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NEUROPHARMACOLOGY

Low Sensitivity of the Positron Emission Tomography Ligand [¹¹C]Diprenorphine to Agonist Opiates

Hammersmith Imanet Ltd., Hammersmith Hospital, London, United Kingdom (S.P.H., V.N., E.H., R.A.); Psychopharmacology Unit, University of Bristol, Bristol, United Kingdom (A.R.L.-H., D.J.N.); and Department of Palliative Medicine, Bristol Haematology and Oncology Centre, Bristol, United Kingdom (A.N.D.)

Received for publication February 20, 2007
Accepted May 7, 2007.

	Abstract

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Previously, we reported minimal opioid receptor occupancy following a clinical dose of the µ-opioid agonist, methadone, measured in vivo using positron emission tomography (PET) with [¹¹C]diprenorphine and subsequently used rats to obtain experimental data in support of a high receptor reserve hypothesis (Melichar et al., 2005). Here, we report on further preclinical studies investigating opioid receptor occupancy with oxycodone (µ- and -receptor agonist), morphine (µ-receptor agonist), and buprenorphine (partial agonist at the µ-receptor and antagonist at the - and -receptors), each given at antinociceptive doses. In vivo binding of [¹¹C]diprenorphine was not significantly reduced after treatment with the full agonists but was reduced by 90% by buprenorphine. In addition, given that [¹¹C]diprenorphine is a non-subtype-specific PET tracer, there was no regional variation that might feasibly be interpreted as due to differences in opioid subtype distribution. The data support minimal competition between the high-efficacy agonists and the non-subtype-selective antagonist radioligand and highlight the limitations of [¹¹C]diprenorphine PET to monitor in vivo occupancy. Alternative means may be needed to address clinical issues regarding opioid receptor occupancy that are required to optimize treatment strategies.

Opioid receptors are classified into at least three subtypes (µ, , ) and are G-protein coupled, with activation by both native peptides and structurally distinct nonpeptide alkaloid ligands (Minami and Satoh, 1995). In the continued presence of agonists, acute actions are followed by diverse regulatory processes, including rapid internalization, that modulate the number and functional activity of the receptors (e.g., Ko et al., 1999). The oripavine, diprenorphine has a high affinity to each subtype (Richards and Sadée, 1985) but, although acting as a partial agonist at (Toll et al., 1998), likely partial agonist at -receptors, is an antagonist at the µ-subtype (Lee et al., 1999). Its high lipophilicity (Shapira et al., 2001) and lack of sensitivity to sodium ions (Lee et al., 1999) facilitates binding of [¹¹C]diprenorphine to internalized (Shapira et al., 2001), but not down-regulated (Ko et al., 1999), opioid receptors. The efficacy of diprenorphine at the different subtypes is important when interpreting its use as a PET tracer, because the likelihood of it being displaceable by an endogenous or exogenous agonist is less likely if diprenorphine is an antagonist. If a PET tracer is sensitive to exogenous or endogenous compounds, increasing their levels will result in reduced radioactive tracer binding. Such sensitivity is most widely exploited in human imaging of the dopaminergic system, e.g., [¹¹C]raclopride and dopamine.

To help explore why in our human [¹¹C]diprenorphine PET study we were unable to detect occupancy by methadone and to inform future human [¹¹C]diprenorphine PET studies, we investigated whether acute doses of an alternative opioid agonist used in opioid addiction or treatment of dependence (morphine, oxycodone, or buprenorphine) were able to block [¹¹C]diprenorphine binding in rat brain. Relative intrinsic efficacies at the µ-opioid receptor subtype of those tested were methadone > oxycodone > morphine > buprenorphine (Adams et al., 1990; Duttaroy and Yoburn, 1995; Cowan et al., 1977). If higher intrinsic activity at the different receptor subtypes is related to a higher receptor reserve in vivo, we hypothesized that regional differences in [¹¹C]diprenorphine blockade (receptor occupancy) might be detected with the different opiates. As with human studies, in vivo rat PET scanning had already demonstrated blockade of [¹¹C]diprenorphine binding with the antagonist, naloxone (Rajeswaran et al., 1991), and ex vivo autoradiographic findings were consistent with [³H]diprenorphine binding being a sensitive index of endogenous agonist concentration (Seeger et al., 1984; Gerrits et al., 1999). In the first rat study of the current series, we determined that specific binding of [¹¹C]diprenorphine was not reduced by acute pretreatment with methadone, at antinociceptive i.v. doses, and this finding was published alongside the human study, in support of the high receptor reserve hypothesis (Melichar et al., 2005).

Here, we report results from acute treatments with the remaining compounds, namely, oxycodone, a µ- (Chen et al., 1991) and putative - (Nielsen et al., 2000) receptor agonist, morphine, a potent agonist acting primarily via the µ-receptor, and buprenorphine, a partial agonist at the µ-receptor and antagonist at the - and -receptors (Lee et al., 1999; Neilan et al., 2004). All doses were chosen from those reported in the literature as being effectively antinociceptive.

	Materials and Methods

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The study was carried out by licensed investigators in accordance with the UK Home Office's Guidance on the Operation of the Animals (Scientific Procedures) Act 1986 (Her Majesty's Stationery Office, February, 1990) and used 36 adult male Sprague-Dawley rats (mean ± S.E.; body weight, 288 ± 7 g). Animals were from Harlan UK Ltd. (Bicester, UK). Oxycodone hydrochloride, quinine hydrochloride, and morphine sulfate were purchased from Sigma-Aldrich (Poole, UK). Buprenorphine hydrochloride (Temgesic; Reckitt and Coleman) was from Central Biological Services (Hammersmith Campus, Imperial College, London, UK). [¹¹C]Diprenorphine was supplied by Hammersmith Imanet on site for human PET, according to the method described by Luthra et al. (1994), and aliquots dispensed for biological studies.

The biodistribution studies were begun within 10 min of end-of-synthesis and used three to four rats per preparation. The radioligand was given i.v. to awake but lightly restrained rats, via a previously catheterized tail vein. The mean ± S.E. injected radioactivity was 11.7 ± 0.4 MBq, with a specific activity of 42 ± 4 MBq/nmol (decay-corrected to the first injection for each experiment). The coinjected stable diprenorphine was 1.3 ± 0.1 nmol/kg, expected to have a minimal "occupancy" effect (Richards and Sadée, 1985; Cunningham et al., 1991). At 60 min after injection of [¹¹C]diprenorphine, rats were euthanized by i.v. Euthatal, and a blood sample (1 ml) was collected from the ventricle of the heart. The brains were removed within 2 min, and 16 regions were sampled, as listed in Table 1. These were chosen to represent not only a range of opioid receptor density but also a differential distribution of the three major classes. Table 1, compiled from various sources, summarizes the relative proportion of the subtypes in each of the regions sampled. Note that -sites are not prevalent in rat brain, representing an estimated 10 to 12% of the total opioid receptor binding capacity, compared with 37% in human brain (Mansour et al., 1988).

View this table: [in this window] [in a new window]   TABLE 1 Expected relative distributions of µ-, -, and -receptor sites in sampled tissues, compiled from in vitro autoradiographic reports, including Mansour et al. (1988) and Tohyama and Takatsuji (1998)

Radioactivity was measured using a Wallac gamma counter, with automatic correction for radioactive decay. Results were normalized for the amount of radioactivity injected and for body weight, giving "uptake units" = (counts per minute per gram wet weight tissue)/(injected counts per minute per gram body weight). A more detailed description and the rationale for the 60-min endpoint are published in Melichar et al. (2005). To account for group differences in clearance of radioactivity from the peripheral circulation, the tissue concentrations were also expressed relative to both individual plasma and cerebellum concentrations. The latter was assumed to approximate to nonspecific binding of the radioligand (Seeger et al., 1984; Schadrack et al., 1999), with the tissue/cerebellum uptake ratio at 60 min giving a measure of the specific binding index (SBI).

Seven groups of animals were used. One group received only the radioligand (controls, n = 5). Six further groups were predosed 5 min before [¹¹C]diprenorphine as follows: 1) i.v. oxycodone HCl at 0.5 mg/kg in physiological saline (0.9% sodium chloride), n = 5; 2) oxycodone HCl as above but preceded by i.p. quinine HCl at 40 mg/kg in saline, 65 min before [¹¹C]diprenorphine to block oxycodone metabolism, n = 5; 3) quinine HCl as above, n = 5; 4) i.v. morphine sulfate at 1 mg/kg in saline, n = 6; 5) morphine sulfate as above but at a dose of 3 mg/kg, n = 5; and 6) i.v. buprenorphine (300 µg/ml in dextrose) at 0.6 mg/kg, n = 5. Doses were consistent with those detailed in the literature, as follows: oxycodone (Cleary et al., 1994; Nielsen et al., 2000), quinine (Cleary et al., 1994), morphine (Nielsen et al., 2000), and buprenorphine (Ohtani et al., 1997). Because the O-demethylated metabolite of oxycodone, oxymorphone, has also been shown to be a potent µ-receptor agonist, a additional group of rats was given quinine before oxycodone, the former acting to inhibit the cytochrome P450 isozyme, CYP2D1 activity in liver (Cleary et al., 1994), such that any measured effects in this group might be considered due to the parent opiate.

Statistical analyses were primarily by two-way ANOVA (group x region) with Bonferroni correction for multiple comparisons, carried out using Prism 4 (GraphPad Software Inc., San Diego, CA). Significance levels are shown as: *, P < 0.05; **, P < 0.01; ***, P < 0.001 (decrease); or , increase.

	Results

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Uptake Data. Concentration of [¹¹C]diprenorphine (uptake units) for all regions sampled is shown in Table 2. As expected, a statistically significant effect was measured between regions (P < 0.0001, two-way ANOVA, for each treatment). The biodistribution was as published previously (Cunningham et al., 1991; Melichar et al., 2005), with highest binding in colliculi, thalamus, periaqueductal gray, and striata, and minimal binding in cerebellum. Except for the group that received only the CYP2D1 inhibitor quinine, each drug effect was also measured as statistically significantly different from control.

Oxycodone resulted in a significant (P < 0.0001) increase in radioactivity concentration (average ± S.D. = 10 ± 5%, with or without cerebellum). When combined with quinine, the effect of oxycodone was greater (average increase = 28 ± 7%) with five of the dissected tissues reaching individual significance, as detailed in Table 2. Rats pretreated with the lower dose of morphine (1 mg/kg) also showed a significant drug effect (P < 0.0001, average increase = 13 ± 5%, with or without cerebellum). After the higher dose (3 mg/kg), statistically significant increases compared with control were measured in 10 of the 16 tissues sampled. In contrast to the increase in binding seen after oxycodone and morphine, a large decrease in binding was seen after pretreatment with buprenorphine. Individually, significant reductions (P < 0.001) were measured in all tissues except cerebellum, with an average percent decrease of 89 ± 3%, compared with control. A slightly lower effect was noted in cervical cord (80% reduction).

The apparent lack of effect of buprenorphine on cerebellum radioactivity concentration supports the use of cerebellum as a reference region. It then follows that the homogeneous increases in tissue radioactivity measured after oxycodone and morphine reflected increases in nonspecific, rather than specific, binding. Compared with control, increases in plasma radioactivity were measured after all drug treatments, consistent with an increase in bioavailability of radioligand during the 60 min required to approach a pseudoequilibrium. Plasma concentration was, however, also increased after predosing with quinine, contrasting with a minimal 4 ± 4% change measured across brain tissues.

Tissue/Plasma Data. Tissue/plasma radioactivity concentrations are presented in Table 3. As noted for the "uptake" data, there was a statistically significant effect of region for each treatment (P < 0.0001, two-way ANOVA). The highly significant effect of buprenorphine was again observed (P < 0.0001), with each tissue except cerebellum showing individual reductions (P < 0.001; average reduction, 93 ± 2%, without cerebellum). In contrast to the uptake data, however, individual tissue/plasma ratios showed no significant increase following any drug treatment. A small overall drug effect remained following predosing with the higher dose of morphine (P < 0.01; average increase 9 ± 6%, with or without cerebellum). As expected from its differential effect on plasma radioactivity, a significant overall effect of quinine was noted (P < 0.0001; average reduction, 27 ± 3%), with individual effects in 6 of the 16 regions sampled.

Tissue/Cerebellum Normalization. Although no drug was shown to cause a statistically significant difference in group cerebellum uptake, normalizing tissue radioactivity concentration to individual cerebellum samples to estimate SBI gave a different pattern of response, as presented in Table 4. Compared with control, no statistically significant effects were obtained following predosing with quinine, oxycodone with quinine, or either dose of morphine. Although oxycodone given alone caused a small overall drug effect (P < 0.001, average increase, 6 ± 5%), only striatum showed an individual tissue effect (P < 0.05, 19% increase compared with control). Blocking with the partial agonist, buprenorphine, was retained, with all regions showing a significant effect (P < 0.001, average reduction 88 ± 3%). As noted for the uptake data, buprenorphine caused proportionally less blocking in cervical cord (80 versus 90%). In all regions, SBI remained above unity (one-sample Student's t test to obtain two-tailed P values).

For further clarity, Fig. 1 illustrates SBI data, selecting three of the regions sampled, namely, anterior cingulate cortex, prefrontal cortex, and hypothalamus, taken to represent tissues that, in vitro, have higher distributions of µ-, -, and -subtypes, respectively (see Table 1). The percent changes compared with control are also shown. Included for comparison are equivalent data obtained after predosing with methadone (0.35 mg/kg), calculated from previously published SBI data (Melichar et al., 2005).

View larger version (22K): [in this window] [in a new window]   Fig. 1. Specific binding indices after i.v. injection of [11C]diprenorphine in anterior cingulate cortex (µ > ) (a), prefrontal cortex ( > µ) (b), and hypothalamus ( > µ) (c). Data are means with SD bars from five or six rats. Results from statistical analyses of drug treatment against control (two-way ANOVA with Bonferroni correction, *) and SBI against unity for the buprenorphine effect (one-sample Student's t test, ) are shown, with significance levels as described in Table 4. Group differences (percentage of control) for anterior cingulate cortex (d), prefrontal cortex (e), and hypothalamus (f) are also shown. Shaded bars, equivalent data from methadone-treated rats (0.35 mg/kg), estimated from SBI data published previously (Melichar et al., 2005), and a separate, time-matched control group (n = 6, for each).

	Discussion

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The overall lack of an acute blocking effect of methadone, morphine, and oxycodone on [¹¹C]diprenorphine specific binding index is entirely consistent with the high receptor reserve hypothesis. For instance, Perry et al. (1982) reported only a 2% occupancy of binding sites for the agonist oripavine, etorphine at the ED₅₀ for the rat tail-flick assay, and a 50% reduction in [³H]etorphine binding after morphine was only seen at a dose up to 900 times greater than the reported analgesic ED₅₀. Likewise, acute etorphine [intrinsic efficacy >> morphine (Duttaroy and Yoburn, 1995)] treatment had no effect on ex vivo binding of [³H]diprenorphine to rat brain membranes. In the continued presence of etorphine, an 68% decrease in [³H]diprenorphine binding was observed after chronic, 3-day exposure (Tao et al., 1987). This is consistent with receptor down-regulation following internalization/endocytosis (Keith et al., 1996). Compared with the low receptor occupancy of pure agonists at antinociceptive doses, the dose of diprenorphine that displaces 50% of bound [³H]etorphine has been reported as close to the antagonist ED₅₀ (Perry et al., 1982). Likewise, the ED₅₀ for naloxone displacement of [¹¹C]diprenorphine in vivo in human brain is consistent with clinical efficacy of the antagonist (Melichar et al., 2003). Alternative hypotheses that may explain the inability of [¹¹C]diprenorphine to be blocked by opiate agonists include that µ-opiate binding sites have high affinity for both agonists and antagonists, but upon binding, agonists cause dissociation from the G protein, resulting in accelerated dissociation only for agonists leaving low-affinity sites.

In contrast to the lack of effect of full agonists, the partial agonist, buprenorphine, caused an almost complete block of [¹¹C]diprenorphine specific binding index. Mutagenesis studies have suggested that partial agonists may have different determinants for binding to the µ-receptor than full agonists and the similar structure of diprenorphine and buprenorphine (differing only at position C-19, with diprenorphine/methyl and buprenorphine/t-butyl) may explain buprenorphine's binding being more characteristic of an antagonist (Bot et al., 1998). Neilan et al. (2004) have additionally referred to a tight lipophilic interaction of the t-butyl group with the µ-opioid receptor, slowing down the dissociation kinetics of buprenorphine, consistent with its long-lasting agonist action. Alternatively, with this very high-affinity and much slower dissociation rate, buprenorphine is likely to have occupied the µ-opioid receptors close to irreversibly over the experimental time course.

It was notable that oxycodone and morphine resulted in a homogeneous increase in radioactivity relative to control. This was indicative of altered nonspecific rather than specific binding and, consistent with this, was almost completely eliminated by normalizing tissue data to individual rat plasma concentration (Table 3 versus Table 2). Increased plasma retention of radioactivity is assumed to reflect either reduced clearance or metabolism of the radioligand or blockade of peripheral binding. We suggest that since high concentrations of opiate binding have been reported in several rodent organs, including liver, kidney, lung, and intestine (Wang et al., 2004), that this is the most likely explanation for the increase in nonspecific binding. With the aim of separating any small, specific effect of drug from nonspecific binding changes, we chose the cerebellum as a reference region, accepting some known limitations of the method since although in vitro studies have generally failed to detect significant levels of cerebellar µ-opioid receptors (Mansour et al., 1988; Schadrack et al., 1999), in vivo specific [³H]diprenorphine binding has been described previously (Cunningham et al., 1991). This can lead to a bias in the estimation of receptor number, with a more marked effect in low-density regions.

What other factors could contributed to reduced [¹¹C]diprenorphine binding that was not due to agonist occupancy? Firstly, we were unable to measure whether any radiolabeled metabolite(s) might have contributed to the signal in the plasma but nevertheless used the tissue/plasma normalization ratio to express our data. Quinine alone resulted in reduced binding of [¹¹C]diprenorphine, and since we do not expect quinine to bind to opioid receptors in the brain, this reduction must be due to increased concentrations of [¹¹C]diprenorphine in the plasma. Because the major route of metabolism of [¹¹C]diprenorphine is via O-demethylation, this "quinine effect" is probably indicative only of the difficulties and inaccuracies that arise from using tissue(parent)/plasma(total) ratio as a measure of volume of distribution. Secondly, [¹¹C]diprenorphine levels in the plasma compared with the brain may differ since opiates (Wang et al., 2004) and, to some extent, quinine (Solary et al., 2003), are P-glycoprotein substrates, and the volume of distribution of [¹¹C]diprenorphine in the brain is highly dependent on ligand extraction. Because the efflux transporter system is saturable (King et al., 2001), the predosing regimes used might alter [¹¹C]diprenorphine transport. Although potential differences in the ability to cause receptor internalization were originally considered, with methadone but not morphine causing some endocytosis (Whistler et al., 1999), other studies have reported that, at "clinically relevant analgesic doses," methadone, like morphine and buprenorphine, shows little detectable internalization (Keith et al., 1996). In any case, [¹¹C]diprenorphine can label, in vitro, opioid receptor populations on the cell surface and those internalized (Shapira et al., 2001).

One of the possible benefits of using [¹¹C]diprenorphine compared with other PET radiotracers is its quoted similar in vitro affinity for each subtype (Pfeiffer and Herz, 1982), so it could potentially inform the relative contribution of a particular subtype to the image. However, following each of the agonist treatments used, we were unable to detect an obvious differential in tissue response to drug that might be interpreted as due to distribution of the opioid receptor subtypes. Although diprenorphine is quoted as having a similar in vitro affinity for each subtype (Pfeiffer and Herz, 1982), early in vivo binding studies in rat reported a 4-fold higher affinity of diprenorphine for µ- relative to -receptor sites (Richards and Sadée, 1985). This suggests that, despite approximately equal numbers of the receptor subtypes in rat cortex, the in vivo signal (a measure of "receptor number/apparent affinity") will predominantly reflect binding to the µ-receptors, and the signal in the striatum will have similar contributions from µ- and -receptor sites, despite the 3:7 distribution in favor of the latter (Tao et al., 1987). Thus, detecting subtle subtype differences in occupancy, such as, for example, comparing regional effects of the putative -receptor agonist, oxycodone compared with the µ-receptor agonist, morphine, could prove difficult. Buprenorphine has a 40-fold lower in vivo affinity to - compared with µ-subtypes (Sadée et al., 1983), which could explain the slightly lower buprenorphine block in [¹¹C]diprenorphine specific binding index in cervical cord compared with brain, we could find no evidence that the -subtype predominates in cord. In addition, when translating such evidence from rats to man, consideration is needed about the evidence that distribution of opioid receptor subtypes and their availability to [¹¹C]diprenorphine differ in some regions, e.g., -receptors are generally less prominent in the human brain than in rodent brains (e.g., Hammers and Lingford-Hughes, 2006).

A limitation of using antagonist radioligands to estimate occupancy by agonist drugs at G-protein-coupled receptors is that they may underestimate receptor occupancy by agonist drugs since such a receptor can exist in high-affinity, coupled with G-protein, and low-affinity, free receptor, states. Antagonists such as diprenorphine or naloxone bind homogeneously to both, but agonists such as morphine or methadone bind with high affinity only to G-protein-coupled receptors (e.g., Bantick et al., 2004). Given that our studies show exogenous agonists at behaviorally active doses do not result in detectable occupancy, it is interesting to note that endogenous peptides, whose in vitro intrinsic efficacies are comparable with morphine, seem to be able to block in vivo binding of radiolabeled diprenorphine. This has been seen with reading epilepsy (Koepp et al., 1998) and in animals (Gerrits et al., 1999). Currently, it is not clear why the sensitivity of [¹¹C]diprenorphine binding to exogenous and endogenous ligands is different. Although long-term receptor changes due to either drugs or pathology must be considered, compelling evidence for specific binding index as a means to monitor receptor occupancy was presented by Seeger et al. (1984). However, the data presented here suggest that the nonselective opioid radioligand [¹¹C]diprenorphine is not an ideal choice for indexing agonist receptor occupancy in vivo when data using a more selective antagonist, [¹⁸F]cyclofoxy (Kling et al., 2000), or agonist, [¹¹C]carfentanil (Zubieta et al., 2000), could prove either more robust or interpretable.

	Footnotes

 
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.121749.

ABBREVIATIONS: PET, positron emission tomography; SBI, specific binding index; ANOVA, analysis of variance.

¹ Current affiliation: Department of Palliative Medicine, Royal Marsden National Health Science Foundation Trust, Surrey, United Kingdom.

Address correspondence to: David J. Nutt, Psychopharmacology Unit, Dorothy Hodgkin Building, Whitson Street, University of Bristol, Bristol, BS1 3NY, UK. E-mail: david.j.nutt{at}bristol.ac.uk

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