Pharmacological Characterization of Levorphanol, a G-Protein Biased Opioid Analgesic.

BACKGROUND: Levorphanol is a potent analgesic that has been used for decades. Most commonly used for acute and cancer pain, it also is effective against neuropathic pain. The recent appreciation of the importance of functional bias and the uncovering of multiple µ opioid receptor splice variants may help explain the variability of patient responses to different opioid drugs. METHODS: Here, we evaluate levorphanol in a variety of traditional in vitro receptor binding and functional assays. In vivo analgesia studies using the radiant heat tail flick assay explored the receptor selectivity of the responses through the use of knockout (KO) mice, selective antagonists, and viral rescue approaches. RESULTS: Receptor binding studies revealed high levorphanol affinity for all the µ, δ, and κ opioid receptors. In S-GTPγS binding assays, it was a full agonist at most µ receptor subtypes, with the exception of MOR-1O, but displayed little activity in ß-arrestin2 recruitment assays, indicating a preference for G-protein transduction mechanisms. A KO mouse and selective antagonists confirmed that levorphanol analgesia was mediated through classical µ receptors, but there was a contribution from 6 transmembrane targets, as illustrated by a lower response in an exon 11 KO mouse and its rescue with a virally transfected 6 transmembrane receptor splice variant. Compared to morphine, levorphanol had less respiratory depression at equianalgesic doses. CONCLUSIONS: While levorphanol shares many of the same properties as the classic opioid morphine, it displays subtle differences that may prove helpful in its clinical use. Its G-protein signaling bias is consistent with its diminished respiratory depression, while its incomplete cross tolerance with morphine suggests it may prove valuable clinically with opioid rotation.

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.

Characterization and distribution of a cloned rat mu-opioid receptor.

We have cloned and expressed a rat brain cDNA, TS11, that encodes a mu-opioid receptor based on pharmacological, physiological, and anatomical criteria. Membranes were prepared from COS-7 cells transiently expressing TS11 bound [3H]diprenorphine with high affinity (KD = 0.23 +/- 0.04 nM). The rank order potency of drugs competing with [3H]diprenorphine was as follows: levorphanol (Ki = 0.6 +/- 0.2 nM) approximately beta-endorphin (Ki = 0.7 +/- 0.05 nM) approximately morphine (Ki = 0.8 +/- 0.5 nM) approximately [D-Ala2, N-Me-Phe4,Gly-ol5]-enkephalin (DAMGO; Ki = 1.6 +/- 0.5 nM) uch much greater than U50,488 (Ki = 910 +/- 0.78 nM) > [D-Pen2,5]- enkephalin (Ki = 3,170 +/- 98 nM) > dextrorphan (Ki = 4,100 +/- 68 nM). The rank order potencies of these ligands, the stereospecificity of levorphanol, and morphine's subnanomolar Ki are consistent with a mu-opioid binding site. Two additional experiments provided evidence that this opioid-binding site is functionally coupled to G proteins: (a) in COS-7 cells 50 microM 5'-guanylylimidodiphosphate shifted a fraction of receptors with high affinity for DAMGO (IC50 = 3.4 +/- 0.5 nM) to a lower-affinity state (IC50 = 89.0 +/- 19.0 nM), and (b) exposure of Chinese hamster ovary cells stably expressing the cloned mu-opioid receptor to DAMGO resulted in a dose-dependent, naloxone-sensitive inhibition of forskolin-stimulated cyclic AMP production. The distribution of mRNA corresponding to the mu-opioid receptor encoded by TS11 was determined by in situ hybridization to brain sections prepared from adult female rats. The highest levels of mu-receptor mRNA were detected in the thalamus, medial habenula, and the caudate putamen; however, significant hybridization was also observed in many other brain regions, including the hypothalamus.

Dextrorphan: an antagonist for phencyclidine receptors.

Radio-binding assay, bioassay and HPLC detection were used to observe the antagonistic effects of dextrorphan on PCP's actions. Dextrorphan displayed high affinity to PCP receptor in the rabbit mesenteric blood vessels. It had weak PCP-like bioactivity, but could antagonize PCP's action dose-dependently in vitro study with the rabbit ear artery preparation and shifted the dose-response curve of PCP to the right. After PCP administration, the content of norepinephrine in the vascular bath medium was increased, which was reversed by dextrorphan. Thus suggests that dextrorphan is an antagonist with very mild agonistic action for PCP receptors.

Differential maturation of mu and delta opioid receptors in the chick embryonic brain.

The developmental profiles of the binding of mu and delta opiate receptors agonists was investigated using the chick embryo brain. Binding of opioids was performed at embryonic days 5, 6, 15, 18, and 20 in the developing chick embryo brain. [3H]dihyromorphine was used as a mu ligand and with 5 X 10(-7) M levorphanol for non-specific binding, and [3H](D-Ala2-D-Leu5)-enkephalin was used as a delta with 5 X 10(-7) M (D-Ser-Gly-Phe-Leu-Thr)-enkephalin for non-specific binding. Crude membranes were prepared from whole brain at days 5, 6 and cerebral hemispheres at days 15, 18, and 20 of embryonic age. Both mu and delta opiate receptors were present during early embryogenesis and as early as day 5. Analysis of binding sites revealed high and low affinity mu sites during early embryogenesis but only one delta site. By 18 days of embryonic age, only one mu site remained. This developmental change is interpreted as a transitory state of the receptor to the adult mu pattern. The presence of only one delta site is constant throughout embryonic age; it is high during early embryogenesis reaching a lower level by 18 days. The presence of a dual binding site pattern for the mu receptor in early embryogenesis is implicated to have a functional significance in the pluripotential role of the endogenous opioids in early development.

Opioid and non-opioid binding of beta-endorphin to bovine adrenal medullary membranes.

Binding of human beta-endorphin (beta h-EP) to bovine adrenal medullary membranes was characterized using [125I]Tyr27-beta h-EP [( 125I]beta h-EP) as a primary ligand. The specific binding of [125I]beta h-EP was time-dependent, saturable and stereospecific. Analysis of a saturation isotherm revealed two apparent classes of specific binding sites with dissociation constants of 2.4 and 34 nM. The extent of maximum inhibition of specific [125I]beta h-EP binding by either levorphanol, morphine, naloxone, dynorphin A (1-13) or D-Ala2-D-Leu5-enkephalin was similar to each other and remained partial (60-70%). Levorphanol eliminated the high affinity component but showed no effect on the low affinity component of [125I]beta h-EP binding. beta h-EP(1-31) displaced completely the [125I]beta h-EP binding. However, beta h-EP(1-23) only partially (approximately 80%) inhibited the [125I]beta h-EP binding. beta h-EP(6-31) showed inhibitory activity on [125I]beta h-EP binding. These results suggest that [125I]beta h-EP binding to bovine adrenal medullary membranes consists of a high affinity opioid-sensitive component and a low affinity non-opioid component. The non-opioid component of [125I]beta h-EP binding may be related to COOH-terminal of the beta h-EP molecule.

Levorphanol: pharmacokinetics and steady-state plasma concentrations in patients with pain.

Plasma concentrations of the narcotic analgesic, levorphanol, have been determined following i.v., i.m. and oral administration of therapeutic doses of the drug to patients with pain. In two patients who received single i.v. doses of levorphanol the plasma concentration-time profile in each subject was best described by a triexponential decline of the concentrations with terminal half-lives (t 1/2) of about 11 hr. Following i.m. and oral administrations, peak plasma concentrations of intact drug were generally reached after about 0.5 and 1 hr, respectively. Conjugated (beta-glucuronidase labile) levorphanol appeared rapidly in plasma following all routes of administration and quickly reached concentrations which were 5 to 10 fold higher than the intact drug. Effective analgesic steady-state concentrations of levorphanol in patients receiving a wide range of chronic oral and i.m. dosages of the drug ranged from about 10 to 100 ng/ml and these concentrations showed no apparent correlation with either the dose or the subjective analgesic response achieved. The latter observations are probably a reflection of extensive and variable inter-subject "first-pass" metabolism of the drug combined with different degrees of pharmacologic tolerance at the receptor level. However, in the non-tolerant patient it appears that a plasma concentration of about 10 ng/ml is associated with a positive analgesic effect. Furthermore it seems that analgesia is often maintained within a narrow plasma concentration range for each subject in that relatively small decreases in plasma concentration in some patients may be associated with either mild or severe pain. Plasma protein binding at steady-state in 10 patients averaged 40 +/- 2.6%. Concentrations of the drug in the cerebrospinal fluid of 2 patients studied were 60 to 70% of the corresponding plasma levels of the drug.

Opiate receptor mediation of ketamine analgesia.

Previous workers have noted that analgesia produced by ketamine can be antagonized by the narcotic antagonist, naloxone. In order to elaborate further the apparent similarity between ketamine- and narcotic-induced analgesia, the authors examined the effects of ketamine in three standard test systems for the opiate receptor. In a radioligand binding assay using 3H-dihydromorphine, ketamine stereospecifically bound to opiate receptors in rat brain homogenate, (+) ketamine being 2-3 times more potent than the (-) enantiomer of ketamine. In a bioassay for the opiate receptor, using the longitudinal muscle-myenteric plexus of the guinea pig ileum, ketamine inhibited the twitch-like muscular contractions, as do narcotics. However, only the inhibitory effects of (+) ketamine, which in this system also was twice as potent as (-) ketamine, could be partially antagonized by naloxone, suggesting that this enantiomer is responsible for the opiate receptor-related effects of ketamine. In vivo, the authors found that ketamine displaces 3H-etorphine, a potent narcotic, from opiate receptors in regional areas of the mouse brain, especially in the thalamic region, but not in the cortex. The results suggest that a significant mechanism of ketamine-induced analgesia is mediated by opiate receptors.

Pharmacodynamic studies with (-)-3-phenoxy-N-methylmorphinan in rats.

Analgesia and brain and plasma concentrations of (-)-3-phenoxy-N-methylmorphinan (PMM) and its metabolites were determined in rats administered 50 mg/kg of 3H-labeled PMM p.o., an approximate ED50. Unchanged PMM and two active metabolites, levorphanol and a different phenol, p-hydroxylated on the 3-phenoxy group (pOH-PMM), were present in brain at concentrations greater than in plasma. Analgesia was observed from 1 to 6 hr and was associated with brain concentrations of 400-1400 ng/g of PMM, 190-300 ng/g of pOH-PMM, and 16-27 ng/g of levorphanol. The presence of 58% of the administered dose as unchanged PMM in the gastrointestinal tract at 6 hr may reflect slow absorption and explain the persisting brain concentrations of PMM and its metabolites as well as the prolonged analgesia. Analgesia may have been due to the presence in brain of only PMM, pOH-PMM or levorphanol, or to the combined activity of two or three of these substances. Administration of the approximate ED50 of 3H-labeled levorphanol (0.1 mg/kg, s.c., or 6 mg/kg, p.o.) resulted in brain levorphanol concentrations (11-18 ng/g) close to those observed when PMM was administered p.o. at 50 mg/kg. After administration of an approximate subcutaneous ED50 of [3H]pOH-PMM of 24 mg/kg, the brains contained pOH-PMM (1500-4100 ng/g) and levorphanol (60-100 ng/g); these levorphanol concentrations were higher than those found after administration of the approximate ED50 of PMM or levorphanol. The findings indicate that brain levorphanol concentrations resulting from administration of PMM or pOH-PMM to rats may account for the analgesic activity observed, i.e. that PMM and pOH-PMM may act as prodrugs for levorphanol

The metabolism of (-)-3-phenoxy-N-methylmorphinan in dogs.

The disposition of radioactive (-)-3-phenoxy-N-methyl[2-3H]morphinan in dogs after oral administration has been investigated. Unchanged drug was not found in bile, urine, or feces. Excretion of total radioactivity in feces ranged from 67 to 78% of an oral dose. Two unconjugated metabolites were isolated from feces and identified by NMR and GC/MS. Both were substituted on the phenoxy group; they were found to be the p-hydroxy (pOH-PMM) and the m-methoxy-p-hydroxy (mOCH3-pOH-PMM) metabolites. Further, levorphanol and norlevorphanol were identified in feces both as free and conjugated metabolites, as well as a small amount of levomethorphan. Urine contained mostly unknown metabolites and conjugated levorphanol and pOH-PMM. Although the glucuronide of mOCH3-pOH-PMM was the major metabolite in bile, smaller amounts of the glucuronide and sulfate conjugated of both levorphanol and pOH-PMM were also found. Estimates for the total urinary and fecal excretion (as percentages of the dose) by two dogs for the five known metabolites were as follows: levorphanol, 18.8-21.5%; pOH-PMM, 14.4-20.6%; mOCH3-pOH-PMM, 14.9%; norlevorphanol, 2.8-6.1%; levomethorphan, 0.5%. Two of these metabolites, pOH-PMM and levorphanol, are potent analgesics.

Purification of the opiate receptor from rat brain.

The opiate receptor was purified from a Triton-solubilized preparation of rat neural membranes by the use of affinity chromatography. The affinity gel was prepared by coupling 14-beta-bromoacetamidomorphine, a newly synthesized ligand, to omega-aminohexyl-Sepharose. After elution of the nonspecific proteins with 50 mM Tris (pH 7.5), the receptor proteins were eluted with 1 microM levorphanol or etorphine. NaDodSO4/polyacrylamide gel electrophoresis revealed three major proteins associated with the opiate receptor, having molecular weights of 43,000, 35,000, and 23,000. The purified receptor binds 10(-11) mol of dihydromorphine/per mg of protein, with a Kd of 3.8 X 10(-9) M. Other opiates, naloxone, and methionine-enkephalin, inhibit [3H]dihydromorphine binding in a manner similar to that observed with intact and solubilized neural membranes.

Cerebellar opiate receptors in lagomorphs. Demonstration, characterization and regional distribution.

The cerebellum of lagomorphs (pika, rabbit, hare) binds 100--200 femtomoles of [3H]etorphine per milligram of protein. This is very high in comparison with the 10--15 fmol/mg found in the cerebellum of rodents (mouse, hamster, rat). In the rabbit cerebellum, the etorphine sites have binding properties indistinguishable from those of genuine opiate receptor sites in brain. They exhibit a high affinity for [3H]etorphine (KD = 1 X 10(-10) M), [3H]naloxone (KD = 9 X 10(-10) M), morphine and levorphanol but not for dextrophan. Moreover, sodium ions enhance binding of naloxone (antagonist response) and diminish binding of etorphine, morphine and levorphanol (agonist response) to cerebellum homogenates. The regional distribution of [3H]etorphine binding sites in the rabbit cerebellum points toward concentrations higher in the neocerebellum (hemispheres) than in the archecerebellum (lingula and flocculonodular lobe). Finally, the specific concentration of opiate receptor sites in the isolated molecular layer is at least two times that in the isolated granular layer and ten times that in white matter.

Evidence for topographical analogy between methionine-enkephalin and morphine derivatives.

Analogues of the endogenous opiate-receptor ligand [5-methionine]enkephalin (H-Tyr-Gly-Gly-Phe-Met-OH) were designed and synthesized for the purpose of testing the proposed similarity in spatial structure between this peptide and morphine derivatives. In the bioassay (inhibition of electrically induced contractions of the mouse vas deferens) [1-O-methyltyrosine,5-methionine]enkephalin, [1-N-methyltyrosine,5-methionine]enkephalin, [4-tryptophan,5-methionine]enkephalin, and [5-methionine sulfoxide]enkephalin possess, respectively, 0.4, 21, 27, and 67% activity of [5-methionine]enkephalin. These morphinomimetic activities correlate well with the opiate receptor affinities determined by displacement of [3H]naloxone in a guinea pig brain membrane preparation. The effects of O-methylation of the tyrosine residue and N-methylation of the terminal amino group on biological activity and receptor affinity support the hypothesis that the latter two moieties in the peptide correspond to the phenol group and the tertiary nitrogen, respectively, in morphine. Determination of the efficiency of energy transfer from tyrosine in position 1 to tryptophan in position 4 in [4-tryptophan,5-methionine]enkephalin from both tyrosine fluorescence quenching and relative enhancement of tryptophan fluorescence by means of a modified procedure permitted the calculation of an average intramolecular tyrosine-tryptophan separation of 10.0 +/- 1.1 A. Inspection of CPK models showed excellent agreement between this value and both the intrafluorophore distance in the 4 leads to 1 and 5 leads to 2 hydrogen bonded betaI-bend models of [4-tryptophan,5-methionine]enkephalin (9-11 A) and the phenol-phenyl separation in the potent morphine derivative 7alpha-(1(R)-hydroxy-1-methyl-3-phenylpropyl)-6,14-endo-ethenotetrahydrooripavine (8-10.5 A). The ensemble of these findings suggests an analogous topography for [5-methionine]enkephalin and morphine-oripavine derivatives.

Binding of an opiate, levorphanol, to intact neuroblastoma cells in continuous culture.

Mouse neuroblastoma cells in continuous culture, incubated for 1 to 2 days in the presence of 10--6 M levorphanol or morphine, were found to become tolerant to and dependent on those biologically active opiates. Examination of the interaction between levorphanol and the whole neuroblastoma cell suggested that levorphanol was binding to stereospecific opiate receptor sites. This binding was time and temperature dependent, and saturable at concentrations greater than 10--5 M levorphanol. Competition by other opiates for levorphanol sites correlated with their biological activity. This is the first evidence for saturable, stereospecific opiate binding in a homogeneous population of unhybridized cells in continuous culture.