Augmentation of cognitive function by NS9283, a stoichiometry-dependent positive allosteric modulator of α2- and α4-containing nicotinic acetylcholine receptors.

BACKGROUND AND PURPOSE: Positive allosteric modulation of α4ß2 nicotinic acetylcholine (nACh) receptors could add a new dimension to the pharmacology and therapeutic approach to these receptors. The novel modulator NS9283 was therefore tested extensively. EXPERIMENTAL APPROACH: Effects of NS9283 were evaluated in vitro using fluorescence-based Ca(2+) imaging and electrophysiological voltage clamp experiments in Xenopus oocytes, mammalian cells and thalamocortical neurons. In vivo the compound was tested in models covering a range of cognitive domains in mice and rats. KEY RESULTS: NS9283 was shown to increase agonist-evoked response amplitude of (α4)(3) (ß2)(2) nACh receptors in electrophysiology paradigms. (α2)(3) (ß2)(2) , (α2)(3) (ß4)(2) and (α4)(3) (ß4)(2) were modulated to comparable extents, but no effects were detected at α3-containing or any 2α : 3ß stoichiometry nACh receptors. Native nACh receptors in thalamocortical neurons similarly displayed DHßE-sensitive currents that were receptive to modulation. NS9283 had favourable effects on sensory information processing, as shown by reversal of PCP-disrupted pre-pulse inhibition. NS9283 further improved performance in a rat model of episodic memory (social recognition), a rat model of sustained attention (five-choice serial reaction time task) and a rat model of reference memory (Morris water maze). Importantly, the effects in the Morris water maze could be fully reversed with mecamylamine, a blocker of nACh receptors. CONCLUSIONS AND IMPLICATIONS: These results provide compelling evidence that positive allosteric modulators acting at the (α4)(3) (ß2)(2) nACh receptors can augment activity across a broad range of cognitive domains, and that α4ß2 nACh receptor allosteric modulation therefore constitutes a promising therapeutic approach to symptomatic treatment of cognitive impairment.

[Life-threatening respiratory depression after pain therapy]. / Lebensbedrohliche Atemdepression nach Schmerztherapie.

The case is reported of a female patient with attack-like neuropathic pain following lower arm injury with repeated respiratory depression and unconsciousness even after several administrations of sufficiently dosed analgetics for suppression of the attacks. Readers are encouraged to make their own diagnosis on the basis of the clinical findings and to discuss the case online (http://www.blogs.springer.com/DerSchmerz). The discussion and the solution will be published in the next issue.

[Hepatitis B associated polyarteriitis nodosa with cerebral vasculitis]. / Hepatitis-B-assoziierte Panarteriitis nodosa mit zerebraler Vaskulitis.

HISTORY AND ADMISSION FINDINGS: A 53-year-old male was admitted with an acute brainstem syndrome. He developed a severe fluctuating psychosis. Because of the worsening neurological symptoms he was admitted to our neurological clinic five months after onset of the disease. On admission he showed signs of a productive psychosis in addition to akinetic-rigid parkinsonism and cerebellar symptoms. INVESTIGATIONS: Laboratory tests revealed a HBeAg-negative hepatitis B. The initial neuroradiolgical studies showed multiple supratentorial and periventricular ischemic and hemorrhagic lesions. MR-angiography and conventional cerebral angiography demonstrated multiple irregularities of the intracranial vessels and vascular occlusions, findings which were compatible with cerebral vasculitis. DIAGNOSIS, THERAPY AND COURSE: The laboratory and neuroradiological studies indicated a hepatitis B-associated polyarteriitis nodosa and cerebral vasculitis. He was given oral immunsuppressive therapy (prednisolone 60 mg daily) and virostatic drug (lamivudine 100 mg daily). When the steroid dosis was reduced to 40 mg prednisolon a severe relapse of the encephalopathy occurred which was treated with the atypical antipsychotic drug risperidon, 3 mg daily, and intravenous methylprednisolone plus plasmaphereses. Later he was given prednisolone (60 mg daily) and lamivudine (100 mg daily) again which has so far stabilized the clinical course. CONCLUSION: The main treatment of the rare hepatitis B-associated polyarteriitis nodosa with cerebral vasculitis consists of oral steroids in combination with antiviral drugs. Depending on the course of the disease an escalating steroid pulse administration and plasmaphereses should be considered.

Synthesis and biological evaluation of clitocine analogues as adenosine kinase inhibitors.

Adenosine kinase (AK) is the primary enzyme responsible for adenosine metabolism. Inhibition of AK effectively increases extracellular adenosine concentrations and represents an alternative approach to enhance the beneficial actions of adenosine as compared to direct-acting receptor agonists. Clitocine (3), isolated from the mushroom Clitocybe inversa, has been found to be a weak inhibitor of AK. We have prepared a number of analogues of clitocine in order to improve its potency and demonstrated that 5'-deoxy-5'-amino-clitocine (7) improved AK inhibitory potency by 50-fold.

Pyridopyrimidine analogues as novel adenosine kinase inhibitors.

A novel series of pyridopyrimidine analogues 9 was identified as potent adenosine kinase inhibitors based on the SAR and computational studies. Substitution of the C7 position of the pyridopyrimidino core with C2' substituted pyridino moiety increased the in vivo potency and enhanced oral bioavailability of these adenosine kinase inhibitors.

Structure-activity studies of 5-substituted pyridopyrimidines as adenosine kinase inhibitors.

The synthesis and SAR of a novel series of non-nucleoside pyridopyrimidine inhibitors of the enzyme adenosine kinase (AK) are described. It was found that pyridopyrimidines with a broad range of medium and large non-polar substituents at the 5-position potently inhibited AK activity. A narrower range of analogues was capable of potently inhibiting adenosine phosphorylation in intact cells indicating an enhanced ability of these analogues to penetrate cell membranes. Potent AK inhibitors were found to effectively reduce nociception in animal models of thermal hyperalgesia and persistent pain.

ABT-702 (4-amino-5-(3-bromophenyl)-7-(6-morpholinopyridin-3-yl)pyrido[2, 3-d]pyrimidine), a novel orally effective adenosine kinase inhibitor with analgesic and anti-inflammatory properties: I. In vitro characterization and acute antinociceptive effects in the mouse.

Adenosine (ADO) is an inhibitory neuromodulator that can increase nociceptive thresholds in response to noxious stimulation. Inhibition of the ADO-metabolizing enzyme adenosine kinase (AK) increases extracellular ADO concentrations at sites of tissue trauma and AK inhibitors may have therapeutic potential as analgesic and anti-inflammatory agents. ABT-702 is a novel and potent (IC(50) = 1. 7 nM) non-nucleoside AK inhibitor that has several orders of magnitude selectivity over other sites of ADO interaction (A(1), A(2A), A(3) receptors, ADO transporter, and ADO deaminase). ABT-702 was 1300- to 7700-fold selective for AK compared with a number of other neurotransmitter and peptide receptors, ion channel proteins, neurotransmitter/nucleoside reuptake sites, and enzymes, including cycloxygenases-1 and -2. ABT-702 was equipotent (IC(50) = 1.5 +/- 0. 3 nM) in inhibiting native human AK (placenta), two human recombinant isoforms (AK(long) and AK(short)), and AK from monkey, dog, rat, and mouse brain. Kinetic studies revealed that AK inhibition by ABT-702 was competitive with respect to ADO and noncompetitive with respect to MgATP(2-). AK inhibition by ABT-702 was demonstrated to be reversible after 4 h of dialysis. ABT-702 is orally active and fully efficacious in reducing acute somatic nociception (ED(50) = 8 micromol/kg i.p.; 65 micromol/kg p.o.) in the mouse hot-plate assay. ABT-702 also dose dependently reduced nociception in the phenyl-p-quinone-induced abdominal constriction assay. The antinociceptive effects of ABT-702 in the hot-plate assay were blocked by the nonselective ADO receptor antagonist theophylline, and by the A(1)-selective antagonist cyclopentyltheophylline (10 mg/kg i.p.), but not by a peripherally selective ADO receptor antagonist 8-(p-sulfophenyl)-theophylline (50 mg/kg i.p.), by the A(2A)-selective antagonist 3, 7-dimethyl-1-propargylxanthine (1 mg/kg i.p.) or the opioid antagonist naloxone (5 mg/kg i.p.). Thus, ABT-702 is a novel and potent non-nucleoside AK inhibitor that effectively reduces acute thermal nociception in the mouse by a nonopioid, non-nonsteroidal anti-inflammatory drug, ADO A(1) receptor-mediated mechanism.

Characterization of the effects of adenosine kinase inhibitors on acute thermal nociception in mice.

Adenosine (ADO) is an inhibitory neuromodulator that can increase the nociceptive threshold in animals exposed to a variety of noxious stimuli. Inhibition of the ADO-metabolizing enzyme, ADO kinase (AK), provides a means of locally enhancing extracellular ADO concentrations. In the present study, the AK inhibitors 5'-amino,5'-deoxy-ADO (NH2dADO), 5-iodotubercidin (5-IT), and 5'-deoxy,5-iodotubercidin (5'd-5IT) were examined for their analgesic efficacy in the hot-plate model of acute somatic nociception. Control and drug-treated adult male mice were placed on a 55 degrees C hot plate and the latency to the 10th jump was recorded via a computer driven infrared-beam photosensor. All three AK inhibitors were found to significantly increase jump latencies in a dose-dependent fashion. 5'd-5IT was the most potent AK inhibitor (approx. ED50 value = 1 micromol/kg, IP), followed by 5-IT (ED50 value = 10 micromol/kg, IP), and NH2dADO (ED50 value = 100 micromol/kg, IP). 5'd-5IT was found to be more potent and equally efficacious to morphine (ED50 value = 5.2 micromol/kg, IP) in this assay. In a model of persistent chemical pain, the phenyl-p-quinone-induced abdominal constriction assay, 5'd-5IT (ED50 value = 1.5 micromol/kg, SC) and morphine (ED50 value = 3.0 micromol/kg, SC) dose dependently reduced nociception. Pretreatment of mice with either the nonselective ADO receptor antagonist, theophylline (56 micromol/kg, IP), but not the peripherally acting antagonist, 8-(p-sulfophenyl)-theophylline (8-PST, 200 micromol/kg, IP) significantly attenuated the antinociceptive effects of 5'd-5IT in the hot-plate assay. Furthermore, the antinociceptive effects of 5'd-5IT were completely blocked by an ADO A1 receptor selective antagonist, DPCPX, while an ADO A2A receptor selective antagonist, ZM 241385, showed markedly less antagonist activity. The analgesic effects of 5'd-5IT were not blocked by the opioid receptor antagonist naloxone; however, 5'd-5IT could produce additive analgesic effects with morphine when both compounds were administered in combination. The apparent efficacy of 2.5 micromol/kg, IP, of 5'd-5IT was not significantly altered following the repeated administration of this dose twice daily for 4 days. The present data provide evidence for an antinociceptive action of AK inhibitors in the hot-plate test, which, at least for 5'd-5IT, is mediated by an enhancement of ADO's actions at the ADO A1 receptor subtype, is nonopioid in nature, and which does not exhibit tolerance following repeated administration.

Nitroaromatic amino acids as inhibitors of neuronal nitric oxide synthase.

Nitric oxide (NO.) is an important biomodulator of many physiological processes. The inhibition of inappropriate production of NO. by the isoforms of nitric oxide synthase (NOS) has been proposed as a therapeutic approach for the treatment of stroke, inflammation, and other processes. In this study, certain 2-nitroaryl-substituted amino acid analogues were discovered to inhibit NOS. Analogues bearing a 5-methyl substituent on the aromatic ring demonstrated maximal inhibitory potency. For two selected inhibitors, investigation of the kinetics of the enzyme showed the inhibition to be competitive with l-arginine. Additionally, functional NOS inhibition in tissue preparations was demonstrated.

Induction by endotoxin of nitric oxide synthase in the rat mesentery: lack of effect on action of vasoconstrictors.

1. Male Sprague-Dawley or Wistar rats were injected with bacterial lipopolysaccharide (LPS; 5 mg kg-1, i.p.) and killed after 1, 3, 6, 15, and 24 h. The brains, mesenteries, spleens, lungs, livers, kidneys, hearts, aortae and diaphragms were removed and frozen immediately. Control rats were injected with sterile saline and killed after 6 h. 2. The organs were homogenized in a semi-frozen state and NO synthase (NOS) activity measured in tissues from both LPS-treated and saline-treated groups by the ability of homogenates to convert [3H]-L-arginine to [3H]-L-citrulline in a NADPH-dependent manner. 3. The NOS activity in all organs taken from control animals was found to be calcium-dependent, with the highest activity being in the brain. After LPS-treatment an induced calcium-independent NOS was detected in all tissues tested, with the exception of the brain. The spleen, lung, mesentery and liver had the highest amounts of LPS-induced NOS activity. No induction of calcium-dependent NOS was detected. 4. Induction of NOS was maximum 6 h after administration of LPS and had returned to control levels in 24 h. 5. The constitutive NOS in brain and mesentery and the LPS-induced activities in the spleen, lung, liver and mesentery were inhibited by NG-monomethyl-L-arginine (L-NMMA) or NG-nitro-L-arginine methyl ester (L-NAME) according to concentration. The IC50 for L-NAME was 2.5 microM against the constitutive NOS from brain, and 20-25 microM against the inducible NOS. For L-NMMA the IC50 was 20-25 microM against either NOS isoform. 7. The vascular responses to endothelin-I (ET-1), the thromboxane A2-mimetic 11 alpha,9 alpha-epoxymethanoprostaglandin F2alpha (U46619), phenylephrine (PE) or 5-hydroxytryptamine (5-HT) were measured in the simultaneously perfused arterial and venous mesenteric vascular beds from both control and LPS-treated(6 h) rats. Vasoconstrictor responses to all agonists tested were unaffected by LPS treatment. In the presence of L-NAME (100 microM) vasoconstrictor responses were potentiated in both the arterial and venous portion of the mesenteric beds from both control and LPS-treated rats. The potentiation of responses to U46619 was significantly greater in beds from LPS-treated rats.8. Injection of LPS i.p. is associated with induction of NOS in all organs tested, except for the brain. In the mesentery this is not accompanied by a hyporesponsiveness to constrictor agents suggesting an increased sensitivity, particularly to U46619. This may explain the poor perfusion and tissue damage in the splanchnic circulation associated with sepsis.

Nitric oxide synthase in ferret brain: localization and characterization.

1. In the present study, we have investigated the distribution of nitric oxide synthase in the ferret brain. Nitric oxide synthase was determined biochemically and immunochemically. 2. In the rat brain, the highest nitric oxide synthase activity has been detected in the cerebellum. However, in the ferret brain, the highest activity was found in the striatum and the lowest in the cerebellum and cerebral cortex. The enzymatic activity was localized predominantly in the cytosolic fractions, it was dependent on NADPH and Ca2+, and inhibited by NG-nitro-L-arginine or NG-methyl-L-arginine. 3. Western blot analysis revealed that all regions of the ferret brain contained a 160 kD protein crossreacting with an antibody to nitric oxide synthase purified from the rat cerebellum, and the levels of relative intensity of staining by the antibody correlated with the distribution of nitric oxide synthase activity. 4. These results indicate that the ferret brain contains a nitric oxide synthase similar to the rat brain, but the distribution of enzymatic activity in the ferret brain differs markedly from the rat brain.

Induction of NADPH-dependent diaphorase and nitric oxide synthase activity in aortic smooth muscle and cultured macrophages.

Lipopolysaccharide (LPS), either alone or in combination with cytokines, induces nitric oxide (NO) synthase activity in cells that normally release little or no NO. In arterial smooth muscle cells and various macrophage cell lines, NO synthase activity is induced after several hours of incubation with LPS. In brain, NADPH-dependent diaphorase activity has been associated with constitutive NO synthase. Here we show that incubation of rat aorta or cultured macrophages with LPS causes a time-dependent induction of NO synthase. The NO synthase activity in both rat aorta and macrophages was calcium independent and inhibited by NG-monomethyl-L-arginine and NG-nitro-L-arginine. We also found that LPS caused a time-dependent induction in NADPH-dependent diaphorase activity in both rat aorta and cultured macrophages. The diaphorase activity was mainly NADPH dependent and NADH independent. NO synthase activity and NADPH-diaphorase activity in crude cytosol from LPS-treated macrophages were found to co-purify, using 2',5'-ADP-Sepharose followed by Superose-6 gel permeation chromatography.

Induced RAW 264.7 macrophages express soluble and particulate nitric oxide synthase: inhibition by transforming growth factor-beta.

RAW 264.7 macrophages induced with lipopolysaccharide and interferon-gamma expressed nitric oxide (NO) synthase. Approximately two-thirds of the total induced NO synthase activity was found in the cytosolic fraction, whereas one-third was associated with the particulate fraction. Both enzymes formed L-citrulline in addition to NO-like material. NO and L-citrulline formation by both enzymes were calcium-independent and inhibited by NG-nitro-L-arginine and NG-methyl-L-arginine. Transforming growth factor-beta 1 prevented the induction of both enzymes.