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An innovative chemical strategy named peptide welding technology (PWT) has been developed for the facile synthesis of tetrabranched peptides. [Dmt1]N/OFQ(1-13)-NH2 acts as a universal agonist for nociceptin/orphanin FQ (N/OFQ) and classical opioid receptors. The present study investigated the pharmacological profile of the PWT derivative of [Dmt1]N/OFQ(1-13)NH2 (PWT2-[Dmt1]) in several assays in vitro and in vivo after spinal administration in monkeys subjected to the tail withdrawal assay. PWT2-[Dmt1] mimicked the effects of [Dmt1]N/OFQ(1-13)-NH2 displaying full agonist activity, similar affinity/potency and selectivity at human recombinant N/OFQ (NOP) and opioid receptors in receptor binding, stimulation of [35S]GTPγS binding, calcium mobilization in cells expressing chimeric G proteins, and BRET studies for measuring receptor/G-protein and receptor/β-arrestin 2 interaction. In vivo in monkeys PWT2-[Dmt1] elicited dose-dependent and robust antinociceptive effects being more potent and longer lasting than [Dmt1]N/OFQ(1-13)-NH2. The analgesic action of PWT2-[Dmt1] was sensitive to the NOP receptor antagonist J-113397, but not naltrexone. Thus, the present study demonstrated that the tetrabranched derivative of [Dmt1]N/OFQ(1-13)-NH2 obtained with the PWT technology maintains the in vitro pharmacological profile of the parent peptide but displays higher potency and longer lasting action in vivo.

Despite high sequence similarity between NOP (nociceptin/orphanin FQ opioid peptide) and opioid receptors, marked differences in endogenous ligand selectivity, signal transduction, phosphorylation, desensitization, internalization and trafficking have been identified; underscoring the evolutionary difference between NOP and opioid receptors. Activation of NOP receptors affects nociceptive transmission in a site-specific manner, with antinociceptive effects prevailing after peripheral and spinal activation, and pronociceptive effects after supraspinal activation in rodents. The net effect of systemically administered NOP receptor agonists on nociception is proposed to depend on the relative contribution of peripheral, spinal and supraspinal activation, and this may depend on experimental conditions. Functional expression and regulation of NOP receptors at peripheral and central sites of the nociceptive pathway exhibits a high degree of plasticity under conditions of neuropathic and inflammatory pain. In rodents, systemically administered NOP receptor agonists exerted antihypersensitive effects in models of neuropathic and inflammatory pain. However, they were largely ineffective in acute pain while concomitantly evoking severe motor side effects. In contrast, systemic administration of NOP receptor agonists to non-human primates (NHPs) exerted potent and efficacious antinociception in the absence of motor and sedative side effects. The reason for this species difference with respect to antinociceptive efficacy and tolerability is not clear. Moreover, co-activation of NOP and μ-opioid peptide (MOP) receptors synergistically produced antinociception in NHPs. Hence, both selective NOP receptor as well as NOP/MOP receptor agonists may hold potential for clinical use as analgesics effective in conditions of acute and chronic pain.

Carrageenan-induced hyperalgesia is a widely used pain model in rodents. However, characteristics of carrageenan-induced hyperalgesia and effects of analgesic drugs under these conditions are unknown in nonhuman primates.The aims of this study were to develop carrageenan-induced hyperalgesia in rhesus monkeys and determine the efficacy and potency of agonists selective for the four opioid receptor subtypes in this model versus acute pain, as compared to non-steroidal anti-inflammatory drugs (NSAIDs).Tail injection of carrageenan produced long-lasting thermal hyperalgesia in monkeys. Systemically administered agonists selective for opioid receptor subtypes, i.e., fentanyl (mu/MOP), U-50488H (kappa/KOP), SNC80 (delta/DOP) and Ro 64-6198 (nociceptin/orphanin FQ/NOP) dose-dependently attenuated carrageenan-induced thermal hyperalgesia with different potencies. In absence of carrageenan, these agonists, except SNC80, blocked acute thermal nociception. Opioid-related ligands, especially Ro 64-6198, were much more potent for their antihyperalgesic than antinociceptive effects. Both effects were mediated by the corresponding receptor mechanisms. Only fentanyl produced scratching at antihyperalgesic and antinociceptive doses consistent with its pruritic effects in humans, illustrating a translational profile of MOP agonists in nonhuman primates. Similar to SNC80, systemically administered NSAIDs ketorolac and naproxen dose-dependently attenuated carrageenan-induced hyperalgesia but not acute nociception.Using two different pain modalities in nonhuman primates, effectiveness of clinically available analgesics like fentanyl, ketorolac and naproxen was distinguished and their efficacies and potencies were compared with the selective KOP, DOP, and NOP agonists. The opioid-related ligands displayed differential pharmacological properties in regulating hyperalgesia and acute nociception in the same subjects. Such preclinical primate models can be used to investigate novel analgesic agents.

Nor-binaltorphimine (nor-BNI) is kappa opioid receptor (KOR) antagonist with the extremely long duration in mice analgesic assay, in vivo . For the evaluation of long-lasting effect of nor-BNI, brain content and serum concentration of nor-BNI were quantified in comparison with those of naloxone (a short-acting non-specific opioid receptor antagonist) by high-performance liquid chromatography with electrochemical detector in mice. After concomitant administration (20 mg/kg, s.c.) of nor-BNI and naloxone, nor-BNI in brain and serum showed biphasic elimination, with a rapid phase for 0.75–4 h and a slow phase for 4–48 h. Elimination rate in brain was slower than that of serum. Naloxone in brain and serum was detected for 3 h and 4 h, respectively. The brain/serum ratio of nor-BNI gradually increased over 0.75–48 h, while that of naloxone rapidly declined. After concomitant administration (30 mg/kg, s.c.) of nor-BNI and naloxone, brain nor-BNI was detected in all mice from day 1 to day 21 and in two of six mice at day 28, while serum nor-BNI was detected in all mice at day 1, three of seven at day 3 and one of six at day 7. After that, serum nor-BNI was not detected. Naloxone in brain and serum was not detected at day 1. These results provide pharmacokinetic support for the long-lasting antagonistic effects of nor-BNI.

Background and Purpose Intrathecally (i.t.) administered nociceptin/orphanin FQ (N/OFQ) evokes antinociceptive effects in rodents. Recent studies in monkeys demonstrated that i.t. co-application of N/OFQ and morphine elicits synergistic antinociceptive actions suggesting mixed N/OFQ peptide (NOP) and μ opioid receptor agonists as innovative spinal analgesics. Thus, novel N/OFQ related peptides were synthesized in order to identify and pharmacologically characterize a mixed NOP/ μ opioid receptor agonist. Experimental Approach The following in vitro assays were used: calcium mobilization in cells expressing the human NOP or classical opioid receptors and chimeric G proteins, receptor and [35S]-GTPγS binding, [35S]-GTPγS binding in rat spinal cord membranes, guinea pig ileum bioassay. In vivo experiments were performed in monkeys using the tail withdrawal assay. Key Results From calcium mobilization studies [Dmt1]N/OFQ(1–13)-NH2 was selected as the most potent and least selective compound. The mixed NOP/opioid full agonist activity and high affinity of [Dmt1]N/OFQ(1–13)-NH2 was confirmed at human recombinant receptors in receptor binding, calcium mobilization and/or [35S]-GTPγS binding studies, at rat spinal cord receptors in [35S]-GTPγS binding experiments, and at guinea pig receptors inhibiting neurogenic contractions in the ileum. In vivo in the tail withdrawal assay in monkeys i.t. [Dmt1]N/OFQ(1–13)-NH2 was able to elicit robust and long-lasting antinociceptive effects. Conclusions and Implications Collectively, these results demonstrate that [Dmt1]N/OFQ(1–13)-NH2 behaves as NOP/opioid receptor universal agonist and substantiate the suggestion that such mixed ligands are worthy of development as innovative spinal analgesics.

Although mu opioid (MOP) receptor agonists are the most commonly used analgesics for the treatment of moderate to severe pain in the clinic, the side effects of MOP agonists such as abuse liability limit their value as a medication. Research to identify novel analgesics without adverse effects is pivotal to advance the health care of humans. The nociceptin/orphanin FQ peptide (NOP) receptor, the fourth opioid receptor subtype, mediates distinctive actions in nonhuman primates which suggests the possibility that activity at this receptor may result in strong analgesia in the absence of virtually all of the side effects associated with MOP agonists. The present review highlights the recent progress of pharmacological studies of NOP-related ligands in primates. Selective NOP agonists, either peptidic or nonpeptidic, produce full analgesia in various assays in primates, when delivered systemically or intrathecally. Yet small molecule NOP agonists do not serve as reinforcers, indicating a lack of abuse liability. Given that NOP agonists have low abuse liability and that coactivation of NOP and MOP receptors produces synergistic antinociception, it is worth developing bifunctional NOP/MOP ligands. The outcomes of these studies and recent developments provide new perspectives to establish a translational bridge for understanding the biobehavioral functions of NOP receptors in primates and for facilitating the development of NOP-related ligands as a new generation of analgesics without abuse liability in humans.

Despite the critical need, no previous research has substantiated safe opioid analgesics without abuse liability in primates. Recent advances in medicinal chemistry have led to the development of ligands with mixed mu opioid peptide (MOP)/nociceptin-orphanin FQ peptide (NOP) receptor agonist activity to achieve this objective. BU08028 is a novel orvinol analog that displays a similar binding profile to buprenorphine with improved affinity and efficacy at NOP receptors. The aim of this preclinical study was to establish the functional profile of BU08028 in monkeys using clinically used MOP receptor agonists for side-by-side comparisons in various well-honed behavioral and physiological assays. Systemic BU08028 (0.001-0.01 mg/kg) produced potent long-lasting (i.e., >24 h) antinociceptive and antiallodynic effects, which were blocked by MOP or NOP receptor antagonists. More importantly, the reinforcing strength of BU08028 was significantly lower than that of cocaine, remifentanil, or buprenorphine in monkeys responding under a progressive-ratio schedule of drug self-administration. Unlike MOP receptor agonists, BU08028 at antinociceptive doses and ∼10- to 30-fold higher doses did not cause respiratory depression or cardiovascular adverse events as measured by telemetry devices. After repeated administration, the monkeys developed acute physical dependence on morphine, as manifested by precipitated withdrawal signs, such as increased respiratory rate, heart rate, and blood pressure. In contrast, monkeys did not show physical dependence on BU08028. These in vivo findings in primates not only document the efficacy and tolerability profile of bifunctional MOP/NOP receptor agonists, but also provide a means of translating such ligands into therapies as safe and potentially abuse-free opioid analgesics.

An innovative chemical strategy named peptide welding technology (PWT) has been developed for the facile synthesis of tetrabranched peptides. [Dmt1]N/OFQ(1-13)-NH2 acts as a universal agonist for nociceptin/orphanin FQ (N/OFQ) and classical opioid receptors. The present study investigated the pharmacological profile of the PWT derivative of [Dmt1]N/OFQ(1-13)NH2 (PWT2-[Dmt1]) in several assays in vitro and in vivo after spinal administration in monkeys subjected to the tail withdrawal assay. PWT2-[Dmt1] mimicked the effects of [Dmt1]N/OFQ(1-13)-NH2 displaying full agonist activity, similar affinity/potency and selectivity at human recombinant N/OFQ (NOP) and opioid receptors in receptor binding, stimulation of [35S]GTPγS binding, calcium mobilization in cells expressing chimeric G proteins, and BRET studies for measuring receptor/G-protein and receptor/β-arrestin 2 interaction. In vivo in monkeys PWT2-[Dmt1] elicited dose-dependent and robust antinociceptive effects being more potent and longer lasting than [Dmt1]N/OFQ(1-13)-NH2. The analgesic action of PWT2-[Dmt1] was sensitive to the NOP receptor antagonist J-113397, but not naltrexone. Thus, the present study demonstrated that the tetrabranched derivative of [Dmt1]N/OFQ(1-13)-NH2 obtained with the PWT technology maintains the in vitro pharmacological profile of the parent peptide but displays higher potency and longer lasting action in vivo.

Neuroinflammation is a pathological condition that underlies diabetes and affects sensory processing. Given the high prevalence of pain in diabetic patients and crosstalk between chemokines and opioids, it is pivotal to know whether neuroinflammation-associated mediators are dysregulated in the central nervous system of diabetic primates. Therefore, the aim of this study was to investigate whether mRNA expression levels of glial markers, chemokines, and opioid receptors are altered in the spinal cord and thalamus of naturally occurring type 2 diabetic monkeys ( n = 7) compared with age-matched non-diabetic monkeys ( n = 6). By using RT-qPCR, we found that mRNA expression levels of both GFAP and IBA1 were up-regulated in the spinal dorsal horn (SDH) of diabetic monkeys compared with non-diabetic monkeys. Among all chemokines, expression levels of three chemokine ligand-receptor systems, i.e., CCL2-CCR2, CCL3-CCR1/5, and CCL4-CCR5, were up-regulated in the SDH of diabetic monkeys. Moreover, in the SDH, seven additional chemokine receptors, i.e., CCR4, CCR6, CCR8, CCR10, CXCR3, CXCR5, and CXCR6, were also up-regulated in diabetic monkeys. In contrast, expression levels of MOP, KOP, and DOP, but not NOP receptors, were down-regulated in the SDH of diabetic monkeys, and the thalamus had fewer changes in the glial markers, chemokines and opioids. These findings indicate that neuroinflammation, manifested as glial activation and simultaneous up-regulation of multiple chemokine ligands and receptors, seems to be permanent in type 2 diabetic monkeys. As chemokines and opioids are important pain modulators, this first-in-primate study provides a translational bridge for determining the functional efficacy of spinal drugs targeting their signaling cascades.

It has been two decades since the peptide, nociceptin/orphanin FQ (N/OFQ), and its cognate (NOP) receptor were discovered. Although NOP receptor activation causes a similar pattern of intracellular actions as mu opioid (MOP) receptors, NOP receptor-mediated pain modulation in rodents are more complicated than MOP receptor activation. In this review, we highlight the functional evidence of spinal, supraspinal, and systemic actions of NOP receptor agonists for regulating pain. In rodents, effects of the N/OFQ-NOP receptor system in spinal and supraspinal sites for modulating pain are bidirectional depending on the doses, assays, and pain modalities. The net effect of systemically administered NOP receptor agonists may depend on relative contribution of spinal and supraspinal actions of the N/OFQ-NOP receptor signaling in rodents under different pain states. In stark contrast, NOP receptor agonists produce only antinociception and antihypersensitivity in spinal and supraspinal regions of nonhuman primates regardless of doses and assays. More importantly, NOP receptor agonists and a few bifunctional NOP/MOP receptor agonists do not exhibit reinforcing effects (abuse liability), respiratory depression, itch pruritus, nor do they delay the gastrointestinal transit function (constipation) in nonhuman primates. Depending upon their intrinsic efficacies for activating NOP and MOP receptors, bifunctional NOP/MOP receptor agonists warrant additional investigation in primates regarding their side effect profiles. Nevertheless, NOP receptor-related agonists display a much wider therapeutic window as compared to that of MOP receptor agonists in primates. Both selective NOP receptor agonists and bifunctional NOP/MOP receptor agonists hold a great potential as effective and safe analgesics without typical opioid-associated side effects in humans.

Buprenorphine is known as a μ-opioid peptide (MOP) receptor agonist, but its antinociception is compromised by the activation of nociceptin/orphanin FQ peptide (NOP) receptors in rodents. The aim of this study was to investigate the roles of MOP and NOP receptors in regulating buprenorphine-induced physiological responses in primates (rhesus monkeys). The effects of MOP antagonist (naltrexone), NOP antagonist [(±)-1-[(3R*,4R*)-1-(cyclooctylmethyl)-3-(hydroxymethyl)-4-piperidinyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (J-113397)], and NOP agonists [(1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4.5] decan-4-one (Ro 64-6198) and 3-endo-8-[bis(2-methylphenyl)methyl]-3-phenyl-8-azabicyclo[3.2.1]octan-3-ol (SCH 221510)] on buprenorphine were studied in three functional assays for measuring analgesia, respiratory depression, and itch in primates. Over the dose range of 0.01 to 0.1 mg/kg, buprenorphine dose-dependently produced antinociception, respiratory depression, and itch/scratching responses, and there was a ceiling effect at higher doses (0.1-1 mg/kg). Naltrexone (0.03 mg/kg) produced similar degrees of rightward shifts of buprenorphine's dose-response curves for all three endpoints. Mean pK(B) values of naltrexone (8.1-8.3) confirmed that MOP receptors mediated mainly buprenorphine-induced antinociception, respiratory depression, and itch/scratching. In contrast, J-113397 (0.1 mg/kg) did not change buprenorphine-induced physiological responses, indicating that there were no functional NOP receptors in buprenorphine-induced effects. More importantly, both NOP agonists, Ro 64-6198 and SCH 221510, enhanced buprenorphine-induced antinociception without respiratory depression and itch/ scratching. The dose-addition analysis revealed that buprenorphine in combination with the NOP agonist synergistically produced antinociceptive effects. These findings provided functional evidence that the activation of NOP receptors did not attenuate buprenorphine-induced antinociception in primates; instead, the coactivation of MOP and NOP receptors produced synergistic antinociception without other side effects. This study strongly supports the therapeutic potential of mixed MOP/NOP agonists as innovative analgesics.

Cocaine is considered to be the most addictive of all substances of abuse and mediates its effects by inhibiting monoamine transporters, primarily the dopamine transporters. There are currently no small molecules that can be used to combat its toxic and addictive properties, in part because of the difficulty of developing compounds that inhibit cocaine binding without having intrinsic effects on dopamine transport. Most of the effective cocaine inhibitors also display addictive properties. We have recently reported the use of cocaine esterase (CocE) to accelerate the removal of systemic cocaine and to prevent cocaine-induced lethality. However, wild-type CocE is relatively unstable at physiological temperatures ({tau}{sub 1/2} {approx} 13 min at 37 C), presenting challenges for its development as a viable therapeutic agent. We applied computational approaches to predict mutations to stabilize CocE and showed that several of these have increased stability both in vitro and in vivo, with the most efficacious mutant (T172R/G173Q) extending half-life up to 370 min. Here we present novel X-ray crystallographic data on these mutants that provide a plausible model for the observed enhanced stability. We also more extensively characterize the previously reported variants and report on a new stabilizing mutant, L169K. The improved stability of thesemore » engineered CocE enzymes will have a profound influence on the use of this protein to combat cocaine-induced toxicity and addiction in humans.« less

Enhancing cocaine metabolism by administration of cocaine esterase (CocE) has been recognized as a promising treatment strategy for cocaine overdose and addiction, because CocE is the most efficient native enzyme for metabolizing the naturally occurring cocaine yet identified. A major obstacle to the clinical application of CocE is the thermoinstability of native CocE with a half-life of only a few minutes at physiological temperature (37 degrees C). Here we report thermostable variants of CocE developed through rational design using a novel computational approach followed by in vitro and in vivo studies. This integrated computational-experimental effort has yielded a CocE variant with a approximately 30-fold increase in plasma half-life both in vitro and in vivo. The novel design strategy can be used to develop thermostable mutants of any protein.

Itch/pruritus is the most common side effect associated with spinal administration of morphine given to humans for analgesia. The aim of this study was to investigate the effectiveness of kappa-opioid receptor (KOR) agonists with diverse chemical structures as antipruritics and to elucidate the receptor mechanism underlying the antipruritic effect in monkeys. In particular, previously proposed non-KOR-1 agonists, including nalfurafine [TRK-820, 17-cyclopropylmethyl-3,14 beta-dihydroxy-4,5 alpha-epoxy-6 beta-[N-methyl-trans-3-(3-furyl)acrylamido]morphinan], bremazocine [(+/-)-6-ethyl-1,2,3,4,5,6-hexahydro-3-[(1-hydroxycyclopropy)-methyl]-11,11-dimethyl-2,6-methano-3-benzazocin-8-ol], and GR 89696 [4-[(3,4-dichlorophenyl)acetyl]-3-(1-pyrrolidinylmethyl)-1-piperazinecarboxylic acid methyl ester] were studied in various behavioral assays for measuring itch/scratching, analgesia, and respiratory depression. Systemic administration of nalfurafine (0.1-1 microg/kg), bremazocine (0.1-1 microg/kg), or GR 89696 (0.01-0.1 microg/kg) dose-dependently attenuated intrathecal morphine (0.03 mg)-induced scratching responses without affecting morphine antinociception. The combination of intrathecal morphine with these KOR agonists did not cause sedation. In addition, pretreatment with effective antiscratching doses of nalfurafine, bremazocine, or GR 89696 did not antagonize systemic morphine-induced antinociception and respiratory depression. The dose-addition analysis revealed that there is no subadditivity for nalfurafine in combination with morphine in the antinociceptive effect. Furthermore, the KOR antagonist study revealed that antiscratching effects of both nalfurafine and a prototypical KOR-1 agonist, U-50488H [trans-(+/-)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]-cyclohexyl)-benzeneacetamide], could be blocked completely by a selective KOR antagonist, nor-binaltorphimine (3 mg/kg). These findings suggest that the agonist action on KOR mainly contributes to the effectiveness of these atypical KOR agonists as antipruritics, and there is no evidence for KOR subtypes or mu-opioid antagonist action underlying the effects of these KOR agonists. This mechanism-based study further supports the clinical potential of KOR agonists as antipruritics under the context of spinal opioid analgesia.

The μ-opioid receptors are G protein-coupled receptors that play a pivotal role in the analgesic effects of opioid receptor agonists used clinically.1,2 Given that intrathecal (i.t.) administration of opioids is one of the most frequently used methods of analgesia in humans,3-5 it is important to study the functions of spinal μ-opioid receptors. In particular, the peptidic μ-opioid receptor agonists are of interest due to their enzymatic degradation and low toxicity.6 Endomorphin-1 and endomorphin-2 are endogenous opioid peptides isolated from bovine and human brains, and both peptides have high affinity and selectivity for μ-opioid receptors.7,8 Endomorphins have been implicated in a broad range of physiological functions including antinociceptive, cardiovascular, respiratory, digestive, rewarding, and endocrine responses.9,10 One important issue is the efficacy of endomorphins in activating μ-opioid receptors and subsequent antinociceptive effects manifested in vivo. Previous studies measuring the degree of agonist-stimulated G protein activation have shown that endomorphins act as partial agonists in the mouse spinal cord membranes and cell lines expressing μ-opioid receptors.11,12 In contrast, other studies indicate that endomorphins are full agonists in the rat thalamic membranes and cell lines expressing μ-opioid receptors.13,14 Although the antinociceptive effects of spinally administered endomorphins have been studied in rodents,15-18 there are few studies that directly compare the relative degrees, potencies, and durations of antinociceptive effects of i.t. endomorphins with those of i.t. [d-Ala,2N-Me-Phe,4Gly5-ol]-enkephalin (DAMGO), a highly effective peptidic μ-opioid receptor agonist.17 In particular, there is no study using both in vivo and in vitro measurements at the same time to compare the intrinsic efficacy between endomorphins and DAMGO at the rat spinal μ-opioid receptors. Therefore, the aim of the study was to systematically investigate and directly compare the spinal antinociceptive effects of DAMGO, endomorphin-1, and endomorphin-2 together in rats by using both in vitro and in vivo assays. Agonist-stimulated [35S] guanosine-5′-O-(3-thio)triphosphate (GTPγS) binding19-22 was used to observe the functional activity at the level of the receptor-G protein in both spinal cord and thalamic membranes. Acute and inflammatory pain models23,24 were used to determine the degree of antinociceptive effects of these peptidic agonists after i.t. administration. With additional antagonist studies, this study used a pharmacological approach to elucidate the relative efficacy and functional selectivity of endomorphins as antinociceptive agents in the spinal cord of rats.

Summary Opioids can modulate neuroendocrine function. Less is known about the involvement of opioid receptor subtypes in the stimulatory effects of opioids on the primate hypothalamic–pituitary–adrenal (HPA) axis. The aim of this study was to investigate the stimulatory effects of opioids selective for each receptor subtype on plasma adrenocorticotropic hormone (ACTH) and cortisol levels in both male and female monkeys. The blood collection procedure was conducted in home-caged and unanesthetized rhesus monkeys that showed low and stable basal ACTH and cortisol levels. Three opioid receptor agonists, fentanyl, U-50488H, and SNC80, were used in behaviorally active doses; they are highly selective for mu, kappa, and delta opioid receptors, respectively. Plasma samples were collected at multiple time points before and after IV administration of each compound and were quantified by radioimmunoassay. Neither fentanyl (0.0003–0.02 mg/kg) nor SNC80 (0.03–0.3 mg/kg) changed either ACTH or cortisol basal levels. In contrast, U-50488H (0.01–1 mg/kg) dose-dependently stimulated ACTH and cortisol release in both male and female monkeys. Importantly, the stimulatory effects of U-50488H on the secretion of ACTH were blocked by a selective kappa opioid receptor antagonist, nor-Binaltorphimine. The antagonist effect of nor-binaltorphimine lasted up to 20 weeks. These results indicate that only synthetic kappa, but not mu or delta opioid receptor agonists stimulate HPA axis activity after acute administration in primates.

kappa-opioid receptor antagonists such as nor-Binaltorphimine (nor-BNI) have been shown to produce antidepressant-like behavioral effects in animal models of depression. The aim of this study was to investigate further the duration of centrally administered nor-BNI-induced antidepressant-like actions measured by both behavior and brain-derived neurotrophic factor (BDNF) gene expression. In addition, antagonist studies were conducted to determine the role of opioid receptor subtypes and the time course of nor-BNI's pharmacological actions. Antidepressant-like behavioral effects were measured by decreased immobility in the rat forced swim test and BDNF mRNA expression was determined by in situ hybridization. Centrally administered nor-BNI (20 microg, i.c.v.) decreased immobility and increased BDNF mRNA expression in the hippocampus on day 1, not on days 3-14, post-administration. Systemic administration of selective mu-, delta- and kappa-opioid receptor antagonists did not block nor-BNI-induced antidepressant-like effects. In contrast, i.c.v. administration of nor-BNI 7 or 14 days earlier significantly blocked subsequent nor-BNI-induced decreased immobility and upregulation of BDNF mRNA expression. Although the duration of nor-BNI's antidepressant-like effects did not synchronize with that of its kappa-opioid receptor antagonist effects, this study is the first to show that centrally administered nor-BNI, like most clinically used antidepressants, can upregulate BDNF mRNA expression in the rat hippocampus. These findings further demonstrate that central kappa-opioid receptor mediates antidepressant-like effects of nor-BNI measured by both behavior and BDNF gene expression.

The bradykinin B(1) receptor plays a critical role in chronic pain and inflammation, although efforts to demonstrate efficacy of receptor antagonists have been hampered by species-dependent potency differences, metabolic instability, and low oral exposure of current agents. The pharmacology, pharmacokinetics, and analgesic efficacy of the novel benzamide B(1) receptor antagonist 7-chloro-2-[3-(9-pyridin-4-yl-3,9-diazaspiro[5.5]undecanecarbonyl)phenyl]-2,3-dihydro-isoindol-1-one (ELN441958) is described. ELN441958 competitively inhibited the binding of the B(1) agonist ligand [(3)H]desArg(10)-kallidin ([(3)H]DAKD) to IMR-90 human fibroblast membranes with high affinity (K(i) = 0.26 +/- 0.02 nM). ELN441958 potently antagonized DAKD (but not bradykinin)-induced calcium mobilization in IMR-90 cells, indicating that it is highly selective for B(1) over B(2) receptors. Antagonism of agonist-induced calcium responses at B(1) receptors from different species indicated that ELN441958 is selective for primate over rodent B(1) receptors with a rank order potency (K(B), nanomolar) of human (0.12 +/- 0.02) approximately rhesus monkey (0.24 +/- 0.01) > rat (1.5 +/- 0.4) > mouse (14 +/- 4). ELN441958 had good permeability and metabolic stability in vitro consistent with high oral exposure and moderate plasma half-lives in rats and rhesus monkeys. Because ELN441958 is up to 120-fold more potent at primate than at rodent B(1) receptors, it was evaluated in a primate pain model. ELN441958 dose-dependently reduced carrageenan-induced thermal hyperalgesia in a rhesus monkey tail-withdrawal model, with an ED(50) approximately 3 mg/kg s.c. Naltrexone had no effect on the antihyperalgesia produced by ELN441958, indicating a lack of involvement of opioid receptors. ELN441958 is a novel small molecule bradykinin B(1) receptor antagonist exhibiting high oral bioavailability and potent systemic efficacy in rhesus monkey inflammatory pain.

How neuropeptides in the primate spinal cord regulate itch and pain is largely unknown. Here we elucidate the sensory functions of spinal opioid-related peptides and gastrin-releasing peptide (GRP) in awake, behaving monkeys. Following intrathecal administration, β-endorphin (10–100 nmol) and GRP (1–10 nmol) dose-dependently elicit the same degree of robust itch scratching, which can be inhibited by mu-opioid peptide (MOP) receptor and GRP receptor (BB2) antagonists, respectively. Unlike β-endorphin, which produces itch and attenuates inflammatory pain, GRP only elicits itch without affecting pain. In contrast, enkephalins (100–1000 nmol) and nociceptin-orphanin FQ (3–30 nmol) only inhibit pain without eliciting itch. More intriguingly, dynorphin A(1–17) (10–100 nmol) dose-dependently attenuates both β-endorphin- and GRP-elicited robust scratching without affecting pain processing. The anti-itch effects of dynorphin A can be reversed by a kappa-opioid peptide (KOP) receptor antagonist nor-binaltorphimine. These nonhuman primate behavioral models with spinal delivery of ligands advance our understanding of distinct functions of neuropeptides for modulating itch and pain. In particular, we demonstrate causal links for itch-eliciting effects by β-endorphin-MOP receptor and GRP-BB2 receptor systems and itch-inhibiting effects by the dynorphin A-KOP receptor system. These studies will facilitate transforming discoveries of novel ligand-receptor systems into future therapies as antipruritics and/or analgesics in humans.

Given its profound analgesic nature, neuraxial opioids are frequently used for pain management. Unfortunately, the high incident rate of itch/pruritus after spinal administration of opioid analgesics reported in postoperative and obstetric patients greatly diminishes patient satisfaction and thus the value of the analgesics. Many endeavors to solve the mystery behind neuraxial opioid-induced itch had not been successful, as the pharmacological antagonism other than the blockade of mu opioid receptors remains elusive. Nevertheless, as the characteristics of all opioid receptor subtypes have become more understood, more studies have shed light on the potential effective treatments. This review discusses the mechanisms underlying neuraxial opioid-induced itch and compares pharmacological evidence in nonhuman primates with clinical findings across diverse drugs. Both nonhuman primate and human studies corroborate that mixed mu/kappa opioid partial agonists seem to be the most effective drugs in ameliorating neuraxial opioid-induced itch while retaining neuraxial opioid-induced analgesia.

Cocaine esterase (CocE) is the most efficient protein catalyst for the hydrolysis of cocaine characterized to date. The aim of this study was to investigate the in vivo potency of CocE in blocking cocaine-induced toxicity in the mouse and to assess CocE's potential immunogenicity. Cocaine toxicity was quantified by measuring the occurrence of convulsions and lethality. Intravenous administration of CocE (0.1-1 mg) 1 min before cocaine administration produced dose-dependent rightward shifts of the dose-response curve for cocaine toxicity. More important, i.v. CocE (0.1-1 mg), given 1 min after the occurrence of cocaine-induced convulsions, shortened the recovery time after the convulsions and saved the mice from subsequent death. Effects of repeated exposures to CocE were evaluated by measuring anti-CocE antibody titers and the protective effects of i.v. CocE (0.32 mg) against toxicity elicited by i.p. cocaine (320 mg/kg) (i.e., 0-17% occurrence of convulsions and lethality). CocE retained its potency against cocaine toxicity in mice after a single prior CocE exposure (0.1-1 mg), and these mice did not show an immune response. CocE retained similar effectiveness in mice after three prior CocE exposures (0.1-1 mg/week for 3 weeks), although these mice displayed 10-fold higher antibody titers. CocE partially lost effectiveness (i.e., 33-50% occurrence of convulsions and lethality) in mice with four prior exposures to CocE (0.1-1 mg/2 week for four times), and these mice displayed approximately 100-fold higher antibody titers. These results suggest that CocE produces robust protection and reversal of cocaine toxicity, indicating CocE's therapeutic potential for acute cocaine toxicity. Repeated CocE exposures may increase its immunogenicity and partially reduce its protective ability.

The aim of this study was to characterize scratching behavior elicited by central administration of morphine or bombesin in rats, and to determine the role of opioid receptors in scratching induced by both pruritogenic agents. Central administration included intracisternal (i.c.), intrathecal (i.t.), and intracerebroventricular (i.c.v.) routes. Scratching events made with hind paws were counted by observers blinded to treatment conditions. Intracisternal morphine (0.01-0.1 microg) produced dose-dependent increases in scratching; the maximum response to i.c. morphine 0.1 microg was approximately 500 scratches within a 1-hour period. Neither i.t. nor i.c.v. morphine significantly increased scratching. Bombesin (0.01-0.32 microg) elicited robust scratching following i.c. administration. The maximum response to i.c. bombesin 0.32 microg was approximately 4000 scratches within a 1-hour period. Both i.t. and i.c.v. bombesin produced profound scratching at similar doses. Antagonist studies confirmed that mu-opioid receptors selectively mediate i.c. morphine-induced scratching. However, selective mu-, kappa-, and delta-opioid antagonists did not attenuate i.c. bombesin-induced scratching. These results demonstrate that morphine and bombesin elicit scratching through different receptor mechanisms, at different central sites, and to different degrees.

Kappa opioid receptor (KOR) agonists such as U-50488H and bremazocine are analgesics and diuretics. In monkeys, the selective KOR antagonist, nor-binaltorphimine (nor-BNI), produces a long-lasting antagonism of the antinociceptive effects of U-50488H but not those of bremazocine, suggesting that KOR-mediated antinociception may occur through two distinct KORs. The aim of this study was to characterize the antagonist effect of nor-BNI against the diuretic effects of U-50488H and bremazocine in monkeys. Urine outputs were collected over 3 h subsequent to i.m. administration of KOR agonists. Both U-50488H (0.032–1 mg/kg) and bremazocine (0.00032–0.01 mg/kg) dose-dependently increased urine output and the diuretic effect reached a plateau at higher doses. The maximum effect of either U-50488H or bremazocine was approximately 15 ml/kg/3 h of urine. Pretreatment with intracisternal nor-BNI 0.32 mg significantly blocked both U-50488H (0.18 mg/kg)- and bremazocine (0.0032 mg/kg)-induced diuresis for 20 weeks. However, the same dose of nor-BNI 0.32 mg given subcutaneously was not effective. These results demonstrate that central KOR mediate KOR agonist-induced diuresis in monkeys. More important, this study provides functional evidence for a homogenous population of KOR underlying KOR-mediated diuresis and illustrates a unique pharmacological profile of nor-BNI-induced ultra-long KOR antagonism in vivo.

The aim of this study was to investigate the relative density of micro -, kappa-, and delta-opioid receptors (MOR, KOR, and DOR) and guanosine 5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding stimulated by full agonists in cortical and thalamic membranes of monkeys. The binding parameters [Bmax (femtomoles per milligram)/Kd (nanomolar)] were as follows: [3H][d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) (MOR; 80/0.7), [3H]U69593 [(5alpha,7alpha,8beta)-(-)-N-methyl-N-(7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl) benzeneacetamide] (KOR; 116/1.3), and [3H][d-Pen2,d-Pen5]-enkephalin (DPDPE) (DOR; 87/1.3) in the cortex; [3H]DAMGO (147/0.9), [3H]U69593 (75/2.5), and [3H]DPDPE (22/2.0) in the thalamus. The relative proportions of MOR, KOR, and DOR in the cortex were 28, 41, and 31% and in the thalamus were 60, 31, and 9%. Full selective opioid agonists, DAMGO (EC50 = 532-565 nM) and U69593 (EC50 = 80-109 nM) stimulated [35S]GTPgammaS binding in membranes of cortex and thalamus, whereas SNC80 [(+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethyl-benzamide] (DOR; EC50 = 68 nM) was only active in cortical membranes. The magnitudes of [35S]GTPgammaS binding stimulated by these agonists were similar in the cortex, ranging from 17 to 25% over basal binding. In the thalamus, DAMGO and U69593 increased [35S]GTPgammaS binding by 44 and 23% over basal, respectively. Opioid agonist-stimulated [35S]GTPgammaS binding was blocked selectively by antagonists for MOR, KOR, and DOR. The amount of G protein activated by agonists was highly proportional to the relative receptor densities in both regions. These results distinguish the ability of opioid agonists to activate G proteins and provide a functional correlate of ligand-binding experiments in the monkey brain. In particular, the relative densities of opioid receptor binding sites in the two brain areas reflect their functional roles in the pharmacological actions of opioids in the central nervous system of primates.

1 Orphanin FQ (OFQ), an endogenous peptide for ORL1 receptors, has been identified. Although the actions of OFQ have much in common with those of opioid peptides at the cellular level, behavioral studies in rodents seem conflicting. 2 The aim of this study was to investigate the potential pronociceptive or antinociceptive function of peripheral ORL1 receptors in primates. Experiments were conducted to verify whether local administration of OFQ can attenuate capsaicin-induced nociception and whether peripheral ORL1 receptors selectively mediate the local action of OFQ in monkeys. 3 Capsaicin (100 mg) was administered subcutaneously in the tail to locally evoke a nociceptive response (thermal allodynia/hyperalgesia), which was manifested as a reduced tail-withdrawal latency in normally innocuous 468C warm water. 4 Co-administration of OFQ (1‐30 mg) with capsaicin in the tail dose-dependently inhibited thermal nociception. However, a locally eAective dose of OFQ (30 mg), when applied in the back, did not inhibit capsaicin-induced nociception. 5 OFQ-induced local antinociception was antagonized by a small dose (10 mg) of J-113397, a selective ORL1 receptor antagonist, in the tail. Similarly, s.c. administration of 10 mg of J-113397 in the back did not antagonize local antinociception of OFQ. 6 In addition, s.c. administration of either OFQ or J-113397 in the tail alone did not change its thermal nociceptive threshold. Local administration of opioid receptor antagonists selective for mu, kappa, and delta opioid receptors did not antagonize OFQ-induced local antinociception. Local administration of J-113397 also did not interfere with the local actions of mu, kappa, and delta opioid agonists in the tail. 7 These results provide the first functional evidence that activation of peripheral ORL1 receptors produces thermal antinociception in primates and this action is independent of antinociception produced at classical opioid receptors. British Journal of Pharmacology (2002) 135, 943‐950

Nociceptin/orphanin FQ peptide (NOP) receptor agonists attenuate morphine-induced conditioned place preference in rodents. However, it is not known whether NOP agonists have reinforcing properties or can inhibit mu opioid receptor (MOP)-mediated reinforcement as measured by drug self-administration in rodents. Further understanding the behavioral effects of NOP agonists could suggest them as having potential in attenuating reinforcing effects of opioids. In the first part of the study, reinforcing properties of selective NOP agonist SCH221510 were determined and compared with the full MOP agonist remifentanil under fixed-ratio 5 (FR5) and progressive-ratio (PR) schedules of drug self-administration. In the second part, effects of systemic and intracisternal pretreatment of SCH221510 were determined and compared with MOP antagonist naltrexone in attenuating reinforcing effects of remifentanil and a non-drug reinforcer (sucrose pellets). Remifentanil self-administration (0.3-10 µg/kg/infusion) generated a biphasic dose-response curve, characteristic of drugs with reinforcing properties. SCH221510 (3-300 µg/kg/infusion) self-administration resulted in flat dose-response curves and early break-points under the PR, indicative of drugs lacking reinforcing value. Intracisternally, but not systemically, administered SCH221510 (0.3-3 µg) attenuated remifentanil self-administration, comparable with systemic naltrexone (0.03-0.3 mg/kg). SCH221510 (1-3 µg), unlike naltrexone (0.03-1 mg/kg), attenuated responding for sucrose pellets. Both effects of SCH221510 were reversed by the NOP antagonist J-113397 (0.3-3 µg). These results suggest that SCH221510 does not function as a reinforcer in rats, and that it can attenuate the reinforcing value of MOP agonists; therefore, the potential utility of NOP agonists for the treatment of drug addiction warrants further evaluation.

Pruritus (itch sensation) was defined 350 years ago by a German physician, Samuel Hafenreffer, as an unpleasant sensation that elicits a desire or reflex to scratch. Itch/pruritus is the most common symptom in skin diseases. At any given time, one of three people in the United States has a skin disease (Johnson 2004; Thorpe et al. 2004). Skin disease is listed as one of the top 15 groups of medical conditions in which prevalence and healthcare spending increased the most between 1987 and 2000 in the United States (Thorpe et al. 2004). The estimated cost of skin diseases, including direct medical cost and the economic burden on quality of life to the American public in a single year 2004, was approximately US$96 billion (Bickers et al. 2006). Actually, the significance of skin diseases is a worldwide issue. For example, 70% of people living in developing countries suffer from skin diseases at some points in their lives, but of these 3 billion, people in 127 countries do not have access to basic skin medications (Ersser and Penzer 2000).According to a recent review of epidemiological data, itch is also one of the key symptoms in patients suffering from a variety of systemic disorders including infectious, uremic, hepatic, and hematological diseases (Weisshaar and Dalgard 2009). Although causes of itch depend on several factors such as disease, age, ethnicity, and characteristics of the regional healthcare systems, the symptom of itch is highly prevalent and represents a medical burden in the global community (Weisshaar and Dalgard 2009). Itch is not life threatening in most conditions. However, chronic or severe itch can cause a great deal of psychological distress, social isolation, and occupational difficulties, posing a significant financial burden in the form of physician visits, hospital care, prescription drugs, and over-the-counter products for treating the symptom (Yosipovitch et al. 2003; Bickers et al. 2006). Given that itch is a significant clinical problem afflicting a large worldwide population of humans, there is a strong need for more research on the cause, prevention, and treatment of itch, especially to advance the discovery of efficacious therapy for the treatment of itch originated from various nervous system disorders. The present review highlights the functions of central opioid receptor subtypes in regulating itch sensation.

Francisco Aboitiz*, F. Xavier Castellanos and Ximena Carrasco Departamento de Psiquiatria, Facultad de Medicina, and Centro Interdisciplinario de Neurociencia, Pontificia Universidad Catolica de Chile, Santiago, Chile New York University Child Study Center, Nathan Kline Institute for Psychiatric Research, New York, NY, USA Servicio de Neurologia y Psiquiatria infantil, Hospital Luis Calvo Mackenna Facultad de Medicina, Universidad de Chile, Santiago, Chile

Bombesin along with several closely related neuropeptides elicit scratching behavior when administered centrally. The first part of the study was designed to determine the antagonistic effects of a novel phyllolitorin analogue [desTrp3,Leu8]phyllolitorin (DTP) on scratching induced by three peptides (bombesin, neuromedin-C, and [Leu8]phyllolitorin). In addition, the binding affinity of each peptide for the bombesin receptor site was determined. DTP (30 μg) inhibited scratching induced by these peptides, but unlike the peptides, DTP had no affinity for the bombesin site, thereby suggesting that DTP is displaying physiological antagonism through an unknown mechanism.

Nociceptin/orphanin FQ peptide receptor (NOP) agonists produce antinociceptive effects in animal models after spinal administration and potentiate μ-opioid receptor (MOP)-mediated antinociception. This study determined the antinociceptive effects of spinally administered bifunctional NOP/MOP ligands and the antinociceptive functions of spinal NOP and MOP receptors in mice. Antinociceptive effects of bifunctional NOP/MOP ligands BU08028 [(2S)-2-[(5R,6R,7R,14S)-N-cyclopropylmethyl-4,5-epoxy-6,14-ethano-3-hydroxy-6-methoxymorphinan-7-yl]-3,3-dimethylpentan-2-ol] and SR16435 [1-(1-(2,3,3α,4,5,6-hexahydro-1H-phenalen-1-yl)piperidin-4-yl)-indolin-2-one] were pharmacologically compared with the putative bifunctional ligand buprenorphine, selective NOP agonist SCH221510 [3-endo-8-[bis(2-methylphenyl)methyl]-3-phenyl-8-azabicyclo[3.2.1]octan-3-ol] and selective MOP agonist morphine in neuropathic and inflammatory pain models. Additionally, the degree of tolerance development to the antiallodynic effects of SR16435 and buprenorphine were determined after repeated intrathecal administration. Our data indicated that BU08028 and SR16435 were more potent than morphine and SCH221510 in attenuating nerve injury-induced tactile allodynia and inflammation-induced thermal hyperalgesia. Coadministration of receptor-selective antagonists further revealed that both NOP and MOP in the spinal cord mediated the antiallodynic effects of BU08028 and SR16435, but intrathecal buprenorphine-induced antiallodynic effects were primarily mediated by MOP. Repeated intrathecal administration of SR16435 resulted in reduced and slower development of tolerance to its antiallodynic effects compared with buprenorphine. In conclusion, both NOP and MOP receptors in the spinal cord independently drive antinociception in mice. Spinally administered bifunctional NOP/MOP ligands not only can effectively attenuate neuropathic and inflammatory pain, but also have higher antinociceptive potency with reduced tolerance development to analgesia. Such ligands therefore display a promising profile as spinal analgesics.

Previous studies have demonstrated the capacity of a long-acting mutant form of a naturally occurring bacterial double mutant cocaine esterase (DM CocE) to antagonize the reinforcing, discriminative, convulsant, and lethal effects of cocaine in rodents and reverse the increases in mean arterial pressure (MAP) and heart rate (HR) produced by cocaine in rhesus monkeys. This study was aimed at characterizing the immunologic responses to repeated dosing with DM CocE and determining whether the development of anti-CocE antibodies altered the capacity of DM CocE to reduce plasma cocaine levels and ameliorate the cardiovascular effects of cocaine in rhesus monkeys. Under control conditions, intravenous administration of cocaine (3 mg/kg) resulted in a rapid increase in the plasma concentration of cocaine (n = 2) and long-lasting increases in MAP and HR (n = 3). Administration of DM CocE (0.32 mg/kg i.v.) 10 min after cocaine resulted in a rapid hydrolysis of cocaine with plasma levels below detection limits within 5 to 8 min. Elevations in MAP and HR were significantly reduced within 25 and 50 min of DM CocE administration, respectively. Although slight (10-fold) increases in anti-CocE antibodies were observed after the fourth administration of DM CocE, these antibodies did not alter the capacity of DM CocE to reduce plasma cocaine levels or ameliorate cocaine's cardiovascular effects. Anti-CocE titers were transient and generally dissipated within 8 weeks. Together, these results suggest that highly efficient cocaine esterases, such as DM CocE, may provide a novel and effective therapeutic for the treatment of acute cocaine intoxication in humans.

Cocaine abuse and toxicity remain widespread problems in the United States. Currently cocaine toxicity is treated only symptomatically, because there is no Food and Drug Administration-approved pharmacotherapy for this indication. To address the unmet need, a stabilized mutant of bacterial cocaine esterase [T172R/G173Q-CocE (DM-CocE)], which hydrolyzes cocaine into inactive metabolites and has low immunogenic potential, has been developed and previously tested in animal models of cocaine toxicity. Here, we document the rapid cocaine hydrolysis by low doses of DM-CocE in vitro and in vivo, as well as the pharmacokinetics and distribution of the DM-CocE protein in rats. DM-CocE at 50.5 μg/kg effectively eliminated 4 mg/kg cocaine within 2 min in both male and female rats as measured by mass spectrometry. We expanded on these findings by using a pharmacologically relevant dose of DM-CocE (0.32 mg/kg) in rats and monkeys to hydrolyze convulsant doses of cocaine. DM-CocE reduced cocaine to below detection limits rapidly after injection; however, elimination of DM-CocE resulted in peripheral cocaine redistribution by 30 to 60 min. Elimination of DM-CocE was quantified by using [35S] labeling of the enzyme and was found to have a half-life of 2.1 h in rats. Minor urinary output of DM-CocE was also observed. Immunohistochemistry, Western blotting, and radiography all were used to elucidate the mechanism of DM-CocE elimination, rapid proteolysis, and recycling of amino acids into all tissues. This rapid elimination of DM-CocE is a desirable property of a therapeutic for cocaine toxicity and should reduce the likelihood of immunogenic or adverse reactions as DM-CocE moves toward clinical use.

No small-molecule therapeutic is available to treat cocaine addiction, but enzyme-based therapy to accelerate cocaine hydrolysis in serum has gained momentum. Bacterial cocaine esterase (CocE) is the fastest known native enzyme that hydrolyzes cocaine. However, its lability at 37°C has limited its therapeutic potential. Cross-linking subunits through disulfide bridging is commonly used to stabilize multimeric enzymes. Herein we use structural methods to guide the introduction of two cysteine residues within dimer interface of CocE to facilitate intermolecular disulfide bond formation. The disulfide-crosslinked enzyme displays improved thermostability, particularly when combined with previously described mutations that enhance stability (T172R-G173Q). The newly modified enzyme yielded an extremely stable form of CocE (CCRQ-CocE) that retained greater than 90% of its activity after 41 days at 37°C, representing an improvement of more than 4700-fold over the wild-type enzyme. CCRQ-CocE could also be modified by polyethylene glycol (PEG) polymers, which improved its in vivo residence time from 24 to 72 h, as measured by a cocaine lethality assay, by self-administration in rodents, and by measurement of inhibition of cocaine-induced cardiovascular effects in rhesus monkeys. PEG-CCRQ elicited negligible immune response in rodents. Subunit stabilization and PEGylation has thus produced a potential protein therapeutic with markedly higher stability both in vitro and in vivo.

Bombesin is a pruritogenic agent that causes intense itch-scratching activity in rodents. Bombesin has high affinity for the gastrin-releasing peptide (GRP) receptor (GRPr) and the neuromedin B (NMB) receptor (NMBr). The aim of this study was to investigate pharmacologically the ability of GRPr and NMBr to elicit scratching behavior in rats. The intracerebroventricular route was selected for drug delivery because the study focused on supraspinal sites of action. The magnitude and duration of scratching produced by the naturally occurring peptides GRP and NMB were characterized. Antagonists selective for GRPr [(d-Tpi6, Leu13Ψ(CH2-NH)-Leu14)Bombesin(6-14) (RC-3095)] and NMBr [(S)-α-methyl-α-[[[(4-nitrophenyl)amino]carbonyl]amino]-N-[[1-(2-pyridinyl)cyclohexyl]methyl]-1H-indole-3-propanamide (PD168368)] were used to define the role of GRPr and NMBr in the scratching response. After intracerebroventricular administration, GRP (0.03–0.3 nmol) and NMB (0.1–1 nmol) dose-dependently elicited marked scratching. There was a tolerance to scratching elicited by daily repeated administration of bombesin, GRP, or NMB. Presession administration of RC-3095 (0.1–1 nmol) and PD168368 (0.3–3 nmol) dose-dependently antagonized scratching elicited by GRP and NMB, respectively. More importantly, 1 nmol of RC-3095 failed to block NMB-elicited scratching, and 3 nmol of PD168368 failed to block GRP-elicited scratching. In addition, pretreatment with effective doses of RC-3095 or PD168368 alone or in combination did not block bombesin-elicited scratching. Through the use of the selective antagonists RC-3095 and PD168368, this study demonstrates that central GRPr and NMBr act independently to elicit scratching behavior and there is an additional, unidentified receptor mechanism underlying bombesin-elicited scratching.

A long-acting mutant form of a naturally occurring bacterial cocaine esterase (T172R/G173Q CocE; double mutant CocE (DM CocE)) has previously been shown to antagonize the reinforcing, convulsant, and lethal effects of cocaine in rodents. However, the effectiveness and therapeutic characteristics of DM CocE in nonhuman primates, in a more clinically relevant context, are unknown. The current studies were aimed at (1) characterizing the cardiovascular effects of cocaine in freely moving rhesus monkeys, (2) evaluating the capacity of DM CocE to ameliorate these cocaine-induced cardiovascular effects when administered 10 min after cocaine, and (3) assessing the immunological responses of monkeys to DM CocE following repeated administration. Intravenous administration of cocaine produced dose-dependent increases in mean arterial pressure (MAP) and heart rate (HR) that persisted throughout the 2-h observation period following a dose of 3.2 mg/kg cocaine. Cocaine failed to produce reliable changes in electrocardiograph (ECG) parameters, body temperature, and locomotor activity. DM CocE produced a rapid and dose-dependent amelioration of the cardiovascular effects, with saline-like MAP measures restored within 5–10 min, and saline-like HR measures restored within 20–40 min of DM CocE administration. Although administration of DM CocE produced increases in anti-CocE antibodies, they did not appear to have a neutralizing effect on the capacity of DM CocE to reverse the cardiovascular effects of cocaine. In conclusion, these findings in monkeys provide strong evidence to suggest that highly efficient cocaine esterases, such as DM CocE, can provide a potential therapeutic option for treatment of acute cocaine intoxication in humans.

Background A longer acting, double mutant bacterial cocaine esterase (CocE T172R/G173Q; DM CocE) has been shown to protect mice from cocaine-induced lethality, inhibit the reinforcing effects of cocaine in rats, and reverse cocaine's cardiovascular effects in rhesus monkeys. The current studies evaluated the effectiveness of DM CocE to protect against, and reverse cocaine's cardiovascular, convulsant, and lethal effects in male and female rats. Methods Pretreatment studies were used to determine the effectiveness and in vivo duration of action for DM CocE to protect rats against the occurrence of cardiovascular changes, convulsion and lethality associated with acute cocaine toxicity. Posttreatment studies were used to evaluate the capacity of DM CocE to rescue rats from the cardiovascular and lethal effects of large doses of cocaine. In addition, male and female rats were studied to determine if there were any potential effects of sex on the capacity of DM CocE to protect against, or reverse acute cocaine toxicity in rats. Results Pretreatment with DM CocE dose-dependently protected rats against cocaine-induced cardiovascular changes, convulsion and lethality, with higher doses active for up to 4 h, and shifting cocaine-induced lethality at least 10-fold to the right. In addition to dose-dependently recovering rats from an otherwise lethal dose of cocaine, post-treatment with DM CocE also reversed the cardiovascular effects of cocaine. There were no sex-related differences in the effectiveness of DM CocE to protect against, or reverse acute cocaine toxicity. Conclusions Together, these results support the development of DM CocE for the treatment of acute cocaine toxicity.