Environmental risk assessment of metformin and its transformation product guanylurea. I. Environmental fate.

Metformin (MET) is a pharmaceutical with very high use worldwide that is excreted in unchanged form, leading to concern about potential aquatic life impacts associated with MET, and its primary transformation product guanylurea (GUU). This study presents, in two companion papers, a risk assessment following internationally accepted guidelines of MET and GUU in surface water based on literature data, previously unpublished studies, and a new degradation test that resolves conflicting earlier results. Previous studies have shown that MET is removed during sewage treatment, primarily through transformation to GUU. In addition, measurements in WWTPs suggest that MET is not only transformed to GUU, but that GUU is further biodegraded. A prolonged inherent biodegradation test strongly suggests not only primary transformation of MET to GUU, but also subsequent full mineralization of GUU, with both degradation phases starting after a clear lag phase. MET may partition from surface water to sediment, where both transformation to GUU and in part mineralization is possible, depending on the presence of competent degrading microorganisms. In addition, MET may form non-extractable residues in sediments (12.8-73.5%). Both MET and GUU may be anaerobically degraded during sludge digestion, in soils or in sediments. Bioconcentration factor (BCF) values in crops and most plants are close to 1 suggesting low bioaccumulation potential, moreover, at least some plants can metabolize MET to GUU; however, in aquatic plants higher BCFs were found, up to 53. Similarly, neither MET nor GUU are expected to bioaccumulate in fish based on estimated values of BCFs ≤3.16.

Synthesis and dual histamine H1 and H2 receptor antagonist activity of cyanoguanidine derivatives.

Premedication with a combination of histamine H1 receptor (H1R) and H2 receptor (H2R) antagonists has been suggested as a prophylactic principle, for instance, in anaesthesia and surgery. Aiming at pharmacological hybrids combining H1R and H2R antagonistic activity, a series of cyanoguanidines 14-35 was synthesized by linking mepyramine-type H1R antagonist substructures with roxatidine-, tiotidine-, or ranitidine-type H2R antagonist moieties. N-desmethylmepyramine was connected via a poly-methylene spacer to a cyanoguanidine group as the "urea equivalent" of the H2R antagonist moiety. The title compounds were screened for histamine antagonistic activity at the isolated ileum (H1R) and the isolated spontaneously beating right atrium (H2R) of the guinea pig. The results indicate that, depending on the nature of the H2R antagonist partial structure, the highest H1R antagonist potency resided in roxatidine-type compounds with spacers of six methylene groups in length (compound 21), and tiotidine-type compounds irrespective of the alkyl chain length (compounds 28, 32, 33), N-cyano-N'-[2-[[(2-guanidino-4-thiazolyl)methyl]thio]ethyl]-N″-[2-[N-[2-[N-(4-methoxybenzyl)-N-(pyridyl)-amino] ethyl]-N-methylamino]ethyl] guanidine (25, pKB values: 8.05 (H1R, ileum) and 7.73 (H2R, atrium) and the homologue with the mepyramine moiety connected by a six-membered chain to the tiotidine-like partial structure (compound 32, pKB values: 8.61 (H1R) and 6.61 (H2R) were among the most potent hybrid compounds. With respect to the development of a potential pharmacotherapeutic agent, structural optimization seems possible through selection of other H1R and H2R pharmacophoric moieties with mutually affinity-enhancing properties.

Antinociceptive activity of CC44, a biotinylated improgan congener.

Improgan, a non-opioid, antinociceptive drug, activates descending analgesic circuits following brain administration, but the improgan receptor remains unidentified. Since biotinylation of drugs can enhance drug potency or facilitate discovery of new drug targets, a biotinylated congener of improgan (CC44) and several related compounds were synthesized and tested for antinociceptive activity. In rats and mice, intracerebroventricular (i.c.v.) administration of CC44 produced dose-dependent reductions in thermal nociceptive (tail flick and hot plate) responses, with 5-fold greater potency than improgan. CC44 also robustly attenuated mechanical (tail pinch) nociception in normal rats and mechanical allodynia in a spinal nerve ligation model of neuropathic pain. Similar to the effects of improgan, CC44 antinociception was reversed by the GABAA agonist muscimol (consistent with activation of analgesic circuits), and was resistant to the opioid antagonist naltrexone (implying a non-opioid mechanism). Also like improgan, CC44 produced thermal antinociception when microinjected into the rostral ventromedial medulla (RVM). Unlike improgan, CC44 (i.c.v.) produced antinociception which was resistant to antagonism by the cannabinoid CB1 antagonist/inverse agonist rimonabant. CC44 was inactive in mice following systemic administration, indicating that CC44 does not penetrate the brain. Preliminary findings with other CC44 congeners suggest that the heteroaromatic nucleus (imidazole), but not the biotin moiety, is required for CC44's antinociceptive activity. These findings demonstrate that CC44 is a potent analgesic compound with many improgan-like characteristics. Since powerful techniques are available to characterize and identify the binding partners for biotin-containing ligands, CC44 may be useful in searching for new receptors for analgesic drugs.

Efficacy of improgan, a non-opioid analgesic, in neuropathic pain.

Improgan, a non-opioid analgesic, is known to act in the rodent brain stem to produce highly effective antinociception in several acute pain tests. However, improgan has not been studied in any models of chronic pain. To assess the efficacy of improgan in an animal model of neuropathic pain, the effects of this drug were studied on mechanical allodynia following unilateral spinal nerve ligation (SNL) in rats. Intracerebroventricular (icv) improgan (40-80 µg) produced complete, reversible, dose-dependent attenuation of hind paw mechanical allodynia for up to 1h after administration, with no noticeable behavioral or motor side effects. Intracerebral (ic) microinjections of improgan (5-30 µg) into the rostral ventromedial medulla (RVM) also reversed the allodynia, showing this brain area to be an important site for improgan's action. The recently-demonstrated suppression of RVM ON-cell activity by improgan may account for the presently-observed anti-allodynic activity. The present findings suggest that brain-penetrating, improgan-like drugs developed for human use could be effective medications for the treatment of neuropathic pain.

Brain P450 epoxygenase activity is required for the antinociceptive effects of improgan, a nonopioid analgesic.

The search for the mechanism of action of improgan (a nonopioid analgesic) led to the recent discovery of CC12, a compound that blocks improgan antinociception. Because CC12 is a cytochrome P450 inhibitor, and brain P450 mechanisms were recently shown to be required in opioid analgesic signaling, pharmacological and transgenic studies were performed in rodents to test the hypothesis that improgan antinociception requires brain P450 epoxygenase activity. Intracerebroventricular (i.c.v.) administration of the P450 inhibitors miconazole and fluconazole, and the arachidonic acid (AA) epoxygenase inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH) potently inhibited improgan antinociception in rats at doses that were inactive alone. MW06-25, a new P450 inhibitor that combines chemical features of CC12 and miconazole, also potently blocked improgan antinociception. Although miconazole and CC12 were weakly active at opioid and histamine H(3) receptors, MW06-25 showed no activity at these sites, yet retained potent P450-inhibiting properties. The P450 hypothesis was also tested in Cpr(low) mice, a viable knock-in model with dramatically reduced brain P450 activity. Improgan (145 nmol, i.c.v.) antinociception was reduced by 37% to 59% in Cpr(low) mice, as compared with control mice. Moreover, CC12 pretreatment (200 nmol, i.c.v.) abolished improgan action (70% to 91%) in control mice, but had no significant effect in Cpr(low) mice. Thus, improgan's activation of bulbospinal nonopioid analgesic circuits requires brain P450 epoxygenase activity. A model is proposed in which (1) improgan activates an unknown receptor to trigger downstream P450 activity, and (2) brainstem epoxygenase activity is a point of convergence for opioid and nonopioid analgesic signaling.

Physiological basis for inhibition of morphine and improgan antinociception by CC12, a P450 epoxygenase inhibitor.

Many analgesic drugs, including µ-opioids, cannabinoids, and the novel nonopioid analgesic improgan, produce antinociception by actions in the rostral ventromedial medulla (RVM). There they activate pain-inhibiting neurons, termed "OFF-cells," defined by a nociceptive reflex-related pause in activity. Based on recent functional evidence that neuronal P450 epoxygenases are important for the central antinociceptive actions of morphine and improgan, we explored the convergence of opioid and nonopioid analgesic drug actions in RVM by studying the effects of the P450 epoxygenase inhibitor CC12 on the analgesic drug-induced activation of these OFF-cells and on behavioral antinociception. In rats lightly anesthetized with isoflurane, we recorded the effects of intraventricular morphine and improgan, with and without CC12 pretreatment, on tail flick latency and activity of identified RVM neurons: OFF-cells, ON-cells (pronociceptive neurons), and neutral cells (unresponsive to analgesic drugs). CC12 pretreatment preserved reflex-related changes in OFF-cell firing and blocked the analgesic actions of both drugs, without interfering with the increase in spontaneous firing induced by improgan or morphine. CC12 blocked suppression of evoked ON-cell firing by improgan, but not morphine. CC12 pretreatment had no effect by itself on RVM neurons or behavior. These data show that the epoxygenase inhibitor CC12 works downstream from receptors for both µ-opioid and improgan, at the inhibitory input mediating the OFF-cell pause. This circuit-level analysis thus provides a cellular basis for the convergence of opioid and nonopioid analgesic actions in the RVM. A presynaptic P450 epoxygenase may therefore be an important target for development of clinically useful nonopioid analgesic drugs.

Neural basis for improgan antinociception.

Improgan, the prototype compound of a novel class of non-opioid analgesic drugs derived from histamine antagonists, attenuates thermal and mechanical nociception in rodents following intracerebroventricular (i.c.v.) administration. Improgan does not bind to known opioid, histamine or cannabinoid receptors, and its molecular target has not been identified. It is known however, that improgan acts directly in the periaqueductal gray and the rostral ventromedial medulla to produce its antinociceptive effects, and that inactivation of the rostral ventromedial medulla prevents the antinociceptive effect of improgan given i.c.v. Here we used in vivo single-cell recording in lightly anesthetized rats to show that improgan engages pain-modulating neurons in the medulla to produce antinociception. Following improgan administration, OFF-cells, which inhibit nociception, became continuously active and no longer paused during noxious stimulation. The increase in OFF-cell firing does not represent a non-specific neuroexcitant effect of this drug, since ON-cell discharge, associated with net nociceptive facilitation, was depressed. NEUTRAL-cell firing was unaffected by improgan. The net response of rostral ventromedial medulla (RVM) neurons to improgan is thus comparable to that evoked by mu-opioids and cannabinoids, well known RVM-active analgesic drugs. This common basis for improgan, opioid, and cannabinoid antinociception in the RVM supports the idea that improgan functions as a specific analgesic agent.

Inhibition of brain [(3)H]cimetidine binding by improgan-like antinociceptive drugs.

[(3)H]cimetidine, a radiolabeled histamine H(2) receptor antagonist, binds with high affinity to an unknown hemoprotein in the brain which is not the histamine H(2) receptor. Improgan, a close chemical congener of cimetidine, is a highly effective pain-relieving drug following CNS administration, yet its mechanism of action remains unknown. To test the hypothesis that the [(3)H]cimetidine-binding site is the improgan antinociceptive target, improgan, cimetidine, and 8 other chemical congeners were studied as potential inhibitors of [(3)H]cimetidine binding in membrane fractions from the rat brain. All compounds produced a concentration-dependent inhibition of [(3)H]cimetidine binding over a 500-fold range of potencies (K(i) values were 14.5 to >8000nM). However, antinociceptive potencies in rats did not significantly correlate with [(3)H]cimetidine-binding affinities (r=0.018, p=0.97, n=10). These results suggest that the [(3)H]cimetidine-binding site is not the analgesic target for improgan-like drugs.

Enantioselective in-line and off-line CE methods for the kinetic study on cimetidine and its chiral metabolites with reference to flavin-containing monooxygenase genetic isoforms.

An in-line screening and an off-line chiral CE method were developed to determine the stereoselectivity of flavin-containing monooxygenase (FMO) isoforms using cimetidine (CIM) as a substrate. The S-oxygenation of CIM was investigated using achiral chemical oxidants and (human supersomes) enzymatic metabolism procedures. In the off-line setup, the chiral selector sulfobutylether-beta-CD was chosen to separate the CIM S-oxide (CSO) metabolites. The electrophoretic migration order of CSO was confirmed to be (+) before (-) through the use of single enantiomers obtained by preparative chromatography. For the electrophoretically mediated microanalysis method, the in-line enzymatic reaction was performed in 100 mM phosphate reaction buffer (pH 8.3), whereas 50 mM phosphate buffer with 30 mM chiral selector (pH 2.5) was used as a BGE. During the screening of FMO isoenzymes by the electrophoretically mediated microanalysis method, formation of the new chiral center on the CIM sulfur was found to be stereoselective. FMO1 produces more (-)-CSO-enantiomer, while FMO3 generates mainly (+)-CSO-enantiomer. On the other hand, FMO5 shows no activity. The kinetic constants of FMO1 and FMO3 were measured by the off-line method. A K(m)=4.31 mM for the formation of the (+)-CSO-enantiomer and a K(m)=4.56 mM for the (-)-CSO-enantiomer are reported for the first time for FMO1.

Non-opioid antinociception produced by brain stem injections of improgan: significance of local, but not cross-regional, cannabinoid mechanisms.

Improgan, a cimetidine derivative which lacks activity at known histamine, opioid or cannabinoid receptors, acts by an unknown mechanism in the periaqueductal gray (PAG) and raphe magnus (RM) to stimulate descending, analgesic circuits. These circuits may utilize cannabinoid mechanisms. To characterize further the nature of these circuits, the effects of intracerebral (i.c.) microinjections of rimonabant (a CB(1) receptor inverse agonist) were studied on antinociceptive responses following i.c. microinjections of improgan and the cannabinoid agonist WIN 55,212 (WIN) in rats. Separate intra-RM injections of improgan (30 microg) and WIN (8 microg) produced near-maximal antinociception on both the hot plate (HP) and tail flick (TF) nociceptive tests. Pretreatment with intra-RM rimonabant (20 microg) antagonized the antinociception produced by both intra-RM improgan and intra-RM WIN, but had no effects when given alone. Similar studies with improgan demonstrated rimonabant-sensitive sites within the dorsal and ventrolateral PAG. However, intra-RM pretreatment with rimonabant had no effect on antinociceptive responses following intra-PAG improgan. These studies show that improgan activates pain-relieving mechanisms in the PAG and the RM, both of which may utilize local cannabinoid mechanisms.

Unexpected products and reaction mechanisms of the aqueous chlorination of cimetidine.

Many pharmaceuticals and personal care products (PPCPs) resist degradation in wastewater treatment plants. Thus, they may be transformed by chemical disinfectants in the final treatment stage, generating products that may possess enhanced toxicity/biological activity relative to the parent compounds. For this reason, the reaction of cimetidine, an over-the-counter antacid, with the frequently used disinfectant, free chlorine, was investigated. Cimetidine degraded rapidly in the presence of excess free chlorine, indicating that it will likely undergo significant transformation during wastewater disinfection. Four major products were isolated and extensively characterized by comparison of liquid chromatographic retention times to known standards, mass spectrometry, 1H- and 2D-nuclear magnetic resonance spectroscopy, and infrared spectroscopy. An expected sulfur oxidation product, cimetidine sulfoxide, was identified along with three unexpected products: 4-hydroxymethyl-5-methyl-1H-imidazole, 4-chloro-5-methyl-1H-imidazole, and a product proposed to be either a beta- or delta-sultam. The last three products are formed by transformations not frequently observed in free chlorine reactions of PPCPs such as C-C bond cleavage and intramolecular nucleophilic substitution. The unexpected transformations yielded compounds with more substantial structural changes than would be observed in common free chlorine reactions such as N-chlorination or electrophilic halogenation. The reaction pathway was elucidated by kinetic analysis and by independently treating isolated intermediates with free chlorine, and reaction mechanisms were proposed. Finally, the predicted no-effect concentration (PNEC) of the chlorination products was estimated, and the products 4-hydroxymethyl-5-methyl-1H-imidazole and 4-chloro-5-methyl-1H-imidazole were estimated to have lower PNECs than cimetidine.

Antinociceptive activity of furan-containing congeners of improgan and ranitidine.

Furan-containing congeners of the histamine H(2) receptor antagonist ranitidine were synthesized and tested for improgan-like antinociceptive activity. The most potent ligand of the series, VUF5498, is the most potent improgan-like agent described to date (ED(50)=25 nmol, icv). This compound is approximately equal in potency with morphine. These non-imidazole, improgan-like pain relievers further define the structural requirements for analgesics of this class and are important tools for ongoing mechanism-based studies.

Significance of cannabinoid CB1 receptors in improgan antinociception.

UNLABELLED: Improgan is a congener of the H(2) antagonist cimetidine, which produces potent antinociception. Because a) the mechanism of action of improgan remains unknown and b) this drug may indirectly activate cannabinoid CB(1) receptors, the effects of the CB(1) antagonist/inverse agonist rimonabant (SR141716A) and 3 congeners with varying CB(1) potencies were studied on improgan antinociception after intracerebroventricular (icv) dosing in rats. Consistent with blockade of brain CB(1) receptors, rimonabant (K(d) = 0.23 nM), and O-1691 (K(d) = 0.22 nM) inhibited improgan antinociception by 48% and 70% after icv doses of 43 nmol and 25 nmol, respectively. However, 2 other derivatives with much lower CB(1) affinity (O-1876, K(d) = 139 nM and O-848, K(d) = 352 nM) unexpectedly blocked improgan antinociception by 65% and 50% after icv doses of 300 nmol and 30 nmol, respectively. These derivatives have 600-fold to 1500-fold lower CB(1) potencies than that of rimonabant, yet they retained improgan antagonist activity in vivo. In vitro dose-response curves with (35)S-GTPgammaS on CB(1) receptor-containing membranes confirmed the approximate relative potency of the derivatives at the CB(1) receptor. Although antagonism of improgan antinociception by rimonabant has previously implicated a mechanistic role for the CB(1) receptor, current findings with rimonabant congeners suggest that receptors other than, or in addition to CB(1) may participate in the pain-relieving mechanisms activated by this drug. The use of congeners such as O-848, which lack relevant CB(1)-blocking properties, will help to identify these cannabinoid-like, non-CB(1) mechanisms. PERSPECTIVE: This article describes new pharmacological characteristics of improgan, a pain-relieving drug that acts by an unknown mechanism. Improgan may use a marijuana-like (cannabinoid) pain-relieving mechanism, but it is shown presently that the principal cannabinoid receptor in the brain (CB(1)) is not solely responsible for improgan analgesia.

Improgan-induced hypothermia: a role for cannabinoid receptors in improgan-induced changes in nociceptive threshold and body temperature.

Improgan, a congener of the H(2) antagonist cimetidine, produces non-opioid antinociception which is blocked by the CB(1) antagonist rimonabant, implying a cannabinoid mechanism of action. Since cannabinoids produce hypothermia as well as antinociception in rodents, the present study investigated the pharmacological activity of improgan on core body temperature and nociceptive (tail flick) responses. Improgan (60, 100 and 140 microg, intraventricular [ivt]) elicited significant decreases in core temperature 3-30 min following injection with a maximal hypothermic effect of -1.3 degrees C. Pretreatment with rimonabant (50 microg, ivt) produced a statistically significant but incomplete (29-42%) antagonism of improgan hypothermia. In control experiments, the CB(1) agonist CP-55,940 (37.9 microg, ivt) induced significant decreases in core temperature (-1.8 degrees C) 3-30 min following injection. However, unlike the case with improgan, pretreatment with rimonabant completely blocked CP-55,940 hypothermia. Furthermore, CP-55,940 and improgan elicited maximal antinociception over the same time course and dose ranges, and both effects were attenuated by rimonabant. These results show that, like cannabinoid agonists in the rat, improgan produces antinociception and hypothermia which is blocked by a CB(1) antagonist. Unlike cannabinoid agonists, however, improgan does not produce locomotor inhibition at antinociceptive doses. Additional experiments were performed to determine the effect of CC12, a recently discovered improgan antagonist which lacks affinity at CB(1) receptors. Pretreatment with CC12 (183 microg, ivt) produced complete inhibition of both the antinociception and the hypothermia produced by improgan, suggesting the possible role of an unknown improgan receptor in both of these effects.

CC12, a high-affinity ligand for [3H]cimetidine binding, is an improgan antagonist.

Improgan, a chemical congener of cimetidine, is a highly effective non-opioid analgesic when injected into the CNS. Despite extensive characterization, neither the improgan receptor, nor a pharmacological antagonist of improgan has been previously described. Presently, the specific binding of [(3)H]cimetidine (3HCIM) in brain fractions was used to discover 4(5)-((4-iodobenzyl)thiomethyl)-1H-imidazole, which behaved in vivo as the first improgan antagonist. The synthesis and pharmacological properties of this drug (named CC12) are described herein. In rats, CC12 (50-500nmol, i.c.v.) produced dose-dependent inhibition of improgan (200-400nmol) antinociception on the tail flick and hot plate tests. When given alone to rats, CC12 had no effects on nociceptive latencies, or on other observable behavioral or motor functions. Maximal inhibitory effects of CC12 (500nmol) were fully surmounted with a large i.c.v. dose of improgan (800nmol), demonstrating competitive antagonism. In mice, CC12 (200-400nmol, i.c.v.) behaved as a partial agonist, producing incomplete improgan antagonism, but also limited antinociception when given alone. Radioligand binding, receptor autoradiography, and electrophysiology experiments showed that CC12's antagonist properties are not explained by activity at 25 sites relevant to analgesia, including known receptors for cannabinoids, opioids or histamine. The use of CC12 as an improgan antagonist will facilitate the characterization of improgan analgesia. Furthermore, because CC12 was also found presently to inhibit opioid and cannabinoid antinociception, it is suggested that this drug modifies a biochemical mechanism shared by several classes of analgesics. Elucidation of this mechanism will enhance understanding of the biochemistry of pain relief.

Cannabinoid-improgan cross-tolerance: Improgan is a cannabinomimetic analgesic lacking affinity at the cannabinoid CB1 receptor.

Improgan is a non-opioid analgesic which does not act at known histamine or cannabinoid receptors. Because improgan antinociception is blocked by low doses of a cannabinoid CB1 antagonist, the present experiments determined if development of cannabinoid tolerance in mice would alter improgan antinociception. Twice-daily injections of Delta9-tetrahydrocannabinol (THC, 10 mg/kg, s.c.) for 3.5 days induced 47-54% and 42-56% reductions in cannabinoid (WIN 55,212-2, 20 microg, i.c.v.) and improgan (30 microg, i.c.v.) antinociception, respectively, as compared with responses from vehicle-treated groups. Because improgan lacks cannabinoid-like side effects in rats, and does not act directly on cannabinoid CB1 receptors, the finding that development of cannabinoid tolerance reduces improgan antinociception suggests that this drug may release endocannabinoids, or activate novel cannabinoid sites. Either possibility offers the potential for developing new types of analgesics.

Mechanisms of inverse agonism at histamine H(2) receptors - potential benefits and concerns.

It is well established that histamine exerts its effects by activating histamine receptors that belongs to the family of seven transmembrane G-protein coupled receptors (GPCRs). Many ligands with important therapeutic actions that had been assumed to be antagonists at histamine H2 receptor have been shown to be inverse agonists. The mechanism whereby these drugs achieve their effects seems to be not unique. Theoretical models predict at least three ways in which inverse agonists can exert their action that are supported by experimental observations. These different mechanisms have crucial consequences on basic pharmacology and clinical treatments. The pharmacological models, the feasible mechanisms of action of H2 inverse agonists, findings about molecular basis of tiotidine inverse agonism in particular, and their impact on clinical protocols will be discussed.

Antinociceptive activity of chemical congeners of improgan: optimization of side chain length leads to the discovery of a new, potent, non-opioid analgesic.

Improgan is a chemical congener of the H2 antagonist cimetidine which shows the profile of a highly effective analgesic when administered directly into the CNS. Although the improgan receptor is unknown, improgan activates analgesic pathways which are independent of opioids, but may utilize cannabinoid mechanisms. To discover selective, potent, improgan-like drugs, seven compounds chemically related to improgan were synthesized and tested for antinociceptive activity in rats after intracerebroventricular (icv) administration. Among a series of improgan congeners in which the alkyl chain length of improgan ((-CH2)3-) was varied, five compounds showed full agonist antinociceptive activity with potencies greater than that of improgan. VUF5420 (containing (-CH2)4-, EC50 = 86.1 nmol) produced maximal antinociceptive activity after doses which showed no motor impairment or other obvious toxicity, and was 2.3-fold more potent than improgan (EC50 = 199.5 nmol). As found previously with improgan, VUF5420-induced antinociception was unaffected by administration of the opioid antagonist naltrexone, but was inhibited by the CB1 antagonist SR141716A, suggesting a non-opioid, cannabinoid-related analgesic action. However, VUF5420 showed very low affinity (Kd approximately 10 microM) on CB1-receptor activation of 35S-GTPgammaS binding, indicating that this drug does not directly interact with the CB1 receptor in vivo. The present results show that VUF5420 is a high potency, improgan-like, non-opioid analgesic which may indirectly activate cannabinoid pain-relieving mechanisms.

Antinociceptive, brain-penetrating derivatives related to improgan, a non-opioid analgesic.

The antinociceptive profile of selected histamine H(2) and histamine H(3) receptor antagonists led to the discovery of improgan, a non-brain-penetrating analgesic agent which does not act on known histamine receptors. Because no chemical congener of improgan has yet been discovered which has both antinociceptive and brain-penetrating properties, the present study investigated the antinociceptive effects of a series of chemical compounds related to zolantidine, a brain-penetrating histamine H(2) receptor antagonist. The drugs studied presently contain the piperidinomethylphenoxy (PMPO) moiety, hypothesized to introduce brain-penetrating characteristics. Following intracerebroventricular (i.c.v.) dosing in rats, six of eight drugs produced dose- and time-related antinociception on both the tail flick and hot plate tests over a nearly eight-fold range of potencies. Ataxia and other motor side effects were observed after high doses of these drugs, but two of the compounds (SKF94674 and loxtidine) produced maximal antinociception at doses which were completely devoid of these motor effects. Consistent with the hypothesis that PMPO-containing drugs are brain-penetrating analgesics, SKF94674 and another derivative (JB-9322) showed dose-dependent antinociceptive activity 15 to 30 min after systemic dosing in mice, but these effects were accompanied by seizures and death beginning 45 min after dosing. Other drugs showed a similar pattern of antinociceptive and toxic effects. In addition, loxtidine produced seizures without antinociception, whereas zolantidine produced neither effect after systemic dosing in mice. Although several of the drugs tested have histamine H(2) receptor antagonist activity, neither the antinociception nor the toxicity was correlated with histamine H(2) receptor activity. The present results are the first to demonstrate the existence of brain-penetrating antinociceptive agents chemically related to zolantidine and improgan, but further studies are needed to understand the mechanisms of both the pain relief and toxicity produced by these agents.

Absence of 5-HT3 and cholinergic mechanisms in improgan antinociception.

Improgan, an analgesic derived from histamine antagonists, acts in the brain stem to activate descending non-opioid, pain-relieving circuits, but the mechanism of action of this drug remains elusive. Because improgan has a moderate affinity for 5-HT(3) receptors, and, since cholinergic and serotonergic drugs can modulate descending analgesic circuits, roles for 5-HT(3), nicotinic and muscarinic receptors in improgan antinociception were presently investigated in rats. Improgan (80 microg, icv) induced nearly maximal inhibition of hot plate and tail flick nociceptive responses, and these actions we unaffected by antagonists of muscarinic (atropine, 5.9 mg/kg, i.p.) and nicotinic (mecamylamine, 2 mg/kg, i.p.) receptors. Control experiments verified that these antagonist treatments were maximally effective against muscarinic and nicotinic antinociception in both tests. In addition, improgan antinociception was unaffected by icv pretreatment with a 5-HT(3) antagonist (ondansetron, 20 microg). When given alone, icv treatment with neither this antagonist nor a 5-HT(3) agonist (m-chlorophenylbiguanide, 1000 nmol, icv) modified thermal nociceptive latencies. These results show no role for supraspinal cholinergic and 5-HT(3) receptors in improgan antinociception. The findings help to narrow the search for the relevant mediators of the action of this novel analgesic agent.