, resulting in a reduction in the potency and/or

efficacy of the orthosteric agonist response Compounds that

bind to the allosteric site with very weak or no PAM or NAM

activity are essentially neutral allosteric ligands or silent

allos-teric modulators (SAMs) These SAMs act as competitive

antagonists at the allosteric site, and are therefore useful for

characterizing the site of action of identified PAMs and

NAMs

The classification of ligands as agonists, partial agonists,

neutral antagonists, inverse agonists, allosteric agonists,

PAMs, NAMs and SAMs is dependent on the cellular system

evaluated, and the particular aspect of signalling being

explored Also, for allosteric ligands, the allosteric

coopera-tivity can be different depending on which particular

orthos-teric agonist (probe) is used (Jager et al., 2007; Koole et al.,

2010) This is referred to as probe dependence Therefore,

defining a specific compound as a PAM or a NAM should only

be done in the context of the cellular system, the agonist

probe and the assay used

Moreover, the situation is even more complex For

example, with homo- and hetero-oligomers (Gomes et al.,

2004; Gupta et al., 2010; Costantino et al., 2012; Stockton

and Devi, 2012) the partnering receptor can be considered

the allosteric modulator (Gomes et al., 2004) causing

confor-mational changes in the target receptor that may affectorthosteric agonist affinity and/or efficacy, as well as possiblesignalling bias It is reasonable to assume both orthostericand allosteric ligands that bind to one receptor in thecomplex will alter this allosteric interaction between GPCRs.Allosteric ligands have several potential advantages overtraditional orthosteric ligands as drugs (Christopoulos and

Kenakin, 2002; Leach et al., 2007; May et al., 2007; Conn

et al., 2009a; Burford et al., 2011; Keov et al., 2011;Langmead, 2012) Because they do not bind to highly con-served orthosteric binding pockets, allosteric ligands canexhibit greater receptor selectivity Additionally, PAMs havekey potential advantages over orthosteric agonist drugs:PAMs can increase the amplitude while maintaining thespatial and temporal fidelity, and the physiological regula-tion, of native signalling patterns – something that orthos-teric agonist drugs cannot come close to doing These keyfeatures of PAMs are illustrated in Figures 2 and 3, and dis-cussed below

Discovery of μ-opioid receptor positive allosteric modulators ( μ-PAMs)

knowl-edge are the first PAMs described in the literature for this

receptor (Burford et al., 2013) Two negative allosteric

modu-lators of opioid receptors have been described previously

shown to be a negative allosteric modulator of agonistbinding toμ- and δ-opioid receptors (Kathmann et al., 2006).

Figure 2

PAMs maintain spatial fidelity of native signalling Endogenous opioid agonist is released at locations in the brain or spinal cord where it is required,maintaining the spatial fidelity of native signalling (A) Exogenous agonist is distributed and can activate target receptors throughout the body.This may lead to ‘on target’ side effects (B) PAMs can enhance the effects of endogenous agonists while still maintaining the spatial fidelity ofnative signalling (C)

BJP N T Burford et al.

280 British Journal of Pharmacology (2015) 172 277–286

agonist (Sheffler and Roth, 2003), but has also been shown to

be a negative allosteric modulator of theμ-opioid receptor,

although with∼100-fold weaker potency than its activity at

κ-opioid receptor (Rothman et al., 2007) μ-PAMs were

iden-tified in a high-throughput screen using aβ-arrestin

recruit-ment assay (PathHunter technology, DiscoveRx Corp.,

Fremont, CA, USA) (Bassoni et al., 2012) in human

osteosar-coma cells (U2OS) cells expressingμ-opioid receptors In this

assay, compounds were tested alone (agonist detection mode)

or in the presence of a low (approximately EC10)

detection mode) Concentration–response curves of the

screening hits were evaluated in U2OS cells expressing

μ-opioid receptors (U2OS-OPRM1 cells) and in U2OS cells

β-arrestin assay, in both agonist and PAM detection modes

Two of the compounds identified (986121 and

BMS-986122) showed no agonist activity, were selective forμ- over

δ-opioid receptors, and produced a sevenfold leftward shift

(αβ = 7) in the potency of endomorphin-I in the β-arrestin

assay in U2OS-OPRM1 cells

These PAMs were further evaluated in an inhibition of

forskolin-stimulated cAMP assay in CHO cells expressing

μ-opioid receptors In this assay, the PAMs produced leftward

shifts in the potency of endomorphin-I as well as two other

μ-opioid receptor agonists, leu-enkephalin and morphine

Interestingly, both PAMs showed some direct agonist activity

in this assay format (τBin Figure 1), although at much weaker

potencies than were observed for PAM activity

came from ligand binding studies and studies with a

radiola-belled, poorly hydrolysed analogue of GTP ([35S]-GTPγS) using

receptors (C6-μ cells) and mouse brain homogenates Bindingstudies in C6-μ cell membranes showed that while the affinity

binding with the selective full agonist [D-Ala2, N-MePhe4,

increased the affinity of DAMGO by sixfold, suggesting thattheseμ-PAMs act, at least in part, by increasing the affinity ofthe orthosteric agonist binding to the receptor (α in Figure 1)

where morphine and endomorphin-I were shown to be

shown to enhance the maximal response of these partialagonists, suggesting that they also can positively modulatethe efficacy of responses to agonists (β in Figure 1)

Compounds similar in structure to BMS-986122 weretested in theβ-arrestin recruitment assay resulting in someinteresting structure activity relationships Small changes instructure resulted in greatly reducedμ-PAM activity, althoughthe EC50of the responses were similar It was subsequentlyshown that some of these compounds were SAMs, binding tothe allosteric site but having no detectable effect in modulat-ing the activity of the orthosteric agonist However, the SAMs

recep-tor PAM activity, suggesting that μ- and δ-opioid receptorsmay share a similar allosteric site, and that selectivitybetweenμ- and δ-opioid receptors can be engineered into thecompounds

Figure 3

PAMs maintain temporal fidelity of native signalling Endogenous agonist can be released and cleared or metabolized quickly, leading to signallingeffects that have temporal fidelity (A) Exogenous agonist occupies receptors constantly, leading to effects that last until the drug is cleared ormetabolized (B) PAMs can enhance the effects of endogenous agonists while still maintaining the temporal fidelity of native signalling (C)

Key features of PAMs compared with

orthosteric agonists

Receptor selectivity

Receptors binding the same native agonist(s) necessarily

exhibit high homology at the orthosteric agonist binding

site Thus, the identification of orthosteric ligands with

selec-tivity between these related receptors can be difficult This

has posed major challenges for drug discovery programmes,

where often one particular receptor type or subtype is the

desired therapeutic target, but activity at related receptors can

lead to undesired side effects Well-known examples include

the metabotropic glutamate receptors, muscarinic receptors

and adenosine receptors, for which selective orthosteric

ligands have remained elusive throughout decades of

research In contrast, allosteric sites on GPCRs do not bind

the native ligand, and therefore are not under the same

evolutionary constraint as orthosteric sites Presumably

because of an increased diversity at allosteric binding pockets,

it has been possible to identify several highly selective

allos-teric agonists and PAMs for the notoriously difficult receptor

targets listed above (Bruns and Fergus, 1990; Gasparini et al.,

2002; Birdsall and Lazareno, 2005; Gao et al., 2005; Conn

et al., 2009b).

For opioid receptors, orthosteric agonist selectivity

between the receptor types has largely been achieved through

decades of medicinal chemistry programmes However,

allos-teric agonists and PAMs may offer new structural scaffolds to

further improve receptor type selectivity

Because of the lack of evolutionary constraint imposed

upon allosteric sites, allosteric ligands may be

species-selective as well as receptor-species-selective This can pose serious

issues for drug development where a compound active at

receptors in mice or rats may have no activity at the human

orthologue, or vice versa Therefore, activity of allosteric

ligands at receptor orthologues should be determined early in

our group, we saw no species selectivity between human, rat

or mouse orthologues of theμ-opioid receptor

Maintenance of temporal and spatial fidelity

Another advantage of PAMs is that they can maintain the

temporal and spatial activity of receptor signalling in vivo.

This is illustrated in Figures 2 and 3

Neuronal signals are closely regulated within the nervous

system with a high degree of temporal and spatial precision

When an orthosteric agonist drug is added systemically, it has

two major disadvantages Firstly, it is available throughout

the body and not just at the specific location where it is

needed This leads to activation of target receptors in other

areas of the brain and in other tissues, which can be

detri-mental to the therapeutic potential of the drug (Figure 2)

Secondly, the added drug activates all the receptors

through-out the body for an extended period of time Usually,

neuro-transmitter release is pulsatile in nature and quickly removed

between bursts of activity Continuous exposure to an

orthos-teric agonist drug for extended periods of time may lead to

receptor desensitization and tachyphylaxis, as well as toxic

side effects mediated by long-term exposure of drug at the

receptor (Figure 3)

These disadvantages of orthosteric drugs may be come with PAM drugs, where activity of an endogenouslyreleased orthosteric agonist are enhanced by the PAM, withthe PAM having no effect at the receptor when the receptor isnot bound with endogenous agonist Such drugs wouldmaintain the native temporal and spatial activity of thereceptor in response to endogenous agonist

over-Based on the pharmacological principle above, one canclearly envisage one potential way thatμ-PAMs could provide

an advantage over current orthosteric opiate analgesic

activity of the endogenous opioid peptide ligands in mediating pathways of the central and peripheral nervoussystem In this way, the temporal and spatial activity of theendogenous opioid peptides would be preserved, and sideeffects resulting from continuous and indiscriminate activa-tion of opioid receptors may be averted This hypothesisraises several key questions: Does significant endogenousopioid signalling occur physiologically (i.e is there enoughendogenous opioid signals to amplify)? Does this endog-enous signalling increase under conditions of injury, orchronic inflammatory or neuropathic pain? Are suchincreases spatially and/or temporally specific? Evidence for anendogenous peptide agonist-induced tone forμ-opioid recep-tor activity does exist For example, inhibition of enkephali-nases, which break down endogenous opioid peptides, results

pain-in antpain-inociception pain-in animal models of pain-inflammatory and

neuropathic pain (Roques et al., 2012) Similarly, naloxone, a

μ-opioid receptor antagonist, increased pain perception whenadministered to post-operative patients who were not takingexogenous opiates, suggesting the endogenous opioid pep-

tides produced a basal analgesic tone (Levine et al., 1978).

Recently, opioid receptor antagonists were also shown toincrease hyperalgesia in acute and chronic inflammatory painmodels in mice that had not been treated with exogenous

opioids (Corder et al., 2013) The authors suggested that

initial release of endogenous opioids leads to constitutiveactivation of the μ-opioid receptor, resulting in long-termendogenous analgesia

The development ofμ-PAMs will allow researchers to testwhether, when administered alone, they will have efficacy inpain relief models, and whether the side effect profiles may bebetter compared with current opiate therapy Of particularinterest is whether tolerance and dependence can be avoided

activated all the time by an exogenous agonist, one canhypothesize there will be less tolerance and dependenceliability

A second potential therapeutic utility forμ-PAMs can beenvisaged: It is possible that administration of a low dose ofopiate with aμ-PAM may also provide therapeutic benefit butwith fewer side effects The combination of a lower dose of

devel-opment of tolerance, which results from long-term exposure

to opiates There is precedence for this behaviour at the

forskolin-induced cAMP formation in recombinant cells decreased afterexposure to a saturating GABA concentration, but not after acombination of a low GABA concentration and the PAMGS39783, which activated the receptor to the same extent(Gjoni and Urwyler, 2008) The authors suggested thatBJP N T Burford et al.

282 British Journal of Pharmacology (2015) 172 277–286

GS39783 has a lower propensity to develop tolerance due to

less receptor desensitization than classical agonists It will be

interesting to see whether a low dose of morphine combined

withμ-PAM can produce similar levels of pain relief as a high

dose of morphine, but with fewer tolerance and dependence

liabilities

Most of the untoward side effects of opiates (e.g

respira-tory depression, constipation) are mediated throughμ-opioid

receptors, and there is no a priori reason to assume that

μ-PAMs would not potentiate these unwanted effects of

opiates as well as their desired therapeutic effects However,

perhaps the on-target side effects might be minimized by

using reduced concentrations of morphine

Finite shifts in orthosteric agonist potency

with increasing concentrations of PAM

Modulation of orthosteric agonist responses by PAMs or

NAMs is finite As modulator concentrations reach the point

where the allosteric binding sites on all available receptors are

occupied, then no additional change in orthosteric agonist

functional potency or efficacy is observed, even when the

concentration of PAM or NAM is increased further Therefore,

allosteric modulators can be designed and selected based on

their ability to produce a defined ‘fold-shift’ in functional

potency of the orthosteric agonist The main advantage of

this is that PAMs with a defined fold-shift of agonist potency

may reduce toxicity or avoid overdosing of the patient

This would clearly be a potential benefit for the use of

μ-PAMs where overdose with opiate drugs is a serious issue,

resulting in many deaths In many of these cases, the need to

take more drug to overcome receptor tolerance issues

com-pounds the problem

Probe dependence

Another important aspect of allostery is the fact that the level

or appearance of allosteric modulation can depend upon the

orthosteric agonist ligand used, as described above This has

important consequences Firstly, when evaluating

com-pounds as PAMs, one should use, whenever possible, the

endogenous ligand This can add a level of complexity to a

drug discovery programme when multiple endogenous

ligands exist Additionally, it is of note that previously

inac-tive or weak potency metabolites of the endogenous ligand

may show significant activity in the presence of a PAM

Therefore, probe dependence is an important consideration

when evaluating the therapeutic potential of a given PAM

(Wootten et al., 2012).

Opioid receptors have multiple endogenous peptide

agonist ligands So it will be important to establish how each

of these ligands is modulated by PAMs Firstly, the selectivity

of the PAM for each of the opioid receptors should be

one must also consider whether peptide agonists that are

receptor activity (e.g dynorphin-A) become more active at

theμ-opioid receptor, and what consequences that has on the

various pathways controlled by endogenous peptides

Simi-larly, metabolites of these peptides, which may not have

much affinity/efficacy for opioid receptors, may produce

sig-nificant activity in the presence of an opioid receptor PAM

Metabolism of morphine and other opiates also produce

However, one must ensure that these metabolites in the

the receptor, and if they do, one must determine theconsequences

Ligand-biased signalling and biased modulation

Historically, receptor pharmacology has been thought of inrelatively simplistic terms, where ligands bind to and activate

a receptor leading to a defined cascade of signalling pathwayswithin the cell However, over the past decade, research hasconvincingly shown that ligands acting at the same receptorcan activate different signalling pathways, with each ligandproducing subtly different changes in conformations of thereceptor when they are bound This feature, commonly calledsignalling bias or functional selectivity, has greatly increasedour understanding of receptor pharmacology and revolution-ized approaches to drug discovery (Kenakin, 2011; Whalen

et al., 2011; Kenakin and Christopoulos, 2013) The

possibil-ity of identifying small molecule orthosteric agonist ligandsthat can preferentially activate certain signalling pathwaysand not others offers the potential to discriminate betweentherapeutically beneficial pathways and unwanted side effectpathways even when the side effects are mediated by thetarget receptor, as is the case for theμ-opioid receptor.Recently, there has been a great deal of interest in signal-ling bias with various opioid receptor ligands, and ligand biashas been observed with respect to agonist-mediated phospho-rylation and internalization of theμ-opioid receptor, inhibi-tion of cAMP accumulation, ion channel activity,β-arrestinrecruitment responses and other non-canonical signalling

pathways (Burford et al., 1998; Mailman, 2007; Violin and Lefkowitz, 2007; Rivero et al., 2010; McPherson et al., 2012; Pradhan et al., 2012; Rives et al., 2012) Based on observations

negative modulators of analgesia, and positive modulators ofsomeμ-opioid receptor-related side effects (including toler-

ance) (Bohn et al., 1999; 2000), it has been hypothesized that

opioid agonists with bias toward the G protein-mediated

may be beneficial in separating the analgesic effects from theside effects Indeed, Trevena have recently identified a G

which is reported to be a potent analgesic but with reducedgastrointestinal and respiratory dysfunctional effects com-

pared with morphine (Dewire et al., 2013).

Very recently, phosphorylation of theμ-opioid receptor atTyr336

by Src has been shown to serve as the trigger for version of a classical Gi/Go-coupled receptor into a receptortyrosine kinase-like entity, resulting in a non-canonicalpathway leading to increased activation of AC even after the

con-original Gi/Go signals are blunted (Zhang et al., 2013).

Above, we have described the potential advantages ofligand bias signalling with respect to orthosteric agonists attheμ-opioid receptor However, it is conceivable that a PAMmay change the active conformation of the receptor in thepresence of agonist, thus changing the signalling cascade to

be biased towards one pathway and away from another This

‘biased modulation’ has been observed for many GPCRs and

these have been recently reviewed elsewhere (Koole et al.,

2010; Keov et al., 2011; Davey et al., 2012; Kenakin and

Christopoulos, 2013; Wootten et al., 2013).

Conclusions

a new and exciting avenue to explore not only novel pain

therapeutics at theμ-opioid receptor, but also therapeutics for

conditions, such as mood disorders, for which there is

mounting evidence that opioid receptors present viable

therapeutic targets (Lambert, 2008; Hegadoren et al., 2009;

Lutz and Kieffer, 2013) With the design of improvedμ-PAMs

with higher affinity for the receptor and better

pharmacoki-netic, pharmacodynamic, and safety profiles, it will be

possi-ble to assess whetherμ-PAMs have efficacy in models of pain

relief and other medical conditions either when administered

alone, thereby modulating endogenous opioid pathways, or

in combination with lower concentrations of exogenous

opiates, such as morphine In either scenario, it will be

impor-tant to know if beneficial actions are enhanced, while sparing

tolerance, dependence and other side effects associated with

current opioid therapies

Nevertheless, there are also a number of challenges for

any drug discovery programme seeking allosteric modulators

of opioid receptors Due to the probe-dependent nature of

allosteric modulation and the non-selectivity of several of

the endogenous opioid peptides and opioid drugs, the

activ-ity of opioid PAMs will need to be assessed across opioid

receptor types and with a variety of endogenous and other

orthosteric agonists, including potentially active metabolites

(Wootten et al., 2012) An additional complication is that

described (Burford et al., 2013) can switch function from

PAMs to NAMs or SAMs with only small changes in structure

(Melancon et al., 2012) However, there is no doubt that the

inherent advantages of PAMs, especially their maintenance

of temporal and spatial signalling fidelity and promise of

biased modulation, in addition to the potential to use lower

doses of opioid drugs, will guide research over the next few

the ‘holy grail’ of opioid research, developing powerful

analgesic drugs devoid of the side effects associated with

morphine

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