Dennis J. Stone

JNJ-20788560 [9-(8-Azabicyclo[3.2.1]oct-3-ylidene)-9H-xanthene-3-carboxylic Acid Diethylamide], a Selective Delta Opioid Receptor Agonist, Is a Potent and Efficacious Antihyperalgesic Agent That Does Not Produce Respiratory Depression, Pharmacologic Tolerance, or Physical Dependence

μ-Opioid analgesics are a mainstay in the treatment of acute and chronic pain of multiple origins, but their side effects, such as constipation, respiratory depression, and abuse liability, adversely affect patients. The recent demonstration of the up-regulation and membrane targeting of the δ-opioid receptor (DOR) following inflammation and the consequent enhanced therapeutic effect of δ-opioid agonists have enlivened the search for δ-opioid analgesic agents. JNJ-20788560 [9-(8-azabicyclo-[3.2.1]oct-3-ylidene)-9H-xanthene-3-carboxylic acid diethylamide] had an affinity of 2.0 nM for DOR (rat brain cortex binding assay) and a naltrindole sensitive DOR potency of 5.6 nM (5′-O-(3-[35S]thio)triphosphate assay). The compound had a potency of 7.6 mg/kg p.o. in a rat zymosan radiant heat test and of 13.5 mg/kg p.o. in a rat Complete Freunds adjuvant RH test but was virtually inactive in an uninflamed radiant heat test. In limited studies, tolerance was not observed to the antihyperalgesic or antinociceptive effects of the compound. Unlike ibuprofen, JNJ-20788560 did not produce gastrointestinal (GI) erosion. Although morphine reduced GI motility at all doses tested and reached nearly full effect at the highest dose, JNJ-20788560 did not retard transit at the lowest dose and reached only 11% reduction at the highest dose administered. Unlike morphine, JNJ-20788560 did not exhibit respiratory depression (blood gas analysis), and no withdrawal signs were precipitated by the administration of opioid (μ or δ) antagonists. Coupled with the previously published lack of self-administration behavior of the compound by alfentanil-trained primates, these findings strongly recommend δ-opioid agonists such as JNJ-20788560 for the relief of inflammatory hyperalgesia.

Gas chromatographic method using nitrogen-phosphorus detection for the measurement of tramadol and its O-desmethyl metabolite in plasma and brain tissue of mice and rats

A method that allows the measurement of plasma and brain levels of the centrally-acting analgesic tramadol and its major metabolite (O-desmethyl tramadol) in mice and rats was developed using gas chromatography equipped with nitrogen-phosphorus detection (GC-NPD). Plasma samples were extracted with methyl tert.-butyl ether (MTBE) and were injected directly into the GC system. Brain tissue homogenates were precipitated with methanol, the resulting supernatant was dried then acidified with hydrochloric acid. The aqueous solution was washed with MTBE twice, alkalinized, and extracted with MTBE. The MTBE layer was dried, reconstituted and injected into the GC system. The GC assay used a DB-1 capillary column with an oven temperature ramp (135 to 179 degrees C at 4 degrees C/min). Dextromethorphan was used as the internal standard. The calibration curves for tramadol and O-desmethyl tramadol in plasma and brain tissue were linear in the range of 10 to 10000 ng/ml (plasma) and ng/g (brain). Assay accuracy and precision of back calculated standards were within +/- 15%.

Unexceptional Seizure Potential of Tramadol or Its Enantiomers or Metabolites in Mice

Tramadol is one of the most widely used centrally acting analgesics worldwide. Because of its multimodal analgesic mechanism (opioid plus nonopioid), the adverse effects profile of tramadol, similar to its analgesic profile, can be atypical compared with single-mechanism opioid analgesics. The comparison is often favorable (e.g., less respiratory depression or abuse), but it is sometimes cited as unfavorable in regard to seizure potential. As part of a broader study of this analgesic, we compared seizure induction in mice produced by administration of tramadol, the enantiomers and metabolites [M1 (O-desmethyl tramadol), M2 (N-desmethyl tramadol), M3 (N,N-didesmethyl tramadol), M4 (O,N,N-tridesmethyl tramadol), and M5 (O,N-didesmethyl tramadol)] of tramadol, and opioid and nonopioid reference compounds. We found that tramadol, its enantiomers, and M1 to M5 metabolites were of intermediate potency in this endpoint (on either a milligram per kilogram or millimole per kilogram basis). The SD50 (estimated dose required to induce seizures in 50% of test group) of tramadol to antinociceptive ED50 ratio was almost identical to that of codeine. The enantiomers of tramadol were about equipotent to tramadol on this endpoint. The M1 to M5 metabolites (and M1 enantiomers) of tramadol were less potent than tramadol. The relative potency of tramadol to opioids was not altered by quinidine (an inhibitor of CYP4502D6), noxious stimulus (48°C hot-plate), multiple dosing, or in reserpinized mice. Tramadol seizures were increased by naloxone, principally at high tramadol doses and due to an effect on the (–)enantiomer that overcame the opposite effect on the (+)enantiomer. No synergistic effect on seizure induction was observed between concomitant tramadol and codeine or morphine.

Differential cholera-toxin sensitivity of supraspinal antinociception induced by the cannabinoid agonists Δ9-THC, WIN 55,212-2 and anandamide in mice

Intracerebroventricular (i.c.v.) administration to mice of delta9-tetrahydrocannabinol (delta9-THC), WIN 55,212-2 or the endogenous cannabinoid anandamide induced dose-related antinociception in the 55 degrees C warm-water tail-flick test. Pretreatment (24 h, i.c.v.) with pertussis toxin dose-dependently reduced the antinociceptive effect of delta9-THC (955 nmol), WIN 55,212-2 (30 nmol) and anandamide (135 nmol) (IC50 = 0.13, 5.5, and 0.32 nmol, respectively). In contrast, pretreatment (24 h, i.c.v.) with cholera toxin (0.1-3.0 mg) reduced the antinociception of WIN 55,212-2, had minimal effect on delta9-THC, and dose-dependently increased the antinociception of anandamide (ED50 = 0.50 nmol). These data suggest differences in the receptor-effector coupling of delta9-THC, WIN 55,212-2 and anandamide in supraspinal-induced antinociception in mice.

Determination of the adsorption of tramadol hydrochloride by activated charcoal in vitro and in vivo.

Although tramadol is one of the most widely used centrally acting analgesics worldwide, no literature is available regarding adsorption of tramadol HCl powder or tablets (Ultram; 50 mg tramadol HCl per tablet) by activated charcoal (AC) for use as potential adjunct treatment of overdose. The present study incorporated a novel combination of in vitro and in vivo methods to investigate this question. Based on a binding curve of tramadol UV absorbance (UV(a); 225 nm) plotted against the amount of AC, the ratio of amount of tramadol completely adsorbed by AC was 0.05 mg/mg. Also based on UV(a), no tramadol was detected in filtrate of slurries in which up to 62 tablets of Ultram were mixed with 50 g AC; 4.6% of unbound tramadol was detected when 100 tablets of Ultram were mixed with AC. The ratio of amount of tramadol completely adsorbed by AC in this test was 0.10. In vivo, co-administration of 0.1 g/ml of AC produced a 13- to 14-fold rightward shift in tramadols antinociceptive dose-response curve and a 1.6-fold rightward shift in tramadols lethality dose-response curve.

The Novel, Orally Active, Delta Opioid RWJ-394674 Is Biotransformed to the Potent Mu Opioid RWJ-413216

Although the mu opioid receptor is the primary target of marketed opioid analgesics, several studies suggest the advantageous effect of combinations of mu and delta opioids. The novel compound RWJ-394674 [N,N-diethyl-4-[(8-phenethyl-8-azabicyclo]3.2.1]oct-3-ylidene)-phenylmethyl]-benzamide]; bound with high affinity to the delta opioid receptor (0.2 nM) and with weaker affinity to the mu opioid receptor (72 nM). 5′-O-(3-[35S]-thio)triphosphate binding assay demonstrated its delta agonist function. Surprisingly given this pharmacologic profile, RWJ-394674 exhibited potent oral antinociception (ED50 = 10.5 μmol/kg or 5 mg/kg) in the mouse hot-plate (48°C) test and produced a moderate Straub tail. Antagonist studies in the more stringent 55°C hot-plate test demonstrated the antinociception produced by RWJ-394674 to be sensitive to the nonselective opioid antagonist naloxone as well as to the delta- and mu-selective antagonists, naltrindole and β-funaltrexamine, respectively. In vitro studies demonstrated that RWJ-394674 was metabolized by hepatic microsomes to its N-desethyl analog, RWJ-413216 [N-ethyl-4-[(8-phenethyl-8-azabicyclo[3.2.1]oct-3-ylidene)-phenylmethyl]-benzamide], which, in contrast to RWJ-394674, had a high affinity for the mu rather than the delta opioid receptor and was an agonist at both. Pharmacokinetic studies in the rat revealed that oral administration of RWJ-394674 rapidly gave rise to detectable plasma levels of RWJ-413216, which reached levels equivalent to those of RWJ-394674 by 1 h. RWJ-413216 itself demonstrated a potent oral antinociceptive effect. Thus, RWJ-394674 is a delta opioid receptor agonist that appears to augment its antinociceptive effect through biotransformation to a novel mu opioid receptor-selective agonist.

α-Subunit G-protein antisense oligodeoxynucleotide effects on supraspinal (i.c.v.) α2-adrenoceptor antinociception in mice

An in vivo antisense strategy was used to examine the involvement of G-protein subunits in supraspinal (intracerebroventricular; i.c.v.) α2-adrenoceptor-mediated antinociception. Mice that were injected with 33-mer antisense oligodeoxyribonucleotides (6 nmol) or vehicle were tested (tailflick) with an agonist (clonidine, guanfacine or BH-T 920) administered i.c.v. 18–24 h later. Gi3α antisense treatment attenuated BH-T 920 and clonidine-induced antinociception. Gi2α antisense produced differential effects on the three agonists. Gi1α and Gsα antisense treatment had no significant effect. Together with the previous demonstration that i.c.v. μ-opioid antinociception is mediated via Gi2α, the present results suggest that different receptors may mediate antinociception via different G-protein subunits and, hence, that specific subunits might offer novel targets for drug discovery.

μ Receptor and Gi2α antisense attenuate [d-Met2]-FMRFamide antinociception in mice

Abstract FMRFamide (Phe-Met-Arg-Phe-NH 2 ) and several analogs produce centrally-mediated, naloxone-reversible antinociception, but have minimal affinity for opioid receptor (sub)types. In the present study, the antinociception in mice (55°C tail-flick test) produced by supraspinal (intracerebroventricular; ICV) administration of [ d -Met 2 ]-FMRFamide (a stable analog of FMRFamide) was attenuated by pretreatment with ICV oligodeoxyribonucleotide antisense to the opioid μ receptor or by antisense to the G i 2α G-protein subunit. These data suggest that [ d -Met 2 ]-FMRFamide produces its antinociception via an opioid interneuron.