Antipruritic and antihyperalgesic actions of loperamide and analogs

Loperamide and three of its analogs were evaluated for their ability to inhibit binding to cloned human opioid receptor subtypes and to produce antipruritis and antinociception following local s.c. administration to rodents. All four compounds were fully efficacious agonists with affinities of 2 to 4 nM for the cloned human mu opioid receptor. Local s.c. injection of loperamide, ADL 01-0001 or ADL 01-0002 at the same site as the introduction of the pruritogenic compound 48/80 resulted in antipruritic activity in a mouse model of itch. Similarly, i.paw or i.pl. administration of compounds ADL 01-0001, ADL 01-0002 and ADL 01-0003 to inflamed paws caused potent antinociception, inhibiting late phase formalin-induced flinching, Freund's adjuvant-induced mechanical hyperalgesia and tape stripping-induced mechanical hyperalgesia. Loperamide and its analogs were efficacious in animal models of itch and inflammatory pain, and may have potential therapeutic utility as antipruritic and antihyperalgesic agents.     

Cholecystokinin and cholecystokinin antagonists enhance postsynaptic excitability in the dentate gyrus

The sulfated and unsulfated octapeptide cholecystokinin (CCK) sequences and the pancreatic CCK antagonists, CR 1409 and benzotript, were applied iontophoretically in the rat dentate gyrus granular layer while the response evoked by single pulse stimulation of the perforant path was recorded. The stimulating current was varied and the resulting relationship between the slope of the response (input) against the population spike amplitude (output) was used as a measure of excitability at the granule cell synapse. All four test compounds shifted the input/output curve to the left indicating an increase in postsynaptic excitability. These results thus imply that endogenous CCK acts at the central type of CCK receptor to modulate cortical input to granule cells by reducing the threshold for synaptic excitation.     

Supraspinal circuitry mediating opioid antinociception: Antagonist and synergy studies in multiple sites

Supraspinal opioid antinociception is mediated by sensitive brain sites capable of supporting this response following microinjection of opioid agonists. These sites include the ventrolateral periaqueductal gray (vIPAG), the rostral ventromedial medulla (RVM), the locus coeruleus and the amygdala. Each of these sites comprise an interconnected anatomical and physiologically relevant system mediating antinociceptive responses through regional interactions. Such interactions have been identified using two pharmacological approaches: (1) the ability of selective antagonists delivered to one site to block antinociception elicited by opioid agonists in a second site, and (2) the presence of synergistic antinociceptive interactions following simultaneous administration of subthreshold doses of opioid agonists into pairs of sites. Thus, the RVM has essential serotonergic, opioid, cholinergic and NMDA synapses that are necessary for the full expression of morphine antinociception elicited from the vIPAG, and the vIPAG has essential opioid synapses that are necessary for the full expression of opioid antinociception elicited from the amygdala. Further, the vIPAG, RVM, locus coeruleus and amygdala interact with each other in synergistically supporting opioid antinociception.     

(+/-)-4-[(N-allyl-cis-3-methyl-4-piperidinyl)phenylamino]-N,N-diethylbenzamide displays selective binding for the delta opioid receptor

Racemic 4-[(N-allyl-cis-3-methyl-4-piperidinyl)phenylamino]-N,N-diethylbenzam ide (3a) was synthesized and found to have good affinity and selectivity for the delta receptor. These compounds can be viewed as an analog of BW373U86 and SNC-80 where an internal piperazine nitrogen has been transposed with a benzylic carbon. Functionally, 3a behaves as an agonist at the delta receptor with no measurable stimulation of either the mu or kappa receptor subtypes and was found to be devoid of any measurable amount of antagonist activity for any opioid receptor. A comparison of 3a to SNC-80 and DPDPE in the [35S]GTPgammaS functional assay suggests that 3a may be more like the peptide DPDPE.     

Pharmacological regulation of opioidergic antinociceptive mechanisms]

Naloxone-depending potentiation of morphine antinociception by some non-opioidergic compounds between different classes of drugs was found in experiments on mice using nociceptive stimuli of different modality. This potentiation can or cannot be bound with elevation of sensitivity of opioid receptors, release of endogenous opioids or destruction of blood-brain barrier function mor morphine peripheral administration. This potentiation named as "release of functional reserve of opioid antinociceptive response" can or cannot be accompanied by an increase of breathing function depression. Taking into account the data of literature about the dissociation of analgetic positive-supporting morphine effects and also the capability of some compounds to lower the narcogenic opiates potential, the supposition about the real possibility of creating combined drugs is made.     

Synthesis and characterizations of novel quinoline derivatives having mixed ligand activities at the κ and μ receptors: Potential therapeutic efficacy against morphine dependence

Based on an established 3D pharmacophore, a series of quinoline derivatives were synthesized. The opioidergic properties of these compounds were determined by a competitive binding assay using ¹²⁵I-Dynorphine, ³H-DAMGO and ¹²⁵I-DADLE for κ, μ, and δ receptors, respectively. Results showed varying degree of activities of the compounds to κ and μ opioid receptors with negligible interactions at the δ receptor. The compound, S4 was the most successful in inhibiting the two most prominent quantitative features of naloxone precipitated withdrawal symptoms - stereotyped jumping and body weight loss. Determination of IC₅₀ of S4 revealed a greater affinity towards μ compared to κ receptor. In conclusion, quinoline derivatives of S4 like structure offer potential tool for treatment of narcotic addictions.     

Spinal administration of selective opioid antagonists in amphibians: evidence for an opioid unireceptor

In mammals, opioids act by interactions with three distinct types of receptors: mu, delta, or kappa opioid receptors. Using a novel assay of antinociception in the Northern grass frog, Rana pipiens, previous work demonstrated that selective mu, delta, or kappa opioids produced a potent antinociception when administered by the spinal route. The relative potency of this effect was highly correlated to that found in mammals. Present studies employing selective opioid antagonists, beta-FNA, NTI, or nor-BNI demonstrated that, in general, these antagonists were not selective in the amphibian model. These data have implications for the functional evolution of opioid receptors in vertebrates and suggest that the tested mu, delta, and kappa opioids mediate antinociception via a single type of opioid receptor in amphibians, termed the unireceptor.     

Antinociceptive pattern of flavone and its mechanism as tested by formalin assay

Flavone, dextrose and long swim stress exhibited antinociception. Degree of antinociception was greater with long swim stress as compared to flavone or dextrose. Combination of these treatments resulted in potentiation of antinociception. Naloxone (opioid antagonist; 5 mg/kg i.p.) antagonised flavone or long stress induced antinociception showing opioid medicated mechanism, however, failed to reverse the potentiated antinociceptive component recorded in long stressed animals which received flavone and dextrose. Antinociceptive activity of flavone, dextrose and long swim stress which was documented by acetic acid assay has been confirmed in the present study. Role for opioid system in this action has been demonstrated. Therefore, formalin test can also be considered as an useful assay procedure for testing flavonoids. However, like acetic acid assay this assay procedure also has the limitation that it is unable to detect minor changes in the degree of antinociception produced by physiological interventions such as long swim and dextrose.     

Importance of histamine H2 receptors in restraint-morphine interactions

The effects of the brain-penetrating H₂ antagonist zolantidine (ZOL, 3 mg/Kg, S.c.) were studied on morphine (MOR, 4 mg/Kg, S.c.) antinociception (tail flick test) in the presence and absence of previous restraint stress. Animals were handled for 3 days (to reduce handling stress), restrained for 1 hr or handled on day 4 and tested 24 hrs later. As found previously, restraint enhanced the intensity and duration of MOR antinociception. ZOL potentiated MOR antinociception in handled, non-restrained animals, but inhibited MOR action in restrained animals. In contrast, ZOL had no effects on nociceptive responses in either handled or stressed subjects in the absence of MOR. The data suggest that, in the absence of restraint, brain HA acts at the H₂ receptor to inhibit MOR antinociception. In contrast, when an animal has been previously restrained, HA enhances MOR antinociception. Thus, brain HA appears to mediate the restraintinduced potentiation of MOR antinociception. Taken with previous results, the present findings suggest that in the presence of MOR, brain HA can provide bidirectional modulation of nociception. The direction of the modulation seems to depend upon the stress experience of the animal.     

Antinociceptive profile of LP1, a non-peptide multitarget opioid ligand

AimsOpioid drugs are the principal treatment option for moderate to severe pain and exert their biological effects through interactions with opioid receptors that are widely distributed throughout the CNS and peripheral tissues. Ligands capable of simultaneously targeting different receptors could be successful candidates for the treatment of chronic pain. Enhanced antinociception coupled with a low incidence of side effects has been demonstrated for ligands possessing mixed mu-opioid receptor (MOR) and delta-opioid receptor (DOR) activity. We previously reported that 3-[(2R,6R,11R)-8-hydroxy-6,11-dimethyl-1,4,5,6-tetrahydro-2,6-methano-3-benzazocin-3(2 H)-yl]-N-phenylpropanamide (LP1) acted as a MOR-DOR ligand in in vitro functional assays and moreover this drug produced a valid antinociception that was longer lasting than that of morphine. The aim of this work was to determine whether the antinociceptive effect produced by LP1 was central or peripheral and to assess which opioid receptor subtypes are involved in its effects.Main methodsWe explored the effects of naloxone methiodide (NX-M), a quaternary opioid antagonist, administered either intracerebroventricularly (i.c.v.) or subcutaneously (s.c.), on LP1-mediated antinociception in male Sprague–Dawley rats. In addition, we administered s.c. selective antagonists for MOR, DOR and kappa-opioid receptor (KOR) to investigate the effects of LP1. To characterise this drug's DOR profile better, we also investigated the effects of LP1 on DPDPE, a selective DOR agonist.Key findingsData obtained by tail flick test showed that LP1 induced predominantly MOR-mediated supraspinal antinociception and was able to counteract DPDPE analgesia.SignificanceLP1, a multitarget opioid ligand, is a supraspinal acting antinociceptive agent that is useful for the treatment of chronic pain.     

The CCK(-like) receptor in the animal kingdom: functions, evolution and structures

In this review, the cholecystokinin (CCK)(-like) receptors throughout the animal kingdom are compared on the level of physiological functions, evolutionary basis and molecular structure. In vertebrates, the CCK receptor is an important member of the G-protein coupled receptors as it is involved in the regulation of many physiological functions like satiety, gastrointestinal motility, gastric acid secretion, gall bladder contraction, pancreatic secretion, panic, anxiety and memory and learning processes. A homolog for this receptor is also found in nematodes and arthropods, called CK receptor and sulfakinin (SK) receptor, respectively. These receptors seem to have evolved from a common ancestor which is probably still closely related to the nematode CK receptor. The SK receptor is more closely related to the CCK receptor and seems to have similar functions. A molecular 3D-model for the CCK receptor type 1 has been built together with the docking of the natural ligands for the CCK and SK receptors in the CCK receptor type 1. These molecular models can help to study ligand-receptor interactions, that can in turn be useful in the development of new CCK(-like) receptor agonists and antagonists with beneficial health effects in humans or potential for pest control.     

Studies on the structure-activity relationship of 2',6'-dimethyl-l-tyrosine (Dmt) derivatives: bioactivity profile of H-Dmt-NH-CH(3)

The 2',6'-dimethyl-l-tyrosine (Dmt) enhances receptor affinity, functional bioactivity and in vivo analgesia of opioid peptides. To further investigate its direct influence on these opioid parameters, we developed a series of compounds (H-Dmt-NH-X). Among them, H-Dmt-NH-CH(3) showed the highest affinity (K(i)mu=7.45 nM) equal to that of morphine, partial mu-opioid agonism (E(max)=66.6%) in vitro and a moderate antinociception in mice.     

Is paradoxical pain induced by sustained opioid exposure an underlying mechanism of opioid antinociceptive tolerance?

Opiates are the primary treatment for pain management in cancer patients reporting moderate to severe pain, and are being increasingly used for non-cancer chronic pain. However, prolonged administration of opiates is associated with significant problems including the development of antinociceptive tolerance, wherein higher doses of the drug are required over time to elicit the same amount of analgesia. High doses of opiates result in serious side effects such as constipation, nausea, vomiting, dizziness, somnolence, and impairment of mental alertness. In addition, sustained exposure to morphine has been shown to result in paradoxical pain in regions unaffected by the initial pain complaint, and which may also result in dose escalation, i.e. 'analgesic tolerance'. A concept that has been gaining considerable experimental validation is that prolonged use of opioids elicits paradoxical, abnormal pain. This enhanced pain state requires additional opioids to maintain a constant level of antinociception, and consequently may be interpreted as antinociceptive tolerance. Many substances have been shown to block or reverse antinociceptive tolerance. A non-inclusive list of examples of substances reported to block or reverse opioid antinociceptive tolerance include: substance P receptor (NK-1) antagonists, calcitonin gene-related peptide (CGRP) receptor antagonists, nitric oxide (NO) synthase inhibitors, calcium channel blockers, cyclooxygenase (COX) inhibitors, protein kinase C inhibitors, competitive and non-competitive antagonists of the NMDA (N-methyl-D-aspartate) receptor, AMPA (alpha-amino-3-hydroxy-5-methyl-4 isoxazolepropionic acid) antagonists, anti-dynorphin antiserum, and cholecystokinin (CCK) receptor antagonists. Without exception, these substances are also antagonists of pain-enhancing agents. Prolonged opiate administration indeed induces upregulation of substance P (SP) and calcitonin gene-related peptide (CGRP) within sensory fibers in vivo, and this is accompanied by an enhanced release of excitatory neurotransmitters and neuropeptides from primary afferent fibers upon stimulation. The enhanced evoked release of neuropeptides is correlated with the onset of abnormal pain states and opioid antinociceptive tolerance. Importantly, the descending pain modulatory pathway from the brainstem rostral ventromedial medulla (RVM) via the dorsolateral funiculus (DLF) is critical for maintaining the changes observed in the spinal cord, abnormal pain states and antinociceptive tolerance, because animals with lesion of the DLF did not show enhanced evoked neuropeptide release, or develop abnormal pain or antinociceptive tolerance upon sustained exposure to opiates. Microinjection of either lidocaine or a CCK antagonist into the RVM blocked both thermal and touch hypersensitivity as well as antinociceptive tolerance. Thus, prolonged opioid exposure enhances a descending pain facilitatory pathway from the RVM that is mediated at least in part by CCK activity and is essential for the maintenance of antinociceptive tolerance.     

Increased level of cholecystokinin in cerebrospinal fluid is associated with chronic pain-like behavior in spinally injured rats.

Cholecystokinin (CCK) is a physiological antagonist of opioid-mediated antinociception and may be involved in some chronic pain states where opioids have reduced effect. We have previously shown in a rat model of central neuropathic pain after spinal cord injury that blockade of CCK-B receptors lead to marked pain relief. In the present study, we showed that spinally injured rats exhibiting chronic pain-like behaviors (aversive reaction to innocuous mechanical and cold stimulation) had significantly elevated level of CCK-like immunoreactivity in cerebrospinal fluid compared to normal rats or spinally injured rats which did not exhibit pain-like behaviors. The increased level of circulating CCK in the cerebrospinal fluid may thus contribute to the maintenance of chronic pain in these rats by reducing the endogenous inhibitory tone provided by opioid peptides and may be involved in the phenomenon of opioid insensitivity.     

Drug design and synthesis of epsilon opioid receptor agonist: 17-(cyclopropylmethyl)-4,5alpha-epoxy-3,6beta-dihydroxy-6,14-endoethenomorphinan-7alpha-(N-methyl-N-phenethyl)carboxamide (TAN-821) inducing antinociception mediated by putative epsilon opioid receptor

Here we report the new drug design and synthesis of a series of 6,14-endoethenomorphinan-7-carboxamide derivatives as a putative epsilon opioid receptor agonist. One of these compounds, 17-(cyclopropylmethyl)-4,5alpha-epoxy-3,6beta-dihydroxy-6,14-endoethenomorphinan-7alpha-(N-methyl-N-phenethyl)carboxamide (TAN-821), showed agonistic activity for a putative epsilon opioid receptor (IC(50) = 71.71nM) in the rat vas deferens (RVD) preparations. TAN-821 stimulated the binding of the nonhydrolyzable guanosine 5'-triphosphate analog, guanosine 5'-(gamma-thio)-triphosphate (GTPgammaS), to the mouse pons/medulla membrane via the activation of putative epsilon opioid receptor. Moreover, TAN-821 given intracerebroventricularly (i.c.v.) produced a marked antinociception in the tail-flick test (ED(50) = 1.73 microg) and the hot-plate test (ED(50) = 2.05 microg) in a dose-dependent manner. The antinociception induced by TAN-821 administered i.c.v. was blocked by the i.c.v.-pretreatment with a putative epsilon opioid receptor partial agonist beta-endorphin [1-27], but not a mu opioid receptor antagonist beta-FNA, a delta opioid receptor antagonist NTI, or a kappa opioid receptor antagonist nor-BNI. The present results suggest that TAN-821 may be a useful tool for the investigation on the pharmacological properties of the putative epsilon opioid receptor.     

Dextromethorphan potentiates morphine-induced antinociception at both spinal and supraspinal sites but is not related to the descending serotoninergic or adrenergic pathways

Morphine is a strong and widely used opioid analgesic in pain management, but some adverse effects limit its clinical use at high doses. The clinically available non-opioid antitussive, dextromethorphan (DM) can potentiate the analgesic effect of morphine and decrease the dose of morphine in acute postoperative pain. However, the mechanism underlying this synergistic phenomenon is still not clear. To examine if the potentiation by DM occurs through the descending pain-inhibitory pathways, ketanserin (a 5-HT₂ receptor antagonist) and yohimbine (an ₂-adrenergic receptor antagonist) were employed and found to have no significant effect on the potentiation by DM. Using local delivery of drugs in rats in the present study, potentiation of morphine-induced antinociception by DM was observed via both intrathecal and intracerebroventricular routes, suggesting that both spinal and supraspinal sites are involved. This suggests that the potentiation of morphine-induced antinociception by DM is not mediated by the serotoninergic or adrenergic descending pain-inhibitory pathways. The present results are consistent with findings in clinical studies, which showed that DM can effectively decrease the consumption of morphine in patients suffering from pain. Since DM has excellent clinical potential as a synergistic agent with morphine, further investigating and clarifying the possible pharmacological mechanism of DM are of great importance for future studies.     

Interaction of copper(II) and nickel(II) with a bis-amide ligand functionalized with pyridine moieties: Thermodynamic and spectroscopic studies in aqueous solution

We synthesized a new bis-amide ligand derived from the l(+)-tartaric acid. We then determined its protonation constants and the stability constants of the copper(II) and nickel(II) chelates by potentiometry as well as ESI-MS and UV-Vis spectroscopy. We found that both metal ions are able to induce the deprotonation and the coordination of an amide nitrogen donor atom. In the case of copper complexes, the data show the formation of two major species: Cu₂(L₂H₋₃)⁺ and Cu₂(LH₋₄). EPR and XAS experiments led us to precise the relative structure of these compounds. In Cu₂(L₂H₋₃)⁺, each metal center is coordinated by pyridinic and amidic nitrogen atoms of one ligand and by nitrogen and oxygen atoms from pyridine and hydroxyl moieties from the other one. In Cu₂(LH₋₄), the copper centers are coordinated by pyridinic and amidic nitrogen atoms, as well as a deprotonated hydroxyl group of the ligand. In this latter complex, the lower value of the Cu-Cu distance determined from EXAFS experiments and compared to the one of the solid species likely involve the formation of an exogeneous hydroxyl bridge between the two copper centers. With Ni(II) ions, the only one major species is the mononuclear Ni(LH₋₂) complex, in which Ni(II) is held in an octahedral environment with the metal center chelated by the two pyridinic and the two amidic nitrogen atoms, and two oxygen atoms from water molecules.     

Synthesis of novel analogues of the delta opioid ligand SNC-80 using AlCl3-promoted aminolysis.

Two focused libraries of delta opioid ligands were synthesised using AlCl3 facilitated aminolysis. Several compounds were identified with DOR binding affinities higher or similar to SNC-80. A novel acyclic derivative of SNC-80 produced antinociception in the acetic acid abdominal constriction test, which is at least partially mediated via the delta-opioid receptor.     

Pharmacological properties and predicted binding mode of arylmethylene quinuclidine-like derivatives at the α3β4 nicotinic acetylcholine receptor (nAChR)

We have carried out a pharmacological evaluation of arylmethylene quinuclidine derivatives interactions with human α3β4 nAChRs subtype, using cell-based receptor binding, calcium-influx, electrophysiological patch-clamp assays and molecular modeling techniques. We have found that the compounds bind competitively to the α3β4 receptor with micromolar affinities and some of the compounds behave as non-competitive antagonists (compounds 1, 2 and 3), displaying submicromolar IC₅₀ values. These evidences suggest a mixed mode of action for these compounds, having interactions at the orthosteric site and more pronounced interactions at an allosteric site to block agonist effects. One of the compounds, 1-benzyl-3-(diphenylmethylene)-1-azoniabicyclo[2.2.2]octane chloride (compound 3), exhibited poorly reversible use-dependent block of α3β4 channels. We also found that removal of a phenyl group from compound 1 confers a partial agonism to the derived analog (compound 6). Introducing a hydrogen-bond acceptor into the 3-benzylidene quinuclidine derivative (compound 7) increases agonism potency at the α3β4 receptor subtype. Docking into the orthosteric binding site of a α3β4 protein structure derived by comparative modeling accurately predicted the experimentally-observed trend in binding affinity. Results supported the notion that binding requires a hydrogen bond formation between the ligand basic nitrogen and the backbone carbonyl oxygen atom of the conserved Trp-149.     

Modification by Cholecystokinin Octapeptide of the Binding ofμ-, δ-, and K-Opioid Receptors

Previous study has shown that cholecystokinin (CCK) octapeptide (CCK-8) suppressed the binding of opioid receptors to the universal opioid agonist [3H]etorphine. In the present study, highly selective tritium-labeled agonists for the mu-[(tryrosyl-3,5-3H][D-Ala2,MePhe4,Gly-ol5]enkephalin ([3H]DAGO], delta- ([tyrosyl-3,5-3H][D-Pen2,5]enkephalin ([3H]DPDPE], and kappa- ([3H]U69,593) opioid receptors were used to clarify which type(s) of opioid receptor in rat brain homogenates is suppressed by CCK-8. In the competition experiments, CCK-8 suppressed the binding of [3H]DAGO and [3H]U69,593 but not that of [3H]DPDPE to the respective opioid receptor. This effect was blocked by the CCK antagonist proglumide at 1 mumol/L. In the saturation experiments, CCK-8 at concentrations of 0.1 nmol/L to 1 mumol/L decreased the Bmax of [3H]DAGO binding sites without affecting the KD; on the other hand, CCK-8 increased the KD of [3H]U69,593 binding without changing the Bmax. The results suggest that CCK-8 inhibits the binding of mu- and kappa-opioid receptors via the activation of CCK receptors.