[11C]-Diprenorphine to Image Opioid Receptor Occupancy by Methadone in Opioid

Home , Diprenorphine

JPET Fast Forward. Published on September 3, 2004 as DOI: 10.1124/jpet.104.072686 JPET FastThis Forward. article has not Published been copyedited on and September formatted. The 3, final 2004 version as mayDOI:10.1124/jpet.104.072686 differ from this version. JPET #72686

Using [11C]-Diprenorphine to image opioid receptor occupancy by methadone in opioid

addiction: clinical and preclinical studies

Jan K Melichar, Susan P Hume, Tim M Williams, Mark R C Daglish, Lindsay G Taylor,

Rabia Ahmad, Andrea L Malizia, David J Brooks, Judith S Myles, Anne Lingford-Hughes &

David J Nutt Downloaded from

Psychopharmacology Unit, University of Bristol, Bristol, UK (JKM, TMW, MRCD, LGT,

ALM, ALH, DJN), Hammersmith Imanet Ltd., Hammersmith Hospital, London, UK (SPH, jpet.aspetjournals.org RA), MRC Clinical Sciences Centre and Division of Neuroscience, Faculty of Medicine,

Imperial College, Hammersmith Hospital, London, UK (DJB), Bristol Specialist Drug

Service, Blackberry Hill Hospital, Bristol, UK (JSM). at ASPET Journals on October 2, 2021

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Copyright 2004 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on September 3, 2004 as DOI: 10.1124/jpet.104.072686 This article has not been copyedited and formatted. The final version may differ from this version. JPET #72686

RUNNING TITLE PAGE

[11C]-Diprenorphine PET in Methadone Addicts and Rats

FULL POSTAL ADDRESS FOR CORRESPONDENCE

Professor David J Nutt

Psychopharmacology Unit

University of Bristol

Dorothy Hodgkin Building

Whitson Street Downloaded from

Bristol, BS1 3NY

Tel +44 117 331 3178 jpet.aspetjournals.org Fax +44 117 331 3180 [email protected]

OTHER DETAILS: at ASPET Journals on October 2, 2021 Number of Text pages: 18

Number of Tables: 3

Number of Figures: 5

Number of References: 40

Number of Words in Abstract: 221

Number of Words in Introduction: 530

Number of Words in Discussion: 1228

Non-Standard Abbreviations: PET: Positron Emission Tomography; DSM-IV:

Diagnostic and Statistical Manual of Mental Disorders - Fourth Edition; MRC: Medical

Research Council; HPLC: High Performance Liquid Chromatography; VD: Volume of

Distribution; VOI: Volume of Interest.

Recommended Section Assignment: Neuropharmacology

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ABSTRACT

Substitute methadone prescribing is one of the main modes of treatment for opioid dependence with established evidence for improved health and social outcomes. However, the pharmacology underpinning the effects of methadone is little studied despite controversies about dosing in relation to outcome. We therefore examined the relationship between methadone dose and occupation of opioid receptors in brain using the Positron Emission

Tomography (PET) radioligand [11C]-Diprenorphine in humans and rats.

Eight opioid dependent subjects stable on their substitute methadone (18-90mg daily) had an Downloaded from

[11C]-Diprenorphine PET scan at predicted peak plasma levels of methadone. These were compared with 8 healthy controls. No difference in [11C]-Diprenorphine binding was found jpet.aspetjournals.org between the groups, with no relationship between methadone dose and occupancy. Adult male

Sprague-Dawley rats that had been given an acute i.v. injection of methadone hydrochloride

(0.35, 0.5, 0.7 or 1.0 mg.kg-1) prior to [11C]-Diprenorphine showed a dose-dependent increase at ASPET Journals on October 2, 2021 in biodistribution but no reduction in [11C]-Diprenorphine binding. We suggest that the lack of a dose dependent relationship between methadone dose, either given chronically in man or acutely in rat, and occupancy of opioid receptor measured with [11C]-Diprenorphine PET is related to efficacy of this opioid agonist at very low levels of opioid receptor occupancy. This has implications for understanding the actions of methadone in comparison with other opioid drugs such as partial agonists and antagonists.

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Opiate addiction is a chronically relapsing illness and a major health problem, with large direct health costs, both psychiatric and physical, as well as significant costs to society in terms of crime, loss of earnings and productivity and social damage (Mark et al., 2001). One of the few proven treatments is the use of substitute or maintenance therapy with the opioid agonist methadone, which has been used successfully for several decades in the USA and the

UK. There is substantial evidence to support the effectiveness of methadone in reducing illicit opioid use, intravenous drug use, crime, unemployment, and improving overall health status, social rehabilitation and quality of life (Marsch, 1998). However, methadone treatment is not Downloaded from without its problems. Apart from the perceived stigma of being addicted to an opioid, many patients still relapse and fundamental questions about the mechanisms of action of methadone jpet.aspetjournals.org remain unanswered. For example, it is not well understood why some patients appear to require larger doses to suppress the desire to use or to help them move away from use of heroin (Maxwell and Shinderman, 2002). Inter-individual differences in the pharmacokinetics at ASPET Journals on October 2, 2021 and pharmacodynamics of methadone globally (Eap et al., 2002) and at the blood brain barrier

(Wang et al., 2004) may only partially explain these differences.

Despite the extensive use of the methadone over twenty years, little is known about the relationship between dose, brain levels and receptor occupation. A long held underlying assumption is that substitution medication, such as methadone, occupies opioid receptors in the brain and thus minimizes withdrawal symptoms and prevents ‘on-top’ heroin use from accessing the receptor, thus reducing its reinforcement. We have previously shown that methadone maintained opioid dependent patients are less sensitive to the objective and subjective effects of hydromorphone, an opioid agonist (Melichar et al., 2003a). Information about the dose:opioid receptor occupancy relationship for methadone should help address

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JPET Fast Forward. Published on September 3, 2004 as DOI: 10.1124/jpet.104.072686 This article has not been copyedited and formatted. The final version may differ from this version. JPET #72686 important clinical issues such as the correlation of dose to outcome and the assessment of patients who claim they are being under-dosed.

[11C]-Diprenorphine has been used extensively as a PET neuroimaging ligand. It has high affinity at µ, κ and δ opioid receptors (Jones et al., 1988; Sadzot et al., 1991), being an antagonist at µ and δ but having weak efficacy at the κ opioid receptor, where it acts as a partial agonist (Lewis and Husbands, 2004). It was originally chosen as a positron emitting Downloaded from radioligand because of its safety, lack of side-effects at tracer doses in human pilot studies, rapid cerebral uptake and high percentage (80-90%) specific binding in animal in vivo studies.

In rat, it is most densely distributed in the basal ganglia and neocortex excluding the occipital jpet.aspetjournals.org cortex (Pert et al., 1975). In man, in vivo, high uptake of [11C]-Diprenorphine has been demonstrated with PET in regions such as the thalamus, caudate nucleus, temporal, frontal and parietal cortices, which are known from post-mortem studies to have high concentrations at ASPET Journals on October 2, 2021 of µ, κ and δ opioid receptors (Jones et al.,1988). The aim of this current study, therefore, was to examine the relationship between methadone dose and occupancy of brain opioid receptor levels, as measured by [11C]-Diprenorphine PET in humans and rats.

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MATERIALS AND METHODS

Human study:

Subjects:

Eight male opioid dependent patients, stabilised on methadone were recruited from patients attending the methadone clinic at the Bristol Specialist Drug Service in Bristol, UK. They fulfilled the DSM-IV criteria for opioid dependence and were only consuming methadone.

They were typically long-standing opioid users, had been on methadone for a mean of 5.9 years (range 1-20 years) with a mean age of 39.9 (range 20-46) and had been on a stable dose Downloaded from for at least 1 month: see Table 1. Subjects were not abusing other drugs, as demonstrated by regular urine drug screens and all smoked nicotine. They had no serious physical or mental jpet.aspetjournals.org illness, as demonstrated by clinical interview and physical assessment. These subjects were compared with 8 age-matched control subjects (6 males, 2 females), mean age 37 years (range

25-56), who had no significant mental or physical illness and were not abusing any drugs, as at ASPET Journals on October 2, 2021 demonstrated by a urine drug screen (2 were nicotine smokers).

Methods:

The opioid dependent patients received under supervision their usual methadone dose 4 hours prior to their [11C]-Diprenorphine PET scan. This was in order to provide a measure of peak methadone occupancy at the opioid receptors. [11C]-Diprenorphine was synthesized according to standard protocols (Luthra et al., 1985). Scans were obtained using a brain dedicated ECAT

953b Siemens/CTI PET scanner in high-sensitivity 3D mode. The dose of [11C]-

Diprenorphine per scan was of the order of 370 MBq given as an intravenous bolus (Jones et al., 1994). An arterial line was inserted into the non-dominant radial artery to allow [11C]-

Diprenorphine metabolites to be measured every 5 to 10 minutes to generate a radiolabelled metabolite corrected input function. The images were acquired over 90 minutes as 18 time

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For six of the methadone group, blood samples were analysed for their (R)-enantiomeric

[active compound] and (S)-enantiomeric methadone levels. Blood samples were taken 4 hours after supervised ingestion of their usual daily dose of methadone, in order to provide a measure of peak methadone levels, just prior to the injection of [11C]-Diprenorphine. The blood samples were analyzed by high performance liquid chromatography (HPLC) with a Downloaded from chiral column as previously described (Eap et al., 1996).

jpet.aspetjournals.org This study was approved by the local Research Ethics Committees and by the UK

Administration of Radioactive Substances Advisory Committee (ARSAC). After a full explanation of the protocol, all volunteers gave written informed consent. at ASPET Journals on October 2, 2021

Animal Study:

The work was carried out by licensed investigators, according to the Home Office’s

“Guidance in the Operation of Animals (Scientific Procedures) Act 1986” and used 34 adult male Sprague-Dawley rats with a body weight of 292 ± 3 g (mean ± s.e.). During the study, the rats were awake but lightly restrained in Bollman cages.

Methods:

Each rat was given ~11 MBq of [11C]-Diprenorphine in 0.20 ml, via a previously catheterised tail vein. At designated times after injection, rats were given an i.v. injection of Euthatal, the brain removed, and tissues rapidly sampled into pre-weighed vials. The tissues were: cervical cord, olfactory tubercles, hypothalamus, thalamus, prefrontal cortex, striatum, somatosensory

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JPET Fast Forward. Published on September 3, 2004 as DOI: 10.1124/jpet.104.072686 This article has not been copyedited and formatted. The final version may differ from this version. JPET #72686 cortex, hippocampus, visual with temporal cortex, inferior colliculi, superior colliculi, medulla with pons and cerebellum.

In the first series of experiments (‘time:course’), sample times were 1, 5, 20, 30, 45, 60 and 90 min after injection for a control group, and for a group of rats given racemic methadone hydrochloride (Sigma Aldrich; 0.35 mg.kg-1, IV), 5 min before [11C]-Diprenorphine. A single animal was used per time point. In 6 of the rats, blood was collected at graded times via a previously catheterised tail artery (3 control and 3 pre-dosed with methadone, 6 samples per rat). The 0.35 mg.kg-1 IV dose was chosen as one which elicits “analgesia with minimum Downloaded from adverse respiratory effects” (Garrido et al., 1999). Using this dose, the authors quote a rat plasma concentration of ~100 ng.ml-1 immediately after injection, falling rapidly to ~50 jpet.aspetjournals.org ng.ml-1 within 5 min and then, more gradually, to ~20 ng.ml-1 at 60 min. The corresponding brain (cortex) concentration was ~350 ng.g-1 at 5 min after injection, falling to ~150 ng.g-1 at

60 min after injection. Their estimate for drug potency for analgesia (tail flick test) was 24 at ASPET Journals on October 2, 2021 ng.ml-1 of plasma, uncorrected for plasma protein binding. In the present study, the pre-dosed rats were pale and non-responsive, with depressed respiration; recovery began at ~40 min.

In the second series (‘dose:effect’), the sample time was 60 min after [11C]-Diprenorphine injection for a control group (n=5) and for 4 further groups of rats given methadone as before, at either 0.35 (n = 5), 0.5 (n = 3), 0.7 (n = 4) or 1.0 mg.kg-1 (n = 3). For presentation, data from control and 0.35 mg.kg-1 dosed rats have been added to the first series, giving n=6, for the 60-min time point.

Data analysis / Statistics:

Human study:

The dynamic images were reconstructed with filtered back projection, measured attenuation and dual energy window scatter corrections. Each reconstructed frame of [11C]-Diprenorphine

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View, Calif, USA) using AnalyzeAVW version 3.1 (Mayo Foundation; Robb & Hanson,

11 1991). Volume of distribution [VD] images of [ C]-Diprenorphine were generated with spectral analysis using ‘receptor parametric mapping’ software developed at the MRC

Cyclotron Unit, which does not require any reference regions to be used (Gunn et al 1997).

Volumes of interest [VOI] were drawn on individual VD maps of the opioid receptors as labelled by [11C]-Diprenorphine with reference to the stereotaxic atlas of Talairach and

Tournoux. The regions studied included the thalamus, caudate nucleus, temporal and frontal Downloaded from cortices, known to have high concentrations of opioid receptors. Mean VD values were calculated for each VOI. These values were then compared between groups using two-tailed t- jpet.aspetjournals.org tests. The relationship between methadone dose, oral daily dose and (R)-enantiomer methadone level, and availability of opioid receptors was explored.

at ASPET Journals on October 2, 2021 Animal study:

Radioactivity in tissue and blood (whole blood and plasma) was counted using a WALLAC gamma-counter with automatic correction for radioactivity decay. Data were normalised for injected radioactivity and body weight, giving:

‘uptake units’ = (cpm.g-1 wet weight tissue).(injected cpm.g-1 body weight)-1.

The methodology has been previously described (Hume et al., 1992). Specific, rather than total, binding was estimated by expressing tissue radioactivity concentration as tissue:cerebellum ratios minus unity, assuming that rat cerebellum represents a tissue with minimal opioid receptors (Cunningham et al., 1991). Where discussed, statistical significance was assessed using either two-factor ANOVA or two-tailed t-test with Bonferroni correction for multiple comparison and a level of significance set at 0.05 (13 tissues and 12 tissue:cerebellum ratios).

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RESULTS

Human study:

The opioid-dependent subjects R-enantiomer methadone levels are shown in Table 2,

11 alongside their mean global [ C]-Diprenorphine VD. Comparison of mean global

Diprenorphine VD showed a non-significant trend toward the methadone group having 12% lower levels of [11C]-Diprenorphine binding than the control group (17.14 + 1.06 vs. 19.54

+1.21 p=0.10 uncorrected, mean + S.E.) (Figure 1). When individual regions were examined,

11 Downloaded from [ C]-Diprenorphine VD was not significantly lower in any region in opioid dependent patients compared to control subjects, nor were there any hemispheric asymmetries: see Table

3 and Figure 1. The data was further analysed using published data (Pfeiffer et al.; 1982, jpet.aspetjournals.org Cross et al.; 1987, Delay-Goyet et al., 1987) on the regional variation in the distribution of the

µ, κ and δ-opioid receptor sub-types assessed in vitro. This did not yield any relationship between receptor sub-type distribution and the effect of methadone on [11C]-Diprenorphine at ASPET Journals on October 2, 2021 binding (data not shown).

There was no clear relationship between oral doses or plasma levels (ng.ml-1) of the R- enantiomer of methadone and [11C]-Diprenorphine binding, both globally and regionally

(Figure 2).

Animal study:

Time-course:

Figure 3 illustrates the effect of 0.35 mg.kg-1 methadone hydrochloride (i.v. 5 min before

[11C]-Diprenorphine injection) on the distribution of radioactivity in two of the tissues dissected, namely thalamus and striatum, chosen as high µ-site density regions. In these tissues, as in all others dissected, the methadone treatment resulted in a higher initial uptake

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JPET Fast Forward. Published on September 3, 2004 as DOI: 10.1124/jpet.104.072686 This article has not been copyedited and formatted. The final version may differ from this version. JPET #72686 and retention of [11C]-Diprenorphine. Also shown in Figure 3, are the equivalent tissue:cerebellum ratios, taken to represent total:non-specific binding of the radioligand.

When expressed in this way, there was no effect of the methadone pre-dosing, implying that the increased radioactivity concentration reflected a difference in bioavailability of the tracer, rather than an increase in the specific component of binding of [11C]-Diprenorphine.

Consistent with increased bioavailability, we measured a higher plasma radioactivity content in the methadone-treated rats, at early times after injection (data not shown).

Figure 4 summarises the 60-minute data for all tissues dissected. Using a two-factor ANOVA Downloaded from with replication, the radioactivity content (Fig. 4(a)) was significantly increased, by an average of 16%, in the methadone treated group (closed bars) compared with control (open jpet.aspetjournals.org bars) (P<0.001). Evaluation of each tissue using individual ANOVA and Bonferroni correction for multiple comparison showed a statistically significant effect in hippocampus and inferior colliculi. Similar analyses for the tissue:cerebellum ratio data (Fig. 4(b)) showed at ASPET Journals on October 2, 2021 no significant group effect and no effect of methadone pre-dosing in any tissue.

Dose:Effect:

As discussed in the Methods section, the 0.35 mg.kg-1 dose was chosen as being pharmacologically active and was expected to result in an acute plasma level comparable with that expected during maintenance treatment in patients. Since, however, there is a marked species difference in the pharmacokinetics of methadone (t1/2=70-90 min in rats compared with 24 hours in man (Zhou et al., 1996)) and the rat studies used a racemic mixture, an additional series was included, increasing the methadone up to 1 mg.kg-1. Again, the methadone was given IV, 5 min before [11C]-Diprenorphine but all rats were euthanased at 60 min after radioligand injection. For the same tissues as shown in Fig. 3, Fig. 5 illustrates a graded increase in radioactivity content, as the methadone dose was increased, but again no reduction in the tissue:cerebellum ratios. Statistical analysis comparing the highest dose group

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JPET Fast Forward. Published on September 3, 2004 as DOI: 10.1124/jpet.104.072686 This article has not been copyedited and formatted. The final version may differ from this version. JPET #72686 with control showed a significant increase in content in all tissues sampled and a significant increase in tissue:cerebellum ratios for thalamus, striatum and colliculi (all tissues with a high

µ-site density). In no tissue, for any of the doses used, was there the expected decrease in specific binding. Downloaded from jpet.aspetjournals.org at ASPET Journals on October 2, 2021

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DISCUSSION

Using [11C]-Diprenorphine PET to quantify opioid receptors we have shown that, over a range of clinically effective doses of methadone, no significant occupancy of opioid receptors in the brain was detectable and no difference in [11C]-Diprenorphine binding between these patients and healthy controls was observed. Parallel animal studies using an acute analgesic dose of methadone [0.35 mg.kg-1] similiarly revealed no detectable reduction in opioid receptor availability. These findings contrast with our previous demonstration that patients on similar doses of methadone showed dose related blockade of opioid agonist effects [Melichar et al., Downloaded from

2003a].

jpet.aspetjournals.org Our finding contrasts with other recent neuroimaging studies in opioid dependent subjects.

Kling et al. (2000) explored the dose-occupancy relationship of methadone using [18F]-

Cyclofoxy PET as a marker of µ and κ opioid receptors in patients scanned just before their at ASPET Journals on October 2, 2021 next methadone dose (30 to 90mg daily). This is similar in dose range to our study, though opposite in timing, as they looked at trough levels of methadone. They found a significant

(19-32%) reduction in [18F]-Cyclofoxy binding throughout the brain, particularly in the thalamus, caudate, anterior cingulate, middle temporal and medial frontal cortices. However, within these areas, only in caudate was there a significant relationship between plasma methadone levels and reduction in [18F]-Cyclofoxy binding.

Another PET radiotracer, [11C]-Carfentanil, a high affinity µ opioid receptor agonist, has also been used to examine availability of opioid receptor in patients taking buprenorphine, a µ partial agonist and κ antagonist increasingly used in the treatment of opioid dependence.

Partial agonists can be used at much higher concentrations than full agonists due to their increased safety and, at high receptor occupancy, act as antagonists to full opioid agonists

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(Nutt, 1997). Greenwald et al. (2003) reported that buprenorphine dose-dependently reduced whole brain [11C]-Carfentanil binding by 41%+ 8, 80% + 2, and 84%+ 2 at 2, 16, and 32 mg respectively. Importantly, this study also demonstrated a direct relationship for buprenorphine between in vivo human brain receptor binding and clinical symptoms. In addition, these reductions were associated with reduced effects of hydromorphone, similar to those reported previously by our group with methadone (Melichar et al 2003a). So, unlike methadone, buprenorphine at clinical doses does appear to significantly occupy opioid receptors, resulting in almost complete blockade at high doses [16mg, 32mg]. Downloaded from

There are several factors that need to be examined to help explain our findings of a lack of jpet.aspetjournals.org occupancy of brain opioid receptors by methadone at clinically effective doses using [11C]-

Diprenorphine PET. Does this reflect the binding properties of the opioid agonist, methadone, is it an insensitivity of [11C]-Diprenorphine PET or is it a function of the opioid dependent at ASPET Journals on October 2, 2021 subjects studied?

With regards to the opioid agonist used, the range of methadone doses prescribed corresponds to those in the study by Kling et al. (2000). In addition, patients in the present study had been stable on their dose for at least 1 month (range 1 month – 8 years) with no on- top heroin use, implying that their dose was ‘clinically effective’. In our study we only measured the active R-enantiomer 4 hours after methadone ingestion whilst Kling (ibid) reported plasma methadone levels 22 hours after ingestion. This makes direct comparison of plasma doses in these two studies difficult due to the complex metabolism of methadone (Eap et al., 2002). Plasma methadone levels are related to cerebrospinal fluid (CSF) levels, with

CSF levels being ~20% of plasma levels (Rubenstein et al., 1978). There is some variability, some of which could be explained by the polymorphic expression of P-glycoprotein: recent

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JPET Fast Forward. Published on September 3, 2004 as DOI: 10.1124/jpet.104.072686 This article has not been copyedited and formatted. The final version may differ from this version. JPET #72686 work looking at the blood brain barrier has shown that the entry of methadone is as a substrate of P-glycoprotein (Wang et al., 2004). One can, even with this potential variability, estimate that, in our sample, given plasma levels of 176-540nM, the concentration of methadone in

CSF available at the µ opioid receptor is ~40 – 100nM (~20%). Since the Ki of methadone is

10.1nM at the µ opioid receptor (Li et al., 1998), this would suggest that the concentration of methadone is sufficient to occupy a significant proportion of brain µ opioid receptors.

Therefore, it is likely that the range of plasma and brain methadone concentrations in the Downloaded from present study are within the same range as in Kling et al (2000), predicting a reduction in

[11C]-Diprenorphine binding similar to that seen with [18F]-Cyclofoxy.

jpet.aspetjournals.org

We used [11C]-Diprenorphine as a tracer to measure opioid receptor binding. It is a non- selective tracer that labels µ, κ and δ opioid receptors with similar high affinity (Lewis and

Husbands, 2004). We have previously shown that the non-selective opioid antagonist, at ASPET Journals on October 2, 2021 naloxone, displaces ~50% of [11C]-Diprenorphine signal at clinically relevant doses

(~13µg/kg) (Melichar et al. 2003b). In addition, in vivo in man [11C]-Diprenorphine does appear to be sensitive to increased levels of endogenous opioids since reduced binding has been shown in response to pain (Jones et al., 1991), reading epilepsy (Koepp et al., 1998) and, in animals, to cocaine and ethanol (Gerrits et al., 1999). However, results from our animal study showed that a single high dose of methadone did not reduce specific [11C]-

Diprenorphine binding, whereas previously we have observed that naloxone (Rajeswaran et al., 1991) and buprenorphine (Hume, unpublished observations) did reduce [11C]-

Diprenorphine binding in the rat brain. Taken together, these data suggest that interactions between an agonist, partial agonist or antagonist and the opioid receptor result in differential ability to displace [11C]-Diprenorphine, perhaps reflecting slightly different interactions with the pharmacophore.

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Methadone is a high efficacy agonist so requires only a small proportion of receptors to exert its effect and it may be that this low occupation is at the limits of detection using [11C]-

Diprenorphine PET. Methadone has affinity for all three receptor subtypes, but its Ki at the µ receptor subtype is 10.1nM (Li et al., 1998) compared with over 1000nM for κ and δ receptor subtypes (Raynor et al., 1994). Even in the thalamus where the µ subtype predominates

(Cross et al., 1987), no reduction in [11C]-Diprenorphine binding was seen. An alternative Downloaded from explanation is the possible internalisation of opioid receptors secondary to agonist

(methadone) exposure (Borgland et al., 2003) and the ability of the different PET radiotracers to label these internalised receptors. [11C]-Diprenorphine has been shown to label, in vitro, jpet.aspetjournals.org internalised opioid receptors (Shapira 2001), which could explain the lack of reduction in

[11C]-Diprenorphine binding whereas data are lacking for the two other tracers.

at ASPET Journals on October 2, 2021

Another consideration is whether chronic methadone administration might have increased the expression of opioid receptors so obscuring any observable effect of methadone occupation of receptors. In animal, chronic opioid exposure with morphine has been reported to increase receptor number (Zadina et al., 1995). However, a post-mortem study of heroin addicts revealed no difference in opioid receptors levels (Gabilondo et al., 1994). We have preliminary data showing a global increase in [11C]-Diprenorphine binding in recently detoxified methadone patients (Daglish et al., 2000) which may suggest that methadone does upregulate receptor number.

In conclusion, we have shown that over a range of clinically effective doses, methadone does not appear to result in significant occupancy of brain opioid receptors. In contrast, in a similar population of methadone maintained opioid dependent patients, we showed tolerance to

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JPET Fast Forward. Published on September 3, 2004 as DOI: 10.1124/jpet.104.072686 This article has not been copyedited and formatted. The final version may differ from this version. JPET #72686 opioids with attenuation of objective and subjective responses to hydromorphone. We therefore suggest that clinically relevant doses of methadone result in low (<10%) levels of occupancy that are below the limits of detection with [11C]-Diprenorphine PET. Our results support evidence that tolerance to methadone is likely to involve changes other than reducing receptor number. Downloaded from jpet.aspetjournals.org at ASPET Journals on October 2, 2021

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REFERENCES

Borgland SL, Connor M, Osborne PB, Furness JB, and Christie MJ (2003). Opioid Agonists

Have Different Efficacy Profiles for G Protein Activation, Rapid Desensitization, and

Endocytosis of Mu-opioid Receptors. J Biol Chem 278(21): 18776-18784.

Cross AJ, Hille C, Slater P. (1987) Subtraction autoradiography of opioid receptor subtypes in human brain. Brain Res 418:343-348.

Downloaded from

Cunningham VJ, Hume SP, Price GR, Ahier RG, Cremer JE, Jones AK. (1991)

Compartmental analysis of Diprenorphine binding to opioid receptors in the rat in vivo and its jpet.aspetjournals.org comparison with equilibrium data in vitro. J Cereb Blood Flow Metab 11(1):1-9.

Daglish MRC, Lingford-Hughes A, Melichar J, Brooks DJ, Myles JS, Nutt DJ. (2000) at ASPET Journals on October 2, 2021 Changes in opioid receptor binding during opioid detoxification. J Psychopharmacol 14 (3):

A17.

Delay-Goyet P, Zajac JM, Javoy-Agid F, Agid Y, Roques BP. (1987) Regional distribution of mu, delta and kappa opioid receptors in human brains from controls and parkinsonian subjects. Brain Res 414(1):8-14

Eap CB, Buclin T, Baumann P. (2002) Interindividual variability of the clinical pharmacokinetics of methadone: implications for the treatment of opioid dependence. Clin

Pharmacokinet 41(14):1153-93.

- 18 -

JPET Fast Forward. Published on September 3, 2004 as DOI: 10.1124/jpet.104.072686 This article has not been copyedited and formatted. The final version may differ from this version. JPET #72686

Eap CB, Finkbeiner T, Gastpar M, Scherbaum N, Powell K, Baumann P. (1996) Replacement of (R)-methadone by a double dose of (R,S)-methadone in addicts: interindividual variability of the (R)/(S) ratios and evidence of adaptive changes in methadone pharmacokinetics. Eur J

Clin Pharmacol 50:385-389.

Gabilondo AM, Meana JJ, Barturen F, Sastre M, Garcia-Sevilla JA.(1994) mu-Opioid receptor and alpha 2-adrenoceptor agonist binding sites in the postmortem brain of heroin addicts. Psychopharmacology 115(1-2): 135-40. Downloaded from

Garrido MJ, Valle M, Calvo R, Troconiz IF. (1999) Altered plasma and brain disposition and jpet.aspetjournals.org pharmacodynamics of methadone in abstinent rats. J Pharmacol Exp Ther 288:179-187.

Gerrits MA, Wiegant VM, Van Ree JM. (1999)Endogenous opioids implicated in the at ASPET Journals on October 2, 2021 dynamics of experimental drug addiction: an in vivo autoradiographic analysis. Neuroscience

89(4): 1219-27.

Greenwald MK, Johanson CE, Moody DE, Woods JH, Kilbourn MR, Koeppe RA, Schuster

CR, Zubieta JK. (2003) Effects of Buprenorphine Maintenance Dose on mu-Opioid Receptor

Availability, Plasma Concentrations, and Antagonist Blockade in Heroin-Dependent

Volunteers. Neuropsychopharmacology 28(11):2000-9.

Gunn RN, Lammertsma AA, Hume SP and Cunningham VJ. (1997) Parametric Imaging of

Ligand-Receptor Binding Using a Simplified Reference Region Model. NeuroImage 6:279-

287.

- 19 -

JPET Fast Forward. Published on September 3, 2004 as DOI: 10.1124/jpet.104.072686 This article has not been copyedited and formatted. The final version may differ from this version. JPET #72686

Hume SP, Lammertsma AA, Bench CJ, Pike VW, Pascali C, Cremer JE, Dolan RJ. (1992)

Evaluation of S-[11C]]citalopram as a radioligand for in vivo labelling of 5-hydroxytryptamine uptake sites. Int J Rad Appl Instrum B 19(8):851-5.

Jones AK, Luthra SK, Maziere B, Pike VW, Loch C, Crouzel C, Syrota A, Jones T. (1988)

Regional cerebral opioid receptor studies with [11C]-Diprenorphine in normal volunteers. J

Neurosci Methods 23:121-129.

Downloaded from

Jones AK, Qi LY, Fujirawa T, Luthra SK, Ashburner J, Bloomfield P, Cunningham VJ, Itoh

M, Fukuda H, Jones T. (1991) In vivo distribution of opioid receptors in man in relation to the jpet.aspetjournals.org cortical projections of the medial and lateral pain systems measured with positron emission tomography. Neurosci Lett 126:25-28.

at ASPET Journals on October 2, 2021 Jones AK, Cunningham VJ, Ha-Kawa SK, Fujiwara T, Liyii Q, Luthra SK, Ashburner J,

Osman S, Jones T (1994) Quantitation of [11C] Diprenorphine cerebral kinetics in man acquired by PET using presaturation, pulse-chase and tracer-only protocols. J Neurosci

Methods 51: 123–124.

Kling MA, Carson RE, Borg L, Zametkin A, Matochik JA, Schluger J, Herscovitch P, Rice

KC, Ho A, Eckelman WC and Kreek MJ (2000) Opioid receptor imaging with positron emission tomography and [(18)F]Cyclofoxy in long-term, methadone-treated former heroin addicts. J Pharmacol Exp Ther 295(3): 1070-6.

Koepp MJ, Richardson MP, Brooks DJ, Duncan JS (1998) Focal cortical release of endogenous opioids during reading-induced seizures. Lancet 352 (9132): 952-955.

- 20 -

JPET Fast Forward. Published on September 3, 2004 as DOI: 10.1124/jpet.104.072686 This article has not been copyedited and formatted. The final version may differ from this version. JPET #72686

Lee KO, Akil H, Woods JH, Traynor JR. (1999) Differential binding properties of oripavines at cloned mu- and delta-opioid receptors. Eur J Pharmacol 378(3):323-30.

Lewis JW, Husbands SM. (2004) The orvinols and related opioids - high affinity ligands with diverse efficacy profiles. Curr Pharm Des 10(7):717-32.#

Li JG, Raffa RB, Cheung P, Tzeng TB and Liu-Chen LY (1998) Apparent thermodynamic Downloaded from parameters of ligand binding to the cloned rat mu-opioid receptor. Eur J Pharmacol 354(2-3):

227-37. jpet.aspetjournals.org

Luthra SK, Pike VW and Brady F (1985) The preparation of carbon-11 labelled

Diprenorphine: a new radioligand for the study of the opioid receptor system in vivo. J Chem at ASPET Journals on October 2, 2021 Soc Chem Commun :1423-1425.

Mark TL, Woody GE, Juday T, Kleber HD (2001) The economic costs of heroin addiction in the United States. Drug Alcohol Depend 61(2): 195-206.

Marsch LA. (1998) The efficacy of methadone maintenance interventions in reducing illicit opioid use, HIV risk behavior and criminality: a meta-analysis. Addiction 93(4): 515-32.

Matthes HW, Maldonado R, Simonin F, Valverde O, Slowe S, Kitchen I, Befort K, Dierich A,

Le Meur M, Dolle P, Tzavara E, Hanoune J, Roques BP, Kieffer BL. (1996) Loss of morphine-induced analgesia, reward effect and withdrawal symptoms in mice lacking the mu- opioid-receptor gene. Nature 383:819-823.

- 21 -

JPET Fast Forward. Published on September 3, 2004 as DOI: 10.1124/jpet.104.072686 This article has not been copyedited and formatted. The final version may differ from this version. JPET #72686

Maxwell S, Shinderman MS. (2002) Optimizing long-term response to methadone maintenance treatment: a 152-week follow-up using higher-dose methadone. J Addict Dis

21(3):1-12.

Melichar JK, Myles JS, Eap CB, Nutt DJ. (2003a) Using saccadic eye movements as objective measures of tolerance in methadone dependent individuals during the hydromorphone challenge test. Addiction Biology 8(1):59-66. Downloaded from

Melichar JK, Nutt DJ, Malizia AL. (2003b) Naloxone displacement at opioid receptor sites jpet.aspetjournals.org measured in vivo in the human brain. Eur J Pharmacol 459:217-219.

Nutt DJ. (1997) The Neurochemistry of Addiction. Hum Psychopharmacol 12: S53-S58. at ASPET Journals on October 2, 2021

Pert CB, Kuhar MJ, Snyder SH. (1975) Autoradiograhic localization of the opioid receptor in rat brain. Life Sci 16(12):1849-53.

Pfeiffer A, Pasi A, Mehraein P, Herz A. (1982) Opiate receptor binding sites in human brain.

Brain Res 248(1):87-96

Rajeswaran S, Hume SP, Cremer JE, Young J, Bailey DL, Ashburner J, Luthra SK, Jones

AKP, Jones T. (1991) Dynamic monitoring of [C-11] Diprenorphine in rat-brain using a prototype positron imaging device J Neurosci Meth 40 (2-3): 223-232.

- 22 -

JPET Fast Forward. Published on September 3, 2004 as DOI: 10.1124/jpet.104.072686 This article has not been copyedited and formatted. The final version may differ from this version. JPET #72686

Raynor K, Kong H, Chen Y, Yasuda K, Yu L, Bell GI, Reisine T. (1994) Pharmacological characterization of the cloned kappa, delta, and mu opioid receptors. Mol Pharmacol 45(2):

330-4.

Robb R, Hanson D. (1991) A Software System for Interactive and Quantitative Visualisation of Multidimensional Biomedical Images. Australian Physical and Engineering Sciences in

Medicine 14(1):9-30.

Downloaded from

Rubenstein RB, Kreek MJ, Mbawa N, Wolff WI, Korn R, Gutjahr CL. (1978) Human spinal fluid methadone levels. Drug Alcohol Depend 3(2):103-6. jpet.aspetjournals.org

Sadzot B, Price JC, Mayberg HS, Douglass KH, Dannals RF, Lever JR, Ravert HT, Wilson

AA, Wagner HN Jr, Feldman MA. (1991) Quantification of human opioid receptor at ASPET Journals on October 2, 2021 concentration and affinity using high and low specific activity [11C]-Diprenorphine and positron emission tomography. J Cereb Blood Flow Metab 11:204-219.

Shapira M, Keren O, Gafni M, Sarne Y (2001). Divers pathways mediate delta-opioid receptor down regulation within the same cell. Mol Brain Res 96 (1-2): 142-150.

Wang JS, Ruan Y, Taylor RM, Donovan JL, Markowitz JS, DeVane CL. (2004) Brain penetration of methadone (R)- and (S)-enantiomers is greatly increased by P-glycoprotein deficiency in the blood-brain barrier of Abcb1a gene knockout mice. Psychopharmacology

173(1-2):132-8.

- 23 -

JPET Fast Forward. Published on September 3, 2004 as DOI: 10.1124/jpet.104.072686 This article has not been copyedited and formatted. The final version may differ from this version. JPET #72686

Zadina JE, Kastin AJ, Harrison LM, Ge LJ, Chang SL (1995) Opiate receptor changes after chronic exposure to agonists and antagonists. Ann N Y Acad Sci 757:353-61.

Zhou Y, Spangler R, Maggos CE, LaForge KS, Ho A, Kreek MJ. (1996) Steady-state methadone in rats does not change mRNA levels of corticotropin-releasing factor, its pituitary receptor or proopiomelanocortin. Eur . Pharmacol 315(1): 31-35.

Downloaded from jpet.aspetjournals.org at ASPET Journals on October 2, 2021

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FOOTNOTES

FINANCIAL SUPPORT:

We are grateful to the Wellcome Trust and the Medical Research Council for financial support.

REPRINT REQUESTS:

Professor David J Nutt

Psychopharmacology Unit

University of Bristol Downloaded from

Dorothy Hodgkin Building

Whitson Street jpet.aspetjournals.org Bristol, BS1 3NY

Tel +44 117 331 3178

Fax +44 117 331 3180 at ASPET Journals on October 2, 2021 [email protected]

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FIGURE LEGENDS

Figure 1

11 Graph showing Volume of Distribution (VD) changes in whole brain [ C]-Diprenorphine binding with opioid use. Note that there is a slight, non-significant reduction in binding in the methadone group compared with the normal controls.

Figure 2

11 Downloaded from Plasma methadone (ng/ml) vs. [ C]-Diprenorphine VD for whole brain and three regions.

Note that there is no observable trend, either in whole brain or regionally. Whole Brain,

Thalamus, R Caudate, Occipital jpet.aspetjournals.org

Figure 3

Radioactivity content (uptake units) or tissue:cerebellum concentration ratios in either at ASPET Journals on October 2, 2021 thalamus or striatum as a function of time after injection of [11C]-Diprenorphine. The data are from either control rats (open symbols) or rats pre-dosed with 0.35 mg.kg-1 methadone hydrochloride (closed symbols). Datum points are from individual animals except for the 60 min time point, which is the mean ± SD from groups of 6 rats.

Figure 4

Radioactivity content (uptake units) or tissue:cerebellum concentration ratios in the tissues indicated, measured 60 min after injection of [11C]-Diprenorphine. The data are from either control rats (open columns) or rats pre-dosed with 0.35 mg.kg-1 methadone hydrochloride

(closed columns). Data are means ± SD from groups of 6 rats. The asterisks denote a statistically significant effect of treatment after Bonferroni correction.

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Figure 5

Radioactivity content (uptake units) or tissue:cerebellum ratios in either thalamus or striatum, measured 60 min after injection of [11C]-Diprenorphine. The data are from either control rats

(open columns, n = 6) or rats pre-dosed with either 0.35, 0.5, 0.7 or 1.0 mg.kg-1 methadone hydrochloride (closed columns). Data are means ± SD from groups of 6, 3, 4 or 3 rats, respectively. The asterisks denote a statistically significant effect of treatment after

Bonferroni correction Downloaded from jpet.aspetjournals.org at ASPET Journals on October 2, 2021

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Table 1

Demographic Information for the eight male methadone addicts. All are nicotine smokers and had previously used heroin as their opioid drug of choice.

Duration of Time on Current

Duration of current current Alcohol

Weight Age Methadone opioid addiction methadone methadone Intake

(kg) (yrs) dose (mg) (yrs) treatment (yrs) dose (yrs) (weekly units)

64 20 18 3 1 0.08 20 Downloaded from

77 46 30 25 8 8 28

71 41 50 23 6 7 28 jpet.aspetjournals.org 85 44 50 25 4 2 0

89 42 50 22 3 3 20

65 37 70 16 2 0.25 4 at ASPET Journals on October 2, 2021 78.5 43 90 27 3 0.17 20

68 46 90 24 20 7 30

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Table 2: Methadone dose, R-enantiomer methadone levels (in ng.ml-1 and nM) and [11C]-

Diprenorphine Whole Brain Volume of Distribution (VD) results in opioid dependent patients maintained on methadone. N/A = not available

Methadone Weight (kg) R-enantiomer R-enantiomer [11C]-Diprenorphine

-1 oral dose (mg) levels (ng.ml ) levels (nM) VD (Whole Brain)

18 64 N/A N/A 19.52

30 77 61 176 14.86 Downloaded from

50 71 187 540 17.62

50 85 96 277 15.97 jpet.aspetjournals.org 50 89 145 419 13.24

70 65 173 500 19.07

90 78.5 156 450 14.61 at ASPET Journals on October 2, 2021 90 68 N/A N/A 22.19

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Table 3

Comparison of [11C]-Diprenorphine Volume of Distribution values between controls and methadone groups

Volume of Volume of Distribution (mean + SE) t-test

Interest Control Methadone Uncorrected p Corrected p

Whole brain 19.54 ± 1.21 17.14 ± 1.06 0.10 1.64

Thalamus 29.87 ± 1.97 25.55 ± 1.82 0.19 3.09 Downloaded from

L putamen 29.99 ± 2.06 26.65 ± 1.80 0.35 5.67

R putamen 29.91 ± 2.20 25.95 ± 1.86 0.27 4.26 jpet.aspetjournals.org L caudate 30.12 ± 1.75 25.75 ± 1.53 0.16 2.53

R caudate 30.60 ± 1.96 24.81 ± 1.35 0.06 1.01

Ant. Cingulate 28.14 ± 1.58 24.18 ± 1.41 0.13 2.04 at ASPET Journals on October 2, 2021 L frontal 27.18 ± 1.66 23.84 ± 1.24 0.13 2.12

R frontal 26.83 ± 1.73 24.04 ± 1.41 0.24 3.92

L orbito-frontal 24.12 ± 2.01 20.76 ± 1.07 0.22 3.47

R orbito-frontal 23.45 ± 2.06 20.67 ± 1.20 0.31 5.02

Occipital 11.80 ± 0.63 10.44 ± 0.84 0.27 4.37

Lmed. Temporal 21.00 ± 1.29 18.65 ± 1.31 0.84 13.38

Rmed. Temporal 21.40 ± 1.48 18.50 ± 1.10 0.78 12.48

L cerebellum 19.87 ± 0.96 18.86 ± 1.93 0.65 10.37

R cerebellum 19.61 ± 0.97 18.26 ± 2.00 0.57 9.19

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JPET Fast Forward. Published on September 3, 2004 as DOI: 10.1124/jpet.104.072686 This article has not been copyedited and formatted. The final version may differ from this version. Figure 1

R cerebellum Methadone L cerebellum Control

R med Temporal

L med Temporal

Occipital Downloaded from

R orbito-frontal

L orbito-frontal jpet.aspetjournals.org

R frontal

L frontal at ASPET Journals on October 2, 2021

Ant. Cingulate

R Caudate

L Caudate

R Putamen

L Putamen

Thalamus

Whole brain

0102030 [11C]-Diprenorphine VD JPET Fast Forward. Published on September 3, 2004 as DOI: 10.1124/jpet.104.072686 This article has not been copyedited and formatted. The final version may differ from this version. Figure 2

20 Downloaded from jpet.aspetjournals.org 10 Whole Brain Thalamus at ASPET Journals on October 2, 2021 [11C]-Diprenorphine VD [11C]-Diprenorphine R Caudate Occipital 0 50 100 150 200 Plasma Methadone Levels (ng/ml) JPET Fast Forward. Published on September 3, 2004 as DOI: 10.1124/jpet.104.072686 This article has not been copyedited and formatted. The final version may differ from this version. Figure 3

5 5 Thalamus Striatum 4 4 3 3

2 2 1

1 Downloaded from Radioactivity Radioactivity (uptake units) 0 0 0 20406080100 0 20 40 60 80 100 jpet.aspetjournals.org 30 30 25 Thalamus 25 Striatum 20 20 15 15 at ASPET Journals on October 2, 2021 10

Ratio 10 5 5 0 0

Tissue:Cerebellum Tissue:Cerebellum 0 20406080100 0 20406080100 Time after [11C]-Diprenorphine injection (minutes) JPET Fast Forward. Published on September 3, 2004 as DOI: 10.1124/jpet.104.072686 This article has not been copyedited and formatted. The final version may differ from this version. Figure 4 cerebellum (a) medulla with pons superior colliculi inferior colliculi * visual with temporal cortex hippocampus * somatosensory cortex striatum Downloaded from pre-frontal cortex thalamus hypothalamus jpet.aspetjournals.org olfactory tubercles cervical cord

0123456 Radioactivity (uptake units) at ASPET Journals on October 2, 2021

cerebellum (b) medulla with pons superior colliculi inferior colliculi visual with temporal cortex hippocampus somatosensory cortex striatum pre-frontal cortex thalamus hypothalamus olfactory tubercles cervical cord

0 5 10 15 20 25 30 Tissue:Cerebellum Ratio JPET Fast Forward. Published on September 3, 2004 as DOI: 10.1124/jpet.104.072686 This article has not been copyedited and formatted. The final version may differ from this version. Figure 5

control control Thalamus Thalamus 0.35 0.35

0.5 0.5 Downloaded from 0.7 * 0.7

1 **1 jpet.aspetjournals.org 0246810 0102030

control control

Striatum Striatum at ASPET Journals on October 2, 2021 0.35 0.35

* 0.5 * 0.5

0.7 * 0.7 *

1 * 1 *

0246810 0102030 Radioactivity (uptake units) Tissue:Cerebellum Ratio