Preclinical evaluation of the 6-O-sulfate ester of morphine for the treatment of multimodal acute and chronic pain

Abstract

Chronic pain is a huge economic burden and a devastating complication requiring therapeutic interventions. This dissertation evaluated the analgesic potency, efficacy, tolerance and cross-tolerance profile of morphine and its ester derivative, morphine-6-O-sulfate (M6S) in a rat model of diabetic neuropathy, and in normal rats utilizing a multimodal pain domain approach (e.g., burning pain, pricking pain, and deep muscle pain). Diabetes was induced in male Sprague-Dawley rats with streptozotocin, 65mg/kg (intraperitoneally). Hot water tail-flick latency, paw pressure, pinprick sensitivity and hot plate withdrawal thresholds (HTL, PPT, PST and HPT) were measured at various time points and doses after i.p. injection (N=6) on days 1, 3, 7, 9, 12, 19, 25 and 29 of treatment in both normal and diabetic rats. Affinity binding studies, GTPγ S assays and intracellular cAMP assays were performed in Chinese hamster ovary cells transfected with human mu (μ) - or delta (δ)-opioid receptors (N=5). HPLC-DAD stability studies were performed in vitro in various pH buffers and biological fluids (N=4). Pharmacokinetic parameters for M6S were studied using intravenous (1 mg/kg), intraperitoneal (i.p. 5.6, 10 mg/kg) and oral routes (10, 30 mg/kg) of drug administration (N=4 to 7 for each dose). Compared to morphine, in diabetic animals, M6S was 12-fold more potent in the HPT test (ED₅₀ M6S = 1.1 ± 0.02 μmol/kg Vs ED₅₀ MOR=12.1 ± 1.8 μmol/kg; p< 0.05), and 20-times more potent in the HTL test (in diabetic rats, ED₅₀ M6S = 1.1 ± 0.02 μmol/kg Vs ED₅₀ MOR = 12.1 ± 1.8 μmol/kg; p< 0.001). In the PST test, M6S was 3-fold more potent than morphine in diabetic rats (ED₅₀ M6S = 6.9 ± 1.46 μmol/kg Vs ED₅₀ MOR = 16.2 ± 1.24 μmol/kg). Similar potency differences were also observed between morphine and M6S in normal rats. Diabetes caused a decrease in potency (about 3-5 fold) of morphine in the HPT and HTF tests and a loss of efficacy in the PST and PPT tests (4^th week of diabetes) without significantly effecting M6S potency and efficacy in all these tests. Furthermore, 9 vs. 28 days of chronic treatment were required for rats to become tolerant to morphine and M6S, respectively, in the HTF and HPT tests. M6S also sustained its potent analgesic effects in morphine-tolerant rats (i.e. no cross-tolerance) and demonstrated clinical potential as an opioid rotation drug in morphine-tolerant subjects. With no significant differences in binding, activation of GTPγ and inhibition of cAMP at the μ-opioid receptor, M6S activated G-proteins via δ-ORs more potently than morphine (e.g., M6S-ED₅₀ = 101 ± 41.6 nM; MOR-ED₅₀ = 785 ± 140 nM), and modulated adenylyl cyclase activity via δ-ORs in intact CHO-hDOR cells more potently than morphine (e.g., M6S-ED₅₀ = 55.1 ± 17.5 nM; MOR-ED₅₀ = 372 ± 11.9 nM). Naltrindole (1mg/kg; i.p.) blocked M6S analgesia by 50% in HTF, HPT tests and 90% in the PST test but was without effect on morphine analgesia. In vitro stability studies and pharmacokinetic studies showed no susceptibility of M6S to hydrolyze to morphine. The bioavailability of M6S after the i.p. route of administration was above 93%, while oral bioavailability was ~ 5%. Our in vitro stability assays and pharmacokinetic studies have demonstrated that M6S is not a prodrug of morphine and that the molecule has difficulty in passing the epithelial barrier of gastrointestinal tract, due to its polar and zwitterionic nature. Our computational and receptor docking studies suggested that the sulfate moiety of M6S and 214 lysine residue of delta opioid receptor is a critical binding interaction. Collectively, our studies have provided a set of direct and indirect evidence supporting the ability of the stable M6S molecule to interact with and activate both μ, and most importantly, δ-opioid receptors to modulate their respective pain control pathways. In addition, evidence has been provided that clearly demonstrates the superiority of M6S over morphine in the treatment of multidomain neuropathic pain.