Naloxone blocks suppression of cough by codeine in anesthetized rabbits.

Opioid receptors which are involved in cough generation are abundantly expressed in the brainstem. Codeine is a potent µ-opioid receptor agonist. In the present study we examined the effects of naloxone, a µ-opioid receptor antagonist, on mechanically-induced tracheobronchial cough and on the cough suppressing effect of codeine in six pentobarbitone anesthetized spontaneously breathing rabbits. A single dose of naloxone (0.4 mg/kg) followed by a single dose of codeine (7 mg/kg) were administered intravenously. The number and amplitude of cough and sneeze reflexes were examined sequentially; before and after naloxone, and then after codeine. We found that neither did naloxone alone nor codeine given after prior naloxone pretreatment appreciably affect coughing or sneezing. Likewise, there were no significant differences in the diaphragm and abdominal muscles electromyographic moving averages, or the inspiratory and expiratory esophageal pressure amplitudes. However, we detected a tendency for the rise in expiratory motor drive during coughing and sneezing after injection of naloxone. The respiratory rate was significantly higher after naloxone in comparison with control (P < 0.001). No significant differences in arterial blood pressure were observed. We conclude that the failure of codeine to suppress the cough reflex on the background of naloxone pretreatment confirms the involvement of µ-opioid mechanism in the central antitussive effect of codeine.

Microsomal codeine N-demethylation: cosegregation with cytochrome P4503A4 activity.

Codeine is metabolized by glucuronidation, by O-demethylation to morphine, and by N-demethylation to norcodeine. The enzyme responsible for the O-demethylation to morphine has been identified as cytochrome P4502D6 (CYP2D6). The purpose of the present study was to identify the specific P450 enzyme responsible for codeine N-demethylation. Microsomal preparations (250 pmol of P450) obtained from 12 human liver donors were incubated with 20 microM codeine and analyzed for norcodeine formation. Codeine N-demethylation activity was linearly correlated with nifedipine oxidation activity (r = 0.90, p < 0.001), a marker of CYP3A4, but not with codeine O-demethylation, a marker of CYP2D6. Preincubation with troleandomycin (50 microM), or gestodene (50 microM) inhibitors of CYP3A4, decreased the rate of production of norcodeine by 60 and 45% compared to control values, respectively. Similarly, ketoconazole (10 microM) and erythromycin (10 microM) inhibited codeine N-demethylation by 75 and 35%, respectively. In contrast, the presence of quinidine, sulfaphenazole, or diethyldithiocarbamate in the incubation mixture had no effect on norcodeine formation. Preincubation with antibodies raised to CYP3A4 (5 mg lgG/nmol P450) caused 96% inhibition of norcodeine production, whereas preimmune IgG or antibodies raised to CYP2A6 and CYP2C had no effect. Additionally, significant norcodeine production was observed with purified CYP3A4 derived from human liver microsomes. In conclusion, codeine N-demethylation activity cosegregates with CYP3A4 activity. Coadministration of codeine with selective inhibitors of CYP3A4 may result in increased morphine production and enhanced pharmacodynamic effects due to shunting down the CYP2D6 pathway.

Conjunctival provocation tests with codeine phosphate. Effect of disodium cromoglycate.

The reactivity of ocular mast cells is poorly characterized in man. Provocation tests with codeine phosphate, a molecule known to activate connective tissue mast cells, were performed in ten normal subjects. Ten-fold increasing concentrations of codeine phosphate (10(-5) to 10(-1) mg/mL) were tested in both eyes until a positive challenge was observed. Schirmer strips were placed under the eyelid and left for five minutes. A negative control was performed ten days later. All subjects had a strongly positive reaction for the same codeine phosphate concentration (10(-1) mg/mL). Histamine was released in 8/10 subjects (control: 7.06 +/- 4.19 nM/L, codeine phosphate: 18.2 +/- 15.7 nM/L, P < .018), PGD2 was released in 8/10 subjects (control: 0 codeine phosphate: 273.3 +/- 408.9 ng/L). Disodium cromoglycate blocked the release of histamine and PGD2. Codeine phosphate is potent at causing mast cell activation in the eye and this effect is blocked by disodium cromoglycate.

Codeine O-demethylation: rat strain differences and the effects of inhibitors.

The oxidative metabolism of more than 20 drugs (e.g. sparteine, debrisoquine, dextromethorphan) is mediated by cytochrome P450IID6. Codeine O-demethylation to morphine was recently demonstrated to co-segregate with the polymorphic metabolism of debrisoquine and dextromethorphan. The female Dark-Agouti rat (DA) is an animal model for the poor metabolizer phenotype (PM) using debrisoquine or dextromethorphan as substrates. Studies were carried out to evaluate codeine metabolism in liver microsomes from female DA and Sprague-Dawley (SD) rats. The intrinsic clearance of codeine to morphine was 10-fold lower in DA rats due to a 5-fold higher Km (287 vs 49 microM) and a 2-fold lower Vmax (48 vs 94 nmol/mg/hr). Nineteen drugs were tested for inhibition of codeine O-demethylation. The four most potent competitive inhibitors were dextromethorphan (Ki = 2.53 microM), propafenone (Ki = 0.58 microM), racemic methadone (Ki = 0.3 microM) and quinine (Ki = 0.07 microM). The differences in morphine formation from codeine between SD and DA rats and the inhibition results show that this animal model appears to be a suitable model for the human EM and PM phenotypes, respectively. These strains could be used to study the pharmacodynamic consequences of the genetic polymorphism in codeine O-demethylation, and the effects of metabolic inhibitors. The outcome of these studies could impact on the therapy of pain control.

Changes in responsiveness to mu and kappa opiates following a series of convulsions.

After a series of seven electroconvulsive shocks, mice (C57BL/6J) showed a marked change in their response to opiates. Although very large doses of mu agonists induce convulsions in normal control mice, our evidence indicated that this was accomplished through nonopiate mechanisms: they could not be blocked by naltrexone and the pattern of drug potencies (codeine greater than morphine greater than levorphanol) was not consistent with an opiate response. In contrast, after electroconvulsive shock small doses of mu agonists induced convulsions that could be blocked by naltrexone and the pattern of drug potency (levorphanol greater than morphine greater than codeine) was consistent with an opiate mechanism. Kappa drugs, on the other hand, produced convulsions in both control and ECS animals, although there was an enhanced responsiveness in the latter. Furthermore, the convulsions produced by kappa drugs were blocked by naltrexone and showed stereoselectivity in both control and ECS animals. The changes in responsiveness to mu and kappa opiates cannot be explained on the basis of a general increase in seizure susceptibility, as sensitivity to the nonopiate convulsant, strychnine, was not enhanced after electroconvulsive shock. The results point to a qualitative change in response to mu agonists after electroconvulsive shock, but only a change in sensitivity to kappa agonists.

RX 336-M, a new chemical tool in the analysis of the quasi-morphine withdrawal syndrome.

RX 336-M (7,8-dihydro-5',6'-dimethylcyclohex-5'-eno-1',2',8',14 codeinone) and four other chemically-diverse agents--AG-3-5 (1-[2-hydroxyphenyl]-4-[3-nitrophenyl]-1,2,3,6-tetrahydropyrimidine-2-one), Sgd 8473 (alpha-[4-chlorobenzylideneamino)-oxy]-isobutyric acid), thyrotropin releasing hormone (TRH), and sodium valproate--each induce signs of withdrawal, most notably 'wet-dog' shaking, after acute i.p. administration in drug-naive rats. They are therefore additions to a recently recognized and, as yet, ill-defined class of behaviorally active compounds. The pharmacological baselines that link these disparate agents together have been studied in the present work, using 'wet-dog' shaking as the behavioral measure and RX 336-M as the reference shake-inducing compound. Peripheral administration of clonidine, haloperidol, d-lysergic acid diethylamide, or morphine suppressed chemically induced shaking: naloxone had no marked effect. Reverse tolerance was associated with TRH-induced shaking whereas tolerance occurred with the other four compounds. Cross-tolerance interactions were asymmetrical. Thus, rats rendered tolerant to RX 336-M were cross-tolerant to AG-3-5, TRH, and sodium valproate but not to Sgd 8473; in contrast, RX 336-M-induced shaking was only significantly reduced in rats made tolerant to Sgd 8473. In view of the unidirectional nature of the cross-tolerance relationships studied, it is concluded that AG-3-5, Sgd 8473, sodium valproate, and TRH initiate 'wet-dog' shaking through neural substrates that differ from the one(s) associated with RX 336-M. Nevertheless, all five compounds may eventually trigger a common shake-inducing mechanism.

Comparative studies of the pharmacological effects of the d- and l-isomers of codeine.

Opiates are known for their stereospecificity. The following studies show that l-codeine was active in the mouse tail-flick test as well as in the hot plate test whether given p.o. or s.c. The ED50 in the first test was 4.09 mg/kg s.c. (2.01-8.34 mg/ kg) and 13.41 mg/kg p.o. (6.91-26.0 mg/kg). In the second antinociceptive test, the ED50 was 20.66 mg/kg s.c. (11.52-37.08 mg/kg) and 20.47 mg/kg p.o. (14.63-28.57 mg/kg). The d-isomer of codeine was inactive ina both tests up to 100 mg/kg but caused hyperexcitability, convulsions and ultimately death. Although l-codeine was more potent than d-codeine inhibiting the cough reflex in the anesthetized cat, the d-compound did have good activity. The ED50 of the l-isomer was 0.27 mg/kgi.v. (0.14-0.47 mg/kg) and that of the d-isomer was 1.61 mg/kg i.v. (0.98-2.65 mg/kg). In these animals, l-codeine did not significantly affect the cardiovascular parameters at the doses tested, whereas d-codeine caused a significant but transient decrease in the blood pressure and heart rate. The specific and nonspecific properties of d- and l-codeine were further delineated in the opiate receptor binding assay. l-Codeine inhibited the stereospecific binding of 2.2 x 10(-9) M [3H]dihydromorphine in mouse brain homogenate with the IC50 being 1.6 x 10(-5) M (1.2 x 10(-5)--2.0 x 10(-5) M). d-Codeine had no effect up to 10(-4) M.

Lysergic acid diethylamide antagonizes shaking induced in rats by five chemically different compounds.

Thyrotropin-releasing hormone (TRH), sodium valproate, AF-3-5 (1-[2-hydroxyphenyl]-4-[3-nitrophenyl]-1,2,3,6-tetrahydropyrimidine-2-one), RX336-M (7,8-dihydro-5',6'-dimethylcyclohex-5'-eno-1',2',8',14 codeinone), and Sgd 8473 (alpha-[4-chlorobenzylideneamino)-oxy]-isobutyric acid) each induced repetitive shaking of the body of rats after intraperitoneal injection. This action of the five diverse chemicals appears to be subserved by a common pharmacological component, because pretreatment with d-lysergic acid diethylamide (0.03--1.0 mg kg-1, s.c.) attenuated the shaking behavior in a dose-related manner, and cross tolerance was found between RX336-M and TRH, sodium valproate, and AG-3-5.