Naltrexone and nalmefene attenuate cocaine place preference in male mice

Accepted Manuscript Naltrexone and Nalmefene Attenuate Cocaine Place Preference in Male Mice

Kyle A. Windisch, Brian Reed, Mary Jeanne Kreek PII:

S0028-3908(18)30403-9

DOI:

10.1016/j.neuropharm.2018.07.025

Reference:

NP 7275

To appear in:

Neuropharmacology

Received Date:

22 March 2018

Accepted Date:

22 July 2018

Please cite this article as: Kyle A. Windisch, Brian Reed, Mary Jeanne Kreek, Naltrexone and Nalmefene Attenuate Cocaine Place Preference in Male Mice, Neuropharmacology (2018), doi: 10.1016/j.neuropharm.2018.07.025

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TITLE: Naltrexone and Nalmefene Attenuate Cocaine Place Preference in Male Mice

AUTHORS: Kyle A Windischa, Brian Reeda, Mary Jeanne Kreeka

AFFILIATIONS: aLaboratory

of the Biology of Addictive Diseases, The Rockefeller University, 1230 York

Avenue, New York, NY 10065, USA

CORRESPONDING AUTHOR: Kyle A Windisch, PhD The Laboratory of the Biology of Addictive Diseases The Rockefeller University 1230 York Ave, Box #171 New York, NY 10065 Email: [email protected]

Declarations of interest: none

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ABSTRACT: Cocaine addiction treatment is difficult due to the current lack of approved pharmacotherapuetics. Several preclinical and clinical studies have demonstrated that the mu opioid receptor (MOPr) antagonist/kappa opioid receptor (KOPr) partial agonist naltrexone (NTX) reduces the subjective effects and self-administration of cocaine. However, very limited research has examined the ability of the structurally similar MOPr antagonist/KOPr partial agonist nalmefene (NMF) to reduce cocaine reward. Here we examine the effect of low (1 mg/kg) and high (10 mg/kg) doses of NTX or NMF on cocaine place preference. In vivo characterization of these NTX and NMF doses were performed to examine their effectiveness at MOPr and KOPr. RESULTS: Both NTX doses and high dose NMF significantly reduced cocaine place preference. Conversely, a significant place avoidance was observed for high dose NTX and both NMF doses. Interestingly, neither NTX nor NMF blocked cocaine-induced hyperlocomotion. High dose NTX and both NMF doses fully blocked MOPr agonist morphine-induced thermal analgesia as well as KOPr agonist U50,488H-induced locomotor discoordination. However, low dose NTX fully blocked morphine analgesia but not U50,488H locomotor discoordination suggesting that low dose NTX is effective at MOPr but not KOPr. CONCLUSION: Both NTX and NMF block the place preference, but not locomotor activating, effects of cocaine. These results suggest that both NTX and NMF may be viable pharmacotheraputics for some aspects of cocaine addiction. This is an important step to understanding the potential mechanism(s) of action of NTX and NMF for the development of more efficacious pharmacological treatments for substance use disorders.

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1. INTRODUCTION Abuse and dependence of cocaine remains a major public health issue with

3

significant social and economic consequences. However, no targeted

4

pharmacotherapeutic option currently exists to combat cocaine addiction. Cocaine acts

5

primarily by increasing extracellular concentrations of dopamine as well as other

6

monoamines (serotonin and norepinephrine) in the brain mesocorticolimbic system (Ritz

7

et al., 1987). As the endogenous opioid system is highly expressed in key regions of the

8

mesocorticolimbic system (Mansour et al., 1987, 1988) and has been shown to regulate

9

monoamine signaling (Di Chiara and Imperato, 1988; Zhang et al., 2004a), the opioid

10

system may be involved in cocaine reinforcement and reward. Prior exposure of mu

11

opioid receptor (MOPr) agonists, such as morphine, have been shown to enhance the

12

reinforcing aspects of cocaine and vice versa, a phenomenon known as cross-

13

sensitization (Lett, 1989; Shippenberg and Heidbreder, 1995; Shippenberg et al., 1998).

14

Opioid receptor ligands, such as naloxone, block cocaine reward (Houdi et al., 1989;

15

Simmons and Self, 2009). The cross-sensitization between opioids and cocaine as well

16

as ability of opioid antagonists to block cocaine reward suggest common

17

neurobiological pathways for opiates and cocaine and, therefore, pharmacotherapies

18

currently approved for the treatment of opiate addiction may be efficacious in treating

19

cocaine addiction.

20

Naltrexone (NTX) is an FDA approved medication for the treatment of opioid use

21

disorder and alcoholism that has been shown in vitro to act as an antagonist at MOPr

22

and partial agonist at kappa opioid receptors (KOPr), albeit with a low maximal effect for

23

KOPr (Ghirmai et al., 2008; Wentland et al., 2009). Several clinical studies have

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demonstrated the potential viability of NTX for the treatment of cocaine addiction in both

25

reducing cocaine use as measured by cocaine-negative urine samples (Kosten et al.,

26

1989; Oslin et al., 1999; Pettinati et al., 2008; Schmitz et al., 2014; Schmitz et al., 2001)

27

and attenuating the subjective effects of cocaine (Comer et al., 2013; Kosten et al.,

28

1992; Sofuoglu et al., 2003) (however see also Hersh et al., 1998; Modesto-Lowe et al.,

29

1997; Schmitz et al., 2004; Schmitz et al., 2009; Walsh et al., 1996). Overall, the prior

30

clinical studies suggest that higher doses of NTX (150 mg/day) than what is typically

31

prescribed for the treatment of alcoholism (50 mg/day) may be necessary to regulate

32

the reinforcing aspects of cocaine in clinical populations, particularly populations with

33

polysubstance dependence (e.g., cocaine and alcohol co-dependence).

34

Several preclinical studies have examined the potential viability of NTX in

35

reducing the rewarding effects of cocaine. Bilsky et al. (1992) found that 56 mg/kg NTX

36

administered 4 hours prior to cocaine conditioning reduced but did not fully block the

37

rewarding effects of cocaine as measured by conditioned place preference (CPP).

38

Lower doses of NTX (2-3 mg/kg, sc) administered 10-15 minutes prior to cocaine fully

39

block cocaine place conditioning (Sala et al., 1995; Suzuki et al., 1992). As well, high

40

dose chronic NTX (120 and 240 mg but not 10 - 60 mg implant) administered via

41

osmotic pump fully blocks cocaine CPP (Mitchem et al., 1999). In preclinical studies of

42

alcohol exposure, doses of NTX as low as 0.1 - 0.3 mg/kg have been shown to reduce

43

seeking and operant self-administration of alcohol (Henderson-Redmond and

44

Czachowski, 2014; Williams and Broadbridge, 2009). Together with the clinical NTX

45

findings, these studies suggest that higher doses of NTX, potentially doses that act at

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both the KOP and MOP receptors, are necessary to block the rewarding effects of

47

cocaine.

48

Nalmefene (NMF) is a MOPr antagonist/KOPr partial agonist (Ghirmai et al.,

49

2008) structurally similar to that of NTX (methylene substitution at 6 position of NTX;

50

see figure 1) that is equipotent to NTX at MOPr and roughly two-fold more potent than

51

NTX at KOPr and delta opioid receptors (deHaven-Hudkins et al., 1990; Michel et al.,

52

1985). Though originally considered an antagonist for KOPr, NMF was recently shown

53

to act as a partial KOPr agonist both in vitro and in vivo (Bart et al., 2005; Stahl et al.,

54

2015). No published preclinical studies to date have examined the effect of NMF on

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cocaine reward. Clinically, Grosshans et al. (2015) reported a reduction in cocaine

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craving with “as needed” nalmefene in a female patient. This case report, along with the

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structural and pharmacological similarity of NMF and NTX, suggests that NMF may

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attenuate the rewarding aspects of cocaine; however, more experiments are needed to

59

clarify the ability of NMF to block cocaine reward.

60

Here we attempted to clarify the ability of the MOPr antagonists/KOPr partial

61

agonists naltrexone and nalmefene to regulate the rewarding aspects of cocaine. For

62

this we examined the ability of low (1 mg/kg) and high (10 mg/kg) doses of NTX or NMF

63

to block the rewarding and locomotor stimulating effects of cocaine using unbiased

64

place conditioning and open field assays. Further, we characterized the NTX and NMF

65

doses for in vivo effectiveness at the mu and kappa opioid receptors.

66 67

2. Materials and Methods

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2.1 Subjects

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Adult male C57Bl/6J mice (10 weeks old on arrival; Jackson Laboratory, Bar

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Harbor, ME) were housed in groups of four with free access to food and water in light

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(12:12 hour light/dark cycle; lights on at 1900 for CPP, thermal analgesia, and rotarod

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assays; lights on at 0700 locomotor activity assay) and temperature (22oC) controlled

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rooms. For all experiments upon arrival animals were allowed 1 week to habituate to the

74

environment prior to the start of the testing. Animals were weighed and handled a

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minimum of three times as well as received two separate mock injections to habituate to

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experimental procedures. Animal care and experimental procedures were conducted

77

according to the National Institutes of Health guide for the care and use of Laboratory

78

animals (NIH Publications No. 8023, revised 1978). The experimental protocols used

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were approved by the Institutional Animal Care and Use Committee of The Rockefeller

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University.

81 82 83

2.2 Drugs The drugs used were cocaine hydrochloride (Sigma-Aldrich, St. Louis, MO),

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morphine hydrochloride (Sigma-Aldrich, St. Louis, MO), U50,488H (Sigma-Aldrich, St.

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Louis, MO), naltrexone hydrochloride (Tocris Bioscience, Minneapolis, MN), and

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nalmefene hydrochloride (Baker Norton Pharmaceuticals, Miami, FL). All drugs were

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dissolved in sterile saline and injected at a volume of 5 mL/kg body weight. Sterile

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saline was used as vehicle treatment for all experiments.

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2.3 Place Conditioning Apparatus

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The mouse place preference chambers have three distinct compartments

92

separated by removable doors (ENV-3013; Med Associate, VT). Movement within each

93

compartment is tracked by individual infrared photobeams on a photobeam strip (six

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beams in the white and black compartments and two beams in the smaller central gray

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compartment). The center compartment had a solid neutral gray floor and gray walls.

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The black and white compartments (16.8 x 12.7 x 12.7 cm) had stainless steel rod and

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mesh floors, respectively.

98 99 100

2.4 Place Conditioning Experiments were performed in a dimly-lit, sound attenuated chamber described

101

above. A 15 mg/kg dose of cocaine was selected based both on previous studies

102

(Bailey et al., 2012; Velazquez-Sanchez et al., 2010) and pilot work demonstrating that

103

this dose is sufficient to produce robust cocaine-induced CPP (data not shown).

104

During the pre-conditioning session (D1), adult mice (12 weeks at start;

105

n=8/group) were placed in the center compartment and allowed free access to all

106

compartments (see Figure 2a: timeline place conditioning experiments). The time spent

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in each compartment was recorded for 30 min. During the conditioning sessions, mice

108

received two injections. Mice were first injected with either saline, NTX, or NMF 30

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minutes prior to the conditioning session and returned to their home cage. Animals were

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then injected with either saline or cocaine (15 mg/kg) and immediately placed into the

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appropriate conditioning compartment for 30 min. Conditioning sessions occurred at the

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same time each day (1000-1600 hours) with animals injected with drug (NTX or NMF +

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cocaine) or saline (saline + saline) treatment on alternate days, for a total of eight

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conditioning sessions (four drug and four saline). Animals did not receive NTX or NMF

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on saline conditioning days. The study used an unbiased design in which drug paired

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compartment (white or black) was counterbalanced across animals. The post-

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conditioning test session was performed on the day after the last conditioning session

118

(24 hours following final drug conditioning session) and was identical to the pre-

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conditioning session. Each mouse had free access to all compartments for 30 minutes.

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Place preference was calculated as percent preference for the drug paired chamber

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during the post-test session. NTX and NMF were tested in separate experiments.

122 123 124

2.5 Open Field Mouse locomotor activity was assessed using an open field arena that consisted

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of a standard rat shoebox cage within a locomotor activity monitoring frame (Kinder

126

Scientific, San Diego, CA). Distance traveled consisted of horizontal movement within

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the arena and data were collected in 5-minute bins. Separate cohorts of drug naïve

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adult male C57Bl/6J mice were used to examine the effect of low (1 mg/kg) dose and

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high (10 mg/kg) NTX or NMF pretreatment on cocaine induced-hyperlocomotion and

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locomotor sensitization (n=8/group). Animals were habituated to the locomotor boxes

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during a single 30-minute session to reduce a potential novelty confound (see Figure

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2b: timeline open field experiments). For locomotor sensitization the injection

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pretreatment time, doses, and days (saline versus drug) were identical to those used for

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the place conditioning experiments; however, separate groups were run for dose [i.e.,

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one group with subgroups pretreated with low dose (1 mg/kg) NTX or NMF and another

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group with subgroups pretreated with high dose (10 mg/kg) NTX or NMF]. Mice were

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first injected with either saline, NTX, or NMF 30 minutes prior to the locomotor session

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and returned to their home cage. Animals were then injected with either saline or

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cocaine (15 mg/kg) and immediately placed into the open field arena and allowed to

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freely explore the arena for 30 minutes. Locomotor sessions occurred at the same time

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each day (1200-1700 hours) with animals injected with drug (NTX or NMF + cocaine)

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and saline (saline + saline) treatment on alternate days, for a total of eight locomotor

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sessions (four drug and four saline).

144 145 146

2.6 Rotarod NMF was shown to act as a KOPr partial agonist both in vitro, in [35S]GTPγS

147

assays, and in vivo, in humans, (Bart et al., 2005), but was shown to antagonize KOPr

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-arrestin-2 recruitment (i.e., extremely biased partial agonist; Stahl et al., 2015). KOPr

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-arrestin-2 recruitment by differentially G-protein biased KOPr agonists in vivo has

150

been associated with motor incoordination in the rotarod assay, as a measure of

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sedation (Dunn et al., 2018; White et al., 2015). Therefore, blockade of KOPr agonist

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U50,488H-induced locomotor discoordination using rotarod was used to characterize

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the effect of NTX and NMF (0, 1, or 10 mg/kg) at the KOPr. Balance and motor

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coordination was measured on an accelerating rotarod (rod diameter 1.25”; IITC Life

155

Science Inc., Woodland Hills, CA). Drug naïve mice were trained on the apparatus twice

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daily for up to 5 days. During the first training session, mice were on the rotarod for at

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least 120 seconds with a constant 3 rpm rotation. For the second session, the rod

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initially rotated at 3 rpm and gradually increased to a maximum of 15 rpm over a 5 min

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period. Mice walked on the accelerating rod for a minimum of 200 seconds. For the

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remaining training sessions, the top rpm was set to 30 and mice were to again remain

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on the rod for a minimum of 200 seconds. During training, if a mouse fell from the rod

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prior to the end of session, they were gently placed back on to the rod. Mice continued

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training until they were able to remain on the rotarod for at least 150 seconds at the last

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training session.

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For testing, the rod initially rotated at 3 rpm and gradually increased to a

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maximum of 30 rpm over a 5 minute period (the maximum length of the trial). The

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latency to fall off the rod was measured by the rotarod timer. Between testing, animals

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were returned to their home cage after falling from the rod. On the testing day, each

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mouse first completed two drug-free baseline trials to determine performance prior to

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drug administration. Animals then received a pretreatment injection of either NTX or

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NMF (0, 1.0, or 10 mg/kg) 30 min prior to an injection of the kappa opioid receptor

172

agonist U50,488H (0 or 10 mg/kg). Rotarod performance was assessed at -10 (+20 min

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following pretreatment), +30, and +60 minutes following U50,488H administration.

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Effects of drug treatment were calculated as percent of the average baseline time on

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the rod before falling.

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2.7 Morphine Thermal Analgesia One week following rotarod assay, mice were tested for blockade of morphine-

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induced analgesia by NTX or NMF (0, 1.0, or 10 mg/kg) using the standard hot-plate

180

method. The apparatus consisted of a clear, vertical, plexiglas cylinder placed on a

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hotplate (IITC Model 39 D; IITC Inc., Woodland Hills; CA). Mice were habituated to the

182

apparatus by placement onto the room temperature (22 ºC) plate for 30 seconds twice

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on each of the two days preceding the testing day. On the day of testing, mice were

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allowed to walk on the hotplate (54ºC ± 0.1°C) for up to 45 seconds (maximum allowed

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latency; to avoid tissue damage). Latency to jump, lick a hind paw, or flutter/flick a hind

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paw (defined as vigorous shaking of paw while standing or stepping such that the paw

187

is extended out of vertical body plane away from body) was recorded. Two baseline

188

measurements separated by 15 min were recorded for each animal to determine

189

performance prior to drug administration. Animals then received a pretreatment injection

190

of either NTX or NMF (0, 1.0, or 10 mg/kg) 30 min prior an injection of the mu opioid

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receptor agonist morphine (0 or 15 mg/kg; i.p.). Thermal analgesia was assessed at -10

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(+20 min following pretreatment), +20, +40, and +60 minutes following morphine

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administration.

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2.8 Statistics Conditioned place preference was calculated as percent preference for the drug

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paired chamber during the post-test session using the following formula: (time drug-

198

paired side post-test)/(time drug-paired side post-test + time saline paired side post-

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test). The CPP experiments were designed to examine dose related differences with

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NTX and NMF run in separate cohorts. The locomotor experiments were designed to

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examine drug related differences with low and high doses run in separate cohorts. A

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conservative approach to the statistical analyses was used to reduce the potential of

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error attributable to environmental differences between cohorts. Differences in CPP

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were analyzed separately for NTX and NMF using two-way analysis of variances

205

(ANOVAs) with pretreatment (saline, 1.0, and 10 mg/kg NTX or NMF) and drug (saline

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or 15 mg/kg cocaine) as factors followed by Tukey post hocs for multiple comparisons.

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Difference in open field locomotor activity across conditioning sessions were analyzed

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separately for low (1 mg/kg) and high (10 mg/kg) dose during drug paired (days 2, 4, 6,

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and 8) sessions using two-way repeated measures ANOVAs with treatment and day as

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factors followed by Tukey post hocs for multiple comparisons for treatment and session.

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U50488H-induced locomotor discoordination were analyzed as percent of baseline time

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(in seconds), with the following standard transformation: [(test latency – average

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baseline latency)/(average baseline latency)] X 100 and analyzed separately for NTX

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and NMF using two-way repeated measures ANOVAs with treatment and time as

215

factors followed by Dunnett post hocs for multiple comparisons. Antinociceptive effects

216

of morphine were analyzed as percent maximum possible effect (%MPE), with the

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following standard transformation: %MPE = [(test latency – baseline latency)/(maximum

218

cutoff latency – baseline latency)] X 100 and were analyzed separately for NTX and

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NMF using two-way repeated measures ANOVAs with treatment and time as factors

220

followed by Dunnett post hocs for multiple comparisons. All analyses were conducted

221

using GraphPad Prism 7.0c (GraphPad Software, La Jolla, CA).

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3. RESULTS

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3.1 Conditioned Place Preference/Aversion

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The mean (SEM) percent preference for the drug paired side during the post-

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test session for NTX is shown in Figure 3a. For NTX, significant effects of both

227

pretreatment [F(2,42)=31.56, p <0.0001] and drug [F(1,42)=30.05, p <0.0001] were

228

observed. No significant pretreatment x drug interaction was found [F(2,42)=1.451, p =

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0.45]. Post hoc tests revealed that a significant place preference was observed for

230

saline + cocaine treated mice compared to saline + saline control mice (p<0.01). Both

231

low (p<0.05) and high (p<0.001) dose NTX significantly attenuated the place preference

232

for the cocaine-paired side. High, but not low, dose NTX alone was observed to induce

233

a significant place aversion compared to saline + saline control (p<0.05), although there

234

was a trend towards aversion in the low dose (Figure 3a).

235

The mean (SEM) percent preference for drug paired side during post-test

236

session for NMF is shown in Figure 3b. For NMF, a significant effect of pretreatment

237

[F(2,42)=21.05, p <0.001] and drug [F(1,42)=37.04, p <0.0001] were observed. No

238

significant pretreatment x drug interaction was found [F(2,42)=1.183, p = 0.55]. Post hoc

239

tests revealed that a significant place preference was observed for saline + cocaine

240

treated mice compared to saline + saline control (p<0.05). In contrast with NTX, high

241

(p<0.05), but not low (p=0.09), dose NMF significant attenuated the place preference for

242

the cocaine-paired side, although there was a trend towards attenuation in the low dose.

243

Both low (p<0.05) and high (p<0.01) dose NMF alone were observed to induce a

244

significant place aversion compared to saline + saline control (Figure 3b).

245 246

3.2 Locomotor Sensitization

247

3.2.1 Low dose NTX or NMF Pretreatment

248

The mean (SEM) distance traveled (in cm) for low (1 mg/kg) dose NTX or NMF

249

plus cocaine across drug exposure days (days 2, 4, 6, and 8) are shown in Figure 4a.

250

For drug exposure sessions, a significant effect of treatment [F(5,126)=61.07, p

251

<0.0001], day [F(3,126)=5.12, p<0.0001], and treatment x day interaction

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[F(15,126)=8.59, p <0.0001] were observed. Post hoc tests revealed a significant

253

increase in locomotion in the saline + cocaine (p<0.01) and low NTX + cocaine (p<0.01)

254

groups compared to saline + saline control on day 2. Additionally, a significant increase

255

in locomotion was observed for the saline + cocaine, low NTX + cocaine, and low NMF

256

+ cocaine groups compared to saline + saline control on day 4 (p<0.01 each group

257

versus control), day 6 (p<0.0001 each group versus control), and day 8 (p<0.0001 each

258

group versus control). A significant increase in locomotion (i.e., locomotor sensitization)

259

was observed on day 6 and day 8 for the saline + cocaine (p<0.01), low NTX + cocaine

260

(p<0.0001), and low NMF + cocaine (p<0.001) groups compared to day 2. No effect of

261

pretreatment by NTX or NMF was observed (Figure 4a).

262 263 264

3.2.2 High dose NTX or NMF Pretreatment The mean (SEM) distance traveled (in cm) for high (10 mg/kg) NTX or NMF

265

plus cocaine across drug exposure days (days 2, 4, 6, and 8) are shown in Figure 4b.

266

For drug exposure days, a significant effect of treatment [F(5,126)=59.91, p <0.0001],

267

day [F(3,126)=4.73, p <0.0001], and treatment x day interaction [F(15,126)=4.68, p

268

<0.0001] were observed. Post hoc tests revealed a significant increase in locomotion in

269

the saline + cocaine (p<0.05), high NTX + cocaine (p<0.01), and high NMF + cocaine

270

(p<0.01) groups compared to saline + saline control on day 2. Additionally, a significant

271

increase in locomotion was observed for the saline + cocaine, high NTX + cocaine, and

272

high NMF + cocaine groups compared to saline + saline control on day 4 (p<0.001 each

273

group versus control), day 6 (p<0.0001 each group versus control), and day 8

274

(p<0.0001 each group versus control). Post hoc tests revealed a significant increase in

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locomotion (i.e., locomotor sensitization) on day 6 (p=0.02) and day 8 (p<0.01) for the

276

saline + cocaine group compared to day 2. A significant increase in locomotion was

277

observed for high NTX + cocaine (p<0.001) and high NMF + cocaine (p<0.001) groups

278

on day 8 compared to day 2. No effect of pretreatment by NTX or NMF was observed

279

(Figure 4b).

280 281

3.3 Effectiveness of NTX and NMF at the Kappa Opioid Receptor (U50,488H

282

locomotor discoordination assay)

283

The average baseline latency before falling, across groups was 224  6 seconds.

284

No group differences were observed for baseline latency [F(5,42)=0.28, p =0.92].

285

Following U50,488H, latency decreased to 123  20 seconds at +30 min maximal time

286

point for the saline + U50 group. The mean (SEM) percent baseline time before falling

287

from the rotarod for low (1 mg/kg) and high (10 mg/kg) NTX (a) and NMF (b) are shown

288

in Figure 5. A significant effect of treatment [F(5,84)=16.43, p <0.05] and time

289

[F(2,84)=9.13, p <0.0001] were observed. No significant treatment x time interaction

290

was observed [F(10,84)=5.96, p = 0.07]. Post hoc tests revealed no significant effect of

291

either dose of NTX or NMF on locomotor discoordination 20 minutes following injection

292

compared to vehicle (p>0.99). A significant locomotor discoordination was observed for

293

the saline + U50 group both 30 minutes (p<0.01) and 60 minutes (p<0.05) following U50

294

administration compared to saline + saline control. As well, a significant locomotor

295

discoordination was observed for the low NTX + U50 group 30 minutes following U50

296

administration compared to saline + saline control (p<0.05). The reduction in percent

297

baseline time before falling for the low NTX + U50 group was not significantly different

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from the saline + U50 group (p=0.76). High dose NTX + U50 and well as both doses of

299

NMF + U50 were not significantly different from saline + saline control at both the 30

300

and 60 minute time point following U50 administration (p> 0.9). As well, these doses

301

(high NTX, low and high NMF) were significantly different from the saline + U50 group at

302

the 30 minute time point (p  0.01).

303 304

3.4 Effectiveness of NTX and NMF at Mu Opioid Receptor (morphine thermal

305

analgesia assay)

306

The average baseline latency, across groups was 15.9  0.5 seconds. No group

307

differences were observed for baseline latency [F(5,42)=0.45, p =0.81]. Following

308

morphine, latency increased to 44.1  0.9 seconds at +20 min maximal time point for

309

the saline + morphine group. The mean (SEM) percent maximal effect for low (1

310

mg/kg) and high (10 mg/kg) NTX (a) and NMF (b) are shown in Figure 6. A significant

311

effect of treatment [F(5,126)=48.58, p <0.0001], time [F(3,126)=8.31, p <0.0001], and

312

treatment x time interaction [F(15,126)=22.85, p <0.0001] were observed. Post hoc

313

tests revealed no significant effect of either dose of NTX or NMF on thermal analgesia

314

20 minutes following injection compared to vehicle (p>0.67). A significant analgesic

315

effect was observed for the saline + morphine group at 20, 40, and 60 minutes

316

(p<0.0001) following morphine administration compared to saline + saline control. No

317

significant difference in thermal analgesia was observed for either dose of NTX or NMF

318

at 20, 40, and 60 minutes following morphine administration compared to saline + saline

319

control (p>0.75).

320

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4. DISCUSSION There are conflicting reports on the effectiveness of the MOPr antagonist/KOPr

323

partial agonist NTX for treating cocaine use disorder depending on the patient

324

population examined. In multiple studies in which the subject population was restricted

325

to individuals with only cocaine use disorder, the standard NTX dose (i.e., 50 mg/day)

326

was sufficient to moderately reduce the positive subjective effects of cocaine (Kosten et

327

al., 1992; Sofuoglu et al., 2003), cocaine craving (Comer et al., 2013), and cocaine use

328

(Kosten et al., 1989; Schmitz et al., 2014; Schmitz et al., 2001). In studies with a subject

329

population with polysubstance dependence, in particular cocaine and alcohol co-

330

dependence, 50-100 mg/day NTX treatment did not significantly reduce cocaine use or

331

craving (Hersh et al., 1998; Modesto-Lowe et al.; Schmitz et al., 2009; Schmitz et al.,

332

2004). However, 150 mg/day NTX significantly reduced cocaine use for patients with

333

cocaine and alcohol co-dependence (Oslin et al., 1999; Pettinati et al., 2008). It should

334

be noted that medication compliance was relatively low for these studies (e.g., Pettinati,

335

et al., 2008; Schmitz, et al., 2001). Therefore, to interpret the positive treatment effects

336

of NTX for reducing cocaine use one should also consider the low study and medication

337

compliance as well as the known side effects of NTX. As well, these potential clinical

338

“complications” may limit the therapeutic benefit of NTX, and by extension NMF, in a

339

cocaine dependence treatment context. Use of extended release NTX (i.e., Vivitrol, a

340

once month intramuscular injection) may reduce the compliance related complications

341

associated with daily oral NTX administration. Unfortunately, however, no clinical

342

studies to date have examined the effectiveness of Vivitrol in reducing cocaine use and

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craving in cocaine dependent patients without other substance dependence (i.e.,

344

cocaine but not alcohol dependent population).

345

Although there is growing preclinical and clinical evidence for the effectiveness of

346

NTX for the treatment of cocaine addiction very limited research has examined the

347

structurally similar MOPr antagonist/KOPr partial agonist NMF for treating cocaine use

348

disorder. No preclinical studies to date have examined the effect of NMF on cocaine

349

reward. Clinically, a single case report has described “as needed” NMF (18 mg)

350

reducing cocaine craving in a female cocaine-dependent patient for 5 months

351

(Grosshans et al., 2015). Here we report, to our knowledge for the first time, direct

352

comparison of NTX and NMF in mouse models. We examined the effectiveness of low

353

(1 mg/kg) and high (10 mg/kg) doses of NTX and NMF on attenuating the rewarding

354

effects of cocaine.

355

As has been reported previously (e.g. Bailey et al., 2012; Velazquez-Sanchez et

356

al., 2010), a significant conditioned place preference was observed for 15 mg/kg

357

cocaine. Pretreatment with NTX or NMF 30 minutes prior to cocaine injection was

358

observed to attenuate the place preference for cocaine for both the low and high doses

359

of NTX and high dose NMF. This is in line with prior preclinical NTX studies which have

360

demonstrated that either acute or chronic NTX pretreatment significantly reduces

361

cocaine place preference (Bilsky et al., 1992; Mitchem et al., 1999; Sala et al., 1995;

362

Suzuki et al., 1992). Importantly, this is the first observation of NMF attenuating cocaine

363

place preference. The interaction between NTX or NMF with cocaine cannot be directly

364

ruled out as being strictly behavioral rather than pharmacological in nature from these

365

experiments. However, prior studies have shown that NTX, but not lithium chloride,

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366

blocks cocaine place preference despite both NTX and lithium chloride having similar

367

conditioned place aversions when administered alone (Sala et al., 1995; Suzuki et al.,

368

1992). This suggests that the blockade of cocaine place preference by NTX, and by

369

extension NMF, observed here may be pharmacological in nature. One potential

370

mechanism may be that NTX and NMF attenuate cocaine-induced striatal dopaminergic

371

efflux via their KOPr partial agonist effects as full KOPr agonists have been observed to

372

fully block cocaine-induced striatal dopamine efflux (Zhang et al., 2004a, b). Further

373

studies are needed to clarify the precise mechanism(s) by which NTX and NMF block

374

the rewarding effects of cocaine.

375

In our in vivo characterization in C57Bl/6 mice, both the low and high doses of

376

NTX fully block MOPr agonist induced analgesia; however, the high, but not low, dose

377

of NTX fully blocked KOPr agonist-induced locomotor discoordination. This suggests

378

that the low (1 mg/kg) dose of NTX is fully active at MOPr, but not KOPr. Similar to the

379

findings with NTX, both the low and high doses of NMF fully block MOPr agonist

380

induced analgesia; however, both NMF doses were observed to fully block KOPr

381

agonist-induced locomotor discoordination. Prior in vitro studies (deHaven-Hudkins et

382

al., 1990; Ghirmai et al., 2008; Michel et al., 1985) suggest that NMF is more potent

383

than NTX at the KOPr. This is supported by our finding that systemic administration of 1

384

mg/kg NMF, but not 1 mg/kg NTX, is able to fully block the KOPr-agonist induced

385

locomotor discoordination.

386

Together with the CPP data, our findings suggest that the rewarding effects of

387

cocaine are reduced by a MOPr selective dose of NTX (1 mg/kg) and fully attenuated by

388

a NTX or NMF dose (i.e., 10 mg/kg) that is active at both mu and kappa opioid

17

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389

receptors. This indicates that for the MOPr antagonists/KOPr partial agonists NTX and

390

NMF, reduction of the rewarding effects of cocaine can be achieved by MOPr selective

391

doses; however, further reductions are observed at doses active at both the MOP and

392

KOP receptors. Systemic and striatal administration of full KOPr agonists (e.g., Dyn A1-

393

17,

394

cocaine place preference in both rats and mice (Crawford et al., 1995; Shippenberg et

395

al., 1996; Zhang et al., 2004a, b). This provides some support for the attenuation of

396

cocaine place preference by high dose NTX/NMF being in part due to their partial

397

agonist effects at the KOPr. However, due to a lack of selective KOPr partial agonists

398

the precise role of KOPr partial agonism on attenuating cocaine reward cannot be

399

determined. As well, the effect of high dose NTX/NMF may be due to other factors

400

beyond KOPr partial agonism (e.g., signaling events downstream of MOPr or action at

401

delta opioid receptors).

402

U50488H, U69593, and R-84760) has been observed to significantly attenuate

In addition to increased potency at KOPr and DOPr, NMF has been shown to

403

have a longer duration of action than NTX across several species with a mean terminal

404

half-life of 11-13 hrs versus 9 hrs in humans, respectively (Dixon et al., 1987; Gal et al.,

405

1986; Ingman et al., 2005; Kyhl et al., 2016; Misra et al., 1976; Teklezgi et al., 2018;

406

Wall et al., 1981). In rats, similar plasma half-lives were observed for NTX (1.4 hr) and

407

NMF (50 min) (Misra et al., 1976; Murthy et al., 1996); it should be noted, however, that

408

different routes of drug administration (ip versus iv, respectively) were utilized for these

409

studies. Similar reports of naltrexone and/or nalmefene in mice have not been reported;

410

but as no significant species difference was observed for plasma NTX binding across

411

species, in particular between rats and mice (20% and 22%, respectively), plasma half-

18

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lives for NTX and NMF may be similar (Ludden et al., 1976). Both compounds are

413

extensively metabolized by the liver with species specific differences in metabolites

414

(Dayton and Inturrisi, 1976; Dixon et al., 1987; Ingman et al., 2005; Misra et al., 1976;

415

Murthy et al., 1996; Wall et al., 1981). In particular, for humans the major metabolites of

416

NTX are the biologically inert glucuronide conjugated NTX and peripherally active opioid

417

receptor antagonist 6--naltrexol (Wall et al., 1981) while rats have major metabolites of

418

glucuronide conjugated NTX, 7,8-dihydro-14-hydroxy-normorphinone, and 7,8-dihydro-

419

14-hydroxy-normorphine with negligible 6--naltrexol observed in plasma (Misra et al.,

420

1976; Rodgers et al., 1980). Conversely, for humans the major metabolite of NMF is

421

glucuronide conjugated NMF (Dixon et al., 1987; Ingman et al., 2005) while in rats NMF

422

is metabolized to the potentially biologically active nornalmefene (Murthy et al., 1996).

423

The biological relevance of these species-specific divergences in NTX and NMF

424

metabolites are predominately unknown.

425

In humans NTX and NMF have been shown to fully block the physiological and

426

behavioral effects of fentanyl up to 48 hrs following acute dosing (Gal et al., 1986).

427

Despite rather rapid plasma clearance (1.4 hrs in humans and 50 min in rats), NMF has

428

been observed to slowly dissociate from central MOPr with a clearance half-life of 28.6

429

hrs (Kim et al., 1997) and ~50-70% occupancy 50 hrs post administration (Ingman et al.,

430

2005). Similar protracted effects of opioid ligands have been observed. For example, in

431

vivo the plant-derived KOPr agonist Salvinorin A has a short duration of action on

432

thermal analgesia in mice with maximal effect at 10-15 min and no effect 30-45 min

433

following administration (Ansonoff et al., 2006; John et al., 2006). However, Salvinorin A

434

has been shown to significantly reduce striatal extracellular dopamine concentration in

19

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435

mice for 10 hours following administration (Zhang et al., 2005). Therefore, although the

436

efficacies of NTX and NMF at MOPr and KOPr were examined here for double the

437

duration of place conditioning and locomotor activity assays (i.e., 60 minutes) it is likely

438

that both compounds are still centrally active far longer than the period measured.

439

Further research is needed to examine the duration of action of NTX and NMF at MOPr

440

and KOPr centrally, in particular regarding striatal dopamine levels.

441

Mixed findings have been reported as to the place conditioned effect of NTX

442

alone. Some studies have shown that 5 – 15 minute pretreatment with low dose NTX

443

(0.01-1 mg/kg) prior to the conditioning session was neutral in the conditioned place

444

preference paradigm, producing neither preference nor aversion for the drug paired

445

compartment (Bespalov et al., 1999; Parker and Rennie, 1992). Others, however, have

446

reported that either low dose NTX (0.1 – 1 mg/kg) administered immediately prior to the

447

conditioning session or higher dose NTX (3-10 mg/kg) produced a conditioned place

448

aversion (Bechara and van der Kooy, 1985; Daniels et al., 2016; Suzuki et al., 1992).

449

The inconsistencies across studies in the literature are not clearly explained by

450

procedural differences alone. Here, a significant place avoidance was found for high

451

(10mg/kg) dose NTX and both doses of NMF (1.0 and 10 mg/kg) with a 30-minute

452

pretreatment using an unbiased conditioning paradigm. Previous studies have shown

453

that blockade of central endogenous opioid activity by MOPr antagonists can induce a

454

CPA (Mucha et al., 1985). As both low and high dose NTX and NMF were found to fully

455

block MOPr-induced thermal analgesia, the observed place aversion for NTX and NMF

456

is consistent with their antagonist action at MOPr.

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457

Systemic administration of NTX has been reported to block cocaine-induced

458

hyperlocomotion (Sala et al., 1995); however, chronic NTX exposure via osmotic pump

459

has been shown to only slightly reduce (Kunko et al., 1998) or even increase cocaine-

460

induced hyperlocomotion (Lesscher et al., 2005). Here, no effect of either NTX or NMF

461

pretreatment on cocaine-induced locomotor stimulation or locomotor sensitization was

462

observed. This was an unexpected, though not unprecedented, finding. Prior studies

463

have demonstrated a dissociation between the rewarding and locomotor activating

464

aspects of various drugs including cocaine (Jocham et al., 2006; Pum et al., 2008;

465

Rademacher et al., 2000; Rademacher and Steinpreis, 2002; Ukai and Holtzman,

466

1988). This suggests that the rewarding effects of cocaine, as measured by conditioned

467

place preference, and the cocaine-induced hyperlocomotion and locomotor sensitization

468

may involve divergent neural pathways. The former pathway may be modulated by the

469

opioid system, with the latter not necessarily directly interacting with the opioid system.

470

It should be noted that only a single dose of cocaine was used for the place conditioning

471

and cocaine-induced hyperlocomotion assays. This dose (15 mg/kg) was selected

472

based both on previous studies (Bailey et al., 2012; Velazquez-Sanchez et al., 2010)

473

and pilot work demonstrating that this dose is sufficient to produce a robust cocaine

474

place preference. The inconsistency between these findings and the literature for NTX

475

blockade of cocaine-induced hyperlocomotion may be due to the dose of cocaine used

476

(15 mg/kg versus 10 mg/kg used by Sala et al. 1995). Further research is needed to

477

clarify the role of NTX/NMF in attenuating cocaine-induced hyperlocomotion and

478

cocaine reward.

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479

Overall, the structurally and pharmacologically similar compounds NTX and NMF

480

were both demonstrated to attenuate the rewarding effects of cocaine in C57Bl/6 mice,

481

as reflected in dose-dependent reduced conditioned place preference to cocaine. Both

482

of these compounds also produced aversive effects in the place conditioning paradigm

483

when administered alone. The cocaine CPP findings suggest that antagonism/partial

484

agonism at the KOPr may further attenuates the rewarding effects of cocaine in non-

485

dependent male C57Bl/6 mice. Further research is necessary to determine the potential

486

mechanism(s) by which high dose NTX or NMF treatment reduces cocaine craving in

487

cocaine and alcohol co-dependent patient populations.

488 489

Acknowledgements: The authors gratefully thank Dr. Eduardo Butelman for his

490

constructive discussion of the data and manuscript. We also thank Aurora Grutman for

491

her assistance with the open field assay.

492

Funding: This work was supported by the Gary R. Helman Postdoctoral Research

493

Fellowship (KAW), the Robertson Therapeutic Development Fund (BR, MJK), and the

494

Dr. Miriam and Sheldon G. Adelson Medical Research Foundation (MJK). Sponsors had

495

no role in study design; collection, analysis, and interpretation of data; writing

496

manuscript; or journal selection for publication.

497

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Figure Captions. Fig 1. Structures of naltrexone and nalmefene. Fig 2. a) Timeline for place conditioning experiments. Following pretest session, mice received four saline (saline + saline) and four drug (NTX or NMF + cocaine) conditioning sessions (8 days total; saline first conditioning session). Mice received pretreatment (saline, NTX, or NMF) 30 minutes prior to saline or cocaine injection after which they were immediately placed into and confined to appropriate conditioning compartment for 30 minutes. Following conditioning, on day 10 mice had a 30-minute non-drug post-test session. Place preference or aversion was calculated as percent preference for the drug paired side during post-conditioning test (time drug pair side)/ (time drug pair side + saline pair side)x100. b) Timeline for cocaine-induced locomotor sensitization assay. Injections and drug exposure were identical to that for the place conditioning experiment. Fig 3. Effect of naltrexone (a) or nalmefene (b) on cocaine condition place preference (n=8/group). Percent preference for drug paired side during 30-minute drug-free postconditioning test session. Cocaine significantly increased preference for the drug paired side (a and b). Both doses of naltrexone (a) and high dose nalmefene (b) significantly decreased percent preference for the cocaine paired compartment. High dose naltrexone and both doses of nalmefene caused significant aversion for the drug paired compartment. (*p<0.05) Fig 4. Effect of naltrexone and nalmefene on locomotor activity across drug exposure days (n=8/group). Locomotor activity in open field is expressed as mean distance traveled in cm (SEM) during 30 min session on drug exposure days. Cocaine alone (a and b), low dose naltrexone or nalmefene + cocaine (a), and high dose naltrexone or nalmefene + cocaine (b) significantly increased locomotor activity compared to saline control (*p<0.05) as well as were significantly increased on final activity session (day 8) compared to first activity session (day 1; # p<0.05). Fig 5. Effect of naltrexone or nalmefene on kappa opioid receptor agonist U50,488H-induced locomotor discoordination (n=8/group). Significant locomotor discoordination was observed for U50,488H (10 mg/kg) alone compared to vehicle control (* p<0.05). High, but not low, dose naltrexone (a) and both doses of nalmefene (b) fully blocked U50,488H-induced locomotor discoordination (# p<0.05; compared to U50,488H group). Fig 6. Effect of naltrexone or nalmefene on mu opioid receptor agonist morphine-induced thermal analgesia (n=8/group). A significant antinociceptive response was observed for morphine (15 mg/kg) alone compared to vehicle control (* p<0.05). Both doses of naltrexone (a) and nalmefene (b) fully blocked morphine induced antinociception.

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Direct comparison structurally similar ligands naltrexone (NTX) and nalmefene (NMF)

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NTX and NMF attenuate cocaine-induced conditioned place preference

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NTX and NMF alone induce conditioned place avoidance

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NTX and NMF have no effect on cocaine-induced hyperlocomotion

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NMF in vivo is more potent at kappa opioid receptors than NTX