Exploration of catalytic properties of CYP2D6 and CYP3A4 through metabolic studies of levorphanol and levallorphan.

CYP2D6 and CYP3A4, two members of the cytochrome P450 superfamily of monooxygenases, mediate the biotransformation of a variety of xenobiotics. The two enzymes differ in substrate specificity and size and characteristics of the active site cavity. The aim of this study was to determine whether the catalytic properties of these isoforms, reflected by the differences observed from crystal structures and homology models, could be confirmed with experimental data. Detailed metabolite identification, reversible inhibition, and time-dependent inhibition were examined for levorphanol and levallorphan with CYP2D6 and CYP3A4. The studies were designed to provide a comparison of the orientations of substrates, the catalytic sites of the two enzymes, and the subsequent outcomes on metabolism and inhibition. The metabolite identification revealed that CYP3A4 catalyzed the formation of a variety of metabolites as a result of presenting different parts of the substrates to the heme. CYP2D6 was a poorer catalyst that led to a more limited number of metabolites that were interpreted in terms to two orientations of the substrates. The inhibition studies showed evidence for strong reversible inhibition of CYP2D6 but not for CYP3A4. Levallorphan acted as a time-dependent inhibitor on CYP3A4, indicating a productive binding mode with this enzyme not observed with CYP2D6 that presumably resulted from close interactions of the N-allyl moiety oriented toward the heme. All the results are in agreement with the large and flexible active site of CYP3A4 and the more restricted active site of CYP2D6.

The antitussive activity of delta-opioid receptor stimulation in guinea pigs.

In this study, the activity of the delta-opioid receptor subtype-selective agonist, SB 227122, was investigated in a guinea pig model of citric acid-induced cough. Parenteral administration of selective agonists of the delta-opioid receptor (SB 227122), mu-opioid receptor (codeine and hydrocodone), and kappa-opioid receptor (BRL 52974) produced dose-related inhibition of citric acid-induced cough with ED(50) values of 7.3, 5.2, 5.1, and 5.3 mg/kg, respectively. The nonselective opioid receptor antagonist, naloxone (3 mg/kg, i.m.), attenuated the antitussive effects of codeine or SB 227122, indicating that the antitussive activity of both compounds is opioid receptor-mediated. The delta-receptor antagonist, SB 244525 (10 mg/kg, i.p.), inhibited the antitussive effect of SB 227122 (20 mg/kg, i.p.). In contrast, combined pretreatment with beta-funaltrexamine (mu-receptor antagonist; 20 mg/kg, s.c.) and norbinaltorphimine (kappa-receptor antagonist; 20 mg/kg, s.c.), at doses that inhibited the antitussive activity of mu- and kappa-receptor agonists, respectively, was without effect on the antitussive response of SB 227122 (20 mg/kg, i.p.). The sigma-receptor antagonist rimcazole (3 mg/kg, i.p.) inhibited the antitussive effect of dextromethorphan (30 mg/kg, i.p.), a sigma-receptor agonist, but not that of SB 227122. These studies provide compelling evidence that the antitussive effects of SB 227122 in this guinea pig cough model are mediated by agonist activity at the delta-opioid receptor.

Analysis of 16,16-dimethylprostaglandin E2-induced diarrhea in cecectomized rats.

The 16,16-dimethylprostaglandin E2 (dmPGE2)-induced diarrhea was analyzed in cecectomized rats prepared by resecting the cecum and its vasculature without disturbing the ileocecal junction. dmPGE2 (0.1-1.0 mg/kg, p.o.) dose-dependently increased the number of defecation episodes and induced a soft and watery stool in cecectomized rats. At 0.3 mg/kg, the diarrhea-inducing effects of dmPGE2 were more pronounced in cecectomized than in control rats. When given i.p., dmPGE2 (0.3 mg/kg) induced a watery stool in cecectomized and control rats with the same efficacy, although these effects were short-lasting as compared to oral administration. Castor oil (4 ml/kg, p.o.) also induced diarrhea, but did not produce a watery stool in cecectomized rats. There were no differences between cecectomized and control rats in basal small intestinal transits or in dmPGE2 (0.3 mg/kg, p.o.)-induced enhancements. Moreover, the basal and dmPGE2-induced jejunal net fluid transfers were the same in cecectomized and in control rats. On the other hand, the enhanced secretion of colonic fluid by dmPGE2, given intraluminally, was only half of that in control rats, whereas the colonic transit-enhancing effect of dmPGE2 in cecectomized rats was more pronounced than in control rats at 15 but not at 30 min after its administration. The basal colonic fluid contents and transits were the same in cecectomized and in control rats. Loperamide and morphine (0.1 and 1.0 mg/kg, s.c.) inhibited the dmPGE2 (0.3 mg/kg, p.o.)-induced diarrhea in cecectomized rats. N-methyllevallorphan (5 mg/kg, s.c.) completely antagonized the inhibitory effect of loperamide and partly antagonized the effect of morphine. These results suggest that oral administration of dmPGE2 induces a more pronounced secretory diarrhea in cecectomized than in control rats, probably due to the lack of the reservoir function of the cecum in the operated animals. This secretory diarrhea model is suitable for evaluating the antidiarrheal activity of drugs.

Identification with liquid chromatography-ionspray mass spectrometry of the metabolites of the enantiomers N-methyl dextrorphan and N-methyl levorphanol after rat liver perfusion.

To gather more information on stereochemical factors in the hepatic disposition of organic cations, mass spectrometry coupled to liquid chromatography was used to determine the identity of the metabolites excreted in bile after isolated rat liver perfusions with the quaternary ammonium derivatives of the enantiomeric drugs dextrorphan and levorphanol. Ionspray mass spectrometry was chosen for its soft ionization and absence of thermal degradation of labile compounds. The drugs were labelled with a stable (2H) isotope and mixed with unlabelled drugs to create an artificial isotope pattern in the mass spectrum and facilitate the recognition of unknown metabolites. In mass spectra that were recorded under normal conditions, fragmentation was absent and metabolites of N-methyl dextrorphan and N-methyl levorphanol were visible as parent-ion 'doublets'. Collision-induced fragmentation studies were performed to support the identification of the metabolites. For N-methyl dextrorphan the glucuronide, the glutathione conjugate and the glucuronide of the N-demethylated metabolite were found in bile. For N-methyl levorphanol the glucuronide, the glutathione conjugate, the sulphate conjugate and the glucuronide of a hydroxylated N-methyl levorphanol were excreted in bile. Thus a remarkable stereoselectivity occurs in the metabolism of these quaternary ammonium compounds in the rat liver.

A synthetic opioid peptide increases plasma growth hormone and prolactin in Holstein calves.

The effect of the synthetic opioid agonist D-Ala2,N-Me-Phe4,Met(O)5-ol enkephalin (DAMME) on plasma growth hormone (GH) and prolactin (PRL) concentrations in Holstein heifer calves was investigated in this study. The possible site of action of DAMME was determined by pretreating calves with an opioid antagonist that crosses the blood-brain barrier poorly if at all (N-methyl levallorphan-methane sulphonate [MLM]) or one that crosses readily (naloxone [NAL]). All calves were assigned to one of three treatment groups: 1) pretreatment with saline, 2) pretreatment with NAL, or 3) pretreatment with MLM. All groups were injected with DAMME 30 min after pretreatments. Plasma PRL increased after injection of DAMME in calves pretreated with saline. Prolactin concentrations were not different before and after injection of DAMME in calves pretreated with either NAL or MLM. Plasma GH increased after injection of DAMME in saline- and MLM-pretreated calves but was unchanged in NAL-pretreated calves. These data show that peripherally administered DAMME increases plasma GH and PRL in Holstein heifer calves and suggest that DAMME mediates GH release through receptors located somewhere inside the blood-brain barrier, but it can induce PRL secretion at a site located outside the barrier.

Central and peripheral control of postprandial pyloric motility by endogenous opiates and cholecystokinin in dogs.

The role of endogenous opiates and cholecystokinin (CCK) in the control of postprandial pyloric myoelectric activity was investigated in conscious dogs with chronically implanted intraparietal electrodes at the gastroduodenal junction. Meals consisted of either 20 g/kg of canned food (standard meal) or the same food supplemented with 0.5 mL/kg of arachis oil (fat meal). During the 6 hours after standard and fat meals, the number of pyloric spike bursts, 2-4 seconds in duration, was 61.8 +/- 15.8 and 49.9 +/- 12.7/15 minutes, respectively. Administered 15 minutes before a fat meal, naloxone (50 micrograms/kg IV) decreased the number of spike bursts by 31.4%, whereas methyl-levallorphan, a peripheral opiate antagonist, increased postprandial spike activity by 22.2% when administered IV (0.5 mg/kg) and decreased it when administered intracerobroventricularly at a dose of 10 micrograms/kg. These two antagonists administered in the same conditions before a standard meal had no effect on the postprandial spike activity. A 1-hour infusion of cholecystokinin octapeptide (CCK-8), 500 ng.kg-1.h-1 IV and 50 ng.kg-1.h-1 intracerebroventricularly, performed 1 hour after a standard meal induced a 19.6% and 15.8% decrease in the number of pyloric spike bursts, respectively. Both naloxone IV (50 micrograms/kg) and methyl-levallorphan intracerebroventricularly (10 micrograms/kg) administered before the infusion of CCK-8 reinforced this pyloric inhibition, which was antagonized by methyl-levallorphan IV (0.5 mg/kg). The CCK antagonist asperlicin, 200 micrograms/kg IV and 20 micrograms/kg intracerebroventricularly, administered before a fat meal increased pyloric spike bursts by 22.0% and 31.5%, respectively. These results indicate that after a fat meal, endogenous opiates exert a peripheral inhibitory and central stimulatory control of pyloric motility; they suggest the involvement of both peripheral and central release of CCK.

Opioid modulation of basal intestinal fluid transport in the mouse: actions at central, but not intestinal, sites.

Central and peripheral sites of opioid action on net basal fluid transport were studied in loops of jejunum in urethane-anesthetized mice. Intracerebroventricular administration of morphine, [D-Ala2, N-MePhe4, Gly-ol]enkephalin (DAMGO), D-Pen2, D-Pen5] enkephalin (DPDPE) or U50,488H produced dose-related increases in net basal intestinal absorption. Intracerebroventricular DAMGO was approximately 2.6, 1278 and 2674 times more potent than morphine, DPDPE and U50,488H, respectively. The increase in net basal fluid absorption mediated by i.c.v. administration of all these compounds, except DPDPE, was antagonized in a dose-related manner by coadministration of i.c.v. naloxone, an opioid antagonist which did not produce any effects when given alone. Neither the increase in net basal fluid absorption produced by DPDPE nor the fluid transport effects produced by the other agonists tested was antagonized by the delta antagonist, N,N-diallyl-Tyr-alpha-aminoisobutyric acid [( Aib]-Aib-Phe-Leu-OH) and no effects were observed with this delta antagonist alone. Intracerebroventricular administration of beta-funaltrexamine (18.8 nmol, 4 hr before testing) blocked the i.c.v. effects of DAMGO, but not those of U50,488H. In contrast to the effects seen following i.c.v. administration of these agonists, no changes in net basal fluid transport were obtained by the i.p. route for DAMGO, DPDPE, [D-Ala2,D-Leu5]enkephalin, [D-Ala2, Met5]enkephalinamide or U50,488H; of the compounds tested, only morphine produced an increase in net basal fluid absorption after i.p. administration. The effects of i.c.v. or i.p. morphine were blocked by i.c.v. SR 58002C, a quaternary opioid antagonist which had no effects alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Non-opioid effects of N-methyl levallorphan in the anaesthetised rat.

1. The effects of the quaternary opioid antagonist N-methyl levallorphan upon cardiovascular responses to non-opioid agonists has been studied in the urethane-anaesthetised rat. 2. N-methyl levallorphan showed an initial nicotinic agonist effect followed by pronounced ganglion blocking activity after intravenous administration. These effects are observed at doses only 2-fold higher than those required to block cardiovascular responses to [Met] enkephalin. 3. The narrow selectivity of N-methyl levallorphan makes this compound inappropriate for opioid antagonist studies where nicotinic receptors may be involved.

Peripheral sympatho-inhibitory cardiovascular effects of opioid peptides in anaesthetized rabbits.

1. Opioid agonists influence isolated cardiovascular tissues from rabbits, as well as the cardiovascular system of pithed rabbits, through presynaptic receptors on postganglionic sympathetic nerve fibres. The present experiments were carried out in order to study effects which result from activation of these receptors in anaesthetized rabbits. 2. In pithed rabbits with electrically stimulated sympathetic outflow, infusion of [D-Ala2-D-Leu5]-enkephalin (DADLE) 10 micrograms kg-1 min-1 and dynorphin-(1-13) (dynorphin) 1 microgram kg-1 min-1 decreased the plasma noradrenaline concentration, mean arterial pressure (MAP) and heart rate. The effects of dynorphin and, less completely, those of DADLE were antagonized by the peripherally selective opioid antagonists N-methyl naloxone bromide (NMN) 1.3 mg kg-1 and N-methyl levallorphan methanesulphonate (NML) 1-3 mg kg-1. 3. In pentobarbitone-anaesthetized rabbits, DADLE 3-30 micrograms kg-1 min-1 and dynorphin 0.3-3 micrograms kg-1 min-1 decreased the plasma noradrenaline concentration and MAP. The highest dose of dynorphin also decreased heart rate, whereas DADLE 10 micrograms kg-1 min-1 caused slight cardioacceleration. The effects of DADLE but not those of dynorphin decreased upon repeated administration. 4. The effects of dynorphin 10 micrograms kg-1 min-1 were abolished or greatly attenuated by NMN 1.3 mg kg-1 and NML 3 mg kg-1. In contrast, the antagonists reduced only slightly the blood pressure-lowering effect of DADLE 10 micrograms kg-1 min-1 and did not reduce significantly the effects of DADLE on the plasma noradrenaline level and heart rate. 5. It was concluded that systemically administered dynorphin produces sympatho-inhibition and an ensuing fall in blood pressure by an action at peripheral receptors, in all probability presynaptic Kappa-receptors on postganglionic sympathetic nerve fibres. The effects of DADLE are more complex and may involve both central and peripheral components.

Antagonism by N-methyl levallorphan-methane sulphonate (SR 58002 C) of morphine-elicited acute and chronic central and peripheral effects.

The peripheral activity of the quaternary narcotic antagonist N-methyl levallorphan-methane sulphonate (SR 58002 C) at opioid sites located in the periphery and in the central nervous system (CNS), was studied by different approaches in rats after subcutaneous injection (s.c.). Pretreatment with SR 58002 C 2,8 or 32 mg/kg s.c. 10, 50 or 110 min before buprenorphine consistently reduced buprenorphine in vivo binding only in the small intestinal longitudinal muscle with attached myenteric plexus (MP), whereas naloxone (1 mg/kg s.c.) 10 min before buprenorphine lowered buprenorphine binding in MP and brain (without cerebellum). Plasma levels were not altered by SR 58002 C or naloxone. The same doses of SR 58002 C injected 10, 50 or 110 min before morphine selectively antagonized the inhibition of transit of a charcoal meal along the small intestine (mainly a peripheral effect) induced by the agonist, but did not antagonize morphine-elicited analgesia in the hot-plate test (central effect). Naloxone (1 mg/kg s.c.) injected 10 min before morphine antagonized both agonist effects simultaneously. In morphine-dependent rats SR 58002 C (0.25, 1, 4 and 32 mg/kg s.c.) induced diarrhea, dose-dependently, in most animals within the first 30 min, while jumping, measured in the same rats, occurred in some animals, not dose-dependently, from 60 min on. Naloxone (1 mg/kg s.c.) induced both effects in most rats. These findings suggest that, although SR 58002 C probably penetrates the blood-brain barrier in some morphine-dependent rats, it discriminates peripheral and CNS opioid effects.

Central and peripheral injection of quaternary antagonist, SR58002C, reduces drinking.

Earlier research has demonstrated that opioid involvement in drinking is primarily mediated centrally [8,10]. Drinking is attenuated by the centrally and peripherally active opioid antagonists naloxone and naltrexone in doses as low as 1.0 mg/kg, but not by the quaternary forms of these antagonists [4]. Peripherally administered quaternary forms of these antagonists fail to suppress drinking in doses as high as 10 mg/kg. We generated dose-response curves for centrally and peripherally administered SR58002C, a "newer" quaternary opioid antagonist purported to have high peripheral selectivity, on drinking in 23.5 hr water deprived rats. SR58002C was administered both intracerebroventricularly (ICV, 0, 10, 40 and 80 micrograms/rat) and intraperitoneally (IP, 0, 10, 40 and 80 mg/kg). Doses of SR58002C above 10 mg/kg IP or 10 micrograms ICV significantly reduced drinking in comparison to controls. However, SR58002C appears to be less potent than quaternary naltrexone in suppressing drinking after ICV administration.

Dissociation of morphine withdrawal diarrhea and jumping in mice by the peripherally selective opioid antagonist SR 58002 C.

Mice were rendered physically dependent by repeated administration of morphine, 25 mg/kg s.c., 5 times daily for 4 days, and on the 5th day, 2 h after the last morphine dose, they were challenged with a s.c. injection of either naloxone, 25 mg/kg, or the peripherally selective opioid antagonist SR 58002 C (N-methyl levallorphan mesilate), 75 mg/kg. Naloxone provoked jumping and diarrhea in all the animals; mice challenged with SR 58002 C presented no significant jumping but a high frequency of withdrawal diarrhea. When naloxone, 12 mg/kg, or SR 58002 C, 50 mg/kg, were given s.c. in combination with repeated morphine as above, mice which had received naloxone with morphine presented virtually no diarrhea or jumping upon naloxone challenge; those repeatedly treated with morphine plus SR 58002 C were substantially protected from naloxone-precipitated diarrhea, but not jumping. These results further support the remarkable selectivity for peripheral opioid receptors of SR 58002 C, even after repeated high-dose treatment, and are strongly consistent with the primary role of a local intestinal mechanism in the development and expression of opioid withdrawal diarrhea in mice. The in vivo dissociation of central and peripheral components of dependence on morphine is illustrated, apparently for the first time.

[Peripheral analgesic actions of opioid peptides and morphine analogues].

Opiates and opioid peptides were administered in the order of 10(-9)-10(-6) mol peripherally, and their action on pain sensitivity was investigated by the modified formalin test which has two characteristic pain responses (the first and the second phase) in the mouse hindpaw. Opioid peptides (20-500 pmol) had dose-dependent analgesia against both first and second phases, and their action ranked dynorphin greater than [D-Ala2, Met5]-enkephalinamide greater than [Met5]-enkephalin. EKC and morphine (0.4-2.5 nmol) inhibited pain response of the first phase, but produced hyperalgesia in the second phase dose-dependently. Lidocaine hydrochloride had peripheral analgesic action, but was about 500-10000 times weaker than these substances. So, these peripheral analgesic actions have a different mechanism from that of local anesthetic action. N-methyl levallorphan which is thought to be a peripherally selective narcotic antagonist reversed these peripheral analgesic actions at the first and second phases and also prevented the hyperalgesic effects of EKC and morphine at the second phase. Naloxone reversed analgesia at only the first phase. These results suggest that an analgesic mechanism by opioids may exist at the peripheral site as well. Furthermore, it is estimated that a receptor exists which is antagonized by N-methyl levallorphan but not by naloxone and that there is a system of hyperalgesia by EKC and morphine in pain modulation.

Different targets for i.v. vs. i.c.v. administered morphine for its effect on colonic motility in dogs.

The effects of intracerebroventricular (i.c.v.) or intravenous (i.v.) administration of morphine on colonic motility were investigated in conscious dogs chronically fitted with a strain gauge transducer sutured to the serosa of the proximal colon. Morphine administered i.v. (100 micrograms/kg) or i.c.v. (10 micrograms/kg) induced similar increases in the motility index for about 3 h. The pattern of colonic contractions after i.v. morphine mainly consisted of an increase in the number of phases of contractile activity each lasting about 5 min. Morphine i.c.v. administered induced a peculiar pattern consisting of short (0.5-1.5 min) phases of contractile waves occurring at a high rate (10-15 per h). The effects of i.v. morphine were abolished after previous i.v. (1 microgram/kg) or i.c.v. (0.1 micrograms/kg) administration of naloxone or methyl-levallorphan, a narcotic antagonist with a high peripheral selectivity (100 micrograms/kg i.v., 10 micrograms/kg i.c.v.). The effects of i.c.v. morphine were not modified by previous i.v. or i.c.v. administration of naloxone (100 micrograms/kg). These results suggest that the stimulatory effect of i.v. morphine on colonic motility involves both central and peripheral components. The i.c.v. administration of morphine does not reproduce its central effect when given i.v. but acts on different central receptors.

Relative affinities of the quaternary narcotic antagonist, N-methyl levallorphan (SR 58002), for different types of opioid receptors.

The relative affinities for different subtypes of opioid receptors (mu, kappa and delta) of the peripheral narcotic antagonist N-methyl levallorphan (SR 58002) have been studied in two in vitro smooth muscle systems (guinea-pig ileum and rabbit vas deferens) and by binding studies (guinea-pig brain and cerebellum membranes) using selective tritiated ligands. All the evidence obtained indicates that SR 58002 is a pure antagonist with relative affinity for mu receptors vs kappa and delta superior to that of the parent tertiary compound, levallorphan.

The peripheral narcotic antagonist N-allyl levallorphan-bromide (CM 32191) selectively prevents morphine antipropulsive action and buprenorphine in-vivo binding in the rat intestine.

The assumed low ability of the quaternary narcotic antagonist N-allyl levallorphan-bromide (CM 32191) to cross the blood-brain barrier and its selectivity in relieving the peripherally-elicited antipropulsive action of morphine while preserving analgesia has been tested. To ascertain the extent of penetration of CM 32191 into the cns, its relative potency in preventing the in-vivo binding of high specific activity [3H]buprenorphine in the rat cns and small intestine was compared. Pretreatment was with CM 32191 at 16, 30 or 60 mg kg-1 s.c., 20, 60 or 120 min before buprenorphine, the concentrations of which in cerebrum and spinal cord were comparable with control values, but were consistently reduced in the intestine (longitudinal muscle with attached myenteric plexus). Pretreatment with naloxone (20 min, 0.5 or 1 mg kg-1 s.c.) lowered buprenorphine binding in intestine and cns. Neither narcotic antagonist produced significant changes in buprenorphine plasma concentrations. The peripheral selectivity of CM 32191, methyl naloxone and naloxone was examined by investigating in the same rats nociception in the hot plate (central opiate-sensitive mechanism) and the transit of a charcoal meal along the small intestine (local opiate-sensitive mechanism). Both effects were inhibited by morphine (5 mg kg-1 i.v.). Naloxone (10 min pretreatment, 0.5 or 1 mg kg-1 s.c.) did not selectively antagonize intestinal action of the morphine since the relief of charcoal transit inhibition was consistently associated with complete loss of analgesia.(ABSTRACT TRUNCATED AT 250 WORDS)

Levallorphan methyl iodide (SR 58002), a potent narcotic antagonist with peripheral selectivity superior to that of other quaternary compounds.

The peripheral selectivity of the newer quaternary narcotic antagonist levallorphan methyl iodide (SR 58002) was found superior in mice to those of the previously available compounds N-allyl levallorphan (CM 32191), N-methyl nalorphine (MNph) and N-methyl naloxone (MNx). SR 58002 and MNph were the most potent (ID50, mg/kg s.c., 3.6 and 3.7) in preventing constipation by s.c. morphine (charcoal meal). Antinociception (hot-plate) of s.c. morphine was completely prevented by MNx, MNph and CM 32191 (ID50, mg/kg s.c., 1.6, 8.6 and 15.2) but only partially antagonized by 30 mg/kg s.c. SR 58002. Constipation elicited centrally by intracerebroventricular (i.c.v.) morphine was prevented by either s.c. MNx or MNph but not by SR 58002 or CM 32191, up to 60 mg/kg.