Analgesia and serum assays of controlled-release dihydrocodeine and metabolites in cancer patients with pain.

Aim of the study was to assess dihydrocodeine (DHC) and metabolites concentrations and their correlations with DHC analgesia in cancer patients with pain. Thirty opioid-naive patients with nociceptive pain intensity assessed by VAS (visual analogue scale) > 40 received controlled-release DHC as the first (15 patients, 7 days) or as the second opioid (15 patients, 7 days). Blood samples were taken on day 2, 4 and 7 at each study period. DHC and its metabolites were assayed by HPLC. DHC provided satisfactory analgesia when administered as the first or the second opioid superior to that of tramadol. When DHC was the first opioid administered, DHC and dihydrocodeine-6-glucuronide (DHC-6-G) concentrations increased in the second and the third comparing to the first assay. A trend of nordihydromorphine (NDHM) level fall between the first and the third assay was noted; trends of dihydromorphine (DHM) level increase in the second relative to the first determination and decrease in the third compared to the second assay were observed. When DHC followed tramadol treatment a trend of DHC concentration increase in the second relative to the first assay was noted. DHC-6-G level increased in the second and in the third comparing to the first determination; NDHM and DHM concentrations were stable. DHC and DHC-6-G concentrations increased similarly during both treatment periods which suggest their prominent role in DHC analgesia. Few significant correlations were found between DHC dose, DHC and metabolites serum concentrations with analgesia suggesting the individual DHC dose titration.

Morphine-related metabolites differentially activate adenylyl cyclase isozymes after acute and chronic administration.

Morphine-3- and morphine-6-glucuronide are morphine's major metabolites. As morphine-6-glucuronide produces stronger analgesia than morphine, we investigated the effects of acute and chronic morphine glucuronides on adenylyl cyclase (AC) activity. Using COS-7 cells cotransfected with representatives of the nine cloned AC isozymes, we show that AC-I and V are inhibited by acute morphine and morphine-6-glucuronide, and undergo superactivation upon chronic exposure, while AC-II is stimulated by acute and inhibited by chronic treatment. Morphine-3-glucuronide had no effect. The weak opiate agonists codeine and dihydrocodeine are also addictive. These opiates, in contrast to their 3-O-demethylated metabolites morphine and dihydromorphine (formed by cytochrome P450 2D6), demonstrated neither acute inhibition nor chronic-induced superactivation. These results suggest that metabolites of morphine (morphine-6-glucuronide) and codeine/dihydrocodeine (morphine/dihydromorphine) may contribute to the development of opiate addiction.

The detection of dihydrocodeine and its main metabolites in cases of fatal overdose.

The levels of dihydrocodeine found in impaired individuals and in fatalities show a wide overlap in the ranges. Among other factors, the genetically controlled metabolism of dihydrocodeine should play an important role in dihydrocodeine toxicity. For the first time, the most important metabolites of dihydrocodeine were investigated in femoral blood from three fatal cases by simultaneous determination using HPLC and native fluorescence for detection. The amount of parent drug always exceeded dihydrocodeine-glucuronide formation and dihydromorphine concentrations ranged from 0.16-0.21 mg/L. The similar binding affinities of dihydromorphine and morphine to mu-opioid receptors suggest similar pharmacological effects and adverse reactions. The determination of the pharmacologically active metabolites should help to clarify the cause of death in fatal cases especially if a relatively low concentration of the parent drug is found.

Mu3 opiate receptor expression in lung and lung carcinoma: ligand binding and coupling to nitric oxide release.

The mu3 opiate receptor subtype is expressed in human surgical specimens of both normal lung and non-small-cell lung carcinoma. Nitric oxide (NO) release is mediated through the mu3 receptor, and in lung carcinoma, morphine-stimulated NO release is significantly higher and prolonged than in normal lung. Using reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot analysis we show that specific mu opioid receptor transcripts are present in lung carcinoma and other cells with the mu3 profile. Our findings identify a unique role for the mu3 opiate receptor in opiate-mediated NO release and suggest that endogenous opiates, through their release of NO, may play a role in cancer progression.

The visceral and somatic antinociceptive effects of dihydrocodeine and its metabolite, dihydromorphine. A cross-over study with extensive and quinidine-induced poor metabolizers.

AIMS: Dihydrocodeine is metabolized to dihydromorphine via the isoenzyme cytochrome P450 2D6, whose activity is determined by genetic polymorphism. The importance of the dihydromorphine metabolites for analgesia in poor metabolizers is unclear. The aim of this study was to assess the importance of the dihydromorphine metabolites of dihydrocodeine in analgesia by investigating the effects of dihydrocodeine on somatic and visceral pain thresholds in extensive and quinidine-induced poor metabolizers. METHODS: Eleven healthy subjects participated in a double-blind, randomized, placebo-controlled, four-way cross-over study comparing the effects of single doses of placebo and slow-release dihydrocodeine 60 mg with and without premedication with quinidine sulphate 50 mg on electrical, heat and rectal distension pain tolerance thresholds. Plasma concentrations and urinary excretion of dihydrocodeine and dihydromorphine were measured. RESULTS: In quinidine-induced poor metabolizers the plasma concentrations of dihydromorphine were reduced between 3 and 4 fold from 1.5 h to 13.5 h after dosing (P < 0.005) and urinary excretion of dihydromorphine in the first 12 h was decreased from 0.91% to 0.28% of the dihydrocodeine dose (P < 0.001). Dihydrocodeine significantly raised the heat pain tolerance thresholds (at 3.3 h and 5 h postdosing, P < 0.05) and the rectal distension defaecatory urge (at 3.3 h and 10 h postdosing, P < 0.02) and pain tolerance thresholds (at 3.3 h and 5 h postdosing, P < 0.05) compared with placebo. Premedication with quinidine did not change the effects of dihydrocodeine on pain thresholds, but decreased the effect of dihydrocodeine on defaecatory urge thresholds (at 1.5 h, 3.3 h and 10 h postdosing, P < 0.05). CONCLUSIONS: In quinidine-induced poor metabolizers significant reduction in dihydromorphine metabolite production did not result in diminished analgesic effects of a single dose of dihydrocodeine. The metabolism of dihydrocodeine to dihydromorphine may therefore not be of clinical importance for analgesia. This conclusion must however, be confirmed with repeated dosing in patients with pain.

Synthesis and structure-activity relationships of 6,7-benzomorphan derivatives as antagonists of the NMDA receptor-channel complex.

We have synthesized a series of stereoisomeric 6,7-benzomorphan derivatives with modified N-substituents and determined their ability to antagonize the N-methyl-D-aspartate (NMDA) receptor-channel complex in vitro and in vivo. The ability of the compounds to displace [3H]-MK-801 from the channel site of the NMDA receptor in rat brain synaptosomal membranes and to inhibit NMDA-induced lethality in mice was compared with their ability to bind to the mu opioid receptor. Examination of structure-activity relationships showed that the absolute stereochemistry is critically important for differentiating these two effects. (-)-1R,9 beta,2"S-enantiomers exhibited a higher affinity for the NMDA receptor-channel complex than for the mu opioid receptor. The aromatic hydroxy function was also found to influence the specificity of the compounds. Shift of the hydroxy group from the 2'-position to the 3'-position significantly increased the affinity for the NMDA receptor-channel complex and considerably reduced the affinity for the mu opioid receptor. From this series of 6,7-benzomorphan derivatives, the compound 15cr.HCl [(2R)-[2 alpha, 3(R*),6 alpha]-1,2,3,4,5,6-hexahydro-3-(2-methoxypropyl)-6,11,11-trimethyl -2,6-methano-3-benzazocin-9-ol hydrochloride] was chosen as the optimum candidate for further pharmacological investigations.

Ligand recognition in mu opioid receptor: experimentally based modeling of mu opioid receptor binding sites and their testing by ligand docking.

For three-dimensional understanding of the mechanisms that control potency and selectivity of the ligand binding at the atomic level, we have analysed opioid receptor-ligand interaction based on the receptor's 3D model. As a first step, we have constructed molecular models for the multiple opioid receptor subtypes using bacteriorhodopsin as a template. The S-activated dihydromorphine derivatives should serve as powerful tools in mapping the three-dimensional structure of the mu opioid receptor, including the nature of the agonist-mediated conformational change that permits G protein-coupling to "second messenger' effector molecules, and in identifying specific ligand-binding contacts with the mu opioid receptor. The analyses of the interactions of some opioid ligands with the predicted ligand binding sites are consistent with the results of the affinity labeling experiments.

Thermodynamic parameters of opioid binding in the presence and absence of G-protein coupling.

We have investigated the thermodynamic parameters of various opioid ligands interacting with their receptors in rat brain membranes. Affinity constants (Ka), enthalpy and entropy values were obtained from homologous displacement experiments performed at 0, 24 and 33 degrees C. It was found that all the opioid agonists tested ([3H]dihydromorphine (DHM) mu alkaloid; [3H]DAMGO mu peptide; [3H]deltorphin-B delta peptide) display endothermic binding accompanied with a large entropy increase, regardless of their chemical structure (alkaloid or peptide), or of their mu or delta receptor selectivity. In contrast, binding of the antagonist naloxone is exothermic, mainly enthalpy driven. Na+ or Mg2+ results only in quantitative changes of the thermodynamic parameters. In the presence of the GTP-analog Gpp(NH)p; or Gpp(NH)p + Na+; or Gpp(NH)p + Na- + Mg2+ the affinity of DHM binding dramatically decreases which might reflect functional uncoupling of the receptor-ligand complex and G-proteins. This altered molecular interactions are also indicated by curvilinear van't Hoff plot and entropy increase. It is concluded that the thermodynamic analysis provides means of determining the underlying driving forces of ligand binding and helps to delineate its mechanism.

The presence of the mu3 opiate receptor in invertebrate neural tissues.

A previous report demonstrated the presence of the newly discovered opiate alkaloid selective and opioid peptide insensitive mu3 receptor in ganglia of several invertebrate- and one vertebrate species as well as in microglial cells that had egressed from these ganglia after their maintenance in culture medium for several days. In the present study carried out in two representatives of invertebrates, the binding densities of this receptor determined in intact ganglia were compared with those in ganglia depleted of microglial cells. The aim was to ascertain whether the differences in binding capacity recorded in those two groups of ganglia might give an indication of the possible presence of this opiate receptor in nonmicroglial components of the nervous tissue, i.e., neurons. Within a period of 72 h of incubation, the gradual reduction in binding density had reached a plateau, in accordance with the termination of the egress of microglia. The fact that at least two thirds of the binding capacity of mu3 receptors were retained by the ganglia strongly suggests that part of this capacity may be attributed to neurons. This view is supported by additional data, in particular the demonstration of endogenous morphine in nervous tissue and its localization within distinct neurons.

Presence of the mu3 opiate receptor in endothelial cells. Coupling to nitric oxide production and vasodilation.

Initial confinement of opiate receptors to the nervous system has recently been broadened to several other cell types. Based on the well established hypotensive effect of morphine, we hypothesized that endothelial cells may represent a target for this opiate substance. Endothelial cells (human arterial and rat microvascular) contain a high affinity, saturable opiate binding site presumed to mediate the morphine effects that is stereoselectively and characteristically antagonized by naloxone. This opiate alkaloid-specific binding site is insensitive to opioid peptides. It is, therefore, considered to be the same subtype of opiate receptor (designated mu3) used in the mediation of morphine in other cell types exhibiting the same binding profile. Experiments with endothelial cultures and the aortic ring of rats cultured in vitro demonstrate that morphine exerts direct modulatory control over the activities of endothelial cells, which leads to vasodilation. It induces the production of nitric oxide, a process that is sensitive to naloxone antagonism and nitric oxide synthase inhibition. In contrast with that of opiates, the administration of opioid peptides does not induce nitric oxide production by endothelial cells. In conclusion, the data presented above reveal a novel site of morphine action, endothelial cells, where a mu3 receptor is coupled to nitric oxide release and vasodilation.

Dihydrocodeine: a new opioid substrate for the polymorphic CYP2D6 in humans.

BACKGROUND: The opioid dihydrocodeine (DHC) is frequently used as an analgesic and antitussive agent. However, until now there have been no detailed data on dihydrocodeine metabolism in humans. We therefore investigated pathways that contribute to elimination of dihydrocodeine, and we tested the hypothesis that dihydrocodeine O-demethylation to dihydromorphine (DHM) is catalyzed by the polymorphic CYP2D6. METHODS: A single oral dose of dihydrocodeine was administered to six extensive (metabolic ratio [MR] < or = 1), two intermediate (1 < MR < 20) and six poor metabolizers (MR > or = 20) of sparteine/debrisoquin. Serum concentrations of dihydrocodeine and dihydromorphine were measured up to 25 hours, and urinary excretion of conjugated and unconjugated dihydrocodeine, dihydromorphine, and nordihydrocodeine were determined. RESULTS: There were no differences in the pharmacokinetics of dihydrocodeine between extensive and poor metabolizers. However, the area under the serum concentration-time curve (AUC), partial metabolic clearance, and total urinary recovery of dihydromorphine were significantly lower in poor metabolizers (10.3 +/- 6.1 nmol.hr/L; 7.0 +/- 4.1 ml/min; 1.3% +/- 0.9% of dose) compared with extensive metabolizers (75.5 +/- 42.9 nmol.hr/L; 49.7 +/- 29.9 ml/min; 8.9% +/- 6.2%; p < 0.01). There was a strong correlation between the AUCDHC/AUCDHM ratio and the urinary metabolic ratio of sparteine (rS = 0.89, p = 0.001). No significant differences between extensive and poor metabolizers were detected in urine for conjugated dihydrocodeine (extensive metabolizers, 27.7% of dose; poor metabolizers, 31.5%), unconjugated dihydrocodeine (extensive metabolizers, 31.1%; poor metabolizers, 31.1%), conjugated nordihydrocodeine (extensive metabolizers, 6.3%; poor metabolizers, 5.4%), or unconjugated nordihydrocodeine (extensive metabolizers, 15.8%; poor metabolizers, 19.5%). CONCLUSIONS: Dihydrocodeine O-demethylation to dihydromorphine is impaired in poor metabolizers of sparteine. The main urinary metabolites after administration of dihydrocodeine are the parent compound and its conjugates in extensive and poor metabolizers.

Binding characteristics of hypothalamic mu opioid receptors throughout the estrous cycle in the rat.

This study presents a detailed analysis of the binding characteristics of hypothalamic mu opioid receptors during the different phases of the estrous cycle of the female rat. Different groups of adult female rats with a regular 4-day estrous cycle were killed by decapitation at 10.00 h of diestrus days 1 and 2, at 10.00, 12.00, 14.00, 16.00, 18.00 and 20.00 h of the day of proestrus and at 10.00, 12.00, 14.00, 16.00 and 18.00 h of the day of estrus. At sacrifice, the hypothalami of the animals were dissected out, plasma membrane preparations were obtained and the binding characteristics (Bmax, Kd) of the specific mu opioid ligand dihydromorphine (DHM) on mu opioid receptors were evaluated. Blood has been collected from the trunk vessels to monitor with specific radioimmunoassays serum levels of LH, estradiol and progesterone. The data obtained indicate that in the hypothalamus of female rats with a regular 4-day estrous cycle, the binding characteristics of DHM for mu receptors show important variations during the different phases of the estrous cycle. In general, the number of mu opioid receptors is elevated during the morning of diestrus day 2 and during the day of proestrus being maximal at 14.00 h and declining significantly at 18.00 and 20.00 h of the same day. At estrus, the number of mu receptors appears high at 10.00 and 16.00 h and low at 12.00, 14.00 and 18.00 h. All these variations take place without any significant change of the affinity (Kd) of DHM for the mu receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

The E. coli envY gene encodes a high affinity opioid binding site.

In an attempt to isolate a cDNA encoding an opioid receptor, a cDNA library was constructed in the lambda ZAP vector using NG108-15 mRNA as template. Using an in vitro transcription-translation assay and a sib selection strategy, a single phage was isolated. An RNA transcribed from this cDNA was able to direct in vitro translation of opioid binding sites. The insert was sequenced and comparison with data banks showed a 100% homology with the E. coli envY gene. We assume that the presence of the envY sequence in the NG108-15 cDNA library was due to a contamination of the lambda ZAP vector with E. coli DNA. A search for opioid binding sites on E. coli strains showed that envY+ strains, but not envY- mutants were able to bind opiates. On envY+ cells, the sites are stereospecific, saturable and of high affinity for the opiate ligands. These sites bind opiate agonists and antagonists but neither mu nor delta opioid peptides. In contrast, rabbit reticulocyte lysate primed with RNA transcribed in vitro from the envY sequence elicited the synthesis of an opioid binding site with mixed mu and delta properties. In addition, transfection of the envY sequence into mammalian cells resulted in the expression of opioid binding sites. Depending on the type of cells transfected, these sites were selective for either the mu or delta ligands.

Differential development of beta-endorphin and mu opioid binding sites in mouse brain.

Mouse brains of various ages from embryonal day 14 (E14) to adult were analyzed for opioid receptor binding using the enkephalin analog Tyr-D-Ala-Gly-NMe-Phe-Gly-ol (DAMGE) and the opiate alkaloid dihydromorphine (DHM) as mu-selective radioligands. Binding parameters were estimated from homologous and heterologous competition binding curves. During the postnatal period, Kd values for [3H]DAMGE did not change but Bmax values (fmol/mg protein) increased 2.7 fold from postnatal day 3 (P3) to P7. Minor receptor density fluctuations were evident from P7 to adult. Similar results were obtained with [3H]DHM. In contrast, estimation of total mu binding sites (fmol/brain) revealed a continuous rise from P3 to the adult. The postnatal developmental profile of total mu binding sites was comparable to the weight gain of mouse brain and the increase in protein content. In contrast, during the same period beta-endorphin immunoreactivity (IR) levels undergo an increase that is inversely proportional to mu opioid receptor Bmax values. [3H]DAMGE binding to E14 membrane preparations was inhibited to a greater extent by Gpp(NH)p than that to P1 or adult. Additional characterization of mu receptors was accomplished by heterologous competition binding assays. IC50 values for beta-endorphin in competition with [3H]DHM and [3H]DAMGE were age dependent and differed for the two radioligands. These results suggest that mu receptor selectivity for mu-specific peptide and alkaloid ligands changes as a function of age.

Synthesis and binding characteristics of the highly delta-specific new tritiated opioid peptide, [3H]deltorphin II.

A radiolabelled form of deltorphin II was synthesized by catalytic tritiation using [p-IPhe3]-deltorphin II as a precursor. The ligand labels rat brain membranes with a Kd value of 1.9 nM, and the Bmax was found to be 92 fmol/mg protein. This new tritiated ligand exhibits high affinity for the delta opioid binding site, whereas its binding to the mu type is weak and extremely low for the kappa type. Mu/delta and kappa/delta selectivity ratios were about 900 and 10,000, respectively. The highly delta selective binding properties of this new radioligand suggest that it could serve as an excellent tool for investigating the delta opioid receptors in various species.

Desipramine modulation of sigma and opioid peptide receptor expression in glial cells.

Exposure of C6 glial cell cultures to desipramine induced the appearance of opioid receptors and up-regulated sigma receptors. Opioid binding was demonstrated with 3H-etorphine and 3H-dihydromorphine (DHM), but was not observed with the mu, delta and kappa ligands 3H-DAMGE, 3H-DADLE or 3H-(-)ethylketocyclazocine in the presence of specific blockers, respectively. Competition experiments with 3H-DHM and either (-)naloxone or (+)naloxone indicated the presence of authentic opioid receptors. In similar studies with beta-endorphin, its truncated form (1-27) or their N-acetyl derivatives, beta-endorphin proved to have the highest affinity. Opioid receptors in glial cell aggregates were primarily kappa, with few mu and delta sites. Desipramine increased Bmax values for kappa but not mu and delta.

Reconstitution of high-affinity opioid agonist binding in brain membranes.

In synaptosomal membranes from rat brain cortex, the mu selective agonist [3H]dihydromorphine in the absence of sodium, and the nonselective antagonist [3H]naltrexone in the presence of sodium, bound to two populations of opioid receptor sites with Kd values of 0.69 and 8.7 nM for dihydromorphine, and 0.34 and 5.5 nM for naltrexone. The addition of 5 microM guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]) strongly reduced high-affinity agonist but not antagonist binding. Exposure of the membranes to high pH reduced the number of GTP[gamma-35S] binding sites by 90% and low Km, opioid-sensitive GTPase activity by 95%. In these membranes, high-affinity agonist binding was abolished and modulation of residual binding by GTP[gamma S] was diminished. High-affinity (Kd, 0.72 nM), guanine nucleotide-sensitive agonist binding was reconstituted by polyethylene glycol-induced fusion of the alkali-treated membranes with (opioid receptor devoid) C6 glioma cell membranes. Also restored was opioid agonist-stimulated, naltrexone-inhibited GTPase activity. In contrast, antagonist binding in the fused membranes was unaltered. Alkali treatment of the glioma cell membranes prior to fusion inhibited most of the low Km GTPase activity and prevented the reconstitution of agonist binding. The results show that high-affinity opioid agonist binding reflects the ligand-occupied receptor-guanine nucleotide binding protein complex.

Modulation of the binding characteristics of hypothalamic mu opioid receptors in rats by gonadal steroids.

Recent evidence suggests that the effects of the opioids on gonadotropin release may depend on the endocrine status existing in the experimental animal. In the brain, the effects of the opioids are exerted through the interaction with different classes of opioid receptors (mu, delta, kappa, etc.). Among these, the mu receptors appear to be particularly relevant to the control of gonadotropin secretion. Different groups of experiments have been performed in the rat in order to analyze whether changes of circulating levels of sex steroids may have an impact on the binding characteristics of hypothalamic mu opioid receptors, as evaluated by a receptor binding assay performed on plasma membrane preparations, using [3H]dihydromorphine as a mu ligand. In a first series of experiments, it has been observed that the ontogenesis of hypothalamic mu opioid receptors is different in male and in female rats: the concentration of mu sites, similar in animals of the two sexes at 16 days of age, increases in females, but not in males, between day 16 and day 26 of life. This sexual difference persists in 60-day old animals, when the brain is fully mature. It has also been observed that the pattern of maturation of hypothalamic mu receptors can be reversed by neonatal castration of males and by neonatal testosterone treatment of females. In a second series of experiments, it has been shown that in the hypothalamus of regularly cycling female rats the concentration of mu receptors varies during the different phases of the estrous cycle. In particular, a rather high density of mu sites during diestrus day 2 and the morning of the day of proestrus was found; this is followed by a progressive decline during the afternoon of the day of proestrus and the day of estrus, with a minimum value of the concentration of mu receptors being recorded in the first day of diestrus. These fluctuations seem to be linked to the physiological changes of serum levels of ovarian steroids: in fact, in a third series of experiments, it has been found that the positive feedback effect on LH release, exerted by the treatment of ovariectomized female rats with estrogens plus progesterone, is accompanied by a significant decrease of the concentration of hypothalamic mu opioid receptors; treatments with estrogens alone, able to induce a negative feedback effect on LH secretion, are not associated with modifications of hypothalamic mu receptors. These data seem to indicate that hypothalamic mu receptors may be involved in the positive but not in the negative feedback control of LH secretion.(ABSTRACT TRUNCATED AT 400 WORDS)

Common stereospecificity of opioid and dopamine systems for N-butyrophenone prodine-like compounds.

The two optical isomers of 1-[3-(p-fluorobenzoyl) propyl]-3-methyl-4-phenyl-4-propionoxypiperidine (FPP) were obtained by resolution of (+/-)-r-3-methyl-4-phenyl-c-4-piperidinol followed by N-alkylation and O-propionylation. These, as well as the racemate, were evaluated for their antinociceptive, opioid, and neuroleptic properties using in vivo and in vitro test systems. The results are remarkable in two respects, namely, the dextrorotatory isomer is consistently the most potent on all tests, and it acts on both opioid (mu) and neuroleptic (D2) receptors.