Evidence on the identity of the CXCR2 antagonist AZD-5069

Crystalline forms A and D of N-[2-[[(2,3-difluoropheny)methyl]thio]-6-{[(1R,2S)-2,3-dihydroxy-1-methylpropyl]oxy}-4-pyrimidinyl]-1-azetidinesulfonamide are claimed. The compound is a previously disclosed CXCR2 chemokine receptor antagonist and is claimed to be useful in the treatment of inflammatory diseases such as chronic obstructive pulmonary disease (COPD).     

Syntheses of a radiolabelled CXCR2 antagonist AZD5069 and its major human metabolite

The CXCR2 antagonist AZD5069 has been synthesized in tritium and carbon‐14‐labelled forms. [³H]AZD5069 was prepared via reductive dehalogenation of an iodinated precursor with tritium gas to provide material with a specific activity of 25.1 Ci/mmol. [¹⁴C]AZD5069 was labelled in the pyrimidine ring from [¹⁴C]thiourea in an overall radiochemical yield of 18%. In addition, a synthetic route to the major metabolite of AZD5069 was developed. The synthesis of this metabolite was achieved from AZD5069 using a chemoselective Lindgren–Pinnick reaction in order to minimize oxidation of the sulphide group.     

In vitro effect of Fansimef on human neutrophil and monocyte function

Summary The effect of Fansimef, a recently registered triple combination of mefloquine, sulfadoxine and pyrimethamine (10:25:1) on human peripheral blood polymorphonuclear and mononuclear leucocyte function was studied. At clinically obtainable concentrations Fansimef had no effect on leucocyte viability, it did inhibit neutrophil chemiluminescence and it had no effect on other major functions, such as chemotaxis, NBT reduction, superoxide production, phagocytosis and the bactericidal activity of phagocytic cells.     

SB-656933, a novel CXCR2 selective antagonist, inhibits ex vivo neutrophil activation and ozone-induced airway inflammation in humans

AIMS

To determine the safety and tolerability of a novel selective CXCR2 antagonist and assess its pharmacodynamic effects using measures of neutrophil activation and function, including CD11b expression in whole blood and ozone-induced airway inflammation in healthy subjects.

METHODS

Flow cytometric determination of ex vivo CXCL1-induced CD11b expression on peripheral blood neutrophils was performed following single dose oral administration of SB-656933 (dose range 2–1100 mg). A subsequent randomized study (placebo, 50 mg and 150 mg) was performed to explore the dose–response for ozone-induced airway inflammation, as measured by sputum biomarkers.

RESULTS

Oral administration of SB-656933 resulted in significant inhibition of CXCL1-induced CD11b expression on peripheral blood neutrophils at single doses greater than or equal to 50 mg. Maximum inhibition (70%) relative to placebo was observed following administration of SB-656933 400 mg (95% CI 60%, 77%). This was sustained up to a dose of 1100 mg. Single doses of SB-656933 reduced ozone-induced airway inflammation in a dose-dependent manner. Relative to placebo, there were 55% (95% CI 20%, 75%) and 74% (95% CI 55%, 85%) fewer neutrophils in the sputum of subjects after a single dose of 50 mg or 150 mg, respectively. There was a corresponding reduction in myeloperoxidase concentrations in the sputum supernatant of 32.8% (95% CI 9.2, 50.3) and 50.5% (95% CI 33.3, 63.3). SB-656933 was safe and well-tolerated at all doses.

CONCLUSIONS

SB-656933 is a CXCR2 antagonist that demonstrates dose-dependent effects on neutrophil activation and recruitment within a well-tolerated dose range. These data suggest that SB-656933 may be an effective agent in neutrophil-predominant diseases.     

Biochemical and pharmacological characterization of AZD1981, an orally available selective DP2 antagonist in clinical development for asthma

Background and Purpose

The discovery of DP₂ as a second receptor for PGD₂ has prompted the search for antagonists as potential novel therapies based on the associations between PGD₂ and disease. Here we describe the biochemical and pharmacological properties of 4-(acetylamino)-3-[(4-chlorophenyl)thio]-2-methyl-1H-indole-1-acetic acid (AZD1981), a novel DP₂ receptor antagonist.

Experimental Approach

Binding to DP₂, functional receptor pharmacology and selectivity were studied in both human and animal systems.

Key Results

AZD1981 displaced radio-labelled PGD₂ from human recombinant DP₂ with high potency (pIC₅₀ = 8.4). Binding was reversible, non-competitive and highly selective against a panel of more than 340 other enzymes and receptors, including DP₁ (>1000-fold selective). AZD1981 inhibited DP₂-mediated shape change and CD11b up-regulation in human eosinophils, shape change in basophils and chemotaxis of human eosinophils and Th2 cells with similar potency. AZD1981 exhibited good cross-species binding activity against mouse, rat, guinea pig, rabbit and dog DP₂. Evaluation in mouse, rat or rabbit cell systems was not possible as they did not respond to DP₂ agonists. Agonist responses were seen in guinea pig and dog, and AZD1981 blocked DP₂-mediated eosinophil shape change. Such responses were more robust in the guinea pig, where AZD1981 also blocked DP₂-dependent eosinophil emigration from bone marrow.

Conclusions and Implications

AZD1981 is a DP₂ antagonist that blocks functional responses in eosinophils, Th2 cells and basophils. It exhibited similar potency irrespective of the cell type, DP₂ agonist or species used. This selective orally active agent is currently under clinical evaluation as a potential therapeutic agent in respiratory diseases including asthma.     

Receptor binding mode and pharmacological characterization of a potent and selective dual CXCR1/CXCR2 non-competitive allosteric inhibitor

BACKGROUND AND PURPOSE

DF 2156A is a new dual inhibitor of IL-8 receptors CXCR1 and CXCR2 with an optimal pharmacokinetic profile. We characterized its binding mode, molecular mechanism of action and selectivity, and evaluated its therapeutic potential.

EXPERIMENTAL APPROACH

The binding mode, molecular mechanism of action and selectivity were investigated using chemotaxis of L1.2 transfectants and human leucocytes, in addition to radioligand and [³⁵S]-GTPγS binding approaches. The therapeutic potential of DF 2156A was evaluated in acute (liver ischaemia and reperfusion) and chronic (sponge-induced angiogenesis) experimental models of inflammation.

KEY RESULTS

A network of polar interactions stabilized by a direct ionic bond between DF 2156A and Lys⁹⁹ on CXCR1 and the non-conserved residue Asp²⁹³ on CXCR2 are the key determinants of DF 2156A binding. DF 2156A acted as a non-competitive allosteric inhibitor blocking the signal transduction leading to chemotaxis without altering the binding affinity of natural ligands. DF 2156A effectively and selectively inhibited CXCR1/CXCR2-mediated chemotaxis of L1.2 transfectants and leucocytes. In a murine model of sponge-induced angiogenesis, DF 2156A reduced leucocyte influx, TNF-α production and neovessel formation. In vitro, DF 2156A prevented proliferation, migration and capillary-like organization of HUVECs in response to human IL-8. In a rat model of liver ischaemia and reperfusion (I/R) injury, DF 2156A decreased PMN and monocyte-macrophage infiltration and associated hepatocellular injury.

CONCLUSION AND IMPLICATIONS

DF 2156A is a non-competitive allosteric inhibitor of both IL-8 receptors CXCR1 and CXCR2. It prevented experimental angiogenesis and hepatic I/R injury in vivo and, therefore, has therapeutic potential for acute and chronic inflammatory diseases.     

Treatment with DF 2162, a non-competitive allosteric inhibitor of CXCR1/2, diminishes neutrophil influx and inflammatory hypernociception in mice

BACKGROUND AND PURPOSE: Neutrophil migration into tissues is involved in the genesis of inflammatory pain. Here, we addressed the hypothesis that the effect of CXC chemokines on CXCR1/2 is important to induce neutrophil migration and inflammatory hypernociception. EXPERIMENTAL APPROACH: Mice were treated with a non-competitive allosteric inhibitor of CXCR1/2, DF 2162, and neutrophil influx and inflammatory hypernociception were assessed by myeloperoxidase assay and electronic pressure meter test, respectively, in various models of inflammation. KEY RESULTS: DF 2162 inhibited neutrophil chemotaxis induced by CXCR1/2 ligands but had no effect on CXCL8 binding to neutrophils. A single mutation of the allosteric site at CXCR1 abrogated the inhibitory effect of DF 2162 on CXCL-8-induced chemotaxis. Treatment with DF 2162 prevented influx of neutrophils and inflammatory hypernociception induced by CXCL1 in a dose-dependent manner. The compound inhibited neutrophil influx and inflammatory hypernociception induced by carrageenan, lipopolysaccharide and zymosan, but not hypernociception induced by dopamine and PGE(2). DF 2162 had a synergistic effect with indomethacin or the absence of TNFR1 to abrogate carrageenan-induced hypernociception. Treatment with DF 2162 diminished neutrophil influx, oedema formation, disease score and hypernociception in collagen-induced arthritis. CONCLUSIONS AND IMPLICATIONS: CXCR1/2 mediates neutrophil migration and is involved in the cascade of events leading to inflammatory hypernociception. In addition to modifying fundamental pathological processes, non-competitive allosteric inhibitors of CXCR1/2 may have the additional benefit of providing partial relief for pain and, hence, may be a valid therapeutic target for further studies aimed at the development of new drugs for the treatment of rheumatoid arthritis.     

Rabbit neutrophil chemotactic protein (NCP) activates both CXCR1 and CXCR2 and is the functional homologue for human CXCL6

Neutrophil chemotactic protein (NCP) is a rabbit CXC chemokine with activating and chemotactic properties on neutrophilic granulocytes. Although its selective activity on neutrophils is demonstrated, its interactions with specific chemokine receptors are not defined. For further functional characterization, NCP was chemically synthesized and was found to be equipotent as natural NCP in neutrophil chemotaxis. To identify its human homologue, we separately expressed two potential rabbit NCP receptors (CXCR1 and CXCR2) in Jurkat cells. Pure synthetic NCP was equally efficient to promote chemotaxis through either rabbit CXCR1 or CXCR2. Moreover, chemotaxis assays on rabbit CXCR1 and CXCR2 transfectants showed that NCP uses the same receptors as interleukin-8 (IL-8), a major rabbit CXC chemokine, but not rabbit GROalpha, which only recognized CXCR2. In addition, specific inhibitors for CXCR1 or CXCR2 reduced rabbit neutrophil chemotaxis induced by NCP and rabbit IL-8. Furthermore, NCP and the structurally related human CXCR1/CXCR2 agonist CXCL6/GCP-2 (granulocyte chemotactic protein-2) cross-desensitized each other in intracellular calcium release assays on human neutrophils, further indicating that both chemokines share the same receptors. The inflammatory role of NCP was also evidenced by its potent granulocytosis inducing capacity in rabbits upon systemic administration. This study provides in vitro and in vivo evidences that NCP is the functional rabbit homologue for human CXCL6/GCP-2 rather than the most related CXCR2 agonist CXCL5/ENA-78 (epithelial cell-derived neutrophil activating peptide-78). It is concluded that the rabbit is a better model to study human neutrophil activation compared to mice, which lack CXCL8/IL-8.     

Characterization of a selective and potent antagonist of human P2X(7) receptors, AZ11645373

BACKGROUND AND PURPOSE: The ATP-gated P2X(7) receptor has been shown to play a role in several inflammatory processes, making it an attractive target for anti-inflammatory drug discovery. We have recently identified a novel set of cyclic imide compounds that inhibited P2X(7) receptor-mediated dye uptake in human macrophage THP-1 cells. In this study the actions and selectivity of one of these compounds, AZ11645373, were characterized. EXPERIMENTAL APPROACH: We measured membrane currents, calcium influx, and YOPRO-1 uptake from HEK cells expressing individual P2X receptors, and YOPRO1 uptake and interleukin-1beta release from THP-1 cells in response to ATP and the ATP analogue benzoylbenzoyl ATP (BzATP). KEY RESULTS: AZ11645373 up to 10 microM, had no agonist or antagonist actions on membrane currents due to P2X receptor activation at human P2X(1), rat P2X(2), human P2X(3), rat P2X(2/3), human P2X(4), or human P2X(5) receptors expressed in HEK cells. AZ11645373 inhibited human P2X(7) receptor responses in HEK cells in a non-surmountable manner with K (B) values ranging from 5 - 20 nM, with mean values not significantly different between assays. K (B) values were not altered by removing extracellular calcium and magnesium. ATP-evoked IL-1beta release from lipopolysaccharide-activated THP-1 cells was inhibited by AZ11645373, IC(50) = 90 nM. AZ11645373 was > 500-fold less effective at inhibiting rat P2X(7) receptor-mediated currents with less than 50% inhibition occurring at 10 microM. CONCLUSIONS AND IMPLICATIONS: AZ11645373 is a highly selective and potent antagonist at human but not rat P2X(7) receptors and will have much practical value in studies of human cells.     

Modulating human proteinase activated receptor 2 with a novel antagonist (GB88) and agonist (GB110)

BACKGROUND AND PURPOSE

Many cells express proteinase activated receptor 2 (PAR2) on their plasma membrane. PAR2 is activated by proteolytic enzymes, such as trypsin and tryptase that cleave the receptor N-terminus, inititating signalling to intracellular G proteins. Studies on PAR2 have relied heavily upon activating effects of proteases and peptide agonists that lack stability and bioavailability in vivo.

EXPERIMENTAL APPROACH

A novel small molecule agonist GB110 and an antagonist GB88 were characterized in vitro against trypsin, peptide agonists, PAR2 antibody, PAR1 agonists and flow cytometry,in seven cell lines using intracellular Ca²⁺ mobilization and examined in vivo against PAR2- and PAR1-induced rat paw oedema.

KEY RESULTS

GB110 is a potent non-peptidic agonist activating PAR2-mediated Ca²⁺ release in HT29 cells (EC₅₀∼200 nM) and six other human cell lines, inducing PAR2 internalization. GB88 is a unique PAR2 antagonist, inhibiting PAR2 activated Ca²⁺ release (IC₅₀∼2 µM) induced by native (trypsin) or synthetic peptide and non-peptide agonists. GB88 was a competitive and surmountable antagonist of agonist 2f-LIGRLO-NH₂, a competitive but insurmountable antagonist of agonist GB110, and a non-competitive insurmountable antagonist of trypsin. GB88 was orally active and anti-inflammatory in vivo, inhibiting acute rat paw oedema elicited by agonist GB110 and proteolytic or peptide agonists of PAR2 but not by corresponding agonists of PAR1 or PAR4.

CONCLUSIONS AND IMPLICATIONS

The novel PAR2 agonist and antagonist modulate intracellular Ca²⁺ and rat paw oedema, providing novel molecular tools for examining PAR2-mediated diseases.     

The antihypertensive effect of a selective central muscarinic cholinergic antagonist: N-(4-diethylamino-2-butynyl)-succinimide

N-(4-Diethylamino-2-butynyl)-succinimide (DKJ-21), a cholinergic antagonist selective for muscarinic receptors in the central nervous system, has an antihypertensive effect in conscious spontaneously hypertensive rats (SHR) but not in normotensive Wistar-Kyoto (WKY) control rats. Intravenous doses over a range of 3.1 to 25.0 mg/kg produced a dose-dependent decrease in systolic blood pressure. This effect was still apparent 24 hr after drug administration, but blood pressure returned to predrug levels by 48 hr after injection of DKJ-21. In a dose–response study, the maximum antihypertensive response in a group receiving 25.0 mg/kg was 43 ± 8 mm Hg. Marked variability of the maximum response was observed in all age groups and with all doses. Decreases in blood pressure up to 75 mm Hg were obtained in individual animals. The magnitude of the antihypertensive effect is not age related since 10-, 15-, 22-, and 36-week-old SHR responded to the same degree after injection of 25 mg/kg DKJ-21. Smaller doses (50 μg) of DKJ-21 decreased blood pressure when administered into the lateral cerebral ventricle of SHR. Vascular responses to norepinephrine, acetylcholine, dimethylphenylpiperazinium, angiotensin I, and tyramine were not inhibited by i.v. injection of DKJ-21; however, the centrally mediated pressor response to physostigmine was reduced by 60%.     

A novel DP2 receptor antagonist (AM-461): a patent evaluation of WO2011085033

This application claims salts and crystalline forms of a previously disclosed DP₂ receptor antagonist (2-[3-[2-(tert-butylsulfanylmethyl)-4-(2,2-dimethylpropanoylamino)phenoxy]-4-methoxy-phenyl]acetic acid (1)). It also claims compositions containing the free acid and its salts, especially the sodium salt and their use in the treatment of inflammatory and respiratory diseases, especially asthma. Notably, the application presents Phase I clinical data on compound (1).     

Pharmacological characterization of the first potent and selective antagonist at the cysteinyl leukotriene 2 (CysLT2) receptor

Background and purpose:

Cysteinyl leukotrienes (CysLTs) have been implicated in the pathophysiology of inflammatory and cardiovascular disorders. Their actions are mediated by CysLT₁ and CysLT₂ receptors. Here we report the discovery of 3-({[(1S,3S)-3-carboxycyclohexyl]amino}carbonyl)-4-(3-{4-[4-(cyclo-hexyloxy)butoxy]phenyl}propoxy) benzoic acid (HAMI3379), the first potent and selective CysLT₂ receptor antagonist.

Experimental approach:

Pharmacological characterization of HAMI3379 was performed using stably transfected CysLT₁ and CysLT₂ receptor cell lines, and isolated, Langendorff-perfused, guinea pig hearts.

Key results:

In a CysLT₂ receptor reporter cell line, HAMI3379 antagonized leukotriene D₄- (LTD₄-) and leukotriene C₄- (LTC₄-) induced intracellular calcium mobilization with IC₅₀ values of 3.8 nM and 4.4 nM respectively. In contrast, HAMI3379 exhibited very low potency on a recombinant CysLT₁ receptor cell line (IC₅₀ > 10 000 nM). In addition, HAMI3379 did not exhibit any agonistic activity on both CysLT receptor cell lines. In binding studies using membranes from the CysLT₂ and CysLT₁ receptor cell lines, HAMI3379 inhibited [³H]-LTD₄ binding with IC₅₀ values of 38 nM and >10 000 nM respectively. In isolated Langendorff-perfused guinea pig hearts HAMI3379 concentration-dependently inhibited and reversed the LTC₄-induced perfusion pressure increase and contractility decrease. The selective CysLT₁ receptor antagonist zafirlukast was found to be inactive in this experimental setting.

Conclusions and implications:

HAMI3379 was identified as a potent and selective CysLT₂ receptor antagonist, which was devoid of CysLT receptor agonism. Using this compound, we showed that the cardiac effects of CysLTs are predominantly mediated by the CysLT₂ receptor.     

LF 16.0335, a novel potent and selective nonpeptide antagonist of the human bradykinin B2 receptor

1. In the present paper, we describe the in vitro pharmacological properties of LF 16.0335 (1-[[3-[(2,4-dimethylquinolin-8-yl)oxymethyl]-2,4-dichloro-phenyl]sulphonyl] -2(S) - [[4 -[4-(aminoiminomethyl)phenylcarbonyl]piperazin-1-yl]carbonyl]pyrrolidine), a novel and potent nonpeptide antagonist of the human bradykinin (BK) B₂ receptor.
2. LF 16.0335 displaced [³H]-BK binding to membrane preparations from CHO cells expressing the cloned human B₂ receptor, INT 407 cells and human umbilical vein with K_i values of 0.84±0.39 nM, 1.26±0.68 nM and 2.34±0.36 nM, respectively.
3. In saturation binding studies performed in INT 407 cell membranes in the presence or absence of LF 16.0335, B_max values of [³H]-BK were not significantly changed suggesting that LF 16.0335 behaves as a competitive antagonist.
4. LF 16.0335 had no affinity for the cloned human kinin B₁ receptor stably expressed in 293 cells. In addition, this compound at 1 μM did not significantly bind to a range of 40 different membrane receptors and eight ion channels except muscarinic M₂ and M₁ receptors for which an IC₅₀ value of 0.9 and 1 μM was obtained.
5. BK stimulates in a concentration-dependent manner phosphoinositosides (IPs) production in cultured INT 407 cells. Concentration-response-curves to BK were shifted to the right in the presence of LF 16.0335 (0.1 μM) without reduction of the maximum. LF 16.0335 inhibited the concentration-contraction curve to BK in the human umbilical vein giving a pA₂ value of 8.30±0.30 with a Schild plot slope that was not different from unity.
6. These results demonstrate that LF 16.0335 is a potent, selective and competitive antagonist of the human bradykinin B₂ receptor.

     

Pharmacology of an original and selective nonpeptide antagonist ligand for the human tachykinin NK2 receptor

The pharmacological outline of a novel and original antagonist at the human tachykinin NK2 receptor is presented, namely MEN13510 (N-N'-bis-[2-(1H-indol-3-yl)-ethyl]-N,N'-bis-(3-thiomorpholin-4-yl-propyl)-phthalamide). MEN13510 retained nanomolar affinity for the human tachykinin NK2 receptor (Ki 6.4 nM), and micromolar affinity for the human tachykinin NK1 and NK3 receptors. A competitive antagonism is indicated by the Schild analysis (pK(B) 7.8, slope -0.94) of concentration-response curves of NKA induced inositolphosphates accumulation in Chinese hamster ovary (CHO) cells expressing the human NK2 receptor in the presence of MEN13510 (30-300 nM concentration range). The MEN13510 interaction with the human NK2 receptor was evaluated by means of heterologous inhibition binding experiments, by using agonist and antagonist radioligands ([125I]NKA, [3H]nepadutant, [3H]saredutant) at a series of mutant receptors having single aminoacidic substitutions of residues located in transmembrane (TM) segments 3, 4, 5, 6, and 7. MEN13510 affinity was not affected by the mutations in TM 3 and 4 (Q109A, F112A, T171A, C167G), and it was reduced by 10-fold at the I202F mutant, but not at the Y206A (TM4). Amongst the investigated mutants bearing the mutated residues in TM6 (F270A, Y266F, W263A) only F270A decreased the MEN13510 affinity by 7-fold. Even mutations in TM7 did reduce MEN13510 affinity by 32-fold (Y289T, but not Y289F) and 13-fold (F293A). Studied mutations represent the human tachykinin NK2 receptor discriminants involved in the binding of previously reported peptidic and nonpeptidic antagonists, against which results obtained with MEN13510 are compared. Results indicate that the binding site of this antagonist is, at least in part, overlapping to that described for NKA or saredutant. Finally we show that MEN13510 retains nanomolar affinity for the recently discovered splice variant of the human tachykinin NK2 receptor, namely beta isoform, as it has been described for the nonpeptide antagonist saredutant.     

The pharmacokinetics after intravenous and oral administration in man of theα 2-adrenoreceptor antagonist idazoxan (RX781094)

Summary A high performance liquid chromatographic method was developed for the quantitative determination of idazoxan in plasma. The assay was used to study the disposition of the drug after intravenous infusion and oral administration to five normal subjects. After i.v. administration the kinetics could be described by a two compartment model with a mean elimination half life of 4.20 h. The mean calculated volume of distribution during the elimination phase was 3.20 l/kg^–1 and the mean plasma clearance was 824 ml min^–1.After oral administration a lag period before onset of absorption was observed in all five volunteers, the plasma levels declining monoexponentially from the peak concentration with a mean elimination half life of 5.58 h. The absolute availability varied between 26% and 41% with a mean value of 34%. Invitro measurements produced a blood/plasma ratio of 1.3 for idazoxan.     

Comparative effects of two bisphosphonates (clodronate and zk 90695) on Hydroxyapatite-Rimed human neutrophil chemiluminescence

Kowolik MJ, Hyvönen PM. Comparative effects of two bisphosphonates (clodronate and ZK 90695) on hydroxyapatite-primed human neutrophil chemiluminescence. Inflammopharmacology. 1994;2:369-376. An established in-vitro model was applied to study the interaction between human neutrophil granulocytes, hydroxyapatite particles (HA) and two bisphosphonate drugs, dichloromethylene bisphosphonate (clodronate), and ZK 90695, an experimental agent. Luminol- and lucigenin-mediated chemiluminescence (CL) assays were used to monitor activation in the oxygen-dependent pathway. Little effect was seen by either drug over a concentration range of 1 × 10^s-13 to 1 × 10^s-5 mol/L, in the absence of HA, when the cells were subjected to a second challenge of serum-treated zympsan or phorbol myristate acetate (PMA). Although beyond the therapeutic range, only at 1 × 10^s-3 mol/L was cell activity abolished as confirmed by trypan blue dye exclusion. Preincubation of neutrophils with serum-coated HA produced a typical priming for the second stimulus but binding of either drug to the mineral particles had minimal effect on priming except at the highest concentrations of 1 × 10^s-4 and 1 × 10^s-3 mol/L. These results support our findings regarding the low toxicity of clodronate in this experimental assay, and provide similar evidence for the experimental bisphosphonate, ZK 90695. In addition to the established use of bisphosphonates, there is increasing interest in studying their anti-inflammatory effects, to which this model is well-suited.     

钙调素拮抗剂E6对大鼠神经细胞内钙的影响

目的　观察钙调素拮抗剂E6对神经细胞静息[Ca2 +]i、KCl (5 0mmol·L-1)和谷氨酸 (glutamicacid ,Glu ,0 1mmol·L-1)引起的神经细胞 [Ca2 +]i 升高的影响。方法　用Ca2 +敏感荧光指示剂Fura 2 /AM负载大鼠脑细胞 ,测定神经细胞内游离Ca2 +浓度 ([Ca2 +]i)。结果　E6可升高神经细胞静息 [Ca2 +]i,EC50 为 6 6 8μmol·L-1;无外Ca2 +条件下 ,也升高 [Ca2 +]i,EC50 为 1 30 μmol·L-1,说明E6主要是通过促进细胞内贮存Ca2 +释放引起内Ca2 +升高。E6抑制KCl升高细胞 [Ca2 +]i 的IC50 为 6 0 μmol·L-1;抑制Glu激发的细胞 [Ca2 +]i 升高的IC50 为 0 15 μmol·L-1。结论　E6可能是通过与细胞内钙调素 (Calmodulin ,CaM)结合后 ,影响兴奋性氨基酸受体的开放和电压依赖性钙通道的活性状态 ,表现出对由去极化或受体激动剂引起的内Ca2 +升高的抑制作用     

Binding of [3H]MSX-2 (3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine) to rat striatal membranes--a new, selective antagonist radioligand for A(2A) adenosine receptors.

The present study describes the preparation and binding properties of a new, potent, and selective A_2A adenosine receptor (AR) antagonist radioligand, [³H]3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine ([³H]MSX-2). [³H]MSX-2 binding to rat striatal membranes was saturable and reversible. Saturation experiments showed that [³H]MSX-2 labeled a single class of binding sites with high affinity (K_d=8.0 nM) and limited capacity (B_max=1.16 fmol·mg⁻¹ of protein). The presence of 100 μM GTP, or 10 mM magnesium chloride, respectively, had no effect on [³H]MSX-2 binding. AR agonists competed with the binding of 1 nM [³H]MSX-2 with the following order of potency: 5′-N-ethylcarboxamidoadenosine (NECA)>2-[4-(carboxyethyl)phenylethylamino]-5′-N-ethylcarboxamidoadenosine (CGS-21680)>2-chloroadenosine (2-CADO)>N⁶-cyclopentyladenosine (CPA). AR antagonists showed the following order of potency: 8-(m-bromostyryl)-3,7-dimethyl-1-propargylxanthine (BS-DMPX)>1,3-dipropyl-8-cyclopentylxanthine (DPCPX)>(R)-5,6-dimethyl-7-(1-phenylethyl)-2-(4-pyridyl)-7H-pyrrolo[2,3-d]pyrimidine-4-amine (SH-128)>3,7-dimethyl-1-propargylxanthine (DMPX)>caffeine. The K_i values for antagonists were in accordance with data from binding studies with the agonist radioligand [³H]CGS21680, while agonist affinities were 3–7-fold lower. [³H]MSX-2 is a highly selective A_2A AR antagonist radioligand exhibiting a selectivity of at least two orders of magnitude versus all other AR subtypes. The new radioligand shows high specific radioactivity (85 Ci/mmol, 3150 GBq/mmol) and acceptable nonspecific binding at rat striatal membranes of 20–30%, at 1 nM.     

Serotonin-2 receptors and human sleep: effect of a selective antagonist on EEG power spectra.

To investigate the effect on the sleep EEG, a 1-mg oral dose of SR 46349B, a novel 5-HT2 antagonist, was administered three hours before bedtime. The drug enhanced slow wave sleep (SWS) and reduced stage 2 without affecting subjective sleep quality. In nonREM sleep (NREMS) EEG slow-wave activity (SWA; power within 0.75-4.5 Hz) was increased and spindle frequency activity (SFA; power within 12.25-15 Hz) was decreased. The relative NREMS power spectrum showed a bimodal pattern with the main peak at 1.5 Hz and a secondary peak at 6 Hz. A regional analysis based on bipolar derivations along the antero-posterior axis revealed significant 'treatment' x 'derivation' interactions within the 9-16 Hz range. In enhancing SWA and attenuating SFA, the 5-HT2 receptor antagonist mimicked the effect of sleep deprivation, whereas the pattern of the NREMS spectrum differed.