Antinociceptive Tests Research Articles

α 2-Adrenoceptor Agonists and Stress-Induced Analgesia in Rats: Influence of Stressors and Methods of Analysis

DE KOCK, M. AND T. F MEERT. α 2-Adrenoceptor agonists and stress-induced analgesia in rats: Influence of stressors and methods of analysis. PHARMACOL BIOCHEM BEHAV 58 [1] 109–117, 1997.—The present experiments were designed to investigate the role of housing and handling conditions during testing, as well as data analysis, on the outcome of antinociceptive testing of α 2-adrenoceptor agonists, fentanyl, and a high dose of chlordiazepoxide in the tail withdrawal reaction test (TWR test) in rats. Dose–response curve data were obtained with fentanyl, clonidine, xylazine, dexmedetomidine, and 40.00 mg/kg chlordiazepoxide and were compared under normal TWR test conditions and during immobilization or immobilization with continuous painful stimulation. Data were analyzed in terms of all-or-none criteria as well as percentage maximum possible effect (%MPE) analysis over the total measurement period or at any specific time point during testing. The results indicate that stress, induced by immobilization and immobilization with long-term-applied paw pressure, unmasked possible antinociceptive properties of the various α 2-adrenoceptor agonists and potentiated the effects of fentanyl. Stress also unmasked the positive effects of benzodiazepines. The manner of data analysis was shown to significantly affect the outcome measured in stress and nonstress conditions. The MPE analysis, particularly at one time point, appeared much more sensitive than the all-or-none criteria. The data indicate that the housing and handling conditions of animals during testing, together with data analysis, may affect the outcome of different classes of compounds in the TWR test, and this knowledge may help control for false positive results.

Peptide Targeting and Delivery across the Blood−Brain Barrier Utilizing Synthetic Triglyceride Esters: Design, Synthesis, and Bioactivity

As an approach to the development of therapeutically useful peptide pharmaceuticals that can penetrate the blood-brain barrier, we have designed and demonstrated the application of a carrier-targeting system. We have developed a prodrug design strategy that is designed to utilize membrane-bound enzymes whereby release of a bioactive peptide from a highly lipophilic triglyceride peptide-carrier is achieved in situ, thus attaining high localized concentrations of the bioactive peptide. Following localization of such a system, normal peptidase and lipase action is utilized to release the active peptide (deltorphin II) intact and in high concentration. At present, the exact mechanisms are unclear, but the observed results in which analgesia is observed following peripheral administration suggest that the active peptide is able to cross the blood-brain barrier and sustain prolonged periods of analgesia as determined by antinociception tests by release of the bioactive peptide. In vitro tests of binding and bioactivity by the peptide conjugate show essentially no potency in either target or control analogues, but potent antinociceptive effects are observed following peripheral administration.

Naratriptan: biological profile in animal models relevant to migraine.

The biological profile of naratriptan (N-methyl-3-(1-methyl-4-piperidinyl)-1H-indole-5-ethane-sulphonamide), a novel 5HT1B/1D receptor agonist, was investigated in a variety of experimental models of relevance to migraine. Naratriptan has high affinity for human recombinant 5HT1B and 5HT1D receptors (pKi = 8.7 +/- 0.03 and 8.3 +/- 0.1, respectively) and causes contractions of dog isolated basilar and middle cerebral artery (EC50 values of 0.11 and 0.07 microM, respectively). Naratriptan causes small contractions of human isolated coronary arteries (EC50 value of 0.17 microM; maximum contraction equivalent to 33% of 5HT maximum). In anaesthetized dogs, naratriptan causes selective vasoconstriction of the carotid arterial bed (CD50 dose = 19 +/- 3 micrograms kg-1) and, in anaesthetized rats, naratriptan selectively inhibits neurogenic plasma protein extravasation in the dura (ID50 = 4.1 micrograms kg-1). In a variety of antinociceptive tests, naratriptan has no effect even at high doses. In conscious rats and dogs, naratriptan has high oral bioavailability (71% and 95%, respectively). The data show that naratriptan is a selective agonist at 5HT1B/1D receptors, with a pharmacological profile very similar to that of sumatriptan, albeit 2-3 fold more potent. These observations, coupled with high oral bioavailability in animals, suggest that naratriptan has the profile of an orally effective anti-migraine drug.

Morphine metabolites.

Morphine is a potent opioid analgesic widely used for the treatment of acute pain and for long-term treatment of severe pain. Morphine is a member of the morphinan-framed alkaloids, which are present in the poppy plant. The drug is soluble in water, but its solubility in lipids is poor. In man, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) are the major metabolites of morphine. The metabolism of morphine occurs not only in the liver, but may also take place in the brain and the kidneys. The glucuronides are mainly eliminated via bile and urine. Glucuronides as a rule are considered as highly polar metabolites unable to cross the blood-brain barrier. Although morphine glucuronidation has been demonstrated in human brain tissue, the capacity is very low compared to that of the liver, indicating that the M3G and M6G concentrations observed in the cerebrospinal fluid (CSF) after systemic administration reflect hepatic metabolism of morphine and that the morphine glucuronides, despite their high polarity, can penetrate into the brain. Like morphine, M6G has been shown to be relatively more selective for mu-receptors than for delta- and kappa-receptors while M3G does not appear to compete for opioid receptor binding. The analgesic properties of M6G were recognised in the early 1970s and more recent work suggests that M6G might significantly contribute to the opioid analgesia after administration of morphine. The analgesic potency of M6G after intracerebroventricular (ICV) or intrathecal (IT) administration in rats is from 45-800 timer greater than that of morphine, depending on the animal species and the experimental antinociceptive test used. Furthermore, the development of a sensitive high-performance liquid chromatography (HPLC) assay for the quantitative determination of morphine, M6G and M3G has revealed that M6G and M3G were present in abundance after chronic oral morphine administration and that the area under the plasma concentration-time curve exceeded that of morphine. M3G has been found to antagonise morphine and M6G induced analgesia and ventilatory depression in the rat, which has led to the hypothesis that M3G may influence the development of morphine tolerance. M3G exhibits no analgesic effect after ICV or IT administration. Some studies do, however, indicate that M3G may cause non-opioid mediated hyperalgesia/allodynia and convulsions after IT administration in rats. These observations led to the hypothesis that M3G might be responsible for side-effects, hyperalgesia/allodynia and myoclonus seen after high-dose morphine treatment.

Pretreatment with protein kinase C activator phorbol 12,13-dibutyrate attenuates the antinociception induced by μ- but not ϵ-opioid receptor agonist in the mouse

The effects of pretreatment with a protein kinase C activator, phorbol 12,13-dibutyrate, on antinociception induced by i.c.v.-administered μ-opioid receptor agonist [ d-Ala 2, NMePhe 4, Gly(ol) 5] enkephalin (DAMGO) or morphine and ϵ-opioid receptor agonist β-endorphin were studied in male ICR mice. The tail-flick responses were used for antinociceptive tests. I.c.v. pretreatment with phorbol 12,13-dibutyrate (50 pmol) for 30 or 60 but not 10 min attenuated antinociception induced by i.c.v. administered DAMGO. I.c.v. pretreatment with phorbol 12,13-dibutyrate (10 and 50 pmol) for 60 min caused a dose-dependent attenuation of DAMGO (19.5 pmol)- or morphine (6.0 nmol)-induced antinociception. The dose-response curve for DAMGO-induced antinociception was shifted to the right by 7.3-fold by i.c.v. pretreatment with phorbol 12,13-dibutyrate (50 pmol) for 60 min. However, the i.c.v.-administered β-endorphin-induced antinociception was not affected by the same pretreatment with phorbol 12,13-dibutyrate. The attenuation of i.c.v.-administered DAMGO- and morphine-induced antinociception by phorbol 12,13-dibutyrate was reversed by concomitant i.c.v. pretreatment with a selective protein kinase C inhibitor calphostin C. These results suggest that activation of protein kinase C by phorbol 12,13-dibutyrate leads to the desensitization of μ-, but not ϵ-opioid receptor-mediated antinociception. These findings also provide additional evidence for differential intracellular modulation on antinociceptive action of μ- and ϵ-opioid receptor agonists.

A novel approach to the pharmacology of analgesics

To date in the United States when a patient has presented with a complaint of pain requiring some form of pharmacologic relief, the physician has had the choice of two broad classes of drugs: peripherally acting (i.e., NSAID) or centrally acting (i.e., opioid) analgesics. The antidepressant monoamine reuptake inhibitors, particularly when combined with an opioid analgesic, have also proven efficacious in treating certain types of pain conditions. A new approach, available for almost 20 years in Europe and recently approved for use in the United States, is the centrally acting synthetic analgesic tramadol HCl. Preclinical evidence suggests that tramadol produces its antinociceptive effect in animals and analgesic effect in humans through a complementary dual mechanism of action. One mechanism relates to its weak affinity for μ-opioid receptors (6,000-fold less than morphine, 100-fold less than d-propoxyphene, 10-fold less than codeine, and equivalent to dextromethorphan). A metabolite ( O-desmethyltramadol; M1) binds to opioid receptors with a greater affinity than the parent compound and could contribute to this component. However, in most animal tests and human clinical trials, the analgesic effect of tramadol is only partially blocked by the opioid antagonist naloxone, suggesting an important monopioid mechanism. This nonopioid mechanism possibly relates to an increase in central neuronal synaptic levels of two neurotransmitters, 5-hydroxytryptamine (5-HT; serotonin) and norepinephrine. The opioid and nonopioid mechanisms appear to combine in a supra-additive manner in several tests of antinociception, but only in an additive or even counteracting manner in measures of adverse-effect liability. In sum, the apparent dual mechanism of action of tramadol suggests a possible new approach to pain relief. Am J Med. 1996;101(suppl 1A):40S–46S.

Interaction between the μ‐agonist dermorphin and the δ‐agonist [D‐Ala2, Glu4]deltorphin in supraspinal antinociception and δ‐opioid receptor binding

1. In rats, the interaction between the mu-opioid agonist dermorphin and the delta-opioid agonist [D-Ala2, Glu4]deltorphin was studied in binding experiments to delta-opioid receptors and in the antinociceptive test to radiant heat. 2. When injected i.c.v., doses of [D-Ala2, Glu4]deltorphin higher than 20 nmol produced antinociception in the rat tail-flick test to radiant heat. Lower doses were inactive. None of the doses tested elicited the maximum achievable response. This partial antinociception was accomplished with an in vivo occupancy of more than 97% of brain delta-opioid receptors and of 17% of mu-opioid receptors. Naloxone (0.1 mg kg-1, s.c.), and naloxonazine (10 mg kg-1, i.v., 24 h before), but not the selective delta-opioid antagonist naltrindole, antagonized the antinociception. 3. In vitro competitive inhibition studies in rat brain membranes showed that [D-Ala2, Glu4]deltorphin displaced [3H]-naltrindole from two delta-binding sites of high and low affinity. The addition of 100 microM Gpp[NH]p produced a three fold increase in the [D-Ala2, Glu4]deltorphin Ki value for both binding sites. The addition of 10 nM dermorphin increased the Ki value of the delta-agonist for the high affinity site five times. When Gpp[NH]p was added to the incubation medium together with 10 nM dermorphin, the high affinity Ki of the delta-agonist increased 15 times. 4. Co-administration into the rat brain ventricles of subanalgesic doses of dermorphin and [D-Ala2, Glu4]deltorphin resulted in synergistic antinociceptive responses. 5. Pretreatment with naloxone or with the non-equilibrium mu-antagonists naloxonazine and beta-funaltrexamine completely abolished the antinociceptive response of the mu-delta agonist combinations. 6. Pretreatment with the delta-opioid antagonists naltrindole and DALCE reduced the antinociceptive response of the dermorphin-[D-Ala2, Glu4]deltorphin combinations to a value near that observed after the mu-agonist alone. At the dosage used, naltrindole occupied more than 98% of brain delta-opioid receptors without affecting mu-opioid-receptors. 7. These data suggest that in the rat tail-flick test to radiant heat, mu- and delta-opioid agonists co-operate positively in evoking an antinociceptive response. Although interactions between different opioid pathways cannot be excluded, in vitro binding results indicate that this co-operative antinociception is probably mediated by co-activation of the delta-opioid receptors at the cellular level by the mu- and delta-agonist.

Behavioural analysis of changes in nociceptive thresholds produced by remoxipride in sheep and rats

The antinociceptive potential of remoxipride was investigated in sheep and rats with concurrent motor function assessments. Previous studies of sheep given intravenous remoxipride have revealed increases in mechanical nociceptive thresholds. Here, further investigation in sheep demonstrated elevated thermal nociceptive thresholds with no effect on subjectively assessed sedation or motor impairment scores. However, in rats, the dose of remoxipride (100 mg/kg i.p.) required to produce nociceptive thresholds similar to those elicited by morphine (30 mg/kg i.p.), itself reduced rotarod performance. Medetomidine (200 μg/kg i.p.) evoked sedation without influencing rotarod performance or antinociception. The antinociceptive, motor deficit and cataleptogenic actions of remoxipride were similar to those induced by two other dopamine antagonists, haloperidol (5 mg/kg) and raclopride (16 mg/kg i.p). Tocainide (100 mg/kg i.p.) induced thermal antinociception with normal rotarod performance and no catalepsy suggesting that Na + channel blockade by remoxipride is not responsible for the changes in nociceptive thresholds. This study emphasizes the importance of motor function assessment during acute antinociceptive testing.

Morphine-3-glucuronide: evidence to support its putative role in the development of tolerance to the antinociceptive effects of morphine in the rat

Antinociceptive tolerance to morphine (MOR) was induced in groups of Sprague-Dawley rats receiving continuous intravenous infusions of morphine sulphate administered by 3 different MOR dosing regimes. At appropriate intervals throughout each infusion period, antinociceptive testing was performed using the tail-flick latency test and blood samples were collected. Groups of saline (SAL)-infused control rats also underwent antinociceptive testing and blood sample collection. Complete antinociceptive tolerance developed during each MOR infusion period and was characterized by a marked decline in the degree of antinociception from values greater than 90% of the maximum possible effect (%MPE) to pre-dosing baseline values. By contrast, %MPE values in SAL-infused control animals and in sham-operated rats were not significantly different from pre-dosing values throughout the infusion period, indicating that the experimental procedures themselves did not contribute to the development of antinociceptive tolerance to MOR. In addition, the rate of MOR tolerance development was inversely proportional to the MOR infusion rate. A very significant inverse relationship was observed between the mean degree of antinociception (%MPE) and the mean plasma molar concentration ratio, [morphine-3-glucuronide]/[MOR], for each of the 3 MOR dosing regimes and for the cumulated data. This relationship showed that near-maximum antinociception was attainable at ratio values less than approximately 0.50, whilst at ratio values above approximately 1.5, little or no antinociception was observed. Although %MPE was highly inversely correlated with the mean plasma morphine-3-glucuronide (M3G) concentrations for rats receiving regimes A and B, this was not the case for rats receiving regime C where antinociceptive tolerance was partially reversed by an increase in the morphine infusion rate part-way through the infusion period. In addition, a poor relationship was observed between %MPE and the mean plasma MOR concentration, possibly due to the confounding presence of M3G in all samples. Thus, we may conclude from this study in Sprague-Dawley rats that irrespective of the rate of antinociceptive tolerance development, the level of antinociception achievable appears to be highly inversely correlated with the mean [M3G]/[MOR] plasma molar concentration ratio and poorly correlated with the plasma MOR concentration, consistent with the notion that it is perhaps the balance between the excitatory effects of M3G and the inhibitory effects of MOR at the functional level which is the important determinant. Further research is required in carefully conducted studies in cancer patients to evaluate the possible contribution of the MOR metabolites. M3G and morphine-6-glucuronide (MbG), to increasing dosing requirements of MOR. Other factors such as disease progression and increasing psychological distress must also be borne in mind, as these will have a major influence on increasing dosing requirements of MOR in cancer patients (Portenoy 1994).

Interactions between the lipophilic opioid sufentanil and clonidine in rats after spinal application

Alpha2-adrenoceptor agonists, such as clonidine, can potentiate the analgesic properties of spinal opioids. In order to further extend this observation to highly lipophilic and potent spinally acting opioids, we tested the interactions between clonidine and sufentanil after both epidural and intrathecal administration in rats. The rats were equipped with spinal catheters. Antinociceptive testing was performed with the Tail Withdrawal Reaction test. Dose-response functions of sufentanil, clonidine and combinations of clonidine plus sufentanil were determined. These indicated that 1) sufentanil, but not clonidine alone, results in a dose-related antinociception after both the epidural and the intrathecal route of administration; 2) In combination with normally inactive doses of sufentanil, the addition of clonidine results in activity; 3) In a particular dose-range of sufentanil, there is a direct relationship between the doses of sufentanil and clonidine needed to produce antinociception; 4) Even at very high doses of clonidine, a minimal amount of sufentanil is needed to produce antinociception and 5) At the conditions tested here, the contribution of clonidine is minimal at high, intrinsic active doses of sufentanil.

CENTRAL NERVOUS SYSTEM ACTIVITY OF RHAZYA STRICTA (DECNE) IN MICE

1. The effects of orally administered aqueous lyophilized extract of the leaves of Rhazya stricta (2, 4 & 8 g/kg) on aspects of nervous system function were investigated in mice. 2. In three antinociceptive tests (hot plate, abdominal constriction, and warm water tail flick tests), the extract exhibited dose-dependent and significant antinociceptive activity. Naloxone was ineffective in antagonizing the analgesic effect of Rhazya stricta on tail-flick and abdominal constriction tests, possibly indicating that this effect occurs via non-opiate pathways. 3.Pretreatment of mice with the xenobiotic metabolizing enzymes inhibitor cimetidine (50 mg/kg) did not significantly alter the antinociceptive action of the extract, indicating that the effect is probably due to the parent compound(s) present in the extract and not to metabolites thereof. 4. Rhazya stricta produce dose-dependent sedation, decreased motor activity, and impaired motor control. Time spent on a rotarod treadmill was significantly decreased after treatment with the extract. 5. Rhazya stricta extract (8 g/kg) produced a degree of sedation comparable to that produced by diazepam (5-10 mg/kg), and also significantly increased the reaction time of the tail-flick test, an action which was not produced by diazepam. 6. Administration of R. stricta extract potentiated pentobarbitone sleeping time in a dose dependent manner. The extract did not significantly antagonize picrotoxin induced convulsions. The extract (4 and 8 g/kg) significantly decreased the rectal temperature of normothermic and hyperthermic mice. 7. Pretreatment with R. stricta extract (8 g/kg) completely prevented the occurrence of aggressive behaviour in male mice. 8. It is concluded that the crude extract of R. stricta has central nervous system depressant properties.

An Innovative Cold Tail-Flick Test

An innovative antinociceptive test, the cold ethanol tail-flick test (CET), was developed for evaluating the actions of opioid analgesics. To select an optimal operation temperature range for the CET, temperatures from -5 degrees C to -30 degrees C were screened. After screening, temperatures ranging between -20 degrees C and -30 degrees C were both strong and effective enough to act as a noxious cold stimulus. In the following study, -20 degrees C was selected as the cold stimulus for the CET. The sensitivity and specificity of this test were challenged by opioid analgesics: an agonist (morphine) and two agonist-antagonists (buprenorphine and nalbuphine), two tranquilizers (droperidol and diazepam), and four nonopioid analgesics (acetaminophen, aspirin, indomethacin, and ketoprofen). The sensitivity of the CET was also compared with the assays using heat (radiant heat and hot water). The AD50 values determined by the CET for morphine, buprenorphine, and nalbuphine were 0.16 mg/kg, 0.22 micrograms/kg, and 0.19 mg/kg, respectively. Naloxone, an opioid antagonist, blocked the antinociceptive effects of these opioids which were determined by the CET. Furthermore, the tranquilizers and nonopioid analgesics did not show any activity in the CET. Our results show that not only can the CET assess the antinociceptive activity of both opioid agonist and mixed agonist-antagonist, it also possess the characteristics of sensitivity, specificity, simplicity, and reproducibility.

Effects of intraventricular injection of morphine and β-endorphin on serotonin release from the spinal cord in rats

Effects of intraventricular (third ventricle) injection of morphine and β-endorphin on the release of serotonin (5-HT; 5-hydroxytryptamine) and 5-HIAA (5-hydroxy indolacetic acid) from the spinal cord were studied using urethane anesthetized spinally perfused rats. Intraventricular injection of morphine (25 μg) increased the 5-HT level in the perfusate about threefold. The increase of 5-HT release reached at peak between 30 and 60 min after the first injection of morphine. However, the levels of 5-HIAA, a metabolite of 5-HT, was not significantly altered by intraventricular injection of morphine. Furthermore, second intraventricular injection of morphine at the same dose did not increase 5-HT level in the spinal perfusate. In contrast to the results with morphine, β-Endorphin (10 μg) administered intraventricularly did not alter the release of 5-HT and 5-HIAA from the spinal cord. In addition, acute antinociceptive tolerance to intraventricular morphine induced by a prior intraventricular injection of morphine was studied in pentobarbital anesthetized rats. Acute tolerance was induced by intraventricular pretreatment with morphine (20 μg) for 120 min and the same dose of morphine was injected intraventricularly. The tail-flick test was used as an antinociceptive test. Pretreatment of rats with morphine intraventricularly reduced inhibition of the tail-flick response to intraventricularly injected morphine. The results support our previous hypothesis that β-endorphin and morphine administered supraspinally activate separate descending systems. Spinopetal serotonergic descending pathway is selectively activated by intraventricularly injected morphine but not β-endorphin. The production of acute antinociceptive tolerance to morphine injected intraventricularly may be due to, at least, a reduction of the release of 5-HT from the spinal cord.

Non-specific effects of the tachykinin NK 1 receptor antagonist, CP-99,994, in antinociceptive tests in rat, mouse and gerbil

We have examined the antinociceptive activity of the potent and selective tachykinin NK 1 receptor antagonist, CP-99,994, and its less active enantiomer, CP-100,263, in a variety of models in rat, mouse and gerbil. Administered systemically to gerbil or mouse CP-99,994 but not CP-100,263 stereo selectively inhibited a caudally directed biting and scratching elicited by intrathecal administration of the tachykinin NK 1 receptor agonist, GR73632. In contrast, both CP-99,994 (ED 50 = 3 (1−6) μmol · kg −1 s.c.) and CP-100,263 (4 (2–10)), were equipotent at inhibiting acetylcholine-inuced abdominal constrictions in mice. Similarly, both enantiomers were also equipotent in reducing formalin-induced licking in gerbil (CP-99,994 (10.1 (5.7–18.6)), CP-100,263 (13.8 (7.8.–27.1)) and rat (100 μmol · kg −1 s.c.). Finally, in the spinalised rat, CP-99,994 dose-dependently and significantly inhibited the flexion reflex evoked by noxious pinch (5.0 (3.3–7.5) μmol · kg −1 i.v.), whereas the less active enantiomer, CP-100,263, was without significant effect when tested up to 30 μmol · kg −1. Our results demonstrate that in the spinal cord, CP-99,994 exhibits a tachykinin NK 1 receptor mediated antinociceptive action.

ChemInform Abstract: (R)‐Methanandamide: A Chiral Novel Anandamide Possessing Higher Potency and Metabolic Stability.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Antinociceptive response induced by mixed inhibitors of enkephalin catabolism in peripheral inflammation

RB101 ( N-(( R,S)-2- benzyl-3[(S)(2- amino-4- methylthio)butyl dithio]-1- ox-opropyl)- l- phenylalanine benzyl ester) is a recently developed full inhibitor of the enkephalin-catabolizing enzymes able to cross the blood-brain barrier, whereas RB38A (( R)-3-(N- hydroxycarboxamido-2- benzylpropanoyl)- l-phenylalanine ) is as potent as RB101 but almost unable to enter the brain. In this study, we have investigated the effects of systemic administration of morphine, RB101 and RB38A on nociception induced by pressure on inflamed peripheral tissues. Antinociceptive test was performed between 4 and 5 days after injection into the rat left hindpaw of Freund's complete adjuvant to produce localized inflammation. Morphine (1, 2 and 4 mg/kg i.v.) induced antinociception in inflamed paws at all the doses used, and only at the highest dose in non-inflamed paws. RB101 (10 and 20 mg/kg i.v.) induced an antinociceptive response only in the inflamed paws. RB38A, also induced an antinociceptive effect in the inflamed paws, but only at the highest dose (20 mg/kg i.v.). The responses induced by morphine and the inhibitors of enkephalin catabolism were antagonized by the systemic administration of naloxone (1 mg/kg) or methylnaloxonium (2 mg/kg) which acts essentially outside the brain. Central injection (i.c.v.) of methylnaloxonium (2 μg) blocked the effect of morphine only in non-inflamed paws, and slightly decreased the response induced by RB101 on inflamed paws. These results indicate that the endogenous opioid peptides, probably enkephalins, are important in the peripheral control of nociception from inflamed tissues.

Antinociceptive activity of peptides related to interleukin-1β-(193–195), Lys-Pro-Thr

A series of closely related peptide analogues of Lys-Pro-Thr [(interleukin-1β-(193–195)] have been investigated in two models of antinociception in mice (acetic acid-induced abdominal constrictions and formalin tests) and compared with morphine, aspirin and indomethacin. Formalin-induced nociceptive responses in the mouse showed early (0–5 min) and late (15–30 min) phases of peak activity. Lys- d-Pro, Lys- d-Pro-Thr, Lys- d-Pro-Arg, Lys- d-Pro-Val, morphine and aspirin, were antinociceptive in both phases after intraperitoneal (i.p.) and oral (p.o.) administrations. Lys- d-Pro-Leu inhibited the early phase response only after i.p. injection. Lys- d-Pro-Val-NH 2, Lys- d-Pro-Gln, Lys- d-Pro-Tyr, Lys- d-Pro-Asn, Asp-Lys- d-Pro-Val and indomethacin were active only against the late phase (ED 50 values of 64, 32, 44, 94, 67 and 25 mg/kg i.p., respectively). Several of the peptides showed good bio-availability, e.g. Lys- d-Pro-Asn (ED 50: 10 mg/kg i.p.,; 11.4 mg/kg p.o.) in the abdominal constrictions test, where two modes of action were apparent, non-opioid and opioid; non-opioid (naloxone-insensitive antinociception) mechanisms were illustrated by Lys- d-Pro-Thr and Lys- d-Pro-Asn; opioid (naloxone-sensitive antinociception) mechanisms by Lys- d-Pro-Val and Lys- d-Pro-Leu. These data identify orally active antinociceptive peptides in both antinociceptive tests with varied relative potency profiles to morphine, indomethacin and aspirin in the mouse formalin test.

Antinociceptive activity of NK1 receptor antagonists: non-specific effects of racemic RP67580.

1. Release of substance P in the dorsal horn is considered a primary event in the perception of pain. The profile of racemic RP67580, a non-peptide antagonist at the NK1 (substance P) receptor, was examined in a range of antinociception tests on rodents. 2. At doses up to 30 mg kg-1, s.c. racemic RP67580 exhibited antinociceptive activity in writhing and formalin paw tests in mice and gerbils. Acetic acid induced writhing and the licking response to formalin were reduced to 40-50% of the level observed in vehicle-treated animals (P < 0.05). However, this agent was not active in mouse tail flick, rat paw pressure or rat and guinea-pig formalin paw tests. 3. Like racemic RP67580, the calcium channel blockers nifedipine (30 mg kg-1, i.p.) and verapamil (10 or 20 mg kg-1, s.c.) inhibited the response to formalin by approximately 60% in gerbils (P < 0.05 compared with vehicle-treated animals). 4. Evidence for calcium channel antagonist activity of RP67580 was obtained in vitro. Racemic RP67580 inhibited calcium entry into depolarized strips of guinea-pig ileum longitudinal muscle myenteric plexus (apparent KB = 587 +/- 115 nM), inhibited [3H]-diltiazem binding to rabbit skeletal membranes (IC50 = 298 nM) and depressed high threshold calcium currents in neurones cultured from rat cortex (10% inhibition at 10 microM). 5. These findings indicate that the acute antinociceptive effects of RP67580 may not be attributable to a specific interaction with NK1 receptors and may be mediated via calcium channel blockade.

Partial antinociceptive cross-tolerance to intracerebroventricular beta-endorphin in mice tolerant to systemic morphine.

The effects of subcutaneous morphine pellet-implantation on antinociception induced by intracerebroventricular (i.c.v.) administration of beta-endorphin or morphine and intrathecal (i.t.) administration of morphine, [D-Pen2,D-Pen5]enkephalin (DPDPE), [D-Ala2,NMePhe4,Gly5-ol]enkephalin (DAMGO), serotonin or norepinephrine were studied in male ICR mice. The tail-flick and hot-plate responses were used for antinociceptive tests. The ED50 values for i.c.v. administered morphine for antinociception in morphine pellet-implanted mice were increased from 3.3- and 2.2-fold at 0 h to 14.2- and 19.0-fold at 4 h and declined to 4.8- and 3.0-fold at 8 h after pellet removal in the tail-flick and hot-plate tests, respectively. On the other hand, the ED50 values for i.c.v. administered beta-endorphin for antinociception were only slightly increased (1.7- to 5.1-fold increases) throughout the same time course. The inhibition of the tail-flick response induced by i.t. injection of morphine, DPDPE and serotonin, but not norepinephrine or DAMGO, was attenuated in morphine pellet-implanted mice. These findings are consistent with previous studies indicating that different neuronal mechanisms are involved in morphine- and beta-endorphin-induced antinociception.

Pseudoirreversible binding characteristics of [D-Ala 2, Glu 4]deltorphin and its Cys 4 substituted derivative to δ-opioid receptors

Following the identification of [D-Ala 2,Glu 4]deltorphin as a selective δ 2-opioid receptor agonist in vivo, we synthesized the Cys 4-substituted analogue as a potential ligand which might bind ‘irreversibly’ at this site through a proposed thil-disulfide exchange mechanism. Previous studies showed that intracerebroventricular (i.c.v.) pretreatment with [D-Ala 2,Cys 4]deltorphin, 24 h prior to antinociceptive testing, produced a selective antagonism of [D-Ala 2,Glu 4]deltorphin-induced antinociception in mice. Surprisingly, however, the Ser 4-analogue (synthesized as a control) and even the parent molecule, [D-Ala 2,Glu 4]deltorphin, had the same antagonistic effect following pretreatment in vivo, while pretreatment with an equiantinociceptive dose of [D-Ser 2,Leu 5,Thr 6]-enkephalin, a structurally unrelated δ 2-opioid receptor agonist did not exhibit long-lasting antinociceptive actions. These data raised questions regarding the mechanism of the antagonism observed in vivo with the deltorphins; the present studies have attempted to explore these issues using radioligand binding techniques. The results demonstrate a decrease in the B max of [ tyrosyl-3′,5′- 3H,D-Pen 2,p-Cl-Phe 4,D-Pen 5]-enkephalin ([ 3H]p-Cl-DPDPE) (δ-opioid receptor ligand) following i.c.v. pretreatment of mice (at −24 h) with [D-Ala 2,Cys 4]deltorphin or [D-Ala 2,Glu 4]deltorphin, but not with [D-Ala 2,Ser 4] deltorphin, suggesting a difference in mechanism of antagonism seen in vivo with these compounds. Incubation of mouse whole brain homogenates in vitro with [D-Ala 2,Cys 4]deltorphin or with [D-Ala 2,Glu 4]deltorphin, also resulted in a decrease in the radioligand binding of [ 3H]p-Cl-DPDPE, but this effect was not prevented by coincubation with dithiothreitol, a thiol-reducing agent. Direct evaluation of binding using [ 3H][D-Ala 2,Glu 4]deltorphin (5 nM) showed that a portion of this ligand (i.e., about 10% of all specific binding) remained specifically and ‘irreversibly’ bound to mouse brain membranes following incubation in vitro and extensive washing. The ‘irreversibly’, specifically bound [ 3H][D-Ala 2,Glu 4]deltorphin could be removed, however, by brief exposure of the membranes to a low pH (2.5), high-salt (0.5 M NaCl) solution. These data suggest that [D-Ala 2,Cys 4] deltorphin and [D-Ala 2,Glu 4]deltorphin bind in a ‘pseudoirreversible’ (no-covalent) manner to an δ-opioid receptor via a mechanism that apparently does not involve thiol-disulfide exchange.