Synergistic analgesic effects between neuronostatin and morphine at the supraspinal level

Neuronostatin, a 13-amino acid peptide, is encoded in the somatostatin pro-hormone. I.c.v. administration of neuronostatin produces a significant antinociceptive effect in the mouse tail-flick test, which is mediated by endogenous opioid receptor. However, the direct functional interaction between morphine and neuronostatin has not been characterized. In the present study, effect of neuronostatin on morphine analgesia was investigated in the tail-flick test. Our findings showed that i.c.v. administration of neuronostatin (0.3 nmol/mouse i.c.v.) significantly enhanced the antinociceptive effect of morphine (2.5, 5 or 10 μg/kg) at the supraspinal level. Results of antagonism experiments suggested that the synergistic analgesia induced by morphine and neuronostatin was mediated by μ- and к-opioid receptors not δ-opioid receptor. In conclusion, there may be a cascade amplification phenomenon when morphine and neuronostatin were co-administered in acute pain model. The above results provide evidence for the potential use of neuronostatin in combination with morphine to control pain and addiction.     

In vivo characterization of the opioid antagonist nalmefene in mice

AimsThe current study assessed the in vivo antagonist properties of nalmefene using procedures previously used to characterize the opioid antagonists naloxone, naltrexone, 6β-naltrexol and nalbuphine.Main methodsICR mice were used to generate antagonist dose–response curves with intraperitoneal (i.p.) nalmefene against fixed A₉₀ doses of morphine in models of morphine-stimulated hyperlocomotion and antinociception. Additional dose–response curves for antagonist precipitated opioid withdrawal were run in mice treated acutely (100 mg/kg, s.c., ? 4 h) or chronically (75 mg pellet, s.c., ? 72 h) with morphine. Comparisons were made between antagonist potency and degree of precipitated withdrawal.Key findingsNalmefene produced dose- and time-related antagonism of morphine-induced increases in locomotor activity with a calculated ID₅₀ (and 95% confidence interval) of 0.014 (0.007–0.027) mg/kg. Nalmefene produced rapid reversal of morphine-induced locomotor activity (5.1 min for 50% reduction in morphine effect). A 0.32 mg/kg dose of nalmefene produced blockade of morphine-induced antinociception in the 55 °C tail-flick test that lasted approximately 2 h. Nalmefene was able to potently precipitate withdrawal in mice treated acutely or chronically with morphine.SignificanceThese results demonstrate that nalmefene is similar to naloxone and naltrexone with respect to its in vivo pharmacology in mice. Specifically, nalmefene produces potent antagonism of morphine agonist effects while precipitating severe withdrawal. The compound has a slower onset and longer duration of action compared to naloxone and naltrexone. The data allows for a more complete preclinical comparison of nalmefene against other opioid antagonists including the putative opioid neutral antagonist 6β-naltrexol.     

Role of the central amygdala GABA-A receptors in morphine state-dependent memory

AimsIn the present experiments, the effects of bilateral microinjections of the GABA-A receptor agonist and/or antagonist into the central amygdala (CeA) on morphine state-dependent memory were examined.Main methodsIn order to assess memory retrieval, a step-through passive avoidance task was used in adult male Wistar rats.Key findingsSubcutaneous (s.c.) administration of morphine (5 and 7.5 mg/kg) immediately after training (post-training) decreased the memory retrieval. Pre-test administration of the opioid (7.5 mg/kg) also induced amnesia. The response induced by post-training morphine (7.5 mg/kg) was significantly reversed by pre-test administration of the drug (5 and 7.5 mg/kg), indicating morphine state-dependent memory. Pre-test intra-CeA microinjection of muscimol, a GABA-A receptor agonist (0.01, 0.02 and 0.03 µg/rat) reduced morphine state-dependent memory. However, the same doses of muscimol by itself had no effect on memory retrieval. Furthermore, pre-test intra-CeA microinjection of bicuculline, a GABA-A receptor antagonist by itself did not alter memory retrieval. The antagonist also did not change post-training morphine (7.5 mg/kg)-induced amnesia, but in combination with a lower dose of morphine (0.5 mg/kg), improved memory performance. Moreover, muscimol's ability to interfere with morphine state-dependent memory was reversed by co-injection of bicuculline.SignificanceThe results suggest that GABA-A receptor mechanism of the CeA may influence morphine state-dependent memory.     

In vivo characterization of the effects of ghrelin on the modulation of acute pain at the supraspinal level in mice

Ghrelin, an acylated peptide produced in the stomach, increases food intake and growth hormone secretion, inhibits pro-inflammatory cascade, etc. Ghrelin and its receptor (GHS-R1a) mRNA were found in the area related to the regions for controlling pain transmission, such as the hypothalamus, the midbrain, the spinal cord, etc. Ghrelin has been shown to have antinociceptive activity and also anti-inflammatory properties in inflammatory pain and chronic neuropathic pain. Therefore, the aim of the present study was to investigate the effects of ghrelin for the first time in the acute pain modulation at the supraspinal level, using the tail withdrawal test and hot-plate test in mice. Intracerebroventricular (i.c.v.) administration of ghrelin (mouse, 0.1–3 nmol) produced a dose- and time-related antinociceptive effect in the tail withdrawal test and hot-plate test, respectively. Antinociceptive effect elicited by ghrelin (i.c.v., 1 nmol) was significantly antagonized by opioid receptor antagonist naloxone (i.c.v., 10 nmol co-injection or i.p., 10 mg/kg, 10 min prior to ghrelin) in both tail withdrawal test and hot-plate test. At these doses, naloxone significantly antagonized the antinociceptive effect induced by morphine (i.c.v., 3 nmol). Ghrelin (i.c.v., 1 nmol)-induced antinociception was significantly antagonized by co-injection with 10 nmol [d-Lys3]-GHRP-6, the selective antagonist of GHS-R1a identified more recently, while [d-Lys3]-GHRP-6 (10 nmol) alone induced neither hyperalgesia nor antinociception. Overall this data indicate that ghrelin could produce antinociception through an interaction with GHS-R1a and with the central opioid system. Thus ghrelin may be a promising peptide for developing new analgesic drugs.     

Attenuation of systemic morphine-induced analgesia by central administration of ghrelin and related peptides in mice

Ghrelin, an acylated 28-amino peptide secreted in the gastric endocrine cells, has been demonstrated to stimulate the release of growth hormone, increase food intake, and inhibit pro-inflammatory cascade, etc. Ghrelin mainly combines with its receptor (GHS-R1α) to play the role in physiological and pathological functions. It has been reported that ghrelin plays important roles in the control of pain through interaction with the opioid system in inflammatory pain and acute pain. However, very few studies show the effect of supraspinal ghrelin system on antinociception induced by intraperitoneal (i.p.) administration of morphine. In the present study, intracerebroventricular (i.c.v.) injection of ghrelin (0.1, 1, 10 and 100 nmol/L) produced inhibition of systemic morphine (6 mg/kg, i.p.) analgesia in the tail withdrawal test. Similarly, i.c.v. injection GHRP-6 and GHRP-2 which are the agonists of GHS-R1α, also decreased analgesia effect induced by morphine injected intraperitoneally in mice. Furthermore, these anti-opioid activities of ghrelin and related peptides were not blocked by pretreatment with the GHS-R1α selective antagonist [d-Lys³]-GHRP-6 (100 nmol/L, i.c.v.). These results demonstrated that central ghrelin and related peptides could inhibit the analgesia effect induced by intraperitoneal (i.p.) administration of morphine. The anti-opioid effects of ghrelin and related peptides do not interact with GHS-R1a. These findings may pave the way for a new strategy on investigating the interaction between ghrelin system and opioids on pain modulation.     

Withania somnifera root extract prolongs analgesia and suppresses hyperalgesia in mice treated with morphine

Previous studies demonstrated that Withania somnifera Dunal (WS), a safe medicinal plant, prevents the development of tolerance to the analgesic effect of morphine.In the present study, we investigated whether WS extract (WSE) (100 mg/kg, i.p.) may also modulate the analgesic effect induced by acute morphine administration (2.5, 5, 10 mg/kg, s.c.) in the tail-flick and in the hot plate tests, and if it may prevent the development of 2.5 mg/kg morphine-induced rebound hyperalgesia in the low intensity tail-flick test. Further, to characterize the receptor(s) involved in these effects, we studied, by receptor-binding assay, the affinity of WSE for opioid (μ, δ, k), cannabinoid (CB₁, CB₂), glutamatergic (NMDA), GABAergic (GABA_A, GABA_B), serotoninergic (5HT_2A) and adrenergic (α₂) receptors.The results demonstrated that (i) WSE alone failed to alter basal nociceptive threshold in both tests, (ii) WSE pre-treatment significantly protracted the antinociceptive effect induced by 5 and 10 mg/kg of morphine only in tail-flick test, (iii) WSE pre-treatment prevented morphine-induced hyperalgesia in the low intensity tail-flick test, and (iv) WSE exhibited a high affinity for the GABA_A and moderate affinity for GABA_B, NMDA and δ opioid receptors.WSE prolongs morphine-induced analgesia and suppresses the development of morphine-induced rebound hyperalgesia probably through involvement of GABA_A, GABA_B, NMDA and δ opioid receptors. This study suggests the therapeutic potential of WSE as a valuable adjuvant agent in opioid-sparing therapies.     

Modulation of delta opioid agonist-induced antinociception by repeated morphine pretreatment in rhesus monkeys

AimsRepeated treatment with morphine increases antinociceptive effects of delta opioid agonists in rodents by a mechanism that may involve increased cell-surface expression of delta receptors. The present study evaluated effects of repeated morphine treatment on behavioral effects of the delta agonist SNC80 and the mu agonist fentanyl in rhesus monkeys.Main methodsIn an assay of schedule-controlled responding, three monkeys responded for food reinforcement under a fixed-ratio 30 schedule. In an assay of thermal nociception, tail-withdrawal latencies were evaluated in three monkeys using thermal stimulus intensities of 48 and 54 °C. In both assays, the effects of SNC80 (0.032–3.2 mg/kg) and fentanyl (0.001–0.056 mg/kg) were evaluated after repeated treatment with saline or a regimen of morphine doses modeled on the regimen that enhanced delta agonist antinociception and apparent delta receptor availability in previous rodent studies.Key findingsBoth SNC80 and fentanyl dose-dependently decreased rates of schedule-controlled responding, and repeated morphine treatment did not significantly alter these effects. In the assay of thermal nociception, SNC80 had little effect on tail-withdrawal latencies from water heated to 48 or 54 °C, whereas fentanyl increased tail-withdrawal latencies at both temperatures. Repeated morphine tended to increase the antinociceptive effects of SNC80 and to decrease the antinociceptive effects of fentanyl, but these effects of repeated morphine were small and were significant only at the higher stimulus intensity (54 °C).SignificanceThese results provide limited support for the proposition that prior stimulation of mu receptors selectively increases the antinociceptive effects of delta agonists in rhesus monkeys.     

High-dose versus low-dose octreotide in the treatment of acute pancreatitis: A randomized controlled trial

To evaluate the therapeutic efficacy of high-dose octreotide in patients with predicted severe acute pancreatitis (SAP) or SAP, two hundred and thirty-six patients with predicted SAP and 136 patients with SAP were randomized into control, high-dose octreotide (High-O) and low-dose octreotide (Low-O) groups. In addition to the conventional managements administrated in control group, High-O group received an intravenous infusion of octreotide at 50 μg/h × 3d + 25 μg/h × 4d, and Low-O group received octreotide at 25 μg/h × 7d. The major primary outcomes included the numbers of predicted SAP patients which developed SAP after intervention and the number of patients with SAP amelioration. Secondary outcomes included APACHE II, SIRS scores, plasma levels of somatostatin (SST), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6). There were no significant differences between the control and Low-O groups in terms of prevention and treatment for SAP. The incidence of SAP in patients with predicted SAP who received High-O was significantly lower than the Low-O group: 37.5% vs. 59.8%, p = 0.005. Compared with Low-O group, the number of SAP patients in the SAP arm in the High-O group was reduced by 29.8%. Plasma levels of SST in both predicted SAP and the SAP patients were efficiently recovered (from 132.71 ± 31.40 pg/ml to 180.00 ± 23.50 pg/ml, p < 0.05) after high-dose octreotide supplementation, which concomitantly reduced TNF-α and IL-6 levels. High-dose octreotide administration within 48 h after AP onset may efficiently reduce the risk of SAP developing and partly attenuate SAP through raising plasma SST to a normal level and decreasing IL-6 and TNF-α.     

Plasma-mediated release of morphine from synthesized prodrugs

Two morphine prodrugs (‘PDA’ and ‘PDB’) were synthesized and the kinetics of esterase-mediated morphine release from these prodrugs were determined when incubated with plasma from different animal species. Morphine was rapidly released from PDA by all species plasma with the maximum reached within 5–10 min; the released morphine was biologically active as determined by an in vitro cAMP assay. The morphine was released from PDB at a slower and species-dependent rate (mouse > rat > guinea pig > human). Morphine’s release from PDB appeared to be mediated by carboxyl esterases as the release was inhibited by the carboxyl esterase inhibitor benzil. PDA nor PDB induce cytotoxicity in the neuronal cell lines SK-NSH and SH-SY5Y. The carboxyl and amino functional moieties present on the linker portions of PDA and PDB, respectively, may facilitate their conjugation to nanoparticles to tailor morphine pharmacokinetics and specific targeting. These studies suggest the potential clinical utility of these prodrugs for morphine release at desired rates by administration of their mixture at selected ratios.     

Involvement of delta and mu opioid receptors in the acute and sensitized locomotor action of cocaine in mice

Analogs of deltorphins, such as cyclo(Nδ, Nδ-carbonyl-d-Orn2, Orn4)deltorphin (DEL-6) and deltorphin II N-(ureidoethyl)amide (DK-4) are functional agonists predominantly for the delta opioid receptors (DOR) in the guinea-pig ileum and mouse vas deferens bioassays. The purpose of this study was to examine an influence of these peptides (5, 10 or 20 nmol, i.c.v.) on the acute cocaine-induced (10 mg/kg, i.p.) locomotor activity and the expression of sensitization to cocaine locomotor effect. Sensitization to locomotor effect of cocaine was developed by five injections of cocaine at the dose of 10 mg/kg, i.p. every 3 days. Our results indicated that DK-4 and DEL-6 differently affected the acute and sensitized cocaine locomotion. Co-administration of DEL-6 with cocaine enhanced acute cocaine locomotion only at the dose of 10 nmol, with minimal effects at the doses 5 and 20 nmol, whereas co-administration of DK-4 with cocaine enhanced acute cocaine-induced locomotion in a dose-dependent manner. Similarly to the acute effects, DEL-6 only at the dose of 10 nmol but DK-4 dose-dependently enhanced the expression of cocaine sensitization. Pre-treatment with DOR antagonist – naltrindole (5 nmol, i.c.v.) and mu opioid receptor (MOR) antagonist, β-funaltrexamine abolished the ability of both peptides to potentiate the effects of cocaine. Our study suggests that MOR and DOR are involved in the interactions between cocaine and both deltorphins analogs. A distinct dose–response effects of these peptides on cocaine locomotion probably arise from differential functional activation (targeting) of the DOR and MOR by both deltorphins analogs.     

The differential extraction and immunoluminometric assay of Urotensin II and Urotensin-related peptide in heart failure

Urotensin II (UTN) is a cyclic eleven amino acid peptide that can induce endothelial independent vasoconstriction and endothelial dependent vasodilatation in human vasculature. The cyclic part of the peptide is composed of six amino acids. Similarly, Urotensin Related Peptide (URP) is only eight amino acids long but shares the identical ring structure to UTN. Plasma UTN has been shown to be raised in patients with chronic heart failure (CHF) suggesting a potential role of the peptide system in the pathophysiology of heart failure. Given their similar structures, techniques measuring plasma UTN may also be simultaneously detecting URP and could provide a misrepresentation of true UTN and URP levels in patients’ plasma. Thus we describe the development of a solid phase extraction technique that can differentially extract UTN and URP from human plasma so that they can be assayed separately using non-radioactive immunoluminometric assays. This reliable and sensitive protocol was utilized to characterise the plasma of 20 healthy controls and 20 patients admitted with acute heart failure (AHF). The groups were age and sex matched. Plasma UTN was significantly raised in patients with AHF on admission when compared to controls (median 1.29 [range 0.50–5.55] pmol/L vs 0.50 [0.50–3.33] pmol/L, p = 0.019). Likewise plasma URP was significantly higher in the heart failure group on admission (8.38 [1.30–66.80] pmol/L vs 2.25 [1.30–14.40] pmol/L, p < 0.005). This suggests a role for both members of the Urotensin peptide system in acute heart failure.     

Acute effects of corticosterone injection on paternal behavior in California mouse (Peromyscus californicus) fathers

Glucocorticoids are thought to mediate the disruption of parental behavior in response to acute and chronic stress. Previous research supports their role in chronic stress; however, no study has experimentally tested the effects of acute glucocorticoid elevation on paternal behavior. We tested the prediction that acute corticosterone (CORT) increases would decrease paternal behavior in California mouse fathers and would lead to longer-term effects on reproductive success, as even short-term increases in CORT have been shown to produce lasting effects on the hypothalamic-pituitary-adrenal axis. First-time fathers were injected with 30 mg/kg CORT, 60 mg/kg CORT or vehicle, or left unmanipulated. Interactions between the male and its pup(s) were recorded 1.5–2 h after injection and scored for paternal and non-paternal behavior. Treatment groups were combined into control (unmanipulated + vehicle, n = 15) and CORT (30 mg/kg + 60 mg/kg, n = 16) for analysis based on resulting plasma CORT concentrations. CORT treatment did not alter paternal or non-paternal behaviors or any long-term measures (male body mass or temperature, pup growth rate, pup survival, interbirth interval, number or mass of pups born in the second litter). Fathers showed a significant rise in body mass at day 30 postpartum, followed by a decrease in body mass after the birth of the second litter; however, this pattern did not differ between the CORT and control groups. In summary, acute elevation of plasma CORT did not alter direct paternal behavior, body mass, or reproductive outcomes, suggesting that acute CORT elevation alone does not overtly disrupt paternal care in this biparental mammal.     

Morphine dependence is associated with changes in neuropeptide S receptor expression and function in rat brain

Neuropeptide S (NPS) is a newly identified ligand for the previously discovered G-protein coupled receptor 154 now named NPSR. Recently, it has been found that NPSR gene expression is altered during ethanol withdrawal. In this study we tried to elucidate if NPSR gene expression is modified in response to morphine withdrawal and its protracted abstinence. To induce opioid dependence Wistar rats were treated for 7 days with morphine. Twelve hours and 7 days after the last morphine administration brains were removed and the expression of NPSR mRNA was analyzed by in situ hybridization (ISH). Successful induction of opioid dependence was confirmed by the naloxone-precipitated withdrawal test 2 h after the last morphine administration. Moreover, 7 days after the last morphine dose animals were checked for signs of anxiety and for intracerebroventricular (ICV) NPS (0.3 and 1.0 nmol) induced anxiolytic effects by elevated plus maze (EPM). Results showed that in morphine treated rats strong somatic signs of naloxone-precipitated withdrawal occurred. ISH data revealed changes in NPSR gene expression in the ventral tegmental area as well as in the basolateral amygdaloid and bed nucleus of stria terminalis at 12 h and 7 days into abstinence, respectively. At 7 days into abstinence post dependent animals showed higher levels of anxiety than controls which were significantly attenuated by NPS. These results demonstrated that morphine dependence induction led to (i) changes in NPSR mRNA expression; (ii) increased anxiety; and (iii) more potent anxiolytic-like effect of NPS.     

Growth performance and starch utilization in common carp (Cyprinus carpio L.) in response to dietary chromium chloride supplementation

A nutrition trial was conducted on juvenile common carp (Cyprinus carpio), initial mean body weight 15 ± 0.4 g within a controlled facility at 25 ± 0.5 °C. Six diets containing various levels of supplementary Cr (0, 0.2, 0.5, 1.0, 1.5, and 2.0) mg Cr/kg of diet as Cr chloride hexahydrate were fed to carp for a period of 10 weeks. Lower growth performance was observed in fish fed on the control diet and the diet supplemented with the highest level of Cr (2.0 mg Cr/kg). Although fish fed 0.5 mg Cr/kg showed the best growth performance, this was not significantly different (P > 0.05) from fish fed 1.0 mg Cr/kg. The regression of plasma glucose concentration was linear (R² = 0.97 and P value = 0.001) as the Cr content of the diet increased (up to 1.5 mg Cr/kg).Cr carcass content was elevated with an increasing level of dietary Cr supplementation up to 1.5 mg Cr/kg; but fish fed on the diet supplemented with the highest level of Cr (2.0 mg Cr/kg) showed a decrease in Cr carcass content.Histological examination to evaluate the impact of different Cr supplementation on liver and gut tissues showed notable changes. The higher level of Cr (2.0 mg Cr/kg) in the diet gave rise to elevated hepatocyte vacuolization and changes in gut tissue morphology.It appeared that Cr chloride significantly improved growth within a defined range (0.2–1.5) mg Cr/kg without any negative impact, while 2.0 mg Cr/kg in carp diet seems to be the threshold for the initiation of toxicity.     

Plasma intermedin levels in patients with acute myocardial infarction

It has been shown that adrenomedullin (ADM) may function as a cardiovascular-regulatory peptide in humans. Intermedin (IMD) is a newly discovered peptide related to ADM and has a greater range of biological effects on the cardiovascular in animal experiments. The purpose of the study was to investigate the pathophysiological role of IMD in patients with acute myocardial infarction (AMI). The present study included twenty patients with acute ST-segment elevation myocardial infarction (STEMI), thirty-three with stable coronary heart disease (SCHD), and eighteen healthy controls. Plasma levels of IMD, malonaldehyde (MDA), and superoxide dismutase (SOD) and cardiac biomarkers were determined at one, two, four and seven days following AMI. Plasma IMD levels were significantly increased on day 1 in AMI patients when compared with SCHD subjects (P = 0.014), and reached a peak of 181.88 ± 9.47 pg/ml at 96 h. Plasma IMD concentrations were correlated with MDA and SOD. Furthermore, patients with severe lesions in their coronary arteries tended to have higher plasma IMD levels (P < 0.05) in AMI patients. A significant increase in plasma IMD following AMI may be associated with oxidative stress, and could be used as a marker to reflect the severity of the coronary stenosis.     

Antinociceptive action of NOP and opioid receptor agonists in the mouse orofacial formalin test

Nociceptin/orphanin FQ (N/OFQ) modulates several biological functions, including pain transmission via selective activation of a specific receptor named NOP. The aim of this study was the investigation of the antinociceptive properties of NOP agonists and their interaction with opioids in the trigeminal territory. The orofacial formalin (OFF) test in mice was used to investigate the antinociceptive potential associated to the activation of NOP and opioid receptors. Mice subjected to OFF test displayed the typical biphasic nociceptive response and sensitivity to opioid and NSAID drugs. Mice knockout for the NOP gene displayed a robust pronociceptive phenotype. The NOP selective agonist Ro 65-6570 (0.1–1 mg kg⁻¹) and morphine (0.1–10 mg kg⁻¹) elicited dose dependent antinociceptive effects in the OFF with the alkaloid showing larger effects; the isobologram analysis of their actions demonstrated an additive type of interaction. The mixed NOP/opioid receptor agonist cebranopadol elicited potent (0.01–0.1 mg kg⁻¹) and robust antinociceptive effects. In the investigated dose range, all drugs did not modify the motor performance of the mice in the rotarod test. Collectively the results of this study demonstrated that selective NOP agonists and particularly mixed NOP/opioid agonists are worthy of development as innovative drugs to treat painful conditions of the trigeminal territory.     

Design,synthesis, and characterization of 6β-naltrexol analogs,and their selectivity for in vitro opioid receptor subtypes

Since the mu opioid receptor (MOR) is known to be involved in the therapeutically relevant pathways leading to the manifestation of pain and addiction, we are currently studying the specific structural characteristics that promote antagonism at the MOR. The opiates 6β-naltrexol and 6β-naltrexamide function as neutral antagonists in in vitro and in vivo systems previously exposed to morphine, and are under investigation as improved treatments for narcotic dependence. In this research, we synthesized and characterized carbamate and sulfonate ester derivates of 6β-naltrexol that do not contain a protic group at C₆, and evaluated these compounds for opioid receptor affinity. In vitro receptor subtype (μ, κ, and δ opioid receptors) binding data of the carbamate and sulfonate derivatives is reported. All four compounds synthesized exhibited affinity for the MOR better than the standard 6β-naltrexol HCl. Based on K_i data, the order of MOR affinity is as follows: 9 > 13 > 14 > 10 > 6β-naltrexol HCl. Carbamate 9 and tosylate 13 displayed subnanomolar affinity for the MOR, while 10 was the most μ-selective compound synthesized. In conclusion, our data indicate that the absence of a hydrogen-bond donor on the C₆ oxygen enhances rather than impedes the in vitro affinity of naltrexol derivatives for the MOR. Additionally, data also suggest that increasing the bulk around C₆ may allow control of subtype selectivity within these compound series.