A new hypothesis to investigate bioequivalence of pharmaceutical inhalation products.

BACKGROUND: This short communication reports a new hypothesis regarding bioequivalence of inhalation products which can potentially provide a reliable means to compare pharmaceutical aerosol formulations and inhalers. METHODS: Available methods regarding the bioequivalence studies, inhaled drugs and advantages of exhaled breath condensate (EBC) samples were reviewed to develop this hypothesis. RESULTS: It is postulated that two inhalation products providing the same drug concentrations in airway lining fluid (ALF) could be considered bioequivalent. The use of EBC tests which reflect ALF composition can be recommended as an alternative to current testing methods for consideration of bioequivalence. CONCLUSION: The methods based on EBC analysis can potentially be applied to bioequivalence study of inhalation products and could reflect drug concentration in ALF. However, experimental studies would be necessary to support or refute this hypothesis on the novel application of EBC to bioequivalence in the future. Graphical abstract In vitro (cascade impactor) and In vivo (EBC concentration) corrolation for inhaled drugs.

Effects of captopril on the cysteamine-induced duodenal ulcer in the rat.

Oxidative stress is important factor underlying in a variety of diseases. Antioxidative enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) are part of the physiological defenses against oxidative stress. Malondialdehyde (MDA) is a lipid peroxidation biomarker and its elevated level in various diseases is related to free radical damage. Cysteamine is a cytotoxic agent, acting through generation of reactive oxygen species (ROS) and may decrease defense activity of antioxidative enzymes against ROS and induce duodenal ulcer. Captopril, acts as free radical scavengers and protect against injuries from oxidative damage to tissues.The aim of this study was the evaluation of the effect of captopril against cysteamine-induced duodenal ulcer by determining duodenal damage, duodenal tissue SOD and GSH-PX activities and plasma MAD level. This study was performed on 3 groups of 7 rats each: saline, cysteamine and cysteamine plus captopril treated groups. The effect of captopril against cysteamine-induced duodenal ulcer is determined by evaluating the duodenal damage, duodenal tissue SOD and GSH-PX activities and plasma MDA level. All animals were euthanized 24h after the last treatment and 2 ml blood and duodena samples were collected for calculation of ulcer index, histopathological assessment and measurement of tissue SOD, GSH-PX activities and plasma MDA level. Cysteamine produced severe duodenal damage, decreased the activity of duodenal tissue SOD and GSH-PX and increased the plasma MDA level compared with saline pretreated rats. Pretreatment with captopril decreased the cysteamine-induced duodenal damage and plasma level of MDA and increased the activities of SOD and GSH-PX in duodenal tissue compared with cysteamine pretreated animal. Our results suggest that captopril protects against cysteamine-induced duodenal ulcer and inhibits the decrease in SOD and GSH-PX activities and lipid peroxidation by increasing antioxidant defenses.

Fluoxetine improves the effect of levodopa on 6-hydroxy dopamine-induced motor impairments in rats.

PURPOSE: Long term L-DOPA therapy in Parkinson's disease is associated with troublesome motor fluctuations such as L -DOPA Induced dyskinesia and wearing off effect. Our recent study showed that activation of 5-HT1A receptors could improve the anti-cataleptic effect of L-DOPA in parkinsonian rats. In this study we investigated the effect of fluoxetine on anti-parkinsonian effect of L-DOPA in 6-hydroxydopamine (6-OHDA)-lesioned rats. METHODS: Catalepsy and motor incoordination were induced by unilateral injection of 6-OHDA (8µg/2µl/rat) into the central region of the sabstantia nigra pars compacta (SNc). After 3 weeks as a recovery period, these rats injected intraperitoneally (i.p.) L-DOPA (15 mg/kg) twice daily for 20 consecutive days, and anti-parkinsonian effect of L-DOPA was investigated by bar-test and rotarod on days 5, 10, 15 and 20. RESULTS: The results showed that L-DOPA is able to improve motor coordination in rotarod only until day 15 and these effects of L-DOPA were abolished on the day 20. On day 21, rats were co-injected with fluoxetine (0.1, 0.5 and 1mg/kg, i.p.) and L-DOPA (15 mg/kg, i.p.). Fluoxetine increased anti-cataleptic effect of L-DOPA at the dose of 1 mg/kg, while fluoxetine had not any impact on the effect of L-DOPA in rotarod test. The effect of fluoxetine (1 mg/kg, i.p.) on anti-cataleptic effect of L-DOPA (15 mg/kg, i.p.) was reversed by 1-(2-methoxyphenyl)-4-(4-phthalimidobutyl) piperazine hydrobromide (NAN-190; 0.5 mg/kg, i.p.), as a 5-HT1A receptor antagonist. CONCLUSION: According to the results, it may be concluded that fluoxetine improves 6-OHDA-induced catalepsy and motor imbalance in L-DOPA- treated rats through activation of 5-HT1A. Further studies should be designed to clarify the precise mechanism of interaction between 5-HT1A and dopaminergic neurons.

Synthesis of a Novel Siliconized Analog of Clofibrate (Silafibrate) and Comparison of their Anti-inflammatory Activities.

Fibrates, as hypolipidemic drugs known as agonists of peroxisome proliferator-activated receptors, diminish inflammatory responses. Studies have shown that incorporation of a silicon atom into a drug structure improves its pharmacological potency, modifies its selectivity toward a given target, or changes its metabolic rate, in addition to increasing the lipophilicity of the compounds. A siliconized analog of clofibrate, ethyl-2-methyl-2-(4-(trimethylsilyl)phenoxy)propionate was synthesized, whereby the chlorine atom in the phenoxy ring was replaced by a trimethylsilyl group. The anti-inflammatory effects of the siliconized analog (silafibrate) were evaluated in an air-pouch model of inflammation and compared with those of clofibrate. Oral administration of both drugs produced a significant anti-inflammatory action by reducing carrageenan induced pouch leukocyte recruitment, exudates production, and granulated tissue weight. The silicon isostere of clofibrate has improved anti-inflammatory properties.

Role of 5-Hydroxytryptamine 1A Receptors in 6-Hydroxydopmaine-induced Catalepsy-like Immobilization in Rats: a Therapeutic Approach for Treating Catalepsy of Parkinson's Disease.

We have shown that buspirone, a partial agonist of 5-hydroxytryptamine 1A (5-HT1A) receptors, improves motor dysfunctions induced by 6-hydroxydopamine (6-OHDA) and haloperidol in rats. The present work extends these findings by investigating the role of 5-HT1A receptors on catalepsy-like immobilization in rats, a model of Parkinson's disease. Catalepsy was induced by unilateral infusion of 6-OH-dopamine (8 µg/2µL/rat) into the central region of the substantia nigra, compact part (SNc) and assayed by bar-test method 5, 60, 120 and 180 min after the drugs administration. The involvement of 5-HT1A receptors in 6-OHDA-induced catalepsy was studied through intraperitoneal (0.25, 0.5 and 1mg/Kg IP) and intrasubstantia nigra, compact part (10 µg/rat, intra-SNc) injection of 8-hydroxy-2-[di-n-propylamino] tetralin (8-OHDPAT) as well as administration of 1-(2-methoxyphenyl)-4-[4-(2-pthalimmido) butyl] piperazine hydrobromide (0.1, 0.5 and 1 mg/Kg, NAN-190, IP). NAN-190 (1 mg/Kg, IP) and 8-OHDPAT (1 mg/Kg, IP and 10 µg/rat, intra-SNc) increased and decreased 6-OHDA-induced catalepsy respectively. In normal (non 6-OHDA-lesioned) rats, NAN-190 (1 mg/Kg, IP) increased the elapsed time in bar-test while 8-OHDPAT did not produce any significant effect. The anticataleptic effect of 8-OHDPAT (1 mg/Kg, IP) was reversed markedly by co-injection with NAN-190 (1 mg/Kg, IP). These findings suggest that 5-HT1A receptors are involved in 6-OHDA-induced catalepsy-like immobilization.

Buspirone improves the anti-cataleptic effect of levodopa in 6-hydroxydopamine-lesioned rats.

In Parkinson's disease (PD), prolonged exposure to L-3,4-dihydroxyphenylalanine (L-DOPA) results in motor fluctuations, such as the on-off phenomenon, and L-DOPA-induced dyskinesia. Previously, we found that activation of 5-HT(1A) in the substantia nigra pars compacta (SNc) decreased catalepsy in parkinsonian rats. In the current investigation, we attempted to evaluate the effect of buspirone on the anti-cataleptic effect of L-DOPA in 6-hydroxydopamine (6-OHDA)-lesioned male Wistar rats. Catalepsy was induced by the unilateral infusion of 6-OHDA (8 µg/2 µl/rat) into the central region of the SNc. After a 3-week recovery period, rats received L-DOPA intraperitoneally (ip; 15 mg/kg) twice daily for 20 days, and the anti-cataleptic effect of L-DOPA was assessed by the bar test at days 5, 10, 15 and 20. The results showed that L-DOPA had an anti-cataleptic effect only until day 15, and its effect was abolished on day 20. On day 21, these rats were co-treated with three different doses of buspirone (0.1, 0.5 and 2.5 mg/kg, ip) and L-DOPA (15 mg/kg, ip). At a dose of 0.5 mg/kg, buspirone improved the anti-cataleptic effect of L-DOPA. Furthermore, the effect of buspirone (0.5 mg/kg, ip) on the anti-cataleptic effect of L-DOPA (15 mg/kg, ip) was reversed by 1-(2-methoxyphenyl)-4-(4-phthalimidobutyl)piperazine hydrobromide (NAN-190; 0.5 mg/kg, ip), a 5-HT(1A) receptor antagonist. From these results, it may be concluded that buspirone improves the anti-cataleptic effect of L-DOPA in a 6-OHDA-induced animal model of PD through the activation of 5-HT(1A) receptors. In this regard, further investigations should be undertaken to clarify the exact mechanism of the interaction between 5-HT(1A) and dopaminergic neurons.

Comparing of the effects of hypericin and synthetic antidepressants on the expression of morphine-induced conditioned place preference.

The effect of hypericin on the expression of morphine-induced conditioned place preference (CPP) was investigated and compared with the effect of the synthetic antidepressants. The CPP paradigms took place over six days using an unbiased procedure. The results demonstrate that intra-peritoneal (IP) injection of morphine sulfate (2.5, 5 and 10 mg/Kg) significantly induce the CPP in rat. Intra-peritoneal and intracerebroventricular (ICV) injection of hypericin and/or synthetic antidepressants augmented morphine-induced CPP. It has been suggested that the adrenergic, serotonergic and dopaminergic neurotransmissions play an important role in mediating the antidepressant effect of hypericin and this effect may be due to its inhibitory effect on the reuptake of neurotransmitters. Morphine produces a reinforcement (reward) effect by activating. The µ-receptors that facilitate dopaminergic transmission. Serotonin is also a potent stimulator of dopamine release in such a way that an increase in brain serotonin could possibly stimulate the dopaminergic system. In conclusion, it may suggest that the augmentation of morphine-induced CPP by hypericin and synthetic antidepressants may be related to the increasing dopamine and serotonin concentrations in synaptic clefts.

Buspirone improves 6-hydroxydopamine-induced catalepsy through stimulation of nigral 5-HT(1A) receptors in rats.

Receptors for 5-HT(1A) are widely distributed throughout the basal ganglia, and their activation results in an inhibition of dopamine (DA) release. This study aimed to investigate the effect of buspirone, as a partial agonist of 5-HT(1A) receptors, on 6-hydroxydopamine (6-OHDA)-induced catalepsy in male Wistar rats. Catalepsy was induced by unilateral infusion of 6-OH-DA (6 microg/2 microl/rat) into the central region of the substantia nigra pars compacta (SNc) and assayed by the bar-test method 60, 120 and 180 min after drug administration. The results demonstrated that intraperitoneal (ip) injection of buspirone at doses of 5, 7.5 and 10 mg/kg decreased catalepsy compared with the control group. In addition, intra-SNc injection of 8-hydroxy-2-[di-n-propylamino]tetralin (8-OH-DPAT; 10 microg/rat), a 5-HT(1A) receptor agonist, decreased 6-OHDA-induced catalepsy. The effects of buspirone (7.5 mg/kg, ip) and 8-OH-DPAT (10 microg/rat, intra-SNc) were abolished by 1-(2-methoxyphenyl)-4-[4-(2-phthalimido) butyl]piperazine hydrobromide (NAN-190; 10 microg/rat, intra-SNc), a 5-HT(1A) receptor antagonist. Our study indicates that buspirone improves catalepsy in a 6-OHDA-induced animal model of Parkinson's disease through activation of nigral 5-HT(1A) receptors. However, further investigations should be undertaken to clarify the exact mechanism of interaction between 5-HT(1A) and DA receptors.

Gastric healing effect of melatonin against different gastroinvasive agents in cholestatic rats.

BACKGROUND AND OBJECTIVE: The frequency of gastrointestinal ulceration is higher in jaundiced patients than in healthy population. The aim of this study was to assess the effect of pretreatment with melatonin, a potent scavenger of reactive oxygen species, on stress-induced gastric ulcers of cholestatic rats. MATERIALS AND METHODS: Cholestasis was induced by surgical ligation of bile-duct and sham-operated rats served as sham animals. The animals received saline or melatonin (1, 3 or 10mg/kg) before stress induction. Three different types of gastroinvasive agents including ethanol, indomethacin or water immersion were used as stress agents to induce gastric ulceration. RESULTS: Gastric mucosal damage induced by different gastroinvasive agents was significantly greater in bile-duct-ligated rats than in sham ones. Melatonin was protective against ethanol-, indomethacin- and water immersion-induced gastric damage in bile-duct-ligated and sham rats, dose-dependently, but the protective effect of melatonin was greater in cholestatic rats than sham rats in all three different series of experiments. CONCLUSIONS: In conclusion, pretreatment of rats with melatonin protected gastric mucosa of cholestatic rats more effectively than the sham ones possibly by a mechanism involving the scavenging of free radicals.

Effects of vitamin C and melatonin on cysteamine-induced duodenal ulcer in a cholestatic rat model: A controlled experimental study.

BACKGROUND: Superoxide dismutase (SOD) is one of the defense mechanisms against free radicals. Cysteamine is a cytotoxic agent, acting through generation of reactive oxygen species (ROS) such as hydrogen peroxide, hydroxyl radical, and superoxide, and may decrease defense activity of SOD against ROS and induce duodenal ulcer. Melatonin is a suicidal antioxidant that has a protective effect against ROS and cytoprotective effect through inhibition of the decrease in SOD activity. OBJECTIVES: The primary aim of this study was to assess the effects of pretreatment with vitamin C and melatonin on cysteamine-induced duodenal ulcer. Secondary aims were to compare the ulcerogenic effect of cysteamine and the antiulcer effects of vitamin C and melatonin. METHODS: This study was performed in male Wistar rats (200-250 g) in 3 groups of equal size (n = 24): bile duct ligation-induced cholestasis (test), sham, and control groups. In the test and sham groups, laparotomy was performed under general anesthesia and the common bile duct was identified; in sham rats, the common bile duct was left in situ, but in test rats, the common bile duct was isolated and doubly ligated to induce cholestasis. Animals in each group were also divided into 4 equal subgroups (n = 6). These subgroups were treated with vitamin C plus cysteamine, melatonin plus cysteamine, cysteamine alone, and saline, respectively. All animals were euthanized via overdose of ether anesthesia 24 hours after the last injection of cysteamine or saline, and 0.5 mL of blood was collected from the heart ventricle. The duodenum was cut open, washed with saline, fixed, and prepared for calculation of ulcer index (Szabo method) and histopathologic assessment. SOD activity was measured using a branded enzyme kit. RESULTS: In all 3 groups, animals treated with cysteamine had significantly increased mean (SE) ulcer index (test, 4.00 [0.10] vs 1.17 [0.30]; sham, 3.83 [0.16] vs 0.50 [0.22]; control, 3.67 [0.21] vs 0 [0]) and decreased SOD activity (test, 146.41 [2.16] vs 299.83 [1.94] U/mL; sham, 154.75 [2.02] vs 303.08 [0.35] U/mL; control, 157.08 [1.67] vs 314.50 [1.14] U/mL) compared with saline-treated rats (all, P < 0.001). In the test rats, ulcer index was significantly increased and SOD activity was significantly decreased compared with the sham and control groups (both, P < 0.001). Pretreatment with vitamin C and melatonin was associated with attenuation of ulcer index and increased SOD activity compared with rats treated with cysteamine alone (P < 0.001). There were no significant differences in ulcer index or SOD activity between groups administered vitamin C or melatonin. CONCLUSIONS: In this experimental study, pretreatment with melatonin or vitamin C in all rats produced significant attenuation of the ulcer index and enhanced SOD activity. Cysteamine-induced duodenal mucosal damage was greater in cholestatic rats compared with sham and control rats.

Alteration in male reproductive system in experimental cholestasis: roles for opioids and nitric oxide overproduction.

Cirrhosis is associated with impairment of the male reproductive system, hypogonadism and feminization. It is important to rule out whether the impairment in the reproductive system exists earlier in the course of cholestatic liver disease to target effective therapies at the best time point. In this study we investigated the role of endogenous opioid and nitric oxide system in alterations of the reproductive system in male rats. We performed sham or bile duct ligation surgery on male Sprague-Dawley rats and treated the animals for seven days with saline, naltrexone, an opioid receptor blocker (20 mg/kg) and N (G)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor (10 mg/kg). We then evaluated the plasma level of testosterone, luteinizing hormone (LH) and follicle stimulating hormone (FSH), sperm count and motility as well as biomarkers of cholestasis and nitric oxide productions. The results showed that following cholestasis, total testosterone level decrease and LH level increase in plasma of cholestatic rats and treatment with L-NAME and naltrexone could improve the plasma level of testosterone. Naltrexone could decrease the elevated level of LH in cholestatic animals. In addition, the weight of seminal vesicles and prostate significantly decreased in cholestasis as compared to the control group and treatment with L-NAME and naltrexone could improve the weights of the two organs in cholestasis. Our results demonstrate for the first time that the male reproductive system is impaired early in cholestasis and that endogenous opioid and nitric oxide system contribute to these impairments in the early course of the disease.

Effect of memantine on plasma concentrations of carbamazepine and phenytoin in rats: A controlled experimental study.

BACKGROUND: Elderly patients, especially those with Alzheimer's disease, may be prescribed memantine and an antiepileptic drug concurrently. OBJECTIVE: The aim of this study was to compare the interaction of memantine with phenobarbital (an enzyme inducer) and chloramphenicol (an enzyme inhibitor) on plasma concentrations of carbamazepine (CBZ), CBZ-10,11-epoxide (CBZE), and phenytoin in an experimental model. METHODS: Eight groups of rats (200-230 g) were treated for 14 days each. In groups 1 and 2, phenobarbital 50 mg/kg was administered daily as an enzyme inducer 60 minutes before CBZ 50 mg/kg or phenytoin 30 mg/kg administration, respectively. In groups 3 and 4, chloramphenicol 300 mg/kg was administered daily as an enzyme inhibitor 60 minutes before CBZ or phenytoin administration, respectively. In groups 5 and 6, memantine 20 mg/kg was administered daily 60 minutes before CBZ or phenytoin, respectively. In group 7, CBZ alone was administered daily; in group 8, phenytoin alone was administered daily. Two hours after the last intragastric gavage, animals were anesthetized with ether and 2 mL of blood was drawn from the heart into a syringe containing EDTA. A validated method developed in this study was used for simultaneous determination of CBZ, CBZE, and phenytoin concentrations in rat plasma. RESULTS: The study comprised 8 groups of 9 male adult Wistar rats each. Compared with groups 7 and 8, concurrent use of CBZ or phenytoin with phenobarbital (groups 1 and 2) was associated with significantly lower mean (SEM) plasma concentrations of CBZ (3.45 [0.16] vs 2.20 [0.21] µg/mL; P < 0.001) and phenytoin (3.68 [0.09] vs 1.63 [0.15] µg/mL; P < 0.001) and a significantly higher plasma CBZE concentration (9.85 [0.29] vs 11.18 [0.29] µg/mL; P < 0.05). Concurrent use of CBZ or phenytoin with chloramphenicol (groups 3 and 4) was associated with significantly higher plasma concentrations of CBZ (4.81 [0.17] µg/mL; P < 0.001) and phenytoin (6.24 [0.22] µg/mL; P < 0.001) and a significantly lower plasma CBZE concentration (3.88 [0.25] µg/mL; P < 0.001). Concurrent use of CBZ or phenytoin with memantine (groups 5 and 6) was not associated with a significant change in the plasma concentration of CBZ, CBZE, or phenytoin. CONCLUSION: Memantine was not associated with a significant change in the plasma concentration of CBZ, CBZE, or phenytoin in this experimental model.

Potentiation of morphine-induced conditioned place preference with concurrent use of amantadine and fluvoxamine by the intraperitoneal and intracerebroventricular injection in rat.

In this study, the effect of concurrent use of fluvoxamine and amantadine on morphine-induced conditioned place preference (CPP) was investigated by the intraperitoneal (i.p.) and intracerebroventricular (i.c.v.) injection in rat. The CPP paradigms took place on 6 consecutive days by using an unbiased procedure. Our results showed that i.p. injection of morphine sulfate (2.5-10mg/kg) induced CPP in rat. On day 6, fluvoxamine (5 and 10mg/kg, i.p.), and amantadine (5 and 10mg/kg, i.p.) both increased morphine-induced conditioned place preference. Intracerebroventricular injection of fluvoxamine (10 microg/rat) and amantadine (10 microg/rat) were also increased morphine-induced conditioned preference significantly. Concurrent use of fluvoxamine (5mg/kg, i.p.; 10 microg/rat i.c.v.) and amantadine (10mg/kg, i.p.; 10 microg/rat, i.c.v.) potentiated morphine-induced conditioned preference significantly. Release of dopamine from neurons cause reinforcing behavior. Morphine produces reinforcement (reward) effect by activation of mu receptors which facilitated dopaminergic transmission through dopamine release. Fluvoxamine, a serotonin reuptake inhibitor, increase serotonin concentration in synaptic clefts, which is a potent stimulator of dopamine release. Amantadine also appears to work by increasing dopamine release from neuron. In conclusion, our results show that concurrent use of fluvoxamine and amantadine potentiate morphine-like effect on CPP through increasing dopaminergic transmission and this combination may simulate the rewarding effect of morphine and can be candidate for controlling the drug compulsive seeking in morphine dependent subjects.

Alpha-2 agonists decrease expression of morphine-induced conditioned place preference.

Opioidergic system can interact with different transmission such as dopaminergic and adrenergic system. It has been shown that alpha-adrenergic system is involved in some effects of opioid including reward. In this study, alpha-2 agonists were used before testing on day 6 to evaluate their effects on the expression of morphine-induced conditioned place preference (CPP). Our results showed that intraperitoneal (i.p.) injections of morphine (5 mg/kg) induced CPP. Administration of alpha(2)-agonists clonidine (0.01, 0.02 and 0.04 mg/kg, i.p.), tizanidine (0.1, 0.2 and 0.4 mg/kg, i.p.) and xylazine (2.5, 5 and 10 mg/kg, i.p.) decreases the expression of morphine-induced CPP. Yohimbine (0.5 mg/kg, i.p.) reversed the inhibitory effect of alpha(2)-agonists. The comparison of potency of inhibitory effect of three agonists showed that ID(50) values for clonidine, tizanidine and xylazine were 0.013, 0.32 and 1.86 mg/kg respectively. Therefore, it is concluded that alpha(2)-agonists decrease the morphine-induced CPP in mice and clonidine is more potent than tizanidine and xylazine. The relative potency of clonidine with respect to tizanidine and xylazine was 30 and 180 respectively.

alpha2-Adrenoceptor subsensitivity in mesenteric vascular bed of cholestatic rats: the role of nitric oxide and endogenous opioids.

Cholestasis is associated with vascular changes and in previous studies decreased response of visceral vessels of cholestatic animals to phenylephrine and acetylcholine has been shown. In the present study, the response of mesenteric vascular bed of cholestatic rats to clonidine (an alpha2-adrenoceptor agonist) was investigated and we also examined the role of endogenous opioids and nitric oxide (NO). Seven-day ligation of bile duct was used as the model to study cholestasis. Six groups of rats, each of which divided into two subgroups (bile duct-ligated and sham-operated), were examined. Three groups of animals were chronically treated with either normal saline, naltrexone (an opioid receptor antagonist, 20 mg/kg/day, s.c.) or aminoguanidine (a selective inducible nitric oxide synthase inhibitor, 150 mg/kg/day, s.c.) for 7 days. After 7 days the response of the mesenteric vascular bed to subsequent doses of clonidine was studied. In other two groups, 7 days after the operation, the response of the mesenteric vascular bed to clonidine in the presence of either yuhimbine, an alpha2-adrenoceptor antagonist, or N(omega)-nitro-L-arginine methyl ester (L-NAME), a non-selective nitric oxide synthase inhibitor, was studied. In the last group, vasodilation response to sodium nitroprusside (an endothelium-independent vasorelaxant) was evaluated. Clonidine caused vasodilation in a dose-dependent manner by acting on endothelial alpha2-adrenoceptors since its effect was antagonized by yohimbine, and this vasodilation was through the L-arginine pathway since there was no response in the presence of L-NAME in the perfusate. Compared to sham-operated rats, there was a significant right shift in the clonidine concentration curves of cholestatic animals. Maximum response in cholestatic rats was significantly lower comparing to the sham group (P<0.01) and the dose of clonidine that causes 50% of maximum response (ED50) was significantly higher in cholestatic rats (P<0.05). Vasodilation response to sodium nitroprusside was the same in cholestatic and sham-operated rats. Seven-day treatment with aminoguanidine recovered the effect of cholestasis. Seven-day treatment with naltrexone caused an increase in maximum response (P<0.01) and a decrease in ED50 (P<0.05) in cholestatic rats, while this treatment in sham-operated rats caused a decrease in the maximum response (P<0.01) and an increase in ED50 (P<0.05). This study showed that cholestasis is associated with decreased responsiveness of mesenteric vascular bed to clonidine and the cholestasis-associated NO overproduction and increased level of endogenous opioids may contribute to this process.

The interaction of cannabinoids and opioids on pentylenetetrazole-induced seizure threshold in mice.

Cannabinoid and opioid receptor agonists show functional interactions in a number of their physiological effects. Regarding the seizure-modulating properties of both classes of receptors, the present study examined the possibility of a functional interaction between these receptors. We used acute systemic administration of cannabinoid selective CB(1) receptor agonist (ACPA) and antagonist (AM251) and opioid receptor agonist (morphine) and antagonists (naltrexone and norbinaltorphimine) in a model of clonic seizure induced by pentylenetetrazole (PTZ). Acute administration of ACPA (1.5-2 mg/kg) increased the PTZ-induced seizure threshold. In contrast, AM251 (0.5-2 mg/kg) dose-dependently decreased the seizure threshold. Low dose of AM251 (0.5 mg/kg), which did not alter seizure threshold by itself, reversed the anticonvulsant effect of ACPA (2 mg/kg), showing a CB(1) receptor-mediated mechanism. Naltrexone (1 or 10 mg/kg) but not specific kappa-opioid receptor antagonist norbinaltorphimine (5 mg/kg) completely reversed the anticonvulsant effect of ACPA (2 mg/kg). Moreover, the combination of the lower doses of AM251 (0.5 mg/kg) and naltrexone (0.3 mg/kg) had an additive effect in blocking the anticonvulsant effect of ACPA. In accordance with previous reports, morphine exerted biphasic effects on clonic seizure threshold with anticonvulsant effect at lower (0.5-1 mg/kg) and proconvulsant effect at a higher (30 mg/kg) doses. The pretreatment with AM251 blocked the anticonvulsant effect of morphine at 1 mg/kg, while pretreatment with ACPA (1 mg/kg) potentiated the anticonvulsant effect of morphine at 0.5 mg/kg. The proconvulsant effect of morphine at 30 mg/kg was also inhibited by AM251 (2 mg/kg). A similar interaction between cannabinoids and opioids was also detected on their anticonvulsant effects against the generalized tonic-clonic model of seizure. In conclusion, cannabinoids and opioids show functional interactions on modulation of seizure susceptibility.

Potentiation of anandamide effects in mesenteric beds isolated from bile duct-ligated rats: role of nitric oxide.

Changes in vascular responsiveness are proposed as the basis for some of the cardiovascular complications in cholestasis. Cholestasis is also associated with accumulation of endogenous opioid peptides and evidence of nitric oxide (NO) overproduction. On the other hand, it is well known that anandamide, an endogenous cannabinoid ligand, causes hypotension and a decrease in systemic vascular resistance. In the present study, the possible role of the cannabinoid system in cholestasis-induced mesenteric vascular bed responsiveness was investigated. Mesenteric arteries of bile duct-ligated and sham-operated rats receiving daily administrations of saline were used for evaluating phenylephrine or anandamide dose-response, acute effects of N(G)-nitro-L-arginine methyl ester (L-NAME, 100 microM), a non-selective inhibitor of NO synthase (NOS), or naltrexone, an opioid receptors antagonist (1 microM). The other groups of bile duct-ligated and sham-operated rats received daily intraperitoneal administration of L-NAME (20 mg/kg/day), aminoguanidine, a selective inducible NOS (iNOS) inhibitor (150 mg/kg/day) or naltrexone (10 mg/kg/day). After 7 days, the superior mesenteric artery was cannulated and the mesenteric vascular bed was perfused according to the McGregor method. Anandamide-induced relaxation was significantly potentiated in mesenteric vascular beds of bile duct-ligated rats. Chronic treatment of bile duct-ligated animals with L-NAME and aminoguanidine blocked this hyperresponsiveness while the hyperresponsiveness was potentiated at large doses of anandamide on chronic treatment of these animals with naltrexone. Although acute L-NAME treatment of mesenteric beds completely blocked the anandamide-induced vasorelaxation in sham-operated rats, this vasorelaxation still was present in bile duct-ligated animals. Anandamide-induced vasorelaxation remained unaffected after acute naltrexone treatment of mesenteric beds in both bile duct-ligated and sham-operated rats. Our results indicate that (1) there is enhanced anandamide-induced vasorelaxation in cholestatic rats, probably due to a defect in cannabinoid or vanilloid receptors and (2) NO overproduction may be involved in cholestasis-induced vascular hyperresponsiveness.

Inhibition by immunophilin ligands of morphine-induced tolerance and dependence in guinea pig ileum.

Immunophilin ligands, cyclosporine A and FK506 (tacrolimus), besides their immunosuppressive action, have several effects on different neural functions, such as modulation of the release of many neurotransmitters, the reduction of nitric oxide (NO) production by the inhibition of dephosphorylation of neuronal nitric oxide synthase (nNOS) and the alteration of the expression of certain genes. Many of these actions apparently occur through the inhibition of calcineurin, a calcium-calmodulin-dependent phosphatase. On the other hand, several studies have shown that NO has a critical role in opioid-induced tolerance and dependence in both in vivo and in vitro models. In the present study, the effect of cyclosporine A and FK506 on the development of tolerance to and dependence on morphine in the guinea pig ileum was assessed. Morphine inhibited electrically stimulated twitch of ileum in a concentration-dependent manner (pD(2)=7.45+/-0.07). Tolerance to this effect was induced by incubation of ileum with 2 x IC(50) or 4 x IC(50) of morphine for 2 h that induced a degree of tolerance of 6.81 and 18.10, respectively. The co-incubation of ileum with morphine along with either cyclosporine A or FK506 reduced the degree of tolerance significantly (P<0.05) and restored the sensitivity of ileum to the morphine inhibitory effect. Dependence was induced by incubation with 4 x IC(50) of morphine for 2 h and was assessed based on naloxone-induced contractions (10(-5) M). Cyclosporine A (10(-9) M) and FK506 (10(-9) M) can attenuate the development of dependence to morphine as shown by the significant decrease in naloxone-induced contractions (P<0.05). These results suggest that immunophilin ligands at very low concentrations (nanomolar) can reduce the induction of acute tolerance to and dependence on morphine in the myenteric plexus of guinea pig ileum.

Evidences for involvement of nitric oxide in the gastroprotective effect of bromocriptine and cyclosporin A on water immersion stress-induced gastric lesions.

Previous studies have been shown that the adrenergic system involves in gastric secretion and pathogenesis of peptic lesion and activation of alpha(2)-adrenoceptors located on the vagus nerve inhibits gastric acid secretion. Bromocriptine, a dopamine receptor agonist, also has alpha(2) agonistic effect and prevents indomethacin-induced gastric ulcer. alpha(2)-Adrenoceptors involve in the release of nitric oxide which has cytoprotective activity in gastric mucosa. Cyclosporin A (CsA) has also been suppressed stress-induced gastric mucosal lesions, dose dependently. The object of this study was to clarify the interaction between the anti-ulcer effect of bromocriptine (2, 4, 8 mg kg(-1)) or cyclosporin A (5, 10, 20 mg kg(-1)) and nitric oxide. Intraperitoneal injections of bromocriptine and cyclosporin A prevented water immersion stress-induced gastric ulcer in rats. L-Nitro-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, increased stress-induced lesions while L-arginine, a precursor of nitric oxide, decreased these lesions. In conclusion, increasing level of nitric oxide by bromocriptine and cyclosporin A may be one of the contributory factors in their protective effects on gastric mucosa.

Effect of NMDA receptor antagonist on naloxone-precipitated withdrawal signs in cholestatic mice.

The ability of NMDA antagonist MK-801 to block the expression of opioid-like withdrawal signs was examined in bile duct-ligated mice and the signs were compared with sham operated and unoperated animals. Administration of MK-801 (0/1 mg/kg), 10 min prior to naloxone challenge, significantly reduced the investigated withdrawal signs (jumping, diarrhoea, grooming and climbing) in bile duct-ligated animals. Chronic administration (five consecutive days) of MK-801 (0/1 mg/kg) also decreased all the withdrawal signs in the experimental animals. In an independent series of experiments, the effect of acute and chronic administration of MK-801 on tail-flick latency was investigated in bile duct-ligated animals. Pretreatment with the drug significantly decreased the antinociception induced by bile duct ligation in the mice. The results of this study support evidence for the involvement of the NMDA receptor in yopioidergic-dependent manifestations in a model of obstructive cholestasis. Copyright 2000 John Wiley & Sons, Ltd.