Alvimopan and COX-2 inhibition reverse opioid and inflammatory components of postoperative ileus.

Our objective was to investigate the therapeutic potential of peripheral opioid antagonism with alvimopan and anti-inflammatory cyclooxygenase 2 (COX-2) inhibition in an animal model of postoperative ileus with pain management. Intestinal manipulation was conducted in mice and rats with or without postoperative morphine injection. Rodents were orally fed non-digestible fluorescein (FITC)-labelled dextran and transit measured after a period of 90 min. The immunomodulatory effects of morphine and alvimopan were determined on nitric oxide released from the organ cultured muscularis externa. Surgical manipulation of the intestine resulted in a delay in gastrointestinal transit after 24 h that worsened with exogenous morphine. Alvimopan did not significantly alter transit of control or manipulated animals, but significantly antagonized the transit delaying effects of morphine. However, when the inflammatory component was robust enough to obscure a further opioid induced delay in gastrointestinal transit, alvimopan ceased to be effective in improving postoperative intestinal function. Cyclooxygenase 2 inhibition significantly diminished the inflammatory component of postoperative ileus. Surgical manipulation resulted in an increased release of nitric oxide from the inflamed isolated muscularis externa in 24-h organ culture which was not altered by morphine or alvimopan. Two distinct mechanisms exist which participate in postoperative bowel dysfunction: a local inflammatory response which is antagonized by COX-2 inhibition, and a morphine-induced alteration in neural function which can be blocked with alvimopan.

Preclinical studies of opioids and opioid antagonists on gastrointestinal function.

Opioid receptors in the gastrointestinal (GI) tract mediate the effects of endogenous opioid peptides and exogenously administered opioid analgesics, on a variety of physiological functions associated with motility, secretion and visceral pain. The studies reviewed or reported here describe a range of in vivo activities of opioid receptor antagonists upon GI function in rodents, focusing on mu receptors. Naloxone, and the peripherally acting mu-opioid receptor antagonists alvimopan and methylnaltrexone, reverse morphine-induced inhibition of GI transit in mice and rats, and morphine- or loperamide-induced inhibition of castor oil-induced diarrhoea in mice. At doses producing maximal reversal of morphine-induced effects upon GI transit, only the central nervous system (CNS) penetrant antagonist naloxone was able to reverse morphine-induced analgesia. Both central and peripheral opioid antagonists may affect GI function and/or visceromotor sensitivity in the absence of exogenous opioid analgesics, suggesting a constitutive role for endogenous opioid peptides in the control of GI physiology. Furthermore, in contrast to naloxone, alvimopan does not produce hypersensitivity to the visceromotor response induced by nociceptive levels of colorectal distension in a rodent model of post-inflammatory colonic hypersensitivity, suggesting that in the periphery endogenous mu-opioid receptor-mediated mechanisms do not regulate colonic sensitivity. The data support the hypothesis that peripherally acting opioid antagonists may be able to selectively block opioid receptors in the GI tract, thereby preserving normal GI physiology, while not blocking the effects of endogenous opioid peptides or exogenous opioid analgesics in the CNS. These findings suggest that the primary sites of action of mu-opioid agonists with respect to inhibition of GI function are in the periphery, whereas analgesic activity resides primarily in the CNS.

Peripherally restricted opioid agonists as novel analgesic agents.

Mediation of antinociception via opioid receptors located in the periphery is a viable strategy to produce analgesia without the occurrence of side effects associated with stimulation of opioid receptors located in the central nervous system. Peripheral opioid receptors are particularly important in inflammatory pain states and in the responses to pruritogenic stimuli, and have been implicated in the transmission of visceral pain. Medicinal chemistry approaches to achieve peripheralization of opioid agonists have started with a centrally acting opioid agonist as a template, and introduced features of lipophilicity, hydrophilicity, or combined lipophilicity and hydrophilicity to achieve amphiphilicity. Quaternarization of centrally acting opioid agonists or identification of compounds that serve as substrates for the mdr transporter to achieve transport out of the brain has also been employed. The in vivo assays used to identify peripherally selective compounds have measured a variety of behavioral and pharmacokinetic endpoints, with varying degrees of predictability. This review focuses on a discussion of these methods, as well as a review of those compounds where sufficient data exist to support a claim of peripheralization in vivo.

Arylacetamides as peripherally restricted kappa opioid receptor agonists.

Analogues of the kappa (kappa) opioid receptor agonist, ICI 199441, were prepared. Ki values for these analogues at the cloned human kappa opioid receptor ranged from 0.058 to 25 nM. Trifluoromethylaryl derivatives were potent analgesics when administered subcutaneously in the rat and were more peripherally restricted than the parent compound, ICI 199441.

Loperamide (ADL 2-1294), an opioid antihyperalgesic agent with peripheral selectivity.

The antihyperalgesic properties of the opiate antidiarrheal agent loperamide (ADL 2-1294) were investigated in a variety of inflammatory pain models in rodents. Loperamide exhibited potent affinity and selectivity for the cloned micro (Ki = 3 nM) compared with the delta (Ki = 48 nM) and kappa (Ki = 1156 nM) human opioid receptors. Loperamide potently stimulated [35S]guanosine-5'-O-(3-thio)triphosphate binding (EC50 = 56 nM), and inhibited forskolin-stimulated cAMP accumulation (IC50 = 25 nM) in Chinese hamster ovary cells transfected with the human mu opioid receptor. The injection of 0.3 mg of loperamide into the intra-articular space of the inflamed rat knee joint resulted in potent antinociception to knee compression that was antagonized by naloxone, whereas injection into the contralateral knee joint or via the i.m. route failed to inhibit compression-induced changes in blood pressure. Loperamide potently inhibited late-phase formalin-induced flinching after intrapaw injection (A50 = 6 microgram) but was ineffective against early-phase flinching or after injection into the paw contralateral to the formalin-treated paw. Local injection of loperamide also produced antinociception against Freund's adjuvant- (ED50 = 21 microgram) or tape stripping- (ED50 = 71 microgram) induced hyperalgesia as demonstrated by increased paw pressure thresholds in the inflamed paw. In all animal models examined, the potency of loperamide after local administration was comparable to or better than that of morphine. Loperamide has potential therapeutic use as a peripherally selective opiate antihyperalgesic agent that lacks many of the side effects generally associated with administration of centrally acting opiates.

[3H]1,3-di(2-tolyl) guanidine binds to a sigma 2 receptor on Jurkat cell membranes, but sigma compounds fail to influence immunomodulatory events in human peripheral blood lymphocytes.

The binding characteristics of the sigma ligand [3H]1.3-di(2-tolyl)guanidine (DTG) were investigated in membranes prepared from the Jurkat T cell line. Binding was saturable with a KD of 56 +/- 3 nM and a Bmax of 11706 +/- 3173 fmol/mg protein (n = 3). The rank order of potency for sigma reference compounds to inhibit binding in the Jurkat cell line was ifenprodil > 1,3-di(2-tolyl)guanidine > haloperidol > carbetapentane > (+)3-(3-hydroxyphenyl)-N-propylpiperidine ((+)3-PPP) > (-)pentazocine > caramiphen > (+)pentazocine, and significantly correlated with potency at sigma 2 binding sites in guinea pig brain (r = 0.90, p < 0.01). The immunomodulatory activities of the sigma ligands 1,3-di(2-tolyl)guanidine, haloperidol. (-)pentazocine and (+)pentazocine on CD3-induced proliferation, IL-2 production and Ca2+ flux in human lymphocytes did not reveal any consistent pharmacology that could be ascribed to potency of these compounds at sigma binding sites. Collectively the data demonstrate that the [3H]1,3-di(2-tolyl)guanidine binding site on Jurkat cell membranes has a pharmacology consistent with sigma receptors, but no modulation of functional activity or intracellular events in human peripheral blood lymphocytes correlating with sigma receptors was found.

Novel benzo[b]quinolizinium cations as uncompetitive N-methyl-D-aspartic acid (NMDA) antagonists: the relationship between log D and agonist independent (closed) NMDA channel block.

A series of permanently charged benzo[b]quinolizinium cations having lower lipophilicity than MK-801 or phencyclidine (PCP) were synthesized. Data relating agonist independent block of N-methyl-D-aspartic acid (NMDA) ion channels to log D are described. Closed channel access is predicted to result in a more noncompetitive profile of antagonism compared to selective open channel blockers, which are uncompetitive inhibitors. Reduced closed channel block may underlie the absence of PCP or MK-801-like behavioral side effects observed for benzo[b]-quinolizinium cations.

Novel NMDA antagonists: replacement of the pyridinium ring of 6,11-ethanobenzo[b]quinolizinium cations with heteroisoquinolinium cations.

Replacement of the pyridinium ring of 6,11-ethanobenzo[b]quinolizinium cations with thiazolium (4a and 4b) and N-methylimidazolium (4c and 4d) resulted in equipotent compounds in the [3H]TCP binding assay. The corresponding N-methyl-1,2,4-triazolium analogs were less potent in this assay. The thiazolium derivative 4b, with a Ki = 2.9 nM, is being evaluated as a possible neuroprotective N-methyl-D-aspartic acid (NMDA) antagonist.

Anticonvulsant activity of caramiphen analogs.

Caramiphen potently blocks maximal electroshock (MES)-induced seizures in mice and rats. The anticonvulsant mechanism has been hypothesized to be due to high-affinity binding to sigma recognition sites in brain. To study the structure-activity relationship for anticonvulsant activity of caramiphen we evaluated 8 analogs in MES-induced seizures in rats and also determined whether a correlation exists between anticonvulsant potency and sigma binding affinity. Some of the analogs potently inhibited sigma binding but were devoid of anticonvulsant activity. Aminocaramiphen 2 (ED50 = 3.4 mg/kg) and N-methyl-4-piperidinyl 1-phenylcyclopentanecarboxylate 9 (ED50 = 4.8 mg/kg) showed anticonvulsant activity comparable to caramiphen (ED50 = 3.1 mg/kg), although in sigma binding assays the affinities were 3-and 30-fold less than caramiphen, respectively. In the presence of 250 microM of phenytoin, caramiphen and p-aminocaramiphen showed 3- to 5-fold increases in affinity for [3H](+)pentazocine binding, whereas piodocaramiphen, which was inactive as an anticonvulsant, showed no change in affinity for sigma binding. These results indicate that anticonvulsant activity of the caramiphen analogs is not due to interaction with sigma binding sites.

Discovery of 6,11-ethano-12,12-diaryl-6,11-dihydrobenzo[b]quinolizinium cations, a novel class of N-methyl-D-aspartate antagonists.

6,11-Ethano-12,12-diaryl-6,11-dihydrobenzo[b]quinolizinium cations 8, a novel class of N-methyl-D-aspartate (NMDA) antagonists acting at the phencyclidine site, have been identified. Structure-activity relationship studies around the lead compound 8a led to the identification of 12g (WIN 67870-2), one of the most potent compounds in this series. Compound 12g has a Ki = 1.8 +/- 0.2 nM vs [3H]TCP binding, has 700-fold selectivity for binding to the open state of the NMDA receptor-ionophore, and was devoid of MK-801- and PCP-like behavioral effects in rats. Compound 12g was neuroprotective in cultured mouse cortical neurons and exhibited antiischemic activity in a rat middle cerebral artery occlusion/reperfusion model of focal ischemia.

RLH-033, a novel, potent and selective ligand for the sigma 1 recognition site.

RLH-033 [2-(4-phenylpiperidinyl)ethyl 1-(4-nitrophenyl)cyclopentanecarboxylate HCl] is a rationally designed ligand that was synthesized and evaluated for its binding affinities at sigma 1 and sigma 2 sites in guinea pig brain. RLH-033 has high affinity (Ki = 50 pM) for sigma 1 sites labeled by [3H](+)-pentazocine, while it was over 2000-fold less affinity at sigma 2 sites labeled by [3H]1,3-di(2-tolyl)guanidine (DTG) in the presence of 500 nM (+)-pentazocine (Ki = 105 nM). Unlike its potent sigma activity, the compound has little affinity for dopamine D1 (Ki = 2.9 microM), D2 (Ki = 0.35 microM), muscarinic M1 (Ki = 0.88 microM) or M2 (Ki = 1.7 microM) receptors, and none at all for N-methyl-D-aspartate, phencyclidine and opioid receptors. Thus, RLH-033 is the most potent sigma 1 ligand reported to date, and its very high affinity suggests it may be a useful radioligand to characterize the pharmacology of sigma 1 recognition sites.

Identification, synthesis, and characterization of a unique class of N-methyl-D-aspartate antagonists. The 6,11-ethanobenzo[b]quinolizinium cation.

A series of novel N-methyl-D-aspartate antagonists acting at the phencyclidine site has been identified. Compound 2 has a Ki = 8 +/- 1 nM (vs [3H]thienylcyclidine, [3H]TCP) as a mixture of enantiomers. Resolution and further testing indicate that (-)-2, Ki = 4 +/- 0.7 nM, is a potent and selective TCP site ligand with neuroprotective activity in cultured neurons in the presence of excitotoxic concentrations of NMDA (IC50 = 26 nM). Compound (-)-2 is > 1000-fold selective for the TCP site vs a panel of receptor types including opiate, adrenergic, serotonergic, dopamine, adenosine, dihydropyridine, and benzodiazepine and displays increased selectivity for the activated (open) NMDA receptor-ion channel complex vs PCP and MK801 as measured by patch recordings in cultured, voltage-clamped neurons. Highly enhanced "open-channel" selectivity leads to tentative classification of these ligands as uncompetitive vs NMDA. Ligands with these characteristics may enable deconvolution of the pharmacologic effects associated with typical noncompetitive NMDA antagonists. We report here on the identification, synthesis, and activity of compounds of this structural class.

Novel (4-phenylpiperidinyl)- and (4-phenylpiperazinyl)alkyl-spaced esters of 1-phenylcyclopentanecarboxylic acids as potent sigma-selective compounds.

A series of novel 4-phenylpiperidinyl and (4-phenylpiperazinyl)alkyl 1-phenylcyclopentanecarboxylates was synthesized and evaluated for affinity at sigma 1 and sigma 2 sites by inhibition of [3H]-(+)-pentazocine (PENT) and [3H]-1,3-di(2-tolyl)guanidine (DTG) binding in guinea pig brain. The phenylpiperidines were more potent sigma ligands than the corresponding piperazines. Structural modifications varying the optimal spatial distance between the piperidine nitrogen and ester functions led to the identification of the propyl compound 24 ([3H]PENT Ki = 0.50 nM; [3H]DTG Ki = 1.17 nM) and the butyl derivative 32 ([3H]PENT Ki = 0.51 nM; [3H]DTG Ki = 0.69 nM) as novel high-affinity sigma-selective agents. An ethylene spacer was optimum with para-substituted analogs. A notable finding was the discovery of 2-(4-phenylpiperidinyl)ethyl 1-(4-nitrophenyl)-cyclopentanecarboxylate hydrochloride (15) (RLH-033), which demonstrated potent affinity for the [3H]PENT-defined sigma site with a Ki of 50 pM, selectivity for sigma 1 over muscarinic M1 (> 17,600-fold), M2 (> 34,200-fold), dopamine D1 (> 58,000-fold), and D2 (> 7000-fold) receptors, and inactivity at phencyclidine, NMDA, and opioid receptors. RLH-033 is a valuable tool which will aid further in understanding the biology of the sigma recognition site. Information from this research has further defined the topography of the sigma recognition site, which may provide an explanation for the diverse structures which bind with relatively high affinity.

sigma recognition sites in brain and peripheral tissues. Characterization and effects of cytochrome P450 inhibitors.

Binding to sigma sites in subcellular fractions of brain and in crude homogenates from peripheral tissues of the guinea pig was characterized with the [3H]ligands (+)pentazocine and di(2-tolyl)guanidine (DTG). The inhibitory effects of representative sigma compounds and cytochrome P450 inhibitors were evaluated in guinea pig tissues, and the effects of cytochrome P450 induction on sigma binding in the rat were investigated. For both ligands, the majority of sites were localized to the microsomal fractions. The KD values for [3H](+)pentazocine- or [3H]DTG-labeled sigma sites in guinea pig liver and testes were 2-fold lower than those in brain and heart. The number of sites labeled by [3H](+)pentazocine varied, with an order of liver > testes > brain > heart. In contrast, the Bmax values for [3H]DTG-defined sigma sites were greatest in testes, followed by liver, brain and heart. The rank order of potency for representative sigma and P450 compounds was similar in brain, liver and testes for both [3H]ligands, and the potency of selective compounds to displace sigma binding in guinea pig liver failed to correlate with their abilities to inhibit cytochrome P450IID1 activity in human liver. Following induction of cytochrome P450IIB1 with phenobarbital or cytochrome P450IA1 with beta-naphthoflavone, neither the affinity nor the number of sigma sites was altered in rat brain or liver. These results suggest that sigma sites in the periphery are similar to those in the brain, and that the sigma binding site is not identical with cytochrome P450IIB1, P450IA1 or P450IID1.

Synthesis and biodistribution of iodine-125-labeled 1-azabicyclo[2.2.2]oct-3-yl alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)-alpha-phenylacetate. A new ligand for the potential imaging of muscarinic receptors by single photon emission computed tomography.

1-Azabicyclo[2.2.2]oct-3-yl alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)- alpha-phenylacetate (IQNP, 3), an analogue of QNB in which a phenyl ring has been replaced with an iodopropenyl substituent, was prepared and evaluated in vitro and in vivo for m-AChR selectivity and specificity. High specific activity [125]IQNP ([125I]-3) was synthesized in greater than 60% yield utilizing an electrophilic iododestannylation reaction with hydrogen peroxide for the oxidation of iodide. In in vitro receptor binding studies, 3 demonstrated high affinity for M1 (Ki = 0.78 nM), M2 (Ki = 1.06 nM), and M3 (Ki = 0.27 nM) subtypes. In vivo biodistribution studies in female rats [125I]-3 demonstrated high uptake in areas rich in muscarinic receptors such as the brain (cortex and striatum) and the heart. Blocking studies were performed with a series of receptor specific agents and demonstrated that the uptake of [125I]-3 was selective and specific for cerebral muscarinic receptor rich areas and that the binding to m-AChR is reversible. The high-yield preparation and specificity and selectivity of high specific activity [125I]IQNP for muscarinic receptors suggest that this is an attractive new agent for potential imaging of cerebral receptors using single photon tomographic imaging (SPECT).

Sigma receptor binding affinity and inhibition of apomorphine-induced climbing.

Several relatively selective compounds with affinity for the sigma binding site were assessed for their ability to inhibit apomorphine-induced climbing in the mouse. Although, the majority of compounds inhibited apomorphine-induced climbing, there was no correlation between the ability to inhibit climbing and potency in sigma binding assays using [3H]1,3-di-o-tolylguanidine (DTG) or [3H](+)-pentazocine as ligands. The potency of the compounds to inhibit binding to muscarinic M1 or M2 receptors correlated with the potency to inhibit apomorphine-induced climbing. However, several of the compounds that inhibit climbing had microM affinity at muscarinic receptors. Whether these concentrations were achieved in vivo is unclear. Our data suggest that sigma activity per se is not responsible for inhibition of apomorphine-induced climbing.

Caramiphen, iodocaramiphen and nitrocaramiphen are potent, competitive, muscarinic M1 receptor-selective agents.

Caramiphen, iodocaramiphen and nitrocaramiphen were examined for affinity at the muscarinic M1, M2 and M3 receptor subtypes in radioligand binding assays. Caramiphen binds with high affinity at the M1 site labeled by [3H]pirenzepine in rat cortex (Ki = 1.2 nM) and displays a 27-fold greater preference for the M1 than the M2 site labeled by [3H](-)-quinuclidinyl benzilate in rat heart, and a 6-fold greater preference for the M1 than the M3 site labeled by [3H]N-methylscopolamine in rat submaxillary gland. Iodocaramiphen binds with high affinity (Ki = 2.1 nM) and selectivity (59-fold) for the M1 vs. M2 subtype, and is 4-fold more selective for the M1 vs. M3 site. Nitrocaramiphen binds with high affinity for M1 sites (Ki = 5.5 nM) and with a 71-fold selectivity over M2, and a 10-fold selectivity for the M1 over the M3 subtype. All three compounds interacted with the M1 binding site in a competitive manner. Nitrocaramiphen and iodocaramiphen are as potent and showed a comparable selectivity for binding to the M1 over the M2 site than the prototypical agent pirenzepine (M1; Ki = 5.2 nM, 51-fold selectivity). Additionally, nitrocaramiphen demonstrates at least a 10-fold selectivity for the M1 over the M3 site. These ester-type antimuscarinics may be better ligands for the study of M1 receptors in brain than the hydrophilic agent pirenzepine.

Opioid agonist properties of two oxime derivatives of naltrexone, NPC 831 and NPC 836.

Two novel oxime derivatives of naltrexone, 6-[2-phenylethyl]-oximino naltrexone (NPC 831) and 6-[3-phenylpropyl]-oximino naltrexone (NPC 836) were potent agonists at opioid receptors. Both compounds inhibited binding to all three opioid receptor subtypes with nanomolar affinities. In vivo, NPC 831 and NPC 836 were equipotent to morphine and more potent than the kappa-selective agonist U-50,488H to produce analgesia. ED50 values of 4.02 mg/kg for NPC 831 and 2.24 mg/kg for NPC 836 were generated for inhibition of the tail-flick response in the rat, and ED50 values of 0.05 mg/kg for NPC 831 and 0.02 mg/kg for NPC 836 were calculated for inhibition of the writhing response in the mouse. Bombesin-induced scratching was used to evaluate NPC 831 and NPC 836 for kappa-agonist properties, and the A50, defined as the percent antagonism of the bombesin-induced response, was 1.86 mg/kg for NPC 831 and 0.08 mg/kg for NPC 836, compared to an A50 of 1.54 mg/kg for U-50,488H. These data suggest that NPC 831 and NPC 836 possess potent mu- and kappa-agonist properties in vivo, with NPC 836 being approximately twice as potent as NPC 831 to produce analgesia and 20 times as potent as NPC 831 to inhibit the scratching response produced by bombesin.

Allosteric modulation of ligand binding to [3H](+)pentazocine-defined sigma recognition sites by phenytoin.

The allosteric modulation of sigma recognition sites by phenytoin (diphenylhydantoin) has been demonstrated by the ability of phenytoin to stimulate binding of various [3H] sigma ligands, as well as to slow dissociation from sigma sites and to shift sigma sites from a low- to a high-affinity state. Phenytoin stimulated the binding of the sigma 1- selective ligand [3H](+)pentazocine in a dose-dependent manner. Stimulation of binding at a final concentration of 250 microM phenytoin was associated with a decrease in the KD. The affinities of the sigma reference compounds caramiphen, dextromethorphan, dextrophan, (+)3-PPP and (+)SKF-10,047 were three- to eight-fold higher, while the affinities of benzetimide, BMY-14802, carbetapentane, DTG and haloperidol were unchanged in the presence of 250 microM phenytoin. The relative sensitivity of sigma compounds to allosteric modulation by phenytoin is not a property of all sigma ligands, and may provide an in vitro basis for distinguishing actions of sigma compounds and predicting sigma effects in vivo.