Julia Timár

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.

The development of tolerance to locomotor effects of morphine and the effect of various opioid receptor antagonists in rats chronically treated with morphine

Behavioural measures are considered to be highly sensitive indices of opioid withdrawal. Opioids, depending on dose and time protocols may induce both reduction and enhancement of locomotor activity and chronic opioid treatment results in tolerance and sensitisation to these effects. In the present study the locomotor activity as experimental model was used to assess the development of tolerance to subcutaneous morphine challenge at different time points following morphine withdrawal in rats exposed to gradually increasing subcutaneous doses of morphine for 11 days. Tolerance developed to the inhibitory action of morphine (10 mg/kg) was observed even 8 weeks after morphine withdrawal, while tolerance to its locomotor activity enhancing effect (3 mg/kg) was detected 18 h after withdrawal, but not 3 weeks later. In the other series of experiments the locomotor activity of animals exposed to chronic morphine treatment was tested 18 h after spontaneous or subcutaneously administrated opioid antagonists precipitated withdrawal. Spontaneous withdrawal resulted in a moderate decrease of locomotion. Both the non-selective antagonist naloxone in low, mu opioid-receptor selective doses and the delta opioid-receptor selective naltrindole induced marked reduction of locomotor activity. The results provide further evidence that both mu and delta opioid-receptors might be affected during chronic morphine treatment.

Behavioral effects of perinatal opioid exposure.

Opioids are among the worlds oldest known drugs used mostly for pain relief, but recreational use is also widespread. A particularly important problem is opioid exposure in females, as their offspring can also be affected. Adverse intrauterine and postnatal environments can affect offspring development and may lead to various disabilities later in life. It is clear that repetitive painful experiences, such as randomly occurring invasive procedures during neonatal intensive care, can permanently alter neuronal and synaptic organization and therefore later behavior. At the same time, analgesic drugs can also be harmful, inducing neuronal apoptosis or withdrawal symptoms in the neonate and behavioral alterations in adulthood. Hence, risk-benefit ratios should be taken into consideration when pain relief is required during pregnancy or in neonates. Recreational use of opioids can also alter many aspects of life. Intrauterine opioid exposure has many toxic effects, inducing poor pregnancy outcomes due to underdevelopment, but it is believed that later negative consequences are more related to environmental factors such as a chaotic lifestyle and inadequate prenatal care. One of the crucial components is maternal care, which changes profoundly in addicted mothers. In substance-dependent mothers, pre- and postnatal care has special importance, and controlled treatment with a synthetic opioid (e.g., methadone) could be beneficial. We aimed to summarize and compare human and rodent data, as it is important to close the gap between scientific knowledge and societal policies. Special emphasis is given to gender differences in the sensitivity of offspring to perinatal opioid exposure.

Changes in adaptability following perinatal morphine exposure in juvenile and adult rats

The problem of drug abuse among pregnant women causes a major concern. The aim of the present study was to examine the adaptive consequences of long term maternal morphine exposure in offspring at different postnatal ages, and to see the possibility of compensation, as well. Pregnant rats were treated daily with morphine from the day of mating (on the first two days 5mg/kgs.c. than 10mg/kg) until weaning. Male offspring of dams treated with physiological saline served as control. Behavior in the elevated plus maze (EPM; anxiety) and forced swimming test (FST; depression) as well as adrenocorticotropin and corticosterone hormone levels were measured at postpartum days 23-25 and at adult age. There was only a tendency of spending less time in the open arms of the EPM in morphine treated rats at both ages, thus, the supposed anxiogenic impact of perinatal exposure with morphine needs more focused examination. In response to 5min FST morphine exposed animals spent considerable longer time with floating and shorter time with climbing at both ages which is an expressing sign of depression-like behavior. Perinatal morphine exposure induced a hypoactivity of the stress axis (adrenocorticotropin and corticosterone elevations) to strong stimulus (FST). Our results show that perinatal morphine exposure induces long term depression-like changes. At the same time the reactivity to the stress is failed. These findings on rodents presume that the progenies of morphine users could have lifelong problems in adaptive capability and might be prone to develop psychiatric disorders.

Pain-inhibiting inhomogeneous static magnetic field fails to influence locomotor activity and anxiety behavior in mice: No interference between magnetic field- and morphine-treatment

OBJECTIVES We wanted to demonstrate (i) in the writhing test in mice, whether there was a prolonged analgesic effect induced by an inhomogeneous static magnetic field (SMF) exposure; (ii) whether SMF had an effect on the analgesic effect induced by 0.5mg/kgs.c. administered morphine, on the behavioral patterns, and on the hyperlocomotion-inducing effect of morphine. DESIGN A magnetic exposure system developed by the present authors was used with peak-to-peak flux densities in the 2-754mT range. The writhing test was used for the assessment of pain. An elevated plus maze and a Conducta System was used for studying the anxiogenic or anxyolitic effect in mice, and the locomotor activity, respectively. OUTCOME MEASURES We looked for the difference in the number of writhings and in the behavioral patterns between treated (s.c. morphine and/or SMF exposure) and control animals, respectively. RESULTS (i) The antinociceptive effect could be identified 10-30min following SMF exposition in the writhing test in mice. (ii) SMF failed to affect the morphine-induced antinociception, the behavioral patterns in either type of tests, and the hyperlocomotion-inducing effect of morphine. CONCLUSIONS (i) The long-lasting antinociceptive effect of SMF allows experiments under conditions, when in situ application of the SMF-producing device would be technically difficult or impossible; or where it would disturb the experiments. (ii) The results of behavioral tests with freely moving mice in or in the vicinity of inhomogeneous SMFs are not affected by the SMF in the applied flux density range. (iii) Morphine in treated subjects is not interacting with the inhomogeneous SMFs in the applied flux density range.

The central versus peripheral antinociceptive effects of μ-opioid receptor agonists in the new model of rat visceral pain

This study describes the antinociceptive effects of μ-opioid agonists, d-Ala(2),N-Me-Phe(4),Gly(5)-ol-enkephalin (DAMGO) and morphine in a model of rat visceral pain in which nociceptive responses were triggered by 2% acetic acid intraperitoneal (i.p.) injections. DAMGO and morphine were administered i.p., to the same site where acetic acid was delivered or intracerebroventricularly (i.c.v.). The antinociceptive actions of i.p. versus i.c.v. administered DAMGO or morphine were evaluated in the late phase of permanent visceral nociceptive responses. Both compounds inhibited the nociceptive responses in a dose-dependent manner and exhibited more potent agonist activity after i.c.v. than i.p. administration. DAMGO and morphine showed comparable ED(50) values after i.p. injections. However, DAMGO was much stronger than morphine after central administration. Co-administration of the peripherally restricted opioid antagonist, naloxone methiodide (NAL-M), significantly attenuated the antinociceptive effects of i.p. DAMGO or morphine. On the other hand, i.c.v. injections of NAL-M partially antagonized the antinociceptive effect of i.p. morphine and failed to affect the antinociceptive action of i.p. DAMGO indicating the partial and pure peripheral antinociceptive effects of morphine and DAMGO, respectively. These results suggest the role of either central or peripheral μ-opioid receptors (MOR) in mediating antinociceptive effects of i.p. μ-opioid agonists in the rat late permanent visceral pain model which closely resembles the clinical situation.

Differences in some behavioural effects of deprenyl and amphetamine enantiomers in rats

Effects of deprenyl and amphetamine enantiomers on different behavioural patterns were compared. Whereas (+)-amphetamine in doses of 1-3 mg/kg SC, (-)-amphetamine, and (+)-deprenyl in doses of 5-20 mg/kg SC increased the locomotor activity and the time the animals displayed stereotyped head movement, enhanced the acquisition of conditioned avoidance responses, and developed positive place preference conditioning, (-)-deprenyl, even in as high a dose as 20 mg/kg SC, failed to show any amphetamine-type behavioural effect. The results provide further proof why (-)-deprenyl, in contrast to other members of the amphetamine family, can be considered as a safe drug.

Peri, pre and postnatal morphine exposure: Exposure-induced effects and sex differences in the behavioural consequences in rat offspring

This study investigated the behavioural consequences of peri, pre and postnatal morphine (MO) exposure in rats. From gestational day 1 dams were treated with either saline or MO subcutaneously once a day (5 mg/kg on the first 2 days, 10 mg/kg subsequently). Spontaneous locomotor activity in a new environment (habituation) and antinociceptive effects of MO were measured separately in male and female pups after weaning and also in late adolescence or adulthood. The rewarding effect of MO was assessed by conditioned place preference in adult animals. Both exposure-induced and sex differences were observed. A significant delay in habituation to a new environment and decreased sensitivity to the antinociceptive effect of MO were found in male offspring of MO-treated dams. In contrast, the place preference induced by MO was enhanced in the MO-exposed adult animals and this effect was more marked in females. Prenatal exposure to MO resulted in more marked changes than the postnatal exposure through maternal milk. The results indicate that a medium MO dose administered once-daily results in long-term consequences in offspring and may make them more vulnerable to MO abuse in adulthood.

Spinal interaction between the highly selective μ agonist DAMGO and several δ opioid receptor ligands in naive and morphine-tolerant mice.

Since the discovery of opioid receptor dimers their possible roles in opioid actions were intensively investigated. Here we suggest a mechanism that may involve the μ-δ opioid heterodimers. The exact role of δ opioid receptors in antinociception and in the development of opioid tolerance is still unclear. While receptor up-regulation can be observed during the development of opioid tolerance no μ receptor down-regulation could be detected within five days. In our present work we investigated how the selective δ opioid receptor agonists and antagonists influence the antinociceptive effect of the selective μ receptor agonist DAMGO in naïve and morphine-tolerant mice. We treated male NMRI mice with 200 μmol/kg subcutaneous (s.c.) morphine twice daily for three days. On the fourth day we measured the antinociceptive effect of DAMGO alone and combined with delta ligands: DPDPE, deltorphin II (agonists), TIPP and TICPψ (antagonists), respectively, administered intrathecally (i.t.) in mouse tail-flick test. In naive control mice none of the δ ligands caused significant changes in the antinociceptive action of DAMGO. The treatment with s.c. morphine resulted in approximately four-fold tolerance to i.t. DAMGO, i.e. the ED₅₀ value of DAMGO was four times as high as in naive mice. 500 and 1000 pmol/mouse of the δ₁ selective agonist DPDPE enhanced the tolerance to DAMGO while 1000 pmol/mouse of the δ₂ selective agonist deltorphin II did not influence the degree of tolerance. However, both δ antagonists TIPP and TICPψ potentiated the antinociceptive effect of i.t. DAMGO, thus they restored the potency of DAMGO to the control level. The inhibitory action of DPDPE against the antinociceptive effect of DAMGO could be antagonized by TIPP and TICPψ. We hypothesize that during the development of morphine tolerance the formation of μδ heterodimers may contribute to the spinal opioid tolerance. δ ligands may affect the dimer formation differently. Those, like DPDPE may facilitate the dimer formation hence inhibit the antinociceptive effect of DAMGO by causing virtual μ receptor down-regulation. Ligands that do not affect the dimer formation do not influence antinociception either but ligands with the presumed capability of disconnecting the dimers may decrease the spinal tolerance to DAMGO.

A Novel µ-Opioid Receptor Ligand with High In Vitro and In Vivo Agonist Efficacy

The aims of this study were to synthesize 14-O-Methylmorphine-6-O-sulfate (14-O-MeM6SU) and examine its opioid properties (potency, affinity, efficacy) in receptor ligand binding and isolated tissues (mouse vas deferens, MVD and rat vas deferens, RVD bioassays). The results were then compared to the parent compounds morphine-6-O-sulfate (M6SU) and morphine, as well as the �- opioid receptor (MOR) selective agonist peptide [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO). An additional objective was to compare the effect of subcutaneously (s.c.) or intracerebroventricularly (i.c.v.) administered 14-O-MeM6SU, M6SU and morphine in thermal nociception, rat tail-flick (RTF) test. In MVD, the EC50 (nM) value was 4.38 for 14-O-MeM6SU, 102.81 for M6SU, 346.63 for morphine and 238.47 for DAMGO. The effect of 14-O-MeM6SU and DAMGO was antagonized by naloxone (NAL) with Ke value 1-2.00 nM. The Emax values (%) were 99.10, 36.87, 42.51 and 96.99 for 14-O-MeM6SU, M6SU, morphine and DAMGO, respectively. In RVD 14-O-MeM6SU and DAMGO but not M6SU or morphine showed agonist activity. In binding experiments the affinity of 14-OMeM6SU, M6SU, morphine and DAMGO for MOR was 1.12, 11.48, 4.37 and 3.24 nM, respectively. The selectivity of 14-O-MeM6SU was κ/μ= 269 and δ/μ= 9. In G-protein activation experiments, 14-O-MeM6SU and DAMGO showed higher Emax values than M6SU or morphine. S.c. or i.c.v-injected 14-O-MeM6SU, M6SU and morphine produced a dose and time-dependent increase in RTF response latency. 14-O-MeM6SU was the most potent. Our results showed that introduction of 14-O-Me in M6SU increased the binding affinity, agonist potency, and most importantly, the intrinsic efficacy (Emax).