Effect of U50488, a selective kappa opioid receptor agonist and levetiracetam against lithium-pilocarpine-induced status epilepticus, spontaneous convulsive seizures and related cognitive impairment.

PURPOSE: Kappa opioid receptor (KOR) agonists have anticonvulsant effect but their antiepileptogenic effect is unknown. U50488, a selective KOR agonist is used to determine its effect on status epilepticus (SE), spontaneous convulsive seizures (SS) and cognitive impairment in rat lithium-pilocarpine SE model. Effect of an antiepileptic drug levetiracetam is also studied. METHOD: Male Wistar rats with SE were divided into three groups namely, LiP, LiP + U50488 (10 mg/kg, i.p.) and LiP + levetiracetam (400 mg/kg, i.p.) group. SE was terminated after 90 min of its onset with diazepam (15 mg/kg, i.p.) and phenobarbitone (25 mg/kg, i.p.). Drug treatment was started after 15 min of onset of SE and repeated once after 4 h. Rats were video monitored 12 h daily (9 AM to 9 PM) to determine severity of SE using modified Racine scale and onset and frequency of SS from day 0 to day 21. Morris water maze (MWM) test was done at baseline i.e. day -1 (before lithium administration) and day 22, to assess cognitive impairment. RESULTS: As compared to LiP, U50488 decreased the severity of SE (1.98 ± 0.13 vs 2.95 ± 0.12; p-value < 0.0001) but not levetiracetam (2.62 ± 0.09; p-value = 0.3112). Survival increased with both U50488 (90%, n = 10) and levetiracetam (81.8%, n = 11) as compared to NS (56.2%, n = 16). No effect on onset and frequency of SS was found in U50488/levetiracetam group. U50488 improved seizures-induced cognitive impairment. Levetiracetam group showed thigmotactic (wall hugging) behaviour in MWM in 8 out of 9 rats. CONCLUSION: Acute treatment with U50488, a kappa opioid receptor agonist has a beneficial effect on SE, SE-related mortality and memory impairment. The dual protective effect of U50488 on seizures and related cognitive impairment is advantageous over currently used antiseizure drugs which are known to cause cognitive impairment.

Beta-lactam antibiotic prevents tolerance to the hypothermic effect of a kappa opioid receptor agonist.

Beta-lactam antibiotics are the only clinically approved drugs which directly increase glutamate uptake. They activate the glutamate transporter subtype 1 (GLT-1), the protein responsible for 90% of glutamate uptake in the mammalian brain. The capacity of GLT-1 to clear extracellular glutamate suggests that glutamate transporter activators be explored for therapeutic approaches to clinical conditions caused by increased glutamatergic transmission. One of the most common drug effects mediated by increased glutamatergic signaling is opioid tolerance. Therefore, we tested the hypothesis that a beta-lactam antibiotic (ceftriaxone), by increasing glutamate uptake, prevents tolerance to hypothermia induced by a kappa opioid receptor agonist (U-50,488H). A single injection of U-50,488H (20mg/kg, s.c.) caused significant hypothermia in rats. Tolerance to the hypothermic effect of U50,488H was induced by injecting U50,488H (20mg/kg) twice daily for 7days. Pretreatment with ceftriaxone (200mg/kg, i.p.) for 7days did not alter the acute hypothermic response to U50,488H (20mg/kg) but did prevent tolerance to U50,488H-induced hypothermia. Central administration of dl-threo-beta-benzyloxyaspartic acid (TBOA) (0.2micromol, i.c.v.), a glutamate transporter inhibitor, abolished the effect of ceftriaxone. These results identify a functional interaction between ceftriaxone and U50,488H in vivo and provide pharmacological evidence that a beta-lactam antibiotic abolishes tolerance to hypothermia induced by a kappa opioid receptor agonist.

The role of nitric oxide in the development of opioid withdrawal induced by naloxone after acute treatment with mu- and kappa-opioid receptor agonists.

The present study investigated the possible role of nitric oxide (NO) in the development of the withdrawal contractures of guinea pig isolated ileum after acute activation of mu- and kappa-opioid receptors. After a 4-min in vitro exposure to morphine (mu-opioid receptor preferring, but not selective, agonist), [D-Ala2-N-methyl-Phe4-Gly5-ol-]enkephalin (DAMGO; highly selective mu-opioid receptor agonist), or trans(+/-)-3,4-dichloro-N-methyl-N-2(1-pyrrolidynyl)cyclohexyl-ben zeneacetamide (U50-488H; highly selective kappa-opioid receptor agonist), the guinea-pig isolated ileum exhibited a strong contracture after the addition of naloxone. L-N(G)-nitro arginine methyl ester (3-300 microM) injected 10 min before the opioid receptor agonists was able dose dependently to reduce the naloxone-induced contraction after exposure to mu- and kappa-opioid receptor agonists whereas D-N(G)-nitro arginine methyl ester at the same concentrations did not affect it. The inhibitory effect of L-N(G)-nitro arginine methyl ester on morphine, DAMGO and U50-488H withdrawal was dose dependently reversed by L-arginine (3-300 microM) but not by D-arginine. Finally, glyceryl trinitrate on its own (3-300 microM) significantly increased the naloxone-induced contraction after exposure to mu- and kappa-opioid receptor agonist and it was also able to reverse the inhibition of opioid withdrawal caused by L-N(G)-nitro arginine methyl ester. These results provide evidence that NO has a role in the development of opioid withdrawal and that mu- or kappa-opioid receptors are involved.

Effects of U-50,488H and U-50,488H withdrawal on catecholaminergic neurons of the rat hypothalamus.

Previous report from our laboratory showed that morphine produces a stimulatory effect of hypothalamic noradrenaline (NA) turnover concurrently with enhanced pituitary-adrenal response after its acute injection and during withdrawal. In the present work we have studied the effects of acute and chronic administration of the kappa agonist U-50,488H as well as the influence of U-50,488H withdrawal on the activity of hypothalamic NA and dopamine (DA) neurons and on the activity of hypothalamic-pituitary-adrenal (HPA) axis. A single dose of U-50,488H (15 mg/kg i.p.) significantly increased hypothalamic NA and decreased DA turnover at the time of an enhanced corticosterone release. Rats rendered tolerant to the kappa agonist by administration of U-50,488H twice a day for 4 days showed no changes in corticosterone secretion. Additionally, a decrease in both hypothalamic MHPG (the cerebral NA metabolite) production and NA turnover was observed, whereas DOPAC concentration and DA turnover were enhanced, which indicate the development of tolerance towards the neuronal and endocrine actions of U-50,488H. After naloxone (3 mg/kg s.c.) administration to U-50,488H-tolerant rats, we found neither behavioural signs of physical dependence nor changes in hypothalamic catecholaminergic neurotransmission. In addition, corticosterone secretion was not altered in U-50,488H withdrawn rats. Present data clearly indicate that tolerance develops towards the NA turnover accelerating and DA turnover decreasing effect of U-50,488H. Importantly and by contrast to mu agonists, present results demonstrate that U-50,488H withdrawal produce no changes in hypothalamic catecholamines turnover or in corticosterone release (an index of the hypothalamus-pituitary-adrenal activity), which indicate the absence of neuroendocrine dependence on the kappa agonist. As has been proposed, this would suggest that the mu and the kappa receptor be regulated through different cellular mechanisms, as kappa agonists have a lower proclivity to induce dependence.

Identification of a κ-opioid agonist as a potent and selective lead for drug development against human African trypanosomiasis.

A resazurin-based cell viability assay was developed for phenotypic screening of the LOPAC 1280 'library of pharmacologically active compounds' against bloodstream forms of Trypanosoma brucei in vitro identifying 33 compounds with EC(50) values <1 µM. Counter-screening vs. normal diploid human fibroblasts (MRC5 cells) was used to rank these hits for selectivity, with the most potent (<70 nM) and selective (>700-fold) compounds being suramin and pentamidine. These are well-known antitrypanosomal drugs which demonstrate the robustness of the resazurin cell viability assay. The most selective novel inhibitor was (+)-trans-(1R,2R)-U50,488 having an EC(50) value of 60 nM against T. brucei and 270-fold selectivity over human fibroblasts. Interestingly, (-)-U50,488, a known CNS-active κ-opioid receptor agonist and other structurally related compounds were >70-fold less active or inactive, as were several µ- and κ-opioid antagonists. Although (+)-U50,488 was well tolerated by the oral route and displayed good pharmaceutical properties, including high brain penetration, the compound was not curative in the mouse model of infection. Nonetheless, the divergence of antinociceptive and antitrypanosomal activity represents a promising start point for further exploratory chemistry. Bioinformatic studies did not reveal any obvious candidate opioid receptors and the target of this cytostatic compound is unknown. Among the other potent, but less selective screening hits were compound classes with activity against protein kinases, topoisomerases, tubulin, as well as DNA and energy metabolism.

Sustained antihypertensive effects of chronic administration of two kappa-opioid agonists in spontaneously hypertensive rats.

The ability of two nonpeptide kappa-opioid receptor agonists, U-50, 488H and U-62,O66E (spiradoline), to lower arterial pressure on chronic administration to 12-week-old male spontaneously hypertensive rats (SHRs) and age-matched normotensive Wistar Kyoto (WKY) rats was assessed. Each drug was infused subcutaneously at approximately 9.6 mg/kg/d, over a 14-day period with Alzet osmotic pumps in each strain of rat. Arterial pressures were determined daily in each rat by photoelectric tail-cuff plethysmography. Control rats received similar infusions of 0.9% NaCl. Body weights and water consumption were also recorded daily. Both drugs effected a 10% to 20% sustained lowering of arterial pressure beginning on the second day of infusion, until explantation of the pumps on day 14. Heart rates similarly were decreased by 10% to 15% in SHRs. By contrast, neither drug caused significant decrements in arterial pressure or heart rate in the normotensive WKY group. Control infusions of 0.9% NaCl had no significant effects on arterial pressure or heart rate in either SHRs or WKY rats. U-62,066E, but not U-50,488H, caused sustained increased water consumption in both SHRs and WKY rats, with a greater effect in the SHR strain. This likely was accompanied by a water diuresis. Body weight gains over the 14-day period were similar for both strains of rats treated with U-50,488H, compared with saline-treated controls, but rats of both strains infused with U-62,066E gained significantly less weight than saline controls over the 14-day period. The results are supportive of further experimental evaluations of the potential antihypertensive use of nonpeptide kappa-opioid agonist drugs.

Effect of multiple injections of U-50, 488H, a kappa-opioid receptor agonist, on the activity of nitric oxide synthase in brain regions and spinal cord of mice.

1. The time course of the effects of multiple injections of U-50,488H, a kappa-opioid receptor agonist, and its subsequent termination on its analgesic action and nitric oxide synthase (NOS) activity was determined in the brain regions and spinal cord of the mouse. 2. Male Swiss-Webster mice were rendered tolerant to U-50,488H by twice-daily injections of the drug (25 mg/kg, IP) for 4 days. Vehicle-injected mice served as controls. 3. In tolerant mice, NOS activity was unchanged in brain regions and the spinal cord after treatment with U-50,488H. During abstinence from U-50,488H, NOS activity was found to be increased in the cortex and remainder of the brain, but no change was noted in the cerebellum, midbrain and spinal cord. 4. These studies demonstrate that withdrawal from the short-term treatment with U-50,488H in mice causes induction of NOS in certain brain regions. However, long-term treatment and withdrawal from U-50,488H are not associated with changes in the central NOS activity and indicate a possible adaptation in the NOS activity.

Disability of development of tolerance to morphine and U-50,488H, a selective kappa-opioid receptor agonist, in neuropathic pain model mice.

We examined the analgesic and anti-allodynic effects of morphine and U-50,488H (trans-(+/-)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]-cyclohexyl)-benzeneacetamide methanesulfonate salt), a selective kappa-opioid receptor agonist, and the development of tolerance to their effects in neuropathic pain model mice induced by sciatic nerve ligation (SNL). In the tail-pinch method, morphine at 10 mg/kg, s.c. produced a weak analgesic effect in SNL mice; however, U-50,488H at 5 mg/kg, s.c. produced an analgesic effect equipotent to that in normal mice. In contrast, morphine produced an adequate analgesic effect when given either intracerebroventricularly (i.c.v.) or intrathecally (i.t.), but U-50,488H only produced analgesia when given i.t. Repeated administration of morphine (either i.c.v. or i.t.) or U-50,488H (either s.c. or i.t.), did not induce tolerance to the effect. In the static allodynia test with an application of von Frey filaments, both compounds given s.c. suppressed the allodynic effect, but in the dynamic allodynia test involving lightly stroking the plantar surface with a cotton bud, only U-50,488H produced an anti-allodynic effect. Repeated administrations of both compounds did not develop tolerance to these anti-allodynic effects. Thus, U-50,488H was found to be a highly effective at blocking hyperalgesia and allodynia in nerve injury, and these findings suggest that kappa-opioid receptor agonists are attractive pharmacological targets for the control of patients with neuropathic pain.