Modification of the effects of naloxone in morphine-dependent mice

Mice were rendered dependent on morphine by mixing morphine with their food (2 mg/g) for three days. Increasing doses of naloxone precipitated dose-dependent withdrawal reactions such as weight loss and jumping. These withdrawal reactions were antagonized by morphine pretreatment. Effects of morphine, such as increased locomotor activity, inhibition of intestinal transport, and analgesia were antagonized by naloxone in both non-dependent and dependent subjects. The antagonist actions of naloxone were increased in dependent subjects; lower doses of naloxone were sufficient to antagonize effects of morphine. The present results confirm earlier studies indicating that precipitation of withdrawal can be antagonized by morphine pretreatment suggesting that withdrawal reactions are due to actions of naloxone at the same receptor at which opioid agonists act. The increased antagonist potency of naloxone in dependent subjects extends earlier results obtained with analgesic effects to several other agonist effects of morphine and is consistent with the interpretation that exposure to an opioid agonist induces a change in the conformation of opioid receptors.     

The effects of pituitary adenylate cyclase-activating polypeptide on acute and chronic morphine actions in mice

The effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on pain sensitivity, on morphine analgesia, on morphine tolerance and withdrawal were investigated in mice. The heat-radiant tail-flick test was used to assess antinociceptive threshold. Intracerebroventricular (i.c.v.) administration of PACAP alone had no effect on pain sensitivity but in a dose of 500 ng, it significantly diminished the analgesic effect of a single dose of morphine (2.25 mg/kg, s.c.). PACAP (500 ng, i.c.v.) significantly increased the chronic tolerance to morphine and enhanced the naloxone (1 mg/kg, s.c.)-precipitated withdrawal jumping. Theophylline (1 mg/kg, i.p.) pretreatment significantly enhanced the effect of PACAP on morphine analgesia but the effects of PACAP on tolerance and withdrawal were unaffected upon theophylline administration. On the grounds of our previous studies with vasoactive intestinal polypeptide (VIP), it appears that different receptors are involved in the effects of PACAP in acute and chronic morphine actions. Our results indicate that PACAP-induced actions likely participate in acute and chronic effects of morphine and suggest a potential role of PACAP in opioid analgesia, tolerance and withdrawal.     

Measuring naloxone antagonism of discriminative opioid stimulus

The numerous studies of opioids as discriminative stimuli, beginning in 1971, have shown specificity, similarity of several opioids, differences in potency (fentanyl greater than heroin greater methadone greater than morphine), and antagonism by naloxone and naltrexone. The discriminative opioid stimulus is differentiated from those of other classes of drugs, such as sedatives and anxiolytics. Greater potency of the opioid stimulus has been found in rats after subcutaneous (s.c.) than intraperitoneal administration. The discriminative opioid stimulus and its antagonism by naloxone or naltrexone have been demonstrated in rats, squirrel monkeys, gerbils, and pigeons. A few studies have quantified the competitive agonist-antagonist interaction at the receptor by calculating the pA2, which reflects the dose of the antagonist that requires doubling the agonist dose to obtain the original agonist response. The pA2 for naloxone is the same in groups of rats trained to discriminate different doses of morphine (1, 2, or 4 mg/kg s.c.) from saline. Higher pA2 values in tests after fentanyl and methadone than after heroin and morphine in rats trained to discriminate fentanyl (0.04 mg/kg s.c.) from saline reflect greater susceptibility of the synthetic than the natural exogenous opioids to antagonism by naloxone. Different pA2 values are usually interpreted as indicating differences among populations of receptors.     

Bremazocine induces antinociception, but prevents opioid-induced constipation and catatonia in rats and precipitates withdrawal in morphine-dependent rats

Some in vivo agonist and antagonist properties of the putative k-compound bremazocine were characterized in rats. Bremazocine, at doses from 0.015-32 mg/kg i.p., delayed nociceptive reaction on a 55 degrees C hot-plate with a dose-response curve not readily fitting a single straight line; this effect was antagonized by high doses of naloxone. In the same rats bremazocine did not delay the intestinal transit of a charcoal meal fed 5 min earlier and prevented morphine-induced constipation. This antagonism appeared to be opioid-specific and competitive, with apparent pA2 value 8.56. Catatonia induced by etorphine (0.004 mg/kg s.c.) and constipation induced by etorphine (0.004 mg/kg s.c.) and D-Ala2-D-Leu5-enkephalin (0.1 mg/kg i.p.) were completely antagonized by bremazocine (0.03-8 mg/kg i.p.). Antinociception induced by morphine (10 mg/kg i.v.) and etorphine (0.004 mg/kg s.c.) was only partly prevented. Naloxone (1 mg/kg) and bremazocine (0.015-1 mg/kg i.p.) precipitated a withdrawal syndrome, evaluated as jumping frequency, in rats rendered dependent to morphine. These data suggest the involvement of more than one opioid receptor population in bremazocine action in vivo.     

Modulation of naloxone-precipitated morphine withdrawal syndromes in rats by neuropeptide FF analogs

Neuropeptide FF (NPFF) has been reported to be an endogenous anti-opioid peptide that has significant effects on morphine tolerance and dependence. In the present study, we examined the chronic effects of NPFF and its synthetic analogs: the putative agonist, PFRFamide, and the putative antagonists, dansyl-PQRamide and PFR(Tic)amide on naloxone-precipitated morphine withdrawal syndromes in rats. After a 5-day co-administration with morphine [5 mg/kg, intraperitoneally (i.p.), twice per day (b.i.d.)] and the tested peptide [intracerebroventricularly (i.c.v.) or i.p., b.i.d.], naloxone (4 mg/kg, i.p.) was given systemically to evaluate the severity of the morphine withdrawal syndromes. Our results revealed that NPFF significantly potentiated the overall morphine withdrawal syndromes and, on the contrary, dansyl-PQRamide attenuated these syndromes. These results clearly indicate that modulation of the NPFF system in the mammalian central nervous system has significant effects on opiate dependence. In addition, morphine withdrawal syndromes could be practically applied as a valid parameter to functionally characterize the putative NPFF agonists and antagonists.     

Orphanin FQ/nociceptin inhibits morphine withdrawal

The influence of orphanin FQ/nociceptin (OFQ/N) on the morphine-withdrawal symptom was investigated. Withdrawal syndrome was induced in the morphine-dependent rats by an intraperitoneal (i.p.) injection of 2 mg/kg naloxone hydrochloride--an opioid receptors antagonist. Wet-dog shakes were used as a measure of the abstinence syndrome. Intraventricular injections of OFQ/N (5-20 microg/animal) caused significant inhibition of the withdrawal signs at doses between 15-20 microg, in the morphine-dependent rats. OFQ/N alone did not change behavior of the morphine-dependent animals. The obtained results indicate that OFQ/N can inhibit the morphine withdrawal symptoms induced by naloxone.     

Finasteride, a 5alpha-reductase inhibitor, potentiates antinociceptive effects of morphine, prevents the development of morphine tolerance and attenuates abstinence behavior in the rat

It has been shown that morphine increases 5alpha-reductase enzyme activity in the rat central nervous system; however importance of this finding on morphine analgesia, tolerance and dependence has not been reported. In the present study, we investigated inhibition of 5alpha-reductase enzyme on morphine effects using finasteride. To determine whether the 5alpha-reductase enzyme interact with morphine analgesia, finasteride (5 mg/kg, i.p.) was administrated with morphine (5 and 7 mg/kg, i.p.). The tail-flick test was used to assess the nociceptive threshold, before and 15, 30, 45, 60 and 90 min after drug administration. In tolerance experiments, morphine 20 mg/kg was injected i.p., twice daily for 4 days. The development and expression of dependence were assessed in the naloxone precipitation test 5 days after the morphine (20-30 mg/kg, i.p.) administration. We found that finasteride could potentiate the antinociceptive effect of morphine. In addition, chronic finasteride administration effectively blocked development of tolerance and dependence to morphine. Following chronic morphine administration, single dose injection of finasteride failed to reverse tolerance but prevented naloxone precipitate withdrawal syndrome. Therefore, it was concluded that there is a functional relationship between 5alpha-reductase enzyme and morphine.     

Effects of naloxone and fentanyl in acutely decerebrated dogs

Cardiovascular, respiratory and analgesic effects of fentanyl and naloxone were studied in normotensive acutely decerebrated dogs. Naloxone (1 mg/kg, i.v.) increased skin twitch reflex latency, mean blood pressure, pulse pressure, respiratory rate and minute volume. Fentanyl (50 μg/kg, i.v.) decreased heart rate and blood pressure while the animals were artificially ventilated. The skin twitch reflex latency was not significantly altered. Nine minutes later, naloxone (1 mg/kg, i.v.) was administered and the fentanyl-induced cardiovascular depression was reversed above the control level. The skin twitch reflex latency remained unchanged. These findings give further evidence that the endogenous opioid system plays an important role in the brainstem control of circulation and respiration. The mechanism of the anomalous analgesic response of the acutely decerebrated dog requires further investigation.     

Naloxone-induced enhancement of carbon dioxide stimulated respiration

In unanesthetized rats, naloxone (5 mg/kg, s.c.) produced an increase in both respiratory frequency and tidal volume as compared to saline administered animals. Maximal respiratory stimulation was observed within 5 minutes after naloxone injection and duration of the response was greater than 30 minutes. Exposure to different atmospheres of carbon dioxide potentiated the increase in ventilation in a step-wise manner as the carbon dioxide concentration was increased. Pretreatment with low doses of morphine sulfate (2 mg/kg daily for 2 days) or naloxone HCl (5 mg/kg daily for 5 days) enhanced respiratory stimulation induced by naloxone. It was concluded that naloxone increases the sensitivity of central ventilatory response to carbon dioxide as a result of displacement of endogenous endorphins from central opioid receptors.     

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.     

Effects of morphine and naloxone on feline colonic transit

The effects of endogenous and exogenous opioid substances on feline colonic transit were evaluated using colonic transit scintigraphy. Naloxone (0.3 mg/kg, i.m.) accelerated emptying of the cecum and ascending colon, and filling of the transverse colon. Endogenous opioid peptides thus appear to play a significant role in the regulation of colonic transit. At a moderate dose of morphine (0.1 mg/kg, i.m.), cecum and ascending colon transit was accelerated, while at a larger dose (1.0 mg/kg, i.m.) morphine had no effect. Since naloxone, a relatively nonspecific opioid antagonist, and morphine, a principally mu opioid receptor agonist, both accelerate proximal colonic transit, a decelerating role for at least one of the other opioid receptors is inferred.     

Dansyl-PQRamide, a possible neuropeptide FF receptor antagonist, induces conditioned place preference

Neuropeptide FF (NPFF) is an endogenous anti-opioid peptide. NPFF could potentiate the naloxone-precipitated morphine withdrawal syndromes in morphine-dependent rats, indicating the possible involvement of the endogenous NPFF system in opioid analgesia and dependence. The present study was performed to examine the effects of dansyl-PQRamide (dns-PQRa), a putative NPFF antagonist, on conditioned place preference (CPP), in addition, its interaction with the opioid system. Two CPP experiments were conducted. First, rats were treated with dns-PQRa (4-13 mg/kg, i.p.) and paired with the non-preferred compartment while the vehicle was paired with the preferred compartment. Second, similar to experiment 1 except naloxone (1 mg/kg, i.p.) was given 10 min prior to each dns-PQRa administration. The post-drug place preference was examined after 4 alternative pairings. Another group of animals after repetitive dns-PQRa treatments were analyzed for levels of neurotransmitters in discrete brain areas. Dns-PQRa (4-13 mg/kg, i.p.) induced a significant dose-dependent CPP. The dns-PQRa-induced CPP was completely blocked by pretreatment with 1 mg/kg i.p. naloxone, while naloxone alone did not induce any place aversion. The chronic dns-PQRa-treated (13 mg/kg, i.p., b.i.d.) rats caused a significant increase in 3,4-dihydroxyphenylacetic acid and 5-hydroxyindoleacetic acid in the olfactory tubercle compared to the vehicle-treated controls. There was also an increase in the turnover of serotonin in the olfactory tubercle, nucleus accumbens and medial prefrontal cortex. These results suggest that blockade of the NPFF system produces rewarding, possibly via an inhibition of the anti-opioid action of NPFF. These results also reveal a close relationship between NPFF, drug rewarding and the dopaminergic and serotoninergic neurons in the mesolimbic system.     

Effects of selective opioid agonists on feline colonic transit.

The mu agonist morphine and the non-specific opioid antagonist naloxone both may accelerate feline colonic transit; the effects of morphine are dose dependent. Kappa and delta receptor function was studied in the present work. Colonic transit of a radionuclide marker instilled into the cecum was quantitated for 6 hr in a crossover study. The delta agonist [D-Pen2,D-pen5]enkephalin (1 mg/kg, i.m.) prolonged the cecum and ascending colon half-emptying time by 337% (P less than 0.05), and delayed the progression of the geometric center over time. The kappa agonist U-50,488 (1 mg/kg, i.m.) had no apparent effect on the cecum and ascending colon, but delayed filling of the descending colon. Loperamide, an antidiarrheal agent, also delayed colonic transit. Thus, selective opioid agonists have both site and functional differences in their effect on feline colonic transit.     

Comparison of the effects of specific and nonspecific inhibition of nitric oxide synthase on morphine analgesia,tolerance and dependence in mice

The effects of L-Canavanine, a selective inducible nitric oxide synthase (NOS) inhibitor and N(G)-nitro-L-arginine methyl ester (L-NAME), a nonselective NOS inhibitor, on pain threshold and morphine induced analgesia, tolerance and dependence in mice were investigated and compared. Morphine was administered by subcutaneous implantation of a pellet containing 40 mg free base and withdrawal was precipitated by intraperitoneal (i.p.) injection of naloxone (2 mg/kg). L-Canavanine (200 mg/kg, i.p.) did not affect the pain threshold, morphine-induced analgesia and the induction and expression phases of morphine tolerance and dependence. L-NAME (20 mg/kg, i.p.) significantly (p < 0.05) enhanced the pain threshold, potentiated morphine-induced analgesia and attenuated the expression phase of morphine dependence which has been characterized by withdrawal signs and body weight loss, but did not modify the induction phase of morphine tolerance and dependence. It is concluded that constitutive NOS isoforms which were inhibited by L-NAME may be involved specifically in the mechanisms of morphine induced analgesia, tolerance and dependence.     

Early Repeated Administration of CXCR4 Antagonist AMD3100 Dose-Dependently Improves Neuropathic Pain in Rats After L5 Spinal Nerve Ligation

AMD3100 is a specific C-X-C chemokine receptor type 4 (CXCR4) antagonist which blocks the interaction between CXCR4 and CXCL12. Multiple lines of evidence suggest that AMD3100 has analgesic effects on many pathological pain states, including peripheral neuropathic pain. However, little is known about the underlying mechanisms. In the current study, we investigated the effect of different doses of AMD3100 on neuropathic pain in rats after L5 spinal nerve ligation. We used naloxone methiodide (NLXM) to further determine whether AMD3100-mediated analgesic effect was opioid-dependent. Behavioral study showed that early repeated administration of AMD3100 (2 and 5 mg/kg, i.p.) dose-dependently alleviates peripheral neuropathic pain. Flow cytometry, immunofluorescence and NLXM experiments showed that AMD3100 alleviates neuropathic pain partially by augmenting leukocyte-derived endogenous opioid secretion. Furthermore, we found that pro-inflammatory cytokines were down-regulated by AMD3100 using Enzyme-linked Immunosorbent Assay. Our data indicate that AMD3100 dose-dependently alleviates neuropathic pain partially by augmenting leukocyte-derived endogenous opioid secretion. This finding suggests that AMD3100 may be a viable pharmacotherapeutic strategy for the treatment of neuropathic pain.     

Modification of antiallodynic and antinociceptive effects of morphine by peripheral and central action of fluoxetine in a neuropathic mice model

We have previously reported that serotonin concentration was reduced in the brain of mice with neuropathic pain and that it may be related to reduction of morphine analgesic effects. To further prove this pharmacological action, we applied fluoxetine, a selective serotonin reuptake inhibitor, to determine whether it suppressed neuropathic pain and examined how its different administration routes would affect antinociceptive and antiallodynic effects of morphine in diabetic (DM) and sciatic nerve ligation (SL) mice, as models of neuropathic pain. Antiallodynia and antinociceptive effect of drugs were measured by using von Frey filament and tail pinch tests, respectively. Fluoxetine given alone, intracerebroventicularly (i.c.v., 15 microg/mouse) or intraperitoneally (i.p., 5 and 10 mg/kg) did not produce any effect in either model. However, fluoxetine given i.p. enhanced both antiallodynic and antinociceptive effects of morphine. Administration of fluoxetine i.c.v., slightly enhanced only the antiallodynic effect of morphine in SL mice. Ketanserine, a serotonin 2A receptor antagonist (i.p., 1 mg/kg) and naloxone, an opioid receptor antagonist (i.p., 3 mg/kg), blocked the combined antinociceptive effect of fluoxetine and morphine. Our data show that fluoxetine itself lacks antinociceptive properties in the two neuropathy models, but it enhances the analgesic effect of morphine in the periphery and suggests that co-administration of morphine with fluoxetine may have therapeutic potential in treatment of neuropathic pain.     

Methadone induced physical dependence in the rat

Although the morphine withdrawal syndrome has been well described in the rat, a syndrome having similar characteristics has not been demonstrated following chronic methadone treatment. In this study we describe the behavioral effects produced by naloxone (4 mg/kg sc) following 72 hours of continuous iv infusion of methadone, (12.2 ug/kg/min), morphine (12.2 to 97.9 ug/kg/min) or saline. The cessation of methadone or morphine but not saline treatment followed by naloxone resulted in graded signs including wet dog shakes, escape attempts, self-stimulation and body weight loss and quantal signs including diarrhea, ear blanching, exophthalmos, ptosis, tachypnea and teeth chattering. These results indicate that this mode of methadone administration produces physical dependence characterized by a morphine-like withdrawal syndrome in the rat.     

Enhancement of opiate-induced depressions in serum luteinizing hormone levels by the prior administration of naloxone in the male rat

The effects of naloxone pretreatment on opiate agonist-induced depressions in serum luteinizing hormone (LH) levels were examined in male rats. Our results demonstrated a pronounced enhancement of morphine's actions 6 hours after the administration of naloxone (0.5 mg/kg). This effect was characterized by a 10 fold reduction in the ED50 (1.26 mg/kg versus 0.13 mg/kg in saline- and naloxone-pretreated rats, respectively) and much greater depressions in serum LH levels at each dose of morphine. The actions of naloxone were not confined to morphine, since similar increased potencies were found for opioid agonists with selectivity for a variety of opioid receptor subtypes. Because naloxone did not alter the uptake of subsequently administered morphine into brain, our results cannot be explained on the basis of an increased availability of the agonist. Rather, it appears that naloxone pretreatment induces a change in the sensitivity of those receptors involved in the effects of opioid agonists on LH.     

Kratom Alkaloids,Natural and Semi-Synthetic,Show Less Physical Dependence and Ameliorate Opioid Withdrawal

Chronic administration of opioids produces physical dependence and opioid-induced hyperalgesia. Users claim the Thai traditional tea “kratom” and component alkaloid mitragynine ameliorate opioid withdrawal without increased sensitivity to pain. Testing these claims, we assessed the combined kratom alkaloid extract (KAE) and two individual alkaloids, mitragynine (MG) and the analog mitragynine pseudoindoxyl (MP), evaluating their ability to produce physical dependence and induce hyperalgesia after chronic administration, and as treatments for withdrawal in morphine-dependent subjects. C57BL/6J mice (n?=?10/drug) were administered repeated saline, or graded, escalating doses of morphine (intraperitoneal; i.p.), kratom alkaloid extract (orally, p.o.), mitragynine (p.o.), or MP (subcutaneously, s.c.) for 5 days. Mice treated chronically with morphine, KAE, or mitragynine demonstrated significant drug-induced hyperalgesia by day 5 in a 48 °C warm-water tail-withdrawal test. Mice were then administered naloxone (10 mg/kg, s.c.) and tested for opioid withdrawal signs. Kratom alkaloid extract and the two individual alkaloids demonstrated significantly fewer naloxone-precipitated withdrawal signs than morphine-treated mice. Additional C57BL/6J mice made physically dependent on morphine were then used to test the therapeutic potential of combined KAE, mitragynine, or MP given twice daily over the next 3 days at either a fixed dose or in graded, tapering descending doses. When administered naloxone, mice treated with KAE, mitragynine, or MP under either regimen demonstrated significantly fewer signs of precipitated withdrawal than control mice that continued to receive morphine. In conclusion, while retaining some liabilities, kratom, mitragynine, and mitragynine pseudoindoxyl produced significantly less physical dependence and ameliorated precipitated withdrawal in morphine-dependent animals, suggesting some clinical value.

     

Blockade of Nitric Oxide Overproduction and Oxidative Stress by <Emphasis Type="Italic">Nigella sativa</Emphasis> Oil Attenuates Morphine-Induced Tolerance and Dependence in Mice

The effects of Nigella sativa oil on morphine-induced tolerance and dependence in mice and possible mechanism(s) of these effects were investigated, for the first time, in this study. Repeated administration of Nigella sativa oil (4 ml/kg, p.o.) along with morphine (5 mg/kg, s.c.) attenuated the development of tolerance, as measured by the hot plate test, and dependence, as assessed by naloxone (5 mg/kg, i.p.)-precipitated withdrawal manifestations. Concomitantly, nitric oxide overproduction and increase in brain malondialdehyde level induced by repeated administration of morphine to mice or by administration of naloxone to morphine-dependent mice were inhibited by co-administration of the oil. Also, the decrease in brain intracellular reduced glutathione level and glutathione peroxidase activity induced by both treatments were inhibited by co-administration of the oil. The increase in brain glutamate level induced by both treatments was not inhibited by concurrent administration of the oil. The inhibitory effect of the oil on morphine-induced tolerance and dependence and on naloxone-induced biochemical alterations in morphine-dependent mice was enhanced by concurrent i.p. administration of the NMDA receptor antagonist, dizocilpine (0.25 mg/kg). Similarly, concurrent i.p. administration of the NO synthase inhibitors; L-N (G)-nitroarginine methyl ester (10 mg/kg), aminoguanidine (20 mg/kg) and 7-nitroindazole (25 mg/kg) or the antioxidant, N-acetylcysteine (50 mg/kg) enhanced this inhibitory effect of the oil. On the other hand, this effect was antagonized by concurrent i.p. administration of the nitric oxide precursor, L-arginine (300 mg/kg). These results provide evidence that Nigella sativa oil, through inhibition of morphine-induced NO overproduction and oxidative stress, appears to have a therapeutic potential in opioid tolerance and dependence.