Norma Selve

Aconitum sp. alkaloids: The modulation of voltage-dependent Na+channels, toxicity and antinociceptive properties

Alkaloids from Aconitum sp., used as analgesics in traditional Chinese medicine, were investigated to elucidate their antinociceptive and toxic properties considering: (1) binding to Na+ channel epitope site 2, (2) alterations in synaptosomal Na+ and Ca2+ concentration ([Na+]i, [Ca2+]i), (3) arrhythmogenic action of isolated atria, (4) antinociceptive and (5) acute toxic action in mice. The study revealed a high affinity group (Ki 1 microM) and a low affinity group (Ki 10 microM) of alkaloids binding to site 2. The compounds of the high affinity group induce an increase in synaptosomal [Na+]i and [Ca2+]i (EC50 3 microM), are antinociceptive (ED50, 25 microg/kg), provoke tachyarrhythmia and are highly toxic (LD50 70 microg/kg), whereas low affinity alkaloids reduce [Ca2+]i, induce bradycardia and are less antinociceptive (ED50 20 mg/kg) and less toxic (LD50 30 mg/kg). These results suggest that the alkaloids can be grouped in Na+ channel activating and blocking compounds, but none of the alkaloids seem to be suitable as analgesics because of the low LD50/ED50 values.

Mode of antinociceptive and toxic action of alkaloids of Aconitum spec..

Abstract Extracts of the plant Aconitum spec. are used in traditional Chinese medicine predominantly as anti-inflammatory and analgesic agents, the latter allegedly equally potent as morphine but without any habit-forming potential. As the only pharmacologically active compounds, the C19 diterpenoid alkaloid aconitine, and some of its derivatives, have been proven to be antinociceptive in different analgesic assays, but the mode of action is unknown. To elucidate the mode of action, ten aconitine-like derivatives were investigated with respect to their affinity for voltage-dependent Na+ channels, the action on synaptosomal Na+ and Ca2+ homoeostasis and their antinociceptive, arrhythmogenic and acute toxic properties. Since aconitine is known to bind to site II of Na+ channels, we determined the affinity of the aconitine-like derivatives in vitro to synaptosomal membranes by the [3H]-batrachotoxinin-binding assay and their properties on intrasynaptosomal concentrations of free Na+ and Ca2+ ([Na+]i and [Ca2+]i), both the latter determined fluorometrically with SBFI and Fura-2 respectively. Furthermore, the alkaloids’ arrhythmogenic potential was investigated in guinea-pig isolated atria and the antinociceptive action on formalin-induced hyperalgesia and the acute toxic action estimated in mice. The results show that the alkaloids could be divided into at least three groups. The first is characterized by a high affinity to the site II of Na+ channels (Ki about 1.2 μM), the ability to enhance [Na+]i and [Ca2+]i (EC50 about 3 μM), a strong arrhythmogenic action that starts at about 30 nM, an antinociceptive effect (ED50 about 0.06 mg/kg) and high acute toxicity (LD50 values about 0.15 mg/kg). To this group belong aconitine, 3-acetylaconitine and hypaconitine. The second group, with lappaconitine as the only member, has an affinity to Na+ channels an order of magnitude lower (Ki = 11.5 μM), less acute toxicity (LD50 about 5 mg/kg), and a two orders of magnitude lower antinociceptive action (ED50 about 2.8 mg/kg) and lower cardiotoxicity (bradycardia observed at 3 μM). Additionally, lappaconitine suppresses the increase in [Ca2+]i of aconitine-stimulated synaptosomes and increases the excitation threshold of left atria, indicating an inhibition of Na+ channels. The other derivatives, i.e. delcorine, desoxydelcorine, karakoline, lappaconidine, lappaconine and lycoctonine, belong to the third group, which has hardly any effects. They have a low affinity to Na+ channels with Ki values in the millimolar range, show no effect on synaptosomal [Na+]i and [Ca2+]i, no arrhythmogenic potential up to 100 μM, no antinociceptive activity and low toxicity with LD50 values greater than 50 mg/kg. For the investigated alkaloids we suggest two different antinociceptive-like modes of action. Aconitine, hypaconitine and 3-acetylaconitine may induce a block of neuronal conduction by a permanent cell depolarisation, whereas lappaconitine might act like local anaesthetics. However, because of the low LD50/ED30 quotients of 2–6, the antinociceptive-like action of the Aconitum alkaloids seems to reflect severe intoxication rather than a specific antinociceptive action. The structure/activity relationship shows that alkaloids that activate or block Na+ channels have a benzoyl ester side chain in the C-14 or C-4 positions respectively, whereas the other compounds lack this group.

Lacosamide displays potent antinociceptive effects in animal models for inflammatory pain.

Lacosamide is a functionalized amino acid which was initially synthesized as an antiepileptic drug. In addition to its broad anti‐seizure activity, lacosamide was shown to display efficacy in animal models for neuropathic pain and is currently in phase III clinical development for the treatment of epilepsy and neuropathic pain. In order to further profile its antinociceptive properties, the effects of lacosamide on inflammatory pain in the formalin test, the carrageenan model and the adjuvant‐induced arthritis model were investigated.

Spinal antinociception by morphine in rats is antagonised by galanin receptor antagonists

Galanin, a 29 amino acid peptide, has been reported to possess antinociceptive properties at the spinal site and to potentiate opioid-induced antinociception. Our aim was to investigate whether also endogenous galanin interacts with an exogenously administered opioid, morphine, in the rat spinal cord. This question was investigated by use of the recently developed galanin receptor antagonists galantide [M-15, galanin(1–13)-substance P-(5 -11) amide] and M-35 [galanin-(1–13)-bradykinin-(2–9) amide].Nociception was assessed in the rat tail-flick test using radiant heat and the rat Randall-Selitto model of inflammatory pain using vocalization as the nociceptive criterion. Intrathecal (i.t.) injections were performed in rats under ether anaesthesia. Morphine was administered either i.t. or intraperitoneally (i.p.), and the antagonists were injected i.t. [125I]Galanin binding experiments were performed on crude synaptosomal membranes of the rat spinal cord.In the rat tail-flick test, i.t. injection of 3 μg morphine evoked antinociception of about 75% of the maximal possible effect (% MPE). Co-injection of either 2 μg galantide or 2 μg M-35 with morphine almost completely abolished the antinociceptive effect of morphine. I.p. injection of 2.15 mg/kg morphine elicited about 80% MPE when given 10 min prior to i.t. saline injection. Injection of the antagonists instead of saline antagonised the antinociceptive effect of morphine partially thus showing the spinal proportion of the overall antinociceptive effect. In the rat Randall-Selitto test, 3 μg morphine, injected i.t., produced antinociception of almost 100% MPE. Coinjection of the antagonists reduced the maximum effect partially by about 25–35%. I.p. injection of 7.5 mg/kg morphine 10 min prior to Lt. injection of saline elicited an antinociceptive effect of 90–100% MPE; injection of the antagonists instead of saline reduced the peak effect to a similar degree as after i.t. injection of 3 μg morphine. To exclude a direct interference by morphine with the galanin receptor, in vitro binding of [125I]galanin to a spinal synaptosomal fraction was assessed. Morphine, 10 μM, did not interfere with the specific [125I]galanin binding. These results provide further evidence that galanin is involved in spinal nociceptive processing. It seems to be involved in the mediation of the effects of morphine at this site, either as a co-transmitter, or subsequent to µ-receptor activation on nerve terminals or on interneurones.

The antinociceptive effects of morphine, desipramine, and serotonin and their combinations after intrathecal injection in the rat

UNLABELLED Antinociception can be produced at the spinal level by activation of opioidergic, noradrenergic, and serotonergic systems. We tested the antinociceptive effects of combined activation of all three systems. Antinociception was assessed in the rat tail-flick test, and drugs were administered via an intrathecal catheter. Morphine, the norepinephrine uptake inhibitor desipramine, and serotonin produced antinociception of their own. The combination of subthreshold doses of morphine 1 microg and of desipramine 3 microg produced pronounced antinociception that was antagonized by yohimbine. The combination of subthreshold morphine with serotonin 50 microg or desipramine with serotonin caused only small antinociceptive effects. When morphine combined with desipramine was decreased to a subthreshold dose, we observed pronounced antinociception when a subthreshold dose of serotonin was added. A complex interaction can be supposed by results obtained with antagonists. The activation of all three neurotransmitter systems with small doses of agonists may represent an effective principle for pain control at the spinal level. IMPLICATIONS Pain sensations are modulated at the spinal level by opioids, noradrenergic drugs, and serotonin. Using a rat model, we showed that the concurrent use of drugs from each of these classes produces good pain control at doses that should avoid the side effects associated with larger doses of each individual drug.

Galanin receptor antagonists attenuate spinal antinociceptive effects of DAMGO, tramadol and non-opioid drugs in rats

The involvement of endogenous galanin to antinociception elicited by intrathecally (i.t.) or systemically administered drugs from different chemical and therapeutic classes was investigated using the rat Randall-Selitto or the rat tail-flick test, in the absence or presence of the i.t. administered galanin receptor antagonists galantide and M-35. Antinociception elicited by i.t. tramadol (24 micrograms), DAMGO (1 microgram), clonidine (48 micrograms), desipramine (6 micrograms) or fenfluramine (60 micrograms) was attenuated by i.t. galantide (2 micrograms); the attenuation reached significance at least at one time point. A partial antagonism by i.t. galantide was also observed against the antinociception of i.p. tramadol (10 mg/kg), i.v. clonidine (1 mg/kg), i.p. desipramine (1 mg/kg), or i.p. dipyrone (1000 mg/kg), but antinociception by i.p. fenfluramine (30 mg/kg) was not affected. Using M-35 (2 micrograms i.t.), the antinociception of i.t. tramadol or DAMGO was attenuated, but no inhibition was observed when clonidine, desipramine or fenfluramine were used i.t. If drugs were administered systemically, only antinociception of i.p. fenfluramine but not that of i.p. tramadol, or i.v. clonidine, or i.p. desipramine or i.p dipyrone was attenuated. In the rat tail flick test, co-injection of either 2 micrograms i.t. galantide or M-35 with i.t. tramadol (12 micrograms) almost abolished the antinociceptive effect, whereas the antinociception of systemically administered tramadol (4.6 mg/kg i.p.) was only partially attenuated by i.t. galantide and not affected by i.t. M-35. Binding studies in dorsal spinal cord tissue showed no affinity of galantide or M-35 to spinal mu-, or delta-, or kappa-opioid receptors and none of the other drugs interfered with the spinal galanin binding site. These data give further support of at least a partial galanin link in spinal processes of antinociception.

Absence of emetic effects of morphine and loperamide in Suncus murinus

The house musk shrew Suncus murinus recently has been introduced for the study of emesis. We investigated the emetic effects of the opioids morphine (0.1-21.5 mg/kg i.p.) and loperamide (0.01-10 mg/kg i.p.) and found a complete lack of emetogenic potential. Nicotine, however, dose dependently induced vomiting in the Suncus with an ED50 of 8.8 mg/kg s.c. and a 100% incidence at 20 mg/kg. This drug-induced vomiting was reduced by morphine or loperamide: ED50 values obtained were 1.2 mg/kg i.p. for morphine and 0.7 mg/kg i.p. for loperamide. Naloxone (2 mg/kg s.c.) antagonised the inhibitory effect of morphine (2 mg/kg i.p.) or loperamide (10 mg/kg i.p.). Serotonin (20 mg/kg s.c.) had less reliable emetogenic potency than nicotine in the Suncus with incidences between 50 and 100%. However, the serotonin-induced vomiting was abolished by morphine and loperamide and this inhibition was antagonised by naloxone. These results suggest that systemically administered opioids are pure antiemetics in Suncus murinus in contrast to other animal models and man. Naloxone antagonism indicates that this antiemetic effect is mediated by opioid receptors.

Increased total body synthesis of prostacyclin in rats with adjuvant arthritis.

In rats with adjuvant arthritis we measured the urinary excretion of 2,3-dinor-6-oxo-PGF1 alpha, 7 alpha-hydroxy-5,11-dioxo-tetranor-prosta-1,16- dioic acid (PGE-M) and 2,3-dinor-thromboxane-B2, reflecting total body synthesis of prostacyclin, thromboxane and the E-prostaglandins, respectively. The urinary prostanoid metabolites were assessed by gas chromatography/tandem mass spectrometry using stable isotope internal standards. We found a more than 10-fold increase of urinary 2,3-dinor-6-oxo-PGF1 alpha excretion and a 5-fold higher urinary excretion of PGE-M in adjuvant arthritic rats as compared to non-arthritic control rats (p < 0.001; n = 12, each). There was no significant difference in urinary 2,3-dinor-thromboxane-B2 excretion between arthritic rats and control animals. Our data show a dramatic increase of urinary 2,3-dinor-6-oxo-PGF1 alpha excretion reflecting increased total body prostacyclin synthesis. It can be assumed that prostacyclin plays a role in generalized inflammatory reactions, comparable to that of the E-prostaglandins.