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A series of arylglycine-based analogs was synthesized and tested for TRPM8 antagonism in a cell-based functional assay. Following structure-activity relationship studies in vitro, a number of compounds were identified as potent TRPM8 antagonists and were subsequently evaluated in an in vivo pharmacodynamic assay of icilin-induced 'wet-dog' shaking in which compound 12 was fully effective. TRPM8 antagonists of the type described here may be useful in treating pain conditions wherein cold hypersensitivity is a dominant feature.

The unexpected observation of a hyperglycemic effect of some tricycle-based delta opioid receptor (DOR) agonists led to a series of studies to better understand the finding. Single administration of two novel tricyclic DOR agonists dose dependently elevated rat plasma glucose levels; 4-week toxicology studies confirmed the hyperglycemic finding and further revealed pancreatic β-cell hypertrophy, including vacuole formation, as well as bone dysplasia and Harderian gland degeneration with regeneration. Similar diabetogenic effects were observed in dog. A review of the literature on the antiserotonergic and antihistaminergic drug cyproheptadine (CPH) and its metabolites revealed shared structural features as well as similar hyperglycemic effects to the present series of DOR agonists. To further evaluate these effects, we established an assay measuring insulin levels in the rat pancreatic β-cell-derived RINm5F cell line, extensively used to study CPH and its metabolites. Like CPH, the initial DOR agonists studied reduced RINm5F cell insulin levels in a concentration-dependent manner. Importantly, compound DOR potency did not correlate with the insulin-reducing potency. Furthermore, the RINm5F cell insulin results correlated with the diabetogenic effect of the compounds in a 5-day mouse study. The RINm5F cell insulin assay enabled the identification of aryl-aryl-amine DOR agonists that lacked an insulin-reducing effect and did not elevate blood glucose in repeated dosing studies conducted over a suprapharmacologic dose range. Thus, not only did the RINm5F cell assay open a path for the further discovery of DOR agonists lacking diabetogenic potential but also it established a reliable, economical, and high-throughput screen for such potential, regardless of chemotype or target pharmacology. The present findings also suggest a mechanistic link between the toxicity observed here and that underlying Wolcott-Rallison Syndrome.

Thermosensation is an essential sensory function that is subserved by a variety of transducer molecules, including those from the Transient Receptor Potential (TRP) ion channel superfamily. One of its members, TRPM8 (CMR1), a ligand-gated, nonselective cation channel, is activated by both cold and chemical stimuli in vitro. However, its roles in cold thermosensation and pain in vivo have not been fully elucidated. Here, we show that sensory neurons derived from TRPM8 null mice lack detectable levels of TRPM8 mRNA and protein and that the number of these neurons responding to cold (18 degrees C) and menthol (100 microM) is greatly decreased. Furthermore, compared with WT mice, TRPM8 null mice display deficiencies in certain behaviors, including icilin-induced jumping and cold sensation, as well as a significant reduction in injury-induced responsiveness to acetone cooling. These results suggest that TRPM8 may play an important role in certain types of cold-induced pain in humans.

Background and purpose: While investigating the effects of systemic urotensin II (U-II), a potent vasoactive peptide acting at the UT receptor, we observed ear pinna flushing after systemic administration to conscious rats. In the present study, U-II-induced ear flushing was quantified in terms of ear pinna temperature change and potential mechanisms were explored. Experimental approach: U-II-induced ear flushing was quantified by measuring lateral ear pinna temperature changes and compared to that of calcitonin gene-related peptide (CGRP), a known cutaneous vasodilator. Further, the effects of a variety of pharmacological agents on U-II-induced ear flushing were explored. Key results: Subcutaneous injection of U-II (9 μg kg−1)produced localized ear pinna flushing with an onset of ∼15 min, a duration of ∼30 min and a maximal temperature change of 9°C. In contrast, CGRP caused cutaneous flushing within multiple cutaneous beds including the ear pinna with a shorter onset and greater duration than U-II. A potent UT receptor antagonist, urantide, blocked U-II-induced ear flushing but did not affect CGRP-induced ear flushing. Pretreatment with indomethacin or L-Nω -nitroarginine methylester (L-NAME) abolished U-II-induced ear flushing. Mecamylamine or propranolol did not affect this response to U-II. Direct intracerebroventricular injection studies suggested that the ear flushing response to U-II was not mediated directly by the CNS. Conclusion and implications: Our results suggest that U-II-induced ear flushing and temperature increase is mediated by peripheral activation of the UT receptor and involves prostaglandin- and nitric oxide-mediated vasodilation of small capillary beds in the rat ear pinna. British Journal of Pharmacology (2007) 150, 415–423. doi:10.1038/sj.bjp.0707006

The use of proteomic analysis to discover proteins (previously identified or unknown) in a tissue sample is a valuable tool. However, there is a limit to the extent one can validate a discovery with any single technology. In an effort to obviate this inherent constraint and to add value and dimension to protein profiling, we have coupled the information obtained through proteomic techniques with the validation provided by in situ hybridization and immunohistochemistry techniques. This approach can be illustrated by our efforts in the discovery of stannin in rat dorsal root ganglia (DRG). In this study, we initially used the Ciphergen ProteinChip® to perform protein profiling on the DRG of rats in a carrageenan-induced paw inflammation study. In an effort to discover new potential targets in inflammatory pain models, we profiled many potential peaks unique to the ipsilateral DRG of interest. One protein, found to bind to a hydrophobic chip at a molecular mass of 9500 Dalton, was preliminarily identified as stannin. To confirm its identification, we performed in situ hybridization and immunohistochemistry on the source DRG tissue to investigate the presence of stannin mRNA and protein expression, respectively. In addition to confirming the presence of stannin in these DRGs, we observed the upregulation of stannin in the DRGs over the course of carrageenan-induced inflammation, suggesting a possible role of stannin in inflammatory hyperalgesia. Taken together, these results illustrate the synergistic benefits of coupling 0 proteomic and histochemical techniques in identifying and validating targets and biomarkers for drug discovery.

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 tramadol's antinociceptive dose-response curve and a 1.6-fold rightward shift in tramadol's lethality dose-response curve.

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.

Distinguishing between pharmacologically additive and synergistic drug combinations requires experimental designs and statistical analyses that often require appreciable numbers of animals and much experimenter time. The current study employed a design in which individual dose-effect data from each drug were translated into theoretically additive total dose combinations, in a fixed drug proportion, in order to produce a composite additive dose-effect relation that could be compared with that of an actual mixture having the same proportion. Results from this approach, using a combination of intrathecal doses of morphine and clonidine, were virtually identical to those using isobolographic analysis of the same data set. Both analyses showed significant synergism for this combination and, in each method, it was not necessary to constrain the drug regression lines to parallelism. In contrast to the isobole approach, the use of the composite additive dose-effect relation also allows observation of the interaction over a range of effects while reducing the size of the data sets needed. © 1997 Elsevier Science Inc.

In vitro studies have demonstrated that FMRFamide-related peptide receptors can be coupled to different G-proteins, mediating opposite stimulatory and inhibitory effects. The present study tested whether this duality might extend to effects in vivo. Antinociception in mice of ICV [ d -Met 2 ]FMRFamide, which produced agonist [ED 50 = 36.3 μg (61.6 nmol)] and antagonist [ID 50 = 0.72 μg (1.22 nmol)] actions, was attenuated by 24-h pretreatment with ICV pertussis toxin (ID 50 = 0.55 μg) or cholera toxin (ID 50 = 0.09 μg), suggesting that [ d -Met 2 ]FMRFamide in vivo effects might also be explained by dual G i G s coupling.

An in vivo antisense strategy was used to examine the involvement of G-protein subunits in supraspinal (intracerebroventricular; i.c.v.) alpha2-adrenoceptor-mediated antinociception. Mice that were injected with 33-mer antisense oligodeoxyribonucleotides (6 nmol) or vehicle were tested (tail-flick) with an agonist (clonidine, guanfacine or BH-T 920) administered i.c.v. 18 - 24 h later. Gi3alpha antisense treatment attenuated BH-T 920 and clonidine-induced antinociception. Gi2alpha antisense produced differential effects on the three agonists. Gi1alpha and G(s)alpha antisense treatment had no significant effect. Together with the previous demonstration that i.c.v. mu-opioid antinociception is mediated via Gi2alpha, 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.

A series of benzothiophene-based phosphonates was synthesized and many analogs within the series were shown to be potent antagonists of the TRPM8 channel. The compounds were obtained as a racemic mixture in 5 synthetic steps, and were tested for TRPM8 antagonist activity in a recombinant, canine TRPM8-expressing cell line using a fluorometric imaging plate reader (FLIPR) assay. Structure–activity relationships were developed initially by modification of the core structure and subsequently by variation of the aromatic substituents and the phosphonate ester. Compound 9l was administered intraperitoneally to rats and demonstrated engagement of the TRPM8 target in both prevention and reversal-modes in an icilin-induced ‘wet-dog’ shake model.

Discovered as part of an effort to identify delta opioid (DOPr or DOR) agonist analgesics, JNJ-20788560 and JNJ-39204880 exhibited high DOR affinity, with K(i) values of 1.7 and 2.0nM, respectively, and were selective for DOR over the mu opioid receptor (MOPr or MOR), with 596- and 122-fold selectivity, respectively. Both compounds stimulated DOR but not MOR induced GTPgammaS binding and were effective antihyperalgesic agents in the complete Freund's adjuvant model of thermal hyperalgesia in the rat, with oral ED(50) values of 13.5 and 35mg/kg, corresponding to plasma levels of 1 and 9microM, respectively. Autoradiographic analysis of DOR and MOR occupancy in sections of brain (striatum) and lumbar spinal cord (L4-L6) was determined ex vivo, using radiolabeled naltrindole or DAMGO. Quantitative image analysis resulted in striatal DOR ED(50) values of 6.9 and 10.7mg/kg, for JNJ-20788560 and JNJ-39204880 respectively, and spinal cord values of 6.4 and 3.2mg/kg, respectively. Neither compound dose-dependently occupied MOR within the dose range studied. Thus, this study confirmed the DOR selectively over MOR of both compounds following their oral administration, and further demonstrated dose-dependent DOR occupancy by each compound across its antihyperalgesic dose range. Importantly, these in vitro, in vivo, and ex vivo data revealed that the greater in vitro potency of JNJ-20788560 was paralleled by its greater in vivo potency, although JNJ-39204880 achieved higher plasma levels following its oral administration. The receptor occupancy levels observed at the pharmacologic ED(50) doses of these compounds suggest the need for greater target engagement by JNJ-39204880 than by JNJ-20788560 to elicit a similar therapeutic response.

Mu-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 delta-opioid receptor (DOR) following inflammation and the consequent enhanced therapeutic effect of delta-opioid agonists have enlivened the search for delta-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-[(35)S]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 Freund's 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 (mu or delta) antagonists. Coupled with the previously published lack of self-administration behavior of the compound by alfentanil-trained primates, these findings strongly recommend delta-opioid agonists such as JNJ-20788560 for the relief of inflammatory hyperalgesia.

A small series of novel, imidazoles 4 have been prepared that exhibit very good binding affinities for the delta and mu opioid receptors (ORs), as well as demonstrate potent agonist functional activity at the delta OR. Representative imidazole 4a (K(i) delta = 0.9 nM; K(i) mu = 55 nM; K(i) kappa = 124 nM; EC(50) delta =13-25 nM) was further profiled for OR related in vivo effects. Compound 4a reduced gastrointestinal (GI) propulsive motility in a dose-dependent and naloxone-reversible manner, based on the results of the mouse glass bead expulsion test (3, 5, and 10 mg/kg, ip) and the mouse fecal pellet output test (1 and 3 mg/kg, ip). Compound 4a showed no analgesic activity as measured by the mouse abdominal irritant test (MAIT) when dosed at 100 mg/kg, sc, but did show significant MAIT activity at doses of both 10 microg (40% inhibition) and 100 microg (100% inhibition) when dosed intracerebroventricularly (icv). Taken together, these in vivo results suggest that 4a acts peripherally when dosed systemically, and that these prototypical compounds may prove promising as medicinal leads for GI indications.

Two parallel synthetic methods were developed to explore the structure-activity relationships (SAR) of a series of potent opioid agonists. This series of tropanylidene benzamides proved extremely tolerant of structural variation while maintaining excellent opioid activity. Evaluation of several representative compounds from this series in the mouse hot plate test revealed potent antinociceptive effects upon oral administration.

The design and synthesis of novel pyrrolidine-containing bradykinin antagonists, II, are described. Conformational analysis suggested that a pyrrolidine moiety could substitute for the N-methyl cis-amide moiety of FR 173657. The in vitro binding data showed that the (S)-isomer of II was potent in the bradykinin B2 receptor-binding assay with a Ki of 33 nM. The opposite isomer, (R)-II, had a Ki of 46 nM. The in vitro binding data confirmed our conformational hypothesis.

The synthesis and structure–activity relationships of a novel series of aroylpyrrole alkylamides as potent selective bradykinin B2 receptor antagonists are described. Several members of this series display nanomolar affinity at the B2 receptor and show activity in an animal model of antinociception.

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%.

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.