Intrathecal delivery of a mutant micro-opioid receptor activated by naloxone as a possible antinociceptive paradigm.

Direct injection of double-stranded adeno-associated virus type 2 (dsAAV2) with a mu-opioid receptor (MOR) mutant [S4.45(196)A], and a reporter protein (enhanced green fluorescent protein) into the spinal cord (S2/S3) dorsal horn region of ICR mice resulted in antinociceptive responses to systemic injection of opioid antagonist naloxone without altering the acute agonist morphine responses and no measurable tolerance or dependence development during subchronic naloxone treatment. To develop further such mutant MORs into a therapeutic agent in pain management, a less invasive method for virus delivery is needed. Thus, in current studies, the dsAAV2 was locally injected into the subarachnoid space of the spinal cord by intrathecal administration. Instead of using the MORS196A mutant, we constructed the dsAAV2 vector with the MORS196ACSTA mutant, a receptor mutant in which naloxone has been shown to exhibit full agonistic properties in vitro. After 2 weeks of virus injection, naloxone (10 mg/kg s.c.) elicited antinociceptive effect (determined by tail-flick test) without tolerance (10 mg/kg s.c., b.i.d. for 6 days) and significant withdrawal symptoms. On the other hand, subchronic treatment with morphine (10 mg/kg s.c., b.i.d.) for 6 days induced significant tolerance (4.8-fold) and withdrawal symptoms. Furthermore, we found that morphine, but not naloxone, induced the rewarding effects (determined by conditioned place preference test). These data suggest that local expression of MORS196ACSTA in spinal cord and systemic administration of naloxone has the potential to be developed into a new strategy in the management of pain without addiction liability.

Neither single-marker nor haplotype analyses support an association between the dopamine transporter gene and heroin dependence in Han Chinese.

Much evidence suggests that dysfunction of dopamine transporter-mediated dopamine transmission may be involved in the pathophysiology of substance abuse and dependence. The aim of this study was to examine whether the dopamine transporter gene (DAT1; SLC6A3) is associated with the development of heroin dependence (HD) and whether DAT1 influences personality traits in patients with HD. Polymorphisms of DAT1 were analyzed in a case-control study of 1046 Han Chinese (615 patients and 431 controls). All participants were screened using a Chinese version of the modified Schedule of Affective Disorder and Schizophrenia-Lifetime and all patients met the criteria for HD. Furthermore, a Chinese version of the Tridimensional Personality Questionnaire (TPQ) was used to assess personality traits in the patient group and examine the association between their personality traits and DAT1 polymorphisms. Of the patient group, 271 completed the TPQ. No statistically significant differences in allele or genotype frequencies of all investigated variants between HD patients and controls were observed. In haplotype analyses, four haplotype blocks of DAT1 were not associated with the development of HD. These DAT1 polymorphisms did not influence novelty seeking and harm avoidance scores in HD patients. This study suggests that the DAT1 gene may not contribute to the risk of HD and specific personality traits in HD among the Han Chinese population.

Prenatal morphine alters the synaptic complex of postsynaptic density 95 with N-methyl-D-aspartate receptor subunit in hippocampal CA1 subregion of rat offspring leading to long-term cognitive deficits.

Infants who are passively exposed to morphine or heroin through their addicted mothers usually develop neurobiological changes. The postsynaptic density 95 (PSD-95) protein, a submembranous cytoskeletal specialization, is dynamically linked with N-methyl-d-aspartate receptors (NMDARs) to form a synaptic complex in postsynaptic neurons. This complex serves important neurobiological functions, including mammalian learning and memory. However, the effects of prenatal morphine exposure on this synaptic complex are not well understood. In this study, we determined whether prenatal morphine exposure altered the synaptic complex association between PSD-95 and three major NMDAR subunits (NR1, NR2A, and NR2B), at the mRNA and protein levels, within the hippocampal CA1 subregion (an important integration area for mammalian learning and memory) of rat offspring along with the performance of long-term cognitive functions. Sprague-Dawley rat offspring from morphine-addicted mothers were studied at a younger age (postnatal day 14; P14) and at an older age (P45). Subsequently, an eight-arm radial maze task was applied to analyze the working and cued reference memory in such offspring (P45). The real-time polymerase chain reaction results showed that prenatal morphine exposure caused significant decreases in mRNA levels of the PSD-95 and three NMDAR subunits (NR1, NR2A, and NR2B) in offspring (P14 and P45). Similarly, at the protein level, immunoblotting showed that decreased whole levels of PSD-95 and NMDAR subunits were seen in offspring subjected with prenatal morphine. Furthermore, the protein interaction of the synaptic complex between the PSD-95 and NMDAR subunit, as indicated by coimmunoprecipitation, was less in prenatal morphine samples than in vehicle controls (P14 and P45). The prenatal morphine group also showed poorer performance for an eight-arm radial maze task than the vehicle-control group. These results are particularly important for a better understanding of certain opioid-mediated neurobehavioral cognitive changes in offspring associated with altered protein interaction between PSD-95 and NMDAR subunits within the developing brain.

Amitriptyline induces nuclear transcription factor-kappaB-dependent glutamate transporter upregulation in chronic morphine-infused rats.

We previously showed that intrathecal co-administration of amitriptyline with morphine upregulates the expression of the glial glutamate transporters glutamate-aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) and restores neuronal glutamate transporter excitatory amino acid carrier 1 (EAAC1) expression in chronically morphine-infused rats. The present study examined the role of nuclear transcription factor-kappaB (NF-kappaB) in the regulation of the expression of GLAST, GLT-1, and EAAC1 following long-term amitriptyline/morphine co-infusion. Male Wistar rats were implanted with two intrathecal catheters with or without a microdialysis probe; one of the catheters was used for continuous infusion of saline (control), morphine (15 microg/h), or morphine plus amitriptyline (both 15 microg/h) for 5 days, while the other was used for a single daily intrathecal injection of the NF-kappaB inhibitor Ro106-9920 (10 microl of 10 microM) for 5 days. We found that amitriptyline co-infusion restored the antinociceptive effect of morphine (4.5-fold right-shift in the morphine dose-response curve compared with a 65-fold right-shift in its absence) and this effect was inhibited by Ro106-9920 administration (48-fold right-shift). Moreover, amitriptyline/morphine co-infusion increased IkappaBalpha phosphorylation and the translocation of NF-kappaB p65 from the cytosol to the nucleus. Daily intrathecal injection of Ro106-9920 prevented the amitriptyline/morphine-induced NF-kappaB p65 translocation and reversed the amitriptyline/morphine-induced GLAST and GLT-1 upregulation and inhibited the restoration of EAAC1 expression. The Ro106-9920 injections abolished the inhibitory effect of amitriptyline on the morphine-evoked release of excitatory amino acids into the spinal cerebrospinal fluid (CSF) dialysates. In conclusion, amitriptyline/morphine co-infusion restores the antinociceptive effect of morphine and upregulates GLAST and GLT-1 expression and restores EAAC1 expression to baseline levels, thus reducing excitatory amino acid levels in the spinal CSF dialysates. The mechanism involves activation of the NF-kappaB pathway, but may also involve other pathways.

dsAAV type 2-mediated gene transfer of MORS196A-EGFP into spinal cord as a pain management paradigm.

We previously reported that mutations in the mu-opioid receptor (MOR), S196L or S196A, rendered MOR responsive to the opioid antagonist naloxone without altering the agonist phenotype. Subsequently, a mouse strain carrying the S196A mutation exhibited in vivo naloxone antinociceptive activity without the development of tolerance. In this study we investigated the possibility of combining the in vivo site-directed delivery of MORS196A and systemic naloxone administration as a paradigm for pain management. Double-stranded adenoassociated virus type 2 (dsAAV2) was used to deliver MORS196A-EGFP by injecting the virus into the spinal cord (S2/S3) dorsal horn region of ICR mice. MORS196A-EGFP fluorescence colocalized with some calcitonin gene-related peptide and neuron-specific protein immunoreactivity in the superficial layers of the dorsal horn 1 week after injection and lasted for at least 6 months. In mice injected with the mutant receptor, morphine induced similar antinociceptive responses and tolerance development or precipitated withdrawal symptoms and reward effects, similar to those in the control mice (saline injected into the spinal cord). Conversely, in the dsAAV2-injected mice, naloxone produced antinociceptive effects at the spinal level but not at the supraspinal level, whereas naloxone had no measurable effect on the control mice. Furthermore, the chronic administration of naloxone to mice injected with dsAAV2-MORS196A-EGFP did not induce tolerance, dependence, or reward responses. Thus, our current approach to activate a mutant receptor, but not the endogenous receptor, with an opioid antagonist represents an alternative to the use of traditional opioid agonists for pain management.

Endomorphin-1 and -2 induce naloxone-precipitated withdrawal syndromes in rats.

In 1997, endomorphin-1 (EM-1) and -2 (EM-2) were identified as the most specific endogenous mu-opioid ligands. These two peptides have shown analgesic effects and many other opioid functions. In the present study, we attempt to investigate the possible ability of endomorphins to induce naloxone-precipitated withdrawal in comparison with that induced by morphine. Using the previously established scoring system in rats, 12 withdrawal signs (chewing, sniffing, grooming, wet-dog shakes, stretching, yawning, rearing, jumping, teeth grinding, ptosis, diarrhea, and penile erection) were observed and scored following naloxone (4 mg/kg, i.p.) challenge. Compared with the sham control, EM-1 and EM-2 (20 microg, i.c.v., b.i.d. for 5 days) both produced significant naloxone-induced withdrawal syndromes with similar severity to that induced by the same dose of morphine. There was no significant difference between EM-1, EM-2, and morphine-treated group for naloxone-induced withdrawal signs, except for grooming. EM-1 and EM-2 induced more grooming than that caused by morphine. Although EM-1 and EM-2 both led to the withdrawal, they displayed different potency for certain signs and suggest their distinct regulations. The present results indicate EM-1 and EM-2 could initiate certain mechanism involved opiate dependence.

Co-administration of dextromethorphan during pregnancy and throughout lactation significantly decreases the adverse effects associated with chronic morphine administration in rat offspring.

In this study, we have focused our investigation of the facts whether co-administration of a NMDA antagonist dextromethorphan (DM) with morphine during pregnancy and throughout lactation could prevent the adverse effects associated with chronic morphine administration in rat offspring. Adult female Sprague-Dawley rats were randomly separated into four groups and were received subcutaneous injection of either saline, morphine, morphine + dextromethorphan or dextromethorphan twice a day and progressively increased 1 mg/kg at 7-day intervals from a beginning dose of 2 mg/kg for both morphine and dextromethorphan. The rats were mated between days 7 and 8. Administration of drugs was continued during pregnancy. After rat offspring were born, the doses of morphine or dextromethorphan injected into the maternal rats were increased by 1 mg/kg every two weeks till the offspring were 30 day old. The results showed that mortality of morphine group is much higher than control group. The offspring of morphine group weighed significantly less than control group on postnatal day 14 (p14), p30 or p60. The antinociceptive effect of morphine on p14 rats was reduced in the morphine group and indicated the development of morphine tolerance. The hippocampal NMDA receptor densities have been shown decreased on p14 rats. The precipitated withdrawal symptoms were assessed on p7 rats. Rats in morphine group showed greater frequency of abdominal stretch and wet dog shake in 2 hr than control group. On the other hand, co-administration of DM with morphine effectively prevented all these adverse effects of morphine to the offspring rats. DM co-administered with morphine also partially prevented the development of morphine tolerance in maternal rats. If this effect of dextromethorphan is applied to clinical pregnant patients with morphine addiction or chronic pain, it will have a great value for the benefit of their children.

Synergistic apoptosis induced by bacterial endotoxin lipopolysaccharide and high glucose in rat microglia.

The present study investigates whether bacterial lipopolysaccharide (LPS) in the presence of increased levels of glucose induced synergistic cytotoxicity in primary cultured microglia. Significant cytotoxicity was only observed while the concentrations of LPS were increased to 10 microg/ml. D-glucose concentration-dependently (25-125 mM) generated cytotoxicity. Synergistic apoptosis of microglia was seen by LPS in the presence of increased levels of D-glucose. This synergistic cytotoxicity was attenuated by the use of superoxide dimutase and catalase, suggesting the involvement of oxidative free radicals. Collectively, the present results suggest that increased ambient levels of glucose rendered microglia vulnerable to LPS insults, and led to a synergistic apoptosis. The findings here may be important in certain patho-physiological implications in which hyperglycemia exacerbated the ambient functions contributed by microglia, and may provide new insight into a novel therapeutic intervention.

Morphine tolerance increases [3H]MK-801 binding affinity and constitutive neuronal nitric oxide synthase expression in rat spinal cord.

N-Methyl-D-aspartate (NMDA) receptor antagonists and nitric oxide synthase (NOS) inhibitors inhibit morphine tolerance. In the present study, a lumbar subarachnoid polyethylene (PE10) catheter was implanted for drug administration to study alterations in NMDA receptor activity and NOS protein expression in a morphine-tolerant rat spinal model. Antinociceptive tolerance was induced by intrathecal (i.t.) morphine infusion (10 micrograms h-1) for 5 days. Co-administered (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) (10 micrograms h-1 i.t.) with morphine was used to inhibit the development of morphine tolerance. Lumbar spinal cord segments were removed and prepared for [3H]MK-801 binding assays and NOS western blotting. The binding affinity of [3H]MK-801 was higher in spinal cords of morphine-tolerant rats (mean (SEM) KD = 0.41 (0.09) nM) than in control rats (1.50 (0.13) nM). There was no difference in Bmax. Western blot analysis showed that constitutive expression of neuronal NOS (nNOS) protein in the morphine-tolerant group was twice that in the control group. This up-regulation was partially prevented by MK-801. The results suggest that morphine tolerance affects NMDA receptor binding activity and increases nNOS expression in the rat spinal cord.

Immunohistochemical study of opioid receptors after chronic morphine treatment in rats.

Previously, we have used the biochemical receptor binding method to investigate whether down-regulation of the opioid receptor is a mechanism for morphine tolerance, and we were led to a negative conclusion. In the current study, we further used immunohistochemistry to reinvestigate this issue. Male Sprague-Dawley rats (250-300 g) were chronically treated with morphine s.c. for 2, 4 or 6 days, using an escalating dosage paradigm (5-45 mg), which resulted in a 1.8 to 4.0-fold increase in AD50. Rat brains were removed, frozen, coronally sectioned (14 microm) and processed for mu- or delta-opioid receptor immunohistochemistry using the Avidin-Biotin Complex (ABC) method. No significant decrease in mu-opioid receptor (MOR) immunodensity was found in most of the brain regions, which were enriched with MOR after chronic treatment with morphine except for the anteroventral thalamic nucleus in the ventrolateral part (AVVL). No significant change in delta-opioid receptor (DOR) immunodensity after chronic treatment with morphine was found either. Therefore, our conclusion is that down regulation of opioid receptors may not be an important mechanism for morphine tolerance.

Pre-incisional epidural ketamine, morphine and bupivacaine combined with epidural and general anaesthesia provides pre-emptive analgesia for upper abdominal surgery.

BACKGROUND: Previous studies have shown that N-methyl-D-asparate (NMDA) receptor antagonists provide a pre-emptive analgesic effect in humans. This study investigated the benefits of pre-emptive analgesia for upper abdominal surgery, using pre-incisional epidural ketamine + morphine + bupivacaine (K+M+B) treatment for achieving postoperative pain relief. METHODS: Sixty ASA 1-2 patients scheduled for upper abdominal surgery were allocated to three groups in a randomized, single-blinded study. Patients in the control group (I) received general anaesthesia followed by an infusion of normal saline. Group II and III patients received general anaesthesia with a continuous epidural infusion of 2% lidocaine. Thirty minutes after the incision in groups I and II, an epidural pain control regimen was administered using ketamine (10 mg) and morphine (1 mg) in 10 ml of 0.085% bupivacaine (K+M+B). Group III patients also received K+M+B, but it was administered 10 min after the 2% lidocaine injection and 30 min before skin incision. All patients received an epidural pain control regimen (q12 h) for 3 days after their first injection. Patient-controlled analgesia (PCA) with morphine was used to control subsequent postoperative pain. During the 3-day period following surgery, duration to PCA trigger (h), morphine consumption (mg), pain intensity at rest and when coughing/moving, and analgesic-related adverse effects were recorded. The VAS scale (0-10) was used to assess pain intensity. RESULTS: Median times to first PCA trigger were 1.2 (0.5-2.0) h, 3.0 (0.7-4.2) h, and 4.0 (2.5-7.5) h for groups I, II, and III, respectively. Both the incident and resting pain scores were consistently lower for group III patients than groups I and II. The number of PCA triggers (all attempts/successful triggers) during the day following surgery were 14.0 (3-30)/8.0 (3-24) times, 10.0 (3-23)/6.0 (2-20) times, and 7.0 (3-12)/4.5 (1-10) times for groups I, II, and III. Total morphine consumption for the 3-day observation period was 12.5 (3-42) mg, 10.5 (2-29) mg, and 6.0 (1-20) for groups I, II, and III, respectively. CONCLUSION: Pre-incisional epidural K+M+B treatment combined with continuous epidural anaesthesia and general anaesthesia provides an ideal pre-emptive analgesic therapy, exhibiting better postoperative pain relief than general anaesthesia and post-incisional K+M+B treatment.

Prenatal exposure to morphine alters kinetic properties of NMDA receptor-mediated synaptic currents in the hippocampus of rat offspring.

Whole-cell patch-clamp recordings of pharmacologically isolated N-methyl-D-asparate (NMDA) receptor-mediated evoked excitatory postsynaptic currents (EPSCs) were made, to study whether prenatal exposure to morphine affected functional properties of synaptic NMDA receptors in hippocampal slices of 2-week-old rat offspring from morphine-addicted mothers. The saturated amplitude of synaptic NMDA receptor-mediated EPSCs from morphine-treated offspring was about twofold larger than that from vehicle-control offspring. The apparent dissociation constant (Kd) values of NMDA receptors for Mg2+ at 0 mV were 7.5 +/- 1.4 and 7.9 +/- 1.3 mM in slices from vehicle-control and morphine-treated offspring, respectively. In addition, no distinguishable changes in the voltage-dependent nature and the reversal potential of NMDA receptors occurred in morphine-treated offspring, suggesting no alterations of Mg2+ blockade and ion selectivity to NMDA receptors. The 10-90% rise times of NMDA receptor-mediated EPSCs in morphine-treated offspring became longer than those in vehicle-control offspring. The decay of NMDA receptor-mediated EPSCs in both morphine-treated and vehicle-control offspring could be described by the sum of a fast and a slow exponential function. The slow, but not fast, decay times of synaptic NMDA receptor-mediated currents in morphine-treated offspring became slower than those in vehicle-control offspring. Collectively, these results suggest that prenatal exposure to morphine altered kinetic properties of synaptic NMDA receptors in hippocampal CA1 pyramidal neurons of rat offspring during early life. The extended duration of synaptic NMDA receptor-mediated currents presumably provided more Ca2+ entry through NMDA receptors in morphine-treated offspring, and its further prolongation by depolarization in such young offspring strengthened NMDA receptor-dependent functions. Thus, in light of pathophysiological implications within the central nervous system of morphine-treated offspring during early life, the present study may provide important insights and serve as a basis for therapeutic intervention in conditions under which NMDA receptors become abnormal.

Study the mechanisms of U-50,488 to prevent the development of morphine tolerance in guinea pigs.

Previously we have shown that low dose of [trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide hydrochloride] (U-50,488) could prevent the development of morphine tolerance in guinea pigs. In the present study we tried to investigate the role of glutamate and nitric oxide in this process. Male Hartley guinea pigs (200-300 g) were chronically treated s.c. with either saline or morphine (15 mg/kg) or morphine + U-50,488 (0.003 mg/kg) twice a day for 7 days. Antinociceptive activity was assessed by hot-plate test on the first, fourth and seventh day. Spinal cord slices (450 microm) were prepared 30 min after drug treatment on eighth day and [3H] glutamate and nitric oxide (NO) released were determined. We found that coadministration of U-50,488 (0.003 mg/kg) suppressed the development of morphine tolerance to antinociceptive effect as we reported before. The percentage of in vitro spinal release of [3H] glutamate by 100 microM morphine was significantly higher in the chronic morphine group than the control group. On the other hand, coadministration of U-50,488 with morphine for 7 days blocked this effect significantly. The basal NO level released from the spinal cord slices was significantly higher in chronic morphine group but not in chronic (morphine + U-50,488) group. In vitro morphine (100 microM) increased the NO level in control group and chronic (morphine + U-50,488) group and also further increased NO in chronic morphine group. From the NMDA-displaced [3H] glutamate binding in guinea pig spinal cord, we found that the Bmax decreased in chronic morphine group but not in the chronic (morphine + U-50,488) group. In conclusion, chronic morphine treatment may activate the NMDA receptors by increasing the release of glutamate which causes the increase of synthesis and release of NO and following uncertain mechanisms to induce the development of morphine tolerance. And the mechanisms of U-50,488 to prevent the development of morphine tolerance may involve the inhibition of glutamate released by chronic morphine and also the decrease of NO induced by chronic morphine.

The role of nitric oxide in the development of morphine tolerance in rat hippocampal slices.

In the present study, we investigated the effects of a nitric oxide (NO) precursor, L-arginine, on the effect of different drugs, [trans-3, 4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamid e hydrochloride] (U-50,488, a kappa-opioid receptor agonist); dPTyr(Me)AVP (a vasopressin receptor antagonist); dizocilpine (MK-801, a N-methyl-D-aspartate (NMDA) receptor antagonist), to block the development of morphine tolerance or NO release in Sprague-Dawley rat hippocampal slices (450 microm). Slices were continuously superfused with artificial cerebrospinal fluid (ACSF) or drugs at 1 ml/min. Nichrome wire electrodes were placed in the Schaffer-collateral pathway and used to deliver biphasic 0.2-ms pulses of 5-30 V (0.033 Hz). A glass microelectrode was placed in the CA1 area to record population spikes. The amount of NO released in the superfusate was measured as nitrite formation. When the slices were superfused with 10 microM morphine, the amplitude of population spikes increased 200%-300% in 30-40 min. However, this effect of morphine decreased, i.e., tolerance developed, after continuous superfusion of morphine for 2-6 h. On the other hand, the nitrite level was increased about 250% of the control level through 6 h of morphine superfusion. Co-superfusion of L-arginine with morphine could further increase the nitrite level and also facilitate the development of morphine tolerance. On the other hand, 3-Br-7-nitroindazole (a neuronal NO synthase inhibitor) decreased the nitrite level significantly and blocked the development of morphine tolerance. When either U-50,488 (200 nM) or dPTyr(Me)AVP (500 pM) or MK-801 (500 pM) was co-superfused with morphine (10 microM), the development of morphine tolerance was blocked significantly and the nitrite level decreased to 100%-150% of the control level. L-arginine (500 nM) significantly reversed the effect of these drugs to block the development of morphine tolerance or to decrease the nitrite level through 6 h of superfusion. These data suggest that NO may play a key role in the development of morphine tolerance. Drugs which suppress the synthesis or release of NO would be expected to block the development of morphine tolerance.

Preincisional dextromethorphan decreases postoperative pain and opioid requirement after modified radical mastectomy.

PURPOSE: To examine whether preincisional dextromethorphan (DM) improved analgesia after modified radical mastectomy (MRM). METHODS: Sixty patients (ASA I-II) scheduled for MRM were included and randomly allocated into two groups. Patients in the treatment group (DM) received 40 mg DM and 20 mg chlorpheniramine maleate (CPM) i.m., and those in the control group received 20 mg CPM i.m. alone 30 min before skin incision. Meperidine, 1 mg x kg(-1) i.m., was given for postoperative pain relief as required. The time to first meperidine injection, total meperidine consumption, worst pain score, bed-rest time, and side effects were recorded every 24 hr for 48 hr after surgery by a resident anesthesiologist on a double-blind basis. RESULTS: A longer time to first meperidine injection (19.2 +/- 1.6 vs 1.5 +/- 0.23 hr, P < 0.001) and lower meperidine consumption (0[10] vs 75[50] mg, median [interquartile range], P < 0.001) were observed in the DM group than in the control group. The bed-rest time was shorter in the DM than in the control group (18.0[4] vs 23.0[19] hr, P < 0.001). No difference was noted in worst VAS pain score. Meperidine-related side effects (nausea, vomiting, pruritus, dizziness, headache) were more frequent in the control (10/30) than in the DM group (3/30, P < 0.05). The number of patients who required meperidine injection for pain relief was lower in the DM (7/30) than in the control group (25/30, P < 0.005). No DM- or CPM-associated side effects were observed. CONCLUSION: Preincisional IM. DM treatment decreased postoperative pain and opioid requirement after MRM surgery.

Studies on quinazolines and 1,2,4-benzothiadiazine 1,1-dioxides. 8.1, 2 synthesis and pharmacological evaluation of tricyclic fused quinazolines and 1,2,4-benzothiadiazine 1,1-dioxides as potential alpha1-adrenoceptor antagonists.

A series of 2-substituted methyl 2,3-dihydroimidazo[1, 2-c]quinazolin-5(6H)-ones (4), 3-substituted methyl 2, 3-dihydroimidazo[1,2-c]quinazolin-5(6H)-ones (5), 3-substituted methyl 2,3-dihydro-5H-thiazolo[2,3-b]quinazolin-5-ones (15a,b), 3-substituted methyl 2,3-dihydroimidazo[2,1-b]quinazolin-5(1H)-ones (16a,b), 3-substituted methyl 2,3-dihydro-1H-imidazo[1,2-b][1,2, 4]benzothiadiazine 5,5-dioxides (33a,b), 2-substituted methyl imidazo[1,2-c]quinazolin-5(6H)-ones (42-45a,b), 3-substituted methyl imidazo[1,2-c]quinazolin-5(6H)-ones (50-53a,b), 3-substituted methyl 5H-thiazolo[2,3-b]quinazolin-5-ones (55-56a,b), and 3-substituted methyl 5-(methylthio)-2,3-dihydroimidazo[1,2-c]quinazoline (57) were synthesized as compound 1conformational rigid congeners for pharmacological evaluation as potential alpha1-adrenoceptor antagonists. Compounds 4, 5, 33a,b, 44a,b, 45a,b, 52a,b, 53a,b, and 57 were found to possess high affinity for the alpha1-adrenoceptor. Compounds 5 and 57 were the most highly selective and potent alpha1 antagonists with Ki = 0.21 +/- 0.02 and 0.90 +/- 0.08 nM, respectively. The S-enantiomers of these two compounds (Ki = 0.13 +/- 0.01 nM for (S)-(-)-5; Ki = 1.0 +/- 0.2 nM for (S)-(+)-57) were 144-200-fold more potent than the R-enantiomers (Ki = 26 +/- 8 nM for (R)-(+)-5; Ki = 144 +/- 23 nM for (R)-(-)-57). Compound 4 showed 8-fold higher affinity to alpha1A-AR better than alpha1B-AR. These compounds possessed weak to no activity against the 5-HT1A receptor.

Immunohistochemical evidence of down-regulation of mu-opioid receptor after chronic PL-017 in rats.

In a previous study, mu-opioid receptor binding was decreased by chronic treatment of rats with a mu-opioid receptor-selective agonist [CH3Phe3, D-Pro4]morphiceptin (PL-017) [Tao, P.L., Lee, H.Y., Chang, L.R., Loh, H.H., 1990. Decrease in mu-opioid receptor binding capacity in rat brain after chronic PL-017 treatment. Brain Res. 526, 270-275]. However, there was a lack of correlation between the time course of receptor down-regulation and the loss of pharmacological effects of the drug. In the current study, we used immunohistochemistry to reinvestigate this issue. Male Sprague-Dawley rats were chronically treated with PL-017 i.c.v. for 1, 3 or 5 days, using an escalating dosage paradigm (0.75-6.0 microg), which resulted in a 1.4 to 32-fold increase in the AD50. Rat brains were removed, frozen, coronally sectioned (14 microm) and processed for mu-, delta- or kappa-opioid receptor immunohistochemistry by the avidin-biotin complex (ABC) method. Significant decreases in OP3 immunodensity were found in many brain regions which are enriched with OP3 after chronic treatment of PL-017. Time-dependent decreases in OP3 were detected and reached a plateau around 3 days of PL-017 treatment. No significant change in OP1 or OP2 immunodensity after chronic treatment with PL-017 was found. Our conclusion is that chronic treatment with PL-017 of rats selectively down-regulates mu-opioid receptors in the brain. This may be an important mechanism for PL-017 tolerance.

Blockade of the development of morphine tolerance by U-50,488, an AVP antagonist or MK-801 in the rat hippocampal slice.

1. In this study, we investigated the effects of different drugs (a kappa-opioid receptor agonist U-50,488, a vasopressin receptor antagonist dPTyr(Me)AVP or an N-methyl-D-aspartate (NMDA) receptor antagonist MK-801) on the development of morphine tolerance in rat hippocampal slices. 2. Hippocampal slices (450 microm) of Sprague-Dawley rats (250-300 g) were used. Slices were continuously superfused with artificial CSF or drugs at 1 ml min(-1). Nichrome wire electrodes were placed in the Schaffer-collateral pathway and used to deliver biphasic 0.2 ms pulses of 5-30 V (0.033 Hz). A glass microelectrode was placed in the CA1 area to record population spikes. 3. When the slices were superfused with 10 microM morphine, the amplitude of population spikes increased 2-3 fold in 30-40 min. However, this effect of morphine decreased, i.e. tolerance developed after continuous superfusion of morphine for 2-6 h. 4. When either U-50,488 (200 nM) or dPTyr(Me) AVP (500 pM) or MK-801 (500 pM) was co-superfused with morphine (10 microM), it significantly blocked the development of morphine tolerance. Nor-BNI (a kappa-opioid receptor antagonist, 200 nM) significantly reversed the inhibitory effect of U-50,488 but not those of dPTyr(Me)AVP or MK-801 on the development of morphine tolerance. 5. These data indicate that kappa-opioid receptors, AVP receptors and NMDA receptors are all involved in the development of morphine tolerance. The suppression of kappa-opioid receptor activity after chronic morphine may occur before the activation of AVP receptors or NMDA receptors during the development of morphine tolerance.

C-alkylated spiro[benzofuran-3(2H),4'-1'-methyl-piperidine-7-ols] as potent opioids: a conformation-activity study.

Among a series of C-alkylated analogs of the weak mu opioid ligand spiro[benzofuran-3(2H),4'-1'-methylpiperidine-7-ol] (1), the 2-methyl, 2-ethyl, and cis 3'-methyl analogs, namely compounds (+/-)2, (+/-)-3, and (+/-)-4, showed much enhanced mu-affinities, with (+/-)-4 being almost as potent as (-)-morphine; while the trans 3'-methyl analog (+/-)-5 remained a weak mu-binder. Energy calculations and nmr data indicated that compounds 2-4 favor phenyl-axial conformations, while compounds 1 and 5 favor phenyl-equatorial conformations.

Alteration in transcripting the gene encoding the delta-opioid receptor in rat brain is not underlying the development of tolerance to [D-Ala2,D-Leu5] enkephalin.

Previous study has demonstrated that chronic treatment of [D-Ala2,D-Leu5] enkephalin (DADLE) induces profound down-regulation of delta opioid receptor in rat brain. We further examined whether this down-regulation of receptor was due to a decrease in the transcription of gene encoding delta-opioid receptor (DOR-1). Rats received daily i.c.v. injection of DADLE for 1, 3, or 5 days and developed significant tolerance to the antinociceptive effect of DADLE after one-day treatment. We measured the level of mRNA in rat brain tissues using in situ hybridization. No significant changes in the mRNA levels of the cortex, striatum, hippocampus, and thalamus on any examined days were found as compared to those of rats received sham operation only. There is only a transient decrease of DOR-1 mRNA level in midbrain region that occurred after a three-day treatment. Thus, the result of this study did not suggest that alteration in transcription of gene-encoding delta-opioid receptor was responsible for the down-regulation of delta-opioid receptor associated with the development of tolerance to DADLE.