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. 2015 Jan;172(2):668-80.
doi: 10.1111/bph.12796. Epub 2014 Nov 5.

BU08073 a buprenorphine analogue with partial agonist activity at μ-receptors in vitro but long-lasting opioid antagonist activity in vivo in mice

T V Khroyan  1 , J Wu, W E Polgar, G Cami-Kobeci, N Fotaki, S M Husbands, L Toll
Affiliations

BU08073 a buprenorphine analogue with partial agonist activity at μ-receptors in vitro but long-lasting opioid antagonist activity in vivo in mice

T V Khroyan et al. Br J Pharmacol. 2015 Jan.

Abstract

Background and purpose: Buprenorphine is a potent analgesic with high affinity at μ, δ and κ and moderate affinity at nociceptin opioid (NOP) receptors. Nevertheless, NOP receptor activation modulates the in vivo activity of buprenorphine. Structure activity studies were conducted to design buprenorphine analogues with high affinity at each of these receptors and to characterize them in in vitro and in vivo assays.

Experimental approach: Compounds were tested for binding affinity and functional activity using [(35) S]GTPγS binding at each receptor and a whole-cell fluorescent assay at μ receptors. BU08073 was evaluated for antinociceptive agonist and antagonist activity and for its effects on anxiety in mice.

Key results: BU08073 bound with high affinity to all opioid receptors. It had virtually no efficacy at δ, κ and NOP receptors, whereas at μ receptors, BU08073 has similar efficacy as buprenorphine in both functional assays. Alone, BU08073 has anxiogenic activity and produces very little antinociception. However, BU08073 blocks morphine and U50,488-mediated antinociception. This blockade was not evident at 1 h post-treatment, but is present at 6 h and remains for up to 3-6 days.

Conclusions and implications: These studies provide structural requirements for synthesis of 'universal' opioid ligands. BU08073 had high affinity for all the opioid receptors, with moderate efficacy at μ receptors and reduced efficacy at NOP receptors, a profile suggesting potential analgesic activity. However, in vivo, BU08073 had long-lasting antagonist activity, indicating that its pharmacokinetics determined both the time course of its effects and what receptor-mediated effects were observed.

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

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Figures

Figure 1
Figure 1
Structures of buprenorphine and novel compounds tested. Full synthetic details and analysis, including microanalysis are provided in the Supporting Information Appendix S1.
Figure 2
Figure 2
BU08073 inhibits N/OFQ-stimulated [35S]GTPγS binding in CHO cells transfected with NOP receptors. Experiments were conducted as described in Materials and Methods. Data in the figure are represented as mean ± SEM for a single representative experiment conducted in triplicate. The EC50 value for stimulation of [35S]GTPγS binding by N/OFQ was 12.7 ± 0.56 nM (mean ± SEM from three individual experiments). To test as an antagonist, the BU08073 dose response was conducted in the presence of 100 nM N/OFQ and the IC50 for BU08073 was 697 ± 20 nM (mean ± SEM from three individual experiments).
Figure 3
Figure 3
The effect of DAMGO, buprenorphine and BU08073 in a whole-cell assay measuring membrane potential changes in CHO cells transfected with μ-opioid receptors. Experiments were conducted using the FlexStation. Data shown in the figure are represented as mean ± SEM from a single representative experiment conducted in quadruplicates. (A) DAMGO-induced membrane potential fluorescence change tracked for 50 s. (B) BU08073-induced membrane potential fluorescence change tracked for 50 s. (C) Agonist dose response curve for each compound. EC50 values were 1.96 ± 0.56, 45.5 ± 19 and 323 ± 140 (mean ± SEM from four individual experiments) for DAMGO, buprenorphine and BU08073 respectively. (D) Antagonist effect of naloxone demonstrating that all compounds are acting through the opioid receptor. IC50 values of naloxone were 63 ± 11, 34 ± 20 and 101 ± 43 140 (mean ± SEM from four individual experiments) when inhibiting 100 nM DAMGO, 500 nM buprenorphine and 2 μM BU08073 respectively.
Figure 4
Figure 4
The effects of a range of doses of BU08073 on tail-flick latency compared with morphine (10 mg·kg−1) and vehicle controls at various post-injection time points (s.c., n = 6–9 per group). Data are mean %MPE ± SEM. *P < 0.05, significantly different from vehicle controls using repeated anova.
Figure 5
Figure 5
The effects of 3 mg·kg−1 BU08073 on morphine- (A) and U50,488-induced antinociception (B). All drugs were given via s.c. route of administration. Values on the X-axis indicate time elapsed from the BU 08073 injection. Vehicle, morphine and U50,488 were given 30 min prior to testing. Different groups of animals were used for each BU08073 pretreatment time point (n = 8 per group). Data are mean %MPE ± SEM. *P < 0.05, significantly different from vehicle controls. †significantly different from morphine or U50,488 alone.
Figure 6
Figure 6
The effects of 3 mg·kg−1 BU08073 (s.c.) on anxiety as measured using the elevated zero maze examining time in the open quadrants (A), latency to enter an open quadrant (B) and number of dips over the side (C). Different groups of animals were used to assess the effects of BU08073 (n = 10 per time point) following different post-injection time points. Data are mean ± SEM. *P < 0.05, significantly different from vehicle controls.
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