Risperidone attenuates the discriminative-stimulus effects of d-amphetamine in humans

Studies conducted with nonhuman laboratory animals have consistently shown that atypical antipsychotics that are mixed dopamine and serotonin antagonists attenuate the discriminative-stimulus effects of amphetamine. In the present experiment, eight healthy humans learned to discriminate 15 mg of oral d-amphetamine. After acquiring the discrimination (i.e., > or = 80% correct responding on four consecutive days), the effects of a range of doses of d-amphetamine (0, 2.5, 5, 10, and 15 mg), alone and after pretreatment with risperidone (0 and 1 mg), a D2 dopamine and 5-hydroxytryptamine (5-HT)2 serotonin antagonist, were assessed. d-Amphetamine alone functioned as a discriminative stimulus and produced stimulant-like self-reported drug effects (e.g., increased ratings of "like drug"). These effects were generally a function of dose. Risperidone alone did not occasion d-amphetamine-appropriate responding, but impaired performance. Risperidone pretreatment significantly attenuated the discriminative-stimulus effects of d-amphetamine, and some of the self-reported drug effects. The results of the present experiment suggest that combining drug-discrimination and self-reported drug-effect questionnaires may be an effective strategy for assessing the behavioral effects of agonist-antagonist interactions. Future studies should compare the behavioral effects of d-amphetamine after pretreatment with a selective D2 dopamine (e.g., haloperidol) or 5-HT2 serotonin (e.g., ritanserin) antagonist to determine the relative contribution of dopamine and serotonin systems in mediating the behavioral effects of stimulants in humans. The results of these studies might guide the development of a pharmacotherapy for the treatment of amphetamine abuse/dependence.     

Efficacy of the acyclic guanosine analog buciclovir [(R)-9-(3,4-dihydroxybutyl)guanine] in experimental genital herpes.

The efficacy of the anti-herpesvirus drug buciclovir [(R)-9-(3,4-dihydroxybutyl)guanine] was investigated in guinea pigs and mice infected intravaginally with herpes simplex virus type 2. Topical treatment initiated early after infection was efficacious, in contrast to topical treatment delayed 24 h or more. Systemic treatment of infected mice could not prevent the spread of virus to the brain and mortality. Systemically administered buciclovir had an effect in guinea pigs, even after delayed onset of treatment, but this effect required high doses of the drug. Our results suggest that buciclovir has only a limited effect against herpesvirus infections once the virus is present in the nervous systems of infected animals.     

Syndecan 1 plays a novel role in enteral glutamine's gut-protective effects of the postischemic gut

Syndecan 1 is the predominant heparan sulfate proteoglycan found on the surface of epithelial cells and, like glutamine, is essential in maintaining the intestinal epithelial barrier. We therefore hypothesized that loss of epithelial syndecan 1 would abrogate the gut-protective effects of enteral glutamine. Both an in vitro and in vivo model of gut ischemia-reperfusion (IR) was utilized. In vitro, intestinal epithelial cells underwent hypoxia-reoxygenation to mimic gut IR with 2 mM (physiologic) or 10 mM glutamine supplementation. Permeability, caspase activity, cell growth, and cell surface and shed syndecan 1 were assessed. In vivo, wild-type and syndecan 1 knockout (KO) mice received ± enteral glutamine followed by gut IR. Intestinal injury was assessed by fluorescent dye clearance and histopathology, permeability as mucosal-to-serosal clearance ex vivo in everted sacs, and inflammation by myeloperoxidase (MPO) activity. In an in vitro model of gut IR, glutamine supplementation reduced epithelial cell permeability and apoptosis and enhanced cell growth. Shed syndecan 1 was reduced by glutamine without an increase in syndecan 1 mRNA. In vivo, intestinal permeability, inflammation, and injury were increased after gut IR in wild-type mice and further increased in syndecan 1 KO mice. Glutamine's attenuation of IR-induced intestinal hyperpermeability, inflammation, and injury was abolished in syndecan 1 KO mice. These results suggest that syndecan 1 plays a novel role in the protective effects of enteral glutamine in the postischemic gut.     

Differential behavioral tolerance to the delta-opioid agonist SNC80 ([(+)-4-[(alphaR)-alpha-[(2S,5R)-2,5-dimethyl-4-(2-propenyl)-1-piperazinyl]-(3-methoxyphenyl)methyl]-N,N-diethylbenzamide) in Sprague-Dawley rats

Nonpeptidic delta-opioid agonists produce a number of behaviors, such as antidepressant-like effects, locomotor stimulation, antinociception, and convulsions. To consider this class of compounds as potential therapeutics for humans, the effects of delta-opioid agonists after repeated administration must be evaluated. Therefore, the present study investigated the effects of repeated delta-opioid agonist, SNC80 ([(+)-4-[(alphaR)-alpha-[(2S,5R)-2,5-dimethyl-4-(2-propenyl)-1-piperazinyl]-(3-methoxyphenyl)-methyl]-N,N-diethylbenzamide), administration on its antidepressant-like effects in the forced swim test, locomotor activity, and convulsions in male Sprague-Dawley rats. Tolerance developed rapidly to the convulsive and locomotor-stimulating effects of SNC80 but not to the antidepressant-like effects. In addition, tolerance was evaluated at the level of the receptor-G protein interaction by measuring 5'-O-(3-[35S]thio)triphosphate binding in brains from rats that were pretreated with SNC80. With various exposure durations to SNC80, some brain regions demonstrated tolerance at different times, suggesting that adaptations in the delta-opioid system may occur during agonist exposure. Overall, the lack of observable tolerance to the antidepressant-like effects of SNC80 indicates that this class of compounds has potential as a novel antidepressant therapy.     

Pharmacological properties of (2R)-N-[1-(6-aminopyridin-2-ylmethyl)piperidin-4-yl]-2-[(1R)-3,3-difluorocyclopentyl]-2-hydroxy-2-phenylacetamide: a novel mucarinic antagonist with M(2)-sparing antagonistic activity

We evaluated the pharmacological profiles of (2R)-N-[1-(6- aminopyridin-2-ylmethyl)piperidin-4-yl]-2-[(1R)-3,3-difluorocyclopentyl]-2-hydroxy-2-phenylacetamide(compound A), which is a novel muscarinic receptor antagonist with M(2)-sparing antagonistic activity. Compound A inhibited [(3)H]NMS binding to cloned human muscarinic m1, m2, m3, m4, and m5 receptors expressed in Chinese hamster ovary cells with K(i) values (nM) of 1.5, 540, 2.8, 15, and 7.7, respectively. In isolated rat tissues, compound A inhibited carbachol-induced responses with 540-fold selectivity for trachea (K(B) = 1.2 nM) over atria (K(B) = 650 nM). In in vivo rat assays, compound A inhibited acetylcholine-induced bronchoconstriction and bradycardia with intravenous ED(50) values of 0.022 mg/kg and >/=10 mg/kg, respectively. Furthermore, in dogs, compound A (0.1-1 mg/kg p.o.) dose dependently shifted the methacholine concentration-respiratory resistance curves. In mice, compound A (10 mg/kg i.v.) did not inhibit oxotremorine-induced tremor. The brain/plasma ratio (K(p)) of compound A (3 mg/kg i.v.) was 0.13 in rats; this K(p) was less than that of scopolamine (1.7) and darifenacin (0.24). The inhibition of compound A (3 mg/kg i.v.) on ex vivo binding in rat cerebral cortex was almost similar to that of NMS. These findings demonstrate that compound A has high selectivity for M(3) receptors over M(2) receptors, displays a potent, oral M(3) antagonistic activity without inhibition of central muscarinic receptors because of low brain penetration. It is well known that central muscarinic antagonists may have diverse CNS effects, and M(2) receptors regulate cardiac pacing and act as autoreceptors in the lung and bladder. Thus, compound A may have fewer cardiac or CNS side effects than nonselective compounds.     

Orphan G protein-coupled receptor GPR56 plays a role in cell transformation and tumorigenesis involving the cell adhesion pathway

GPR56 is an orphan G protein - coupled receptor, mutations of which have recently been associated with bilateral frontoparietal polymicrogyria, a rare neurologic disease that has implications in brain development. However, no phenotype beyond central nervous system has yet been described for the GPR56-null mutations despite abundant GPR56 expression in many non - central nervous system adult tissues. In the present study, we show that higher GPR56 expression is correlated with the cellular transformation phenotypes of several cancer tissues compared with their normal counterparts, implying a potential oncogenic function. RNA interference-mediated GPR56 silencing results in apoptosis induction and reduced anchorage-independent growth of cancer cells via increased anoikis, whereas cDNA overexpression resulted in increased foci formation in mouse fibroblast NIH3T3 cell line. When GPR56 silencing was induced in vivo in several xenograft tumor models, significant tumor responses (including regression) were observed, suggesting the potential of targeting GPR56 in the development of tumor therapies. The expression profiling of GPR56-silenced A2058 melanoma cell line revealed several genes whose expression was affected by GPR56 silencing, particularly those in the integrin-mediated signaling and cell adhesion pathways. The potential role of GPR56 in cancer cell adhesion was further confirmed by the observation that GPR56 silencing also reduced cell adhesion to the extracellular matrix, which is consistent with the observed increase in anoikis and reduction in anchorage-independent growth phenotypes. The oncogenic potential and apparent absence of physiologic defects in adult human tissues lacking GPR56, as well as the targetable nature of G protein - coupled receptor by small molecule or antibody, make GPR56 an attractive drug target for the development of cancer therapies.     

Differential antiherpesvirus and antiretrovirus effects of the (S) and (R) enantiomers of acyclic nucleoside phosphonates: potent and selective in vitro and in vivo antiretrovirus activities of (R)-9-(2-phosphonomethoxypropyl)-2,6-diaminopurine.

The (S)- and (R)-enantiomers of acyclic purine nucleoside phosphonate analogs (i.e., 3-hydroxy-2-phosphonomethoxypropyl [HPMP] derivatives, 3-fluoro-2-phosphonomethoxypropyl [FPMP] derivatives, and 2-phosphonomethoxypropyl [PMP] derivatives of adenine [A], 2-aminopurine, 2,6-diaminopurine [DAP], and guanine [G]) have been synthesized and evaluated for antiviral activity. As a rule, the HPMP derivatives proved effective against DNA viruses but not RNA viruses or retroviruses. In particular, (S)-HPMPA, (S)-HPMPDAP, and (R)- and (S)-HPMPG were exquisitely inhibitory to herpes simplex virus type 1 (50% effective concentrations, 0.63, 0.22, 0.10, and 0.66 microM, respectively). The FPMP and PMP derivatives showed marked inhibitory activities against retroviruses but not DNA viruses. The (S)-enantiomer of FPMPA and the (R)-enantiomer of PMPA were approximately 30- to 100-fold more effective against human immunodeficiency virus and Moloney murine sarcoma virus (MSV) than their enantiomeric counterparts. In contrast, both (S)- and (R)-enantiomers of the DAP and G derivatives proved equally effective against retroviruses, except for (R)-PMPDAP, which was 15- to 40-fold more inhibitory than (S)-PMPDAP. (R)-PMPDAP emerged as the most potent and selective inhibitor of MSV-induced transformation of murine C3H/3T3 cells and human immunodeficiency virus-induced cytopathicity in MT-4 and CEM cells (50% effective concentration, approximately 0.1 to 0.6 microM). When administered intraperitoneally at a single dose as low as 2 mg/kg, (R)-PMPDAP efficiently decreased MSV-induced tumor formation in newborn NMRI mice and significantly increased the survival time of MSV-infected mice. In addition, upon oral administration to MSV-infected mice, (R)-PMPDAP showed marked antiretroviral efficacy.     

The spleen plays a central role in primary humoral alloimmunization to transfused mHEL red blood cells

BACKGROUND: Several differences exist between antigens on transfused red blood cells (RBCs) and other immunogens, including anatomical compartmentalization. Whereas antigens from microbial pathogens and solid organ transplants drain into local lymph nodes, circulating RBCs remain segregated in the peripheral circulation, where they are consumed by antigen-presenting cells (APCs) in the spleen and liver. Accordingly, it was hypothesized that the splenic APCs play a central role in primary alloimmunization to transfused RBCs.
STUDY DESIGN AND METHODS: Recipient mice were splenectomized and transfused with transgenic RBCs expressing the membrane-bound hen egg lysozyme (mHEL) model RBC antigen. In some experiments, mHEL-specific CD4+ T cells were adoptively transferred into recipient mice to allow investigation of helper T-cell responses. Unmanipulated or sham-splenectomized mice served as controls. Recombinant murine cytomegalovirus expressing mHEL (mHEL-MCMV) was used as a control non-RBC immunogen. Humoral responses were measured by mHEL-specific enzyme-linked immunosorbent assay and flow cytometric–based RBC cross-match.
RESULTS: Control animals synthesized detectable anti-HEL immunoglobulin (Ig)G after a single mHEL RBC transfusion. mHEL-specific CD4+ T cells underwent robust expansion, and adoptive transfer of CD4+ T cells resulted in a 1000-fold increase in anti-HEL IgG. In contrast, minimal anti-HEL IgG was detectable in splenectomized mice, mHEL-specific CD4+ T cells did not proliferate, and adoptive transfer did not increase anti-HEL IgG. However, anti-HEL IgG response after exposure to mHEL-MCMV was equivalent in control and splenectomized mice.
DISCUSSION: Together, these findings illustrate the distinct properties of transfused RBCs as immunologic stimuli, with the spleen playing a critical role in primary RBC alloimmunization at the level of CD4+ T-cell activation.     

Effects of the beta 3-adrenergic receptor agonist disodium 5-[(2R)-2-[[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL-316243) on bladder micturition reflex in spontaneously hypertensive rats

The present study investigated whether beta3-adrenoceptor activation acts on the bladder afferent pathway by examination of the visceromotor reflex (VMR) and pressor responses to urinary bladder distension (UBD) and whether beta3-adrenoceptor activation produces urinary bladder relaxation in hyperactive spontaneously hypertensive rats (SHRs) in comparison with their normotensive control rats [Wistar-Kyoto (WKY)]. Using the VMR responses to noxious UBD as a measure of bladder afferent signal transmission, SHRs did not present a sensitized bladder phenotype. However, reduced bladder compliance accompanied by a reduced void threshold was detected in the SHR detrusor. Furthermore, the selective beta3-adrenoceptor agonist disodium 5-[(2R)-2-[[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL-316243) (i.v.) failed to attenuate VMR or pressor responses to UBD in either SHRs or WKY rats, but it dose-dependently inhibited rhythmic contraction (RC) in SHRs. The minimal effective dose was 0.001 mg/kg. Using the same model in WKY rats, CL-316243 did not elicit significant inhibition of contractions in the bladder RC assay. These results suggest that SHRs represent abnormal efferent/detrusor function (detrusor overactivity) without mechanosensory afferent hypersensitivity. The beta3-adrenoceptor agonist CL-316243 acts on the detrusor muscle to increase urine storage in SHRs.     

SSR240612 [(2R)-2-[((3R)-3-(1,3-benzodioxol-5-yl)-3-[[(6-methoxy-2-naphthyl)sulfonyl]amino]propanoyl)amino]-3-(4-[[2R,6S)-2,6-dimethylpiperidinyl]methyl]phenyl)-N-isopropyl-N-methylpropanamide hydrochloride], a new nonpeptide antagonist of the bradykinin B1 receptor: biochemical and pharmacological characterization

The biochemical and pharmacological properties of a novel non-peptide antagonist of the bradykinin (BK) B(1) receptor, SSR240612 [(2R)-2-[((3R)-3-(1,3-benzodioxol-5-yl)-3-[[(6-methoxy-2-naphthyl)sulfonyl]amino]propanoyl)amino]-3-(4-[[2R,6S)-2,6-dimethylpiperidinyl]methyl]phenyl)-N-isopropyl-N-methylpropanamide hydrochloride] were evaluated. SSR240612 inhibited the binding of [(3)H]Lys(0)-des-Arg(9)-BK to the B(1) receptor in human fibroblast MRC5 and to recombinant human B(1) receptor expressed in human embryonic kidney cells with inhibition constants (K(i)) of 0.48 and 0.73 nM, respectively. The compound selectivity for B(1) versus B(2) receptors was in the range of 500- to 1000-fold. SSR240612 inhibited Lys(0)-desAr(9)-BK (10 nM)-induced inositol monophosphate formation in human fibroblast MRC5, with an IC(50) of 1.9 nM. It also antagonized des-Arg(9)-BK-induced contractions of isolated rabbit aorta and mesenteric plexus of rat ileum with a pA(2) of 8.9 and 9.4, respectively. Antagonistic properties of SSR240612 were also demonstrated in vivo. SSR240612 inhibited des-Arg(9)-BK-induced paw edema in mice (3 and 10 mg/kg p.o. and 0.3 and 1 mg/kg i.p.). Moreover, SSR240612 reduced capsaicin-induced ear edema in mice (0.3, 3 and 30 mg/kg p.o.) and tissue destruction and neutrophil accumulation in the rat intestine following splanchnic artery occlusion/reperfusion (0.3 mg/kg i.v.). The compound also inhibited thermal hyperalgesia induced by UV irradiation (1 and 3 mg/kg p.o.) and the late phase of nociceptive response to formalin in rats (10 and 30 mg/kg p.o.). Finally, SSR240612 (20 and 30 mg/kg p.o.) prevented neuropathic thermal pain induced by sciatic nerve constriction in the rat. In conclusion, SSR240612 is a new, potent, and orally active specific non-peptide bradykinin B(1) receptor antagonist.     

The delta-opioid receptor agonist SNC80 [(+)-4-[alpha(R)-alpha-[(2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl]-(3-methoxybenzyl)-N,N-diethylbenzamide] synergistically enhances the locomotor-activating effects of some psychomotor stimulants, but not direct dopamine agonists, in rats

The nonpeptidic delta-opioid agonist SNC80 [(+)-4-[alpha(R)-alpha-[(2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl]-(3-methoxybenzyl)-N,N-diethylbenzamide] produces many stimulant-like behavioral effects in rodents and monkeys, such as locomotor stimulation, generalization to cocaine in discrimination procedures, and antiparkinsonian effects. Tolerance to the locomotor-stimulating effects of SNC80 develops after a single administration of SNC80 in rats; it is not known whether cross-tolerance develops to the effects of other stimulant compounds. In the initial studies to determine whether SNC80 produced cross-tolerance to other stimulant compounds, it was discovered that amphetamine-stimulated locomotor activity was greatly enhanced in SNC80-pretreated rats. This study evaluated acute cross-tolerance between delta-opioid agonists and other locomotor-stimulating drugs. Locomotor activity was measured in male Sprague-Dawley rats implanted with radiotransmitters, and activity levels were recorded in the home cage environment. Three-hour SNC80 pretreatment produced tolerance to further delta-opioid receptor stimulation but also augmented greatly amphetamine-stimulated locomotor activity in a dose-dependent manner. Pretreatments with other delta-opioid agonists, (+)BW373U86 [(+)-4-[alpha(R)-alpha-[(2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl]-3-hydroxybenzyl]-N,N-diethylbenzamide] and oxymorphindole (17-methyl-6,7-dehydro-4,5-epoxy-3,14-dihydroxy-6,7,2',3'-indolomorphinan), also modified amphetamine-induced activity levels. SNC80 pretreatment enhanced the stimulatory effects of the dopamine/norepinephrine transporter ligands cocaine and nomifensine (1,2,3,4-tetrahydro-2-methyl-4-phenyl-8-isoquinolinanmine maleate salt), but not the direct dopamine receptor agonists SKF81297 [R-(+)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide] and quinpirole [trans-(-)-(4alphaR)-4,4a, 5,6,7,8,8a,9-octahydro-5-propyl-1H-pyrazolo[3,4-g] quinoline monohydrochloride]. In conclusion, SNC80 enhanced the locomotor-stimulating effects of monoamine transporter ligands suggesting that delta-opioid receptor activation might alter the functional activity of monoamine transporters or presynaptic monoamine terminals.     

Antiherpes effects and pharmacokinetic properties of 9-(4-hydroxybutyl) guanine and the (R) and (S) enantiomers of 9-(3,4-dihydroxybutyl)guanine.

Three acyclic guanosine analogs with similar structures, the (R) and (S) forms of 9-(3,4-dihydroxybutyl)guanine and 9-(4-hydroxybutyl)guanine, were compared for antiherpes activity in vivo and in vitro. The three guanosine analogs were viral thymidine kinase-dependent inhibitors of virus multiplication. In cell cultures, (S)-9-(3,4-dihydroxybutyl)guanine was the least active of these three drugs against a variety of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) strains. This was also the case for a certain HSV-1 or HSV-2 strain in different cell lines. In cell cultures, (R)-9-(3,4-dihydroxybutyl)guanine and 9-(4-hydroxybutyl)guanine had similar antiherpes activities. However, in vivo in cutaneous HSV-1 infections in guinea pigs treated topically and in systemic HSV-2 infections in mice treated orally or intraperitoneally, only (R)-9-(3,4-dihydroxybutyl)guanine had a therapeutic effect. The extremely short half-life in plasma and the high clearance of 9-(4-hydroxybutyl)guanine as compared with those of (R)-9-(3,4-dihydroxybutyl)guanine probably made 9-(4-hydroxybutyl)guanine inefficacious when given intraperitoneally or orally to mice infected with herpesvirus. On the other hand, no kinetic differences between (R)-9-(3,4-dihydroxybutyl)guanine and 9-(4-hydroxybutyl)guanine were observed in penetration through guinea pig skin ex vivo, and no preferential metabolism of 9-(4-hydroxybutyl)guanine in skin was noted. We deduced that high thymidine levels in guinea pig skin preferentially antagonize the antiviral effect of 9-(4-hydroxybutyl) guanine in cutaneous HSV-1 infections.     

BCH-1868 [(-)-2-R-dihydroxyphosphinoyl-5-(S)-(guanin-9'-yl-methyl) tetrahydrofuran]: a cyclic nucleoside phosphonate with antitumor activity

Nucleoside phosphonates are widely used therapeutic agents with a broad spectrum of antiviral activity. However, only a few of them are reported to have antitumor activity. In this study, we show that a tetrahydrofuran phosphonate analogue of guanosine, (-)-2-R-dihydroxyphosphinoyl-5-(S)-(guanin-9'-ylmethyl) tetrahydrofuran (BCH-1868), previously reported as having antiviral activity, also displays antitumor activity. In vitro, BCH-1868 inhibited the proliferation of several murine and human cancer cell lines with IC50s in the microM range independently of the tissue type or the presence of multidrug resistance protein MRP/gp190. In vivo, BCH-1868 was active against a variety of human tumor xenograft models (Caki-1, HT-29, DU 145, COLO 205, and CCRF-CEM). In all tumors tested, a significant tumor growth inhibition was noted at 40-50 mg/kg (daily x 5), but no tumor regression was observed in the settings used. To better understand these results, we partially characterized, at the cellular level, the mechanism of action of this new cyclic nucleoside phosphonate and investigated its pharmacokinetic characteristics in mice. We showed that BCH-1868 exerts its antitumor activity by an inhibitory mechanism at the level of DNA polymerase a, resulting in arrest of DNA synthesis and a block of cell division at the S phase of the cell cycle. Low-circulating plasma concentration (Cmax = 87 microM; area under the curve = 1138 micromol x min/liters; after a bolus i.v. injection of 10 mg/kg) and rapid clearance of the drug (terminal half-life, t1/2 = 16 min) may contribute to the modest antitumor efficacy observed in vivo.     

In vitro and in vivo pharmacological characterization of 5-[(R)-2-(5,6-diethyl-indan-2-ylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one (indacaterol), a novel inhaled beta(2) adrenoceptor agonist with a 24-h duration of action

Here, we describe the preclinical pharmacological profile of 5-[(R)-2-(5,6-diethyl-indan-2-ylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one (indacaterol), a novel, chirally pure inhaled beta(2) adrenoceptor agonist, in comparison with marketed drugs. Indacaterol is close to a full agonist at the human beta(2) adrenoceptor (E(max) = 73 +/- 1% of the maximal effect of isoprenaline; pEC(50) = 8.06 +/- 0.02), whereas salmeterol displays only partial efficacy (38 +/- 1%). The functional selectivity profile of indacaterol over beta(1) human adrenoceptors is similar to that of formoterol, whereas its beta(3) adrenoceptor selectivity profile is similar to that of formoterol and salbutamol. In isolated superfused guinea pig trachea, indacaterol has a fast onset of action (30 +/- 4 min) similar to formoterol and salbutamol, and a long duration of action (529 +/- 99 min) comparable with salmeterol. In the conscious guinea pig, when given intratracheally as a dry powder, indacaterol inhibits 5-hydroxytryptamine-induced bronchoconstriction for at least 24 h, whereas salmeterol, formoterol, and salbutamol have durations of action of 12, 4, and 2 h, respectively. When given via nebulization to anesthetized rhesus monkeys, all of the compounds dose-dependently inhibit methacholine-induced bronchoconstriction, although indacaterol produces the most prolonged bronchoprotective effect and induces the lowest increase in heart rate for a similar degree of antibronchoconstrictor activity. In conclusion, the preclinical profile of indacaterol suggests that this compound has a superior duration of action compatible with once-daily dosing in human, together with a fast onset of action and an improved cardiovascular safety profile over marketed inhaled beta(2) adrenoceptor agonists.     

Inhibition of murine AIDS by a heterodinucleotide of azidothymidine and 9-(R)-2-(phosphonomethoxypropyl)adenine

Tenofovir [9-(R)-2-(phosphonomethoxypropyl)adenine (PMPA)] and zidovudine [azidothymidine (AZT)] are potent anti-HIV agents that have shown a strong synergy in in vitro studies. In this paper we have investigated both the potentiality of this synergy in vivo and the possibility to administer AZT and PMPA simultaneously as a single drug AZTpPMPA. The pharmacokinetic studies reported here have shown that AZTpPMPA administered intraperitoneally in mice performs as a prodrug, providing a slow delivery of AZT and PMPA in circulation. C57BL/6 mice infected with the retroviral complex LP-BM5 were used to evaluate the efficacy of AZTpPMPA in inhibiting disease progression. Furthermore, the effectiveness of the heterodinucleotide was compared with that of AZT and PMPA, administered as single drugs, or as a combination (AZT plus PMPA). The results obtained showed that AZTpPMPA is able to reduce lymphoadenopathy (88%), splenomegaly (64%), lymph node BM5 proviral DNA content (49%) and hypergammaglobulinaemia (40%). However, upon AZT plus PMPA administration, similar (splenomegaly and lymphoadenopathy reduction) or better results (64% hypergammaglobulinaemia reduction and 75% lymph node BM5 proviral DNA content inhibition) were obtained. Furthermore, these results overlapped those obtained upon PMPA administration. Thus, no synergy between PMPA and AZT was observed in murine AIDS and administration of AZT does not improve the antiviral results obtained by PMPA administration.     

A-995662 [(R)-8-(4-methyl-5-(4-(trifluoromethyl)phenyl)oxazol-2-ylamino)-1,2,3,4-tetrahydronaphthalen-2-ol], a novel,selective TRPV1 receptor antagonist,reduces spinal release of glutamate and CGRP in a rat knee joint pain model

The TRPV1 antagonist A-995662 demonstrates analgesic efficacy in monoiodoacetate-induced osteoarthritic (OA) pain in rat, and repeated dosing results in increased in vivo potency and a prolonged duration of action. To identify possible mechanism(s) underlying these observations, release of neuropeptides and the neurotransmitter glutamate from isolated spinal cord was measured. In OA rats, basal release of glutamate, bradykinin and calcitonin gene-related peptide (CGRP) was significantly elevated compared to naïve levels, whereas substance P (SP) levels were not changed. In vitro studies showed that capsaicin-evoked TRPV1-dependent CGRP release was 54.7 ± 7.7% higher in OA, relative to levels measured for naïve rats, suggesting that TRPV1 activity was higher under OA conditions. The efficacy of A-995662 in OA corresponded with its ability to inhibit glutamate and CGRP release from the spinal cord. A single, fully efficacious dose of A-995662, 100 μmol/kg, reduced spinal glutamate and CGRP release, while a single sub-efficacious dose of A-995662 (25 μmol/kg) was ineffective. Multiple dosing with A-995662 increased the potency and duration of efficacy in OA rats. Changes in efficacy did not correlate with plasma concentrations of A-995662, but were accompanied with reductions in spinal glutamate release. These findings suggest that repeated dosing of TRPV1 antagonists enhances therapeutic potency and duration of action against OA pain, at least in part, by the sustained reduction in release of glutamate and CGRP from the spinal cord.     

Mitogen-activated protein kinase phosphorylation in the rostral ventrolateral medulla plays a key role in imidazoline (i1)-receptor-mediated hypotension

Our previous study showed that rilmenidine, a selective I(1)-imidazoline receptor agonist, enhanced the phosphorylation of mitogen-activated protein kinase (MAPK)(p42/44), via the phosphatidylcholine-specific phospholipase C pathway in the pheochromocytoma cell line (PC12). In the present study, we tested the hypothesis that enhancement of MAPK phosphorylation in the rostral ventrolateral medulla (RVLM) contributes to the hypotensive response elicited by I(1)-receptor activation in vivo. Systemic rilmenidine (600 microg/kg i.v.) elicited hypotension and bradycardia along with significant elevation in MAPK(p42/44), detected by immunohistochemistry, in RVLM neurons. To obtain conclusive evidence that the latter response was I(1)-receptor-mediated, similar hypotensive responses were elicited by intracisternal (i.c.) rilmenidine (25 microg/rat) or the highly selective alpha(2)-agonist alpha-methylnorepinephrine (4 microg/rat). An increase in RVLM MAPK(p42/44) occurred only after rilmenidine. Furthermore, pretreatment with efaroxan (0.15 microg/rat i.c.), a selective I(1)-imidazoline receptor antagonist, or with PD98059 (2'-amino-3'-methoxyflavone) (5 microg/rat i.c.), a selective extracellular signal-regulated kinase 1/2 inhibitor, significantly attenuated the hypotensive response and the elevation in RVLM MAPK(p42/44) elicited by i.c. rilmenidine. The findings suggest that MAPK phosphorylation in the RVLM contributes to the hypotensive response induced by I(1)-receptor activation and presents in vivo evidence that distinguishes the neuronal responses triggered by the I(1)-receptor from those triggered by the alpha(2)-adrenergic receptor.