A single compound alternative to a buprenorphine/naltrexone combination

Recent studies suggest the combination of buprenorphine and naltrexone is a promising drug lead to prevent relapse to drug taking [1]. However it has proven to be a challenge to design a single formulation of this combination due to different routes of administration needed in the clinical setting. In this project, we targeted buprenorphine analogues as single compound mimics of the combination, having lower efficacy at mu opioid receptors (MOR) than buprenorphine, but retaining buprenorphine's activity at kappa (KOR) and ORL-1 receptors. Novel buprenorphine analogues were synthesized and screened initially using [35S]GTPgS and radioligand binding assays and a number of candidate compounds (BU127, BU10101, BU10136, BU10112 and BU10119) were then more fully studied in isolated tissue assays. Rats (male, 300-325 g), Sprague Dawley strain) were killed using CO2, vasa deferentia were excised and suspended in a tissue bath (3 ml volume) under 1 g tension in standard Krebs-bicarbonate solution at 37◦C (95%O2/5%CO2). Nerve-evoked muscle contractions were induced with single square pulses (0.1 ms duration, 0.1 Hz) and recorded isometrically. To study the effects of candidate compounds on ORL-1 receptors, cumulative concentration response curves for the ORL-1 receptor agonist nociceptin (1 mcM-30 mcM) were constructed in the absence and presence of increasing concentrations of buprenorphine, BU127, BU10101, BU10136, BU10112 and BU10119 (1 mcM-30 mcM, n - 4-8 tissues per compound). The antagonist potency for these compounds at ORL-1 receptor were compared against the standard ORL-1 antagonist SB612111 (10 nM-100 nM, n = 8). To study the effects of candidate compounds on MORs, similar experiments were performed using the MOR agonist DAMGO (10 nM-100 mcM) in the presence of increasing concentrations of buprenorphine, BU127, BU10101 and BU10119 (0.1 nM-30 nM, n = 4-6 tissues per compound) and the standard MOR antagonist naltrexone (1 nM-30 nM, n = 5). All test compounds were added to the organ bath 15-20 min prior to agonist treatment. A single high concentration of each candidate compound (30 - 100 mcM) confirmed their lack of ORL-1 or MOR agonist activity in this isolated organ preparation. In ORL-1 studies, increasing concentrations of buprenorphine, BU127, BU10101, BU10112, BU10119 and Bu10136 produced rightward shifts in the concentration-response curve for nociceptin. Schild plots were constructed to determine the pA2 values. For the standard ORL-1 antagonist, buprenorphine and some test compounds, competitive reversible antagonism was observed with the following pA2 values: SB612111 (8.08, 95% CI 7.99, 8.17), buprenorphine (6.02, 95% CI 5.85, 6.19), BU127 (5.88, 95% CI 5.73, 6.04) and BU10119 (5.87, 95% CI 5.79, 5.96). The Gaddum equation was used to estimate the PA2 value compounds showing pseudo-irreversible or non-competitive antagonism and the estimated pA2 values (mean ± SEM) were 5.97 ± 0.13 for BU10101, 5.65 ± 0.18 for BU10136 and 5.14 ± 0.10 for BU10112. In the MOR studies, a similar approach was taken. Naltrexone, BU127 and BU10119 showed competitive reversible MOR antagonism with the following pA2 values: naltrexone (8.90, 95% CI 8.68, 9.12), BU127 (10.24, 95% CI 10.07, 10.41), BU10119 (10.24, 95% CI 10.14, 10.33). The other compounds tested displayed pseudo-irreversible or non-competitive antagonism, with estimated pA2 values of: buprenorphine (9.66 ± 0.10), BU10101 (9.79 ± 0.03). Studies into the activity of these compounds at KOR are ongoing, but these data suggesr a range of novel compounds with mixed antagonist activity at MOR and ORL-1 receptors, which may mimic a combination of buprenorphine and naltrexone.