English:
[0001] This application claims priority to U.S. Patent Application No. 11/367,449, filed on March 6, 2006, which is a continuation-in-part application of U.S. Patent Application No. 10/802,644, filed on March 17, 2004, which is a continuation application of U.S. Patent Application No. 10/180,753; filed June 26, 2002, now abandoned, which claims priority to U.S. Provisional Application No. 60/300,933, filed June 26, 2001, the disclosures of which are incorporated herein by reference in their entirety.
[0002] This invention was made with support from the United States Federal government under grant number ROl HL36982 from the National Institutes of Health. The United States government may have certain rights in this invention.
[0008] Despite the obvious pathophysiological importance of this process, intracellular signaling mechanisms linking stimulation at the cell surface to mucin granule release has only recently been elucidated. See, Li et al., Journal of Biological Chemistry, 276: 40982- 40990 (2001). It is known that a wide variety of agents and inflammatory/humoral mediators provoke mucin secretion. These include cholinergic agonists, lipid mediators, oxidants, cytokines, neuropeptides, ATP and UTP, bacterial products, neutrophil elastase, and inhaled pollutants. See, Adler et al, Res. Immunol. 149, 245-248 (1998). Interestingly, many of these mucin secretagogues are also known to activate several protein kinases, and studies examining the regulation of excess secretion of mucin by airway epithelial cells from various species have consistently implicated involvement of either protein kinase C (PKC) or cGMP- dependent protein kinase (PKG) in the secretory process. See, e.g., Ko et al, Am. J. Respir. Cell MoI. Biol. 16, 194-198 (1997); Abdullah et al., Am. J. Physiol. 273, L201-L210 (1997); Abdullah et al, Biochem. J. 3\6, 943-954 (1996); Larivee et al. Am. J. Respir. Cell MoI. Biol. 11 , 199-205 (1994); and Fischer et al, Am. J. Respir. Cell MoI. Biol. 20, 413-422 (1999). Coordinated interactions or "cross-talk" between these two protein kinases in regulation of mucin secretion has only recently been demonstrated to involve the MARCKS proteins. See, Li et al, Journal of Biological Chemistry, 276: 40982-40990 (2001). However, signaling events downstream of the coordinated action of these protein kinases that ultimately leads to the exocytotic release of mucin granules have not been fully elucidated.
[0009] MARCKS, a protein of approximately 82 kD, has three evolutionarily-conserved regions (Aderem et al., Nature 1988; 332:362-364; Thelen et al., Nature 1991 ; 351 :320-322; Hartwig et al., Nature 1992; 356:618-622; Seykora et al., J Biol Chem 1996; 271 :18797- 18802): an N-terminus, a phosphorylation site domain (PSD), and a multiple homology 2 (MH2) domain. The N-terminus, a 24 amino acid sequence with a myristic acid moiety attached to a terminal glycine residue is involved in binding of MARCKS to membranes (Seykora et al., J Biol Chem 1996; 271 :18797-18802) and possibly to calmodulin (Matsubara et al., J Biol Chem 2003; 278:48898-48902). This 24 amino acid sequence is known as the MANS peptide. The MANS peptide and active fragments thereof, can compete with native MARCKS in cells for membrane binding. Involvement of MARCKS protein in release of inflammatory mediators from the granules of infiltrating leukocytes is relevant to inflammation in diseases in all tissues and organs, including lung diseases characterized by airway inflammation, such as asthma, COPD and cystic fibrosis. However, inflammation and mucus secretion in the airways are two separate and independent processes (Li et al., J Biol Chem 2001 ; 276:40982-40990; Singer et al., Nat Med 2004; 10:193-196). While mucus production and secretion can be provoked by a number of factors, including mediators released by inflammatory cells, there is no known direct link between excess mucus and inflammation.
[000109] Antisense Oligonucleotide Studies- -To demonstrate further MARCKS as a key signaling component of the mucin secretory pathway, the effect of an antisense oligonucleotide directed against MARCKS on mucin secretion was examined. As illustrated in FIG. 3, this antisense oligonucleotide down-regulated both mRNA and protein levels of MARCKS in NHBE cells and significantly attenuated mucin secretion induced by PMA+8- Br-cGMP, whereas a control oligonucleotide had no effect.
[00079} In one aspect of this invention, administration of a degranulation-inhibiting amount of MANS peptide or an active fragment thereof as described herein to a site of inflammation in a subject, which site of inflammation has resulted from the onset of entry of a disease, a condition, a trauma, a foreign body, or a combination thereof at the site of inflammation in the subject, can reduce the amount of a mediator of inflammation released from infiltrating leukocytes at the site of inflammation, where the leukocytes are preferably granulocytes. The administration of the MANS peptide and/or at least one active fragment thereof can reduce the amount of a mediator of inflammation released from leukocytes such as granulocytes infiltrating into the site of inflammation. The degranulation-inhibiting amount of MANS peptide, or the degranulation-inhibiting amount of an active fragment thereof, is sufficient to reduce or inhibit the exocytotic release of inflammatory mediators from granules contained within the inflammatory cells infiltrating into the site. Degranulation-inhibiting efficacy is measured at a time after administration of the MANS peptide or the fragment thereof by comparison of the percent of inhibition (i.e., percent of reduction) of the release of mediators of inflammation from said cells (leukocytes or granulocytes or other inflammatory cells) relative to the level or amount or concentration of said mediators of inflammation released or produced at approximately the same time in the absence of MANS peptide and/or in the absence of the active fragment thereof. Additionally, a skilled clinician can determine whether inflammation at the tissue site has been reduced by measuring symptoms and parameters of inflammation known as indicators of the disease to determine whether a sufficient or therapeutically effective amount MANS peptide and/or an active fragment thereof has been administered. A sufficient degranulation-inhibiting amount is the percentage of reduction of a mediator of inflammation released from a granulocyte, at the site of inflammation, which is from about 1 % to about 99%, preferably from 5% to about 99%, more preferably from about 10% to about 99%, even more preferably from about 25% to 99%, and even more preferably from about 50% to about 99% of the amount of said mediator of inflammation released from said granulocyte in the absence of MANS peptide or an active fragment thereof tested under the same conditions.

中文:
[0001]本申请要求享有2006年3月6日提交的美国专利申请11/367,449的优先权,后一申请是2004年3月17日提交的美国专利申请10/802,644的部分延续申请,后一申请是2002年6月26日提交、现已放弃的美国专利申请10/180,753的继续申请,后一申请要求享有2001年6月26日提交的美国临时申请60/300,933的优先权,上述申请公开的内容在此完整引入作为参考。
[0002]本发明是在美国联邦政府的资助下完成的,国立卫生研究院项目号R01 HL36982。美国政府对本发明享有一定权利。
[0008]尽管该过程具有明显的病理生理重要性,但只是最近才阐明了连接细胞表面刺激与粘蛋白颗粒释放的胞内信号机制。参见,Li等,Journal ofBiological Chemistry,276:40982-40990(2001)。众所周知多种试剂和炎性/体液介质引起粘蛋白的分泌。这些包括胆碱能激动剂,脂介质,氧化剂,细胞因子,神经肽,ATP和UTP,细菌产物,中性粒细胞弹性蛋白酶,和吸入污染物。参见,Adler等,Res.Immunol.149,245-248(1998).有趣的是,这些粘蛋白促分泌剂中的许多已知还能够活化数种蛋白激酶,利用来自不同种属的气道上皮细胞来检测粘蛋白过量分泌调节的研究一直涉及在分泌过程中使用蛋白激酶C(PKC)或者cGMP-依赖的蛋白激酶(PKG)。参见,例如,Ko等,Am.J.Respir.Cell Mol.Biol.16,194-198(1997);Abdullah等,Am.J.Physiol.273,L201-L210(1997);Abdullah等,Biochem.J.316,943-954(1996);Larivee等Am.J.Respir.Cell Mol.Biol.11,199-205(1994);和Fischer等,Am.J.Respir.Cell Mol.Biol.20,413-422(1999)。只是最近才证明这两种蛋白激酶在调节粘蛋白分泌中的协调相互作用或者“交互作用(cross-talk)”涉及MARCKS蛋白。参见,Li等,Journal of Biological Chemistry,276:40982-40990(2001)。但是,还没有完全阐明这些蛋白激酶协同作用下游的信号转导事件,这最终导致粘蛋白颗粒的胞吐释放。
[0009]MARCKS,一种大约82KD的蛋白,具有3个进化-保守区(Aderem等,Nature 1988;332:362-364;Thelen等,Nature 1991;351:320-322;Hartwig等,Nature 1992;356:618-622;Seykora等,J Biol Chem 1996;271:18797-18802):N-端,磷酸化位点结构域(PSD)和多重同源2(MH2)结构域。所述N-端(24氨基酸序列,具有与末端甘氨酸残基连接的肉豆蔻酸部分)涉及MARCKS与膜的结合(Seykora等,J Biol Chem 1996;271:18797-18802),并可能涉及MARCKS与钙调蛋白的结合(Matsubara等,J Biol Chem 2003;278:48898-48902)。该24氨基酸序列被称为MANS肽。在细胞中MANS肽和其活性片段能够与天然的MARCKS竞争膜结合。MARCKS蛋白在浸润白细胞的颗粒释放炎性介质过程中的参与和所有组织和器官中疾病的炎症有关,包括特征为气道炎症的肺疾病,例如哮喘、COPD和囊性纤维化。但是,气道中炎症和粘液分泌是两个分离并且独立的过程(Li等,J Biol Chem 2001;276:40982-40990;Singer等,Nat Med 2004;10:193-196)。尽管许多因子包括炎性细胞释放的介质都能引起粘液产生和分泌,但是在过量粘液和炎症之间还没有已知的直接联系。
[000109]反义寡核苷酸研究--为了进一步证明MARCKS是粘蛋白分泌途径的关键信号组分,检测了针对MARCKS的反义寡核苷酸对粘蛋白分泌的作用。如图3所示,该反义寡核苷酸下调NHBE细胞中MARCKS的mRNA和蛋白水平,并且显著减弱PMA+8-Br-cGMP诱导的粘蛋白分泌,而对照寡核苷酸没有效果。
[00079]在本发明的一方面,给药脱颗粒抑制量的本文所述MANS肽或者其活性片段到对象的炎症部位(所述炎症部位源于疾病、病症、创伤、异物或者其组合开始进入对象的炎症部位),能够降低炎症部位浸润白细胞释放的炎症介质的量,其中所述白细胞优选的是粒细胞。MANS肽和/或至少一种其活性片段的给药能够降低白细胞例如渗入炎症部位的粒细胞释放的炎症介质的量。脱颗粒抑制量的MANS肽,或者脱颗粒抑制量的其活性片段,足以减少或者抑制炎性介质的胞吐释放,所述炎性介质来自渗入所述部位的炎性细胞内包含的颗粒。在给药MANS肽或者其片段后测量脱颗粒抑制效果,通过比较所述细胞(白细胞或者粒细胞或者其他炎性细胞)释放的炎症介质相对于不存在MANS肽和/或其活性片段的条件下大约相同时间释放或者产生的所述炎症介质的水平或者量或者浓度的抑制百分比(即,降低百分比)。此外,熟练的临床医生通过检查被称为疾病指标的炎症症状和参数能够确定组织部位的炎症是否减轻,从而确定是否已经给药了足够量或者治疗有效量的MANS肽和/或其活性片段。足够的脱颗粒抑制量是炎症部位粒细胞释放的炎症介质的降低百分比为所述粒细胞在不存在MANS肽或者其活性片段、相同条件下测出的释放所述炎症介质的量的从大约1%到大约99%,优选的从5%到大约99%,更优选的从大约10%到大约99%,甚至更优选的从大约25%到99%,并且甚至更优选的从大约50%到大约99%的。