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Background: There is substantial evidence that cholinomimetic drugs increase pain threshold. However, the profound side effects of these agents have limited their clinical use either as analgesics or as analgesic adjuncts. A delivery system that would assure a slow and sustained drug release may be of value in ameliorating the problem of untoward effects. Methods: The acetylcholinesterase inhibitor neostigmine was encapsulated into multilamellar lipid vesicles composed of phosphocholine and cholesterol. Three doses of plain or liposomal neostigmine were administered by the intrathecal route to mice (n=8–10/group), and analgesic duration was quantified by tail flick test. The doses were chosen based on preliminary experiments, which showed the maximum tolerated intrathecal doses of plain and liposomal neostigmine formulation were 0.625 μg and 80 μg, respectively. Two other doses for each formulation were then derived by 1:1 serial dilutions. Results were compared using survival analysis. Results: The median antinociceptive duration for plain neostigmine was 0.33, 0.99 and 1.0 h for the 0.115, 0.312 and 0.625 μg doses, respectively. For the liposomal formulation, the median antinociceptive duration was 1.0, 1.5 and 6.0 h for the 20, 40 and 80 μg doses, respectively. Conclusions: Liposomal neostigmine provides prolonged spinal antinociception, and permits the safe administration of a relatively large dose, because drug is gradually released from the liposomal depot. This technology holds promise for the development of a clinically useful neostigmine formulation to provide spinal analgesia.

We evaluated the intensity of the withdrawal symptoms after the discontinuation of the morphine infusion in rats. Opiate addiction was induced by progressively increasing intraperitoneal morphine infusion rates. The control group (Group 1) received normal saline. The initial morphine rates were 1, 4, and 16 mg kg −1 h for Groups 2, 3, and 4, respectively. Infusion rates were gradually increased by a factor of 1.4, 2, 2.8, and 4 on the second, third, fourth, and fifth days, respectively. The last rate was used for 48 h and then infusions were disconnected. Weight reduction, food consumption, and water intake were used for evaluation of withdrawal. All morphine groups showed a significant reduction of body weight during the 4 postdiscontinuation days and a decline in food and water intake on the first postdiscontinuation day. All changes were dependent on the morphine infusion concentration. No changes were observed in the control group. We suggest that the rat model used in this study may be utilized for quantification of spontaneous withdrawal.

Purpose. To evaluate the dehydration-rehydration technique to prepare a formulation of liposomal bupivacaine, and to assess its analgesic efficacy.

Exposure to opioids results in the activation of opioid receptors; this is follow ed by receptor endocytosis. Previously, we showed that delta opioid receptors undergo rapid agonist-mediated internalization and that mutations in the C-tail result in a substantial loss of agonist-mediated internalization. In this study, we investigated the fate of receptors following rapid internalization. We found that the majority of the wild type receptors recycled back to the surface after acute agonist treatment. The kinetics of internalization and recycling of the receptor were virtually identical to the kinetics of internalization and recycling of the radiolabeled agonist. In contrast, the kinetics of internalization and recycling of a C-tail mutant receptor were substantially altered, suggesting an involvement of the C-tail in the recycling process. It is possible that in addition to agonist-mediated internalization, opioid receptors undergo constitutive, agonist-independent internalization. We directly examined this possibility using an antibody-prebinding assay. The wild type delta opioid receptors exhibited agonist-independent internalization via the clathrin-coated pit pathway. We also examined the role of receptor internalization and recycling in the modulation of its function by quantitating the level of opioid-stimulated phosphorylation of MAP kinase (MAPK) under conditions of receptor internalization and recycling. We found that agonist treatment caused a rapid increase in the level of phosphorylated MAPK that was rapidly desensitized. The removal of the agonist, which results in receptor recycling, led to the resensitization of the receptor, as evidenced by the agonist’s ability to reinduce MAPK phosphorylation. Mutant receptors that underwent rapid recycling exhibited enhanced resensitization, suggesting a role for receptor recycling in the re-sensitization process. Taken together, these results indicate that agonist-mediated internalization and recycling modulate opioid receptor function and that the receptor C-tail plays an important role in both processes.

A sensitive high performance liquid chromatographic (HPLC) assay was developed to quantitate the organic amine proton-acceptor, tris(hydroxymethyl)aminomethane (THAM) in human plasma, erythrocytes,...

To elucidate the biochemical mechanisms of spinal anesthesia, we studied the effects of procaine and tetracaine on protein phosphorylation in the mouse spinal cord. Mice were injected intrathecally with either procaine, tetracaine (67 mM/approximately 2%, 10 microL, N = 5/drug), or saline (N = 4/group). Five minutes after injection, animals were killed with a guillotine, and the spinal cord was removed. The caudal 3-cm cord segment was homogenized and centrifuged, and an aliquot of the supernatant was used for phosphorylation assays. Calcium-dependent phosphorylation was initiated by incubating the samples in buffer containing [gamma-32P]ATP at 37 degrees for 30 min. The proteins were electrophoresed using slab gel and two-dimensional electrophoresis, and phosphorylated proteins were visualized by autoradiography. The data demonstrated that spinal anesthesia changes the phosphorylation state of five endogenous substrate proteins with apparent molecular masses of 130 (protein-a), 105 (protein-b), 55 (protein-c), 47 (protein-d), and 33 (protein-e) kDa. In two-dimensional electrophoresis, protein-a resolved into two proteins (a1 and a2). Analysis of variance of the densitometric data suggested a significant effect for the treatment (F(2,16) 735, P < 0.00005). Post hoc comparisons with the saline-treated controls, using the Newman-Keuls test, indicated that local anesthetics significantly affected phosphoproteins (P < 0.05) except for protein-al in the tetracaine-treated group. Further characterization of these phosphoproteins should aid in determining their role in the signal transduction cascade affected by spinal anesthesia.

N omega-nitro-L-arginine (NG-nitro-L-arginine) is a potent nitric oxide synthase inhibitor which crosses the blood brain barrier and does not undergo extensive metabolism in vivo. In this study, effect of chronic pretreatment of N omega-nitro-L-arginine (75 mg/kg, i.p., twice daily for 7 days) on the harmaline- (100 mg/kg, s.c.), picrotoxin- (4 mg/kg, s.c.), pentylenetetrazole- (50 mg/kg, i.p.), and L-glutamic acid- (400 micrograms/10 microliters/mouse, i.c.v.) induced increase in cerebellar cGMP was assessed. All the four drugs produced significant increase in cerebellar cGMP in vehicle pretreated control animals. Cerebellar cGMP increased induced by harmaline, picrotoxin, and L-glutamic acid was attenuated in N omega-nitro-L-arginine pretreated animals. These results indicate that in vivo cerebellar cGMP levels are increased by the prototype excitatory amino acid receptor agonist, L-glutamic acid and also by the drugs which augment the excitatory amino acid transmission. Furthermore, parenteral chronic administration of N omega-nitro-L-arginine blocks NO synthase in the brain and hence cerebellar cGMP response in chronic N omega-nitro-L-arginine treated animals could be used as a tool to assess the physiological functions of nitric oxide in vivo.

In the brain, nitric oxide (NO) has been identified as a messenger molecule and a mediator of excitatory amino acid-induced neurotoxicity. In this study, the effects of NO on serum-induced mitogenesis and cell proliferation of the cerebellar glial cells were assessed. NO-generating agent, S-nitroso-N-acetylpenicillamine (SNAP) increased intracellular cyclic guanosine monophosphate (cGMP) levels. Furthermore, 2 chemically dissimilar NO-generating agents, SNAP and sodium nitroprusside (SNP) inhibited serum-induced thymidine incorporation and cell proliferation. The antimitogenic effect of NO was mimicked by 8-bromo-cGMP and blocked by hemoglobin, a known inhibitor of NO. The effect of NO was not cytotoxic, since the cells were not stained with Trypan blue and did not show increased release of lactate dehydrogenase in the culture supernatants. However, NO-treated cells showed decreased conversion of tetrazolium to blue formazan suggesting that NO inhibited mitochondrial activity in the glial cells. These results demonstrate that NO inhibits serum-induced mitogenesis and cell proliferation of cultured rat cerebellar glial cells.

Intracerebroventricular (ICV) administration of kappa-agonists (PD 117302, U-50488H and U-69593) induced convulsions in a dose-related manner in mice. The dose at which 50% of animals convulsed (CD50) was in nmol ranges for all opioids. Among the opioids used, PD 117302 was the most potent convulsant. ICV administration of either vehicle alone or U-53445E, a non-kappa-opioid (+) enantiomer of U-50488H did not induce convulsions. The convulsive response of kappa-agonists was differentially susceptible for antagonism by naloxone and/or MR 2266. Collectively, these findings support the view that convulsions induced by kappa-agonists in mice involve stereospecific opioid receptor mechanisms. Furthermore, the convulsant effect of kappa-agonists could not be modified by pretreatment with MK-801, ketamine, muscimol or baclofen. It is concluded that kappa-opioid but not NMDA or GABA receptor mechanisms are involved in convulsions induced by kappa-agonists. These results are the first experimental evidence implicating stereospecific kappa-receptor mechanisms in opioid-induced convulsions in mice.

The effects of the benzomorphan kappa-opiate antagonist MR 2266 and its dextro enantiomer MR 2267 were assessed on thermonociception in male Swiss Webster mice. Experimental diabetes was induced by injecting streptozotocin (200 mg/kg IP, 7-8 days before). Animals with dextrose treatment (5 g/kg, IP, at the time of opiate injection) were used as acute hyperglycemic controls. Nociception was assessed by supraspinal nociceptive reflex (licking and jumping in hot plate test) indicative of higher cognitive process as well as a predominantly lower spinal monosynaptic reflex (tail immersion test). In normoglycemic, acute hyperglycemic and diabetic mice, MR 2266 decreased, while MR 2267 increased, the reaction latencies. The results indicate tonic stereospecific kappa-opiate receptor-mediated spinal and supraspinal thermonociceptive reactions are not modulated by experimental diabetes and thus are distinct from those of naloxone-sensitive mu-opiate receptor sites.

The effects of kappa-opiate agonists on gastrointestinal motility was assessed in Swiss Webster, C57BL/6, BALB/c and DBA/2 strains of mice. The kappa-agonists, PD 117302, U-69593 and U-50488H (3 mg kg-1), were injected subcutaneously and the distance travelled by a charcoal meal in the gastrointestinal tract was measured. All kappa-agonists induced significant inhibition of charcoal meal transit; however, there were significant strain differences in the antitransit effect. It is concluded that kappa-receptors are involved in the inhibition of gastrointestinal transit and that the negative data reported in the literature may be due to a genotype-dependent effect of kappa-agonists.

The effects of the benzomorphan kappa opiate MR 2266 and its dextro enantiomer MR 2267 were assessed on thermonociception in Swiss Webster, C57BL/6, BALB/c and DBA/2 strains of mice. In the hot plate (60 +/- 0.5 degrees C, cut-off time 120 s and tail immersion tests, MR 2266 (10 mg/kg, s.c., 15 min before) decreased, while MR 2267 (10 mg/kg s.c., 15 min before) increased the reaction latencies. In the hot plate test, the sensitivities for the effects of MR 2266 and MR 2267 on jump latency in different strains of mice were as follows: MR 2266; BALB greater than Swiss greater than C57BL greater than DBA and MR 2267; DBA greater than BALB = Swiss greater than C57BL. In the immersion test, for the hyperalgesic response of MR 2266, the rank order of strains was; BALB greater than C57BL and DBA greater than or equal to Swiss while the rank order for the analgesic effect of MR 2267 was; Swiss greater than DBA and BALB. The results indicate the presence of tonic kappa-receptor-mediated regulation of the spinal and supra-spinal thermonociceptive reactions which is stereospecific and strain dependent.

Background Currently available local anesthetics have relatively brief durations of action. An ultralong-acting local anesthetic would benefit patients with acute and chronic pain. The authors prepared and characterized a novel liposomal bupivacaine formulation using remote loading of bupivacaine along an ammonium sulfate gradient and assessed its efficacy in humans. Methods A large multivesicular liposomal bupivacaine formulation was prepared by subjecting small unilamellar vesicles to successive freeze-and-thaw cycles. Bupivacaine hydrochloride was then remotely loaded into the liposomes along an ammonium sulfate gradient ([(NH4)2SO4)]intraliposome/[(NH4)2SO4)]medium > 1000). The liposomes were then characterized for size distribution; drug-to-phospholipid ratio; in vitro release profile at 4 degree, 21 degree C, and 37 degree C; sterility; and pyrogenicity. Six subjects each received six intradermal injections in the lower back with 0.5 ml of 0.5, 1.0, and 2% liposomal bupivacaine; 0.5% standard bupivacaine; saline; and "empty" liposomes. Duration of analgesia was assessed using pinprick testing of the skin directly over the injection sites. Results were compared using the log-rank test. Results The mean large multivesicular vesicle size was 2439 +/- 544 nm, with a drug-to-phospholipid ratio of 1.8, fivefold greater than results previously reported. In vitro release was slowest at 4 degree C. The median duration of analgesia with 0.5% standard bupivacaine was 1 h. The median durations of analgesia after 0.5, 1.0, and 2.0% liposomal bupivacaine were 19, 38, and 48 h, respectively. Neither saline nor "empty" liposomes produced analgesia. Conclusions This novel liposomal formulation had a favorable drug-to-phospholipid ratio and prolonged the duration of bupivacaine analgesia in a dose-dependent manner. If these results in healthy volunteers can be duplicated in the clinical setting, this formulation has the potential to significantly impact the management of pain.

1. The objective of the present study was to assess the time-course profile of analgesia and bupivacaine concentrations at the site of injection after subcutaneous administration of a single dose of standard bupivacaine or a novel controlled-release liposomal bupivacaine formulation. 2. Groups of mice were injected subcutaneously with 0.2 mL of 0.5% standard bupivacaine or 0.5, 1 or 2% liposomal bupivacaine. 3. A prolonged duration of analgesia occurred in mice receiving liposomal bupivacaine. In the liposomal groups, the bupivacaine remained at the injection site for more than 96 h, compared with approximately 8 h in groups injected with standard bupivacaine. 4. These results confirm that the prolonged analgesia observed after injection of the liposomal formulation is associated with sustained higher levels of bupivacaine at the site of injection.

We describe a mouse model for evaluation of skin anesthesia after infiltration of local anesthetic. The method involves subcutaneous injection of the anesthetic over the abdomen, and monitoring the vocalization response to electrical stimulus as a measure of analgesia. Prior to drug injection, the vocalization threshold was determined. Mice that vocalized at < or = 8 mA were included in the study. The model was tested using representative agents of the two classes of local anesthetics, bupivacaine, an amide, and chloroprocaine, an ester. The time course and dose response were assessed after injection. The median analgesic time was 15, 40, and 55 min for 0.015%, 0.0625%, and 0.25% bupivacaine and 30, 50, and 55 min for 0.125%, 0.25%, and 2.0% chloroprocaine, respectively. Statistical analysis of the data showed that this method is sufficiently sensitive to detect differences between the dose and duration of local anesthesia (p

Background The in vitro adaptive responses of delta opiate receptors (DOR) to chronic ethanol treatment have been well documented. The acute effects of ethanol on these receptors are not well characterized beyond its effect on ligand binding. The aim of this study was to evaluate the acute effects of clinically relevant concentrations of ethanol (50-200 mm) on the saturation binding kinetics, receptor/ligand internalization, and agonist stimulation of G-protein coupling in N18TG2 cells expressing the Flag epitope-tagged mouse DOR. Methods Confocal microscopy was used to localize Flag epitope-tagged DOR in N18TG2 cells. Saturation binding assays at 4 degrees C and 37 degrees C were conducted in the absence or presence of ethanol on cells not pretreated or pretreated with ethanol for 30 min at 37 degrees C. Highly specific delta agonist, DPDPE ([D-Pen2,D-Pen5]enkephalin), was used in these studies. The effect of ethanol on agonist stimulation of G-protein coupling was examined using [35S]GTPgammaS (guanosine-5'-O-(3-thio)triphosphate) binding to membranes. Agonist-mediated receptor internalization was examined using flow cytometry of cells labeled with the antiserum directed against the Flag epitope, and the ligand internalization was examined using [3H]DPDPE. Results Ethanol decreased the binding of the agonist [3H]DPDPE, and not the antagonist [3H]diprenorphine, in a dose-dependent manner. These effects were temperature-dependent. Ethanol reversibly inhibited agonist stimulation of [35S]GTPgammaS binding. In non-pretreated cells, ethanol decreased the rate of receptor/ligand internalization, but this effect was not seen in ethanol pretreated cells. Taken together, these results suggest that pretreatment of N18TG2 cells with ethanol induces compensatory mechanisms that allow the receptor to function efficiently in its presence. Conclusion Acute ethanol decreased the binding, agonist-mediated functional coupling and receptor/ligand internalization in N18TG2 cells expressing epitope-tagged DOR. In these cells, 30-min pretreatment with ethanol was sufficient to reverse these effects.

To understand the biochemical mechanisms involved in spinal anesthesia, we measured protein kinase C (PKC) activity and expression of immediate early oncogene protein, c-Fos, in the spinal cord. Spinal anesthesia was induced in mice using intrathecal injection of either 10 microL procaine or tetracaine (0.067 M/approximately 2%). Control groups were treated with either saline or ethanol. Animals were killed at 1, 5, and 15 min after the injection and the caudal 3 cm of the spinal cord was processed for biochemical analysis. PKC activity was measured by the transfer of a phosphate group from [gamma-32P]adenosine 5'-triphosphate to the threonine group on a synthetic peptide specific for PKC. Western blot analysis was used to detect changes in c-Fos protein expression. When compared to saline-treated controls, PKC activity was increased significantly (P < 0.0005) in procaine- and tetracaine-treated groups whereas ethanol decreased PKC activity. The less lipid-soluble procaine produced a larger increase in PKC activity than did the more lipid-soluble tetracaine. Moreover, parallel to the effect on PKC activity, procaine was more potent than tetracaine as a c-Fos inducer. These results implicate some role for a PKC- and c-Fos-dependent pathway in the mechanism of spinal anesthesia. However, these results also demonstrate a lack of correlation between an increase in PKC levels and either potency or lipid solubility of the anesthetics. The increased PKC activity may not be the sole mechanism for spinal anesthesia. These data on the effects of local anesthetics on PKC activity and c-Fos in vivo are of relevance for studies aimed at delineating the biochemical basis of spinal and epidural anesthesia.

Background Propofol has been used to treat status epilepticus, but its use in patients with seizure disorders remains controversial, because of concerns that it produces paroxysmal motor phenomenon. Chemoconvulsants act by known discrete mechanisms and neurotransmitters, and therefore, they are useful tools for screening anticonvulsant activity. The main objective of this study was to characterize the effect of propofol pretreatment on convulsions induced by picrotoxin, bicuculline, and strychnine, all which decrease inhibitory neurotransmission, and by N-methyl-D-aspartic acid, kainic acid, and quisqualic acid, which enhance excitatory neurotransmission. Methods Groups of male Swiss Webster mice (n > or = 10/group) were given either vehicle (intralipid, 10 ml.kg-1, control groups) or propofol (50 mg.kg-1, test groups) injected intraperitoneally. Five min after injection, convulsions were induced with either bicuculline (1.36-5.44 nmoles), picrotoxin (0.21-1 nmol), N-methyl-D-aspartic acid (0.51-2 nmol), quisqualic acid (1-10 nmol), kainic acid (0.252-2 mole), or strychnine (1.35-10.78 nmol) injected intracerebroventricularly. The number of animals with convulsions after each dose was recorded. Analysis of statistical significance was based on the log-probit lines of the quantal dose-response for the respective control and test groups, calculated 50% effective doses (ED50), the potency ratios (ED50higher/ED50lower) and their 95% confidence limits. Results Propofol pretreatment decreased the potency ratio of both bicuculline (0.47, 95% confidence interval = 0.23-0.94) and picrotoxin (0.61, 0.47-0.79), signifying an anticonvulsant effect. Conversely, propofol pretreatment significantly enhanced the convulsive potency of kainic acid (potency ratio and 95% confidence interval = 1.66, 1.21-2.29), quisqualic acid (3.17, 1.98-5.09), and strychnine (1.76, 0.79-3.89). Conclusions Current results suggest that propofol augments the paroxysmal motor phenomenon induced by kainic acid, quisqualic acid, and strychnine. This action may be, at least partly, responsible for the motor manifestations reported after propofol administration. These in vivo results on modulation of gamma-aminobutyric acid, glycine, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and kainate receptor-mediated transmission may be of significance in understanding the mechanism of propofol action at the excitatory and inhibitory amino acid receptors.

In mice pretreated intracerebroventricularly (i.c.v.) with either saline (10 microliters/mouse), pertussis (1 microgram/mouse) or cholera (2.5 micrograms/mouse) toxins, effect of kappa-opioid receptor agonists on the colonic temperature and charcoal meal transit time were assessed. The kappa-opioid receptor agonist, trans-(+)-3,4-dichloro-N-methyl-[2-(1- pyrrolidinyl)cyclohexyl]-benzeneacetamide methane sulfonate hydrate (U-50488H, 50, 100 and 200 micrograms/mouse, i.c.v.) produced dose dependent hypothermia. Pertussis toxin pretreatment (72 and/or 144 h before) antagonized (P < 0.05) the hypothermic effect of U-50488H (100 micrograms/mouse) and (+)-trans-N-methyl-N-[2-(1- pyrrolidinyl)cyclohexyl[benz[b]-thio-phene-4-acetamide (PD 117302, 30 micrograms/mouse). In contrast, cholera toxin pretreatment (48 and/or 96 h before) did not antagonize the hypothermic effect of the kappa-opioid receptor agonists. Moreover, both i.c.v. and intrathecal (i.t.) administration of kappa-opioid receptor agonists, U-50488H, }[5R-(5 alpha,7 alpha,8 beta)]-(+/-)-N-methyl-N-[7-(1- pyrrolidinyl)-1-oxaspiro[4,5]dec-8-yl]-benzeneacetamide? (U-69593) and PD 117302, produced dose dependent inhibition of the charcoal meal transit. Cholera toxin pretreatment (48 and 96 h before) augmented (P < 0.05) the antitransit effect of i.c.v. administered U-50488H (100 micrograms/mouse), U-69593 (100 micrograms/mouse) and PD 117302 (50 micrograms/mouse). However, pertussis toxin pretreatment did not affect the gastrointestinal inhibitory effect of the kappa-opioid receptor agonists. The present results extend our previous results on the effect of kappa-selective agonists on gastrointestinal motility and indicate, like the prototype opiate agonist morphine, kappa-opioid receptor agonists are effective in inhibiting the gastrointestinal motility when administered either by intrathecal or intracerebroventricular routes.(ABSTRACT TRUNCATED AT 250 WORDS)

In light of the recent recognition of the physiological significance of nitric oxide, there is considerable interest in the methodological variables that can confound the results of the cerebellar cGMP analysis from in vivo experiments. In this study, using male Swiss Webster mice, the effect of such methodological variables as 1) weight of the animals; 2) tissue extraction procedures used in radioimmunoassay for cGMP; and 3) the commercial source of the assay kit on, harmaline-, pentylenetetrazole- or SNAP-induced increase in cerebellar cGMP in vivo were evaluated. Results indicate that mice in the 15- to 19-g weight range are most sensitive and best suited for in vivo drug effects on cerebellar cGMP. Furthermore, for the extraction of cerebellar cGMP, use of ice-cold 0.5 N hydrochloric acid and subsequent dilution of the sample in assay buffer is the simplest and fastest method. Present data also indicate that the source of the radioimmunoassay kit has a significant effect on the cerebellar cGMP results. Based on the present results, the protocol developed and the guidelines drawn are timely and of high practical significance for research in the area of pharmacology of nitric oxide.

Dynorphin A-(1–13)-Tyr-Leu-Phe-Asn-Gly-Pro (Dyn la; 1–8 nmol) injected intracerebroventricularly in the mouse produces two independent behavioraeffects: (1) a norbinaltorphimine (κ opioid antagonist)-reversible analgesia in the acetic acid-induced writhing test and (2) motor dysfunction characterized by wild running, pop-corn jumping, hindlimb jerking and barrel rolling and antagonized by the irreversible phencyclidine (PCP) and sigma (σ) receptor antagonist, metaphit and the non-competitive N- methyl- d -aspartate (NMDA) receptor antagonists, dextromethorphan and ketamine. The specific involvement of the PCP receptorin the motor effects of Dyn Ia is supported by the direct competitive interaction of the peptide with the binding of [3H]MK-801 (Ki: 0.63 μM) and [3H]TCP (Ki: 4.6 μM) to mouse brain membrane preparations.

The effect of buprenorphine pretreatment on the acute cocaine toxicity was assessed in male Swiss Webster mice. Buprenorphine pretreatment (0.15 or 0.30 mg/kg ip, 30 mins before) significantly attenuated the lethal effects of cocaine (60–140 mg/kg ip). The dose of cocaine which resulted in 50% mortality (LD50) in saline pretreated group was 100.61 mg/kg while the LD50 of cocaine in buprenorphine (0.15 and 0.3 mg/kg) pretreated groups were 113.57 and 118.16 mg/kg respectively. There was no significant change in the ratio of brain/plasma levels of cocaine in buprenorphine pretreated group when compared to the ratio from saline treated controls. Furthermore, neither naloxone (10 mg/kg ip, 15 mins before) nor naltrexone (3 mg/kg ip, 15 mins before) pretreatment affected the LD50 of cocaine. When tested 0.5, 1, 2, 4, 8 and 24 hrs after cocaine administration, sublethal dose of cocaine (80 mg/kg ip) injection resulted in significant increase in the plasma lactate dehydrogenase (LDH) levels. Buprenorphine pretreatment significantly attenuated cocaine-induced release of LDH. These results suggest that buprenorphine could be of potential advantage over naloxone in the management of cocaine and heroin (“speed ball”) toxicity and in studies on the pharmacotherapy of cocaine-induced toxicity, LDH levels may be used as a biochemical marker to assess the protective effects of drugs.

The thermic responses of highly selective k-(U-69593 and U-50488H) and mu-(sufentanil and fentanyl) agonists were assessed in normoglycaemic and hyperglycaemic mice. Hyperglycaemia was induced by streptozotocin injection. The opiates were injected subcutaneously and colonic temperatures monitored for 90 min. after injection. In normoglycaemic animals the k-agonists induced a significant hypothermic response and hyperglycaemia did not modify this effect. In normoglycaemic mice, low doses of mu-selective agonists predominantly produced hypothermia, while in high doses resulted in hyperthermia. In chronic hyperglycaemic mice, hyperthermia was the predominant effect of mu-opiates. The present results characterize the time course and dose response of some of the currently available highly selective kappa- and mu-opiate agonists for the first time in mice and suggest that hyperglycaemia differentially affects the opiate receptor subtypes that mediate thermoregulatory events.

Local anesthetic partition coefficients correlate with drug potencies in vitro, but in vivo data have not always complimented in vitro results. Despite extensive studies on intrathecal anesthetic action, whether there is correlation between the partition coefficient and local anesthetic potency has not been addressed. Mice (n = 150) were randomly allocated into 15 groups. Intrathecal injections of etidocaine (E), tetracaine (T), bupivacaine (B), lidocaine (L), or procaine (P) were administered and analgetic effect was measured using tail-flick (TF) test. Concentration-response regressions were constructed for each drug; EC50 values were calculated and compared at 95% confidence intervals. The EC50 values between E (0.017%), T (0.019%), and B (0.012%) were not significantly variant. The EC50 of L (0.098%) and P (0.229%) were significantly different from each other and from E, T, and B. The EC50 values were converted to ED50 in nmols. Relative anesthetic potency, defined as the inverse value of ED50 of drug was 23:16:15:2.4:1 for B, E, T, L, and P, respectively. ED50 showed high correlation (R = 0.978) with partition coefficients of local anesthetics. This study implies that the partition coefficient is a predictor of intrathecal local anesthetic potency. We suggest that the mouse model is reliable for evaluation of intrathecal local anesthetic action.

Subcutaneous (s.c.) administration of azepexole (BHT 933) at doses between 4 and 40 mg/kg produced dose-dependent antinociceptive effects in mice as assessed by tail-immersion, tail-pinch and acetic acid writhing tests. The ED16s were 5.6 +/- 0.4, 6.7 +/- 1.2 and 2.96 +/- 0.2 mg/kg respectively. Similarly, morphine produced analgesia in the same tests with ED16s of 0.87 +/- 0.03, 0.47 +/- 0.1 and 0.45 +/- 0.01 mg/kg respectively. In all instances naloxone (0.1 mg/kg s.c.) shifted the dose-response curves to morphine to the right in a parallel manner. Naloxone (0.1 and 1 mg/kg s.c.) partially antagonized the effect of azepexole in the tail-immersion and tail-pinch tests but significantly decreased the slope of the dose-response curve suggesting that a competitive interaction at the level of the opioid receptors did not occur. Naloxone had no effect on the antinociceptive action of azepexole in the acetic acid writhing test.

The effects of morphine, fentanyl, sufentanil and droperidol on motor coordination in mice were studied. Animals were trained to complete successfully the rotarod test before assessing the effects of drugs. Administration of analgesic doses of the mu agonists morphine, fentanyl and sufentanil did not inhibit motor coordination. Droperidol produced a dose related inhibition of motor coordination. When a subthreshold dose of droperidol was administered followed by an opiate, a significant inhibition of motor coordination was observed. The results indicate that although analgesic doses of mu opioid agonists do not affect motor coordination, their combination with droperidol results in motor incoordination. The mechanisms and/or opioid receptor sub-types involved in this in vivo interaction remain to be established.

The effect of two commonly used methods to immobilize the animals, viz. tube restrainer and wrapping in a diaper (chux) on the tail flick latency in immersion test, was evaluated in mice using a stimulus temperature of 50 degrees C. The animals were immobilized either in the tube or chux briefly (25-30 sec) during the tail flick measurements. The basal tail flick latency was 2.8 +/- 0.2 in the tube restrained and 5.5 +/- 0.3 sec in chux restrained groups (p less than 0.001). The analgesic effect of morphine (1, 3, 4, 7, and 10 mg/kg) was significantly higher in the chux-restrained animals as indicated by the dose ratio of 2.16 for the 50% analgesic response in the chux versus tube restrained mice. The tail flick latency, 15 min after naloxone injection (1 and 3 mg/kg), expressed as % of predrug latency was significantly reduced in the chux- but not the tube-restrained group. The hyperalgesic effect of naloxone could not be detected in chux-restrained animals, when the water temperature was increased to 55 degrees C. The results demonstrate that the restraining procedure will influence the analgesic effects of test drugs in tail immersion test. Furthermore, the stimulus temperature appears to be an important variant that could influence the results in this test. The present results demonstrate the hyperalgesic effect of naloxone after systemic administration in the tail immersion test and supports the concept that tail flick response is tonically inhibited by endogenous opioid systems.

1. 1. The effect of droperidol pre-treatment on the analgesic potency of morphine, fentanyl and sufentanil was assessed in mice. 2. 2. Acetic acid writhing test and tail immersion test were used to measure the analgesic response. 3. 3. The neuroleptic augments the effects of sufentanil and fentanyl. 4. 4. Thus, the dose-response curves for fentanyl and sufentanil were shifted to the left and the ED50 of the analgesics lowered in droperidol pre-treated animals. 5. 5. However, morphine analgesia was not influenced by droperidol. 6. 6. The results suggest that combination of sufentanil or fentanyl with droperidol may be better than morphine to produce neuroleptanalgesia and anesthesia.

Because of the complexities of the central nervous system compounded with the ambiguities of interpreting the sensation of and reaction to “pain” in animals, available techniques for experimental evaluation of nociception to various types of stimuli are crude. Simple methods are often associated with artifacts that affect both the qualitative and the quantitative outcome of the test. It is therefore mandatory to control every detail of the experimental protocol. Further, the acuity expected from the experimenter to observe the animals during the test cannot be overemphasized. Additionally the test procedure should not inflict excessive suffering to the animals. It is the purpose of this review to highlight some of the “safeguard” measures to be adopted in order to avoid false-positive and false-negative results and erroneous interpretations and conclusions.

The effects of yohimbine (0.1, 1, 3 and 10 mg/kg SC) on nociceptive threshold were tested in mice using the tail-immersion and hot-plate tests. The tail-flick (withdrawal) latency, a monosynaptic spinal nociceptive response, was significantly lowered by yohimbine. This hyperalgesic response was at its peak 0.5 hr after yohimbine injection. The tail-flick latencies expressed as % of basal latencies were, 95 +/- 8, 100 +/- 10, 62 +/- 10, 33 +/- 7 and 28 +/- 4 in vehicle and 0.1, 1, 3 and 10 mg/kg in yohimbine-treated groups respectively. Yohimbine-induced hyperalgesia was observed when stimulus temperature was either 50 degrees C or 45 degrees C; however, the opiate antagonist naloxone (3 mg/kg SC) induced a hyperalgesic response at 50 degrees C and an analgesic response at 45 degrees C stimulus temperature. Streptozotocin-induced hyperglycemia did not influence the hyperalgesic response of yohimbine. In the hot-plate (60 degrees C) test, which involves higher centers and a polysynaptic nociceptive reflex, yohimbine did not modify the jump latency. The data provide evidence for the presence of a tonic spinal noradrenergic inhibitory control of nociceptive mechanism(s) which does not appear to be readily altered by hyperglycemia.

1. The effects of intravenous (i.v.) morphine on adrenocorticotrophic hormone (ACTH), beta-endorphin (beta-END), total catecholamines (CA) and histamine (HIS) plasma concentrations, were determined in anaesthetized dogs at 30 degrees C and 37 degrees C. 2. Hypothermia initially increased CA levels by 29%, but the values returned to baseline after 2 h. Morphine (1 mg/kg, i.v.) produced a significant decrease in CA both at 37 degrees C and 30 degrees C (34% and 54%, respectively). Subsequent administration of naloxone (1 mg/kg, i.v.) significantly increased CA levels in both groups. 3. Hypothermia per se had no effect on ACTH, beta-END, and HIS concentrations. Morphine produced a significant increase in pituitary hormones and HIS, in hypothermic but not in normothermic animals. Morphine concentrations were significantly higher at 30 degrees C during the first 45 min. 4. The results suggest that the effects of morphine on hormonal and histamine release observed at 30 degrees C are concentration-dependent and related to changes in morphine pharmacokinetics.

Using morphine as a prototype opiate anesthetic, the dispositional changes and cardiovascular effects during hypothermia (30 degrees C) and hyperthermia (40 degrees C) in dogs under isoflurane anesthesia was assessed. Single intravenous bolus injection of 1 mg/kg morphine resulted in a significant and sustained decrease in mean arterial pressure in hypothermic, but not in hyperthermic or normothermic (37 degrees C) conditions. Hypothermic dogs showed significantly higher levels of morphine both in plasma and in cerebrospinal fluid. In contrast, hyperthermia did not affect these levels. Body temperature did not affect the t1/2 alpha, however t1/2 beta and mean residence time were significantly increased while volume of distribution at steady state and total body clearance were decreased during hypothermia. The results provide evidence that hypothermia is likely to be associated with a sustained increase in opiate levels and might be associated with a enhanced side effects. The results suggests the need for a controlled clinical trial to assess the dose of opiate anesthetics during hypothermia.

In mice with streptozotocin-induced hyperglycemia, nociception was tested after naloxone administration in hot plate and tail immersion tests. The choice of these two tests was to include a supra-spinal nociceptive reflex indicative of higher cognitive process (hot-plate test) as well as a reflex which predominantly represents lower spinal motor mechanisms (tail immersion test). Naloxone-induced hyperalgesia was attenuated in both tests in mice with streptozotocin-induced diabetes. In mice with hyperglycemia induced by intraperitoneal dextrose administration, naloxone hyperalgesia was significantly enhanced in the hot plate test. The basal nociceptive threshold in streptozotocin-treated animals was decreased in the immersion but not in the hot plate test. These results indicate that hyperglycemia per se does not adequately explain the changes in naloxone hyperalgesia in experimental models of diabetes. They also suggest that acute hyperglycemia may modify the interaction of endogenous opioid peptides with their receptors only at supra-spinal sites. However, chronic hyperglycemia appears to affect endogenous opioid peptides both at spinal and supra-spinal levels and their interaction with the opiate receptors.

Systemic administration of highly selective kappa opiate agonists, U-50488H and U-69593 (3, 10 and 30 mg/kg i.p.) produced significant inhibition of the gastrointestinal transit in mice as assessed by charcoal meal test. In contrast, the (+) stereoisomer of U-50488H, U-53455E did not inhibit the gastrointestinal motility. Furthermore, the kappa-selective antagonist, Mr 2266 (3 mg/kg) when administered along with the agonists, reversed the effects of the agonists. These results suggest that stereospecific kappa opiate receptors are involved in inhibition of gut motility in mice.

The effects of mu and kappa opiate agonists were assessed on the pupillary size in normoglycemic and hyperglycemic conditions in mice. The mu agonists used were, morphine (10, 30 and 100 mg/kg), fentanyl (50, 100 and 150 micrograms/kg) and sufentanil (5, 10 and 20 micrograms/kg). The highly selective kappa opiate agonists used were, U-50488H and U-69593 (30 mg/kg). Chronic hyperglycemia was induced by streptozotocin injection (200 mg/kg i.p) 7-8 days before the experiment. Acute hyperglycemia was induced by intraperitoneal glucose (5 g/kg i.p) injection at the time of subcutaneous injection of opiates. In the normoglycemic mice, the mu agonists produced significant mydriasis (P less than 0.05), while kappa agonists had no effect on the pupil. However, both acute and chronic hyperglycemic condition did not affect the mydriatic ratio of the mu opiate agonists. These results indicate that mu opiate receptors induce mydriasis in mice and suggest that the hyperglycemia is not the mechanism involved in the opiate induced mydriasis in mice. With the dose of kappa agonists used, the involvement of kappa receptors in the pupillary effects in mice were not clear. These results support the hypothesis that hyperglycemia is not the primary mechanism for the altered sensitivity of opiates in the animal models of diabetes.

The effects of naloxone on the electrically stimulated vas deferens from mice implanted with morphine (tolerant) or placebo (naive) pellets were studied. In tolerant vas deferens, naloxone produced an 86.6 % increase of the twitch contractions when the preparations were stimulated with 15 V, while only a 12 % increase was observed with supramaximal voltage (40 V). Naloxone had no effect at either voltage in preparations from naive animals. The effect of morphine in naive vas deferens stimulated with 15 V, was reversed by naloxone without further increase over baseline contractions. The results suggest that opioid receptors other than those located in the neuronal soma and/or coupled to adenylate cyclase may be involved in the development of dependence.