Helmut Schmidhammer

Involvement of the κ-opioid receptor in the anxiogenic-like effect of CP 55,940 in male rats

We have studied the possible interaction between three selective opioid-receptor antagonists, nor-binaltorphimine (NB: kappa) (5 mg/kg), cyprodime (CY: mu) (10 mg/kg) and naltrindole (NTI: delta) (1 mg/kg), and the cannabinoid receptor agonist CP 55,940, in the modulation of anxiety (plus-maze) and adrenocortical activity (serum corticosterone levels by radioimmunoassay) in male rats. The holeboard was used to evaluate motor activity and directed exploration. CP 55,940 (75 microg/kg, but not 10 microg/kg) induced an anxiogenic-like effect, which was antagonised by NB. The other effects of CP 55,940 (75 microg/kg), a decreased holeboard activity and stimulation of adrenocortical activity, were not antagonised by any of the three opioid receptor antagonists. CY and NTI, when administered alone, induced marked reductions in motor activity, anxiogenic-like effects and stimulation of adrenocortical activity. The selective kappa-opioid receptor antagonist NB, on its own, did not modify the level of anxiety but stimulated adrenocortical activity. We provide the first pharmacological evidence about the involvement of the kappa-opioid receptor in the anxiogenic-like effect of CP 55,940.

Involvement of μ- and κ-, but not δ-, opioid receptors in the peristaltic motor depression caused by endogenous and exogenous opioids in the guinea-pig intestine

Opiates inhibit gastrointestinal propulsion, but it is not clear which opioid receptor types are involved in this action. For this reason, the effect of opioid receptor – selective agonists and antagonists on intestinal peristalsis was studied. Peristalsis in isolated segments of the guinea‐pig small intestine was triggered by a rise of the intraluminal pressure and recorded via the intraluminal pressure changes associated with the peristaltic waves. μ‐Opioid receptor agonists (DAMGO, morphine), κ‐opioid receptor agonists (ICI‐204,448 and BRL‐52,537) and a δ‐opioid receptor agonist (SNC‐80) inhibited peristalsis in a concentration‐related manner as deduced from a rise of the peristaltic pressure threshold (PPT) and a diminution of peristaltic effectiveness. Experiments with the δ‐opioid receptor antagonists naltrindole (30 nM) and HS‐378 (1 μM), the κ‐opioid receptor antagonist nor‐binaltorphimine (30 nM) and the μ‐opioid receptor antagonist cyprodime (10 μM) revealed that the antiperistaltic effect of ICI‐204,448 and BRL‐52,537 was mediated by κ‐opioid receptors and that of morphine and DAMGO by μ‐opioid receptors. In contrast, the peristaltic motor inhibition caused by SNC‐80 was unrelated to δ‐opioid receptor activation. Cyprodime and nor‐binaltorphimine, but not naltrindole and HS‐378, were per se able to stimulate intestinal peristalsis as deduced from a decrease in PPT. The results show that the neural circuits controlling peristalsis in the guinea‐pig small intestine are inhibited by endogenous and exogenous opioids acting viaμ‐ and κ‐, but not δ‐, opioid receptors.

Peripheral versus central antinociceptive actions of 6-amino acid-substituted derivatives of 14-O-methyloxymorphone in acute and inflammatory pain in the rat.

Opioid analgesics with restricted access to the central nervous system represent a new approach to the treatment of severe pain with an improved safety profile. The objective of this study was to investigate the peripheral and central components of the antinociceptive actions of the 6-amino acid conjugates (glycine, alanine, and phenylalanine) of 14-O-methyloxymorphone. Their antinociceptive activities were compared with those of the centrally penetrating μ-opioid agonists morphine, fentanyl, and 14-O-methyloxymorphone. In the tail-flick test in rats, the 6-amino acid conjugates were 45- to 1170-fold more potent than morphine after i.c.v. administration and 19- to 209-fold after s.c. administration. They showed potencies similar to fentanyl after s.c. administration and were more potent after i.c.v. application. The time course of action was different between s.c. and i.c.v. administration, with significant long-lasting effects after i.c.v. administration. Systemic administration of the peripherally selective opioid antagonist naloxone methiodide antagonized the effects after s.c. but not after i.c.v. administration in the tail-flick test. Subcutaneous 6-amino acid derivatives also elicited antihyperalgesic effects in the formalin test in rats, which were reversed by systemically administered naloxone methiodide. Although morphine exerts its analgesic effects by central and peripheral mechanisms, the investigated new opioids interact primarily with peripheral opioid receptors after s.c. administration. The present data indicate that the 6-amino acid conjugates of 14-O-methyloxymorphone have limited access to the central nervous system and can mediate antinociception at peripheral sites. Also, they might find clinical application when the central actions of opioids are unwanted.

Modulation of basal and stress‐induced amygdaloid substance P release by the potent and selective NK1 receptor antagonist L‐822429

It has been shown that anxiety and stress responses are modulated by substance P (SP) released within the amygdala. However, there is an important gap in our knowledge concerning the mechanisms regulating extracellular SP in this brain region. To study a possible self‐regulating role of SP, we used a selective neurokinin‐1 (NK1) receptor antagonist to investigate whether blockade of NK1 receptors results in altered basal and/or stress‐evoked SP release in the medial amygdala (MeA), a critical brain area for a functional involvement of SP transmission in enhanced anxiety responses induced by stressor exposure. In vitro binding and functional receptor assays revealed that L‐822429 represents a potent and selective rat NK1 receptor antagonist. Intra‐amygdaloid administration of L‐822429 via inverse microdialysis enhanced basal, but attenuated swim stress‐induced SP release, while the low‐affinity enantiomer of L‐822429 had no effect. Using light and electron microscopy, synaptic contacts between SP‐containing fibres and dendrites expressing NK1 receptors was demonstrated in the medial amygdala. Our findings suggest self‐regulatory capacity of SP‐mediated neurotransmission that differs in the effect on basal and stress‐induced release of SP. Under basal conditions endogenous SP can serve as a signal that tonically inhibits its own release via a NK1 receptor‐mediated negative feedback action, while under stress conditions SP release is further facilitated by activation of NK1 receptors, likely leading to high local levels of SP and activation of receptors to which SP binds with lower affinity.

Opioid binding profiles of new hydrazone, oxime, carbazone and semicarbazone derivatives of 14-alkoxymorphinans

Several hydrazone, oxime, carbazone and semicarbazone derivatives of 14-alkoxycodeinones and 14-alkoxydihydrocodeinones were synthesised [1] and characterised in in vitro radioligand binding assays in rat brain membrane preparations. The tested compounds show the highest affinity for the mu opioid binding sites and most of them have agonist character. Subtype analysis of the binding shows mu2 specificity. However, some of these ligands are able to block partially (40-60%) the high affinity (putative mu1) opioid binding sites while all of them act as reversible ligands at the low affinity (putative mu2) sites.

Highly potent novel opioid receptor agonist in the 14-alkoxymetopon series

The newly synthesized 14-alkoxymetopon derivatives, 14-methoxymetopon, 14-ethoxymetopon, 14-methoxy-5-methyl-morphinone, exhibit high affinity for the naloxone binding sites in rat brain. A substantial decrease in affinity was observed, in the presence of NaCl indicating a high degree of agonist activity. All three 14-alkoxymetopon derivatives displayed high affinity for [3H][D-Ala2,(Me)Phe4,Gly-ol5]enkephalin ([3H]DAMGO) binding sites, much less potency toward delta sites and were the least effective at kappa sites. Isolated tissue studies using the guinea pig ileum preparation confirmed their high agonist potency. Following administration the new compounds produced naloxone reversible antinociceptive effects and were 130-300 times more potent than morphine in the acetic acid induced abdominal constriction model in the mouse, and the hot plate and tail flick tests in the rat. The compounds also produced dose-dependent muscle rigidity, and potentiated barbiturate-induced narcosis. The in vivo apparent pA2 values for naloxone against 14-ethoxymetopon and morphine were similar in analgesia, suggesting an interaction with the same (mu) receptor site. The dependence liability of 14-alkoxymetopon derivatives in the withdrawal jumping test was less pronounced than that of morphine in either rats or mice, similar to tolerance to the their analgesic action. It is concluded that the 14-alkoxymetopon derivatives studied are selective and potent agonists at mu opioid receptors, with reduced dependence liability.

14-methoxymetopon, A Potent Opioid, Induces No Respiratory Depression, Less Sedation, and Less Bradycardia than Sufentanil in the Dog

Opioids of the &mgr;-receptor type depress respiration and induce addiction. At 10-min intervals 14-methoxymetopon (HS-198), which is 20,000 times more potent than morphine in the acethylcholine-writhing test, was given in graded IV doses (3, 6, and 12 &mgr;g/kg) to awake, trained canines (n = 7). The following variables were derived: Pao2, Paco2, heart rate (lead II of the electrocardiogram), mean arterial blood pressure, relative changes in the &dgr; domain and the &bgr; domain of the electroencephalogram, the somatosensory evoked potential, and the skin-twitch reflex to electrical stimuli. Thereafter, 20 &mgr;g/kg naltrexone was given for reversal. After a washout period, the same animals were exposed to similar doses of sufentanil (SUF) followed by naltrexone. Both opioids induced a dose-related bradycardia and hypotension. The maximal bradycardic effect was 19% after HS-198 and 42% after SUF (P < 0.005). The maximal hypotension was 6% after HS-198 and 20% after SUF (P < 0.01). In the electroencephalogram, power in the &dgr; band increased by 288% after HS-198 and by 439% after SUF (P < 0.01); simultaneously, power in the &bgr; band decreased by 71% and by 95.7%, respectively (P < 0.01). Pao2 decreased by 41% after SUF and by 4% after HS-198, and Paco2 increased by 56.8% and 6.6% in SUF and HS-198, respectively (P < 0.001). Both opioids induced a dose-related depression in the somatosensory evoked potential and increased tolerance to skin-twitch. The maximal effect was 92.7% after SUF and 81.3% after HS-198 was not significant. Naltrexone reversed all changes back to control. Compared with SUF, HS-198 does not induce hypoxia and hypercarbia, induces less hypotension and bradycardia, and induces less sedative effects. Implications Compared with sufentanil, 14-methoxymetopone does not induce hypoxia and hypercarbia, induces less hypotension and bradycardia, and induces less sedative effects (electroencephalogram). Antinociception is similar to sufentanil (skin-twitch method, amplitude depression in the evoked potential). All effects are reversed by naltrexone. Interaction of &kgr;-receptor is suggested.

Morphinans and isoquinolines: Acetylcholinesterase inhibition, pharmacophore modeling, and interaction with opioid receptors

Following indications from pharmacophore-based virtual screening of natural product databases, morphinan and isoquinoline compounds were tested in vitro for acetylcholinesterase (AChE) inhibition. After the first screen, active and inactive compounds were used to build a ligand-based pharmacophore model in order to prioritize compounds for biological testing. Among the virtual hits tested, the enrichment of actives was significantly higher than in a random selection of test compounds. The most active compounds were biochemically tested for their activity on mu, delta, and kappa opioid receptors.

μ-Opioid receptor specific antagonist cyprodime : characterization by in vitro radioligand and [35S]GTPγS binding assays

The use of compounds with high selectivity for each opioid receptor (μ, δ and κ) is crucial for understanding the mechanisms of opioid actions. Until recently non-peptide μ-opioid receptor selective antagonists were not available. However, N-cyclopropylmethyl-4,14-dimethoxy-morphinan-6-one (cyprodime) has shown a very high selectivity for μ-opioid receptor in in vivo bioassays. This compound also exhibited a higher affinity for μ-opioid receptor than for δ- and κ-opioid receptors in binding assays in brain membranes, although the degree of selectivity was lower than in in vitro bioassays. Cyprodime has recently been radiolabelled with tritium resulting in high specific radioactivity (36.1 Ci/mmol). We found in in vitro binding experiments that this radioligand bound with high affinity (Kd 3.8±0.18 nM) to membranes of rat brain affording a Bmax of 87.1±4.83 fmol/mg. Competition studies using μ, δ and κ tritiated specific ligands confirmed the selective labelling of cyprodime to a μ-opioid receptor population. The μ-opioid receptor selective agonist [d-Ala2,N-MePhe4,Gly5-ol]enkephalin (DAMGO) was readily displaced by cyprodime (Ki values in the low nanomolar range) while the competition for δ- ([d-Pen2,d-Pen5]enkephalin (DPDPE)) and κ- (5α,7α,8β-(−)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl]-benzene-acetamide (U69,593)) opioid receptor selective compounds was several orders of magnitude less. We also found that cyprodime inhibits morphine-stimulated [35S]GTPγS binding. The EC50 value of morphine increased about 500-fold in the presence of 10 μM cyprodime. These findings clearly indicate that cyprodime is a useful selective antagonist for μ-opioid receptor characterization.

The µ Opioid Receptor and Ligands Acting at the µ Opioid Receptor, as Therapeutics and Potential Therapeutics

Although the µ opioid receptor (MOR) was pharmacologically and biochemically identified in binding studies forty years ago, its structure, function, and true complexity only have emerged after its cloning in 1993. Continuous efforts from many laboratories have greatly advanced our understanding of MORs, ranging from their anatomic distribution to cellular and molecular mechanisms, and from cell lines to in vivo systems. The MOR is recognized as the main target for effective pain relief, but its involvement in many other physiological functions has also been recognized. This review provides a synopsis on the history of research on MORs and ligands acting at the MOR with the focus on their clinical and potential use as therapeutic drugs, or as valuable research tools. Since the elucidation of the chemical structure of morphine and the characterization of endogenous opioid peptides, research has stimulated the development of new generations of MOR ligands with distinct pharmacological profiles (agonist, antagonist, mixed agonist/antagonist and partial agonist) or site of action (central/peripheral). Discovery of therapeutically useful morphine-like drugs and innovative drugs with new scaffolds, with several outstanding representatives, is discussed. Extensive efforts on modifications of endogenous peptides to attain stable and MOR selective analogs are overviewed with stimulating results for the development of peptide-based pharmaceuticals. With pharmacophore modeling as an important tool in drug discovery, application of modern computational methodologies for the development of morphinans as new MOR ligands is described. Moreover, the crystal structure of the MOR available today will enable the application of structure-based approaches to design better drugs for the management of pain, addiction and other human diseases, where MORs play a key role.