Comparative biotransformation of morphine,codeine and pholcodine in rat hepatocytes: identification of a novel metabolite of pholcodine

1. Pholcodine (3-morpholinoethylmorphine), a semi-synthetic alkaloid, is widely used as an antitussive agent. 2. Norpholcodine [7,8-didehydro-4,5alpha-epoxy-3-(2-morpholinoethoxy)morphinan-6alpha-ol] (NP) and pholcodine-N-oxide [1(9a)-dehydro-(4aR,5S,7aR,9cS,12S)-4a,5,7a,8,9,9a-hexahydro-5-hydroxy-12-methyl-3-morpholinoethoxy-1H-8,9,c-(iminoethano)phenanthro[4,5-bcd] furan-12-oxide] (PNOX) were identified in incubations of pholcodine with freshly isolated rat hepatocytes by liquid chromatography/electrospray-mass spectrometry (LC/ESI-MS). 3. Synthesized NP and PNOX were characterized by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. 4. N-oxidation was the major metabolic pathway for pholcodine, producing a previously unreported metabolite. 5. The metabolism of morphine and codeine was also determined using freshly isolated hepatocytes. 6. For morphine, 3-glucuronidation was the major metabolic pathway, whilst for codeine it was dealkylation (O- and N-). 7. Neither morphine nor its metabolites were metabolites of pholcodine. 8. This observation supports the hypothesis that the absence of analgesic activity with pholcodine may be due to less O-dealkylation in vivo. 9. Together with the slow biotransformation of pholcodine (k(met) = 0.021 microM min(-1)) in comparison with morphine (k(met) = 0.057 microM min(-1)) and codeine (k(met) = 0.112 microM min(-1)), the results obtained were consistent with its low addiction potential and suggest that its antitussive efficacy is mediated by the parent drug or one of its metabolites other than morphine.     

Metabolism of pholcodine in man

This paper describes studies on the metabolism of the antitussive 3-O-(-2'-morpholinoethyl)-morphine (pholcodine, Tussokon) in man. The metabolites were identified after cleavage of conjugates, extraction and derivatization by acetylation in human urine using gas chromatography-mass spectrometry. The following seven metabolites could be identified besides the unchanged pholcodine (P): Nor-P, desmorpholino-hydroxy-P, nor-desmorpholino-hydroxy-P, hydroxy-P, oxo-P, nor-oxo-P and morphine in traces. Therefore, the following four partly overlapping phase I metabolic pathways can be postulated: N-demethylation, N-desalkylation at the morpholino ring followed by reduction of the resulting aldehyde to the desmorpholino-hydroxy metabolite, oxidation of the morpholino ring to the hydroxy and oxo metabolite, and O-desalkylation to morphine. With the exception of morphine, pholcodine and its phase I metabolites were not converted to enzyme-hydrolysable conjugates. Pholcodine itself could be detected in urine 5-7 weeks after ingestion, the desmorpholino-hydroxy metabolite 1-2 weeks and the other metabolites only in the first few hours. It can be concluded that the low metabolism of the rather lipophilic pholcodine is the reason for the very slow elimination.     

Comparative disposition of codeine and pholcodine in man after single oral doses.

Four healthy male subjects received single oral doses of 15, 30 and 60 mg of codeine and pholcodine according to a balanced cross-over design with an interval of 7 days between the six treatments. Blood samples were collected for 8 h after each drug administration. In phase 2 of the study six different male volunteers received single oral doses of 60 mg of codeine and pholcodine with a 14 day interval between successive drug treatments. Blood was sampled for 12 h after codeine and 121 h after pholcodine administration. Plasma concentrations of free (unconjugated) and total (unconjugated plus conjugated) codeine, pholcodine and morphine were determined by radioimmunoassay and selected pharmacokinetic parameters were derived from these data. Pharmacokinetics of both drugs were independent of dose. Codeine was absorbed and eliminated relatively rapidly [elimination t1/2 = 2.3 +/- 0.4 h (mean +/- s.d.)]. While codeine kinetics were adequately described by a one-compartment open model with first-order absorption, a two-compartment model was required to describe pholcodine elimination from plasma (t1/2,z = 37.0 +/- 4.2 h). Plasma concentrations of conjugated codeine were much greater than those of the unconjugated alkaloid. By contrast, pholcodine appeared to undergo little conjugation. Biotransformation of codeine to morphine was evident in all subjects, although the extent of this metabolic conversion varied considerably between subjects. Morphine was not detectable in the plasma of any subject after pholcodine administration.     

Structure-antitussive activity relationships of naltrindole derivatives. Identification of novel and potent antitussive agents

We have previously reported antitussive effects of naltrindole (NTI), a typical delta opioid receptor antagonist, in a rat model. The ED50 values of NTI by intraperitoneal and peroral injections were 104 microg/kg and 1840 microg/kg, respectively, comparable to those of codeine. Codeine, one of the most reliable centrally acting antitussive drugs, has micro agonist activity and thus the same side effects as morphine, e.g., constipation, dependency, and respiratory depression. Because NTI is a delta opioid antagonist, its derivatives have potential as highly potent antitussives, free from the mu opioid agonist side effects. We attempted to optimize the NTI derivatives to develop novel antitussive agents. On the basis of the studies of structure-antitussive activity relationships of alkyl substituted NTI derivatives, we designed NTI derivatives with extra ring fused structures. As a clinical candidate, we identified a highly potent new compound, (5R,9R,13S,14S)-17-cyclopropylmethyl-6,7-didehydro-4,5-epoxy-5',6'-dihydro-3-methoxy-4'H-pyrrolo[3,2,1-ij]quinolino[2',1':6,7]morphinan-14-ol (5b) methanesulfonate (TRK-850) which was effective even by oral administration (ED50 6.40 microg/kg).     

3H-Codeine binding in the guinea pig lower brain stem

Saturable binding of (-)-3H codeine was found in the guinea pig medulla (KD = 5.6 x 10(-7) M, Bmax = 1.4 pmol/mg protein), whereas little stereospecific binding was detected (KD = 4.4 x 10(-5) M). The saturable binding of (-)-3H codeine was slightly enhanced by Na+ and by Mg++ but not by Li++ and Ca++. The enhancement appears to be due to an increase in the number of receptor sites. (-)-3H-Codeine binding was displaced by (-)- and (+)-codeine, morphine, (-)- and (+)-methadone but not by barbiturates. Naloxone, at a high concentration (1 x 10(-5) M), inhibits the binding by only 40%. This agrees with our previously published data which shows that the optical isomers of codeine had significant antitussive effects in the cat, these effects not being antagonized by naloxone. A class of opiate antitussive receptors, which are less naloxone-sensitive and less stereoselective than the mu receptors, is implicated.     

Urinary concentrations of codeine and morphine after the administration of different codeine preparations in relation to doping analysis.

A capillary GC method with nitrogen-specific detection is described for the analysis of codeine and morphine in urine. Both drugs were determined after enzymatic hydrolysis of the urine. Morphine was derivatized with trifluoroacetic anhydride. For 5-ml samples of urine, the lower detection limits for accurate quantitation were 50 ng ml-1 and 100 ng ml-1 for morphine and codeine, respectively. Both codeine and morphine were already detectable in urine 1 h after the intake of the analgesic preparation Perdolan. Codeine excretion and concentration peaked 2 h after administration of a dose. The percentage of the dose excreted as codeine was 3.0-6.2%. Administration of the antitussive preparation Bisolvon Griblettes resulted in detectable codeine and morphine levels for at least one day; 5.6-9% was excreted as total codeine over 24 h, the conjugated metabolite morphine accounting for 1.7-7.4% of the dose. Nearly the same amounts of codeine and morphine were excreted after administration of the antitussive syrup Bronchodine. The maximum excretion rate of codeine occurred after 1 h. Generally codeine and morphine remained detectable for 12 h. The results of these administration studies are discussed in relation to the codeine and morphine threshold levels recently introduced by the International Cyclist Union.     

Effects of codeine, morphine and a novel opioid pentapeptide BW443C, on cough, nociception and ventilation in the unanaesthetized guinea-pig.

1. Antitussive, antinociceptive and respiratory depressant effects of codeine, morphine and H.Tyr.D-Arg.Gly.Phe(4-NO2) Pro.NH2 (compound BW443C) were investigated in unanaesthetized guinea-pigs. Antagonism of the antitussive and antinociceptive effects was investigated by the use of nalorphine and N-methylnalorphine. Naloxone was used to antagonize respiratory depression. 2. Antitussive ED50s (with 95% confidence limits) for inhibition of cough induced by citric acid vapour were for codeine, morphine and BW443C respectively, 9.1(5.8-15), 1.3(0.7-2.4) and 1.2(0.6-2.6) mg kg-1 s.c. and 8.7(4.2-12), 1.6(1.2-1.9) and 0.67(0.002-3.3) mg kg-1, i.v. The antitussive effects of subcutaneous codeine (25 mg kg-1) morphine (8.1 mg kg-1) and BW443C (2.5 mg kg-1) were significantly antagonized by subcutaneous nalorphine (3.0 mg kg-1) and N-methylnalorphine (3.0 mg kg-1). 3. In the multiple toe-pinch test, the antinociceptive ED50s (with 95% confidence limits) of codeine and morphine were 18(16-22) and 2.3(0.4-4.3) mg kg-1, s.c., respectively. Compound BW443C was ineffective in doses of 2.5 and 10 mg kg-1 s.c., a result consistent with its lacking penetration into the CNS. Subcutaneous nalorphine (3.0 mg kg-1) antagonized the antinociceptive action of codeine (25 mg kg-1) and morphine (8.1 mg kg-1). In contrast, N-methylnalorphine (3.0 mg kg-1) had no significant effect on the antinociceptive action of codeine and morphine, suggesting lack of penetration of the CNS by N-methylnalorphine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacokinetics of pholcodine in healthy volunteers: single and chronic dosing studies.

1. The pharmacokinetics of pholcodine after two single doses and after chronic administration were studied in healthy human volunteers. 2. Six subjects received single oral doses of 20 and 60 mg of pholcodine according to a balanced cross-over design with an interval of 3 weeks between the two treatments. Blood and saliva samples and all urine were collected over 168 h after each dosage administration. Subsequently, the same subjects received 20 mg pholcodine 8 hourly orally for 10 days. Blood and saliva samples and all urine were collected during an 8 h dosing interval after the last dose on day 11. 3. Plasma, saliva and urine concentrations of pholcodine were determined by a high performance liquid chromatographic assay. 4. After the single doses, pholcodine was absorbed rapidly (tmax = 1.6 +/- 1.2 h) and eliminated slowly with a mean half-life of 50.1 +/- 4.1 h. The renal clearance of pholcodine was 137 +/- 34 ml min-1 and was inversely correlated with urine pH (r = 0.60) but not with urine flow rate. 26.2 +/- 3.3% of the dose was excreted as unchanged pholcodine after both doses. The concentration of pholcodine in saliva was 3.6 times higher than in plasma. 5. After chronic administration, the pharmacokinetics of pholcodine were not statistically different from the single dose parameters. 6. Pholcodine did not appear to undergo conjugation. The plasma protein binding was 23.5%. Morphine, in unconjugated or conjugated form, was not detected in the urine of any subject after pholcodine administration.     

Potentiation of antitussive effect of codeine by some 1-dimethoxyphenyl-3-alkylaminobutanols in guinea pigs

Newly synthesized 1-(2',5'-dimethoxyphenyl)-1-n-butyl-3-diethylaminobutanol (compd. 4) and its analogs enhanced the antitussive effect of codeine and morphine as tested on the cough induced by mechanical stimulation of the trachea in guinea pigs. This effect was illustrated to be a potentiation on the Gaddum's diagram. The following parameters were affected little or to a small extent: 1. analgesic effect of codeine and morphine in guinea pigs and mice, 2. duration of anesthesia induced by hexobarbital in mice, 3. respiratory depression caused by codeine in guinea pigs, and 4. LD50 of codeine in guinea pigs and mice. Explorations of the mechanism of potentiating action suggested some peripheral mechanism, but the exact one remained to be elucidated.     

Simultaneous determination of codeine and ethyl morphine HCL in tablet formulations using LC.

A reverse phase high performance liquid chromatography (HPLC) method was developed for the simultaneous determination of codeine (methyl morphine) and dionin (ethyl morphine hydrochloride) in antitussive analgesic tablet formulations. A C(18) column and methanol-water (1:2) mixture mobile phase (pH 3.0) were used. Spectrophotometric detection was carried out at 210 nm. The total elution time was shorter than 7 min. This method was found to be quite precise and reproducible. This proposed method was successfully applied to the determination of codeine and ethyl morphine hydrochloride in tablets produced by the Turkish Army Drug Factory.     

Impurity profiling of pholcodine by liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS)

Previously, a dimorpholinoethyl pholcodine manufacturing impurity was reported to be present in some samples of pholcodine. Apart from this impurity and morphine, other unknown impurities were detected in all the samples analysed by HPLC and micellar electrokinetic capillary chromatography. In this study, liquid chromatography mass spectrometry (LC-MS) analysis of samples of pholcodine showed that two of the previously unidentified compounds had mass spectra with molecular ions which differed from pholcodine by 16 amu. From this observation and other experimental data it was concluded that they are hydroxy derivatives of pholcodine. 10-S-hydroxy-pholcodine, which was synthesized by the oxidation of pholcodine with chromic acid, had the same chromatographic properties as one of these compounds. An early eluting compound in the LC-MS chromatograms of pholcodine was identified as pholcodine-N-oxide by matching chromatographic and mass spectral data of a synthesized pholcodine-N-oxide standard. The reaction of pholcodine with m-chloroperoxybenzoic acid not only produced the mono N-oxide, but also pholcodine-di-N,N'-oxide.     

Antitussive properties of levodropropizine

The antitussive activity of levodropropizine (S(-)-3-(4-phenyl-piperazin-1-yl)-propane-1,2-diol, DF 526), was evaluated in anaesthetized guinea-pigs and rabbits and in unanaesthetized guinea-pigs. Levodropropizine was shown to have good antitussive activity. Intravenously, it was 1/10 to 1/20 as active as codeine and comparable to dropropizine, from which it is derived, on mechanically and electrically induced coughing in rabbits and guinea-pigs. After oral administration to the guinea-pig the antitussive activity of levodropropizine was comparable with those of both dropropizine and codeine against coughing induced by irritant aerosols.     

Effects of Ethanol on Ethylmorphine Metabolism in Isolated Rat Hepatocytes: Characterization by Means of a Multicompartmental Model

Abstract: Hepatic cytochrome P-450 enzymes mediate at least two important biotransformation pathways of codeine and ethylmorphine starting with either N-demethylation or O-dealkylation, producing polar metabolites which are then subsequently glucuronidated. The present study was designed to characterise the acute effects of ethanol on the metabolism of ethylmorphine and to compare it with the effects on codeine in suspensions of freshly isolated rat hepatocytes. Isolated rat hepatocytes from male Wistar rats were prepared by a collagenase perfusion method. Ethylmorphine, codeine and their metabolites were quantified by HPLC with UV detection. The total ethylmorphine elimination rate was reduced by 12% at 5 mM and 38% at 100 mM ethanol. The corresponding percentages for codeine were 16 and 43%. In the presence of ethanol the concentrations of several intermediate and end products of ethylmorphine and codeine changed markedly from the control situation. The experimental data were applied to a mathematical compartmental linear model to estimate the influence of ethanol on the separate reaction rates in the two main metabolic pathways. The ratios between reaction rate constants in the ethylmorphine experiments at 100 and 0 mM ethanol were 0.65 for ethylmorphine→norethyl-morphine, 0.63 for norethylmorphine→normorphine, 0.56 for ethylmorphine→morphine, 0.49 for morphine→normor-phine, 0.31 for normorphine→normorphine-3-glucuronide and 0.49 for morphine→morphine-3-glucuronide. Almost similar effects of ethanol on codeine metabolism were found. In additional experiments, norethylmorphine or norcodeine (50 μM) was incubated with 5 mM to 100 mM of ethanol and the metabolism of both norethylmorphine and norcodeine was found to be inhibited by ethanol in a concentration-dependent manner. The glucuronidation of morphine and normorphine added in separate experiments was also inhibited by ethanol, from 22 to 36% for morphine-3-glucuronide and 30 to 60% for normorphine-3-glucuronide, respectively, in the presence of 5 mM to 100 mM of ethanol. It was concluded that all steps in the metabolism of ethylmorphine (and codeine) leading to the end products morphine-3-glucuronide and normorphine-3-glucuronide were inhibited by ethanol, and that the glucuronidation processes were the ones most affected by ethanol.     

Evidence for peripheral mechanisms mediating the antitussive actions of opioids in the guinea pig.

1. Comparisons were made between the doses required of aerosol and intraperitoneally administered morphine, dextromethorphan, codeine and the specific peripherally acting mu-receptor agonist DALDA (H-Tyr-D-Arg-Phe-Lys-NH2) to suppress citric acid-induced coughing in conscious guinea pigs. 2. Estimated ID50s for inhibition of numbers of coughs induced by an aerosol of 5% citric acid were 1.0 and 2.4 mg/kg for intraperitoneally administered morphine and dextromethorphan, respectively. 3. The estimated ID50s after inhalation of morphine and dextromethorphan as aerosols were approximately 2.2 and approximately 12 micrograms/kg, respectively. 4. Aerosilized codeine (approximately 72 micrograms/kg, n = 5) significantly inhibited coughing by 62 +/- 23% whereas 3 mg/kg, i.p. was required to significantly reduce coughing by a similar degree (60 +/- 6%, n = 7). 5. Inhalation of DALDA (approximately 7.2 micrograms/kg, n = 7) also significantly inhibited coughing. 6. The antitussive effect of inhaled morphine (approximately 7.2 micrograms/kg, n = 11) was inhibited after administration of 3 mg/kg of either naloxone hydrochloride or naloxone methylbromide intraperitoneally. 7. The results support the hypothesis that effects at a peripheral site can make a major contribution to the antitussive actions of these drugs.     

Comparison of the antinociceptive effect of morphine, methadone, buprenorphine and codeine in two substrains of Sprague-Dawley rats

Sprague-Dawley rats from two different vendors, M?lleg?rd, Denmark and B&K Universal, Sweden, have been tested for the antinociceptive effect of morphine, methadone, buprenorphine and codeine on the hot plate. Morphine and methadone had significantly weaker effect in M?lleg?rd rats compare to B&K rats. In contrast, the effect of buprenorphine was stronger in M?lleg?rd rats than in B&K rats and the effect of codeine was similar in the two substrains. Plasma levels of morphine, morphine-6-glucuronide, morphine-3-glucuronide, buprenorphine and norbuprenorphine were determined at two time points after drug injection. M?lleg?rd rats had significantly lower mean plasma level of morphine and significantly higher ratio of morphine-3-glucuronide/morphine at 30 min, compared to B&K rats. No difference was seen for the metabolism of buprenorphine in the two substrains. The results suggest that M?lleg?rd rats metabolize morphine to morphine-3-glucuronide to a greater extent than B&K rats, and this may at least partly underlie the substrain difference in the effect of morphine. It is also suggested that the antinociceptive mechanisms of buprenorphne may be different from those of morphine and methadone.     

Dextromethorphan differentially affects opioid antinociception in rats

Opioid drugs such as morphine and meperidine are widely used in clinical pain management, although they can cause some adverse effects. A number of studies indicate that N-methyl-D-aspartate (NMDA) receptors may play a role in the mechanism of morphine analgesia, tolerance and dependence. Being an antitussive with NMDA antagonist properties, dextromethorphan (DM) may have some therapeutic benefits when coadministered with morphine. In the present study, we investigated the effects of DM on the antinociceptive effects of different opioids. We also investigated the possible pharmacokinetic mechanisms involved. The antinociceptive effects of the mu-opioid receptor agonists morphine (5 mg kg(-1), s.c.), meperidine (25 mg kg(-1), s.c.) and codeine (25 mg kg(-1), s.c.), and the kappa-opioid agonists nalbuphine (8 mg kg(-1), s.c.) and U-50,488H (20 mg kg(-1), s.c.) were studied using the tail-flick test in male Sprague-Dawley rats. Coadministration of DM (20 mg kg(-1), i.p.) with these opioids was also performed and investigated.The pharmacokinetic effects of DM on morphine and codeine were examined, and the free concentration of morphine or codeine in serum was determined by HPLC.It was found that DM potentiated the antinociceptive effects of some mu-opioid agonists but not codeine or kappa-opioid agonists in rats. DM potentiated morphine's antinociceptive effect, and acutely increased the serum concentration of morphine. In contrast, DM attenuated the antinociceptive effect of codeine and decreased the serum concentration of its active metabolite (morphine).The pharmacokinetic interactions between DM and opioids may partially explain the differential effects of DM on the antinociception caused by opioids.     

Antitussive effect of RU-20201--central and peripheral actions

The antitussive effect of the new compound 1, 2, 3, 4a, 9b-hexahydro-8, 9b-dimethyl-4-[3-(4-methyl-piperazine-1-yl) propionamide] dibenzofuran-3-one dihydrochloride (RU-20201) was investigated in dogs and guinea pigs, including its sites of action. The antitussive effect of RU-20201 was about 1/10 as potent as that of codeine phosphate in dogs with the puncture electrode-induced cough (PEC) method and about 1/12 and 1/4 as potent as that of codeine phosphate in guinea pigs with the PEC and chemical stimulation methods, respectively. When RU-20201 was administered in a dose range of 1 to 10 mg into the vertebral artery toward the brain in lightly anesthetized dogs, no antitussive effect was observed against the coughing elicited by electrical stimulation of the central cut end of the superior laryngeal nerve. However, a stimulative effect on respiration, especially on respiratory rate occurred. The peripheral effect of RU-20201 on the cough was investigated using the in situ upper trachea perfusion preparation which allows a direct drug administration to the local site around the tracheal mucosa, this site being electrically stimulated to induce coughing. A close i.a. infusion of RU-20201 in doses of 1 and 3 mg/min into the tracheal vascular bed for 5 min inhibited the cough response elicited by mucosal stimulation. The above findings suggest that RU-20201 has a significant antitussive activity, the site of action being probably, at least, at the cough receptor level.     

Synthesis and antitussive activity of 3-azabicyclo[3.2.2]nonane derivatives

Mannich bases derived from a number of substituted acetophenones and propiophenones and 3-azabicyclo[3.2.2]nonane have been evaluated for antitussive activity. One of these compounds was as potent as codeine and dextromethorphan in its antitussive activity. The most potent compound of the series, 3-(3-azabicyclo[2.2.2]nonan-3-yl)-4'-benzyloxy-2-methyl propiophenone, also exhibited antimorphine activity. There was no direct correlation between the antitussive effect and antimorphine activity.     

The role of bronchoconstriction in cough reflex.

To investigate the role of bronchoconstriction in the cough reflex, we compared antitussive effects of several drugs with their ability to effect the respiratory tract (i.e. bronchodilation vs. bronchoconstriction). Antitussive activities of five drugs administered either intravenously or orally on electrically-induced cough were evaluated in the non-anesthetized dog. The antitussive activities were as follows: morphine, 0.1 mg/kg (i.v.) and 0.5 mg/kg (p.o.); codeine, 1.0, 4.0; picoperidamine, 2.0, 9.8; piclobetol, 7.6, 9.0; HH-197, 12.5, 143.0, respectively. Morphine, codeine and HH-197 caused bronchoconstriction, but picoperidamine and picrobetol caused bronchodilation. The antitussive and bronchodilatation effects of isoproterenol were abolished by propranolol. Each bronchoconstricting drug (i.e. morphine, codeine and HH-197) was administered concurrently with isoproterenol (10 mug/kg, i.v., and non-antitussive activity), and the cough reflex was observed. Compared with the single administration of each drug, respiratory resistance was decreased and the antitussive effect was increased. These results indicate a strong correlation between bronchodilatation and increased antitussive activity.     

The antitussive activity of a novel compound RU 20201

A novel compound, 1,2,3,4,4a,9b-hexahydro-8,9b-dimethyl-4-[3-(4-methylpiperazin-1-yl)propionamido]dibenzofuran-3-one dihydrochloride (RU 20201), has been shown to have comparable antitussive activity to that of codeine phosphate in animal experiments after oral administration. RU 20201 was also shown to have local antitussive activity when given by aerosol. This effect took place immediately. Initial observations suggest that RU 20201 is an antitussive compound that exerts its activity in the lungs, probably by direct inhibition of superficial receptors in the respiratory tract.