A consideration of the patentability of enantiomers in the pharmaceutical industry in the United States

During the last thirty years, concern over stereoselectivity of drug action has drawn a great deal of interest within the pharmaceutical field due to an improved understanding of the pharmacology and pharmacokinetics of enantiomers. Developing single enantiomers versus racemates or introducing a single enantiomer following the development of the racemic mixture appears to be the new trend. The intellectual property status of single enantiomers from racemates may be unclear. Drug discoverers and patent attorneys must examine the examples of the past to establish an appropriate pathway towards the development and intellectual property protection of chiral drugs. The review will focus on the patenting of an enantiomer in view of the prior art disclosure for the racemic mixture.     

S-nitrosylation is required for β2AR desensitization and experimental asthma

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Type II estrogen binding site agonist: synthesis and biological evaluation of the enantiomers of methyl-para-hydroxyphenyllactate (MeHPLA)

A high-affinity ligand for the type II estrogen binding site (EBS) was identified. Methyl para-hydroxyphenyllactate (MeHPLA) was observed to suppress the cellular proliferation of estrogen-sensitive MCF-7 breast cancer cells in vitro and to suppress the growth of rat uteri in vivo. The high affinity of MeHPLA for the type II EBS suggests that this interaction is responsible for the observed suppression of cell growth. In this study, the enantiomers of MeHPLA were synthesized and separated by three methods and evaluated for biological activity. When the methods were compared, it was found that the method using an optically pure amine to form the diastereomers of the enantiomers gave the superior yield. Binding studies for the enantiomers to the type II EBS showed that the S-MeHPLA had a higher affinity for the binding site. However, higher binding affinity did not translate into superior cell growth suppression. Both enantiomers suppressed cell growth equally.     

Stereoselectivity and affinity in molecular pharmacology. III. Structural aspects in the mode of action of natural and synthetic auxins

Analysis of available potency estimates for 35 pairs of enantiomeric arylcarboxylic acids with auxin activity (flax-root-growth inhibition test) revealed extensive correlations between the activity of the more potent and less potent isomers, as well as between the log of the ratio of potencies and the log potency of the more active isomer when structurally similar analogs are compared. 5 structural subgroups were discernible (n, eudismic-affinity quotient (EAQ), r2); (1) arylpropionic acids (6, -0.36, 0.66); (2) 2-naphthoxy-carboxylic acids (6, +1.07, 0.99); (3) 1-naphthoxycarboxylic acids (3, +1.56, 0.96); (4) ortho-substituted phenoxycarboxylic acids (10, +0.97, 0.96) and (5) ortho-unsubstituted phenoxycarboxylic acids (10, +0.56, 0.70). For achiral lower homologs such as auxin itself 3-indolyl-acetic acid (IAA), phenoxyacetic acid and 1-naphthoxyacetic acid, extrapolated potencies were found to agree well with experimental values. On the basis of these observations an auxin receptor is postulated and binding arrangements are described which explain most of the experimental data available. A 3-point attachment when allowed is the only binding mode compatible with the reported data.     

Olfactory sensitivity for enantiomers and their racemic mixtures--a comparative study in CD-1 mice and spider monkeys

Using a conditioning paradigm, the olfactory sensitivity of six CD-1 mice for the enantiomers of carvone and of limonene as well as for their racemic mixtures was investigated. With all six stimuli, the animals significantly discriminated concentrations 相似文献   

Effect of a novel leukotoriene synthesis inhibitor, BAY x1005, on the antigen- and LPS-induced airway hyperresponsiveness in guinea pigs

Due to the inhibition of 5-lipoxygenase-activating protein (FLAP), BAY x1005 is a new selective inhibitor of leukotriene synthesis. The effects of BAY x1005 on the antigen- and bacterial lipopolysaccharide (LPS)-induced airway hyperresponsiveness in guinea pigs were investigated. Six times provocation of aeroantigen caused biphasic increases in airway resistance which peaked at 1 hr (immediate phase reaction) and 4 hrs (late phase reaction). It also caused airway hyperreactivity to acetylcholine. BAY x1005 at doses of 10mg/kg and 30mg/kg significantly inhibited antigen-induced increase in respiratory resistance (Rrs) at 1 and 4 hrs after the last antigen challenge. Simultaneously, BAY x1005 inhibited the antigen-induced airway hyperresponsiveness at doses of 10 and 30mglkg and airway eosinophilia (bronchoalveolar lavage study) at a dose of 30 mg/kg. In addition, BAY x1005 at a dose of 30mg/kg inhibited bacterial LPS-induced airway hyperreactivity to acetylcholine. In this model, BAY x1005 did not affect the increase of the number of leukocytes in bronchoalveolar lavage fluid.These results suggest that BAY x1005 is a potent anti-asthmatic agent with an inhibitory action to airway hyperreactivity.     

Influences of PON1 on airway inflammation and remodeling in bronchial asthma

This study aims to explore the influences of Paraoxonase‐1 (PON1) involved in airway inflammation and remodeling in asthma. Mice were divided into control, asthma, asthma + PON1 and asthma + NC groups, and asthma models were established via aerosol inhalation of ovalbumin (OVA). HE, Masson, and PAS stains were used to observe airway inflammation and remodeling, Giemsa staining to assess inflammatory cells in bronchoalveolar lavage fluid (BALF), qRT‐PCR and Western blot to detect PON1 expression, lipid peroxidation and glutathione assays to quantify malondialdehyde (MDA) activity and glutathione peroxidase (GSH) levels, ELISA to determine inflammatory cytokines and immunoglobulin, and colorimetry to detect PON1 activities. Additionally, mice lung macrophages and fibroblasts were transfected with PON1 plasmid in vitro; ELISA and qRT‐PCR were performed to understand the effects of PON1 on inflammatory cytokines secreted by lung macrophages, MTT assay for lung fibroblasts proliferation and qRT‐PCR and Western blot for the expressions of PON1, COL1A1, and fibronectin. After overexpression of PON1, the asthma mice had decreased inflammatory cell infiltration, fibrosis degree, and airway wall thickness; inflammatory cells and inflammatory cytokines in BALF were also reduced, expressions of OVA‐IgE and IgG1, and MDA activity were decreased, but the expressions of OVA‐IgG2a and INF‐γ and GSH levels were increased. Besides, PON1 significantly inhibited microphage expression of LPS‐induced inflammatory cytokines, lung fibroblast proliferation, and COL1A1 and fibronectin expression. Thus, PON1 could relieve airway inflammation and airway remodeling in asthmatic mice and inhibit the secretion of LPS‐induced macrophage inflammatory cytokines and the proliferation of lung fibroblasts.     

Selenium and asthma

Se is a potent nutritional antioxidant important for various aspects of human health. Because asthma has been demonstrated to involve increased oxidative stress, levels of Se intake have been hypothesized to play an important role in the pathogenesis of asthma. However, significant associations between Se status and prevalence or severity of asthma have not been consistently demonstrated in human studies. This highlights both the complex etiology of human asthma and the inherent problems with correlative nutritional studies. In this review, the different findings in human studies are discussed along with results from limited intervention studies. Mouse models of asthma have provided more definitive results suggesting that the benefits of Se supplementation may depend on an individual’s initial Se status. This likely involves T helper cell differentiation and the mechanistic studies that have provided important insight into the effects of Se levels on immune cell function are summarized. Importantly, the benefits and adverse effects of Se supplementation must both be considered in using this nutritional supplement for treating asthma. With this in mind new approaches are discussed that may provide more safe and effective means for using Se supplementation for asthma or other disorders involving inflammation or immunity.     

Randomised,double-blind,placebo-controlled crossover study to investigate different dosing regimens of olodaterol delivered via Respimat? in patients with moderate to severe persistent asthma

Background

A Phase II, multicentre, randomised, double-blind, placebo-controlled, crossover trial comparing the 24-h forced expiratory volume in 1 s (FEV₁) time profile after 3 weeks’ treatment with once-daily (QD) or twice-daily (BID) olodaterol (at the same total daily dose) versus placebo delivered via Respimat® in patients with moderate to severe asthma.

Methods

Patients were randomised to different sequences of olodaterol with 2-week washout, either as a total daily dose of 5 μg (5 μg QD [AM] or 2.5 μg BID) or placebo, or 10 μg (10 μg QD [AM] or 5 μg BID) or placebo. Primary end point was FEV₁ area under the curve from 0 to 24 h (AUC_0–24) response (defined as change from study baseline FEV₁) after 3 weeks. Key secondary end points were FEV₁ AUC_0–12 and AUC_12–24 responses.

Results

Two hundred and six patients received treatment. All olodaterol treatments demonstrated statistically significant improvements in FEV₁ AUC_0–24 response at 3 weeks versus placebo (p < 0.0001); adjusted mean treatment difference versus placebo was 0.191 L for olodaterol 2.5 μg BID (95 % confidence interval [CI] 0.152, 0.229), 0.150 L for 5 μg QD (95 % CI 0.111, 0.189), 0.228 L for 5 μg BID (95 % CI 0.190, 0.266) and 0.209 L for 10 μg QD (95 % CI 0.170, 0.247). These results were supported by the key secondary end points. Olodaterol 5 μg QD provided numerically lower mean values for 24-h bronchodilation than olodaterol 2.5 μg BID (p = 0.0465), with no statistically significant difference between treatment with olodaterol 10 μg QD and 5 μg BID. No relevant differences in morning and evening peak expiratory flow or Asthma Control Questionnaire scores at 3 weeks were observed between different doses and regimens. Adverse events were generally mild to moderate and comparable between groups.

Conclusions

All doses and dose frequencies provided adequate 24-h bronchodilation superior to placebo. Based on the results of this study, it would be reasonable to include both posologies of 5 μg olodaterol daily (5 μg QD or 2.5 μg BID, both delivered in two puffs per dose from the Respimat® inhaler) in subsequent studies. Further studies are necessary to confirm the optimum dosing regimen in asthma. No safety concerns were identified.

Trial registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01311661","term_id":"NCT01311661"}}NCT01311661

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0243-1) contains supplementary material, which is available to authorized users.     

Upregulation of a disintegrin and metalloproteinase-33 by VEGF in human airway smooth muscle cells: Implications for asthma

Asthma is a chronic respiratory disease characterized by reversible airway obstruction with persistent airway inflammation and airway remodeling. Features of airway remodeling include increased airway smooth muscle (ASM) mass. A disintegrin and metalloproteinase (ADAM)–33 has been identified as playing a role in the pathophysiology of asthma. ADAM-33 is expressed in ASM cells and is suggested to play a role in the function of these cells. However, the regulation of ADAM-33 is not fully understood. Vascular endothelial growth factor (VEGF) has been implicated in inflammatory and airway blood vessel remodeling in asthmatics. Although VEGF was initially thought of as an endothelial-specific growth factor, recent reports have found that VEGF can promote proliferation of other cell types, including ASM cells. To investigate the precise mechanism of VEGF's effect on ASM cell proliferation, we tested the expression of ADAM-33, phospho-extracellularsignal-regulated kinase 1/2 (ERK1/2), and phospho-Akt in VEGF-stimulated ASM cells. We found that VEGF up-regulates ADAM-33 mRNA and protein levels in a dose- and time-dependent manner as well as phosphorylation of ERK1/2 and Akt. We also found that VEGF-induced ASM cell proliferation is inhibited by both ADAM-33 knockdown and a selective VEGF receptor 2 (VEGFR2) inhibitor (SU1498). Furthermore, VEGF-induced ADAM-33 expression and ASM cell proliferation were suppressed by inhibiting ERK1/2 activity, but not by inhibiting Akt activity. Collectively, our findings suggest that VEGF enhances ADAM-33 expression and ASM cell proliferation by activating the VEGFR2/ERK1/2 signaling pathway, which might be involved in the pathogenesis of airway remodeling. Further elucidation of the mechanisms underlying these observations might help develop therapeutic strategies for airway diseases associated with smooth muscle hyperplasia such as asthma.     

Isoprostanes and asthma

Isoprostanes are prostaglandin (PG)-like compounds generated in vivo following oxidative stress by non-enzymatic peroxidation of polyunsaturated fatty acids, including arachidonic acid. They are named based on their prostane ring structure and by the localization of hydroxyl groups on the carbon side chain; these structural differences result in a broad array of isoprostane molecules with varying biological properties. Generation of specific isoprostanes is also regulated by host cell redox conditions; reducing conditions favor F₂-isoprostane production while under conditions with deficient antioxidant capacity, D₂- and E₂-isoprostanes are formed. F₂-isoprostanes (F₂-isoP) are considered reliable markers of oxidative stress in pulmonary diseases including asthma. Importantly, F₂-isoP and other isoprostanes function as ligands for PG receptors, and potentially other receptors that have not yet been identified. They have been reported to have important biological properties in many organs. In the lung, isoprostanes regulate cellular processes affecting airway smooth muscle tone, neural secretion, epithelial ion flux, endothelial cell adhesion and permeability, and macrophage adhesion and function. In this review, we will summarize the evidence that F₂-isoP functions as a marker of oxidative stress in asthma, and that F₂-isoP and other isoprostanes exert biological effects that contribute to the pathogenesis of asthma. This article is part of a Special Issue entitled Biochemistry of Asthma.     

Murine model of allergen induced asthma

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world (1). More than 8% of the US population has asthma, with the prevalence increasing (2). As with other diseases, animal models of allergic airway disease greatly facilitate understanding of the underlying pathophysiology, help identify potential therapeutic targets, and allow preclinical testing of possible new therapies. Models of allergic airway disease have been developed in several animal species, but murine models are particularly attractive due to the low cost, ready availability, and well-characterized immune systems of these animals (3). Availability of a variety of transgenic strains further increases the attractiveness of these models (4). Here we describe two murine models of allergic airway disease, both employing ovalbumin as the antigen. Following initial sensitization by intraperitoneal injection, one model delivers the antigen challenge by nebulization, the other by intratracheal delivery. These two models offer complementary advantages, with each mimicking the major features of human asthma (5). The major features of acute asthma include an exaggerated airway response to stimuli such as methacholine (airway hyperresponsiveness; AHR) and eosinophil-rich airway inflammation. These are also prominent effects of allergen challenge in our murine models (5,6), and we describe techniques for measuring them and thus evaluating the effects of experimental manipulation. Specifically, we describe both invasive (7) and non-invasive (8) techniques for measuring airway hyperresponsiveness as well as methods for assessing infiltration of inflammatory cells into the airways and the lung. Airway inflammatory cells are collected by bronchoalveolar lavage while lung histopathology is used to assess markers of inflammation throughout the organ. These techniques provide powerful tools for studying asthma in ways that would not be possible in humans.     

谷胱甘肽通过抑制重型儿童哮喘巨噬细胞凋亡发挥保护作用

目的探究GSH对重型儿童哮喘气道巨噬细胞凋亡的影响。方法通过GSH检测试剂盒检测重型及轻型哮喘患儿巨噬细胞AM内的GSSG水平,并采用MTT法检测巨噬细胞AM的细胞活力,Western blot法检测凋亡相关蛋白Cleaved caspase 3的表达情况。结果 (1)重型哮喘患儿的吞噬细胞内GSSG水平显著高于轻型患儿;(2)哮喘患儿巨噬细胞AW的细胞活力与轻型哮喘患儿相比显著降低,且体外补充的GSH可以逆转细胞活力降低;(3)体外补充GSH可以逆转重型哮喘患儿巨噬细胞凋亡蛋白Cleaved caspase 3的表达增多。结论体外补充GSH可以抑制重型哮喘患儿气道巨噬细胞凋亡,发挥保护作用。     

艾司洛尔和右旋美托咪啶对减轻患者插管 和喉镜检查的拟交感反应分析

目的：比较艾司洛尔和右旋美托咪啶减弱神经外科患者插管和喉镜检查的拟交感反应的疗效。方法：选取在我院神经外科拟 接受选择性神经外科手术的90 例患者。患者平均随机分为三组。对照组：静注给予20 mL生理盐水;右旋美托咪啶组：1 ug/kg右 旋美托咪啶使用生理盐水稀释至20 mL静注;艾司洛尔组：1.5 mg/kg 艾司洛尔使用生理盐水稀释至20 mL静注。所有患者均接 受2 min 的麻醉诱导,所有药物均在10 min 内滴注完毕。在给药后、诱导后及气管插管后1、2、3、5、10、15 min 后对患者心率 （HR）、收缩压、舒张压、平均动脉压进行记录,同时记录患者基线值。结果：与对照组相比,右旋美托咪啶组插管后心率和血压未见 显著性差异;艾司洛尔组插管后1、2、3 min 后血压及插管后5 min 的心率显著上升。结论：对减弱神经外科患者插管和喉镜检查 的拟交感作用,右旋美托咪啶比艾司洛尔更为有效。     

Viruses and asthma

Background

Viral respiratory infection has long been known to influence the occurrence of asthma exacerbations. Over the last 20 years much effort has been put into clarifying the role that viral respiratory infections play in the eventual development of asthma.

Scope of review

In this review we give a general background of the role of viruses in the processes of asthma exacerbation and asthma induction. We review recent additions to the literature in the last 3 years with particular focus on clinical and epidemiologic investigations of influenza, rhinovirus, bocavirus, respiratory syncytial virus, and metapneumovirus.

Major conclusions

The development of asthma emerges from a complex interaction of genetic predisposition and environmental factors with viral infection likely playing a significant role in the effect of environment on asthma inception. This article is part of a Special Issue entitled: Biochemistry of Asthma.

General significance

Further understanding of the role that viruses play in asthma exacerbation and inception will contribute to decreased asthma morbidity in the future. This article is part of a Special Issue entitled: Biochemistry of Asthma.     

颗粒蛋白前体通过抑制IL-6表达减轻哮喘气道炎症

目的: 探究颗粒蛋白前体(PGRN)在过敏性哮喘中的作用及机制。方法: 分别在野生鼠和IL-6 缺陷鼠中设置对照组和哮喘模型组,每组8只。模型组中,在第0日和第7日致敏小鼠(腹腔注射OVA 100 μg),从第14日起连续激发8 d(5％OVA雾化吸入,30 min/d,每日1次),末次激发24 h后取标本;对照组用PBS代替OVA做相同处理。采集支气管肺泡灌洗液(BALF)进行白细胞计数和分类计数;HE染色观察肺组织病理情况;Q-PCR及ELISA检测小鼠肺匀浆、血清和BALF中细胞因子水平。用IL-13刺激A549或BEAS-2B细胞建立体外哮喘炎症模型,每组3个复孔,共4组:PBS处理组、IL-13处理组、IL-13与重组人PGRN蛋白(rhPGRN)共同处理组及p38磷酸化抑制剂(SB203508)处理组。0 min~48 h后收集细胞及上清,用Q-PCR及ELISA检测PGRN和IL-6的表达;Western blot检测p38的磷酸化。结果: 与对照组相比,哮喘组小鼠肺匀浆和BALF中PGRN均显著降低(P＜ 0.01),血清PGRN有降低的趋势,然而哮喘小鼠BALF中IL-6显著升高(P＜0.05)。与野生鼠哮喘组相比,IL-6缺陷鼠哮喘组BALF中白细胞总数降低(P＜0.05),中性粒细胞数降低(P＜0.05),PGRN显著升高(P＜0.05),肺部病理损伤也减轻。体外实验中,IL-13处理组与PBS处理组相比,PGRN显著降低(P＜0.05),IL-6显著增高(P＜ 0.05),p38的磷酸化增加;p38抑制剂处理组比未处理组中IL-6水平降低(P＜0.05)。IL-13与rhPGRN共同处理组的IL-6显著低于IL-13处理组(P＜0.05),p38的磷酸化降低(P＜0.05)。结论: PGRN通过抑制p38磷酸化降低IL-6水平从而减轻哮喘小鼠气道炎症。