Mei-Chuan Ko

Most Efficient Cocaine Hydrolase Designed by Virtual Screening of Transition States

Cocaine is recognized as the most reinforcing of all drugs of abuse. There is no anticocaine medication available. The disastrous medical and social consequences of cocaine addiction have made the development of an anticocaine medication a high priority. It has been recognized that an ideal anticocaine medication is one that accelerates cocaine metabolism producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway, i.e., cocaine hydrolysis catalyzed by plasma enzyme butyrylcholinesterase (BChE). However, wild-type BChE has a low catalytic efficiency against the abused cocaine. Design of a high-activity enzyme mutant is extremely challenging, particularly when the chemical reaction process is rate-determining for the enzymatic reaction. Here we report the design and discovery of a high-activity mutant of human BChE by using a novel, systematic computational design approach based on transition-state simulations and activation energy calculations. The novel computational design approach has led to discovery of the most efficient cocaine hydrolase, i.e., a human BChE mutant with an approximately 2000-fold improved catalytic efficiency, promising for therapeutic treatment of cocaine overdose and addiction as an exogenous enzyme in human. The encouraging discovery resulted from the computational design not only provides a promising anticocaine medication but also demonstrates that the novel, generally applicable computational design approach is promising for rational enzyme redesign and drug discovery.

How best to fight that nasty itch – from new insights into the neuroimmunological, neuroendocrine, and neurophysiological bases of pruritus to novel therapeutic approaches

While the enormous clinical and psychosocial importance of pruritus in many areas of medicine and the detrimental effects of chronic ‘itch’ on the quality of life of an affected individual are widely appreciated, the complexity of this sensation is still often grossly underestimated. The current Controversies feature highlights this complexity by portraying pruritus as a truly interdisciplinary problem at the crossroads of neurophysiology, neuroimmunology, neuropharmacology, protease research, internal medicine, and dermatology, which is combated most successfully if one keeps the multilayered nature of ‘itch’ in mind and adopts a holistic treatment approach – beyond the customary, frequently frustrane monotherapy with histamine receptor antagonists. In view of the often unsatisfactory, unidimensional, and altogether rather crude standard instruments for pruritus management that we still tend to use in clinical practice today, an interdisciplinary team of pruritus experts here critically examines recent progress in pruritus research that future itch management must take into consideration. Focusing on new insights into the neuroimmunological, neuroendocrine, and neurophysiological bases of pruritus, and discussing available neuropharmacological tools, specific research avenues are highlighted, whose pursuit promises to lead to novel, and hopefully more effective, forms of pruritus management.

Thermostable Variants of Cocaine Esterase for Long-Time Protection against Cocaine Toxicity

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°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 ∼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.

Design, preparation, and characterization of high-activity mutants of human butyrylcholinesterase specific for detoxification of cocaine.

Cocaine is a widely abused drug without a U.S. Food and Drug Administration-approved medication. There is a recognized, promising anticocaine medication to accelerate cocaine metabolism, producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway [i.e., cocaine hydrolysis catalyzed by butyrylcholinesterase (BChE) in plasma]. An ideal, therapeutically valuable mutant of human BChE should have not only a significantly improved catalytic activity against (−)-cocaine but also certain selectivity for (−)-cocaine over neurotransmitter acetylcholine (ACh), such that one would not expect systemic administration of the BChE mutant to interrupt cholinergic transmission. The present study accounting for the mutation-caused changes of the catalytic activities of BChE against both (−)-cocaine and ACh by means of molecular modeling and site-directed mutagenesis has led to identification of three BChE mutants that have not only a considerably improved catalytic efficiency against (−)-cocaine but also the desirable selectivity for (−)-cocaine over ACh. Two representative BChE mutants have been confirmed to be potent in actual protection of mice from acute toxicity (convulsion and lethality) of a lethal dose of cocaine (180 mg/kg). Pretreatment with the BChE mutant (i.e., 1 min before cocaine administration) dose-dependently protected mice against cocaine-induced convulsions and lethality. In particular, all mice pretreated with the mutant (e.g., 0.02 mg or more of A199S/F227A/S287G/A328W/E441D BChE) survived. The in vivo data reveal the primary factor (i.e., the relative catalytic efficiency), determining the efficacy in practical protection of mice from the acute cocaine toxicity and future direction for further improving the efficacy of the enzyme in the cocaine overdose treatment.

Behavioral Effects of a Synthetic Agonist Selective for Nociceptin/Orphanin FQ Peptide Receptors in Monkeys

Behavioral effects of a nonpeptidic NOP (nociceptin/orphanin FQ Peptide) receptor agonist, Ro 64-6198, have not been studied in primate species. The aim of the study was to verify the receptor mechanism underlying the behavioral effects of Ro 64-6198 and to systematically compare behavioral effects of Ro 64–6198 with those of a μ-opioid receptor agonist, alfentanil, in monkeys. Both Ro 64-6198 (0.001–0.06 mg/kg, s.c.) and alfentanil (0.001–0.06 mg/kg, s.c.) produced antinociception against an acute noxious stimulus (50°C water) and capsaicin-induced allodynia. An NOP receptor antagonist, J-113397 (0.01–0.1 mg/kg, s.c.), dose-dependently produced rightward shifts of the dose–response curve of Ro 64-6198-induced antinociception. The apparent pA2 value of J-113397 was 8.0. Antagonist studies using J-113397 and naltrexone revealed that Ro 64-6198 produced NOP receptor-mediated antinociception independent of μ-opioid receptors. In addition, alfentanil dose-dependently produced respiratory depression and itch/scratching responses, but antinociceptive doses of Ro 64-6198 did not produce such effects. More important, Ro 64-6198 did not produce reinforcing effects comparable with those of alfentanil, cocaine, or methohexital under self-administration procedures in monkeys. These results provide the first functional evidence that the activation of NOP receptors produces antinociception without reinforcing effects in primates. Non-peptidic NOP receptor agonists may have therapeutic value as novel analgesics without abuse liability in humans.

Functional plasticity of the N/OFQ-NOP receptor system determines analgesic properties of NOP receptor agonists.

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.

Antinociceptive Effects of Nociceptin/Orphanin FQ Administered Intrathecally in Monkeys

UNLABELLED Nociceptin/orphanin FQ (N/OFQ) is the endogenous peptide for the NOP receptors. Depending on the doses, intrathecal administration of N/OFQ has dual actions (ie, hyperalgesia and antinociception) in rodents. However, the pharmacological profile of intrathecal N/OFQ is not fully known in primates. The aim of this study was to investigate behavioral effects of intrathecal N/OFQ over a wide dose range and to compare its effects with ligands known to produce hyperalgesia or antinociception in monkeys. Intrathecal N/OFQ from 1 fmol to 1 nmol did not produce any hyperalgesic or scratching responses. In contrast, intrathecal substance P 100 nmol produced hyperalgesia, and intrathecal DAMGO 10 nmol produced antinociception. At the dose range between 10 nmol and 1 micromol, intrathecal N/OFQ dose-dependently produced thermal antinociception against a noxious stimulus in 2 intensities. More importantly, N/OFQ in combined with intrathecal morphine dose-dependently potentiated morphine-induced antinociception without inhibiting morphine-induced itch/scratching. Taken together, this study is the first to provide a unique functional profile of intrathecal N/OFQ over a wide dose range in primates. Intrathecal N/OFQ produces thermal antinociception without anti-morphine actions or scratching responses, indicating that N/OFQ or NOP receptor agonists represent a promising target as spinal analgesics. PERSPECTIVE Intrathecal administration of N/OFQ only produced thermal antinociception, not hyperalgesia, in monkeys. In addition, intrathecal N/OFQ does not have anti-morphine actions or itch/scratching responses. This study strongly supports the therapeutic potential of N/OFQ or NOP receptor agonists as spinal analgesics for clinical trials.

Orphanin FQ inhibits capsaicin-induced thermal nociception in monkeys by activation of peripheral ORL1 receptors

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. 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. Capsaicin (100 μg) 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 46°C warm water. Co‐administration of OFQ (1 – 30 μg) with capsaicin in the tail dose‐dependently inhibited thermal nociception. However, a locally effective dose of OFQ (30 μg), when applied in the back, did not inhibit capsaicin‐induced nociception. OFQ‐induced local antinociception was antagonized by a small dose (10 μg) of J‐113397, a selective ORL1 receptor antagonist, in the tail. Similarly, s.c. administration of 10 μg of J‐113397 in the back did not antagonize local antinociception of OFQ. 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. 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.

Local administration of Δ9-tetrahydrocannabinol attenuates capsaicin-induced thermal nociception in rhesus monkeys: a peripheral cannabinoid action

Abstract  Rationale: Cannabinoids can reduce nociceptive responses by acting on peripheral cannabinoid receptors in rodents. Objectives: The study was conducted to evaluate the hypothesis that local administration of Δ9-tetrahydrocannabinol (Δ9-THC) can attenuate capsaicin-induced nociception in rhesus monkeys. Methods: Capsaicin (100 µg) was applied locally in the tail of rhesus monkeys to evoke a nociceptive response, thermal allodynia, in normally innocuous 46°C water. Δ9-THC (10–320 µg) was coadministered with capsaicin in the tail to assess local antinociceptive effects. In addition, a local antagonism study was performed to confirm the selectivity of Δ9-THC action. Results:Δ9-THC dose-dependently inhibited capsaicin-induced allodynia. This local antinociception was antagonized by small doses (10–100 µg) of the cannabinoid CB1 antagonist, SR141716A, applied in the tail. However, 100 µg SR141716A injected subcutaneously in the back did not antagonize local Δ9-THC. Conclusions: These results indicate that the site of action of locally applied Δ9-THC is in the tail. It provides functional evidence that activation of peripheral cannabinoid CB1 receptors can attenuate capsaicin-induced thermal nociception in non-human primates and suggests a new approach for cannabinoids in pain management.

Roles of μ-Opioid Receptors and Nociceptin/Orphanin FQ Peptide Receptors in Buprenorphine-Induced Physiological Responses in Primates

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 buprenorphines dose-response curves for all three endpoints. Mean pKB 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.